US 20180362598A1 ( 19) United States ( 12) Patent Application Publication (10 ) Pub . No. : US 2018 /0362598 A1 CARLSON et al. ( 43 ) Pub . Date: Dec . 20 , 2018 (54 ) CLEAVABLE PEPTIDES AND INSECTICIDAL CO7K 5 / 103 ( 2006 .01 ) AND NEMATICIDAL PROTEINS CO7K 14 /435 (2006 .01 ) COMPRISING SAME CO7K 14 /325 ( 2006 .01 ) (71 ) Applicant: VESTARON CORPORATION , A01N 63 /02 ( 2006 .01 ) KALAMAZOO , MI (US ) C12N 15 /82 (2006 .01 ) (72 ) Inventors : ALVAR R . CARLSON , (52 ) U . S. CI. KALAMAZOO , MI (US ) ; CPC ...... CO7K 14 /415 (2013 .01 ) ; CO7K 7 / 06 ALEXANDRA M . HAASE , MARTIN , (2013 .01 ) ; CO7K 5 / 101 (2013 . 01 ) ; CO7K MI (US ) ; ROBERT M . KENNEDY , 14 / 43518 (2013 .01 ) ; CO7K 14 / 325 (2013 . 01 ) ; DEXTER , MI (US ) CO7K 2319 / 04 ( 2013 .01 ); C12N 15 /8285 ( 2013 . 01 ) ; C12N 15 /8286 ( 2013 .01 ); COZK ( 73 ) Assignee : VESTARON CORPORATION , 2319 /50 (2013 .01 ) ; CO7K 2319 /55 (2013 .01 ) ; KALAMAZOO , MI (US ) A01N 63 / 02 (2013 .01 ) ( 21 ) Appl. No. : 15 /727 , 277 ( 22 ) Filed : Oct . 6 , 2017 (57 ) ABSTRACT Related U . S . Application Data A peptide comprised of either a binary or a tertiary peptide, (60 ) Provisional application No. 62 /411 , 117 , filed on Oct. the peptide contains at least 4 amino acids and up to a 21 , 2016 . maximum of 16 amino acids, comprised of 2 or 3 different regions, wherein the binary peptides have 2 different regions Publication Classification and the tertiary peptides have 3 different regions; wherein , (51 ) Int . Cl. the peptide can be cleaved by both an animal gut protease CO7K 14 /415 ( 2006 .01 ) and an insect or nematode gut protease . CO7K 7 / 06 ( 2006 .01 ) Specification includes a Sequence Listing .
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FIG . 9 US 2018 /0362598 A1 Dec. 20 , 2018
CLEAVABLE PEPTIDES AND INSECTICIDAL [ 0007 ] Bt proteins, or Bt peptides, are effective insecti AND NEMATICIDAL PROTEINS cides used for crop protection in the form of both plant COMPRISING SAME incorporated protectants and foliar sprays . Commercial for mulations of Bt proteins are widely used to control insects CROSS REFERENCE TO RELATED at the larval stage . Inhibitory cysteine knot motif ( ICK ) APPLICATIONS proteins are also a class of insecticidal peptides which are derived from arthropod origins , such as the venoms of [0001 ] This application claims the benefit of, and priority scorpions or spiders . to , U . S . Provisional Application Ser. No . 62 /411 , 117 , filed [0008 ] Other classes and types of potential peptides have on Oct. 21 , 2016 , the disclosure of which is incorporated by been identified , such as Trypsin modulating oostatic factor reference herein in its entirety . ( TMOF ) peptides. TMOF peptides have to be delivered to their physiological site of action for insecticidal activity to SEQUENCE LISTING ensue . TMOF peptides have been identified as potential 10002 ] This application incorporates by reference in its larvicides, with great potential, see D . Borovsky , Journal of entirety the Sequence Listing entitled “ FAM _ O _ US _ Experimental Biology 206 , 3869 - 3875 , but like nearly all 225312 _ 419533 _ SEQ _ LIST_ 2017 _ 10 _ 06 _ ST25. txt” ( 5 .37 other insecticidal peptides , TMOF has not been commer MB ), which was created on Oct. 6 , 2017 , and filed elec cialized or widely used by farmers and there are reasons for tronically herewith .” this . [ 0009 ] The ability to successfully produce insecticidal FIELD OF THE INVENTION peptides on a commercial scale , with reproducible peptide formation and folding , at a reasonable and economical price , [0003 ] New insecticidal and nematicidal proteins , cleav can be a significant obstacle to commercialization . The wide able peptides, DNA constructs encoding same, transgenic variety , unique properties and special nature of insecticidal proteins , their expression in plants, methods of producing peptides , combined with the huge variety of possible pro the transgenic proteins, new processes , production tech duction techniques, can present an overwhelming number of niques , new peptides, new formulations, and combinations approaches to peptide application and production , but few , if of new and known insecticidal and nematicidal proteins any, are commercially successful. contained within transgenic proteins that produce greater [0010 ] There are several reasons why so few of the mul pest resistance than would be expected of related insecticidal titude insecticidal peptides that have been identified have and nematicidal proteins for the control of insects and ever made it to market . First, most insecticidal peptides are nematodes are described and claimed . either too delicate or not toxic enough to be used commer cially. Second , insecticidal peptides are difficult and costly BACKGROUND to produce commercially . Third , many insecticidal peptides [0004 ] The global security of food produced by modern quickly degrade and have a short half- life . Fourth , very few agriculture and horticulture is challenged by insect pests . insecticidal peptides fold properly when then are expressed Farmers rely on insecticides to suppress insect damage, yet by a plant, thus they lose their toxicity in genetically commercial options for safe and functional insecticides modified organisms (GMOs ) such as genetically modified available to farmers are diminishing through the removal of plants . Fifth , most of the identified insecticidal peptides are dangerous chemicals from the marketplace and the evolution blocked from systemic distribution in the insect and / or lose of insect strains that are resistant to all major classes of their toxic nature when consumed by insects. Bt proteins are chemical and biological insecticides . New insecticides are an exception to this last problem and because they disrupt necessary for farmers to maintain crop protection . insect feeding they have been widely used . [0005 ] Insecticidal and nematicidal peptides, polypeptides [0011 ] The present invention provides several solutions to and proteins are peptide and protein polymers that are toxic these major problems which have prevented commercial to their targets , usually insects or nematodes . The insecti ization and wide spread use of insecticidal peptides. The cidal peptides or toxins commonly derive from arthropod present invention provides transgenic proteins that enable origins such as from scorpions or spiders . The toxins can be the synthesis and release of insecticidal and nematicidal delivered directly to the insect ' s gut or internal organs by toxic peptides in relatively large numbers at the site of injection or by inducing the insect to consume the toxin from activity , i . e . , the gut or other organs of the insect or nema its food , for example an insect feeding upon a transgenic tode. The synthesis in the host is facilitated by directed plant. These toxins have been known to have the ability to expression in regions of the host cell that enables proper inhibit the growth , impair the movement, or even kill an folding of the transgenic protein and enhances synthesis in insect when the toxin is delivered to the insect by spreading the host cell . the toxin to the locus inhabited by the insect or to the insect' s environment by spraying , or other means. Through direct SUMMARY OF THE EXEMPLARY exposure , or ingestion , the insect comes into some form of EMBODIMENTS OF THE INVENTION contact with the toxic peptide, polypeptide or protein . [0012 ] The present invention provides cleavable peptides [0006 ] Insecticidal peptides however have enormous that are fused to toxic insecticidal and / or nematicidal toxic problems reaching the commercial market and to date there peptides and proteins that can be expressed and synthesized have been few if any insecticidal peptides approved and in a host cell , for example a plant cell that is subject to marketed for the commercial market , with one notable consumption by an insect or nematode pest. The cleavable exception , peptides derived from Bacillus thuringiensis or peptides may be cleaved in the gut environment and /or Bt. There is presently , great concern over rising insect hemolymph environment of an insect and / or nematode and resistance to Bt proteins. in the gastrointestinal system of a human being thereby US 2018 /0362598 A1 Dec. 20 , 2018 releasing its payload comprising one or more insecticidal toxic protein and / or a repeat construct ( L - TP ) , or ( TP - L ) , and /or nematicidal toxic peptides and proteins. wherein n is an integer ranging from 1 to 200 , or from 1 to [0013 ] In a first aspect, the present invention provides 100 , or from 1 to 10 . In various embodiments , an exemplary cleavable peptides comprising a binary or a tertiary peptide insecticidal and/ or nematicidal protein can include a protein that contains at least 4 amino acids and up to a maximum of construct comprising : (ERSP ) - ( TP - L ) n , or (ERSP ) - ( L ) - ( TP 16 amino acids, comprised of 2 or 3 different regions. In one L ) , , or ( ERSP ) - ( L - TP ) , or (ERSP ) - ( L - TP ) , - ( L ) , wherein n example , the binary peptides have two different regions ( an is an integer ranging from 1 to 200 . In various related insect or nematode cleavable region and an animal ( e . g . embodiments described above , TP is a toxic protein , L is a human ) gut cleavable region , and the tertiary peptides have binary or tertiary peptide and n is an integer ranging from 1 the same two regions of a binary peptide but also includes to 200 , preferably an integer ranging from 1 to 100 , and one additional region not found in a binary peptide ; and more preferably an integer ranging from 1 to 10 . In some wherein the binary and tertiary peptide can be cleaved by embodiments , the insecticidal and /or nematicidal protein both an animal gut protease and an insect or nematode gut may contain TPs that are the same or different, and binary protease . In some embodiments , the cleavable peptides of and /or tertiary peptides that are the same or different. In the present invention (binary and tertiary cleavable peptides some embodiments , the C - terminal TP is fused or unfused at ( L ) ) cannot be cleaved in a plant cell with an endogenous its C - terminus with a binary or tertiary peptide . In some plant protease , or by a naturally occurring plant protease . embodiments , the N - terminal TP is fused or unfused at its [ 0014 ] In a related embodiment , exemplary binary pep N - terminus with a binary or tertiary peptide . tides comprise an amino acid sequence of X -X2 - Y - Y2, [0017 ] In another embodiment, the present invention pro X -X2 - X3 - Y - Y2, X - X2- X3- X4- Y | - Y2, X7- X2- Y - Y2- Y3, vides an insecticidal and / or nematicidal protein having two X - X2- X3 - Y - Y2- Y3, X7- X2- Xz- X4 - Y1 -Y2 - Y3, X7- X2- Y1 or more cleavable peptides, wherein the insecticidal and/ or Y2- Y3- Y4, X -X2 - X3- Y - Y2- Y3- Y4, X -X2 - X3- X4 - Y - Y2 nematicidal protein comprises a stabilizing domain (STA ) Y3- Y4, Y -Y2 - X , -X2 , Y - Y2 - X -X2 - X3, Y | - Y2 - X -X2 - Xz fused in frame with either the N - terminus of a binary or X4, Y - Y2- Y3- X -X2 , Y - Y - Yz- X , - X - Xz, Y - Y2- Y3- X , tertiary peptide , or the N - terminus of a construct compris X - Xz -X4 , Y - Y2- Y3- Y4- X , -X2 , Y -Y2 - Y3 -Y4 - X - X2- X3 , ing: ( L - TP ) n . ( L - TP ), - L , ( TP - L )n , or L - ( TP - L ) n . In some Y - Y2 - Y3 - Y4- X ,- X2- X2 -X4 , wherein each X , and each Y , is related embodiments, an insecticidal and / or nematicidal an amino acid and wherein X , is selected from the group protein comprises an ERSP fused in frame with the N -ter consisting of: ( all single letter amino acid format) A , D , E , minus of a stabilizing domain (STA ) , which is fused in frame G , I , K , L , N , P , Q , R , and V ; X , is selected from the group with either the N - terminus of a binary or tertiary peptide , consisting of A , F , G , I, K , L , N , P , Q , R , S , T , V , and Y ; X3 being fused to the N - terminus of a toxic protein , or the is selected from the group consisting of A , D , E , F , G , I, K , N -terminus of a construct comprising : ( L - TP )n , ( L - TP ), - L , L , N , O , R , S , T , V , and Y ; X , is selected from the group ( TP - L ) n , or L - ( TP - L ) n . consisting of: R , H , K , D , E , S , T , N , Q , G , A , I, L , M , F , W , [ 0018 ] In various embodiments, an exemplary insecticidal Y , and V . In some embodiments , Y is selected from the and /or nematicidal protein can include a construct compris group consisting of: (all single letter amino acid format ) A , ing: (ERSP ) - (STA ) - ( L - TP ) , or ( ERSP ) - (STA ) - ( L - TP ) ,, - ( L ) , D , E , F , G , I , L , N , Q , S , T , V , and Y ; Y , is selected from the or ( ERSP ) -( STA )- ( TP - L )n , or (ERSP ) -( STA )- ( L )- ( TP -L ) n , group consisting of F , G , I, L , and Y ; and Y2 is selected from or (STA ) - ( L - TP ) n , or (STA ) - ( L - TP ) n - ( L ), or ( STA ) - ( TP - L )n , the group consisting of A , D , E , F , G , I, K , L , R , S , T , V , and or (STA ) - ( L ) - ( TP - L ) . . In some related embodiments , an Y ; and Y4 is selected from the group consisting of: R , H , K , insecticidal and /or nematicidal protein comprises an ERSP D , E , S , T , N , Q , G , A , I, L , M , F , W , Y , and V . fused in frame with either the N - terminus of a binary or [ 0015 ] In another related aspect , a tertiary peptide of the tertiary peptide , being fused to the N -terminus of a toxic present invention contains at least 5 amino acids up to a protein , or the N -terminus of a construct comprising : ( L - TP ) maximum of 16 amino acids; wherein the tertiary peptide m (L - TP ), , - L , ( TP - L ), , or L - ( TP - L ) . . In various embodi comprises a binary peptide and a spacer of 1 to 4 amino ments , an exemplary insecticidal and / or nematicidal protein acids . In some embodiments , the spacer comprises the can include a construct comprising : (ERSP ) -( L - TP )n , or amino acid sequence GS and is fused in frame to the (ERSP ) - ( L - TP ) - ( L ) , or (ERSP ) - ( TP - L ) , or ( ERSP ) - ( L ) N - terminus of a binary peptide , or to the C -terminus of the ( TP - L ) n . In various embodiments as described herein and binary peptide , or both to the N - terminus and the C - terminus above, TP is defined as a toxic protein , that is toxic to an of a binary peptide as disclosed herein . insect and / or a nematode, L is a binary or tertiary peptide [0016 ] In a second aspect , the present invention provides and n is an integer ranging from 1 to 200 , preferably an an insecticidal and /or nematicidal protein having a cleavable integer ranging from 1 to 100 , and more preferably an peptide ( a binary or a tertiary peptide ) fused in frame with integer ranging from 1 to 10 . In some embodiments , the an insecticidal or nematicidal toxic protein . In another C -terminal TP of a construct described herein and above is embodiment, the present invention provides an insecticidal fused or unfused at its C - terminus with a binary or tertiary and / or nematicidal protein having two or more cleavable peptide . In some embodiments , the N - terminal TP is fused or peptides , wherein the insecticidal and /or nematicidal protein unfused at its N - terminus with a binary or tertiary peptide . comprises a binary or tertiary peptide , the binary or tertiary [0019 ] In another related aspect, the present invention peptide is fused in frame with a construct comprising provides a plant, or part thereof comprising an insecticidal ( TP -L )n , wherein n is an integer ranging from 1 to 200 , or and / or nematicidal protein as exemplified herein . In related from 1 to 100 , or from 1 to 10 . In another embodiment, the embodiments , the present invention provides a plant, or part insecticidal and / or nematicidal protein described herein thereof comprising a polynucleotide operably linked to an comprises an endoplasmic reticulum signal peptide ( ERSP ) operable promoter for example , a homologous or heterolo fused in frame with : a binary peptide or tertiary peptide , gous promoter, in a DNA construct, for example , an expres which is fused in frame with an insecticidal or nematicidal sion vector, wherein the polynucleotide encodes : an insec US 2018 /0362598 A1 Dec. 20 , 2018 ticidal and / or nematicidal protein comprising a peptide to 20 , or from 20 to 80 , or from 30 to 60 , or from 30 to 50 comprising either a binary or a tertiary peptide , the binary and preferably around or about or specifically 20 , 25 , 30 , 35 , and / or the tertiary peptide containing at least 4 amino acids 40 , 45 or 50 . In some embodiments , the insecticidal and /or and up to a maximum of 16 amino acids , and is comprised nematicidal protein may contain TPs that are the same or of 2 or 3 differentregions , wherein the binary peptide has 2 different, and binary and /or tertiary peptides that are the different regions and the tertiary peptide has 3 different same or different. In some embodiments , the C -terminal TP regions; wherein , the binary and / or the tertiary peptide can is fused or unfused at its C - terminus with a binary or tertiary be cleaved by both an animal gut protease ( for example a peptide . human gut protease) and an insect or nematode gut protease , [0020 ] In a third aspect , the present invention provides a and wherein the binary or tertiary peptide ( L ) is fused in method of controlling a pest infection or infestation of a frame to a toxic protein ( TP ) . In some embodiments , the plant. The method comprises providing in a diet of said pest, insecticidal and /or nematicidal protein comprises a repeat a plant, or part thereof, or a plant cell , said plant, part construct ( L - TP ) , or ( TP - L ) , wherein n is an integer ranging thereof , or plant cell comprising an insecticidal and /or from 1 to 200 , or from 1 to 100 , or from 1 to 10 . In related nematicidal protein as exemplified herein , and /or a DNA embodiments , the binary and / or the tertiary peptide is oper construct comprising at least one polynucleotide operably ably fused at its N - terminus to the C -terminus of an Endo linked to an operable promoter, for example , a homologous plasmic Reticulum Signal Peptide (ERSP ) and at its C - ter or heterologous promoter, wherein the polynucleotide minus operably fused to the N - terminus of a TP . See FIGS. encodes an insecticidal and /or nematicidal protein having 4 , 5 and 6C . In a related embodiment, the plant or part one or more cleavable binary and /or tertiary peptides. In a thereof, comprises an insecticidal and /or nematicidal protein related embodiment, the plant or part thereof, comprises a and / or a polynucleotide that encodes an insecticidal and /or DNA construct that encodes an insecticidal and / or nemati nematicidal protein as exemplified herein , wherein the cidal protein , wherein the insecticidal and /or nematicidal insecticidal and /or nematicidal protein comprises a binary or protein comprises a binary or tertiary peptide which is fused tertiary peptide, the binary or tertiary peptide is fused in in frame with a construct comprising ( TP - L ), or a repeat frame with a construct comprising ( TP - L ) n , or the insecti construct (L - TP )n , ( L - TP )n - L , ( TP -L ) n , or L -( TP -L )n cidal and /or nematicidal protein comprises a repeat construct wherein n is an integer ranging from 1 to 200 , or from 10 to ( L - TP ) , or ( TP - L ) . , wherein n is an integer ranging from 1 175 , or from 20 to 150 , or from 30 to 100 , or from 40 to 100 , to 200 , or from 1 to 100 , or from 1 to 10 . In a related or from 20 to 70 , or from 20 to 60 , or from 20 to 50 , or from embodiment, the plant or part thereof, comprises an insec 20 to 40 , or from 30 - 40 . In some embodiments , n is an ticidal and / or nematicidal protein and / or a polynucleotide integer ranging from 1 to 20 , or from 5 to 20 , or from 10 to that encodes an insecticidal and /or nematicidal protein as 20 , or from 20 to 80 , or from 30 to 60 , or from 30 to 50 and exemplified herein , wherein the insecticidal and / or nemati preferably around or about or specifically 20 , 25 , 30 , 35 , 40 , cidal protein comprises an ERSP fused in frame with the 45 or 50 . In a related embodiment, the plant or part thereof, N - terminus of a binary or tertiary peptide, the binary or comprises a DNA construct that encodes an insecticidal tertiary peptide is fused in frame with a construct comprising and / or nematicidal protein , wherein the insecticidal and / or ( TP - L )n or the insecticidal and /or nematicidal protein com nematicidal protein comprises an ERSP fused in frame with prises an ERSP fused in frame with a binary peptide and / or the N - terminus of a binary or tertiary peptide , the binary or a repeat construct ( L - TP ) , or ( TP - L ) . . See FIGS . 4 , 5 , and tertiary peptide is fused in framewith a construct comprising 6C . In various embodiments , the exemplary insecticidal ( TP - L ) n or the insecticidal and /or nematicidal protein com and / or nematicidal protein can include the construct: prises an ERSP fused in frame with a binary peptide and / or ( ERSP ) - ( TP - L ) , or (ERSP ) - ( L ) - ( TP - L ) w , or (ERSP ) - ( L - TP ) a repeat construct ( L - TP ) , or ( TP - L ) . . In various embodi m , or ( ERSP ) - ( L - TP ) , - ( L ) . In another embodiment, a plant, ments , the exemplary insecticidal and /or nematicidal protein or part thereof comprises a polynucleotide operably linked can include : ( ERSP )- ( TP - L ) n , or ( ERSP ) - ( L ) - ( TP - L )n , or to an operable promoter, for example , a homologous or (ERSP ) - ( L - TP ) , or ( ERSP ) - ( L - TP ) ,, - ( L ) , wherein n is an heterologous promoter, wherein the polynucleotide encodes integer ranging from 1 to 200 , or from 10 to 175, or from 20 an insecticidal and / or nematicidal protein comprising an to 150 , or from 30 to 100 , or from 40 to 100 , or from 20 to ERSP fused in frame with the N - terminus of a stabilizing 70 , or from 20 to 60 , or from 20 to 50 , or from 20 to 40 , or domain ( STA ) . See FIG . 3 , FIG . 5 , and FIG . 6C . The STA from 30 - 40 . In some embodiments, n is an integer ranging is fused in frame with either the N - terminus of a binary or from 1 to 20 , or from 5 to 20 , or from 10 to 20 , or from 20 tertiary peptide, or the N - terminus of a construct compris to 80 , or from 30 to 60 , or from 30 to 50 and preferably ing : ( L - TP ) , ( L - TP ) , - L , ( TP - L ) . , or L - ( TP - L ) . . In various around or about or specifically 20 , 25 , 30 , 35 , 40 , 45 or 50 . embodiments , the exemplary insecticidal and /or nematicidal 10021] In another embodiment the plant or part thereof, protein can include : (ERSP ) - (STA ) - ( L - TP ) n , or ( ERSP ) comprises a DNA construct that encodes an insecticidal (STA ) -( L - TP )n -( L ), or (ERSP ) -( STA )- ( TP -L )n , or (ERSP ) and /or nematicidal protein , wherein the insecticidal and /or ( STA ) - ( L ) - ( TP - L ) n , or ( STA ) - ( L - TP ) n , or ( STA ) - ( L - TP ) n nematicidal protein comprises an ERSP fused in frame with ( L ) , or (STA )- ( TP - L ) , or (STA ) - ( L ) - ( TP - L ) . . In various the N - terminus of a stabilizing domain (STA ) . The STA is embodiments , TP is toxic protein , which may be an insec fused in frame with either the N - terminus of a binary or ticidal protein and / or a nematicidal protein as described and tertiary peptide , or the N - terminus of a construct compris exemplified in the present disclosure, L is a binary or tertiary ing : ( L - TP ) . , ( L - TP ) . - L , ( TP - L ) , or L - ( TP - L ) . In various peptide and n is an integer ranging from 1 to 200 , or from embodiments , the exemplary insecticidal and / or nematicidal 10 to 175 , or from 20 to 150 , or from 30 to 100 , or from 40 protein can include a construct from N - terminus to C - ter to 100 , or from 20 to 70 , or from 20 to 60 , or from 20 to 50 , minus: (STA ) -( TP -L )n , or ( STA )- (L )- ( TP - L )n , or (STA )- ( L or from 20 to 40 , or from 30 - 40 . In some embodiments , nis TP ) , or (STA ) - ( L - TP ) , ( L ) , wherein n is an integer ranging an integer ranging from 1 to 20 , or from 5 to 20 , or from 10 from 1 to 200 , or from 10 to 175 , or from 20 to 150 , or from US 2018 /0362598 A1 Dec. 20 , 2018
30 to 100 , or from 40 to 100 , or from 20 to 70 , or from 20 portion of the TP from the active or mature form of the TP, to 60 , or from 20 to 50 , or from 20 to 40 , or from 30 -40 . In rendering the cleaved active or mature portion insecticidal some embodiments , n is an integer ranging from 1 to 20 , or and /or nematicidal . In some embodiments , insecticidal and / from 5 to 20 , or from 10 to 20 , or from 20 to 80 , or from 30 or nematicidal proteins comprise a Bt TP and /or a modified to 60 , or from 30 to 50 and preferably around or about or Bt protein , one or more binary and /or tertiary peptides and specifically 20 , 25 , 30 , 35 , 40 , 45 or 50 . In various embodi one or more of an ERSP , and a STA as described above . In ments , the exemplary insecticidal and /or nematicidal protein some exemplary embodiments as shown in FIG . 3B , a can include a construct from N - terminus to C - terminus : modified TP can include a Bt TP , for example , a cry , a cyt ( ERSP ) - (STA ) - ( L - TP ) . , or (ERSP ) - (STA ) - ( L - TP ) ,, - ( L ) , or or a VIP protein , in which the TP inactive is a Bt( inactive ) (the ( ERSP ) - (STA ) - ( TP - L ) n , or (ERSP ) - ( STA ) - ( L ) - ( TP - L ) n , or inactive portion of a Bt TP ) and the TP active is a Bt active ) ( the (STA ) - ( L - TP ) n , or (STA ) - ( L - TP ) - - ( L ) , or (STA ) - ( TP - L ) n , or active portion of a Bt TP ) are separated , and fused in frame (STA ) - ( L ) - ( TP - L ) n . In various embodiments , TP is toxic with one or more binary and / or tertiary peptides designated protein , L is a binary or tertiary peptide and n is an integer as L as shown in FIG . 3B ) . Upon exposure to the gut ranging from 1 to 200 , preferably an integer ranging from 1 environment and/ or hemolymph environment of an insect to 100 , and more preferably an integer ranging from 1 to 10 . and / or nematode and in the gastrointestinal system of an In some embodiments , the insecticidal and /or nematicidal animal subject , for example a human subject , the binary or protein may contain TPs that are the same or different, and tertiary peptide is cleaved , thus separating the inactive binary and / or tertiary peptides that are the same or different. portion of the TP from the active or mature portion of the TP , In some embodiments , the C - terminal TP is fused or unfused rendering the cleaved active or mature portion insecticidal at its C - terminus with a binary or tertiary peptide. In each of and / or nematicidal in the insect and / or nematode . In some the exemplified embodiments above, the pest may be an related embodiments , the modified TP as described above , insect or a nematode as described in the present disclosure . for example , an exemplary Bt TP , may also optionally In various embodiments , the DNA construct can be a vector, include an ERSP fused in frame to the N -terminal portion of for example , a cloning vector or an expression vector the modified TP. containing a coding sequence comprising a polynucleotide encoding an insecticidal and / or nematicidal protein of the BRIEF DESCRIPTION OF THE DRAWINGS present invention and one or more control sequences that permits the replication and / or expression of the coding 100231 FIG . 1 . Demonstration of the FRET molecules in sequence in a host cell , for example , a bacterial cell, a yeast FRET kit ( Cat . No . PSREPLI005 , Mimotopes) to detect the cell or a plant cell. In some of the embodiments above , the specificity of amino acid sequence based cleavage by human binary and /or tertiary peptides of the present invention simulated gastrointestinal fluid (SGF ) and insect gut diges cannot be cleaved in a plant cell by an endogenous plant tive enzymes . This figure is borrowed from the Mimotopes protease , or by a naturally occurring plant protease . FRET kit to illustrate how cleavage generates detectable fluorescence . [ 0022 ] In a fourth aspect, the present invention provides a 10024 ] FIG . 2 . Demonstration of first and second aspects modified toxic peptide or protein ( TP ) that comprises a that are exemplary embodiments of the invention . The first binary or tertiary peptide inserted ( e . g . fused in frame) aspect is the graphical representation of a binary and /or between the active and inactive portions of the toxic peptide . tertiary cleavable linker peptide designated ( L ) . The second In one example , a binary or tertiary peptide is inserted aspect illustrates various combinations of L fused to a toxic between the cleavable pro inactive portion of the peptide and peptide or protein (TP ) ( from 1- 200 repeats ). mature active portion of the peptide . In another example , a modified toxic protein ( TP ) includes a stabilizing domain [0025 ] FIG . 3A . Demonstration of addition of an optional fused in frame with a binary or tertiary peptide of the present stability domain or stability protein ( STA ) , fused to L fused invention which is fused in frame with the active portion of to TP . This figure demonstrates an illustrative usage of a the toxic protein ( TP ) . In another example , the modified STA positioned in the N terminal position and fused in frame toxic protein ( TP ) is a Bacillus thuringiensis or B . thuringi to an L and a TP to form an illustrative STA - L - TP construct . ensis (“ Bt" ) derived protein , known herein as a “ Bt protein " 10026 ] FIG . 3B depicts an embodiment in which a modi and “ Bt peptide ” which are used interchangeably and fied TP comprising a TP inactive portion of the TP is fused in include peptides produced by Bt, collectively referred to frame with a cleavable ( L ) peptide which is fused in frame herein as Bt toxic proteins or “ Bt TPs ” . Such peptides and to the TPortive portion of the TP . As illustrated in FIG . 3B , proteins are frequently written as “ cry ” , “ cyt” or “ VIP ” . upon exposure to the animal and insect and /or nematode gut proteins encoded by the cry , cyt and vip genes. Bt TPs are environment , the cleavable L peptide is cleaved , thus sepa more usually attributed to insecticidal crystal proteins rating the TP inactive portion and the TP active portion , thus encoded by the cry genes . Bt TPs are examples of PFIPS liberating the active portion of the TP in the insect and /or ( Pore Forming Insecticidal Proteins ). In various embodi nematode . In various embodiments , the modified TP is a Bt ments, a modified TP can include a Bt protein as described TP as described herein . above and herein , in which the stabilizing domain (STA ) is [0027 ] FIG . 4 . Demonstration of the Binary or Tertiary fused in frame with a binary or tertiary peptide or a construct Cleavable Linking Peptide in an insecticidal and / or nemati containing one or two or more binary or tertiary peptides of cidal protein with two or more TPs and an ERSP. This figure the present invention . The binary or tertiary peptide is fused illustrates various embodiments from FIG . 2 with the addi in frame with the active fragment of the Bt protein , such that tion of an N - terminal fusion of an ERSP . upon exposure to the gut environment and/ or hemolymph f0028 ] FIG . 5 . Demonstration of the Binary or Tertiary environment of an insect and / or nematode and in the gas Cleavable Linking Peptides of an exemplary insecticidal trointestinal system of a human being , the binary or tertiary and /or nematicidal protein with two or more TPs and an peptide is cleaved , thus separating the stabilizing or inactive ERSP fused to the N - terminal of the STA . This figure US 2018 /0362598 A1 Dec. 20 , 2018 illustrates various embodiments from FIG . 2 , with the addi comprising an exemplary insecticidal and /or nematicidal tion of a STA and an N -terminal fusion of an ERSP to that protein as described in FIG . 6A after transformation by a STA . vector described in FIG . 8 . [ 0029 ] FIG . 6A depicts an illustrative insecticidal and /or nematicidal protein comprising a BAAS ERSP, fused in DETAILED DESCRIPTION OF THE frame to ubiquitin which is fused to a U + 2 - ACTX -Hvla TP INVENTION which is fused to 3 L - TP fusion constructs ( each repeating construct comprising a linking cleavable peptide L , wherein Definitions L is a tertiary cleavable linking peptide , having three regions [0035 ] " ACTX ” or “ ACTX peptide ” means a Family of (XYZ ), and wherein the Z region is the dipeptide glycine insecticidal ICK TPs that have been isolated from an Aus serine GS , and the TP is a U - ACTX - Hvla ) and where the tralian funnel -web spider belonging to the Atracinae sub last TP U -ACTX -Hvla has a fused 10 His tag . family . One such spider is known as the Australian Blue Mountains Funnel -web Spider, which has the scientific [0030 ] FIG . 6B depicts an illustrative insecticidal and /or name Hydronyche versuta . Three examples of ACTX pep nematicidal protein comprising a BAAS ERSP , fused in tides from this species are the Omega , Kappa , and U frame to a 3 repeating construct , the construct comprising a peptides . U -ACTX -Hvla TP fused to a linker ( L ) which may be a [0036 ] “ Agroinfection ” means a plant transformation binary or tertiary cleavable linking peptide . The last method where DNA is introduced into a plant cell by using U - ACTX - Hvla TP has a 10 His tag . Agrobacteria A . tumefaciens or A . rhizogenes . [0031 ] FIG . 6C depicts a graphical representation of an [0037 ] “ BAAS ” means barley alpha - amylase signal pep illustrative insecticidal and /or nematicidal protein shown in tide. It is an example of an ERSP . the form of a construct, wherein each depiction of the ERSP , [0038 ] “ Binary vector” or “ binary expression vector ” STA , ( TP - L ) and TP are fused in frame. In this construct, means an expression vector which can replicate itself in both ERSP is fused to the N -terminus of a STA domain , which is E . coli strains and Agrobacterium strains. Also , the vector fused to a repeating construct, the construct comprises units contains a region of DNA (often referred to as t - DNA ) of TP fused to a linker peptide ( L ) , wherein each TP in the bracketed by left and right border sequences that is recog repeating construct may be the same or different and each L nized by virulence genes to be copied and delivered into a in the repeating construct may be the same or different. plant cell by Agrobacterium . Finally , the last repeating unit of the TP - L construct is fused [0039 ] “ Bt, ” also known as Bacillus thuringiensis or B . to the N -terminus of a TP. In this exemplary embodiment, N . thuringiensis, means a gram - positive soil bacterium that has can be 1 to 10 repeating units , for example , 1 , 2 , 3 , 4 , 5 , 6 , been used worldwide for more than sixty years to control 7 , 8 , 9 , or 10 units , such that upon cleavage in an insect agricultural, forestry , and public health insect pests . and / or nematode gut environment, each L is cleaved , thus [ 0040 ] “ Bt proteins” and “ Bt peptides” are used inter releasing multiple copies of TP which is insecticidal and /or changeably and include peptides produced by Bt are collec nematicidal to the insect or nematode that ingested the tively referred to herein as Bt toxic proteins or “ Bt TPs” . insecticidal and/ or nematicidal protein . Such peptides are frequently written as “ cry ” , “ cyt” or “ VIP ” [0032 ] FIG . 7 . Demonstration of detection of cleavage of proteins encoded by the cry , cyt and vip genes . Bt TPs are the tertiary linking peptides , ALKLFVGS (SEQ ID NO : more usually attributed to insecticidal crystal proteins 1660 ) and IFVRLRGS (SEQ ID NO : 1674 ) , in all digestive encoded by the cry genes. Bt TPs are examples of PFIPS enzyme conditions compared to the control peptide , IGER , ( Pore Forming Insecticidal Proteins ) see definition below . which only cleaves in the CEW gut extract. This figure Examples of PFIPS and other Bt proteins are provided in the contains the three digestive enzyme conditions ( rows ) and sequence listing . three different proteins ( columns ) that each contain four TP [0041 ] “ Chimeric gene ” means a DNA sequence that units ( U - ACTX -Hvla ) with intervening tertiary linking pep encodes a gene derived from portions of one or more coding tides or the control peptide , IGER . The cleavage reactions sequences to produce a new gene . are stopped by the addition of denaturing conditions ( chemi [0042 ] “ Cleavable Linker ” see Linker . cal and heat ) at the time points listed below each gel image . [0043 ] “ Conditioned medium ” means the cell culture A His -tagged western blot detection illustrates the digestion medium which has been used by cells and is enriched with and disappearance of the protein band for tertiary peptides cell derived materials but does not contain cells . ALKLFVGS (SEQ ID NO : 1660 ) and IFVRLRGS ( SEO ID [0044 ] “ Conversion ” or “ converted ” refers to the process NO : 1674 ) in all digestive conditions, or the retention of the ofmaking an HP peptide . intact protein band across the time points for the control 100451 “ CRIP ” and “ CRIPS ” are abbreviations for Cyste peptide IGER in both the SCR gut extract and SGF. ine Rich Insecticidal Protein and Proteins respectively . Cys teine rich insecticidal peptides (CRIPS ) are peptides rich in [ 0033 ] FIG . 8 . An example of a stable corn transformation cysteine which form disulfide bonds . CRIPS contain at least vector . This figure depicts one example of a stable corn four ( 4 ) sometimes six (6 ) and sometimes eight (8 ) cysteine transformation vector. The white arrows depict the basic amino acids among proteins or peptides having at least 10 backbone components of the vector. The black arrows depict amino acids where the cysteines form two ( 2 ) , three ( 3 ) or the T -DNA portion of the vector which is transformed into four ( 4 ) disulfide bonds. The disulfide bonds contribute to the corn genome and results in the selection and regenera the folding , three - dimensional structure , and activity of the tion of transgenic corn plants expressing the insecticidal insecticidal peptide. The cysteine -cysteine disulfide bonds protein containing cleavable linking peptides. and the three dimensional structure they form play a sig [ 0034 ] FIG . 9 . An example of efficacy data of a field trial nificant role in the toxicity of these insecticidal peptides . A of corn plants expressing the open reading frame (ORF ) CRIP is exemplified by both inhibitory cysteine knot toxic US 2018 /0362598 A1 Dec. 20 , 2018 proteins or ICK TPs (usually having 6 -8 cysteines ) and by [0056 ] “GFP ” means a green fluorescent protein from the examples of toxic peptides having disulfide bonds but that jellyfish Aequorea victoria . It is an example of a transla are not considered ICK TPs (Non - ICK CRIPS ) . Examples of tional stabilizing protein . an ICK would be an ACTX peptide from a spider and [0057 ] “ High Production peptide” or “ HP peptide ” means defined above. Representative example of ICK proteins a peptide which is capable of being made , or is “ converted , " include U -ACTX insecticidal peptides, Omega -ACTX according to the procedures described herein and which , insecticidal peptides and Kappa - ACTX insecticidal pep once converted can be produced at increased yields , or tides . Examples of a Non - ICK CRIP would be a peptide like higher rates of production , or in greater than normal Av2 and Av3 which are toxic peptides first identified from amounts , in a biological system . The higher rates of pro sea anemones . These peptides are examples of a class of duction can be from 20 to 400 % or greater than can be compounds that modulate sodium channels in the insect achieved with a peptide before conversion , using the same peripheral nervous system (PNS ) . Non - ICK CRIPS can have or similar production methods that were used to produce the 4 - 8 cysteines which form 2 - 4 disulfide bonds. These cyste peptide before conversion . ine -cysteine disulfide bonds stabilized toxic peptides [0058 ] “ Homology ” when used in relation to nucleic acids ( CRIPS ) can have remarkable stability when exposed to the refers to a degree of complementarity . There may be partial environment. Many CRIPS are isolated from venomous homology or complete homology ( i. e ., identity ). “ Sequence animals such as spiders, scorpions, snakes, sea snails and sea identity ” refers to a measure of relatedness between two or anemones and they are toxic to insects . Additional descrip more nucleic acids or proteins , and is given as a percentage tion is provided below . with reference to the total comparison length . The identity [ 00461 “ Defined medium ” means a medium that is com calculation takes into account those nucleotide or amino acid posed of known chemical components but does not contain residues that are identical and in the same relative positions crude proteinaceous extracts or by -products such as yeast in their respective larger sequences . Calculations of identity extract or peptone . may be performed by algorithms contained within computer [0047 ] “ Disulfide bond ” means a covalent bond between programs such as “ GAP ” (Genetics Computer Group , Madi two cysteine amino acids derived by the coupling of two son , Wis . ) and “ ALIGN ” (DNAStar , Madison , Wis . ). A thiol groups on their side chains . partially complementary sequence is one that at least par [ 0048 ] “ Double transgene peptide expression vector” or tially inhibits (or competes with ) a completely complemen " double transgene expression vector” means a yeast expres tary sequence from hybridizing to a target nucleic acid is sion vector which contains two copies of the insecticidal referred to using the functional term “ substantially homolo peptide expression cassette . gous. ” The inhibition of hybridization of the completely [0049 ] “ ELISA ” or “ ELISA ” means a molecular biology complementary sequence to the target sequence may be protocol in which the samples are fixed to the surface of a examined using a hybridization assay ( Southern or Northern plate and then detected as follows: a primary antibody is blot, solution hybridization and the like ) under conditions of applied followed by a secondary antibody conjugated to an low stringency . A substantially homologous sequence or enzyme which converts a colorless substrate to colored probe will compete for and inhibit the binding ( i . e . , the substrate which can be detected and quantified across hybridization ) of a sequence which is completely homolo samples . During the protocol, antibodies are washed away gous to a target under conditions of low stringency . This is such that only those that bind to their epitopes remain for not to say that conditions of low stringency are such that detection . The samples, in our hands , are proteins isolated non - specific binding is permitted ; low stringency conditions from plants , and ELISA allows for the quantification of the require that the binding of two sequences to one another be amount of expressed transgenic protein recovered . a specific (i . e ., selective ) interaction . The absence of non [ 0050 ] “ Expression ORF” means a nucleotide encoding a specific binding may be tested by the use of a second target protein complex and is defined as the nucleotides in the which lacks even a partial degree of complementarity ( e . g . , ORF. less than about 30 % identity ) ; in the absence of non -specific [0051 ] “ ER ” or “ Endoplasmic reticulum ” is a subcellular binding the probe will not hybridize to the second non organelle common to all eukaryotes where some post trans complementary target . lation modification processes occur. [0059 ] In preferred embodiments, hybridization condi [0052 ] “ ERSP ” or “ Endoplasmic reticulum signal pep tions are based on the melting temperature ( Tm ) of the tide ” is an N -terminus sequence of amino acids that during nucleic acid binding complex and confer a defined “ strin protein translation of the mRNA molecule encoding an gency ” The term “ hybridization ” refers to the pairing of insecticidal and / or nematicidal protein is recognized and complementary nucleic acids. Hybridization and the bound by a host cell signal -recognition particle , which strength of hybridization ( i. e ., the strength of the association moves the protein translation ribosome/ mRNA complex to between the nucleic acids ) is impacted by such factors as the the ER in the cytoplasm . The result is the protein translation degree of complementary between the nucleic acids, strin is paused until it docks with the ER where it continues and gency of the conditions involved , the Tm of the formed the resulting protein is injected into the ER . hybrid , and the G : C ratio within the nucleic acids . A single [ 0053 ] “ ersp ” means a nucleotide encoding the peptide , molecule that contains pairing of complementary nucleic ERSP . acids within its structure is said to be “ self -hybridized .” [0054 ] “ ER trafficking ” means transportation of a cell [0060 ] The term “ Tm ” refers to the “ melting temperature ” expressed protein into ER for post -translational modifica of a nucleic acid . Themelting temperature is the temperature tion , sorting and transportation . at which a population of double - stranded nucleic acid mol [ 0055 ] “ FECT” means a transient plant expression system ecules becomes half dissociated into single strands. The using Foxtail mosaic virus with elimination of coating equation for calculating the Tm of nucleic acids is well protein gene and triple gene block . known in the art. As indicated by standard references , a US 2018 /0362598 A1 Dec. 20 , 2018
simple estimate of the Tm value may be calculated by the Funnel -web Spider , Hydronyche versuta , and is a dual equation : Tm = 81 . 5 + 0 .41 % G + C ) , when a nucleic acid is in antagonist to insect voltage -gated Ca2 + channels and volt aqueous solution at 1 M NaCl. The term “ stringency ” refers age - gated K + channels . to the conditions of temperature , ionic strength , and the [0066 ] “ IGER ” means a name for a short peptide , based on presence of other compounds such as organic solvents , its actual sequence of one letter codes. It is an example of a under which nucleic acid hybridizations are conducted . With peptide that is only cleaved in insects . “ high stringency ” conditions, nucleic acid base pairing will [0067 ] “ ICK motif, ” “ ICK motif protein ,” “ ICK polypep occur only between nucleic acid fragments that have a high tides” , “ ICK proteins” , “ inhibitor cystine knot motif, ” frequency of complementary base sequences. Thus , condi “ Toxic insect ICK TPs, " " ICK motif TPs, ” “ CK ” peptides, " tions of “ low ” stringency are often required with nucleic " cystine knot motif ," or " cystine knot peptides ” (all used acids that are derived from organisms that are genetically interchangeably herein ), means a 16 to 60 amino acid diverse , as the frequency of complementary sequences is peptide with at least 6 half - cystine core amino acids having usually less . three disulfide bridges , wherein the 3 disulfide bridges are 10061] “ Low stringency conditions” when used in refer covalent bonds and of the six half -cystine residues the ence to nucleic acid hybridization comprise conditions covalent disulfide bonds are between the first and fourth , the equivalent to binding or hybridization at 42° C . in a solution second and fifth , and the third and sixth half- cystines , of the consisting of 5xSSPE (43 . 8 g / 1 NaC1, 6 . 9 g / l NaH2PO4. H20 six core half -cystine amino acids starting from the N - termi and 1 .85 g / l EDTA , pH adjusted to 7 . 4 with NaOH ) , 0 . 1 % nal amino acid . In general this type of peptide comprises a SDS, 5xDenhardt' s reagent [50xDenhardt ' s contains per beta -hairpin secondary structure , normally composed of 500 ml: 5 g Ficoll ( Type 400 , Pharmacia ), 5 g BSA (Fraction residues situated between the fourth and sixth core half V ; Sigma ) ] and 100 ug /mL denatured salmon sperm DNA cystines of the motif , the hairpin being stabilized by the followed by washing in a solution comprising 5xSSPE , structural crosslinking provided by the motif s three disul 0 . 1 % SDS at 42° C . when a probe of about 500 nucleotides fide bonds. Note that additional cysteine / cystine or half in length is employed . cystine amino acids may be present within the inhibitor [0062 ] “Medium stringency conditions” when used in cystine knot motif. reference to nucleic acid hybridization comprise conditions 10068] Examples are provided in the sequence listing . equivalent to binding or hybridization at 42° C . in a solution Representative example of ICK proteins include U - ACTX consisting of 5xSSPE (43 .8 g / l NaC1, 6 .9 g /1 NaH2PO4 .H2O insecticidal peptides, Omega - ACTX insecticidal peptides and 1 .85 g /1 EDTA , pH adjusted to 7 . 4 with NaOH ), 0 .5 % and Kappa -ACTX insecticidal peptides. SDS, 5xDenhardt' s reagent and 100 ug /mL denatured [0069 ] “ ick ” means a nucleotide encoding an ICK motif salmon sperm DNA followed by washing in a solution protein . comprising 10xSSPE , 1. 0 % SDS at 42° C . when a probe of 10070 ] " ICK motif protein expression ORF ” or “ expres about 500 nucleotides in length is employed . sion ORF ” means a nucleotide encoding an ICK motif [0063 ] " High stringency conditions” when used in refer protein complex and is defined as the nucleotides in the ence to nucleic acid hybridization comprise conditions ORF . equivalent to binding or hybridization at 42° C . in a solution [0071 ] “ ICK motif protein expression vector” or “ ICK consisting of 5 .times . SSPE (43 . 8 g /l NaCl, 6 . 9 g /1 expression vector” , or “ ICK motif expression vector, " NaH2PO4 .H20 and 1. 85 g /1 EDTA , pH adjusted to 7 .4 with means a binary vector which contains an expression ORF . NaOH ) , 0 . 5 % SDS , 5xDenhardt ' s reagent and 100 ug /mL The binary vector also contains the necessary transcription denatured salmon sperm DNA followed by washing in a promoter and terminator sequence surrounding the expres solution comprising 0 . 1 XSSPE , 1 . 0 % SDS at 42° C . when a sion ORF to promote expression of the ORF and the protein probe of about 500 nucleotides in length is employed . it encodes . [0064 ] It is well known that numerous equivalent condi [ 0072 ] “ Insect includes all organisms in the class tions may be employed to comprise low stringency condi “ Insecta ” . The term “ pre - adult ” insects refers to any form of tions ; factors such as the length and nature (DNA , RNA , base composition ) of the probe and nature of the target an organism prior to the adult stage , including , for example , (DNA , RNA , base composition , present in solution or eggs , larvae, and nymphs . immobilized , etc . ) and the concentration of the salts and [0073 ] “ Insect gut environment” or “ gut environment” other components ( e . g . , the presence or absence of forma means the specific pH and protease conditions found within mide, dextran sulfate , polyethylene glycol) are considered the fore, mid or hind gut of an insect or insect larva . and the hybridization solution may be varied to generate [ 0074 ] “ Insect hemolymph environment” means the spe conditions of low stringency hybridization different from , cific pH and protease conditions of found within an insect or but equivalent to , the above listed conditions. In addition , insect larva . the art knows conditions that promote hybridization under [ 0075 ] As used herein , the term “ insecticidal ” is generally conditions of high stringency (e . g ., increasing the tempera used to refer to the ability of a polypeptide or protein used ture of the hybridization and /or wash steps, the use of herein , to increase mortality or inhibit growth rate of insects . formamide in the hybridization solution , etc . ). As used herein , the term “ nematicidal” refers to the ability [0065 ] “ Hybrid peptide ,” aka “ Hybrid ”, aka “ hybrid of a polypeptide or protein used herein , to increase mortality toxin , ” aka " hybrid -ACTX - Hvla ,” aka " native hybri or inhibit the growth rate of nematodes. In general, the term DACTX -Hvla , " as well as “ U peptide, " aka “ U toxin ," aka “ nematode” comprises eggs , larvae , juvenile and mature " native U , ” aka “ U - ACTX -Hvla , " aka " native U -ACTX forms of said organism . Hvla ,” all refer to an ACTX peptide, which was discovered [0076 ] “ Insecticidal activity ” means that on or after expo from a spider known as the Australian Blue Mountains sure of the insect to compounds or peptides, the insect either US 2018 /0362598 A1 Dec. 20 , 2018
dies stops or slows its movement or its feeding, stops or which are peptides first identified from sea anemones. These slows its growth , fails to pupate , cannot reproduce or cannot anemone peptides are examples of a class of compounds that produce fertile offspring . modulate sodium channels in the insect peripheral nervous [0077 ] “ Insecticidal peptide ” or “ Insecticidal protein " or system ( PNS ) . " toxic polypeptide” or “ toxic protein ” ( used interchangeably [0089 ] “ Non - Polar amino acid ” is an amino acid that is herein ) means a protein or polypeptide having insecticidal weakly hydrophobic and includes glycine , alanine, proline , activity when ingested by, in contactwith , or injected into an valine, leucine, isoleucine , phenylalanine and methionine . insect. Glycine or gly is the most preferred non - polar amino acid for [ 0078 ] " Insecticidal peptide production strain screen " the dipeptides of this invention . means a screening process that identifies the higher- yielding [0090 ] “ Normalized peptide yield ” means the peptide insecticidal peptide production yeast strains from the lower yield in the conditioned medium divided by the correspond yielding strains. In the described methods herein , it refers to ing cell density at the point the peptide yield is measured . screens that use reverse - phase HPLC or the housefly injec The peptide yield can be represented by the mass of the tion bioassay . produced peptide in a unit of volume, for example, mg per [0079 ] “ Integrative expression vector” or “ integrative vec liter or mg/ L , or by the UV absorbance peak area of the tor” means a yeast expression vector which can insert itself produced peptide in the HPLC chromatograph , for example , into a specific locus of the yeast cell genome and stably mAu .sec . The cell density can be represented by visible light becomes a part of the yeast genome. absorbance of the culture at wavelength of 600 nm (OD600 ) . [ 0080 ] “ Known peptide ” means a peptide known to have [ 0091 ] " One letter code” means the peptide sequence biological activity and may be a mature peptide or any which is listed in its one letter code to distinguish the various version or fragment thereof including pre and pro peptides amino acids in the primary structure of a protein . alanine = A , and conjugates of active peptides. A preferred known pep arginine = R , asparagine = N , aspartic acid = D , asparagine or tide is one with insecticidal activity . aspartic acid = B , cysteine = C , glutamic acid = E , [0081 ] “ L ” in the proper contextmeans a cleavable inter glutamine = Q , glutamine or glutamic acid = Z , glycine = G , vening binary or tertiary peptide . When referring to amino histidine = H , isoleucine = I , leucine = L , lysine = K , acids , “ L ” can also mean leucine . methionine = M , phenylalanine = F , proline = P , serine = S , [ 0082] “ Linker, Cleavable Linker , or Peptide Linker” threonine = T , tryptophan = W , tyrosine = Y , valine = V . means a short peptide sequence ( a binary or tertiary peptide ) [0092 ] " Omega peptide ” also referred to herein as “ omega that is the target site of at least two types of proteases one toxin , ” which includes the example , " omega - ACTX -Hvla , " of which is an insect and / or nematode protease and the other aka “ native omegaACTX - Hvla ,” all refer to a class of one of which is a human protease such that the linker can be ACTX peptides which were first isolated from a spider separated by both types of protease that can cleave and known as the Australian Blue Mountains Funnel- web Spi separate the protein into two parts or a short DNA sequence der , Hydronyche versuta , and which is an antagonist to the that is placed in the reading frame in the ORF and encoding insect voltage - gated Ca2 + channel. a short peptide sequence in the protein that is the target site [0093 ] “ ORF ” or “ Open reading frame” or “ peptide of an insect and / or nematode and an animal ( e . g . human ) expression ORF ” means that DNA sequence encoding a protease that can cleave and separate the protein into two protein which begins with an ATG start codon and ends with parts . a TGA , TAA or TAG stop codon . ORF can also mean the [0083 ] “ Lepidopteran gut environment” means the spe translated protein that the DNA encodes . cific pH and protease conditions of found within the fore , mid or hind gut of a lepidopteran insect or larva . [0094 ] “ Operably linked ” means that the two adjacent [0084 ] “ Lepidopteran hemolymph environment” means DNA sequences are placed together such that the transcrip the specific pH and protease conditions of found within tional activation of one can act on the other. lepidopteran insect or larva . [0095 ] “ PEP ” means Plant Expressed Peptide . 100851 “ Multiple ICK motif protein domain ” means a [ 0096 ] “ Peptide expression cassette ” , or " expression cas protein composed of multiple ICK motif TPs which are sette ” means a DNA sequence which is composed of all the linked by multiple intervening linker peptides. The ICK DNA elements necessary to complete transcription of an motif TPs in the multiple ICK motif TP domain can be same insecticidal peptide in a biological expression system . In the or different , and the intervening linker peptides in this described methods herein , it includes a transcription pro domain can also be the same or different. moter, a DNA sequence to encode an a -mating factor signal [0086 ] “Nematicidal peptide” or “ nematicidal protein ” sequence and a Kex 2 cleavage site , an insecticidal peptide means a peptide or protein having nematicidal activity when transgene, a stop codon and a transcription terminator. ingested by, in contact with , or injected into a nematode and [0097 ] “ Peptide expression vector” means a host organism is used as an example of a toxic peptide and toxic protein . expression vector which contains a heterologous insecticidal [0087 ] “ Nematicides” and “ nematicidal” refers to the abil peptide transgene . ity of a nematicidal peptide or nematicidal protein to [ 0098 ] “ Peptide expression yeast strain ” , “ peptide expres increase mortality or inhibit the growth rate of nematodes. In sion strain ” or “ peptide production strain ” means a yeast general, the term “ nematode ” comprises eggs , larvae , juve strain which can produce a heterologous insecticidal pep nile and mature forms of said organism . tide. 10088 ] “ Non - ICK CRIPS ” can have 4 - 8 cysteines which [ 0099 ] “ Peptide made special” means a peptide previously form 2 - 4 disulfide bonds. Non - ICK TPs include cystine knot having low peptide yield from a biological expression peptides that are not ICK TPs. Non - ICK TPs may have system that becomes an HP peptide because of the methods different connection orders of the cystine bonds than ICKs. described herein used to increase its yield . Examples of a Non - ICK CRIP are peptides like Av2 and Av3 [ 0100 ] “ Peptide Linker ” see Linker. US 2018 /0362598 A1 Dec. 20 , 2018
[0101 ] “ Peptide transgene” or “ insecticidal peptide trans - described herein , such as for example “GS .” In some gene ” means a DNA sequence that encodes an insecticidal embodiments , the so called tertiary peptides of the present peptide and can be translated in a biological expression invention have three regions , they are : the X and Y region system . and the third region is called either a “ spacer” or “ Z ” region . [0102 ] “ Peptide yield ” means the insecticidal peptide con In some embodiments , each of X and Y can be 2 - 4 amino centration in the conditioned medium which is produced acids in length , and Z can be an additional 1 -4 amino acids from the cells of a peptide expression yeast strain . It can be in length per Z with 1 or 2 Z regions possible if one Z is represented by the mass of the produced peptide in a unit of before and one Z is after the binary peptide, thus allowing volume, for example , mg per liter or mg/ L , or by the UV for a 16 amino acid linker . absorbance peak area of the produced peptide in the HPLC [0114 ] “ STA ” , or “ Translational stabilizing protein ” , or chromatograph , for example , mAu . sec . “ stabilizing domain " , or “ stabilizing protein ” , (used inter [ 0103] “ Peritrophic membrane” means a lining inside the changeably herein ) means a protein with sufficient tertiary insect gut that traps large food particles can aid in their structure that it can accumulate in a cell without being movement through the gut while allowing digestion , but also targeted by the cellular process of protein degradation . The protecting the gut wall . protein can be between 5 and 50 amino acids ( aa ) . In some [ 0104 ] “ Pest " includes, but is not limited to : insects , fungi, embodiments used throughout the exemplary embodiments bacteria , nematodes, mites, ticks , and the like . herein , a STA may include an ICK -motif protein ), 50 to 250 0105 “ PFIP ” means a protein that can form a pore or aa (GNA ) , 250 to 750 aa ( e . g ., chitinase ) and 750 to 1500 aa channel in the cells that line an insect gut, such as gut ( e . g . , enhancin ) . The translational stabilizing protein is epithelium cells. Examples of PFIPS are Bt toxic peptides coded by a DNA sequence for a protein that is fused in frame (Bt TPs) such as cry , crt and VIP other PFIP examples can with a sequence encoding an insecticidal protein in the ORF . be found in the sequence listing . The fusion protein can either be upstream or downstream of [0106 ] “ Plant regeneration media ” means any media that the toxic protein and can have any intervening sequence contains the necessary elements and vitamins for plant between the two sequences as long as the intervening growth and plant hormones necessary to promote regenera sequence does not result in a frame shift of either DNA tion of a cell into an embryo which can germinate and sequence . The translational stabilizing protein can also have generate a plantlet derived from tissue culture . Often the an activity which increases delivery of the ICK motif TP media contains a selectable agent to which the transgenic across the gut wall and into the hemolymph of the insect . cells express a selection gene that confers resistance to the Such a delivery can be achieved by actively trafficking the agent. entire ORF across the gut wall , or by cleavage within the gut [0107 ] “ Plant transgenic protein ” means a protein from a environment to separate the ICK motif TP while the trans heterologous species that is expressed in a plant after the lational stabilizing protein damages the peritrophic mem DNA or RNA encoding it was delivered into one or more of brane and /or gut wall to increase diffusion of the ICK motif the plant cells. TP into the hemolymph . [0108 ] “ Polar amino acid ” is an amino acid that is polar [0115 ] " sta ” means a nucleotide encoding a translational and includes serine , threonine , cystine , cysteine , asparagine , stabilizing protein . glutamine, histidine , tryptophan and tyrosine ; preferred [011 ] “ TMOF ” “ TMOF motif, ” or “ TMOF TPs” means polar amino acids are serine , threonine , cystine , cysteine , “ trypsin modulating oostatic factor ” protein sequences. asparagine and glutamine; with serine being most highly Examples are provided in the sequence listing . Numerous preferred . examples and variants are provided herein . SEQ ID NO : 708 [ 0109 “ Post- transcriptional gene silencing ” , or “ PTGS ” , is the wild type TMOF sequence . Other non - limiting vari means a cellular process within living cells that suppress the ants are provided in SEQ ID NOs: 709 -721 . Other examples expression of a gene . would be known or could be created by one skilled in the art. [0110 ] “ Protein ” has the samemeaning as “ Peptide” in this [0117 ] “ Toxic peptide” or “ toxic protein ” or “ TP ” (all used document. interchangeably ) means a peptide or protein having insec [0111 ] “ Recombinant vector ” means a DNA plasmid vec ticidal and / or nematicidal activity when ingested by , in tor into which foreign DNA has been inserted . contact with , or injected into an insect or nematode respec [0112 ] “ Selection gene ” means a gene which confers an tively . TPs of the present invention also include modified advantage for a genomically modified organism to grow TPs, mutant TPs or derivative TPs . In some embodiments , a under the selective pressure . mutant or variant TP encompasses a TP that may have one [0113 ] “ Spacer ” or “ spacers ” means a peptide comprising or more conservative amino acid substitutions, that when 1 to 4 amino acids that is fused in frame to either the combined , the conservative amino acid substitutions do not N - terminus of a binary peptide , or to the C - terminus of the substantially diminish the insecticidal and / or nematicidal binary peptide , or both to the N - terminus and the C - terminus activity of the mutated TP in relation to its non -mutated TP , of a binary peptide as is disclosed herein , thus forming a i . e . retains the insecticidal and / or nematicidal activity of at tertiary peptide . Tertiary peptides differ from binary peptides least 90 % , or at least 80 % , or at least 70 % , or at least 60 % , in that they are like a binary peptide only with an additional or at least 50 % , or at least 40 % , or at least 30 % of the region called a spacer comprising any 1 to 4 amino acids, insecticidal and / or nematicidal activity of the non -mutated more often 2 to 4 amino acids, sometimes 1 , sometimes 2 , TP . TPs may also include peptides and proteins encoded by sometimes 3 and sometimes 4 amino acids and is also a polynucleotide having one or more point mutations , for sometimes called the “ Z ” region . In some specific embodi- example , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 point mutations or ments , the spacer may be referred to as “ + 2” or “ plus - 2” more , such that when the mutated TP encoding polynucle which is in reference to the portion of the peptide that is a otide is transcribed and / or translated , the expressed mutated dipeptide comprising any two amino acid peptides as TP peptide or protein retains insecticidal and /or nematicidal US 2018 /0362598 A1 Dec. 20 , 2018 activity that is at least 90 % , or at least 80 % , or at least 70 % , [0126 ] “ U + 2 peptide, ” “ U + 2 protein ” , “ U + 2 toxin ," or or at least 60 % , or at least 50 % , or at least 40 % , or at least “ U + 2 ,” or “ U + 2 - ACTX - Hvla ,” all refer to either a toxin , 30 % of the insecticidal and/ or nematicidal activity of the which has an additional dipeptide operatively linked to the peptide or protein encoded by the corresponding non -mu native peptide, and may refer to the spider toxin which is tated TP polynucleotide . TPs of the present invention may sometimes called the U peptide and other names noted also include TP peptides and proteins that have at least 80 % , above . The additional dipeptide that is operatively linked to or at least 85 % , or at least 90 % , or at least 95 % , or at least the U peptide and thus indicated as “ + 2 ” or “ plus 2 ” can be 96 % or at least 97 % or at least 98 % or at least 99 % amino selected among several peptides , any of which may result in acid sequence identity to any TP disclosed in SEQ ID NO : a “ U + 2 peptide” with unique properties as discussed herein . 5 - 1593 and 1761- 1775 and 1761 - 1775 , and still retains at These are also sometimes called “ high production peptides ." least at least 90 % , or at least 80 % , or at least 70 % , or at least When the term “ U + 2 - ACTX - Hvla ” is used , it refers to a 60 % , or at least 50 % , or at least 40 % , or at least 30 % of the specific high production toxic peptide , comprising a natu insecticidal and / or nematicidal activity of the corresponding rally occurring peptide from the Australian Blue Mountains TP set forth in SEQ ID NO : 5 - 1593 and 1761- 1775 . TPs of Funnel -web Spider , which has the scientific name Hydro the present invention may also include TP peptides and nyche versuta . proteins having 1 - 6 amino acid extensions fused in frame to [0127 ] “ VIP ” proteins were discovered from screening the the N - terminus or C - terminus or both termini of any TP supernatant of vegetatively grown strains of Bt for possible having an amino acid sequence as set forth in SEQ ID NOs: insecticidal activity . They have little or no similarity to cry 5 - 1593 and 1761- 1775 . proteins and they were named Vegetative Insecticidal Pro [0118 ] “ TSP ” or “ total soluble protein ” means the total teins or VIP . Of particular use and preference for use with amount of protein that can be extracted from a plant tissue this document are what have been called VIP3 , Vip3 proteins sample and solubilized into the extraction buffer. or Vip toxins which have Lepidopteran activity . They are [0119 ] “ Transgene” means a heterologous DNA sequence thought to have a similar mode of action as Bt cry peptides . encoding a protein which is transformed into a plant. In this document VIP proteins are categorized as a PFIP type 10120 ] " Transgenic host cell ” means a cell which is trans of protein . 10128 ] “ Yeast expression vector, " or " expression vector” , formed with a gene and has been selected for its transgenic or “ vector, " means a plasmid which can introduce a heter status via an additional selection gene . ologous gene and / or expression cassette into yeast cells to be [0121 ] " Transgenic plant” means a plant that has been transcribed and translated . derived from a single cell that was transformed with foreign 10129 ] “ Yield ” refers to the production of a peptide , and DNA such that every cell in the plant contains that trans increased yields can mean increased amounts of production , gene. increased rates of production , and an increased average or [ 0122] “ Transient expression system ” means an Agrobac median yield and increased frequency at higher yields . terium tumefaciens -based system which delivers DNA [0130 ] ( 1 ) . Cleavable Linking Groups for Insecticidal and / encoding a disarmed plant virus into a plant cell where it is or Nematicidal Proteins expressed . The plant virus has been engineered to express a [0131 ] The present invention provides binary and tertiary protein of interest at high concentrations , up to 40 % of the peptides that are operable to be cleaved in the insect, or TSP . In the technical proof, there are two transient expres nematode gut or hemolymph environment and in an animal sion systems used , a TRBO and a FECT system and the plant gastrointestinal system , for example , a human , gastrointes cells are leaf tissue of a tobacco plant " Nicotiana bentha tinal system . The present invention therefore provides cleav miana . ” able binary and tertiary peptides that when linked to other [ 0123 ] “ TRBO ” means a transient plant expression system components of a larger insecticidal or nematicidal protein , using Tobacco mosaic virus with removal of the viral may be cleaved by certain proteases and peptidases found in coating protein gene . the human , insect and nematode gut environments . Advan [ 0124 ] “ Trypsin cleavage ” means an in vitro assay that tageously , multiple copies of insecticidal and nematicidal uses the protease enzyme trypsin (which recognizes exposed peptides can be joined or linked , each copy being separated lysine and arginine amino acid residues ) to separate a by one or more binary or tertiary peptides , that upon cleavable linker at that cleavage site . It also means the act of ingestion or contact with the insect and / or nematode pepti the trypsin enzyme cleaving that site . dase or protease in the gut environment, the binary or [ 0125 ] “ U -ACTX peptide ,” or a “ U protein ” or a “ U tertiary peptides are cleaved releasing the active forms of the toxin , " which may include examples of “ native U , " or linked insecticidal and nematicidal peptides (See for “ U -ACTX -Hvla , ” or “ native U -ACTX -Hvla , " which example FIG . 2 , second aspect) In these illustrative embodi includes the representative example , “ Hybrid peptide, ” aka ments , N as shown in FIG . 2 second aspect , can be any “ hybrid toxin ,” aka " hybrid -ACTX -Hvla ,” aka " native integer ranging from 1 to 200 , or for example , 1 to 100 , or hybrid ACTX -Hvla , ” all refer to a native protein or native preferably from 1 to 10 and any integer therebetween . In toxin , that can be found in nature or is otherwise known as various embodiments of the present invention , the binary examples of U -ACTX peptides , which in the case of and / or tertiary peptides may be linked to 1 or as many as 200 “ U -ACTX -Hvla ," aka " native U -ACTX -Hvla ,” is a native toxic peptides ( i . e . insecticidal and /or nematicidal peptides ) , spider toxin , that was first discovered from a spider with and any number there between , for example , 1 to 100 , or origins in the Australian Blue Mountains and is dual antago more preferably 1 to 10 , thus forming an insecticidal and /or nist against insect voltage gated Ca2 + channels and K + nematicidal transgenic protein , that upon exposure to the channels . The spider from which the toxin was discovered is insect, nematode or human gastric gut environment will known as the Australian Blue Mountains Funnel -web Spi- cleave at specific substrate sequences in the binary and /or der , which has the scientific name Hydronyche versuta . tertiary peptides , thereby liberating a plurality of toxic US 2018 /0362598 A1 Dec. 20 , 2018 peptides from a polymer insecticidal and / or nematicidal gut environments may include : (1 ) serine proteases ; ( 2 ) protein structure . In various embodiments , polynucleotides cysteine proteases; (3 ) aspartic proteases , and ( 4 ) metallo encoding insecticidal and /or nematicidal transgenic proteins proteases . can be used to transform plant cells . In some embodiments, [0135 ] The two predominant protease classes in the diges the insecticidal and /or nematicidal transgenic proteins may tive systems of phytophagous insects are the serine and be formulated into compositions that can be sprayed or cysteine proteases . Murdock et al. ( 1987 ) carried out an otherwise applied in any manner known to those skilled in the art to the surface of plants or parts thereof. Accordingly, elaborate study of the midgut enzymes of various pests DNA constructs are provided herein , operable to encode one belonging to Coleoptera , while Srinivasan et al. ( 2008 ) have or more insecticidal and /or nematicidal transgenic proteins reported on the midgut enzymes of various pests belonging under the appropriate conditions in a host cell , for example , to Lepidoptera . Serine proteases are known to dominate the a plant cell. Methods for controlling a pest infection by a larval gut environment and contribute to about 95 % of the parasitic insect, or a parasitic nematode of a plant cell total digestive activity in Lepidoptera , whereas the Coleop comprises administering or introducing a polynucleotide teran species have a wider range of dominant gut proteases , encoding an insecticidal and / or nematicidal transgenic pro including cysteine proteases . The papain family contains tein as described herein to a plant, plant tissue , or a plant cell peptidases with a wide variety of activities, including endo by recombinant techniques and growing said recombinantly peptidases with broad specificity ( such as papain ), endopep altered plant, plant tissue or plant cell in a field exposed to tidases with very narrow specificity ( such as glycyl endo the pest . Alternatively , the insecticidal and /or nematicidal peptidases ) , aminopeptidases, dipeptidyl -peptidase , and transgenic protein can be formulated into a sprayable com peptidases with both endopeptidase and exopeptidase activi position and applied directly to susceptible plants by direct ties (such as cathepsins B and H ). Other exemplary protei application , such that upon ingestion of the insecticidal nases found in the midgut of various insects include trypsin and / or nematicidal transgenic protein by the infectious like enzymes , e .g . trypsin and chymotrypsin , pepsin , insect and/ or nematode , one or more copies or monomers of carboxypeptidase - B and aminotripeptidases . the insecticidal and nematicidal peptide is cleaved from the 101361 Serine proteases are widely distributed in nearly all insecticidal and / or nematicidal protein ingested by the infec animals and microorganisms ( Joanitti et al. , 2006 ) . In higher tious insect and /or nematode and produces its effect to organisms, nearly 2 % of genes code for these enzymes destroy the insect and / or nematode. ( Barrette - Ng et al ., 2003 ) . Being essentially indispensable to the maintenance and survival of their host organism , serine [0132 ] (1A ). Cleavable Binary and Tertiary Linking Pep proteases play key roles in many biological processes . tides Serine proteases are classically categorized by their sub [0133 ] In various embodiments, the present invention pro strate specificity , notably by whether the residue at P1 : vides cleavable binary and tertiary linking peptides that are trypsin - like (Lys / Arg preferred at P1) , chymotrypsin - like cleaved in the presence of an animal, insect and nematode ( large hydrophobic residues such as Phe / Tyr /Leu at P1) , or protease or peptidase found in the gastrointestinal system of elastase - like (small hydrophobic residues such as Ala /Val at an animal, for example , a human , an insect and/ or a nema P1 ) ( revised by Tyndall et . al. , 2005 ) . Serine proteases are a tode . As used herein , the terms gastrointestinal and gut class of proteolytic enzymes whose central catalytic machin environment are used interchangeably , and is meant to ery is composed of three invariant residues , an aspartic acid , include its common meaning , i . e . the location where a histidine and a uniquely reactive serine , the latter giving ingested food items are proteolytically degraded . In the rise to their name, the “ catalytic triad ” . The Asp - His -Ser human model, the gastrointestinal environment that is triad can be found in at least four different structural contexts capable of breaking down peptides and proteins can include (Hedstrom , 2002 ) . These four clans of serine proteases are the mouth , stomach and small intestine . typified by chymotrypsin , subtilisin , carboxypeptidase Y , [0134 ] Some of the available proteases and peptidases and Clp protease . The three serine proteases of the chy found in insect and nematode gut environment are depen motrypsin -like clan that have been studied in greatest detail dent on the life - stage of the insect as these enzymes are often are chymotrypsin , trypsin , and elastase . More recently , ser spatially and temporally expressed . The digestive system of ine proteases with novel catalytic triads and dyads have been the insect is composed of the alimentary canal and associ discovered for their roles in digestion , including Ser- His ated glands. Food enters the mouth and is mixed with Glu , Ser -Lys / His , His - Ser- His , and N - terminal Ser . secretions that may or may not contain digestive proteases [0137 ] All major types of peptidases have been described and peptidases . The foregut and the hind gut are ectodermal in nematodes . Aspartic peptidases have been described pri in origin . The foregut serves generally as a storage depot for marily in functions related to the digestion of nutrients . In raw food . From the foregut discrete packages of food pass invertebrates it is thought that , along with the cysteine into the midgut (mesenteron or ventriculus ) . The midgut is peptidases , these have the same role as aspartic and serine the site of digestion and absorption of food nutrients . Gen peptidases in vertebrates . In parasitic nematodes , the cyste erally , the presence of certain proteases and peptidases in the ine peptidases cover virtually all functions in which pepti midgut follow the pH of the gut . Certain proteases and dases are involved in parasitic nematodes. Accordingly , peptidases in the human gastrointestinal system may Cathepsins B and L are types of cysteine peptidases belong include: pepsin , trypsin , chymotrypsin , elastase , carboxy ing to the papain family , and have been comprehensively peptidase , aminopeptidase , and dipeptidase . Insect and studied in nematodes . High variability has been found nematode gut environment, include the regions of the diges among the cathepsins B from different species of nematodes tive system in the herbivore species where peptides and regarding optimum temperature and pH , and substrate affin proteins are degraded during digestion . Some of the avail - ity . It is thought that their main role is to digest nutrients and able proteases and peptidases found in insect and nematode that the high interspecific variability observed is due to the US 2018 /0362598 A1 Dec. 20 , 2018
nematode adapting to the ecological niche it occupies . and tertiary peptides contain at least 4 amino acids and up to Cathepsins L also seem to be involved in the digestion of a maximum of 16 amino acids comprised of 2 or 3 different nutrients . regions. Accordingly , binary peptides have two different [ 0138 ] One class ofwell -studied digestive enzymes found regions and tertiary peptides have three different regions. In in the gut environment of insects and nematodes is the class various embodiments, the binary peptides and the tertiary of cysteine proteases . The term " cysteine protease ” is peptides of the present invention are all cleavable by a intended to describe a protease that possesses a highly human protease within one region and an insect and /or reactive thiol group of a cysteine residue at the catalytic site nematode protease in the second region . Tertiary peptides of the enzyme. There is evidence that many phytophagous differ from binary peptides in that they have an additional insects and plant parasitic nematodes rely, at least in part, on region called a spacer comprising 1 to 4 amino acids . In midgut cysteine proteases for protein digestion . These some embodiments , the spacer can be a “ + 2 ” or “ plus - 2 ” include but are not limited to Hemiptera, especially squash dipeptide comprising any two amino acids as described bugs ( Anasa tristis ) ; green stink bug (Acrosternum hilare ) ; herein . Riptortus clavatus ; and almost all Coleoptera examined to [0141 ] As used herein , simply for describing the different date, especially, Colorado potato beetle ( Leptinotarsa deae permutations and combinations of amino acids that can form mlineata ); three - lined potato beetle (Lema trilineata ); the binary and tertiary peptides, for illustrative purposes asparagus beetle (Crioceris asparagi) ; Mexican bean beetle only , the first region of the binary and tertiary peptide is ( Epilachna varivestis ); red flour beetle ( Triolium cas named “ X ” and “ X ” is a substrate for an insect and / or a taneum ); confused flour beetle ( Tribolium confusum ) ; the nematode protease , and the second region of the binary and flea beetles (Chaetocnema spp ., Haltica spp ., and Epitrix tertiary peptide is called “ Y ” , which is a substrate for an spp . ) ; corn rootworm (Diabrotica Spp . ); cowpea weevil animal for example , a human protease , for example, a (Callosobruchus aculatue ) ; boll weevil ( Antonomus gran human protease found in the human gastrointestinal system . dis ) ; rice weevil ( Sitophilus oryza ) ; maize weevil ( Sitophilus In some embodiments, the tertiary peptides of the present zeamais ) ; granary weevil ( Sitophilus granarius) ; Egyptian invention have an X and Y region and the third region is alfalfa weevil (Hypera postica ); bean weevil (Acanthoseel called a spacer or “ Z ” region . In some embodiments , each of ides obtectus ) ; lesser grain borer (Rhyzopertha dominica ) ; X and Y can be 2 - 4 amino acids in length , and Z can be 1 - 4 yellow meal worm ( Tenebrio molitor ); Thysanoptera , espe amino acids in length . In some embodiments , the binary cially , western flower thrips (Franklini ella occidentalis ); and /or tertiary cleavable peptides cannot be cleaved by a Diptera , especially , leafminer spp . ( Liriomyza trifolii ) ; plant plant protease , especially when the insecticidal and /or nem parasitic nematodes especially the potato cyst nematodes aticidal protein is expressed in a plant, or part thereof. In (Globodera spp . ) , the beet cyst nematode (Heterodera these embodiments, the insecticidal and / or nematicidal pro schachtii ) and root knot nematodes (Meloidogyne spp . ) . tein expressed by the plant cannot be cleaved by a plant [0139 ] Another class of digestive enzymes is the aspartic protease in situ , but can be cleaved by a human or insect or proteases. The term “ aspartic protease” is intended to nematode gut protease when the plant or part thereof describe a protease that possesses two highly reactive aspar expressing the insecticidal and /or nematicidal protein is tic acid residues at the catalytic site of the enzyme and which ingested by the human or insect or nematode . is most often characterized by its specific inhibition with [ 0142 ] ( 1B ). Descriptions and Examples of Binary Pep pepstatin , a low molecular weight inhibitor of nearly all tides known aspartic proteases. There is evidence that many [0143 ] Accordingly , the binary peptide can comprise or phytophagous insects rely , in part , on midgut aspartic pro consist of X ( 2 - 4 ) Y ( 2 -4 ) , or Y ( 2- 4 ) X (2 - 4 ) , and an exemplary teases for protein digestion most often in conjunction with tertiary peptide can comprise X ( 2- 4 ) Y ( 2- 4 ) Z ( 1- 4 ), or Z ( 1- 4 ) cysteine proteases . These include but are not limited to X (2 - 4 ) Y (2 - 4 ), or Y (2 - 4 )X (2 -4 )Z (1 - 4 ), or Z (1 - 4 ) Y (2 - 4 ) X (2 -4 ), or Hemiptera especially (Rhodnius prolixus ) and bedbug ( Ci Z (1 -4 ) X (2 - 4 ) Y (2 - 4 )Z (1 - 4 ), or Z (1 -4 ) Y ( 2- 4 ) X ( 2- 4 ) Z (1 - 4 ), mex spp . ) and members of the families Phymatidae , Pen wherein each number in the parenthesis denotes the number tatomidae, Lygaeidae and Belostomatidae; Coleoptera, in of amino acids for that region . For example X ( 2 -4 ) denotes the families of the Meloidae , Chrysomelidae, Coccinelidae an X region with two to four amino acids, for example , X , X , and Bruchidae all belonging to the series Cucujiformia , ( two amino acids) or X X X3 (three amino acids ) or especially , Colorado potato beetle (Leptinotarsa decemlin X X XzX4 ( four amino acids) . For example , a binary pep eata ) three -lined potato beetle (Lematri lineata ); southern tide can include the amino acid sequence : X - X2- Y - Y2, and western corn rootworm (Diabrotica undecimpunctata X , - X2- Xz- Y - Y2, X , - X2- X3 -X4 - Y , - Y2, X , -X2 - Y - Y2- Y3, and D . virgifera ) , boll weevil ( Anthonomus grandis ), squash X - X7- X2 - Y - Y2- Y3 , X . -X2 - X3- 84 - Y - Y2 -Y3 , X , - X2- Y , bug ( Anasatristis ); flea beetle ( Phyllotreta crucifera ), Y2- Y3 - Y4, X - X2- Xz - Y - Y2- Y3- Y4, X - X2- X3 - X4- Y - Y2 bruchid beetle (Callosobruchus maculatus ) , mexican bean Yz- Y4, Y -Y2 - X , - X2, Y - Y2- X -X2 - X3 , Y - Y2- X , - X7- X , beetle (Epilachna varivestis ), soybean leafminer (Odontota X4, Y - Y2- Y3- X - X2, Y - Y2- Y3- X7 - X , -X3 , Y - Y2- Y3- X , horni ) , margined blister beetle ( Epicauta pestifera ) and the X2- X3- X4 , Y - Y2 - Y3- Y4 - X -X2 , Y - Y2 - Y3- Y4- X7 -X2 - X3, red flour beetle ( Triolium castaneum ) ; Diptera , especially Y - Y2- Y3 - Y4- X -X2 - X3 -X4 , wherein each X , and each Yn is housefly (Musca domestica ) ( Terra and Ferreira ( 1994 ) an amino acid , and wherein X , is selected from the group Comn. Biochem . Physiol. 109B : 1 -62 ; Wolfson and Mur consisting of: ( all single letter amino acid format) A , D , E , dock ( 1990 ) J. Chem . Ecol. 16 : 1089 - 1102 ). G , I, K , L , N , P , Q , R , and V ; X , is selected from the group [0140 ] In various embodiments , the present invention pro consisting of A , F , G , I, K , L , N , P, Q , R , S , T, V , and Y ; XZ vides binary and tertiary peptides that act as substrates for is selected from the group consisting of A , D , E , F , G , I, K , human , insect and nematode proteinases , proteases and L , N , Q , R , S , T , V , and Y ; X4 is selected from the group peptidases ( collectively referred to herein as “ proteases ' ) as consisting of: R , H , K , D , E , S , T , N , R , G , A , I , L , M , F , W , described above . In various embodiments , binary peptides Y, and V . In some embodiments , Y , is selected from the US 2018 /0362598 A1 Dec. 20 , 2018 13 group consisting of: ( all single letter amino acid format ) A , VKT, VKY, VLG , VLK , VLN , VLQ , VLR , VPG , VPK , D , E , F , G , I , L , N , Q , S , T , V , and Y ; Y , is selected from the VPR , VR , VRF, VRG , VRI, VRK , VRL , VRN , VRQ , VRR , group consisting of F , G , I , L , and Y ; and Yz is selected from VRS , VRT, VRY, VSG , VSK , VSR , VTG , VTK , VTR , the group consisting of A , D , E , F , G , I, K , L , R , S , T , V, and VVG , VVK , VVR , VYG , VYK , or VYR . Y ; and Y4 is selected from the group consisting of: R , H , K , [ 0145 ] In various embodiments , exemplary X region pep D , E , S , T , N , Q , G , A , I , L , M , F , W , Y , and V . tide sequences , i. e ., peptide sequences that may be cleaved [0144 ] In various embodiments , exemplary X region pep by one or more insect and / or nematode proteases found in tide sequences, i . e . , peptide sequences that may be cleaved the insect and /or nematode gut/ hemolymph environment by one or more insect and /or nematode proteases found in may include : AAK , AAR , AFK , AFR , AIK , AIN , AIQ , AIR , the insect and/ or nematode gut/ hemolymph environment AKF, AKI, AKK , AKL , AKN , AKQ , AKR , AKS , AKT, may include : AAG , AAK , AAR , AFG , AFK , AFR , AGF , AKY, ALK , ALN , ALQ , ALR , APK , APR , ARF, ARI, ARK , AGI, AGK , AGL , AGN , AGQ , AGR , AGS , AGT, AGY, ARL , ARN , ARO , ARR , ARS , ART, ARY, ASK , ASR , ATK . AIG , AIK , AIN , AIQ , AIR , AKF, AKG , AKI, AKK , AKL , ATR , AVK , AVR , AYK , AYR , DIK , DIR , DLK , DLR , EIK , AKN , AKQ , AKR , AKS , AKT, AKY, ALG , ALK , ALN , EIR , ELK , ELR , IIK , IIR , IKA , IKF, IKI, IKK , IKL , IKN , ALQ , ALR , APF, APG , APK , APR , ARF , ARG , ARI, ARK , IKO , IKR , IKS , IKT, IKV , IKY , ILK , ILR , INA, INK , INR , ARL , ARN , ARQ , ARR , ARS , ART, ARY , ASG , ASK , ASR , INV , IPK , IPR , IQA , IQK , IQR , IQV , IRA , IRF , IRI, IRK , ATG , ATK , ATR , AVG , AVK , AVR , AYG , AYK , AYR , IRL , IRN , IRO , IRR , IRS , IRT, IRV , IRY, ISK , ISR , ITK , DGK , DGR , DIG , DIK , DIR , DLG , DLK , DLR , EGK , ITR , KAF, KAI, KAK , KAL , KAR , KAY , KFK , KFR , KIK , EGR , EIG , EIK , EIR , ELG , ELK , ELR , ER , FVR , GAF , KIN , KIQ , KIR , KKA , KKD , KKE , KKF, KKN , KKQ , GAI, GAK , GAL , GAR , GAY , GFK , GFR , GIK , GIN , GIQ , KKS , KKT, KKV, KKY , KLK , KLN , KLQ , KLR , KNK , GIR , GKA , GKD , GKE , GKF, GKI, GKK , GKL , GKN , KNR , KPK , KPR , KQK , KOR , KRA , KRD , KRE , KRF , GKQ , GKR , GKS , GKT , GKV, GKY, GLK , GLN , GLQ , KRN , KRQ , KRS, KRT, KRV , KRY, KSI, KSK , KSL , KSR , GLR , GNA , GNK , GNR , GNV , GPK , GPR , GQA , GQK , KTI, KTK , KTL , KTR , KVF , KVI, KVK , KVL , KVR , GOR , GOV , GRA , GRD , GRE , GRF, GRI, GRK , GRL , KVY , KYK , KYR , LIK , LIR , LKA , LKF, LKI, LKK , LKL , GRN , GRO , GRR , GRS , GRT, GRV , GRY, GSI, GSK , GSL , LKN , LKO , LKR , LKS , LKT, LKV, LKY, LLK , LLR , LNA , GSR , GTI, GTK , GTL ,GTR , GVF , GVI, GVK , GVL , GVR , LNK , LNR , LNV , LPK , LPR , LQA , LQK , LQR , LOV, GVY , GYK , GYR , IGA , IGF, IGI, IGK , IGL , IGN , IGO , LRA , LRF , LRI, LRK , LRL , LRN , LRQ , LRR , LRS , LRT, IGR , IGS , IGT, IGV , IGY, IIG , IIK , IIR , IKA , IKF, IKG , IKI, LRV , LRY , LSK , LSR , LTK , LTR , NIK , NIR , NLK , NLR , IKK , IKL , IKN , IKO , IKR , IKS, IKT, IKV, IKY, ILG , ILK , PIK , PIR , PKK , PKR , PLK , PLR , PRK , PRR , QIK , QIR , ILR , INA , ING , INK , INR , INV, IPG , IPK , IPR , IQA , IQG , QLK , QLR , RAF , RAI, RAK , RAL , RAR , RAY, RFK , RFR , IQK , IQR , IQV, IRA , IRF, IRG , IRI, IRK , IRL , IRN , IRQ , RIK , RIN , RIQ , RIR , RKA , RKD , RKE , RKF , RKN , RKQ , IRR , IRS , IRT, IRV , IRY, ISG , ISK , ISR , ITG , ITK , ITR , RKS , RKT, RKV, RKY , RLK , RLN , RLQ , RLR , RNK , KAF , KAG , KAI, KAK , KAL , KAR , KAY, KFF, KFG , RNR , RPK , RPR , ROK , ROR , RRA , RRD , RRE , RRF , KFK , KFR , KGA , KGD , KGE , KGF, KGI, KGK , KGL , RRN , RRQ , RRS , RRT, RRV , RRY , RSI, RSK , RSL , RSR , KGN , KGQ , KGR , KGS, KGT, KGV, KGY, KIG , KIK , RTI, RTK , RTL , RTR , RVF, RVI, RVK , RVL , RVR , RVY , KIN , KIO , KIR , KKA , KKD , KKE , KKF, KKG , KKN , RYK , RYR , VAK , VAR , VFK , VFR , VIK , VIN , VIQ , VIR , KKO , KKS , KKT, KKV, KKY, KLG , KLK , KLN , KLO , VKF, VKI, VKK , VKL , VKN , VKQ , VKR , VKS , VKT, KLR , KNG , KNK , KNR , KPG , KPK , KPR , KQG , KQK , VKY, VLK , VLN , VLQ , VLR , VPK , VPR , VRF, VRI, VRK , KQR , KRA , KRD , KRE , KRF , KRG , KRN , KRQ , KRS , VRL , VRN , VRQ , VRR , VRS , VRT, VRY, VSK , VSR , KRT , KRV, KRY, KSG , KSI, KSK , KSL , KSR , KTG , KTI, VTK , VTR , VVK , VVR , VYK , or VYR . KTK , KTL , KTR , KVF , KVG , KVI, KVK , KVL , KVR , [0146 ] In various embodiments , exemplary Y region pep KVY , KYG , KYK , KYR , LFR , LGA , LGF, LGI, LGK , tide sequences , i . e ., peptide sequences that may be cleaved LGL , LGN , LGQ , LGR, LGS, LGT, LGV, LGY, LIG , LIK , by one or more animal , for example human proteases found LIR , LK , LKA , LKF , LKG , LKI, LKK , LKL , LKN , LKO , in the animal, for example , a human gastrointestinal envi LKR , LKS , LKT, LKV, LKY, LLG , LLK , LLR , LNA , LNG , ronment may include : AFF , AFG , AFI, AFL , AFV , AFY, LNK , LNR , LNV, LPG , LPK , LPR , LOA , LOG , LOK , AGF, AGL , AIF , AIL , ALF, ALG , ALI, ALL , ALY , AYF, LOR , LOV , LRA , LRF, LRG , LRI, LRK , LRL , LRN , LRQ , AYL , DFF , DFG , DFI, DFL , DFY, DGF , DGL , DIF , DLF , LRR , LRS , LRT, LRV , LRY , LSG , LSK , LSR , LTG , LTK , DLG , DLY, DYF , DYL , EFF , EFG , EFI, EFL , EFY, EGF, LTR , NGK , NGR , NIG , NIK , NIR , NLG , NLK , NLR , PGK , EGL , EIF , ELF , ELG , ELY , EYF , EYL , FF , FFA , FFD , FFE , PGR , PIG , PIK , PIR , PKG , PKK , PKR , PLG , PLK , PLR , FFF, FFG , FFI, FFK , FFL , FFR , FFS , FFT, FFV, FFY, FGA , PRG , PRK , PRR , OGK , OGR , QIG , QIK , QIR , QLG , OLK , FGD , FGE, FGF, FGI, FGK , FGL , FGR , FGS , FGT, FGV , OLR , RAF, RAG , RAI, RAK , RAL , RAR , RAY , RFF , RFG , FGY, FLV, FLR , FYA , FYD , FYE , FYF, FYG , FYI, FYK , RFK , RFR , RGA, RGD , RGE , RGF, RGI, RGK , RGL , FYL , FYR , FYS , FYT, FYV , FYY, GFA , GFD , GFE , GFF , RGN , RGO , RGR , RGS , RGT, RGV, RGY, RIG , RIK , RIN , GFI, GFK , GFL , GFR , GFS, GFT, GFV , GFY, GGL , GIF , RIQ , RIR , RKA , RKD , RKE , RKF, RKG , RKK , RKN , GIL , GLF , GLI, GLL , GLY , GYF, GYL , IF , IFA , IFF, IFG , RKQ , RKS, RKT, RKV, RKY, RLF , RLFL , RLG , RLK , IFI, IFL , IFV , IFY , IGF, IGL , IIF, IIL , ILF , ILG , ILI, ILL , RLN , RLQ , RLR , RNG , RNK , RNR , RPG , RPK , RPR , ILY, IYF , IYL , LFA , LFF , LFG , LFI, LFL , LFV , LFY , LG , ROG , ROK , ROR , RRA , RRD , RRE , RRF, RRG , RRK , LGA , LGE , LGF , LGI, LGL , LGV , LGY, LIF , LIG , LII , LIL , RRN , RRO , RRR , RRS , RRT, RRV , RRY , RSG , RSI, RSK , LIY , LLF , LLG , LLI, LLL , LLY , LYA , LYF , LYG , LYI , LYL , RSL , RSR , RTG , RTI, RTK , RTL , RTR , RVF, RVG , RVI , LYV , LYY , NFF, NFG , NFI, NFL , NFY, NGF, NGL , NIL , RVK , RVL , RVR , RVY, RYG , RYK , RYR , VAG , VAK , NLG , NLI, NLL , NYF, NYL , QFF, QFG , QFI, QFL , QFY , VAR , VFG , VFK , VFR , VGF, VGI, VGK , VGL , VGN , QGF, QGL, QIL , QLG , QLI, QLL , OYF , OYL , SFF , SFG , VGQ , VGR , VGS, VGT, VGY, VIG , VIK , VIN , VIQ , VIR , SFI, SFL , SFY , SGF, SGL , SIF , SIL , SLF, SLG , SLI, SLL , VKF , VKG , VKI, VKK , VKL , VKN , VKQ, VKR , VKS , SLY , SYF , SYL , TFF, TFG , TFI, TFL , TFY , TGF, TGL , TIF , US 2018 /0362598 A1 Dec. 20 , 2018 14
TIL , TLF, TLG , TLI, TLL , TLY , TYF , TYL , VFF , VFG , VFI, NO : 1601 ) , ALKFF (SEQ ID NO : 1602 ) , ALKFLV (SEQ ID VFL , VFY, VGF, VGL , VIF , VIL , VLF, VLG , VLI, VLL , NO : 1603 ) , ALKIFV (SEO ID NO : 1604 ) , ALKLFV (SEO VLY, VYF. VYL , YFA , YFD , YFE , YFF , YFG , YFI, YFK , ID NO : 1605 ) , FFADIK (SEQ ID NO : 1606 ) , FFALK (SEQ YFL , YFR , YFS , YFT, YFV , YFY , YGF, YGL , YYF , or ID NO : 1607 ), FFLK (SEQ ID NO : 1608 ) , FFLR ( SEQ ID YYL NO : 1609) , FFRLR ( SEQ ID NO : 1610 ) , FGYRIK ( SEQ ID [ 0147 ] In various embodiments , exemplary Y region pep NO : 1611 ), FLRLF (SEQ ID NO : 1612 ), FYARR (SEQ ID tide sequences, i. e ., peptide sequences that may be cleaved NO : 1613 ) , GGLRKK (SEO ID NO : 1614 ) , IFVALK (SEO by one ormore animal, for example a human protease found ID NO : 1615 ), IFVLK (SEQ ID NO : 1616 ) , IFVLR (SEQ in the animal, for example , a human gastrointestinal envi ID NO : 1617 ) , IFVR (SEQ ID NO : 1618 ) , IFVRLR (SEQ ronment may include : AFF , AFI, AFL , AFY , AIF , AIL , ALF , ID NO : 1619 ) , ILFNIK (SEQ ID NO : 1620 ) , LFAAPF (SEQ ALI, ALL , ALY , AYF , AYL , DFF , DFI, DFL , DFY, DIF , ID NO : 1621 ), LFVALK (SEQ ID NO : 1622 ) , LFVLK (SEQ DLF, DLY , DYF, DYL , YFL , EFF , EFI, EFL , 0 , EFY, EIF , ID NO : 1623 ) , LFVLR (SEQ ID NO : 1624 ) , LFVR (SEQ ID ELF , ELY, EYF, EYL , FFA , FFD , FFE , FFF, FFI, FFK , FFL , NO : 1625 ) , LFVRLR (SEQ ID NO : 1626 ) , LFVRVFL (SEQ FFR , FFS , FFT, FFV , FFY, FYA , FYD , FYE , FYF , FYI, ID NO : 1627 ) , LGER ( SEQ ID NO : 1628 ) , LKALF (SEQ FYK , FYL , FYR , FYS , FYT, FYV , FYY , IFA , IFF, IFI, IFL , ID NO : 1629 ) , LKFF ( SEO ID NO : 1630 ) , LKIFV (SEQ ID IFV , IFY , IIF , IL , ILF, ILI, ILL , ILY, IYF, IYL , LFA , LFF , NO : 1631) , LKLFV (SEQ ID NO : 1632) , LRALF (SEQ ID LFI, LFL , LFV, LFY, LIF , LII , LIL , LIY , LLF, LLI, LLL , NO : 1633 ), LRFF ( SEQ ID NO : 1634 ), LRIFV (SEQ ID LLY , LYA , LYF , LYI, LYL , LYV , LYY, NFF , NFI, NFL , NO : 1635 ) , LRLFV (SEQ ID NO : 1636 ) , RALF ( SEQ ID NFY , NIL , NLI, NLL , NYF, NYL , OFF , OFI, OFL , OFY, NO : 1637 ) , RIFV ( SEQ ID NO : 1638 ) , RLFV (SEQ ID NO : QIL , QLI, QLL , QYF , QYL , SFF , SFI, SFL , SFY , SIF , SIL , 1639) , RLRALF ( SEQ ID NO : 1640 ) , RLRFF (SEQ ID NO : SLF, SLI, SLL , SLY, SYF, SYL , TFF , TFI, TFL , TFY, TIF , 1641 ), RLRIFV (SEQ ID NO : 1642 ) , RLRLFV ( SEQ ID TIL , TLF, TLI, TLL , TLY , TYF , TYL , VFF , VFI, VFL , VFY, NO : 1643 ) , RRKAFV ( SEO ID NO : 1644 ) , RRKLIF (SEO VIF, VIL , VLF, VLI, VLL , VLY, VYF, VYL , YFA , YFD , ID NO : 1645 ) , RRRFFA (SEQ ID NO : 1646 ), VFGRKG YFE , YFF, YFI, YFK , YFL , YFR , YFS , YFT, YFV , YFY , (SEQ ID NO : 1647 ), and YFVRK (SEQ ID NO : 1648 ). YYF , or YYL . [0151 ] In various embodiments , exemplary binary pep [0148 ] In various embodiments , exemplary X or Y region tides for use in the insecticidal and / or nematicidal polypep peptide sequences, i . e . peptide sequences that may be tides and proteins, DNA constructs and incorporated into cleaved by one or more animal, for example , human pro transgenic plants or parts thereof, of the present invention tease found in the animal, for example , a human gastroin may include one or more cleavable binary peptides selected testinal environment, and one or more insect and /or nema from the group of or consisting of: IFVRLR (SEO ID NO : tode proteases found in the insect and /or nematode gut/ 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID hemolymph environment may include : AFG , AGF, AGL , NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF ALG , DLG , ELG , GFK , GFR , IGF, IGL , ILG , LGA , LGF , (SEQ ID NO : 1621 ). LGI, LGL , LGV, LGY, LIG , LLG , NLG , QLG , VFG , VGF, [0152 ] ( 1C ) . Descriptions and Examples of Tertiary Pep VGL , or VLG . tides [0149 ] In some illustrative examples , a binary peptide of [0153 ] In various embodiments , tertiary peptides can the present invention can comprise the formula : X7- X2- Y - include all of the illustrative binary peptides illustrated Y2, X -X2 - X3 - Y - Y2, X , - X2- X3- XX- Y - Y2, X , -X2 - Y - Y2 above with the addition of 1 to 4 amino acids fused in frame Y3, X -X2 - X3 - Y - Y2- Y3, X - XZ- X3- X4- Y - Y2- Y3, X1- X2 to the N -terminus , the C - terminus or both termini of the Y - Y - Y3- Y4 , X7 -X2 - X3- Y - Y2- Y3- Y4, X -X2 - X3- X4 - Y binary peptide . In some embodiments , the tertiary peptides Yz- Y3- Y4, Y - Y2- X , - X2, Y - Y2- X -X2 - X3 , Y , - Y2- X , -X2 of the present invention can include all of the illustrative X3- X4 , Y - Y2- Y3- X , - X2, Y - Y2- Y3- X -X2 - X3 , Y - Y2- Y3 binary peptides disclosed above having one , two, three or X -X2 - X3 -X4 , Y - Y2 - Y3 - Y4 -X -X2 , Y - Y2- Y3- Y4- X - X2 four amino acids fused in frame to the N -terminus , the X3, Y - Y2- Y3 - Y4- X , -X2 - X3 -X4 , wherein X , is selected C - terminus, or both termini of an exemplary binary peptide . from the group consisting of: ( all single letter amino acid In another example , an illustrative tertiary peptide can format ) A , D , E , G , I , K , L , N , P , Q , R , and V ; X , is selected include an X ( 2- 4 ) Y ( 2- 4 ), or Y (2 -4 ) X (2 -4 ), with the addition of from the group consisting of A , F , G , I, K , L , N , P , Q , R , S , a Z ( 1 -4 ) positioned in frame with the N - terminus , or the T , V , and Y ; X , is selected from the group consisting of A , C - terminus or both termini , of a binary peptide as defined D , E , F , G , I, K , L , N , Q , R , S , T , V , and Y ; X4 is selected above . In some illustrative examples , various tertiary pep from the group consisting of: R , H , K , D , E , S , T , N , Q , G , tide examples can include the amino acid sequence : Z - X , A , I, L , M , F , W , Y , and V . In some embodiments , Y , is X2- Y - Y2, Z , - X , - X2- Xz- Y , - Y2, Z , - X , -X2 - Xz -X4 - Y , - Y2, selected from the group consisting of : (all single letter amino Z - X - X2- Y - Y2- Y3, Z1- X - X2- Xz - Y - Y2- Y3, Z - X -X2 acid format ) A , D , E , F , G , I, L , N , R , S , T , V , and Y ; Y , is Xz- X4 - Y - Y2- Y3 , 27- X , -X2 - Y - Y2 - Y3 - Y4, 27- X , -X2 - Xz selected from the group consisting of F , G , I, L , and Y ; and Y - Y2- Y3- Y4 , Z - X , - X2- X7- X4 - Y - Y - Y3 - Y4, Z -Z2 - X , Y , is selected from the group consisting of A , D , E , F , G , I , X2- Y - Y2, Z -Z2 - X - X2 -Xz - Y - Y2, Zj -Z2 - X -X2 - X3- X4 K , L , R , S , T, V , and Y ; and Y4 is selected from the group Y - Y2, Z -Z2 - X , -X2 - Y -Y2 - Y3, Z -Z2 - X , -X2 - Xz - Y - Y2 consisting of: R , H , K , D , E , S , T , N , Q , G , A , I, L , M , F , W , Y3, Z -Z2 - X7- X2- X3 - X4 - Y - Y2- Y3, Z1 -Z2 - X - X - Y - Y2 Y , and V . Yz- Y4, 27- 22 - X , - X - X3- Y - Y -Y3 - Y4 , Z - Z2- X , -X7 - X , [0150 ] In some embodiments , illustrative binary peptides X4 - Y - Y2- Yz - Y4, 21- 22 -23 - X , -X2 - Y - Y2, Z - Z2- 23 - X , (XY or YX ) comprise the following amino acid sequences: X2- X3 - Y , -Y2 , Z7 -22 - 23 - X - X -X3 - X4 - Y , - Y2, Z7- 22 - 23 AFVRLF (SER ID NO : 1594 ) , AKLFV (SER ID NO : X7- X2- Y - Y2- Y3 , Z1- Z2 -23 - X - X2- Xz - Y - Y2- Y3, Z -Z2 1595 ) , ALFALK (SEO ID NO : 1596 ) , ALFLK (SEO ID NO : Zz- X , -X2 - Xz -X4 - Y - Y2- Y3, Z7 -22 - 23 - X -X2 - Y - Y2- Yz 1597 ) , ALFLR (SEQ ID NO : 1598 ) , ALFR (SEQ ID NO : Y4, Z _ -Z2 -23 - X -X2 - X3 - Y - Y - Y3- Y4, Z1- Z2- 23 - X - X2 1599 ), ALFRLR (SEQ ID NO : 1600 ), ALKALF (SEQ ID X3- X4 - Y - Y2- Y3 - Y4, Z - Z2 - 23- 24 - X , -X2 - Y - Y2, Z , -Z2 - 23 US 2018 /0362598 A1 Dec. 20 , 2018 15
24- X - X2- X3 - Y - Y2 Z -Z2 - 23 - 24 - X -X2 - X3 - X4- Y -Y2 ; 27- X , - X2- Xz- Y - Y2- Y3- 21 - 22 - 23 , Z , - X , -X2 - X3 - X4- Y - Z ; - Z2- 23 - 24 - X , -X2 - Y - Y2- Y3, Z -Z2 - 23 - 24 - X , - X2- Xz - Y - Y2- Y3 - 2 -Z2 - Z3 , Z , - X7 -X2 - Y - Y2- Y3- Y4 - 2 , -22 - Z3, Z , - X - Y2- Y3, Z1- Z2- 23- 24 -X -X2 -X3 -X4 - Y - Y2 - Y3, Z1- Z2 - 23- 24 X2- X3- Y - Y2- Y3- Y4- Z2- Z3, Z1 -X , -X2 - X3 -X4 - Y - Y2- Y3 X -X2 - Y - Y2- Y3- Y4, Z _ -Z2 - 23 - 24 - X - X2- X3- Y - Y2- Y3 Y4- 2 - Z2 - Z3, Z1- Z2 - X -X2 - Y - Y2- 2 , -22 - Z3 , Z -Z2 - X - X2 Y4, Z -Z2 - 23- 24 - X , - X2- X3- X4 - Y , - Y2- Y3- Y4, X , - X2- Y - Xz- Y - Y2- 2 , - Z2- Z3, Z1- Z2- X , - X2- Xz- X4- Y - Y - Z - Z2- 23, Y2- Z1 , X . -X2 - Xz- Y - Y2- Z1 , X7 -X2 - X3- X4 - Y - Y2- 21, Z1- Z2 - X , -X2 - Y - Y2- Y3- 2 ; -Z2 - Z3 , Z7 -Z2 - X7- X2 - Xz- Y -Y2 X - X - Y - Y2- Y3- Z1 , X , -X2 - Xz- Y - Y - Y3- Z1 , X , -X2 - Xz Yz - Z -Z2 - Z3 , Z , -Z2 -X7 - X2- Xz- X4 - Y - Y2- Y3- Zi -Z2 - 23 , X4- Y - Y2- Yz- Z1 , X - X2- Y - Y2- Y3- Y4- Z1 , X - X2- X3 - Y - Z -Z2 - X -X2 - Y - Y2- Y3- Y4- 2 , -22 - Z3 , Z1- Z2 - X7 -X2 - X3- Y - Yz- Y3 - Y4 - 21, X7 - X , -X3 - X4 - Y -Y2 - Y3 - Y4 - 21, Z , - X -X2 Y2- Y3 - Y4 - 27 - 22- 23, 2 , - 22- X , - X2- X3- X4 - Y , -Y2 - Y3 - Y4 - 2 , Y - Y2- 21 , 21- X , -X2 - X3 - Y - Y2- 21, Z ,- X ,- X2- X2- X4- Y , Zz- Z3 , 2 , - 22- 23- X , - X , - Y , -Y2 - 2 , - 22- 23, 2 , -22 - 23 - X -X2 Y2- 21, 21- X - X2- Y - Y2- Y3- Z1 , Z , -X -X2 - X3 - Y - Y2- Yz X3 - Y - Y2- Z -Z2 - Z3 , Z7 -Z2 - 23- X - X2- X7 - X4- Y -Y2 - 2 , -22 Z1, Z7 - X , - X2- X3 - X4- Y - Y2- Y3 -21 , Z7 - X , - X2- Y - Y2- Yz Z3, Z1- Z2 - 23 - X7 -X2 - Y - Y2- Y3- 21 - 22- 23 , Z1 - Z2- 23 - X - X2 Y4- 21 , Z ,- X , -X2 - Xz- Y - Y - YZ- YA- Z1, Z , -X , - X - X2 -X4 Xz- Y - Y2- Yz- Z - Z2- Z3 , Z , -Z2 - Zz - X , -X2 - X3 -X4 - Y - Y2- Yz Y - Y - Y3- Y4- 21 , 21- 22 - X7 - X , - Y -Y2 - 21, 21- 22 - X -X2 Z - Z2- Z3 , Z1- Z2 - 23- X , -X2 - Y -Y2 - Y3 -Y4 - 2 , -Z2 - Z3, Z -Z2 X3 - Y - Y2- 21 , 21- 22 - X , - X - X3- X4 - Y - Y2- 21 , Z , - Z2- X , 23- X -X2 - Xz - Y - Y2- Yz- Y4- 21 - 22 - 23 , 2 ,- 22 - 23- X - X2- Xz X - Y - Y2- Yz- Z1 , Z , -Z2 - X - X2- Xz - Y - Y2- Yz- Z1, Z -Zz X4- Y - Y - Y3- Y4- 21- 22- 23, Z1- 22- 23- 24- X -X2 - Y - Y2- 21 X - X2- Xz - X4- Y - Y2- Y3- Z1 , Z -Z2 - X , - X2- Y - Y2- Y3- Y4 Z2- Z3 , Z1- Z2 - 23 - 24- X , -X2 - Xz - Y - Y2- 24 - Z2 - Z3 , Z -Z2 - Zz Zi, Z , - 22- X , -X2 - X3 - Y - Y2- Y3- Y4- 21 , 21 - 22- X , - X -Xz 24- X , - X - X7- X4- Y -Y2 - 27 - Z2- 23, Z - Z2- 23- 24 - X , - X2- Y , X4- Y -Y2 - Y3 - Y4 - 21, 21- 22 - 23- X -X2 - Y - Y2- 21 , 21 -22 - 23 Y2- Y3- 2 - - 23 , Z - Z2- 23 - 24- X7 - X2- X3 - Y - Y2- Y3- 2 -22 X , -XZ - X3 - Y , - Y2- Z1 , Z , -Z2 - Zz - X , - X - X7 -X4 - Y - Y2- 21 , Z3, Z -Z2 - 23 - 24 - X7- X , - X7- X4 - Y - Y2- Yz- Z -Z2 - Z3 , Z -Z2 Z -Z2 - 23 - X - X2- Y - Y2- Y3- , - 22- - X - X2- Xz - Y - Y2 Z3- 24 - X - X2- Y - Y2- Y3- 94- 21 - 22- Z3 , Z , -Z2 - 23- 24 - X - X2 Y3- 21 , -22 - 23 - X -X2 - X3 - X4- Y -Y2 - Yz -Z1 , Z -Z2 - 23 - X - Xz- Y - Y2- Y3 - Y4- 27 - Z2- Z3 , Z -Z2 - 23 - 24- X - X2- X3 - X4- Y - X2- Y - Y2 - Y3- Y4- 21, 21- 22 -23 - X - X2- X3 - Y - Y2- Y3 - Y4 Y2- Y3 - 94- 21 - 22 -23 , X7- X2 - Y - Y2 - 24- Z2 - 23 - 24 , X7- X2- Xz 21, 2 , -22 - 23 - X - X - X3- X4 - Y - Y2- Yz- Y4 - 21, 21- 22- 23 - 24 Y - Y - Z -Z2 - 23 -24 , X -X2 - X3- X4 - Y - Y - Z -Z2 - 23 - 24 , X7- X , - Y - Y2- Z1 , Z , -Z2 - 23 - 24- X , -XZ - X3 - Y , - Y2- Z1 , Z , -Z2 X -X2 - Y -Y2 - Y3- 2 , - 22- 23- 24, X , - X2- X3 - Y , -Y2 - Y3 - 2 , -22 Z3- 24- X - X2- Xz - X4- Y -Y2 - Z1 , Z , -Z2 - 23 - 24 - X - X - Y - Y - Zz- Z4 , X7- X2- X3- X4 - Y - Y2- Y3 - 21 - 22 - 23- 24 , X - X2- Y - Yz- 21, 21- 22 - 23 - 24 - X , -X2 - X2 - Y - Y - Yz- 21 , 21- 22- 23 -24 Y2- Y3- Y4- 2 , -22 - 23 - 24, X , - X ,- X3- Y - Y2- Y3- Y4- 2 , -22 - 23 X - X2- X3 - X4- Y - Y2- Y3- 21 , 21- 22 - 23 - 24 -X7 - X2- Y - Y2 Z4, X , -X2 - X3 - X4 - Y - Y2- Y3- Y4- 2 , -22 - 23- 24, 27- X , - X2 Yz- Y4- 21 , Z -Z2 - 23- 24 - X7 - X2- Xz- Y -Y2 - Y3 -Y4 - Z , Z -Zz Y - Y2- 2 , -22 - 23 - 24 , 27 - X , -X2 - X3- Y - Y2- 2 , -22 - 23 - 24 , Z3- 24- X - X2- Xz - X4- Y -Y2 - Yz -Y4 - 21 , X -X2 - Y - Y2- 2 2 , - X , - X2- XZ- X4 - Y - Y2 - 2 , -Z2 - 23- 24, 27- X , - X2- Y - Y2 - Yz Z2, X , - X - Xz- Y - Y2- 2 ,- Z2, X - X2- X3- X4 - Y - Y2- 2 , -22 , Z -Z2 - 23- 24 , 27- X -X7 - X3 - Y - Y - Y3- 21- 22- 23- 24, 27- X , X , -X2 - Y - Y2 - Yz- Z , -Z2 , X , - X2- X3 - Y - Y2- Y3- 27 - , X2- X3 - X4 - Y , - Y2- Yz- Z -Z2 - 23- 24 , 27 - X , - X2- Y - Y - Yz X -X2 - Xz -X4 - Y - Y2- Y3 -24 - Z2 , X , - X2- Y - Y2- Y3- 94 -21 Y4- 27 -Z2 - 23- 24 , Z - X - X2- Xz- Y - Y2 - Y3- Y4- 21 - 22 - 23- 24 , Z2, X , - X2- Xz- Y - Y2 - Y3 -Y4 - Z1 - Z2 , X , -X2 - Xz -X4 - Y - Y2 21- X -X2 - X3- X4 - Y - Y - Y3- Y4- 2 , - 22- 23 - 24, 27- 22- X , -X2 Y3 - Y - Z -Z2 , Z7- X , -X2 - Y - Y2- 22 - 22 , Z - X7 -X2 - X3- Y - Y - Y - 2 -22 - 23- 24 , Z - Z2- X , - X2- Xz- Y - Y2- 27 - - 23- 24, Y2- 2 , -22 , 27- X , -X2 - X3 - X4 - Y - Y2- 27 - 22, - X , -X2 - Y - Z - Z2- X - X2- X3- X4- Y - Y2- 2 , - 22- 23 -24 , Z -Z2 - X , - X2- Y , Y2- Yz- Z1 - Z2 , Z , -X , -X2 -X3 - Y , - Y2- Y3 -Z -Z2 , Z ,- X ,- X2 Y2- Yz- Z -Z2 - 23 -24 , Z / -Z2 - X7 - X , - X3- Y - Y - Y3- 2 , -22 - Zz X3- X4 - Y - Y2 - Y3- 2 - Z2, Z7 - X , - X2- Y - Y - Y3- Y4- 27 - 22, 24, 20- 22 - X , - X2- X3- X - Y - Y2- Y3- 2 ; - 22- 23- 24, 2 , -22 - X , Z - X , - X2- Xz - Y - Y2- Y3- Y4- 2 , -22 , Z7 - X , - X2- Xz - X4- Y , X - Y - Y2- Y3- Y4- 2 - 22- 23- 24, 27- 22- X , - X7- X , - Y - Y - Yz Y2 - Yz - Y4- Z -Z2 , Zj -Z2 - X - X2 - Y - Y2- 2 ,- Z2, Z -Z2 - X - X2 Y4- 21 - 22 - 23- 24 , Z - Z2- X -X2 - X3 - X4 - Y - Y2- Y3- Y4- 21 - 22 X3- Y -Y2 - 2 , - 22, 2 / - Z2- X , -X2 - X3 - X4 - Y - Y2- 2 , -22 , 27 -22 Z3- 24 , 27- 22- 23 - X7 - X , - Y -Y2 - 2 , - 22- 23- 24, Z7 - 22- 23 - X - X7- X2- Y - Y2- Y3- 2 ; - Z2, Z1- Z2- X - X2- X3 - Y - Y2- Y3- 21- 22, X2- Xz - Y - Y2 -27 -Z2 - 23- 24 , Z _ -Z2 - 23 - X -X2 - X3- X4 - Y - Y2 27 -Z2 - X , -X2 - X3- X4 - Y - Y2- Y3 - 24 - Z2 , Z -Z2 - X7 -X2 - Y - Y2 Z7 -22 - 23- 24 , 27 -22 - 23- X , -X2 - Y - Y2- Y3- 2 , -22 - 23 - 24, Y3- Y4- 27 -Z2 , Z -22 - X , - X7- X3 - Y - Y2- Y3 - Y4- 27 -Z2 , Z -Z2 Z -Z2 - 23- X7- X ,- X , - Y - Y - Y3- 2 , - 22- 23- 24, Z -Z2 - 23 - X , X -X2 - X3- X4- Y - Y2- Y3 - Y4- 27 - 22, 27- Z2- Z2- X7 - X , - Y , X -X3 - 84 - Y - Y - Y3- 21- 22 - 23- 24, Z1 - Z2- 23- X -X2 - Y - Y2 Y2- 27 - Z2 , Z7 -Z2 - 23- X , -X2 - X2 - Y - Y2 - 2 , -Z2 , Z -Z2 - Z2 - X , Yz- Y4- 2 , -22 - 23 - 24 , Z - Z2- Z2 - X , - X2- XZ - Y - Y2- Yz - Y4- 2 , X2- X3 - X4- Y - Y2- 2 , - Z2, Z -Z2 - Zz - X7- X2 - Y - Y2- Y3- 2 , - 22, Z2- 23 - 24 , Z _ -Z2 - 23 - X7 -X2 - X3- X4 - Y - Y2- Y3- Y4- 2 , -22 - Zz 2 , -22 - 23 - X , - X2- X3- Y - Y2- Y3- 2 ,- 22, 22- 22- 23- X -X2 - Xz Z4, Z , -Z2 - 23 -24 - X - X - Y - Y2- 2 , - -23 - 24 , Z - Z2- 23 -24 X4- Y - Y2- Yz- Z - Z2, Z -Z2 - 23 - X -X2 - Y - Y2- Y3- Y4- 22- 22 , X -X2 - X3- Y - Y2- Z - Z2- 23 - 24, Z1 - Z2- 23 - 24 - X7 -X2 - X3- X4 Zj- Z2- 23 - X , - X - Xz- Y - Y2- Y3- Y4 - 2 - 22, 22- 22 - 23 - X - Xz Y - Y2- 20 - 22- 23- 24 , Z _ - Z2- 23- 24 - X ,- X2 - Y - Y2- Y3 -2 , -22 X2- X4- Y - Y - Y3- Y4- 2 , - Z2, Z - Z2- Z2 - 24 - X , - X - Y - Y2- 2 Zz- Z4 Z -Z2 - 23 - 24- X , - X ,- X3- Y - Y - Y3- 2 , -Z2 - 23 - 24 , Z2, Z - Z2- 23 - 24 - X , -X2 - Xz - Y - Y2- 24- Z2 , Z1- Z2 - 23 - 24 - X - Z -Z2 - 23 - 24- X - X2- Xz - X4- Y - Y2 - Y3- 2 , -22 - 23 -24 , Z _ -22 X2- Xz - X4- Y - Y2- 2 , - Z2, Z - Z2- 23 - 24 - X , -X2 - Y - Y2- Y3- 2 , Z3- 24 - X , -X2 - Y - Y - Y3- Y4- 2 - 22- 23- 24 , Z ; - -23 - 24 - X , Z2, Z -Z2 - 23 - 24- X , -X2 - Xz - Y - Y2- Y3- 2 - 22, Z ; -Z2 - 23 - 24 X -X3 - Y -Y2 - Y3- Y4- 21- 22- 23- 24 , 27- 22- 23- 24- X -X2 - Xz X -X2 - Xz - X4 - Y - Y2- Yz- Z -Z2 , Z , - Z2- 23 - 24- X -X2 - Y - Y2 X4- Y - Y - Y3- Y4- 2 -22 - 23- 24, Z - Y - Y2- X -X2 , Z - Y , Yz - Y4- Z2 - Z2 , Z - Z2- 23 - 24 - X7 -X2 - Xz- Y , - Y2- Y3- Y4- 27 - 22 , Y2- X , -X2 - Xz, Z - Y - Y2- X7 - X2- Xz - X4, Z - Y - Y2- Y3- X , Z -Z2 - 23 -24 - X - X2- X3- X4- Y - Y2- Y3- Y4- 2 , -22 , X , - X2- Y X2, Z - Y - Y2- Y3- X , -X2 - Xz, Z - Y -Y2 - Y3 - X -X2 - X3- X4 , Y2- 2 , -22 - 23, X7 -X2 - Xz- Y - Y2- 2 , - 22- 23 , X , - X2- Xz - X4 Z - Y - Y2- Y3- Y4- X , - X2, Z - Y - Y2- Y3- Y4- X , -X7 - X3 , Y - Y2 - 2 - 2 - 23, X -X2 - Y - Y2 - Y3- 21 - 22- 23 , X - X2- Xz Z - Y - Y2 - Y3- Y4- X7 -X2 - X3- X4 , Z -Z2 - Y - Y2- X -X2 , Y , - Y2- Y3- 2 , - 22- 23 , X - X _- X3- X4 - Y - Y - Y3- 2 , -22 - 23 , Z -Z2 - Y - Y2- X , -X2 - Xz , Z -Z2 - Y - Y2- X , -X2 - Xz - X4 , X -X2 - Y - Y2 - Y3 -Y4 - Z1 - Z2 - Z3, X - X2- Xz - Y - Y2- Y3- Y4 Z1- Z2 - Y - Y2 - Y3- X - X2, Z -Z2 - Y - Y2- Y3- X7 -X2 - X3, Zj- Z2 - Z3 , X7 -X2 - X3- X4 - Y - Y2 - Y3- Y4 - 2 , -22 - Z3 , Z1- X - X2 Z -Z2 - Y - Y2- Y3- X , - X2- X3 - X4, Z _ -Z2 - Y - Y2- Y3- Y4- X - Y - Y2 - 2 -22 - Z3 , Z1- X -X2 - X3- Y - Y2- 21 -22 -Z3 , Z1- X7 - X2 X2, Z , -22 - Y - Y - Y3 - Y4 -X7 - X , -X3 , Z -Z2 - Y - Y2- Y3- 94 X3- X4 - Y - Y2 -24 - Z2 - Z3 , Z4- X - X2- Y - Y2- Y3- 24- Z2 - Z3 , X7- X2- X3 - X4, Z _ -Z2 - 23 - Y - Y2- X7 - X2, Z -Z2 -Z2 - Y - Y2 US 2018 /0362598 A1 Dec. 20 , 2018 16
X -X2 - Xz, Z / -Z2 - Zz - Y - Y2- X - X2- Xz - X4, Z -Z2 - Zz - Y= Y - Y2- Y3 - X , -X2 - X3 - X4- 24 - Z2 -23 , Y - Y2- Y3- Y4- X7 - X2 Y2- Yz - X ,- X2, Z , -22 - 23 - Y -Y2 - Yz- X , - X -Xz , Z - ZZ- Zz Z7 -22 -23 , Y -Y - Y3 - Y4- X , - X ,- X2- 27 -22 - 23 , Y - Y - Yz Y1- Y2- Y3- X - X2- X3- X4, 21- 22- 23 - Y - Y2 - Y3 - Y4 -X -X2 , Y4- X -X2 - X3 -X4 - 24 - Z2- Z3, Z - Y - Y2- X -X2 - 27- Z2 -23 , Z - Z2- 23 - Y - Y - Y3- Y4- X - X2- X3, Z1- Z2 - 23 - Y - Y2- Y3- Y4+ Z - Y - Y2- X , - X2- X3- Z - Z2- Z3 , Z - Y -Y2 - X , -X2 - X3 -X4 - 24 X , -X2 - X7 -X4 , Z _ -Z2 - 23 - 24- Y -Y2 - X , - X2, Z , -Z2 - 23 - 24 - Y , Zz- Z3 , Z / - Y - Y - Y3- X , - X - 2 , -22 - Z3 , Z4 - Y - Y - Y3- X , -X2 Y2- X , -X2 - X3, Z - Z2- 23 - 24- Y -Y2 - X -X2 - X3 - X4, Z1- Z2 - Zz Xz- Z1 - Z2 - Z3, Z - Y -Y2 - Y3- X , -X2 - X3- X4 - 2 , -Z2 - Z3, Z - Y1 Z4- Y - Y2 - Y3- X - X2, Z -Z2 - 23 -24 - Y - Y2 - Y3- X , - X2- X3 , Y2- Y3- Y4- X , - X2- Z -Z2 - Z3 , Z7 - Y , - Y2- Y3 - Y4- X - X2- Xz Z -Z2 - 23 -24 - Y - Y - Y3- X , - X2- X3 - X4, Z - 22- 23 -24 - Y - Y2 21 -22 - 23 , 21- Y1- Y2- Y3- Y4 -X1 - X2 - X3 - X4 - 21 - 22 - Z3 , Z1- Z2 Yz- Y4 - X , - X2, Z ,- Z2- 23 - 24 - Y - Y2- Y3- Y4- X , -X2 - X3, Y - Y2- X - X2- 21- 22- 23, Z1- Z2- Y - Y2- X - X2- X3- 2 , -22 -23 , Z -Z2 - 23- 24- Y - Y2- Y3- Y4- X , -X2 - Xz- X4, Y , - Y2- X , - X2- 21 , Z -Z - Y - Y2- X ,- X - X2 -X4 - Z ,- Z2- Z3 , Z ,- Z2- Y - Y2- Y3 - X , Y - Y2 - X -X2 - X3 -21 , Y - Y2- X - X2- X3- X4 -21 , Y - Y2- Y3 X2- Z1- Z2- Z3, Z1- Z2 - Y - Y2- Y3- X - X2- Xz- Z - Z2- Z3, Z -Z2 X -X2 - 21 , Y - Y2- Y3- X -X2 - X3 - 21 , Y - Y2- Y3- X -X2 - Xz Y - Y2- Y3 - X , -X2 - Xz - X4- 24 - Z2 - Z3, Z1 -Z2 - Y - Y2- Y3- Y4 X4 -21 , Y - Y2- Y3- Y4- X - X2- 21 , Y - Y2- Y3- Y4- X - X2- Xz X -X2 - 27 -22 - 23, Z - Z2- Y - Y - Y3- Y4- X , - X2- X3 - 2 , -22 - 23, Zi, Y - Y - Y3- Y4- X -X2 - X3 - X4 - 21 , Z - Y - Y2- X - X2- 21 , Z -Z2 - Y - Y2- Y3- Y4- X -X2 - X3- X4 - 2 , -22 - Z3 , Z -Z2 - 23 - Y - Z - Y , -Y2 - X7 - X ,- Xz- 21, 21 - Y - Y2- X -X2 - X3 - X4 - 21 , Y2- X7- X , -X2 - Z2 - Z2 - Z3, Z1- Z2 -23 - Y - Y2 - X -X2 - X3- 2 , -22 Z - Y - Y2- Y3 - X - X2- 21 , 21- Y - Y2- Y3 - X - X2- Xz- 21, Z3, Z -Z2 - 23- Y - Y2- X - X - Xz- X4- 21- 22 -Z3 , Z1- Z2- 23 - Y - Z - Y - Y2 - Y3- X - X2- X3 - X4- 21 , Z7 - Y , -Y2 - Y3 - Y4- X , - X2 Y2- Y3- X - X2- Z -Z2 - Z3 , Z1- Z2 - Zz - Y - Y2- Yz -X7 - X2- X3- 21 Z1, Z - Y -Y2 - Y3 - 84 - X , -X2 - Xz- Z1 , Z - Y - Y2- Y3- Y4- X , 22- 23 , 2 - 22- 23- Y - Y2- Y3- X , -X2 - X2 - X - 27 -Z2 - Z3, Z7- 22 X2- X3 -X4 - Z1 , Z -Z2 - Y - Y2 - X -X2 - Z1 , Z -Z2 - Y - Y2- X , Zz- Y - Y - Y3- Y4- X7- X2- 27 - Z2- Z3, Z1- Z2- 23 - Y - Y2 -Y3 - Y4 X2- Xz- Z1 , Z -Z2 - Y - Y2- X , - X - X7 -X4 - 21 , Z / -Z2 - Y - Y2 X - X -X3 - 2 , -22 - 23, 27 - 22- 23- Y - Y - Y3- Y4- X , - X -X3 - X4 Y3- X , -X2 - Z1 , Z1- Z2 - Y - Y2 - Y3- X - X2- Xz - Z1 , Z -Z2 - Y - Z -Z2 - Z3 , Z -Z2 - 23 - 24- Y -Y2 - X - X2- 27 - Z2 -Z3 , Z1- Z2 - 23 Y2- Y3 - X , -X2 - Xz -X4 - Z1 , Z7 -Z2 - Y - Y2- Y3- Y4- X -X2 - 21, 24- Y , - Y2- X , - X2- X3- 21- 22- 23, 21- 22- 23- 24- Y - Y2- X , - X2 21- 22- Y - Y2- Y3- Y4 - X -X2 - Xz- Z1, 2 ,- Z2- Y - Y2- Y3 - Y4 Xz- X4 -24 - Z2 - Z3, Z , - Z2- 23 - 24 - Y - Y2- Y3 - X , -X2 - 27 - 22 -23 , X - X2- Xz- X4- 21 , 21- 22- 23 - Y -Y2 - X , - X2- 21, 27- Zz - Zz - Y - Z -Z2 - 23 - 24 - Y - Y2- Y3- X , - X - X3- 2 , -Z2 -Z3 , Z -Z2 - 23 -24 Y2- X , - X - Xz -Z1 , Z7 -Z2 - Zz- Y -Y2 - X , -X2 - X7 - X4- 21 , Z , -Z2 Y , -Y2 - Y3 - X , - X ,- X3 -X4 - 2 , - 22- 23, 2 , - 22- 23- 24- Y - Y -Yz Z3- Y - Y2- Y3- X -X2 - 21, 22- 22- 23- Y - Y - Y3- X - X2- X3- 21, Y4 - X - X2- X3- 21- 22- Z3, Z / -Z2 - 23- 24 - Y - Y2- Y3 - Y4- X -X2 Z -Z2 - 23 - Y , - Y2 - Y3- X -X2 - Xz- X4 - 21, Z -Z2 -Z2 - Y - Y2- Yz X3 - 2 , - 22 -21 - 22- 23 - 24 - Y - Y2- Y3- Y4- X , -X2 - X3 -X4 - 2 , -22 Y4- X7 -X2 - 21, 21- 22- 23 - Y - Y2- Y3 - Y4- X7- X2- Xz- 21 , - Z3, Y - Y2- X , -X2 - 27- 22- 23- 24, Y - Y2- X7- X ,- X2- 27 - 22- 23 Zz- Y - Y - Y3- Y4- X -X2 - X2 - X4 - 21 , 22- 22 - 23- 24- Y - Y - X , 24, Y - Y2- X - X -X3 - X4 - 2 , -22 - 23 - 24, Y - Y2- Y3- X , - X2 X2- Z1 , Z ; - Z2- 23 -24 - Y - Y2- X - X - Xz- 21 , Z - Z2- 23 -24 - Y - Z -Z2 - 23 - 24, Y - Y2- Y3- X , - X2- Xz - 2 , -22 - 23 - 24 , Y - Y2- Yz Y2- X -X2 - X3 -X4 - Z1, Z -Z2 - 23- 24 - Y - Y2 - Y3- X - X2- 21, X - X - X7- X4- 24 -Z2 - 23- 24 , Y - Y - Y3 - Y - X , -X2 - 2 , -22 - 23 Z - Z2- 23 - 24 - Y - Y - Y3- X , - X2- Xz - Z1 , Z , -Z2 - 23 - 24 - Y - Y - Z4, Y - Y - Y3- Y4- X , - X2- X2- 27 -22 - 23 - 24 , Y - Y2- Y3- 94 Yz- X , -X2 - X3 - X4 -21 , Z -Z2 - 23- 24 - Y - Y2- Y3- Y4- X , - X2- 21 , X - X2- Xz - X4 - Z -Z2 - 23 - 24, Z - Y - Y2- X - X2- Z - Z2- 23 - 24 , 2 ,- 22 - 23 - 24 - Y - Y2- Y3- Y4 - X - X - Xz- Z1 , Z - Z2- 23 - 24 - Y - Z - Y - Y2- X , -X2 - Xz- Z -Z2 - 23 - 24, Z - Y - Y2- X - X2- Xz - X4 Y2- Y3- YA - X , - X2- X3- X4 -21 , Y - Y2- X , -X2 - 2 , - 22, Y - Y2 Z - Z2 - 23 -24 , Z - Y - Y2- Y3- X - X2- 21 - 22 -23 - 24 , Z - Y - Y2 X - X2- X3 - 22 - 22 , Y - Y2- X7 - X , -X3 - X4- 2 , - 22, Y - Y2- Y3 Y3- X -X2 - X3 -24 - Z2 - 23 - 24, Z - Y - Y2 -Y3 - X -X2 - X3 - X4- 21 X -X2 - 27 -22 , Y - Y - Y3- X - X - X2- 27 - 22, Y - Y2- Y3- X , Zz- Zz - Z4 , Z - Y - Y - Y3- Y4- X , - X2- 27 - 22 -23 - 24 , Z - Y , - Y2 X2- X3- X4 - 2 , - 22, Y - Y2- Y3- Y4- X - X2- 27 - 22, Y - Y2- Y3 Yz- Y4- X -X7 - X2 - 27- 22- 23- 24, 2 - Y - Y2- Y3- Y4- X , - X2 Y 4 -X7 - X , -X2 - 2 , - 22, Y - Y2- Y3- Y4- X , -X2 - X3- X4- 24 - 22, Xz- X4 - 2 , - 22- 23 - 24 27 - Z2- Y - Y2- X , - X2- 27 - 22- 23- 24 , 2 , - Y - Y2- X -X2 - 27- 22, 22- Y - Y - X , - X2- X3- 2 -22 , 2 - Y - Zi -Z2 - Y - Y2- X - X2- Xz - Z -Z2 - 23- 24, Z , - Z2- Y - Y2- X -X2 Y2- X , - X2- X3 - X4 - 2 , - Z2 , Z - Y - Y2- Y3 - X , - X2- 2 -22 , X3- X4- 2 , -22 - 23 -24 , 27- 22 - Y - Y2 - Yz - X -X2 - 2 , -22 - 23 - 24 , Z - Y - Y2 - Y3- X - X2- X3 - 2 , -22 , Z7 - Y , - Y2- Y3 - X7 -X2 - Xz Z -Z2 - Y - Y2- Y3- X - X2- X3 - 2 , -22 - 23 -24 , Z _ -Z2 - Y - Y2- Yz X4- 24 - Z2 , Z7 - Y , - Y2- Y3- Y4- X - X2- 27 - Z2, Z7 - Y , - Y2- Yz X - X - Xz- X4 -21 - 22 -23 - 24, Z _ -Z2 - Y - Y2- Y3 - Y4- X - X2- 21 Y4- X , - X2- Xz - Z -Z2 , Z7 - Y , - Y2- Y3 - Y4- X , - X2- Xz - X4- 24 22- 23 - 24 , Z - Z2- Y - Y2- Yz- Y4- X , - X2- X3 - 2 , -22 - 23 -24 , Z2, Z , -Z2 - Y - Y2- X7 - X2- 27 -Z2 , Z ,- Z2 - Y - Y2- X - X - X3 -21 Z -Z2 - Y - Y2- Y3- Y - X , - X ,- X3- X4- 2 , - 22- 23- 24, Z -Z2 - 23 Z2, Z ,- Z2 - Y - Y2- X , - XZ- X3 -X4 - Z ,- Z2, Z ,- Z2 - Y - Y - Yz- X , Y -Y2 - X , -X2 - 2 , - 22- 23- 24, 27- 22 - 23- Y -Y2 - X , -X2 - X2 - 2 , X2- Z1- Z2, Z1- Z2 - Y - Y2- Y3- X - X2- Xz- Z - Z2, Z -Z2 - Y - Y2 Z2- 23 - 24 , Z - Z2- 23- Y - Y2- X - X2- Xz - X4- 24 - Z2 - 23 -24 , Y3- X7 -X2 - X3 - X4 - 2 , - 22, 2 , - 22- Y - Y - Y3- Y4- X - X2- 2 , - 22, Z -Z2 - 23 - Y - Y2- Yz- X7- X2 - 27- Z2 - 23 - 24 , Z -Z2 - Zz - Y - Y2 Z - Z2- Y - Y - Y3 - Y4- X - X2- X2 - 2 , -22 , 20- 22 - Y - Y - Y3- Y4+ Y3- X , -X2 - X3- 2 , -22 - 23 - 24 , Z _ - Z2- 23 - Y - Y2- Y3- X ,- X2- Xz X -X2 - X3 - X4- 24 - Z2 , Z -Z2 - 23 - Y - Y2- X , - X2- 27 - Z2 , Z -Z2 X4 - 2 , - 22 -23 - 24, Z - Z2- 23 - Y - Y2- Y3 - Y4 - X -X2 - 21- 22 - 23 Zz- Y - Y2- X -X2 - X3 - 2 , - 22, 27- 22 - 23- Y -Y2 - X , -X2 - X3 - X4 Z4, Z -Z2 - Zz - Y - Y - Y3- Y4- X , - X2- X2 - 27 - -23 - 24 , Z -Z2 Z - Z2, Z -Z2 - 23 - Y -Y2 - Y3- X7- X2 - 24- Z2 , Z1- Z2 - Zz - Y - Y2 Zz- Y - Y2- Y3 - Y4- X - X2- Xz - X4- 2 , - -23 - 24 , Z - Z2- 23 -24 Yz- X , -X2 - X3 - 2 , - Z2, Z1- Z2 - Z2 - Y - Y - Yz- X , -X2 - X3 -X4 - 2 , Y -Y2 - X , -X2 - 2 , - 22- 23- 24 , 27- 22- 23 - 24- Y - Y2- X , -X7 - Xz Z2, Z -Z2 - 23- Y -Y2 - Y3 - Y4 - X7 -X2 - 27- 22, - 22- 23 - Y - Y2 2 , -22 - 23 - 24 , 27 - 22- 23- 24- Y - Y2- X , -X2 - X3- X4- 24 - Z2 - Zz Yz- Y4- X , - X2- X3- 2 , - 22, 27- 22- 23 - Y , -Y2 - Y3 - Y4 - X , -X2 24, Z - Z2- 23 - 24- Y - Y2- Y3 -X7 - X2- 27 -Z2 - 23- 24 , Z - Z2- 23 X3- X4 - 24 - 22 , Z1- Z2 -23 - 24- Y - Y2- X -X2 - 2 - 22, 22- 22 - 23 Z4- Y - Y2- Y3 - X , -X2 - Xz- Z - Z2- 23 - 24 , Z - Z2- 23 - 24 - Y - Y2 Z4- Y1- Y2- X -X2 - Xz - Z -Z2 , Z - Z2- 23- 24- Y1- Y2- X7- X2- Xz Y3- X - X2- Xz - Xz -X4 - 24 - Z2 - 23- 24 , Z _ -Z2 - 23 -24 - Y - Y2- Yz X4- 2 , -Z2 , Z - Z2- 23- 24 - Y - Y - Y3- X , - X2- 27 - 22, Z -Z2 - 23 Y4- X , - X2- 2 , - 22 -23 - 24, Z - Z2- 23 - 24 - Y - Y2- Y3- Y4- X , -X2 24- Y - Y2- Y3 - X , - X2- X3 - Z -Z2 , Z , -Z2 - 23 - 24 - Y - Y2- Y3- X , X3 - 2 , - 22- 23 -24 , Z _ - Z2 - 23- 24 - Y - Y2- Y3 - Y4- X - X2- X3 -X4 X -X3 -X4 - 2 , - 22, 27- 22- 23- 24- Y - Y - Y3- Y4- X , -X2 - 27 - 22, 27 -22 - 23 - 24 , 27- X , - X2- Y - Y2, 27- X , - X2- X3 - Y , -Y2 ; Z - Z2- 23 - 24 - Y - Y2 - Y3- Y4- X -X2 - Xz- Z -Z2 , Z , -Z2 - 23- 24 Z - X - X2- X3- X4- Y - Y2, Z , - X , -X2 - Y - Y2- Y3, Z - X -X2 Y - Y2- Y3 - Y4 -X7 - X2 - X3- 84 - 24- Z2 , Y - Y2- X7- X2- Z -Z2 - 23 , Xz - Y - Y2- Y3 , Z1- X , -X2 - Xz -X4 - Y - Y2- Y3, Z1- X - X2- Y1 Y - Y2- X -X2 - X3- 2 , -Z2 - 23, Y - Y2- X -X2 - X3- X4 - 24- Z2 - 23 , Y2- Y3 -Y4 , Z - X , -X2 - X3 - Y - Y - Y3- Y4, Z _ -X7 - X2 -X3 - X4 Y - Y2 - Y3- X - X2- 21 - 22- 23 , Y - Y2- Y3- X - X2- X3 - 2 - 2 - 23, Y - Y2- Y3- Y4 , Z - Z2- X -X2 - Y -Y2 , Z -Z2 - X -X2 - X3- Y - US 2018 /0362598 A1 Dec. 20 , 2018 17
Y2, Z1- Z2 - X - X2- X3- X4- Y - Y2, Z -Z2 - X , - X2- Y - Y2- Y3 , Y - Y2- Y3 - Y4 -X7 - X2 -24 -Z2 - Zz , Y - Y2- Y3- Y4- X , - X - Xz Z ; - Z2- X - X2- Xz - Y - Y2- Y3, 24- Z2 - X , - X2- Xz - X4- Y - Y2 Z - Z2 -Zz , Y , -Y2 - Y3 -Y4 - X , -X2 - Xz - X4- 24 -Z2 -Zz , Y , -Y2 Y3, Z1- Z2 - X -X2 - Y - Y2- Y3- Y4 , Z _ - Z2- X7 - X2- X3 - Y - Y2 X - X2- 21 - 22- 23- 24, Y - Y2- X - X2- Xz- Z -Z2 - 23- 24, Y - Y2 Y3- Y4, Z -Z2 - X - X2 -X3 - X4- Y - Y2- Y3- Y4, Z - Z2- 23 - X - X -X2 - X3- X4 -24 - Z2 - 23 - 24, Y - Y2 - Y3- X - X2- 27 - 22- 23 -24 , X2- Y , - Y2, Z ,- Z2- 23 -X , -X2 - Xz - Y , - Y2, Z ,- Z2- Zz- X ,- X2 Y , - Y2- Y3 - X , -X7 - Xz - Z , -Z2 - 23 - 24 , Y - Y - Y3- X , -X2 - Xz Xz- X4- Y - Y2, Z1- Z2 - 23 - X - X2- Y - Y2- Y3, Z1- Z2 -23 - X , X4- 2 - 22- 23 - 24, Y - Y2- Y3- Y4- X -X2 - 21 - 22 - 23- 24 , Y - Y2 X2- Xz- Y - Y2- Y3, Z1- Z2- 23 -X -X2 -X3 -X4 - Y - Y2 - Y3, Y3- Y4 - X , - X2- X3- 21 - 22 - 23- 24 , Y - Y2- Y3- Y4- X , -X2 - Xz Z - Z2- 23 - X - X2- Y - Y - Y3- Y4, Z _ -Z2 - 23- X -X2 - X3- Y - Y2 X4 -21 -22 - 23 - 24 , 21- X1 - X2 - Y - Y2, 21- X1 - X2 - X3 - Y - Y2, Y3- Y4, 27- 22- 23- X7 - X ,- X3 - X4 - Y -Y2 - Y3 - Y4 , 27 -22 - 23 - 24 27- X , - X2- X3 - X4- YA- Y2, Z , -X7 - X2 - Y - Y - Y3, Z7 - X -X2 X -X2 - Y -Y2 , Z - Z2 - 23- 24- X -X2 - X3 - Y - Y2, 27- 22- 23 - 24 X2 - Y - Y2- Y3, Z , -X , -X2 - X7- X4 - Y - Y2- Y3, Z , - X , -X2 - Y , X -X2 - Xz -X4 - Y -Y2 Z1- Z2- 23- 24 - X , -X2 - Y - Y2- Y3, Y2 - Y3 - Y4, Z _- X7- X2- Xz- Y - Y2- Y3- Y4, Z1- X - X2- X3- X4 27 -Z2 - 23- 24 - X - X2- Xz- Y - Y2- Y3, Z1- Z2 - 23 -24 - X - X2- Xz Y - Y2- Y3 - Y4 , Z -Z2 - X -X2 - Y - Y2, Z -Z2 - X - X2- Xz - Y1 X4- Y - Y2- Y3, 2 , - 22- 23 - 24- X , -X2 - Y - Y2 - Y3 -Y4 , Z _ -Z2 - Zz Y2, 27 - Z2- X , -X2 - X3 - X4 - Y , - Y2, Z , - Z2- X , -X2 - Y - Y2- Y3, Z4- X -X2 - X3- Y - Y2- Y3- Y4, Z -Z2 - 23 - 24 -X7 - X2- X3 - X4 2 , - 22- X ,- XZ- X3- Y - Y2- Y3, Z1 -Z2 - X , -X2 - X3 -X4 - Y - Y2 Y - Y2- Y3- Y4, 27 - Y , -Y2 - X , -X2 , 27- Y , - Y2- X7 - XZ - X3 , Y3 , Z1- Z2- X , -X2 - Y - Y2 - Y3 -Y4 , Z _ -22 - X -X2 - X3- Y - Y2 Z - Y - Y2- X - X2- X3- X4, Z - Y - Y2- Y3 - X -X2 , Z1- Y |- Y2 Yz - Y4, Z _ -Z2 - X , -X2 - Xz -X4 - Y - Y2- Y3 -Y4 , Z - Z2- Zz - X - Y3- X , -X2 - X3 , Z - Y - Y2- Y3 - X , -X2 - X3 -X4 , Z - Y - Y2- Yz X - Y - Y2, Z , -Z2 - Zz - X , -X2 - Xz- Y - Y2, Z / -Z2 - Zz - X - X2 Y 4 - X , -X2 , Z - Y - Y2- Y3- Y4- X , - X - X3, 2 , - Y - Y2- Y3- Y4 X7- X4 - Y - Y2, Z -Z2 - 23- X , -X2 - Y - Y - Y3, Z -Z2 - 23 - X , X -X2 - X , -X4 , Z - Z2- Y - Y2- X , -X2 , Z / - Z2- Y - Y2- X -X2 X2- X3 - Y -Y2 - Y3 , Z1- Zz- Zz- X , - X2- X3- X4 - Y -Y2 - Y3 , Xz, Z - Z2- Y - Y ,- X , - X - X3- X4 , 27- 22 - Y , -Y2 - Yz - X , -X2 , Z -Z2 - Zz - X , - X - Y - Y - Y3- Y4 , Z _ -Z2 - 23 - X , - X - X - Y - Y2 Z -Z2 - Y - Y2- Y3- X -X2 - X3 , 2 , - 22- Y - Y2- Y3- X , -X2 - Xz Y3- Y4, Z7 -Z2 -23 - X -X2 - Xz -X4 - Y -Y2 - Y3 -Y4 , Z _ -Z2 - 23 - 24 X4, Z -Z2 - Y - Y2- Y3- Y4- X , -X2 , Zj- Z2 - Y - Y2- Y3- Y4- X - X - X2- Y - Y2, Z -Z2 - 23 - 24 - X -X2 - X3- Y - Y2, Z , -Z2 - 23- 24 X2- X3 , Z1- Z2- Y - Y2- Yz- Y4- X , - X2- X3- X4, Z -Z2 - 23 - Y - X -X2 - Xz - X4 - Y - Y2, Z - Z2- 23 - 24- X -X2 - Y - Y2- Y3 , Y2- X , -X2 , Z , - Z2- 23- Y - Y2- X , - X2- X3, Z7 -22 - 23 - Y - Y2N 21- 22- 23- 24- X - X2- X3 - Y - Y2- Y3, Z -Z2 -23 - 24 - X -X2 - Xz X , - X2- X2 - X4, Z -Z2 -Zz - Y - Y - Y3- X , -X2 , Z -Z2 - 23 - Y X4 - Y , - Y2- Y3 , Z -Z2 - 23 - 24 - X7- X , - Y - Y2- Y3 - Y4, Z7 -Z2 -Zz Y2- Y3- X , -X2 - Xz, Z -Z2 - 23 - Y - Y2- Y3- X - X2- X3 - X4, Z4- X -X2 - Xz- Y - Y2- Y3- Y4, 27 - Z2 -23 - 24 - X - X2- Xz - X4 Z ; - Z2- 22 - Y - Y - Y3- Y4- X -X2 , Z -Z2 - 23- Y - Y2 - Y3- Y4- X , Y - Y2- Y3 - Y4 , X - X - Y - Y2- Z1, X - X2- Xz - Y - Y2- Z1 , X -X3 , Z7 - 22- 23- Y - Y2- Yz- Y4- X -X2 - Xz- X4, Z - Z2- 23 - 24 X -X2 - X3 - X4- Y -Y2 - 21 , X , -X2 - Y - Y2- Y3 -Z1 , X2- X2 -X3 Y - Y2- X , -X2 , Z , - Z2- 23 - 24- Y -Y2 - X - X2- X3 , Z - Z2- 23 -24 Y - Y2- Y3- 21 , X , -X2 - X3- X4 - Y - Y2- Yz- Z1, X , - X2- Y - Y2 Y - Y2- X , - X2- Xz- X4, Z - Z2- 23- ZA - Y - Y2 - Y2- X , -X2 , Yz - Y4- 21 , X , - X - X2- Y - Y - Y3- Y4- 2 , X , - X2- X7 - X4- Y , 2 , -22 - 23 - 24 - Y , - Y2- Yz- X7- X ,- X3, 2 -22 - 23- 24- Y - Y2- Yz Y2- Y3- Y4- 21 , 21 - X -X2 - Y - Y - Z1, Z , - X , -X7 - X - Y - Y2 X -X2 - X3 - X4 , 2 , - 22- 23- 24 - Y , -Y2 - Yz -Y4 - X , - X2, 27 -22 - 23 Z1, Z , - X , - X2- Xz- X4- Y - Yz- Z1 , Z , - X , - X - Y - Y - Yz- Z1 , 24- Y -Y2 - Y3 -Y4 - X - X2- X3 , Z1- Z2- 23- 24 - Y - Y2- Y3 - Y4 27- X , - X2- X3- Y - Y2- Yz- 21 , 27- X , -X2 - X3 - X - Y - Y2- Y3 X -X2 - Xz- X4 , X - X2- Y - Y2- Z1 , X - X2- Xz - Y - Y2- Z1, Zi, Z - X -X2 - Y - Y2- Y3- 94- 21 , 21- X , - X -X3 - Y - Y2- Y3 X - X2- X3 - X4- Y - Y2- Z1 , X - X2- Y - Y2- Y3- 21 , X -X2 - Xz Y4- 21 , 27- X , -X2 - X3 -X4 - Y - Y2- Y3 - Y4- 21 , Z -Z2 - X - X2 Y -Y2 - Y3 - Z1, X , -X2 - X3 - X4- Y - Y2- Yz- Z1 , X -X2 - Y - Y2 Y - Y2- 21, 21 -22 - X7 - X2- X3 - Y - Y2- 21, 21 -22 - X - X2- Xz Y3- Y4 -Z1 , X , - 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Y2- 2 , -22 , Z , - X7 -X2 - Y - Y2- Y3- 2 , -22 , Z7 - X , - X2- Xz Y -Y2 - Y3 - X , -X2 - X3 - 2 , - 22 Y - Y -Y3 - X , - X ,- X5- X4- 2 , Y - Y - Y3- 2 -22 27- X , - X2- X3- X4 - Y , -Y2 - Y3- 27 - 22, Z2, Y - Y2- Y3- Y4- X -X2 - 2 , -22 , Y - Y - Y3- Y4- X , -X2 - Xz 27- X , - X2- Y - Y2- Y3- 94- 21- 22, 27- X , -X2 - Xz - Y - Y2- Yz Z -Z2 , Y - Y2- Y3- Y4- X7 - X2- Xz - X4 - 2 , - 22, Y - Y2- X , -X2 Y4- Z -Z2 , Z7- X , -X2 - X3- X4 - Y - Y2- Y3- Y4 - 2 , -Z2 , Z1- Z2 - X - Zj- Z2 - Zz , Y - Y2- X , - X2- Xz- Z -Z2 - 23, Y - Y2- X , -X2 - Xz X2- Y - Y2- Z -Z2 , Z , -Z2 - X7 -X2 - X3- Y , - Y2- Z -Z2 , Z , -Z2 - X - X4 - 2 , - 22 -23 , Y - Y - Y3- X , -X2 - Z2 - - 23, Y - Y2- Y3 - X , X2- X3 -X4 - Y -Y2 - 21 - Z2 Z - Z2- X -X2 - Y - Y - Y3- 2 , -22 , X2 -X3 - ZA - Z2 - 23 , Y - Y2- Y3- X -X2 - X3- X4- 21 - 22 - 23 , Z -Z2 - X - X2- X3- Y - Y2 - Y3- Z - Z2, Z1- Z2- X -X2 - X3 -X4 - Y - US 2018 /0362598 A1 Dec. 20 , 2018
Y2- Y3 -27 -Z2 , Z - Z2- X , -X2 - Y -Y2 - Y3 -Y4 - 24 - Z2 , Z -Z2 - X , X , - X2- X3- X4- Y -Y2 - 2 , - 22- 23- 24, 27- 22- 23- X , -X2 - Y / - Y2 X2- X3 - Y - Y2- Y3 - 94 -21 - 22 , Z , -Z2 - X7- X2- Xz- X4- Y1- Y2 Y3- 2 , - 22- 23 - 24 , Z - Z2- Z2 - X - X2- Xz - Y - Y2- Y3- 2 , -22 - 23 Y3- Y4- 2 - Z2, Z1- Z2 - 23 - X - X2- Y - Y2- 21 - 22, 21- 22 - 23 - X - 24 , Z _ -Z2 - 23 - X -X2 - X3 -X4 - Y -Y2 - Y3 - 2 , -Z2 - 23 - 24, Z _ -22 X2- X3 - Y1- Y2- 21 - 22 , Z1- 22 - 23 - X -X2 - X3 - X4 - Y - Y2- 21 - 22 , Zz- X - X2- Y - Y2- Y3- 94- 21 - 22- 23 - 24 , Z _ -22 -23 - X - X2 -Xz Z -Z2 - 23 - X , - X2- Y - Y - Y3- 2 , -Z2 , Z , -Z2 - 23 - X , -X7 - X , - Y - Y - Y -Y3 - Y4 - 2 , - 22- 23- 24 , 27- 22 - 23- X , -X2 - X3 - X4 - Y - Y2 Y2- Y3 - Z -Z2 , Z1- Z2- 23 - X -X2 - X3 -X4 - Y - Y - Y3 -21 - 22 , Yz - Y4- 21 - 22 -23 - 24 , Z _ -Z2 - 23- 24- X , -X2 - Y - Y2- Z -Z2 - 23 Z -Z2 - 23 - X - X2- Y - Y2- Y3- Y4- 24 - Z2 , Z1- Z2 -23 - X - X2- Xz 24, Z -22 - 23 - 24 - X , - X2- Xz - Y - Y2- 2 - - 23- 24, Z _ -Z2 - 23 Y - Y2- Y3- 94- 21 - 22, 21 -22 -23 - X -X2 - X3 -X4 - Y - Y2- Y3 Z4- X -X2 - X3- X4 - Y - Y - Z -Z2 - 23- 24, Z _ -Z2 -23 - 24- X7 - X2 Y4- 2 - Z2 , Z -Z2 -23 - 24- X7 - X2- Y - Y2- 27 -Z2 , Z -Z2 - 23- 24 Y - Y - Y3- 2 -22 - 23- 24, 27 - Z2- 23- 24- X -X7 - X3 - Y - Y2- Yz X - X2- Xz - Y - Y2- 2 , -22 , 24- Z2 -23 - 24 - X - X2- X3 - X4- Y - Y2 Z ; - Z2- Zz- Z4, Z -Z2 - 23- 24 -X , - X2- X2 -X4 - Y - Y2- Y3 -2 , -Z2 Z2, Z1- Z2 - 23 - 24 - X - X2- X3 - X4- Y1- Y2- 24 - Z2 , Z1- Z2 - 23- 24 Zz- Z4 Z1- Z2 - 23 - 24 - X - X2- Y - Y2- Y3- Y4- Z1 - Z2- 23- 24, X -X2 - Y -Y2 - Y3- 2 - 22, Z1- Z2 - 23- 24- X -X2 - X3 - Y | - Y2- Y3 Z -Z2 - 23 - 24- X - X2- X3 - Y - Y - Y3- Y4- 2 , -22 - 23 -24 , Z _ -22 2 , -Z2 , Z -Z2 - 23- 24 - X , -X2 - X3 - X4 - Y , - Y2- Y3- 2 , - 22, Z , -22 Zz- Z4 - X7 -X2 - X3- X4 - Y - Y2- Y3- Y4- 2 , -22 - 23 - 24, Z - Y - Y2 Z3- 24 - X -X2 - Y - Y - Y3- Y4- 27- 22 , Z - Z2- 23- 24- X -X2 - Xz X -X2 , Z - Y - Y2- X -X2 - Xz, Z - Y -Y2 - X7 - X2- X3 - X4, Y - Y2- Yz- Y4- 2 ,- Z2 , Z , - Z2- 23 - 24- X - X2- X3- X4- Y - Y2- Y3 Z - Y - Y - Y3- X - X2, Z - Y - Y2- Y3 -X -X2 - X3, Z - Y - Y2 Y4- 27 - Z2 , X - X2- Y - Y - Z -Z2 - Z3 , X7- X2- X3 - Y - Y2- 27 - Yz - X , - X2- X3- X4, Z - Y - Y - Y3- Y4- X7 - X2, Z - Y - Y2- Yz Z3, X7- X2- Xz - X4 - Y - Y2- Z -Z2 - Z3 , X7 -X2 - Y - Y2- Y3- 21 Y4- X - X2- X3 , Z - Y - Y2- Y3 - Y4- X7 - X2- X3 - X4 , Z _ -Z2 - Y - Zz- Z3 , X , - X7- X , - Y -Y2 - Y3 - 2 , -22 - 23 , X . -X2 - X3 - X4 - Y , Y2- X , - X2, Z -Z2 - Y - Y2- X , -X2 - Xz, Z -Z2 - Y - Y2- X , -X2 Y2- Y3 - 2 , -22 - Z3 , X -X2 - Y - Y2- Y3- Y4- 27 - Z2- Z3, X -X2 N X3- X4 , Z / - Z2- Y - Y2- Y3- X7 - X2 , Z - 22 - Y - Y2- Y3- X7- X , X2 - Y - Y - Y3 - Y4- Z , -Z2 - Z3 , X , - X - X7- X4 - Y - Y2- Y3- Y4 Xz, Z -Z2 - Y - Y2- Y3- X , - X2- X2 - X4, Z -Z2 - Y - Y2- Y3- Y4 Zj- Z2 - Z3 , Z1- X7 -X2 - Y - Y2- 2 , -22 - Z3 , Z1- X -X2 - Xz - Y - Y2N X7- X2 , Z -Z2 - Y - Y2 - Y3 -Y4 - X , -X2 - X3 , Z -Z2 - Y - Y2- Yz Z -Z2 - Z3 , Z , - X - X2- X7 - X4- Y - Y - Z -Z2 - Z3 , Z1- X - X2- Y1 Y4- X - X2- Xz - X4, Z -Z2 - Zz - Y - Y2- X - X2, Z / -Z2 - Zz - Y Y2- Y3- 21 - 22 - 23 , Z1- X7 - X2- X3 - Y - Y2- Y3- 2 -22 - Z3 , Z1- X - Y2- X , -X2 - X3, 21 - 22 -23 - Y - Y2- X7 - X2 -X3 - X4, Z1- Z2- 23 X - Xz- X4 - Y - Y3- 2 , -22 - Z3 , Z - X -X2 - Y - Y - YZ- Y4- 2 , -22 N Yi - Y2- Y3- X - X2, Z , -Z2 -23 - Y - Y2- Y3- X - X2- X3, Zj -Z2
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Z3, Z , - X , -XZ - X3 - Y , - Y2- Y3 - Y4 - 2 , -Z2 - Z3 , Z , - X , - X - Xz. Zz- Y - Y2 - Y3- X , - X2- X2 - X4, Z -Z2 - 23- Y - Y2 - Y3- Y4- X , X4- Y - Y2- Y3 - 94- 21 - 22- 23 , Z1- Z2- X7 -X2 - Y - Y2- 27 -22 - 23, X2, Z , -Z2 - Zz - Y - Y2 - Y3- Y4 - X , -X2 - Xz, Z -Z2 - 23 - Y - Y2 2 , -22 - X -X2 - X3 - Y - Y - 2 -22 - 23, 2 , - 22- X , - X , - X , -X4 - Y , Y3- Y4 - X , -X2 - X , -X4 , Z - Z2- 23- 24 - Y -Y2 - X , - X2, Z / -Z2 - 23 Y2- 2 , - 2 -23 , Z - Z2- X , -X2 - Y - Y2- Y3- 21- 22- 23, 27 -Z2 - X 24- Y - Y2- X , - X2- X3, 27- Z2- 23 - 24- Y - Y2- X -X2 - Xz - X4 , X2- Xz- Y - Y - Y3- 2 , -22 -23 , Z - Z2 - X - X2- Xz - X4- Y -Y2 Z -Z2 - 23 - 24 - Y - Y2- Yz- X , - X2, 27- Z2 - 23- 24- Y - Y2- Y3- X , Yz - Z , - Z2 -Z3 , Z - Z2- X , - X2- Y - Y - Yz- Y4- 2 , - 22- Z3 , Z -Z2 X -X3 , Z7 - 22- 23- 24- Y - Y - Y3- X -X7 - X , -X4 , 27 -Z2 - 23 - 24 X -X7 - X - Y - Y2- Yz- Y4 - 2 , - 22 -23 , 2 , -22 - X , - X - X3- X4 Y -Y2 - Y3 - Y4 - X7 -X2 , 27- 22- 23 - 24- Y - Y2- Y3- Y4- X -X2 Y - Y - Y3- Y4- 2 , - Z2- Z3 , Z - ZZ- Z2 - X , - X2- Y - Y - Z - Z2- 23 , Xz, Z - Z2- 23 - 24- Y - Y - Y3 -Y4 - X , - X2- X7- X4, Y - Y2- X , 2 , -22 - 23 - X , - X2- X3- Y - Y2- 2 , - 22- 23, 21- 22- 23- X -X2 - Xz X2- Z , Y , - Y2- X , - X2- Xz - Z1, Y - Y2- X , X2- X7 - X4- 21 , X4- Y / - Y2- 2 , - 22- 23 , 2 , - Z2- 23- X , -X2 - Y - Y2- Y3- 2 -22 - 23, Y - Y2- Yz- X , -X2 - 21 , Y -Y2 - Y3 - X , -X2 - X3- 21, Y - Y2- Yz Z -Z2 - 23- X , -X2 - X3 - Y - Y - Y3- 2 - 2 - 23 , Z - Z2- 23 - X -X2 X - X2- X3 - X4- 21 , Y - Y2- Y3 - Y4- X - X2- 21 , Y - Y2- Y3- Y4 X3- X4 - Y -Y2 - Y3 - 21- 22- 23, 21- 22 - 23- X -X2 - Y - Y2- Y3- Y4 X -XZ - X3 - 21 , Y - Y2- Y3- Y4- X -X2 - X3 - X4 - 21, Z - Y - Y2 Z - Z2- Z3 , Z -Z2 - 23- X , -X7 - X , - Y - Y - Y3- 94- 21 - 22- 23 , X , -XZ - Z1 , Z - Y - Y2- X , - X2- Xz - Z1 , Z / - Y , - Y2- X , - X2- Xz 2 , -22 - 23 - X , - X2- X3- X - Y - Y2- Y3- Y4- 21 - 22- 23, 2 , -22 - 23 X4- 21 , Z - Y - Y2- Y3- X -X2 - 21 , 2 - Y - Y2- Y3- X , - X2- Xz 24- X , - X , - Y - Y2- 2 , - 22- Z3 , Z - Z2- 23- 24- X , - X , - X , - Y - Y , Z1, Z , - Y - Y2- Y3- X -X7 - X3 - X4 - Z1 , Z - Y -Y2 - Y3 - Y4 - X , 2 , -23 - Z3 , Z1- Z2- 23- 24- X -X2 - X3 - X4 - Y - Y - Z -Z2 - Zz, X2- Z1 , Z - Y - Y2- Y3 - Y4- X -X2 - Xz- Z1 , Z - Y - Y2- Y3 - Y4 Z -Z2 - 23- 24- X , - X - Y - Y2- Y3- 2 - 2 -23 , 2 ; - 22 -23 -24 - X , X , - X2- X3- X4 -Z1 , Z -Z2 - Y - Y2- X , -X2 - Z1, Z ,- Z2- Y - Y2 X2- X3 - Y -Y2 - Y3- 2 - 2 - 23, Z1- Z2 - 23- 24- X -X2 - X3 - X4 - Y1 X - X2- Xz - Z1 , Z -Z2 - Y - Y2 - X , -X2 - Xz -X4 - Z1 , Z -Z2 - Y1 Y2- Y3- 2 - 22- Z3, Z - Z2- 23- 24- X , -X2 - Y - Y - Y3- Y4- 2 , - 22 Y2- Y3- X , -X2 - Z1 , Z -Z2 - Y - Y2- Y3 - X , - X2- Xz- Z1 , Z -Z2 Z3, Z , -Z2 - 23 - 24 - X , - X , - X3 - Y - Y2- Y3 - Y4 - 2 , - 22- 23, Z -Z2 Y - Y2- Y3 - X , -X2 - X2 -X4 -21 , 2 ,- 22- Y , - Y2- Y3- YA- X , - X2 Zz -Z4 - X ,- X2 -X3 - X4 - Y - Y - YZ- YA- Z -Z2 - Z3 , X .- X2- Y - Z1, Z / - Z2- Y - Y2- Y3- 94- X -X2 - Xz - 21 , 21- 22 - Y - Y -Yz Y2- 21 - 22 -23 - 24 , X , - X2- X3- Y - Y - Z - Z2- 23 - 24 , X , - X2- Xz Y4- X , - X2- X3- X4 -Z1 , Z , -Z2 - 23 - Y - Y2- X , - X - Z1, Z7 -Z2 -Zz X4- Y1- Y2- 21- 22- 23- 24 X1- X2- Y - Y2- Y3- 21- 22- 23 -24 , Y - Y2- X7 - X2- Xz - Z1 , Z , -Z2 - Zz - Y - Y2- X , -X2 - X2- Xz - X4 X - X2- Xz - Y - Y2- Yz- Z -Z2 - 23 - 24 X - X2- X3 - X4- Y - Y2 Z1, Z1- Z2 - Zz- Y -Y2 - Yz- X7 -X2 - Z1 , Z7 -Z2 - Zz - Y - Y2- Y3- X - Y3- 21 - 22 - 23- 24 , X -X2 - Y -Y2 - Y3 -Y4 - 2 -22 -23 - 24 , X7- X2 X2- X3- 21 , 21 -22 - 23 - Y - Y2- Y3- X7 - X2- X3 - X4- 21 , 21- 22- 23 X3 - Y - Y2- Y3 - Y4 -21 - 22 - 23 -24 , X -X2 - Xz- X4 - Y - Y2- Y3 Y - Y2- Yz- Y4 - X -X2 -21 , - 22- - Y - Y2- Y3 - Y4 - X -X2 Y4- 2 , -22 - 23- 24 , 27- X , - X , - Y - Y2 - 2 , -Z2 - 23 - 24 , Z7 - X , - Xz Xz -Z1 , Z , -Z2 - 23 - Y - Y2- Y3- Y4- X , - X - X7- X4 - 21, 27 -Z2 -Zz Xz- Y - Y2- 21 - 22 -23 - 24 , Z / -X7 - X2- Xz- X4 - Y - Y2- Z -Z2 - Zz Z4- Y - Y2- X - X2- Z1 Z , -Z2 - 23- 24 - Y - Y2- X - X2- Xz - Z1 , Z4, Z - X7 -X2 - Y - Y2- Y3- 2 , -22 - 23 - 24 , Z - X , - X2- Xz - Y - Y2 Z -Z2 - 23 - 24- Y -Y2 - X - X2- Xz - X4- 21 , Z , -Z2 - 23- 24 - Y - Y2 Y3- 21 - 22 -23 - 24 , Z _ - X , -X2 - X3- X4 - Y - Y2- Y3- Z - Z2- 23 - 24, Y3- X , - X - Z1, Z -Z2 - 23 - 24 - Y - Y2- Y3- X -X2 - Xz -Z1 , Z -Z2 27 - X -X2 - Y - Y2- Y3- Y4- 2 , - 22- 23- 24, 27 - X , -X2 - Xz - Y - Y2 Z3- 24- Y - Y - Y3- X , - X2- X3 -X4 - Z1, Z , - Z2- 23 -24 - Y - Y2- Y3 Yz - Y4- Z2 - Z2 - 23 -24 , Z _ - X - X2- X7 - X4- Y - Y2- Y3 - Y4- Z -Z2 Y4- X , -X2 - 21 , 21- 22- 23 -24 - Y - Y2- Y3- Y4- X , -X2 - Xz -Z1 , Zz- Z4 , Z _ -Z2 - X - X2- Y - Y2- 21 - 22 -23 - 24, Z _ -Z2 - X - X2- Xz Z1- Z2 -23 - 24 - Y - Y2- Y3- Y4- X , -X2 - X3- X4 - 21 , Y1- Y2- X - Y -Y2 - 2 , -22 - 23 - 24 , Z - Z2- X , - X2- X3- X - Y - Y2- 21 - 22- 23 X2- 27 - 22 , Y - Y ,- X , - X2- X3 - 2 , -22 , Y - Y2- X , - X2- X3- X4 Z4, Z -Z2 - X -X2 - Y - Y - Y3- 21- 22 - 23- 24, Z - Z2- X -X7 - Xz 2 - 22, Y -Y2 - Y3 - X -X2 - 2 , - 22, Y - Y2- Y3- X -X2 - X3 - 2 - 22, Y , - Y2- Y3- 27 - 22- 23- 24 , 27- 22- X , - X -X3 - X4 - Y , - Y2- Y3- 2 , Y , -Y2 - Yz - X , - X ,- X3 - X4 - 2 , - 22, Y - Y2- Y3- Y4- X , - X2- 2 , Z2- 23- 24 , Z _ -Z2 - X7- X2 - Y - Y2- Y3- Y4- 2 , -22 - 23 -24 , Z _ -Z2 22, Y - Y2- Y3- 94- X -X2 - X3 - 2 , -22 , Y - Y - Yz- Y4- X -X2 X7- X2- Xz- Y -Y2 - Y3 -Y4 - 24 - Z2 -23 - 24, Z -Z2 - X7 - X2- Xz - X4 X3- X4 - 27 -Z2 , Z - Y - Y2 - X7 -X2 - 2 , -Z2 , Z - Y - Y2- X7- X2 Y - Y - Y -Y4 - 21 -22 - 23 -24 , 27 - Z2 -23 - X - X - Y - Y - 2 ; - 22 X3- 2 - Z2 , Z - Y - Y2 - X , -X2 - X3 -X4 - 24 - Z2 , Z - Y - Y2- Y3 Zz- Z4, Z -Z2 - 23- X - X2- X3 - Y - Y - Z -Z2 -23 - 24, Z _ -Z2 - 23- X - X2- 27 - Z2, Z - Y -Y2 - Y3 - X -X2 - X3- 2 -Z2 , Z - Y - Y2 US 2018 /0362598 A1 Dec. 20 , 2018
Y3- X - X2- X3 - X4- 24 - Z2, Z , - Y - Y2- Y3 - Y4- X - X2- 21 - 22 Z4, Z _ -Z2 - Y - Y2- Y3- X - X2- Xz - X4 -24 - Z2 -23 -24 , Z _ -Z2 - Y1 Z - Y - Y - Yz - Y4- X - X2- X3 - 2 , -22 21- Y - Y2- Y3- Y4- X , Y2- Y3- Y4 - X - X2 - 2 , -22 - 23 - 24, Z _ -Z2 - Y - Y2 - Y3- Y4- X -X2 X2- X3- X4- 21 - 22, 21- 22 - Y - Y2- X -X2 - 21 - Z2 , Z1 -Z2 - Y - Y2 Xz - Z -Z2 - 23 - 24, Z _- Z2 - Y - Y2- Y3- Y4- X -X2 - Xz- X4 -2 -22 X - X2- X3 - 2 -22 , Z - Z - Y - Y -X7 - X - X7- X4 - 2 , -22 , Z - Z - Z3- 24 , Z - Z2- 23 - Y -Y2 - X , -X2 - 27 - 22- 23- 24 , 27 - 22- 23 - Y , Y , - Y2- Y3- X , - X2- 2 ,- Z2, Z ,- Z2- Y - Y2- Y3- X , - X - Xz- Z2- 22, Y2- X , - X - Xz - Z -Z2 - 23 - 24, Z _ -Z2 - Zz - Y - Y2- X - X2- Xz - X4 Zj- Z2 - Y - Y - Y3- X7 -X2 - X3- X4 - 27 -Z2 , Z -Z2 - Y - Y2- Y3- Y4 21- 22- 23 - 24 , 27- 22- 23- Y - Y2- Y3- X7 - X , -Z2 - X2 - 23- 24, X - X2- 27 - Z2, Z7 -22 - Y - Y2- Y3 - Y4 -X7 - X2 - Xz- Z -Z2 , Z , -Z2 Z - Z2- Zz - Y - Y - Y3- X - X2- X3 - 24- Z2 -23 -24 , Z -Z2 -23 - Y1 Y - Y2- Y3- Y4 - X - X2 -X3 - X4 -24 - Z2, Z - Z2 -23 - Y - Y2- X1 Y2- Y3- X , -X2 - X3 -X4 - 2 , -22 - 23- 24 , Z _ -Z2 -23 - Y - Y2- Y3- Y4 X2- 27 - 22 , Z7 -Z2 - 23- Y - Y2- X , -X2 - X3 - 2 ; - Z2 , Z -Z2 - 23- Y - X -X2 - Z -Z2 - 23- 24 , Z ,- Z2 -23 - Y ,- Y2 - Yz- Y4- X ,- X2 -Xz - Z , Y2- X - X - Xz- X4 - 2 , - Z2, Z , - Z2 -Zz - Y - Y2- Yz- X , - X - Z -Z2 , 22- 23 - 24 , Z - Z2- 23- Y - Y - Yz- Y4- X - XZ- X3- X4- 2 , - - 23 Z1- Z2- 23 - Y - Y2- Y3- X - X2- X3- Z -Z2 , Z1- Z2 -23 - Y - Y2- Y3 Z4, Z - Z2- 23 -24 - Y - Y2- X , -X2 - 27 -22 - 23 -24 , Z , -Z2 - 23- 24 X - X2- Xz - X4 -24 - Z2, Z -Z2 - 23 - Y - Y2- Y3- Y4- X7 -X2 - 27 -22 , Y - Y2 - X7 -X2 - X3- 21 - 22- 23 - 24 , Z _ - Z2- 23 - 24 - Y - Y2- X -X2 Z ; - Z2- Z2 - Y - Y2- Yz- Y4- X - X2- X2- 2 , - Z2, Z ; - Z2- Z2 - Y - Y2 X7 -X4 - Z -Z2 - 23 -24 , Z _ -Z2 - 23 -24 - Y - Y2 -Y3 - X , - X2- 27 -Z2 Y3- Y4 - X -X2 - X3- X4 - 24 - Z2 , Z , -Z2 - 23- 24- Y - Y2- X7 - X2- 21 23- 24 Z - Z2- 23- 24- Y - Y2- Y3- X - X2- X3- 2 - 22- 23- 24, Z2, Z - Z2 - 23- 24 - Y - Y2- X -X2 - X3- 24 - Z2, Z -Z2 - 23- 24 - Y1 Z -Z2 - 23 - 24 - Y -Y2 - Y3- X - X2- X3- X4 - 2 , -22 - 23 - 24 , Z _ -Z2 Y2 - X -X2 - Xz- X4- Z - Z2, Z - Z2- 23 -24 - Y - Y2- Y3- X - X2- Z Z3- 24 - Y - Y2- Y3- Y4- X - X2- 27 - 22- 23- 24, 27 -22 - 23 - 24 - Y , Z2, Z1- Z2 - 23 - 24 - Y - Y2 -Y3 - X - X2- Xz - Z , -Z2 , Z , -Z2 - 23- 24 Y2- Y3 - Y4 - X7 - X ,- X2 - 27 - 22- 23- 24, 27- 22- 23- 24 - Y -Y2 - Yz Y -Y2 - Y3 - X , -X2 - X , -X4 - 2 , - 22, 27- 22 - 23- 24- Y , - Y , - Y3- Y4 Y4- X -X2 - X3 - X4 - 27 - 22 - 23 - 24 , wherein X , is selected from X -X2 - 27 - Z2 , Z - Z2- 23- 24- Y - Y2- Y3- Y4- X , - X2- X3- 2 , - 22, the group consisting of: (all single letter amino acid format ) Zj- Z2- 23 - 24 - Y - Y - Yz- Y4- X , - X - X7 - X4- Z -Z2 , Y - Y2- X , A , D , E , G , I, K , L , N , P , Q , R , and V ; X , is selected from X2- Z -Z2 - Zz , Y - Y2- X - X2- Xz - Z -Z2 - 23, Y - Y2- X , -X2 the group consisting of A , F , G , I , K , L , N , P, Q , R , S , T, V, X3 -X4 -21 - 22 - 23 , Y - Y2 - Y3 -X -X2 -Z -Z2 - Zz , Y - Y2- Y3 and Y ; X , is selected from the group consisting of A , D , E , X7- X2- X3- 2 , -Z2 - 23, Y - Y2- Y3- X - X2- X3- X4 -24 - Z2- 23, F , G , I , K , L , N , Q , R , S , T , V , and Y ; X , is selected from the Y - Y2 - Y3 - Y4- X - X2- 21- 22 -23 , Y - Y2- Y3- Y4- X - X2- Xz group consisting of: R , H , K , D , E , S , T , N , Q , G , A , I , L , 27 -Z2 - 23, Y - Y - Y3- Y4- X , - X2- X7 - X4- 24 - Z2 -Z3 , Z - Y - Y2 M , F , W , Y , and V . In some embodiments, Y , is selected from X - X2- 27 - Z2- Z3 , Z - Y - Y2- X7- X2 - Xz- Z -Z2 -Z3 , Z4 - Y , - Y2 the group consisting of: (all single letter amino acid format ) X - X2- Xz - X4 - 2 , - 22- Z3 , Z - Y - Y2 - Y3- X - X2- 2 , - 22- 23 , A , D , E , F , G , I , L , N , Q , S , T , V , and Y ; Y , is selected from 2 - Y - Y2- Y3- X7 - X , -X3 - 21- 22- 23 , Z - Y - Y2- Y3- X -X2 the group consisting of F , G , I, L , and Y ; and Y3 is selected Xz- X4- 24 - Z2- Z3 , Z - Y - Y2 -Y3 - Y4 - X -X2 - 2 , -Z2 - Z3 , Z - Y - from the group consisting of A , D , E , F , G , I , K , L , R , S , T , Y2- Y3 - Y4 - X7 - X , -X2 - 2 , - 22- 23, 2 - Y - Y , - Y3- Y4- X -X2 V , and Y ; and Y4 is selected from the group consisting of: R , X7- X4 - 2 , - 22- 23 , Zi- Z2- Y - Y2- X -X2 - 27 -Z2 - Z3, Z1- Z2 - Y - H , K , D , E , S , T , N , Q , G , A , I , L , M , F , W , Y , and V . Specific Yz- X , -X2 - X3 - 2 , - 22- 23, 2 , - 22- Y - Y2- X , -X2 - X3 - X4 - 2 , - 22 exemplary tertiary peptides selected from the above groups Z3, Z -Z2 - Y -Y2 - Yz- X7 -X2 - 2 , -Z2 - Z3 , Z1- Z2 - Y -Y2 - Y3- X , of tertiary peptides are provided below . X2- X3- 2 - 2 -23 , , - 22- Y - Y2- Y3- X , -X2 - X3 - 84 - 21 - 22 - 23, [0154 ] In some illustrative embodiments , the binary pep Zj- Z2- Y - Y2- Y3 - Y4- X7 - X2- 27 - 22- Z3 , Z -Z2 - Y - Y2- Y3- 94 tide can contain at least 4 amino acids and up to a maximum X -X2 - X3 - 2 , - 22- Z3 , Z -Z2 - Y - Y - Y3- Y4 - X -X2 - X3- X4 - 2 , of 8 amino acids , whereas the tertiary peptide can contain at Zz- Z3, Z -Z2 - 23 - Y - Y - X , - X - 2 , -22 - Z3, Z -Z2 - Zz - Y - Y - least 5 amino acids up to a maximum of 16 amino acids. X -X2 - Xz- Z - Z2 -Z3 , Z1- Z2 - Zz - Y - Y2- X7 -X2 - X3- X4 - 24 -22 [0155 ] In related embodiments , cleavable binary linker Z3, Z , -Z2 - 23 - Y - Y2- Y3- X , -X2 - 27- 22 - Z3, Z - Z2- Z2 - Y - Y - peptides ( L ) comprising an X region and a Y region are Y3- X -X2 - X2 - 2 - 2 - 23, 27- 22 - 23- Y - Y2- Y3- X -X2 - X3 - X4 described above , and exemplary Z regions ( 1 - 4 amino acid 2 , -22 - 23 , 2 , - 22- 23 - Y - Y2 - Y3 - Y4- X , - X2- 2 , - 22- 23, Z4 -22 sequences , for example , 2 amino acid sequences ) that may Zz- Y - Y2 - Y3- Y4- X , -X2 - Xz- Z - Z2 -Z3 , Z -Z2 - 23 - Y - Y2- Y3 be fused in frame to the X and / or Y regions ( either to the Y4- X , -X2 - Xz -X4 - ZA - Z2 - Z3, Z - Z2- 23 - 24 - Y - Y2- X , -X2 - 21 N - terminal sequence of an X or Y region , or the C - terminal Zz- Z3, Z ; -Z2 -23 - 24 - Y - Y2- X7 -X2 - X2- 2 -22 -23 , Z1- Z2- Zz sequence of an X or Y region ) described above form the Z4- Y - Y2 - X -X2 - X3- X4 - Z -Z2 - Z3, Z1- Z2 - 23 -24 - Y - Y2- Y3 tertiary cleavable linking peptides. Illustrative Z or spacer X , - X2- 27 - Z2- Z3 , Z -Z2 - 23- 24 - Y , - Y2- Y3 - X , -X7 - X2- 27 - 22 amino acid sequences may include : AA , AF , AM , AN , AQ , Z3, Z1 -Z2 - 23- 24 - Y , - Y2- Y3 - X , -X2 - X3- X3 - X4- 24 - Z2- 23 , AV , AW , AY , DA , DD , DE , DF, DG , DI, DL , DP, DS , DT , 2 , -22 - 23 - 24 - Y3- Y4- X , - X2- 21- 22- 23, 2 ; - 22- 23- 24 - Y - Y2 DV , DW , DY, EA , ED , EE , EF, EG , EI, EL , EP, ES , ET, EV , Yz- Y4 - X7 -X2 - X3 - 2 , - 22- 23, Z - Z2- 23- 24- Y , -Y2 - Y3- Y4- X , EW , EY , FA , FD , FE , FF , FI, FK , FL , FM , FN , FQ , FR , FS , X - X3- X4 - 24 - 22 - 23, Y - Y - X , - X2- 2 , -22 - 23 - 24 , Y - Y2- X , FT, FV , FW , FY , GA , GD , GE , GF, GI, GL, GM , GN , GQ , X2- Xz - Z -Z2 - 23 -24 , Y , - Y2- X , - X ,- X3- X4 -24 - Z2 - 23 - 24 , GS , GV, GW , GY, HA , HD , HE , HF, HH , HI, HK , HL , HN , Y - Y2- Y3- X , - X -ZA - Z2 - 23- 24 , Y - Y2- Y3 - X , -X2 - Xz- Z -Z2 HP, HO , HR , HS , HT, HV, HY, IA , ID , IE , IG , IH , II, IK , IL , Zz- Z4, Y - Y2- Yz -X7 - X2- X3- X4 - 2 , - 22- 23 -24 , Y - Y2- Y3 IM , IN , IP , IQ , IR , IS , IT, IV , IW , KA , KD , KE , KF , KI, KL , Y4- X7 -X2 - 2 , - 22- 23 - 24 , Y - Y2- Y3- Y4- X , - X2- X3- 21 - 22- 23 KN , KO , KV, KY, LA , LD , LE , LG , LH , LI, LK , LL , LM . 24, Y - Y2- Y3- Y4- X ,- XZ- X3- X4- 24- Z2- 23- 24, Z ,- Y - Y2 LN , LP , LQ , LR , LS , LT ,LV LV, , EWLW . CL, MA , .MF LL , MGLEKO , MI IKI, MK KI , X -X2 - Z2 - Z2 - 23 -24 , Z - Y - Y2 - X , - X2- Xz - Z -Z2 - 23 - 24 , ML , MM , MN , MO , MR , MS, MT, MV, MY, NA , ND , NE , Z - Y - Y2 - X7 -X2 - X3- X4 - 27 -Z2 - 23- 24 , Z - Y - Y2 - Y3- X - X2 NF, NI, NL , NM , NN , NO , NS , NT, NV , NY, OA , OD , OE , Z ; - Z2- 23 - 24 , Z , - Y -Y2 - Y3 - X -X2 - X2- 27 -Z2 - 23 -24 , Z - Y - QF , QI, QL , QM , QN , QQ , QS , QT , QV , QY , RA , RD , RE , Y2- Y3 - X7 -X2 - X2 - X4 - 21- 22- 23- 24 , Z - Y -Y2 - Y3- Y4- X , RF , RI, RL , RN , RO , RV , RY , SA , SF , SG , SK , SN , SP , SO , X2- 2 , -22 - 23- 24 , Z - Y , - Y2- Y3- Y - X , -X2 - X3- 2 - - -24 , SR , SS , ST , SV , SW , SY , TA , TF , TG , TK , TN , TP , TO , TR , Z - Y - Y2 - Y3 - Y4 - X - X2- X3- X4 - Z -Z2 -23 - 24 , Z _ -Z2 - Y - Y2 TS , TT , TV , TW , TY , VA , VF , VM , VN , VQ , VV , VW , VY , X - X -ZA - Z2 - 23 -24 , Z -Z2 - Y - Y2 - X , - X2- Xz - Z -Z2 - Zz - Z4 , WA , WF, WI, WK , WL , WN , WP, WO , WR , WS , WT, WV , 21- 22 - Y - Y2- X -X2 - X3 - X4 - 21 - 22 - 23 - 24, Z _ -Z2 - Y - Y2- Y3 WW , WY, YA , YD , YE , YF , YI, YK , YL , YM , YN , YQ , YR , X , - X2- 2 ,- 22- 23- 24, Z - Z2- Y - Y2- Y3- X -X2 - X3- 2 ,- 22- 23- YS , YT, YV , YW , or YY . In exemplary embodiments , US 2018 /0362598 A1 Dec. 20 , 2018 individual tertiary peptides of the present invention can NO : 1702 ) , YFVRKGS (SEQ ID NO : 1703 ) , GSAFVRLF include a two amino acid sequence, fused in frame to the ( SEQ ID NO : 1704 ) , GSAKLFV (SEQ ID NO : 1705 ) , N - terminus and / or the C -terminus of an illustrative binary GSALFALK (SEQ ID NO : 1706 ) , GSALFLK (SEQ ID NO : peptide disclosed above, wherein the two amino acid 1707 ) , GSALFLR ( SEQ ID NO : 1708 ), GSALFR (SEQ ID sequence is AM , LH , MN , ES , WQ, or DT. NO : 1709 ) , GSALFRLR (SEO ID NO : 1710 ) , GSALKALF [0156 ] In exemplary embodiments , individual tertiary (SEQ ID NO : 1711 ) , GSALKFF ( SEQ ID NO : 1712 ), peptides of the present invention can include a two amino GSALKFLV (SEO ID NO : 1713 ) , GSALKIFV (SEO ID acid sequence , fused in frame to the N - terminus and / or the NO : 1714 ) , GSALKLFV (SEQ ID NO : 1715 ) , GSFFADIK C - terminus of an illustrative binary peptide disclosed above , (SEQ ID NO : 1716 ), GSFFALK (SEQ ID NO : 1717 ) , wherein the two amino acid sequence is GS, WN , WQ, MA GSFFLK ( SEQ ID NO : 1718 ) , GSFFLR ( SEQ ID NO : or MV. 1719 ) , GSFFRLR (SEQ ID NO : 1720 ) , GSFGYRIK ( SEQ [0157 ] In some embodiments , the spacer portion of the ID NO : 1721 ) , GSFLRLF (SEO ID NO : 1722 ) , GSFYARR tertiary peptide ( Z region ) comprises the amino acid ( SEQ ID NO : 1723 ) , GSGGLRKK (SEO ID NO : 1724 ) , sequence GS , which may be fused in frame to the N - termi GSIFVALK ( SEQ ID NO : 1725 ) , GSIFVLK (SEQ ID NO : nus or C - terminus of the binary peptide or both . 1726 ) , GSIFVLR (SEQ ID NO : 1727) , GSIFVR (SEQ ID [0158 ] In various embodiments , exemplary binary pep NO : 1728 ), GSIFVRLR (SEQ ID NO : 1729 ) , GSILFNIK tides for use in the insecticidal and /or nematicidal polypep (SEQ ID NO : 1730 ), GSLFAAPF (SEQ ID NO : 1731) , tides and proteins, DNA constructs and incorporated into GSLFVALK (SEO ID NO : 1732 ) , GSLFVLK (SEO ID NO : transgenic plants or parts thereof , of the present invention 1733 ) , GSLFVLR (SEQ ID NO : 1734 ) , GSLFVR (SEQ ID may include one or more cleavable tertiary peptides com NO : 1735 ), GSLFVRLR (SEQ ID NO : 1736 ) , prising, or selected from the group of, or consisting of : a GSLFVRVFL (SEQ ID NO : 1737 ) , GSLGER (SEQ ID NO : binary peptide selected from IFVRLR (SEQ ID NO : 1619) , 1738 ) , GSLKALF (SEO ID NO : 1739 ) , GSLKFF (SEO ID ALKLFV (SEQ ID NO : 1605 ) , ALKFLV ( SEQ ID NO : NO : 1740 ) , GSLKIFV (SEQ ID NO : 1741 ) , GSLKLFV 1603 ), RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF (SEQ ID (SEQ ID NO : 1742 ), GSLRALF (SEQ ID NO : 1743 ) , NO : 1621 ) , fused in frame either on the N - terminus, C - ter GSLRFF (SEQ ID NO : 1744 ) , GSLRIFV ( SEQ ID NO : minus or both termini with a Z region or spacer sequence , 1745 ) , GSLRLFV (SEQ ID NO : 1746 ), GSRALF (SEQ ID comprising, or selected from the group of, or consisting of: NO : 1747 ) , GSRIFV ( SEQ ID NO : 1748 ) , GSRLFV (SEQ GS, WN , WQ , MA , MV, AM , LH , MN , ES , WQ , or DT. ID NO : 1749 ) , GSRLRALF (SEQ ID NO : 1750 ) , GSRLRFF [0159 ] In some embodiments , exemplary tertiary linking (SEQ ID NO : 1751) , GSRLRIFV (SEQ ID NO : 1752) , peptides ( L ) for use in the insecticidal and /or nematicidal GSRLRLFV ( SEQ ID NO : 1753 ) , GSRRKAFV (SEQ ID polypeptides and proteins, DNA constructs and incorporated NO : 1754 ) , GSRRKLIF (SEQ ID NO : 1755 ) , GSRRRFFA into transgenic plants or parts thereof, of the present inven (SEQ ID NO : 1756 ), GSVFGRKG (SEQ ID NO : 1757 ) , and tion may include (ZXY or XYZ ) comprising the following GSYFVRK (SEQ ID NO : 1758 ) . amino acid sequences : AFVRLFGS (SEQ ID NO : 1649 ) , [0160 ] In various embodiments , a representative binary AKLFVGS (SEQ ID NO : 1650 ) , ALFALKGS (SEQ ID NO : peptide of the present invention can include: IFVRLR (SEQ 1651) , ALFLKGS (SEQ ID NO : 1652 ), ALFLRGS ( SEQ ID ID NO : 1619 ) , ALKLFV (SEO ID NO : 1605 ) , ALKFLV NO : 1653 ) , ALFRGS (SEQ ID NO : 1654 ) , ALFRLRGS (SEQ ID NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or (SEQ ID NO : 1655 ) , ALKALFGS (SEQ ID NO : 1656 ) , LFAAPF (SEQ ID NO : 1621 ) . ALKFFGS (SEO ID NO : 1657 ) , ALKFLVGS (SEO ID NO : 10161] ( 1D ) . INSECTICIDAL AND /OR NEMATICIDAL 1658 ), ALKIFVGS ( SEQ ID NO : 1659 ) , ALKLFVGS (SEQ PROTEINS ID NO : 1660 ) , FFADIKGS (SEO ID NO : 1661 ) , FFALKGS [0162 ] In various embodiments of the present invention , ( SEO ID NO : 1662 ) , FFLKGS (SEO ID NO : 1663 ) , FFL the present disclosure provides an insecticidal and / or nem RGS ( SEQ ID NO : 1664 ) , FFRLRGS (SEQ ID NO : 1665) , aticidal protein having a cleavable peptide ( L ) ( a binary or FGYRIKGS (SEO ID NO : 1666 ) , FLRLFGS ( SEO ID NO : a tertiary peptide ) as described above fused in frame with an 1667 ), FYARRGS (SEQ ID NO : 1668 ), GGLRKKGS (SEQ insecticidal or nematicidal toxic protein ( TP ). ID NO : 1669) , IFVALKGS (SEQ ID NO : 1670 ), IFVLKGS [0163 ] In another embodiment, the present invention pro (SEO ID NO : 1671 ), IFVLRGS (SEQ ID NO : 1672 ) , vides an insecticidal and / or nematicidal protein having two IFVRGS ( SEQ ID NO : 1673 ) , IFVRLRGS (SEQ ID NO : or more cleavable peptides , wherein the insecticidal and / or 1674 ) , ILFNIKGS (SEO ID NO : 1675 ) , LFAAPFGS (SEO nematicidal protein comprises a binary or tertiary peptide , ID NO : 1676 ), LFVALKGS (SEQ ID NO : 1677 ) , LFV - the binary or tertiary peptide is fused in frame with a LKGS (SEQ ID NO : 1678 ) , LFVLRGS ( SEQ ID NO : construct comprising , consisting essentially of, or consisting 1679) , LFVRGS ( SEQ ID NO : 1680 ), LFVRLRGS (SEQ ID of: (L - TP )n , or ( L - TP ). -L , or ( TP - L )n , or L -( TP - L ) n , wherein NO : 1681) , LFVRVFLGS ( SEQ ID NO : 1682 ) , LGERGS TP is a toxic peptide or protein , L is a binary or tertiary (SEQ ID NO : 1683 ) , LKALFGS (SEQ ID NO : 1684 ) , cleavable peptide , and n is an integer ranging from 1 to 200 , LKFFGS (SEQ ID NO : 1685 ) , LKIFVGS (SEQ ID NO : or from 1 to 100 , or from 1 to 10 . In another embodiment, 1686 ), LKLFVGS (SEQ ID NO : 1687) , LRALFGS ( SEQ ID the insecticidal and / or nematicidal protein described herein NO : 1688 ) , LRFFGS ( SEQ ID NO : 1689 ) , LRIFVGS ( SEQ comprises an endoplasmic reticulum signal peptide ( ERSP ) ID NO : 1690 ), LRLFVGS (SEO ID NO : 1691 ) , RALFGS fused in frame with : a binary peptide or tertiary peptide , ( SEQ ID NO : 1692 ) , RIFVGS ( SEQ ID NO : 1693 ) , which is fused in frame with an insecticidal and /or nemati RLFVGS (SEO ID NO : 1694 ) , RLRALFGS ( SEO ID NO : cidal toxic protein and / or a repeat construct ( L - TP ), or 1695 ) , RLRFFGS (SEQ ID NO : 1696 ) , RLRIFVGS (SEQ ( TP - L ) . , wherein n is an integer ranging from 1 to 200 , or ID NO : 1697 ) , RLRLFVGS (SEQ ID NO : 1698 ), RRKAF from 1 to 100 , or from 1 to 10 . In various embodiments , an VGS (SEQ ID NO : 1699 ) , RRKLIFGS (SEQ ID NO : 1700 ), exemplary insecticidal and / or nematicidal protein can RRRFFAGS (SEQ ID NO : 1701 ), VFGRKGGS (SEQ ID include a protein construct comprising : (ERSP )- ( TP -L ) n, or US 2018 /0362598 A1 Dec. 20 , 2018 21
( ERSP ) - ( L ) -( TP - L )n , or (ERSP ) - ( L - TP ) n , or ( ERSP ) - ( L - TP ) least 100 % sequence identity or sequence homology to a n -( L ), wherein n is an integer ranging from 1 to 200 . In peptide having an amino acid sequence as set forth in SEQ various related embodiments described above , TP is a toxic ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771- 1773 , and variants protein , L is a binary or tertiary peptide and n is an integer thereof. ranging from 1 to 200 , preferably an integer ranging from 1 [0166 ] (2 ). Endoplasmic Reticulum Signal Peptide to 100 , and more preferably an integer ranging from 1 to 10 . (ERSP ) In some embodiments , the insecticidal and /or nematicidal 0167 ] In various embodiments, the binary and /or tertiary protein may contain TPs that are the same or different, and cleavable linking peptides may be linked , or fused , or binary and /or tertiary peptides that are the same or different. coupled ( i .e . , connected in frame, and used interchangeably In some embodiments , the C - terminal TP is fused or unfused herein ) with one or more additional components. In some at its C - terminus with a binary or tertiary peptide. In some embodiments , the binary and /or tertiary cleavable linking embodiments , the N -terminal TP is fused or unfused at its peptides are linked to an endoplasmic reticulum signal N - terminus with a binary or tertiary peptide . peptide (ERSP ) as further described herein . [0164 ] In another embodiment, the present invention pro 10168 ] In various embodiments , the N - terminus of an vides an insecticidal and /or nematicidal protein having two illustrative binary and /or tertiary cleavable linking peptide is or more cleavable peptides, wherein the insecticidal and /or linked to the C -terminus of the ERSP . nematicidal protein comprises a stabilizing domain (STA ) 0169 ) As used herein , an ERSP refers to a short continu fused in frame with either the N -terminus of a binary or ous stretch of amino acids residues at the amino - terminus of tertiary peptide , or the N - terminus of a construct compris secreted and membrane -bound proteins. The signal peptide ing : ( L - TP ) n , ( L - TP ) n - L , ( TP - L ) n , or L - ( TP - L ) n . In some targets the protein to the secretory pathway and is cleaved related embodiments, an insecticidal and / or nematicidal from the nascent chain once translocated in the endoplas protein comprises an ERSP fused in frame with the N -ter matic reticulum membrane. The signal peptide consists of minus of a stabilizing domain (STA ) , which is fused in frame three regions : an amino - terminal polar region ( N region ) , with either the N -terminus of a binary or tertiary peptide, where frequently positive charged amino acid residues are being fused to the N - terminus of a toxic protein , or the observed , a central hydrophobic region (H region ) of 7 -8 N - terminus of a construct comprising: ( L - TP ) n , ( L - TP ), - L , amino acid residues and a carboxy - terminal region ( C ( TP -L )n , or L - ( TP - L )n . region ) that includes the cleavage site. The cleavage of the [0165 ] In various embodiments , an exemplary insecticidal signal peptide from the mature protein or polypeptide occurs and / or nematicidal protein can include a construct compris at this cleavage site . An exemplary ERSP is between 3 to ing : ( ERSP )- ( STA )- (L - TP )n , or (ERSP )- ( STA )- (L - TP )n -( L ), 100 amino acids in length , or between 5 to 50 amino acids or ( ERSP ) - (STA ) - ( TP - L ) , or (ERSP ) - ( STA ) - ( L ) - ( TP - L ) . in length , or between 20 to 30 amino acids in length . The or ( STA ) - ( L - TP ) n , or (STA ) - ( L - TP ) n - ( L ) , or (STA ) - ( TP - L ) ne ERSP is a signal peptide so called because it directs the or (STA ) - ( L ) - ( TP - L ) . In some related embodiments , an transportation of a protein . Signal peptides may also be insecticidal and / or nematicidal protein comprises an ERSP called targeting signals , signal sequences , transit peptides , or fused in frame with either the N -terminus of a binary or localization signals . The signal peptides for ER trafficking tertiary peptide , being fused to the N -terminus of a toxic are often 15 to 30 amino acid residues in length and have a protein , or the N - terminus of a construct comprising: ( L - TP ) tripartite organization , comprised of a core of hydrophobic no ( L - TP ). - L , ( TP - L ) n , or L - ( TP - L ) n . In various embodi residues flanked by a positively charged amino terminal and ments , an exemplary insecticidal and/ or nematicidal protein a polar , but uncharged carboxy terminal region . (Zimmer can include a construct comprising : ( ERSP ) - ( L - TP ) , or mann , et al. , “ Protein translocation across the ER mem (ERSP ) - ( L - TP ), - ( L ) , or ( ERSP ) - ( TP - L ) , or ( ERSP ) - ( L ) brane ” , Biochimica et Biohysica Acta , 2011 , 1808 : 912 ( TP - L ) . In various embodiments as described herein and 924 ) . above , TP is defined as a toxic protein , that is toxic to an [ 0170 ] Many ERSPs are known . Many plant ERSPs are insect and / or a nematode , L is a binary or tertiary peptide known . In various embodiments , illustrative ERSPs include and n is an integer ranging from 1 to 200 , preferably an the ERSP be derived from a plant ERSP . In some embodi integer ranging from 1 to 100 , and more preferably an ments , illustrative ERSPs also include non -plant ERSPs , integer ranging from 1 to 10 . In some embodiments , the which are operable with the procedures described herein . C - terminal TP of a construct described herein and above is Many plant ERSPs are however well known and certain fused or unfused at its C - terminus with a binary or tertiary exemplified plant derived ERSPs are useful in the present peptide . In some embodiments , the N -terminal TP is fused or invention . As used herein , " plant ERSPs” include naturally unfused at its N - terminus with a binary or tertiary peptide . occurring ERSPs from plant origin . They do not include In some of the above embodiments , the TP used in the non - plant originated ERSPs that have been transfected into insecticidal and / or nematicidal protein can be a peptide or plants or plant cells and expressed in plants . In one illus protein having an amino acid sequence as set forth in SEQ trative embodiment, a plant ERSP includes the barley alpha ID NOs 5 - 1593 and 1761 - 1775 , or a variant thereof. In amylase signal peptide (BAAS ) , for example , is derived various embodiments , the TP may be the same or different, from the plant , Hordeum vulgare , and has the amino acid and the linker ( L ) may be the same or different. In some sequence as follows: MANKHLSLSLFLVLLGLSASLASG related embodiments, TPs useful in the various insecticidal ( SEQ ID NO : 4 ) . and/ or nematicidal proteins, polynucleotides and DNA con [0171 ] In other embodiments , an exemplary ERSP can structs encoding these insecticidal and /or nematicidal pro include a yeast a -mating factor prepro peptide signal leader teins , and transgenic plants or plant cells containing these consisting of a 19 -amino acid signal (pre ) sequence followed insecticidal and / or nematicidal proteins, include peptides by a 67 - residue ( pro ) sequence containing three consensus having at least 80 % , at least 85 % , at least 90 % , at least 95 % , N - linked glycosylation sites and a dibasic Kex2 endopepti % , at least 97 % , at least 98 % , at least 99 % , or at dase processing site . In some embodiments , an illustrative US 2018 /0362598 A1 Dec. 20 , 2018
ERSP can include a mature yeast a -mating factor. In these plant tissue or a plant seed . In some embodiments , insecti embodiments , an exemplary yeast a -mating factor prepro cidal and /or nematicidal proteins of the present disclosure peptide signal leader and mature yeast a -mating factor can include an ERSP . be found in Pichia pastoris and Saccharomyces cerevisiae [0179 ] In related embodiments , the insecticidal and /or yeast strains . nematicidal proteins include an ERSP, two ormore binary or [0172 ] Plant ERSPs , which are selected from the genomic tertiary cleavable linker peptides and one or more TPs sequence for proteins that are known to be expressed and positioned adjacent to a binary or tertiary cleavable linker released into the apoplastic space of plants , and a few peptides . Other multimers and polymers comprising binary examples include : barley alpha -amylase signal peptide, car and / or tertiary cleavable linker peptides and TP subunits rot extensin , tobacco PR1. The following references provide may be synthesized and are described further herein . In further descriptions, and are incorporated by reference related embodiments , the multimers and polymers compris herein in their entirety . De Loose, M . et al. “ The extensin ing binary or tertiary cleavable linker peptides and TP signal peptide allows secretion of a heterologous protein subunits may also comprise an ERSP at the N -terminus of from protoplasts ” Gene, 99 ( 1991) 95 - 100 . De Loose, M . et the insecticidal and / or nematicidal protein , and may also al. described the structural analysis of an extensin - encoding comprise a stabilizing domain (STA ) . In some embodiments , gene from Nicotiana plumbaginifolia , the sequence of which a STA may also include a TP , for example , an ICK TP . (See contains a typical signal peptide for translocation of the for example , FIGS. 3A and 5 , 6A and 6C ) . protein to the endoplasmic reticulum . Chen , M . H . et al . [0180 ] As used herein a “ construct ” includes a polypeptide “ Signal peptide - dependent targeting of a rice alpha -amylase or protein that is made up of various polypeptide units , for and cargo proteins to plastids and extracellular compart example , ERSP , binary or tertiary cleavable linker peptides , ments of plant cells” Plant Physiology , 2004 July ; 135 ( 3 ) : TPs and STA , each of these subunits are fused in frame and 1367 - 77 . Epub 2004 Jul. 2 . Chen , M . H . et al. studied the operable to be synthesized as a single protein . These con subcellular localization of a -amylases in plant cells by structs which may be written in short form designated as L analyzing the expression of a - amylase , with and without its for binary or tertiary cleavable linker peptides or " linker ” , signal peptide , in transgenic tobacco . These references and TP for toxic proteins, ERSP for endoplasmic reticulum others teach and disclose the signal peptide that can be used signal peptide , and STA for stabilization domain . In various in the methods , procedures and peptide, protein and nucleo embodiments , the insecticidal and /or nematicidal proteins of tide complexes and constructs described herein . the present invention may comprise TPs that may be the [ 0173 ] In some embodiments , the ERSP can be a barley same or they may be different. alpha - amylase signal peptide, or a tobacco extensin signal [0181 ] In addition , in some embodiments , the insecticidal peptide , or a modified tobacco extensin signal peptide , or a and /or nematicidal proteins of the present invention may Jun a 3 signal peptide of Juniperus ashei or J. ashei or a comprise binary or tertiary cleavable linker peptides that signal peptide of potato proteinase inhibitor (II ) (pinII ). may be the same or different. In various embodiments , the 101741 PCR primers operable to isolate the ERSP insecticidal and / or nematicidal proteins of the present inven sequences from plants and other eukaryotic cells are known tion may have the same types of TPs fused with the same in the art and are commercially available . type of linkers , as described in the various examples of 101751. In related embodiments , the binary and /or tertiary insecticidal and / or nematicidal proteins disclosed and illus cleavable linking peptides coupled to an ERSP is linked to trated herein , and as represented by the various constructs , the N - terminus of a toxic peptide ( TP ) (ERSP - TP ). insecticidal and /or nematicidal proteins and polynucleotides [ 0176 ] ( 3 ) . Toxic Peptides ( TP ) encoding all of the aforementioned insecticidal and /or nem [0177 ] A TP of the present invention includes any poly aticidal proteins of the present invention . In some embodi peptide or protein having insecticidal and / or nematicidal ments , in the case where a DNA construct or a polynucle activity when ingested by , in contact with , or injected into an otide contains an open reading frame with a single TP , the insect or nematode respectively . In various embodiments , TP can include Bt TPs , in addition to all of the other several classes of TPs that are polymers of amino acids , i . e . , described TPs, with the proviso that if the DNA construct or proteinaceous materials can be employed and synthesized as polynucleotide contains an open reading frame containing single units or multimers of the same TP species or a mixture two or more TPs , the two or more TPs cannot be Bt TPs. of TP species contained within one transgenically expressed [0182 ] An exemplary class of TPs include the Cysteine protein , wherein each of the plurality of TPs are flanked by Rich Insecticidal Proteins (CRIPS ) . The CRIPS have 4 , 6 or one or two cleavable binary or tertiary peptides as described 8 cysteines and 2 , 3 or 4 disulfide bonds. One example of this above . In various embodiments , exemplary classes of TPS class of TPs are called inhibitor cysteine knot ( ICK ) motif useful in the insecticidal and / or nematicidal proteins protein . ICK motif TPs are a class of proteins with at least described herein can include : CRIP TPS, ICK TPs , non - ICK six cysteine residues that form a specific ICK tertiary TPs, sea anemone peptides, TMOF peptides and Bt proteins . structure . Covalent cross - linking of the cysteine residues in In various embodiments , the insecticidal and / or nematicidal the ICK motif TPs form disulfide bridges that result in a proteins of the present disclosure , are non -natural , man tertiary structures that makes the protein relatively resistant made constructs comprising at least one binary or tertiary to proteases and sometimes to extreme physical conditions cleavable linker peptide and at least one TP . (pH , temperature , UV light, etc . ), and confers activity 10178 ]. The insecticidal and / or nematicidal proteins of the against ion channels , which may be specific to insects . Many present disclosure may be synthesized chemically, made ICK motif TPs have evolved in the venom of invertebrates recombinantly , and in some embodiments , are synthesized and vertebrates that use the ICK motif TPs as a toxin to as an insecticidal and /or nematicidal protein from a poly immobilize or kill their predators or prey . Such insecticidal nucleotide that is inserted into a yeast cell , a plant, plant peptides often have scorpion , spider and sometimes snake tissue or is expressed under appropriate conditions in a plant, origins . In nature, toxic peptides can be directed to the US 2018 /0362598 A1 Dec. 20 , 2018 insect' s gut or to internal organs by injection . In the case of cystine knot peptides ” FASEB J. , 2003 Sep . 17 , ( 12) : 1765 -7 , a TP of the present invention , the delivery of TPs is usually Epub 2003 Jul. 3 . The sequence - specific assignment and the via the insect ' s consumption of insecticidal and / or nemati secondary structure identification of BgK , a K + channel cidal protein expressed in plant tissue . Upon this consump blocking toxin from the sea anemone Bunodosoma granu tion of the toxin from its food , for example an insect feeding lifera , was disclosed by Dauplais , M . et . al. “ On the con upon a transgenic plant , the ICK motif TP may have the vergent evolution of animal toxins ” Journal of Biological ability to inhibit the growth , impair the movement, or even Chemistry . 1997 Feb . 14 ; 272 ( 7 ) : 4302 - 9 . A review of the kill an insect. composition and pharmacology of spider venoms with 10183 ] ICK motif TPs have been shown by others to lose emphasis on polypeptide toxin structure , mode of action , their toxicity when they are expressed in plants . Unless the and molecular evolution showing cysteine bridges , cysteine ICK motif TP is expressed as a properly folded protein it knot formations and the “ knotting - type” fold was published cannot successfully protect a plant or crop from insect by Escoubas, P . et al. “ Structure and pharmacology of spider damage. In some cases a plant expressed peptide will need venom neurotoxins” Biochimie , Vol. 82 , Issues 9 - 10 , 10 to be activated by cleavage within the insect or during Sep . 2000 , pp . 893 - 907 . The purified peptide, iberiotoxin , an expression process in a plant in order to be active . Plant inhibitor of the Ca2 + -activated K + channel , from scorpion expressed inhibitory cysteine knot (ICK ) motif proteins (Buthus tamulus ) venom was disclosed in Galvez , A . et al. from spiders and scorpions have been described (Khan et al. , “ Purification and characterization of a unique , potent, pep Transgenic Res ., 2006 , 15 : 349 - 357 ; Hernandez - Campuzano tidyl probe for the high conductance calcium -activated et al ., Toxicon . 2009 January ; 53 ( 1 ) : 122 - 8 . ) ICK motif TP, ” potassium channel from venom of the scorpion Buthus or " inhibitor cystine knotmotif protein " , which is a 16 to 60 tamulus” Journal of Biological Chemistry, 1990 Jul . 5 ; amino acid peptide with at least 6 half -cysteine core amino 265 ( 19 ) : 11083 - 90 . acids having three disulfide bridges , wherein the 3 disulfide [ 0187 ] The purified peptide, charybdotoxin , an inhibitor of bridges are covalent bonds and of the six half - cystine the Ca2 + -activated K + channel , from the venom of the residues the covalent disulfide bonds are between the first scorpion Leiurus quinquestriatus was disclosed in Gimenez and fourth , the second and fifth , and the third and sixth Gallego , G . et al. “ Purification , sequence , and model struc half - cystines , of the six core half -cystine amino acids start ture of charybdotoxin , a potent selective inhibitor of cal ing from the N -terminal amino acid . The ICK motif TP also cium - activated potassium channels ” Proc. Natl. Acad . Sci. , comprises a beta -hairpin secondary structure , normally com 1988 May ; 85 (10 ): 3329 - 3333. posed of residues situated between the fourth and sixth core [0188 ] From these and other publications , one skilled in half- cysteines of the motif , the hairpin being stabilized by the art should be able to readily identify proteins and the structural crosslinking provided by the motifs three peptides having what we describe as the ICK motif TP disulfide bonds . Note that additional cysteine /cysteine or (which is used interchangeably with “ ICK motif” , “ ICK half- cystine amino acids may be present within the inhibitor motif protein ” and the " inhibitor cystine knot motif '' ) . cysteine knot motif . [0189 ] In some examples , ICK motif TPs have between [0184 ] The ICK motif is common in peptides isolated 26 -60 amino acids in length . Some ICK motif TPs are from the venom of numerous species . Invertebrate species between 16 - 48 amino acids in length . Some ICK motif TPs include spiders , scorpions, cone snail , sea anemone etc ., are between 26 - 48 amino acids in length . Some ICK motif other examples are numerous , even snake venom has been TPs are between 30 -44 amino acids in length . ICK motif TPs known to have peptides having the ICK motif . with natural insecticidal activity are preferred but ICK motif [0185 ] An example of ICK TPs include ACTX peptides TPs with other types of activity such as salt and frost ( for example , a U - ACTX peptide , an Omega - ACTX peptide resistance are known to those skilled in the art and are or a Kappa - ACTX peptide ) from the Australian Blue Moun claimed herein . Examples of ICK motif TPs include the tains Funnel -web Spider . Other ICK proteins described ACTX peptides and genes, and including all of the peptides herein may be used to synthesize insecticidal and / or nem and their coding genes known as Magi6 . aticidal proteins described herein . [0190 ] Specific examples of ICK motif TPs and peptides [0186 ] Examples of TPs with the ICK motif can be found disclosed for purposes of providing examples and not in the following references . The N - type calcium channel intended to be limiting in any way , are the TPs and their blocker w - Conotoxin was reviewed by Lew , M . J . et al. homologs and peptide variants thereof as described above . “ Structure - Function Relationships of w - Conotoxin GVIA ” In some embodiments , some preferred TPs are those par Journal of Biological Chemistry , Vol. 272 , No. 18 , Issue of ticular peptides and nucleotides which originate from the May 2 , pp . 12014 - 12023 , 1997. A summary of numerous venoms of Australian Funnel- web spiders . In various arthropod toxic peptides from different spider and scorpion embodiments , illustrative TPs for use in the preparation of species was reviewed in , Quintero -Hernandez , V . et al . an insecticidal and / or nematicidal protein , composition , “ Scorpion and Spider Venom Peptides : Gene Cloning and insecticidal and / or nematicidal protein , or polynucleotide Peptide Expression ” Toxicon , 58 , pp . 644 -663 , 2011. The encoding the same, can include one or more TPs selected three -dimensional structure of Hanatoxinl using NMR spec from the group : U - ACTX TPs, Kappa - ACTX TPs and troscopy was identified as an inhibitor cysteine knotmotif in Omega -ACTX TPs and polynucleotides encoding the same. Takahashi, H . et al. “ Solution structure of hanatoxinl, a The following documents are incorporated by reference in gating modifier of voltage -dependent K + channels : common the United States in their entirety , are known to one skilled surface features of gating modifier toxins” Journal of in the art, and have all been published . They disclose Molecular Biology, Volume 297 , Issue 3 , 31 Mar. 2000 , pp . numerous ICK motif TPs which , their full peptide sequence , 771 - 780 . The isolation and identification of cDNA encoding their full nucleotide sequence , are specifically disclosed and a scorpion venom ICK toxin peptide , Opicalcinel, was are incorporated by reference , and in addition the full published by Zhu , S . et al. “ Evolutionary origin of inhibitor disclosures are incorporated by reference including all of US 2018 /0362598 A1 Dec. 20 , 2018 24.
their sequence listings . See the following references : (all toxic peptides suitable for plant expression as insecticidal incorporated herein by reference in their entireties ) : U . S . and / or nematicidal proteins by themethods disclosed in this Pat. No. 7 ,354 , 993 B2 , issued Apr. 8 , 2008 , specifically the invention . peptide and nucleotide sequences listed there as SEQ ID 0194 ] The following species are specifically named : NOs: 1 - 39 , from U .S . Pat. No . 7 , 354 , 993 B2, and those Atrax formidabillis, Atrax infensus, Atrax robustus, Hadro named U - ACTX polypeptides, and these and other toxins nyche infensa , Hadronyche versuta . Any TPs derived from that can form 2 to 4 intra - chain disulfide bridges, and any of the genus and species listed above and / or genus variants thereof, and the peptides appearing on columns 4 to species and may be homologous to the U peptide are suitable 9 and in FIG . 2 of U . S . Pat . No . 7 , 354 ,993 B2 . for plant expression as insecticidal and nematicidal proteins [0191 ] Other specific sequences can be found in EP patent according to the present invention . Other TPs suitable for 1 812 464 B1, published and granted Aug . 10 , 2008 , see use in the various insecticidal and/ or nematicidal polypep Bulletin 2008 /41 , specifically the peptide and nucleotide tides and proteins described herein , include : Kappa ACTX sequences listed in the sequence listing , and those the other TPs, Omega - ACTX TPs and U - ACTX TPs. toxins that can form 2 to 4 intra - chain disulfide bridges , and f0195 ] In some embodiments , specific TPs can be cloned those sequences listed there as 1 -39 , and sequences named into proper expression vectors and along with the ERSP and U -ACTX polypeptides , and variants thereof, and the pep cleavable linking groups disclosed herein can be made into tides appearing in paragraphs 0023 to 0055 , and appearing an insecticidal and /or nematicidal protein suitable for trans in FIG . 1 of EP patent 1 812 464 B1, the disclosures of all forming a plant, or a part thereof, or the insecticidal and /or of the specifically enumerated patent references are incor nematicidal protein may be formulated with appropriate porated by reference herein in their entireties . excipients to be applied to the surface of a plant, or plant [0192 ] Described and incorporated by reference herein as part. In some embodiments, a suitable TP can include an examples of insecticidal and / or nematicidal proteins and ICK motif TP : QYCVP VDQPC SLNTQ PCCDD ATCTQ peptides are TPs including the following : peptides and ERNEN GHTVYYCRA (SEQ ID NO : 6 ) , named variants thereof found in , isolated from , or derived from “ U - ACTX - Hvla, ” it has disulfide bridges at positions : 3 -18 , spiders of the genus Atrax or Hadronyche , including the 10 - 23, 17 - 37 . The molecular weight is 4426 . 84 Daltons . In genus species, Hadronyche versuta , or the Blue Mountain another embodiment , specific TPs can be cloned into proper funnel web spider , Atrax robustus, Atrax formidabilis , Atrax expression vectors and along with the ERSP and cleavable infensus, including TPs known as U - ACTX polypeptides , linking groups disclosed herein can be made into an insec which include: U -ACTX -Hvla , U + 2 - ACTX -Hvla , ticidal and / or nematicidal protein suitable for transforming a rU - ACTX -Hvla , rU -ACTX -Hvlb , or mutants or variants , plant, or a part thereof, or the insecticidal and / or nematicidal especially peptides of any of these types and especially those protein may be formulated with appropriate excipients to be less than about 200 amino acids but greater than about 10 applied to the surface of a plant, or plant part . In some amino acids, and especially peptides less than about 150 embodiments , a suitable TP can include an ICK motif TP : amino acids but greater than about 20 amino acids, espe GSQYC VPVD PCSLN TOPCC DDATC TOERN cially peptides less than about 100 amino acids but greater ENGHT VYYCRA (SEQ ID NO : 5 ) , named “ U + 2 - ACTX than about 25 amino acids , especially peptides less than Hvla , " it has disulfide bridges at positions : 5 - 20 , 12 - 25 , about 65 amino acids but greater than about 25 amino acids, 19 - 39 . The molecular weight is 4564 . 85 Daltons. Other ICK especially peptides less than about 55 amino acids but TPs useful in the various insecticidal and /or nematicidal greater than about 25 amino acids, especially peptides of proteins, polynucleotides and DNA constructs encoding about 37 or 39 or about 36 to 42 amino acids , especially these insecticidal and / or nematicidal proteins , and trans peptides with less than about 55 amino acids but greater than genic plants or plant cells containing these insecticidal about 25 amino acids, especially peptides with less than and /or nematicidal proteins, include peptides or proteins about 45 amino acids but greater than about 35 amino acids , having at least 80 % , at least 85 % , at least 90 % , at least 95 % , especially peptides with less than about 115 amino acids but at least 96 % , at least 97 % , at least 98 % , at least 99 % , or at greater than about 75 amino acids , especially peptides with least 100 % sequence identity or sequence homology to a less than about 105 amino acids but greater than about 85 peptide or protein having an amino acid sequence as set forth amino acids, especially peptides with less than about 100 in SEQ ID NOs: 5 - 1593 and 1761 - 1775 . In related embodi amino acids but greater than about 90 amino acids , including ments , ICK TPs useful in the various insecticidal and /or peptide toxins of any of the lengths mentioned here that can nematicidal proteins, polynucleotides and DNA constructs form 2 , 3 and or 4 or more intrachain disulfide bridges , encoding these insecticidal and / or nematicidal proteins , and including toxins that disrupt calcium channel currents, transgenic plants or plant cells containing these insecticidal including toxins that disrupt potassium channel currents , and /or nematicidal proteins, include peptides or proteins especially toxins that disrupt insect calcium channels or Us having at least 80 % , at least 85 % , at least 90 % , at least 95 % , thereof, especially toxins or variants thereof of any of these at least 96 % , at least 97 % , at least 98 % , at least 99 % , or at types , and any combination of any of the types of toxins least 100 % sequence identity or sequence homology to a described herein that have oral or topical insecticidal and / or peptide or protein having an amino acid sequence as set forth nematicidal activity , can be made special by the processes in SEQ ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771 - 1773 , and described herein . variants thereof. [0193 ] The U peptides from the Australian Funnel Web [0196 ] ( 3A ) . Bt TPs Spider , genus Atrax and Hadronyche are particularly suit [0197 ] In some embodiments , illustrative TPs can also able and work well when treated by the methods , procedures include those TPs expressed , or isolated from Bacillus or processes described by this invention . Examples of such thuringiensis , (Bt ) . This ubiquitous Gram - positive spore suitable peptides tested and with data are provided herein . forming bacterium provides a valuable resource due to its The following species are also specifically known to carry ability to synthesize crystal parasporal inclusions during US 2018 /0362598 A1 Dec. 20 , 2018 25 sporulation . These crystals, which include insecticidal pro - (0199 ] Specific examples of Bt TPs can include peptides teins called 6 - endotoxins have been extensively used as where the Bt Protein is a Cry or Cyt protein , and /or the Bt biological insecticides against insect pests of commercial Protein is selected from the group consisting of a Cryl , interest . Bt crystal toxins include the Cry proteins ( crystal Cry3 , TIC851, CryET70 , Cry22 , TIC901 , TIC201 , TIC407 , toxins ) and Cyt proteins ( cytolytic toxins ). The Cry toxins TIC417 , a binary insecticidal protein CryET80 , and are important virulence factors allowing for the development CryET76 , a binary insecticidal protein TIC100 and TIC101, a combination of an insecticidal protein ET29 or ET37 with of the bacterium in dead or weakened insect larvae. The an insecticidal protein TIC810 or TIC812 and a binary largest group of Cry toxins has three distinct structural insecticidal protein PS149B1. The Bt TP can be selected domains; the so - called three -domain (3D ) Cry toxins pro from a Cry protein , a Cry1A protein or a Cry1F protein , or vide the primary focus for this review . The Cyt toxins have a combination Cry1F - Cry1A protein , or it comprises an in vitro cytolytic activity , in addition to in vivo activity amino acid sequence at least 90 % identical to SEQ ID NOs: against various insects including mosquitos [ 5 ] . Cyt toxins 10 , 12 , 14 , 26 , 28 , or 34 of U . S . Pat . No . 7 , 304 , 206 . We which are active against certain Diptera , synergize the describe a transgenic plant wherein the Bt TP is Dipel and toxicity of Cry proteins against mosquitoes and delay the we describe a transgenic plant wherein the Bt TP is Thuri expression of resistance to the latter Bt proteins, or Bt cide . In another example of an insecticidal and / or nemati peptides ( collectively referred to herein as Bt TPs) , are cidal polypeptide and protein described herein , is a Cry Bt effective insecticides used for crop protection in the form of TP called Cry51. This Bt TP is disclosed in U . S . Pat . No . both plant incorporated protectants and foliar sprays. Com 8 ,609 ,936 B2 ( issued on 12 / 17 , 2013 , the disclosure of mercial formulations of Bt proteins are widely used to which is incorporated by reference herein in its entirety ) . control insects at the larval stage. The Bt Cry and Cyt This Cry Bt TP is insecticidal against the genus of lygus proteins require solubilization in the insect midgut to pro - plant- feeding insects . The exemplified Cry Bt TP is useful in duce protoxins that are typically about 130 kDa , 70 kDa or the present invention , for example , as a TP in an insecticidal 27 kDa for Cyt. These in turn are proteolytically cleaved at and / or nematicidal protein , or a plant or plant part thereof the C - terminus and / or at the N - terminus by midgut pro comprising said insecticidal and / or nematicidal polypeptide teases, generating the activated core toxin . Cry protoxins are and protein , for example a plant cell , a plant tissue or plant proteolytically activated to produce the mature active toxin . seed . Processing of the Cry protoxin into its active form is [0200 ] In various embodiments , isolated nucleic acid mol essential for toxin activity . Processing is mediated by insect ecules corresponding to Bt TP encoding nucleic acid proteases that cleave the protoxin polypeptide at specific sequences are provided . Additionally , amino acid sequences sequences . The toxin then crosses the peritrophic matrix and corresponding to the polynucleotides are encompassed . In binds to receptors in the apical membrane of the midgut particular, the present invention provides for an isolated cells , with receptor binding being an important determinant nucleic acid molecule comprising a nucleotide sequence of toxin specificity . Toxin insertion into the epithelial mem encoding the amino acid sequence shown in US 2009 / brane forms ion channels or pores , leading to lysis of the 0099081, published on Apr. 18 , 2009, all of which is herein cells , damage to themidgut epithelial tissue, and death of the incorporated by reference in its entirety , and all sequences larva . identified by number specifically incorporated by reference . [ 0198 ] Among susceptible species in the orders Lepidop Various insecticidal polypeptides and polynucleotides tera and Diptera , the maj or gut proteases are of the serine encoding same as disclosed in US 2009 /0099081 , for type , while in Coleoptera the major proteases are cysteine example , SEQ ID NOS: 9 , 11, 13 , 15 , or 18 , or a nucleotide and aspartic proteases, although some use cathepsin G serine sequence set forth in SEO ID NOs: 1 , 2 , 4 , 6 , 7 , 8 , 10 , 12 , proteases . Since activation is a crucial step to achieve 14 , 16 , or 17 , as well as variants and fragments thereof are toxicity , it has been suggested that the type and /or abun incorporated herein by reference in its entirety . Nucleotide dance of insect proteases is important in contributing to sequences that are complementary to a nucleotide sequence toxin specificity . In some cases , certain insects lack the of the invention , or that hybridize to a sequence of the ability to cleave the inactive portion from the activated or invention are also encompassed . " active " portion of the Bt TP. The present invention provides [0201 ] Nucleotide sequences encoding the proteins of the for certain insecticidal and /or nematicidal proteins that have present invention include the sequences set forth in US a binary or teriary cleavable linker positioned between the 2009 /0099081 , published on Apr. 18 , 2009 , including SEQ inactive portion and the activated or " active " portion of the ID NOs: 1 , 2 , 4 , 6 , 7 , 8, 10 , 12 , 14 , 16 , or 17 , and variants , Bt TP (See FIG . 3B ) and insecticidal and / or nematicidal fragments , and complementary nucleotide sequences thereof proteins that include multiple copies ( for example , 2 - 10 of US 2009 /0099081 are incorporated herein by reference in copies of the Bt TP having an intervening cleavable linker their entirety. The term " complementary sequence ” is positioned between the inactive and active portions of the Bt intended a nucleotide sequence that is sufficiently comple TP ) in the form of insecticidal and / or nematicidal proteins mentary to a given nucleotide sequence such that it can and polynucleotides that encode the Bt TPs and insecticidal hybridize to the given nucleotide sequence to thereby form and / or nematicidal proteins referenced above . In some a stable duplex . Illustrative Bt protein amino acid sequences embodiments , in the case where a DNA construct or a that may be used as a TP for the compositions , transgenic polynucleotide contains an open reading frame with a single molecules (polynucleotides and proteins ) are set forth in the TP, the TP can include Bt TPs , in addition to all of the other present disclosure set forth as SEQ ID NOs: 33 - 533 . described TPs , with the proviso that if the DNA construct or [0202 ] Nucleic acid molecules that are fragments of these polynucleotide contains an open reading frame containing Bt protein encoding nucleotide sequences are also encom two or more TPs , the two or more TPs cannot be two or more passed by the present invention ( for example, US 2009 / Bt TPs. 0099081, published on Apr. 18 , 2009, all of which is herein US 2018 /0362598 A1 Dec. 20 , 2018 incorporated by reference in its entirety , and all sequences described herein using standard parameters. One of skill in identified by number specifically incorporated by reference the art will recognize that these values can be appropriately from US 2009/ 0099081 . adjusted to determine corresponding identity of proteins 10203 ] SEQ ID NO : 8 is a fragment of SEQ ID NOs: 4 and encoded by two nucleotide sequences by taking into account 12 ; SEQ ID NO : 4 is a fragment of SEQ ID NO : 2 as codon degeneracy, amino acid similarity , reading frame disclosed in US 2009 /0099081 ) . By " fragment” is intended positioning , and the like . a portion of the nucleotide sequence encoding a Bt protein . A fragment of a nucleotide sequence may encode a biologi [0207 ] The invention also encompasses variant nucleic cally active portion of a Bt protein , or it may be a fragment acid molecules ( for example , US 2009/ 0099081 , published that can be used as a hybridization probe or PCR primer on Apr. 18 , 2009 , all of which is herein incorporated by using methods disclosed below . Nucleic acid molecules that reference in its entirety , and all sequences identified by are fragments of a Bt protein nucleotide sequence comprise number specifically incorporated by reference , sequence 2 is at least about 50 , 100 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , a variant of sequence 1; sequences 7 and 8 are variants of 900 , 1000 , 1050 , 1100 , 1150 , 1200 , 1250 , 1300 , 1350 , 1400 , sequence 6 ; sequence 10 is a variant of sequences 4 and 12 ; 1450 , 1500 , 1550 , 1600 , 1650 , 1700 , 1750 , 1800 , 1850 , and sequence 14 is a variant of sequence 12 ). “ Variants ” of 1860 , 1870 , 1880 , 1885 contiguous nucleotides, or up to the the Bt protein encoding nucleotide sequences include those number of nucleotides present in a full - length Bt- protein sequences that encode the Bt protein disclosed herein but encoding nucleotide sequence disclosed herein ( for that differ conservatively because of the degeneracy of the example , 1890 nucleotides for US 2009 / 0099081, published genetic code as well as those that are sufficiently identical as on Apr. 18 , 2009, Here these are provided as SEQ ID NOs: discussed above . 1 and 2 , 1806 nucleotides for SEQ ID NO : 4 , 1743 nucleo [0208 ] Naturally occurring allelic variants can be identi tides for SEQ ID NOs: 6 , 7 , 8, and 16 , 1809 nucleotides for fied with the use of well -known molecular biology tech SEQ ID NO : 10 , and 1752 nucleotides for SEQ ID NOs: 12 niques, such as polymerase chain reaction (PCR ) and and 14 , in the sequence listing of US 2009/ 0099081 ) . By hybridization techniques as outlined below . Variant nucleo " contiguous” nucleotides is intended nucleotide residues tide sequences also include synthetically derived nucleotide that are immediately adjacent to one another . Fragments of sequences that have been generated , for example , by using the nucleotide sequences of the present invention will site -directed mutagenesis but which still encode the Bt encode protein fragments that retain the biological activity proteins disclosed in the present invention as discussed of the Bt protein and , hence , retain pesticidal activity . By below . Variant proteins encompassed by the present inven “ retains activity ” is intended that the fragment will have at tion are biologically active , that is they continue to possess least about 30 % , at least about 50 % , at least about 70 % , the desired biological activity of the native protein that is , 80 % , 90 % , 95 % or higher of the pesticidal activity of the Bt retaining insecticidal and / or nematicidal activity (also protein . Methods for measuring pesticidal activity are well known as pesticidal activity ) . By “ retains activity ” is known in the art. See , for example, Czapla and Lang ( 1990 ) intended that the variant will have at least about 30 % , or at J . Econ . least about 40 % , or at least about 50 % , or at least about60 % , [0204 ] Entomol. 83 : 2480 - 2485 ; Andrews et al. (1988 ) or at least about 70 % , or at least about 80 % , or at least about Biochem . J . 252 : 199 - 206 ; Marrone et al . ( 1985 ) J . of Eco 90 % of the pesticidal activity of the native protein . Methods nomic Entomology 78 :290 - 293 ; and U . S . Pat . No. 5 ,743 , for measuring pesticidal activity are well known in the art . 477 , all of which are incorporated herein by reference in See , for example , Czapla and Lang ( 1990 ) J . Econ . Entomol. their entireties , and all sequences identified by number 83 : 2480 - 2485 ; Andrews et al. (1988 ) Biochem . J . 252 : 199 specifically incorporated by reference . 206 ; Marrone et al. ( 1985 ) J . of Economic Entomology [ 0205 ] A fragment of a Bt protein encoding nucleotide 78 :290 -293 ; and U . S . Pat. No. 5 ,743 ,477 , all of which are sequence that encodes a biologically active portion of a incorporated herein by reference in their entireties , and all protein of the invention will encode at least about 15 , 25 , 30 , sequences identified by number specifically incorporated by 50 , 75 , 100 , 125 , 150 , 175 , 200 , 250 , 300, 350 , 400 , 450 , reference . 500, 550 , 560 , 570 , 575 , 580 , 585 , 590 , 595 , 600 contiguous amino acids , or up to the total number of amino acids present 0209 ]. In one aspect of the invention , some TPs that can in a full - length Btprotein of the invention (for example , 580 be employed in the design and synthesis of insecticidal amino acids for SEQ ID NO : 41 , 602 amino acids for SEQ and /or nematicidal proteins containing cleavable linking ID NO : 43 , and 583 amino acids for SEQ ID NOs: 45 and molecules ( i. e . binary and tertiary peptides ) can include one 47 ). or more synthetic axmi- 004 Bt protein sequences , such as [ 0206 ] Preferred Bt protein proteins of the present inven those disclosed in US 2009/ 0099081 , published on Apr. 18 , tion are encoded by a nucleotide sequence sufficiently 2009 , all of which are incorporated herein by reference in identical to the nucleotide sequence of US 2009/ 0099081 , their entireties , and all sequences identified by number published on Apr. 18 , 2009 , all of which is herein incorpo specifically incorporated by reference , ( sequence 1 ) and rated by reference in its entirety , and all sequences identified synaxmi- 004B ( sequence 2 ). These synthetic sequences by number specifically incorporated by reference, sequences have an altered DNA sequence relative to the axmi-004 1 , 2 , 4 , 6 , 7 , 8 , 10 , 12 , 14 , 16 , or 17 . By “ sufficiently sequence (sequence 3 ) recited in U . S . Pat. No . 7 ,355 ,099 , all identical” is intended an amino acid or nucleotide sequence of which is herein incorporated by reference in its entirety , that has at least about 60 % or 65 % sequence identity , about and all sequences identified by number specifically incor 70 % or 75 % sequence identity , about 80 % or 85 % sequence porated by reference ) , and encode the original AXMI- 004 identity, about 90 % , 91 % 92 % 93 % 94 % , 95 % , 96 % , 97 % protein . 98 % , 99 % or greater sequence identity compared to a [0210 ] Likewise , synaxmi- 004B - 2M (sequence 4 ) was reference sequence using one of the alignment programs designated and encodes the axmi- 004 alternate start site US 2018 /0362598 A1 Dec. 20 , 2018 27
( herein referred to as axmi- 004B - 2M and set forth in TABLE 1 - continued sequence 5 ) originally identified in U . S . patent application Ser. No. 10 / 782 , 020 . ( e ) Bt Toxins [0211 ] Additional TPs included as possible designated axmi- 004B - 3M (US 2009 /0099081 , published on Apr. 18 , Toxin Patents or Patent Publication Number 2009 , all of which is herein incorporated by reference in its Cryl US2007061919 , Cry1 US2007061919 , CN1401772 , U . S . Pat. No . 6 , 063 ,605 , entirety, and all sequences identified by number specifically Cry1 US2007061919 , AU784649B , U . S . Pat. No. 5 , 723, 758 , incorporated by reference , sequence 16 ) and encodes the U . S . Pat . No. 5 ,616 , 319 , U . S . Pat . No. 5 , 356 ,623 , U . S . Pat . No. AXMI- 004B - 3M amino acid sequence set forth in sequence 5 , 322 ,687 9 . Synthetic sequences encoding the AXMI- 004B -3M pro Cry1 U . S . Pat. No . 5 ,723 ,758 Cry2 CN1942582 , WO9840490 , tein were also designated . These synthetic nucleotide US2007061919 , UA75570 , sequences were designated synaxmi- 004B - 3M , synaxmi MXPA03006130 , US2003167517 , 004C - 3M , and synaxmi- 004D - 3M and are set forth in U . S . Pat. No. 6 , 107 ,278 , U . S . Pat. No. 6 , 096 , 708, U . S . Pat. No . sequences 6 , 7 , and 8 , respectively . In another aspect of the 5 ,073 ,632 , U . S . Pat . No . 7 , 208 ,474 , U . S . Pat . No. 7 ,244 ,880 , invention , modified versions of the nucleotide sequence Cry3 US2002152496 , RU2278161, encoding AXMI- 004B -3M protein were designed such that US2003054391, additional N -terminal residues are added to the encoded Cry3 U . S . Pat . No. 5 , 837 , 237 , U . S . Pat . No . 5 ,723 ,756 , U . S . Pat . No. protein . These sequence are designated synaxmi- 004B - 3M 5 ,683 ,691 , U . S . Pat. No . 5 , 104 ,974 , U . S . Pat . No . 4 ,996 , 155 , alt1 (US 2009 / 0099081 , published on Apr. 18, 2009 , Cry3 U . S . Pat . No . 5 , 837 , 237 , U . S . Pat. No . 5 ,723 , 756 , sequence 10 ) , synaxmi- 004B - 3M - alt2 ( sequence 12 ) , syn Cry5 W09840491 , US2004018982 , U . S . Pat . No. 6 , 166 , 195 , axmi- 004B - 3M - alt3 ( sequence 14 ), and synaxmi- 004B - 3M US2001010932, U . S . Pat. No . 5 ,985 , 831 , U . S . Pat. No . alt4 (sequence 17 ) . 5 ,824 ,792 , U . S . Pat . No. 528 , 153 [ 0212 ] The encoded proteins are designated AXMI- 004B Cry5 WO2007062064 , US2001010932 , 3M - ALT1 (sequence 11 ) , AXMI- 004B - 3M - ALT2 (sequence U . S . Pat . No . 5 ,824 ,792 , 13 ) , AXMI- 004B - 3M -ALT3 (sequence 15 ), and AXMI Cry6 WO2007062064 , US2004018982 , 004B - 3M - ALT4 ( sequence 18 ) . U . S . Pat . No . 5 ,973 ,231 , U . S . Pat . No. 5 ,874 ,288 , U . S . Pat . No . 5 ,236 ,843 , [0213 ] Other Bt proteins and gene descriptions can be U . S . Pat . No. 683 , 106 found in the following . Each and every patent publication Cryo US2004018982, U . S . Pat. No . 6 ,166 , 195 , referred to below with a note as to the Bt toxin to which the Cry7 U . S . Pat. No . 6 ,048 ,839 , U . S . Pat. No. 5 ,683 ,691 , U . S . Pat. No . publication refers to , is hereby incorporated by reference in 5 , 378 ,625 , U . S . Pat. No . 518 , 709 its entirely . These documents have also published and they Cry7 CN195215 and their sequences are in the public domain . Cry8 [0214 ] Additional examples of Bt genes, proteins suitable Cry8 as polynucleotides encoding TPs and TPs of the present Cry8 US200301796 Cry8 WO2006053473 , US2007245430 , invention , and the patent documents that describe them are Cry8 WO200605347 found in Tables 1 , 2 , and 3 below . The patent documents in Cry9 US2007061919 , Tables 1 , 2 , 3 , in particular the US Patents and US applica Cry9 WO200506620 Cry9 US2007061919 , U . S . Pat. No . 6 , 448 ,226 , tions, are hereby incorporated by reference herein in their US2005097635 , WO2005066202 , entireties. U . S . Pat . No . 6 , 143 , 550 , U . S . Pat. No . 6 ,028 , 246 , U . S . Pat. No . 6 ,727 ,409 , TABLE 1 Cry9 US2005097635 , WO2005066202, Cry9 U . S . Pat. No. 6 , 570 ,005 , ( e ) Bt Toxins Cry9 AU784649B , US2007074308 , U . S . Pat . No. 736 , 180 Toxin Patents or Patent Publication Number Cry11 MXPA0200870 Cry12 US2004018982 , U . S . Pat . No. 6 , 166 , 195 , Cry1 US2003046726 , U . S . Pat. No. 6 ,833 ,449 , U . S . Pat . No . 6 ,077 ,937 , U . S . Pat . No. 5 ,824 , 792 , U . S . Pat . No . CN1260397 , US201026939 , 5 , 753 , 492 , US2006174372 , US2006174372 , Cry13 US2004018982 , U . S . Pat . No . 6 ,166 , 195, U . S . Pat . No. 642 , 241, U . S . Pat . No . 6 ,229 ,004 , U . S . Pat . No . 6 ,077 ,937 , U . S . Pat . No . 5 , 824 ,792 , U . S . Pat. No . US2004194165, U . S . Pat. No. 6 , 573 ,240 , 5 ,753 ,492 , U . S . Pat. No. 5 , 424 , 409 , U . S . Pat . No . 5 ,407 ,825 , Cry14 JP2007006895 , U . S . Pat . No . 5 ,831 , 011 , U . S . Pat . No . 5 , 135 , 867 , U . S . Pat. No. 5 ,055 , 294 , Cry21 U . S . Pat . No . 5 ,831 ,011 , U . S . Pat . No . 5 ,670 , 365 , Cry1 WO2007107302 , U . S . Pat . No. 6 ,855 ,873 , Cry22 US2006218666 , US2001010932 , WO2004020636 , US2007061919 , MXPA01004361, U . S . Pat . No . 5 ,824 ,792 , U . S . Pat. No . 6 ,048 , 839 , US2007061919 , Cry22 US2003229919 , AU784649B , US2007061919 , U . S . Pat . No . 6 , 150 ,589 , Cry23 US2006051822 , US2003144192 , U . S . Pat. No . 5 ,679 , 343 , U . S . Pat. No. 5 ,616 , 319 , U . S . Pat. No. UA75317 , U . S . Pat . No . 6 , 399 , 330 , U . S . Pat . No. 6 , 326 , 351, 5 , 322 ,687 , U . S . Pat . No. 6 ,949 ,626 , Cry1 WO2007107302, US2006174372 , Cry26 US200315001 US2005091714 , US2004058860 , Cry28 US200315001 US2008020968 , U . S . Pat. No . 6 ,043 ,415 , U . S . Pat. No . Cry31 CA2410153 , 5 , 942 ,664 , Cry34 US200316752 Cry1 WO2007107302, US2007061919 , Cry35 US2003167522 , U . S . Pat . No . 6 , 172, 281 , Cry37 US2006051822, US2003144192 , Cry1 W003082910 , MX9606262 , U . S . Pat. No . 5 ,530 , 195 , UA75317 , U . S . Pat . No. 6 , 399 , 330 , U . S . Pat. No. 6 , 326 ,351 , U . S . Pat. No . 5 , 407 ,825 , U . S . Pat . No . 5 , 045 , 469 , U . S . Pat . No . 6 ,949 ,626 , Cryl US2006174372 , Cry43 US200527164 Cry1 US2007061919 , Cyt1 WO2007027776 , US 2018 /0362598 A1 Dec. 20 , 2018 28
TABLE 1 - continued rU -ACTX -Hvlb , or mutants or variants , Omega- ACTX polypeptides and variants and Kappa - ACTX polypeptides ( e ) Bt Toxins and variants . Toxin Patents or Patent Publication Number 0220 ] In some embodiments, illustrative CRIP TPs include a Non - ICK CRIPTP , for example, a Non - ICK CRIP Cyti U .S . Pat. No . 6 , 150 , 165 , Cyt2 US2007163000 , EP1681351 , TP derived from , or originates from , animals having Non U . S . Pat. No . 6 ,686 ,452 , U . S . Pat . No . 6 ,537 , 756 , ICK CRIPS such as sea anemones , sea urchins and sea slugs and variants thereof. In one exemplary embodiment, a Non - ICK CRIP TP can be a TP derived from the sea anemone named Anemonia viridi, optionally including the TABLE 2 TPs named Av2 and Av3 especially TPs similar to Av2 and ( f ) Hybrid Insecticidal Crystal Proteins and Patents Av3 including such peptides listed in the sequence listing of the present applicationor mutants or variants thereof. Patents Holotype Toxin [0221 ] In other embodiments , other TPs that can be used US2008020967 Cry29A in the recombinant constructs and insecticidal and / or nem US2008040827 Cryica aticidal proteins and plants described herein can include one US2007245430 Cry8A US2008016596 Cry8A or more of an ACTX protein , and /or a TMOF protein . US2008020968 Cry1?? TMOF motif, ” or “ TMOF proteins ” include trypsin modu lating oostatic factor peptide . Numerous examples and vari ants of TMOF TPs are provided . SEQ ID NO : 708 in the present application is an exemplary wild type TMOF TABLE 3 sequence . Other non - limiting variants are provided in SEQ ID NOs: 709- 721 . Other examples are known or could be ( g ) Patents Relating to Other Hybrid Insecticidal Crystal Proteins created guided by the knowledge known in the field by one Source toxinsa Patents skilled in the art . Cry1A , Cry1C U . S . Pat. No . 5 ,593 ,881 , U .S . Pat . No. 5 , 932 ,209 [ 0222 ] ( 3C ). Cleavable Bt TPs Cry1c , Cry1A , Cry1F U . S . Pat . No. 6 , 962 , 705 , U . S . Pat . No. 7 , 250 ,501 , US2004093637 [0223 ] In some embodiments , a representative Bt toxic Cry23A , Cry37A U . S . Pat. No. 7 ,214 ,788 protein ( TP ) can include a cleavable Bt TP as provided Cry1A U . S . Pat. No. 7 ,019 , 197 herein that comprises a binary or tertiary peptide inserted Cry1A , Cry1B U . S . Pat . No . 6 , 320 , 100 Cry1A , Cry1c AU2001285900B between the active and inactive portions of the Bt TP . In one Cry23A , Cry37A US2007208168 example , a binary or tertiary peptide is inserted between the Cry3A , Cry1l, Cry1B W00134811 cleavable pro inactive portion of the peptide and mature Cry3A , Cry3B , Cry3C US2004033523 active portion of the peptide . In another example , a Bt TP Cry1A , Cry1c , Cry1E , U . S . Pat . No . 6 ,780 , 408 includes a stabilizing domain fused in framewith a binary or Cry1A , Cry1F US2008047034 tertiary peptide of the present invention which is fused in frame with the active portion of the Bt TP . In another [ 0215 ] The sequence listing of the present disclosure example , the Bt TPs are frequently written as " cry ” , “ cy?" or includes exemplary Bt TP amino acid sequences SEQ ID “ VIP ” proteins encoded by the cry , cyt and vip genes . Bt TPs NOs: 33 -533 . These amino acid sequences include examples are more usually attributed to insecticidal crystal proteins of Bt protein Cry and Cyt protein sequences . Examples are encoded by the cry genes. Bt TPs are examples of PFIPS numerous and one skilled in the art would know of many ( Pore Forming Insecticidal Proteins ) . In various embodi other examples of various Bt sequences that are suitable ments, an insecticidal and / or nematicidal protein may substitutes for those in this disclosure . include a protein that includes two or more binary or tertiary [0216 ] (3B ). TMOF TPs peptides and at least one Bt TP fused in frame with the one [0217 ] In some embodiments , another source of TPs can or more binary and/ or tertiary peptides, In some embodi include : Trypsin modulating oostatic factor ( TMOF ) pep ments , the insecticidal and / or nematicidal protein useful to tides . TMOF peptides have to be delivered to their physi provide insecticidal and / or nematicidal compositions when ological site of action in various ways , and TMOF peptides ingested and exposed to gut or hemolymph environments have been identified as a potential larvicides, with great can contain a Bt TP and / or a cleavable Bt TP as described potential, see D . Borovsky, Journal of Experimental Biology above, and further exemplified herein , containing a binary or 206 , 3869- 3875, the disclosure of which is incorporated tertiary peptide inserted between the cleavable pro ( inactive ) herein by reference in its entirety . portion of the TP and mature ( active ) portion of the TP. 0218 ] In various embodiments , illustrative TPs include [0224 ] For example , a binary or tertiary peptide when the ICK motif TP which can be any polypeptide or protein inserted between the cleavable pro inactive portion of the Bt with the ICK motif ranging in length between 16 and 60 TP and mature active portion of the Bt TP provides a amino acids , with at least 6 cysteine residues that create cleavable Bt TP that upon ingestion and contact with an covalent cross - linking disulfide bonds in the proper order. insect or nematode gut protease results in the cleavage of the 10219 ] In various embodiments , illustrative TPs include cleavable Bt TP ( See FIG . 3B ) . In some embodiments , an the CRIP TPs for example the ICK TPs for example , an ICK insecticidal and/ or nematicidal protein containing cleavable TP derived from , or originates from , Hadronyche versuta , or and / or non -cleavable Bt TPs can be encoded by a DNA the Blue Mountain funnel web spider , Atrax robustus, Atrax construct that when transformed into plants , or parts thereof, formidabilis , Atrax infensus, including TPs known as or when applied directly to the surface of plants provide U - ACTX polypetides , U - ACTX -Hvla , rU - ACTX -Hvla , insecticidal and /or nematicidal activity when the insecticidal US 2018 /0362598 A1 Dec. 20 , 2018 and / or nematicidal protein is ingested and exposed to the gut 95 % , or at least 96 % or at least 97 % or at least 98 % or at and / or hemolymph environment of a target insect and /or least 99 % amino acid sequence identity to any TP disclosed nematode . in SEQ ID NO : 5 - 1593 and 1761- 1775 , and still retains at [ 0225 ] Described and incorporated by reference to the TPs least : 95 % , or 90 % , or 80 % , or 70 % , or 60 % , or 50 % of the identified herein are variants ormutant peptides and proteins insecticidal and /or nematicidal activity of the corresponding of the TP amino acid sequences mentioned above , specifi TP set forth in SEQ ID NO : 5 - 1593 and 1761- 1775 . cally , peptides and proteins having an amino acid sequence [0227 ] The variant TPs may further comprise one or more as shown in SEQ ID NO : 5 - 1593 and 1761 - 1775 , having additional substitutions at one or more ( e . g . , several other homology or sequence identity to such sequences or referred positions . The amino acid changes may be of a minor nature , to herein , which are also identified and claimed as suitable that is conservative amino acid substitutions or insertions for making special according to the processes described that do not significantly affect the folding and / or activity of herein , including all homologous sequences having at least the peptide or the protein ; small deletions , typically of 1 - 30 any of the following percent identities to any of the amino acids ; small amino - or carboxyl- terminal extensions , sequences disclosed here or to any sequence incorporated by such as an amino -terminal methionine residue ; or a small reference : 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , extension that facilitates purification by changing net charge 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % or greater amino acid or another function , such as a poly -histidine tract, an anti sequence identity or 100 % amino acid sequence identity to genic epitope or a binding domain . Examples of conserva any and all sequences identified in the patents noted above , tive substitutions are within the groups of basic amino acids and to any other sequence identified herein , including each ( arginine , lysine and histidine ) , acidic amino acids ( glutamic and every sequence in the sequence listing of this applica acid and aspartic acid ) , polar amino acids ( glutamine and tion . When the term homologous or homology is used herein asparagine ) , hydrophobic amino acids ( leucine , isoleucine with a number such as 50 % or greater , then what is meant and valine ) , aromatic amino acids (phenylalanine , trypto is percent identity or percent similarity between the two phan and tyrosine ) , and small amino acids ( glycine, alanine , peptides . When homologous or homology is used without a serine, threonine and methionine ) . Amino acid substitutions numeric percent then it refers to two peptide sequences that that do not generally alter specific activity are known in the are closely related in the evolutionary or developmental art and are described , for example , by H . Neurath and R . L . aspect in that they share common physical and functional Hill, 1979 , In , The Proteins, Academic Press , New York . aspects, like topical toxicity and similar size (i . e ., the Common substitutions are Ala / Ser, Val/ Ile , Asp /Glu , Thr/ homolog being within 100 % greater length or 50 % shorter Ser, Ala /Gly , Ala / Thr, Ser / Asn , Ala / Val, Ser /Gly , Tyr/ Phe , length of the peptide specifically mentioned herein or iden Ala /Pro , Lys/ Arg , Asp / Asn , Leu / lle , Leu / Val , Ala /Glu , and tified by reference herein as above ) . TPs of the present Asp /Gly . Alternatively, the amino acid changes are of such invention also include variants of TPs specifically disclosed a nature that the physico - chemical properties of the poly or described herein , The term “ variant” means a polypeptide peptides are altered . For example , amino acid changes may or protein having insecticidal and / or nematicidal activity improve the thermal stability of the polypeptide, alter the comprising an alteration , i. e . , a substitution , insertion , and / or substrate specificity , change the pH optimum , and the like . deletion , at one or more ( e . g . , several) positions. A substi [ 0228 ] Essential amino acids in a peptide or protein can be tution means replacement of the amino acid occupying a identified according to procedures known in the art , such as position with a different amino acid ; a deletion means site -directed mutagenesis or alanine -scanning mutagenesis removal of the amino acid occupying a position ; and an (Cunningham and Wells , 1989 , Science 244 : 1081 - 1085 ) . In insertion means adding an amino acid adjacent to and the latter technique , single alanine mutations are introduced immediately following the amino acid occupying a position . at every residue in the molecule, and the resultant mutant The TP variants of the present invention have at least 20 % , molecules are tested for insecticidal or nematicidal activity e . g ., at least 40 % , at least 50 % , at least 60 % , at least 70 % , to identify amino acid residues that are critical to the activity at least 80 % , at least 90 % , at least 95 % , or at least 100 % of of the molecule . See also , Hilton et al. , 1996 , J . Biol. Chem . the insecticidal and /or nematicidal activity of the polypep 271 : 4699 -4708 . The active site of insecticidal and / or nem tides and proteins of SEQ ID NO : 5 - 1593 and 1761- 1775 aticidal peptide or protein or other biological interaction can and the other specifically identified TPs disclosed herein . also be determined by physical analysis of structure, as The variants of the present invention have at least 20 % , e . g ., determined by such techniques as nuclear magnetic reso at least 40 % , at least 50 % , at least 60 % , at least 70 % , at least nance, crystallography, electron diffraction , or photoaffinity 80 % , at least 90 % , at least 95 % , or at least 100 % of the labeling , in conjunction with mutation of putative contact insecticidal and /or nematicidal activity of any one polypep site amino acids . See , for example , de Vos et al . , 1992 , tide or protein of SEQ ID NOs : 5 -1593 and 1761 - 1775. Science 255 :306 -312 ; Smith et al . , 1992 , J . Mol. Biol. [ 0226 ] TPs may also include variant TP peptides and 224 : 899 - 904 ; Wlodaver et al . , 1992 , FEBS Lett. 309 : 59 - 64 . proteins encoded by a polynucleotide having one or more The identity of essential amino acids can also be inferred point mutations, for example, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 from an alignment with a related TP peptide or protein as point mutations or more , such that when the mutated TP disclosed herein and/ or as set forth in SEQ ID NOs: 5 - 1593 encoding polynucleotide is transcribed and / or translated , the and 1761- 1775. expressed mutated TP peptide or protein retains insecticidal [0229 ] TPs of the present invention may also include TP and / or nematicidal activity that is at least : 90 % , or 80 % , or peptides and proteins having 1 - 6 amino acid extensions 70 % , or 60 % , or 50 % of the insecticidal and /or nematicidal fused in frame to the N -terminus or C - terminus or both activity of the peptide or protein encoded by the correspond termini of any TP described herein , for example a TP having ing non -mutated TP polynucleotide . TPs of the present an amino acid sequence as set forth in SEQ ID NOs: 5 - 1593 invention may also include TP peptides and proteins that and 1761- 1775 . The variants can be prepared using any have at least 80 % , or at least 85 % , or at least 90 % , or at least mutagenesis procedure known in the art , such as site US 2018 /0362598 A1 Dec. 20 , 2018 30 directed mutagenesis , synthetic gene construction , semi of genes may thus be synthesized de novo , while other synthetic gene construction , random mutagenesis , shuffling , regions may be amplified using site -specific mutagenic etc . primers, while yet other regions may be subjected to error [0230 ] Site -directed mutagenesis is a technique in which prone PCR or non -error prone PCR amplification . Poly one or more ( e. g ., several ) mutations are introduced at one nucleotide subsequences may then be shuffled . or more defined sites in a polynucleotide encoding the [ 0235 ] For purposes of describing the insecticidal and / or parent. Site - directed mutagenesis can be accomplished in nematicidal peptides , proteins , polynucleotides encoding vitro by PCR involving the use of oligonucleotide primers such insecticidal and /or nematicidal peptides and proteins , containing the desired mutation . Site - directed mutagenesis DNA constructs encoding such insecticidal and/ or nemati can also be performed in vitro by cassette mutagenesis cidal peptides and proteins , transgenic plants or parts thereof involving the cleavage by a restriction enzyme at a site in the containing such insecticidal and /or nematicidal peptides and plasmid comprising a polynucleotide encoding the parent proteins, and methods for controlling a pest infection or and subsequent ligation of an oligonucleotide containing the infestation and methods for increasing the yield of a crop mutation in the polynucleotide . Usually the restriction incorporating such insecticidal and /or nematicidal peptides enzyme that digests the plasmid and the oligonucleotide is and proteins , TPs of the present invention also encompass the same, permitting sticky ends of the plasmid and the ( includes within its definition ) variant and mutant and insert to ligate to one another. See, e . g. , Scherer and Davis , mutated TPs as described above within the definition of a TP 1979, Proc . Natl. Acad . Sci. USA 76 :4949 - 4955 ; and Barton or toxic peptide . In various embodiments , a TP specifically et al. , 1990 , Nucleic Acids Res. 18 : 7349 -4966 . Site - directed described herein include any TP having an amino acid mutagenesis can also be accomplished in vivo by methods sequence as set forth in SEQ ID NOs: 5 - 1593 and 1761 known in the art . See , e . g . , U . S . Patent Application Publi 1775 , any TP recited in any patent or non - patent reference cation No . 2004 /0171154 ; Storici et al. , 2001, Nature Bio described herein and any variant or mutant or mutated technol. 19 :773 - 776 ; Kren et al. , 1998 , Nat. Med . 4 : 285 peptide or protein thereof. IN various embodiments , an 290 ; and Calissano and Macino , 1996 , Fungal Genet . insecticidal and / or nematicidal proteins described herein , Newslett. 43 : 15 - 16 . Any site - directed mutagenesis proce include a TP having at least 80 % , at least 85 % , at least 90 % , dure can be used in the present invention . There are many at least 95 % , at least 96 % , at least 97 % , at least 98 % , at least commercial kits available that can be used to prepare variant 99 % , or at least 100 % sequence identity or sequence homol TPs of the present invention . ogy to a peptide having an amino acid sequence as set forth [ 0231 ] Synthetic gene construction entails in vitro synthe in SEQ ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771 - 1773 , and sis of a designed polynucleotide molecule to encode a variants thereof. polypeptide of interest. Gene synthesis can be performed [0236 ] ( 4) . Recombinant Insecticidal & Nematicidal Pep utilizing a number of techniques , such as the multiplex tide DNA Compositions microchip -based technology described by Tian et al . (2004 , [0237 ] In various embodiments , the present invention pro Nature 432 : 1050 - 1054 ) and similar technologies wherein vides insecticidal and nematicidal polypeptides , proteins and oligonucleotides are synthesized and assembled upon photo DNA constructs and polynucleotides encoding such poly programmable microfluidic chips. peptides and proteins. DNA constructs encoding such insec [0232 ] Single or multiple amino acid substitutions , dele ticidal and nematicidal polypeptides , and proteins may tions, and / or insertions can be made and tested using known encompass a single polynucleotide containing the open methods of mutagenesis , recombination , and /or shuffling , reading frame (ORF ) encoding one long fusion protein , or followed by a relevant screening procedure , such as those may encompass a number of polynucleotides which when disclosed by Reidhaar -Olson and Sauer, 1988 , Science 241 : provided in a vector enables the transcription of mRNA that 53 - 57 ; Bowie and Sauer, 1989 , Proc . Natl . Acad . Sci . USA encodes such insecticidal and nematicidal polypeptides as 86 : 2152 - 2156 ; WO 95 / 17413 ; or WO 95 /22625 . Other described herein . The DNA construct may also contain the methods that can be used include error - prone PCR , phage necessary regulatory elements such as promoters , termina display ( e . g ., Lowman et al. , 1991, Biochemistry 30 : 10832 tion sequences , enhancers and antibiotic resistance genes 10837 ; U . S . Pat . No . 5 , 223 ,409 ; WO 92 / 06204 ) and region that permit the DNA construct or portions thereof to be directed mutagenesis (Derbyshire et al . , 1986 , Gene 46 : 145 ; efficiently transcribed and translated in a host cell . In some Ner et al. , 1988 , DNA 7 : 127 ) . embodiments , a DNA construct of the present invention can [ 0233] Mutagenesis /shuffling methods can be combined include a polynucleotide operably linked to an operable with high - throughput , automated screening methods to promoter , for example , a homologous or heterologous pro detect activity of cloned , mutagenized polypeptides moter, wherein the polynucleotide encodes: an insecticidal expressed by host cells (Ness et al. , 1999 , Nature Biotech and /or nematicidal peptide comprising either a binary or a nology 17 : 893 -896 ) . Mutagenized DNA molecules that tertiary peptide, the binary or a tertiary peptide containing at encode active polypeptides can be recovered from the host least 4 amino acids and up to a maximum of 16 amino acids , cells and rapidly sequenced using standard methods in the and is comprised of 2 or 3 different regions, wherein the art. These methods allow the rapid determination of the binary peptide has 2 different regions and the tertiary peptide importance of individual amino acid residues in a peptide or has 3 different regions . The binary or a tertiary peptide can protein . be cleaved by both an animal gut protease and an insect [ 0234 Semi- synthetic gene construction is accomplished and / or nematode gut protease . In various embodiments the by combining aspects of synthetic gene construction , and /or binary or a tertiary peptide is operably fused ( i . e . fused in site - directed mutagenesis , and / or random mutagenesis , and frame ) to the C -terminus of an Endoplasmic Reticulum or shuffling. Semi- synthetic construction is typified by a Signal Peptide (ERSP ) as described herein and is operably process utilizing polynucleotide fragments that are synthe - fused to the N - terminus of a TP , for example a CRIPTP . (See sized , in combination with PCR techniques . Defined regions for example, FIGS. 4 -6C ) . In related embodiments of the US 2018 /0362598 A1 Dec. 20 , 2018 31 above , TPs useful in the manufacture of a recombinant may encode an insecticidal and /or nematicidal protein com insecticidal and / or nematicidal protein , which may include prising a cleavable binary peptide having the following one or more ICK TPs , for example , a TP selected from an amino acid sequence : AFVRLF (SEQ ID NO : 1594 ), “ ACTX ” or “ ACTX peptide” family of insecticidal ICK AKLFV (SEQ ID NO : 1595 ) , ALFALK (SEQ ID NO : peptides that have been isolated from an Australian funnel 1596 ), ALFLK (SEQ ID NO : 1597 ) , ALFLR (SEQ ID NO : web spiders belonging to the Atracinae subfamily , for 1598 ) , ALFR ( SEQ ID NO : 1599 ) , ALFRLR (SEQ ID NO : example , one or more TPs selected from an Omega - ACTX 1600 ), ALKALF (SEQ ID NO : 1601 ) , ALKFF (SEQ ID NO : TP , a Kappa -ACTX TP and an U -ACTX TP , for example , 1602 ) , ALKFLV (SEQ ID NO : 1603 ) , ALKIFV (SEQ ID U + 2 -ACTX -Hvla , U - ACTX - Hvla , rU - ACTX -Hvla , NO : 1604 ) , ALKLFV (SEQ ID NO : 1605 ) , FFADIK (SEQ rU - ACTX -Hvlb , or a TP having at least 80 % , at least 85 % , ID NO : 1606 ) , FFALK (SEO ID NO : 1607 ) , FFLK ( SEO ID at least 90 % , at least 95 % , at least 96 % , at least 97 % , at least NO : 1608 ) , FFLR (SEQ ID NO : 1609) , FFRLR (SEO ID 98 % , at least 99 % , or at least 100 % sequence identity or NO : 1610 ) , FGYRIK (SEQ ID NO : 1611 ), FLRLF (SEQ ID sequence homology to a peptide or protein having an amino NO : 1612 ) , FYARR (SEQ ID NO : 1613 ) , GGLRKK (SEQ acid sequence as set forth in SEQ ID NOs: 5 , 6 and 1761, ID NO : 1614 ) , IFVALK (SEQ ID NO : 1615 ) , IFVLK ( SEQ 1762 , 1767, 1771- 1773 , and variants thereof, (wherein the ID NO : 1616 ), IFVLR (SEQ ID NO : 1617 ) , IFVR ( SEQ ID TPs, if more than one, may be the same or different) and NO : 1618 ), IFVRLR (SEQ ID NO : 1619 ), ILFNIK (SEQ ID each insecticidal and / or nematicidal protein further compris NO : 1620 ) , LFAAPF (SEQ ID NO : 1621) , LFVALK (SEQ ing one or more tertiary peptides (designated above as L ), ID NO : 1622) , LFVLK ( SEQ ID NO : 1623) , LFVLR (SEQ which may include the amino acid sequence of a binary ID NO : 1624 ) , LFVR (SEQ ID NO : 1625 ) , LFVRLR (SEQ peptide selected from IFVRLR (SEQ ID NO : 1619 ) , ID NO : 1626 ) , LFVRVFL (SEQ ID NO : 1627 ) , LGER (SEO ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID NO : ID NO : 1628 ), LKALF ( SEQ ID NO : 1629 ) , LKFF (SEQ ID 1603 ), RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF ( SEQ ID NO : 1630 ) , LKIFV (SEQ ID NO : 1631 ) , LKLFV (SEQ ID NO : 1621) , fused in frame either on the N - terminus , C - ter NO : 1632 ) , LRALF (SEQ ID NO : 1633 ) , LRFF (SEQ ID minus or both termini of the binary peptide with a Z region NO : 1634 ) , LRIFV (SEQ ID NO : 1635 ), LRLFV (SEQ ID or spacer sequence selected from the group of or consisting NO : 1636 ) , RALF (SEO ID NO : 1637 ) , RIFV (SEO ID NO : ofGS , WN , WQ , MA ,MV , AM , LH , MN , ES , WQ, or DT. 1638 ) , RLFV (SEQ ID NO : 1639 ), RLRALF (SEQ ID NO : [ 0238 ] (4A ). DNA Constructs Encoding Insecticidal and 1640 ) , RLRFF (SEQ ID NO : 1641) , RLRIFV (SEQ ID NO : Nematicidal Transgenic Proteins 1642 ) , RLRLFV (SEQ ID NO : 1643 ) , RRKAFV (SEO ID [ 0239 ] In various embodiments , an illustrative DNA con NO : 1644 ) , RRKLIF ( SEQ ID NO : 1645 ) , RRRFFA (SEQ struct encoding the insecticidal and / or nematicidal protein ID NO : 1646 ) , VFGRKG ( SEQ ID NO : 1647 ) , and YFVRK may be codon optimized for the species in which the DNA (SEQ ID NO : 1648 ). construct will be introduced . For example , where the DNA [0242 ] In various embodiments , an exemplary DNA con construct will be transformed into plants , the DNA construct struct may encode an insecticidal and /or nematicidal protein can be codon optimized for expression and replication in comprising a cleavable tertiary peptide having the following plant cells. amino acid sequence construct : Z7- X , -X2 - Y - Y2, Z - X , [0240 ] In some embodiments , the DNA construct com X2- Xz - Y - Y2, Z ,- X - X2- X3- X4 - Y - Y2, Z ,- X ,- X2- Y - Y2 prises a polynucleotide that encodes a binary peptide com Y3, Z7 - X , -X2 - Xz- Y - Y2- Y3, Z - X , - X2- X7 - X4- Y - Y - Y3, prising the amino acid sequence: X - X2- Y - Y2, X -X2 - Xz 27 - X , - X2- Y - Y - Y3- Y4, 27 - X , -X2 - Xz- Y -Y2 - Y3 -Y4 , Y - Y2, X -X2 - X3 -X4 - Y | - Y2, X - X2- Y - Y2- Y3, X - X2 27- X , - X7- X , - X4- Y -Y2 - Y3 - Y4 , 2 , - Z2- X -X2 - Y - Y2, X3- Y -Y2 - Y3 , X -X2 - X3 - X4 - Y - Y2- Y3, X , -X2 - Y - Y2- Y3 Z -Z2 - X - X2- X3- Y - Y2 Z -Z2 - X , -X2 - X3 -X4 - Y -Y2 , Y4, X , - X2- X3 - Y - Y - Y3- Y4 , X , -X2 - X2 - X4- Y - Y - Y3 -Y4 , 2 , -22 - X -X2 - Y - Y2- Y3, 2 , - 22- X , - X2- Xz - Y - Y2- Y3, Y - Y2- X , - X2, Y - Y2- X , -X2 - Xz, Y - Y2- X -X7 - X7 - X4 , Z -Z2 - X , - X2- X3 - X4- Y , - Y2- Y3 , Z -Z2 - X , -X2 - Y - Y2- Yz Y -Y2 - Y3 - X , -X2 , Y - Y2- Y3- X , - X2- X3, Y - Y - Y3- X -X2 Y4, Z _ -Z2 - X - X2- Xz - Y - Y2 - Y3- Y4, Z _ -Z2 - X - X2- Xz - X4 Xz - X4, Y - Y2- Y3 - Y4- X - X2, Y - Y2- Y3 - Y4- X -X2 - X3 , Y - Y2- Yz- Y4, 21- - 23- X , - X - Y -Y2 Z -Z2 - 23 - X -X2 Yi - Y2 - Yz- Y4 - X - X2- X3 - X4 , wherein each X , and each Y , is X3- Y -Y2 , Z1- Z2 -23 - X , -X2 - X3 -X4 - Y - Y2 , Z -Z2 -23 - X , an amino acid , and wherein each Xn and each Yn is an X - Y - Y2- Y3, Z1 -Z2 -Z2 - X , - X2- X2 - Y - Y2- Y3, Z1- Zz- Zz amino acid , and wherein Xi is selected from the group X -X2 - X3 -X4 - Y - Y2- Y3, Z7 -Z2 - 23- X - X2- Y -Y2 - Y3- Y4, consisting of: (all single letter amino acid format ) A , D , E , Z -Z2 - 23 - X - X2- Xz- Y - Y2- Y3- Y4, Z _ -Z2 - Zz - X - X2- Xz - X4 G , I, K , L , N , P , Q , R , and V ; X2 is selected from the group Y - Y - Y3- Y4, Z7 - 22- 23- 24- X -X2 - Y - Y2, 27 -Z2 - 23 - 24 - X , consisting of A , F , G , I, K , L , N , P , Q , R , S , T , V , and Y ; X , X - X3 - Y - Y2, 2 -Z2 - 23 - 24 -X -X2 - X3- X4 - Y - Y2, Z -Z2 - 23 is selected from the group consisting of A , D , E , F , G , I, K , Z4- X , - X - Y - Y2- Y3, Z -Z2 -23 - 24- X7 - X , - X Y , - Y2- Y3 , L , N , Q , R , S , T , V , and Y ; X4 is selected from the group Z -Z2 - 23 - 24- X - X2- Xz - X4- Y - Y2 - Y3, Z -Z2 - 23- 24 - X7 - X2 consisting of: R , H , K , D , E , S , T , N , Q , G , A , I, L , M , F , W , Y - Y2- Y3 - Y4 2 , -22 - 23 - 24 - X -X2 - Xz- Y -Y2 - Yz- Y4, groupY, and consistingV . In some of : embodiments(all single letter , Y? amino is selected acid formatfrom ) theA , Zj - Z2- 23- 24- X -X2 - X3 -X4 - Y - Y2- Y3 - Y4, X7- X2 - Y -Y2 D , E , F , G , I , L , N , R , S , T , V , and Y ; Y , is selected from the Y - Y2- Y3 - Z1 , X -X2 - X3 - Y - Y2- Y3- Z1 , X , -X2 - X3- X4 - Y1 group consisting of F , G , I, L , and Y ; and Y2 is selected from Y2- Yz -Z1 , X7- X2- Y - Y2- Y3 - Y4- Z1 , X - X2- Xz - Y - Y2- Yz the group consisting of A , D , E , F , G , I , K , L , R , S , T , V , and Y4- 21 , X , -X2 - Xz- X4- Y - Y2 - Y3- Y4- 21 , 27 - X , - X - Y - Y2 Y ; and Y4 is selected from the group consisting of: R , H , K , Zi, Z7 - X , - X - XZ- Y - Y2- 21 , 21- X7- X ,- X7- X4 - Y - Y - Z1, D , E , S , T , N , Q , G , A , I, L , M , F , W , Y , and V . Z - X , - X - Y - Y - Yz- Z1 Z7 - X , - X2- X2 - Y - Y2- Yz- Z1 , [0241 ] In some embodiments , an illustrative DNA con struct may encode an insecticidal and /or nematicidal protein Z1, Z7- X , -X2 - Xz- Y -Y2 - Y3 -Y4 - Z1 , Z -X7 - X2 - X3- 84 -Y1 comprising one or more illustrative binary peptides ( XY or Y2- Y3 - Y4- Z1 , Z -Z2 - X -X2 - Y - Y2- Z1 , Z -Z2 - X -X2 - Xz YX ). In some embodiments , an exemplary DNA construct Y - Y2 - 21 , Z -Z2 - X -X2 - X3- X4 - Y - Y2 - 21, Z -Z2 - X -X2 US 2018 /0362598 A1 Dec. 20 , 2018 32
Y - Y2- Y3- Z1 , Z -Z2 - X , -X2 - Xz- Y - Y - Y3- Z1 , Z / -Z2 - X1 X2- X3- X4- Y -Y2 - Y3 - Y4 - 2 , - 22- 23, 2 - 22- 23 - 24- X -X2 - Y / X2- X3- X4- Y - Y2- Y3- 21 , 21- 22- X7- X2- Y - Y2- Y3- Y4- 21, Y2- 2 , -22 - 23 , 27 - 22- 23- 24- X , - X2- X3- Y - Y2- 2 , -22 - 23, Z -Z2 - X -X2 - X3- Y - Y2- Y3- Y4- 21 , Z , -Z2 - X -X2 - X3- X4 21- 22 - 23- 24- X , -X2 - X3 - X4 - Y , - Y2- 21- 22- 23 , Z1- 22- 23- 24 Y - Y2- Y3 - Y4 -21 , Z -Z2 - 23- X - X2- Y - Y2- 21 , 21- 22- 23 - X - X7- X , - Y , -Y2 - Y3 - 27 - 22- 23, 27- 22- 23- 24 - X7 - X ,- X3- Y -Y2 X2- Xz- Y - Y - Z1, Z ,- Z2- 23 -X -X2 - X3 -X4 - Y , - Y2 -Z1 , Z -Z2 Yz- 2 , -22 - 23 , Z ; - Z2 - Zz -Z4 - X , -X7 - X , -XA - Y - Y - Y3- 2 - 2 - Zz- X7- X2- Y - Y2- Y3- Z1, Z1- Z2- 23- X - X2- X3- Y - Y2- Y3- 21, Z3 , Z1- Z2 - 23- 24 - X , - X2- Y - Y2- Y3 - Y4 -24 - Z2 - Z3 , Z / -Z2 - Zz Z1- Z2- 23- X - X2- X3- X4 - Y - Y2- Y3- Z1, Z -Z2 -23 -X -X2 - Y , Z4- X - X2- Xz- Y - Y2- Y3- Y4- 27 -Z2 - Z3, Z - Z2- 23 -24 - X , -X2 Y2- Y3- Y4 -21 , 21- 22 - 23- X -X2 - X3 - Y - Y2 -Y3 - Y4- 21, Z -Z2 X3 -X4 - Y - Y2 - Y3 - Y4- 2 , -22 - 23, X -X2 - Y - Y - Z -Z2 - 23 - 24 , Zz- X , - X2- Xz- X4 - Y - Y - Y3 - Y4 - 21, 21- 22- 23- 24- X , - X2- Y - X -X7 - X , -Y -Y2 -2 , -22 - 23 - 24 , X , -X -X3 - X4 - Y - Y2- 21 - 22 Y2 -21 , Z , - Z2- 23 - 24- X -X2 - X2 - Y - Y2- Z1 , Z -Z2 - 23 -24 - X , - Zz- Z4, X , - X2- Y - Y2- Y3- 21- 22 - 23- 24, X7 - X2- Xz - Y - Y2 X2- X3 -X44Y1 - Y2- Z1, Z -Z2 - 23 - 24 - X -X2 - Y - Y2- Y3- 21 , Y3- 2 , -22 - 23 -24 , X - X2- X3- X4- Y - Y2- Y3- 27 -Z2 - 23- 24 , 27 -22 - 23- 24 - X - X2- X3 - Y - Y2- Yz- Z1 , Z1- Z2 - 23- 24 - X , -X2 X -X2 - Y - Y2 - Y3 - Y4- 21 - - 23- 24 , X7 -X2 - Xz- Y - Y2- Yz X2- X - Y - Y - Yz- Z1 , Z -Z2 - 23 - 24 - X , - X - Y - Y - YZ- Y4- 21, Y4 - 2 , - 22 -23 - 24, X , - X -X2 -X4 - Y - Y - Yz - Y4 - 2 , -22 - 23 Z -Z2 - 23- 24 - X , -X2 - X3- Y - Y2- Y3 - Y4 -21 , Z -Z2 - 23- 24 - X , 24, Z -X7 -X2 - Y - Y2- 21- 22- 23- 24, Z - X -XZ - X3 - Y - Y2- 21 X2- X3 - X4 - Y -Y2 - Y3 - Y4 - Z1, X , -X2 - Y -Y2 - 2 , - 22, X . -X2 Z2- 23- 24 , Z _ - X -X2 - X3 - X4 - Y - Y2- 2 - 22- 23 - 24, 27 - X , -X2 Xz- Y - Y2- 2 , -Z2 , X - X2- Xz - X4- Y - Y2- 2 , - Z2, X , -X2 - Y1 Y - Y2- Y3- 2 ,- 22- 23- 24, Z , - X , - X2- X3 - Y - Y2- Y3 -21 -22 - 23 Y2- Y3- Z -Z2 , X - X2- Xz - Y - Y2- Yz- Z - Z2, X , -X2 - X3- X4 Z4, Z7 - X , - X2- X3 - X4- Y - Y2- Y3 -27 - Z2 -23 - 24 , Z _ - X -X2 - Y1 Y - Y - Y3- 2 -22 , X , - X , - Y -Y2 - Y3 - Y4 - 2 , - 22, X , -X2 - Xz Y2- Y3- Y4- 2 - - 23- 24, Z , - X , -X7 - X - Y - Y2- Y3- Y4- 2 -22 Y - Y - Yz- Y4- 24- 22 X7- X ,- X3- X4- Y -Y2 - Y3 - Y4- 2 , - 22, Zz- Z4 , Z -X7 - X , -X3 - X4 - Y - Y2- Y3- 94- 21- 22- 23- 24 , 27- 22 Z7 - X , - X - Y - Y2- 27 - Z2, Z7 - X , -X2 - X3- Y -Y2 - 27 -Z2 , Z7 - X , X -X2 - Y -Y2 - 2 , - 22- 23- 24, 27- 22 - X , - X2- X3 - Y , -Y2 - 2 , -22 X2- X3- X4 - Y - Y2- Z -Z2 , Z - X7 -X2 - Y - Y2- Y3- 2 - 22 , Zz- Z4, Z -Z2 - X , -X2 - X3- X4 - Y - Y2- 27 -22 - 23 -24 , Z _ -Z2 - X Z ; - X7 -X2 - Xz- Y - Y2- Y3- 27 -Z2 Z7 - X , - X2- X3 - X4- Y - Y2 X - Y - Y2- Yz- Z -Z2 - 23 - 24, Z _ -Z2 - X , -X2 - Xz - Y - Y2- Y3 - 21 Y3- 21 - 22 , Z -X7 - X2- Y - Y2- Y3 - Y4- 2 -22 , Z -X7 -X2 - Xz Z2- 23 - 24 , 27- 22- X - X2- X3 - X4- Y - Y2- Y3- 24- Z2 - 23 - 24 , Yi - Y2 - Yz- Y4 - 2 , -22 , 24- X , - X2- X3 - X4- Y - Y - Y3- Y4- 21 Z -Z2 - X7- X ,- Y , - Y2- Yz- Y4- 27 - 22- 23- 24, Z _ - Z2- X -X2 - Xz Z2, Z1- Z2 - X , -X2 - Y - Y - Z -Z2 , Z , -Z2 - X , - X2- X2 - Y - Y2- 21 Y - Y - Y3- 94- 21- 22 - 23- 24, 27- 22- X - X -X3 - X4 - Y , - Y2- Yz Z2, Z - Z2 - X -X2 - X3 - X4- Y -Y2 - 27 -Z2 , Z1- Z2 - X7 -X2 - Y - Y2 Y4- 21 - 22 - 23- 24 , Z - Z2- 23- X - X2- Y - Y2- 27 - Z2- 23 - 24 , Yz - Z , - Z2, Z , -Z2 - X7 -X2 - X3- Y - Y - Y3- 2 , -22 , Z -Z2 - X , -X2 Z -Z2 - 23 - X , - X2- Xz - Y - Y2- 2 -Z2 - 23 - 24 , Z -Z2 - Zz - X -X2 X3- X4 - Y2- Y3 - 2 -22 Z - Z2- X - X2- Y - Y2- Y3- Y4- 2 , - 22, X3 -X4 - Y - Y2 -22 - 22 - 23- 24 , Z -Z2 - 23- X -X2 - Y -Y2 - Y3 -21 Z ; - Z2- X - X2- Xz - Y - Y2 -Y3 - Y4 - 2 , -22 Z -Z2 - X , -X2 - Xz Zz- Zz -Z4 , Z - Z2- 23- X - X2- Xz - Y - Y2- Y3- 2 , - 22- 23 -24 , X4 - Y - Y2- Yz - Y4 - 2 -Z2 , Z -Z2 - 23 - X -X2 - Y - Y2- 2 , -22 , Z -Z2 - Zz - X -X2 - Xz- X4 - Y - Y2- Y3- 21- 22- 23 - 24 , - 22- 23 2 , -22 - 23 - X , - X2- Xz- Y - Y2- 27- 22, 2 ;- 22- 23- X -X2 - X3 - X4 X -X2 - Y - Y - Y3- 94- 21- 22 - 23- 24, 27 - Z2- 23- X -X2 - X3 - Y , Y - Y - Z -Z2 , Z , -Z2 - 23 - X7- X ,- Y - Y2- Y3- 2 -Z2 , Z , -Z2 - Zz Y2- Y3- YA- Z -Z2 - 23 - 24, Z -ZZ - Z2 - X , - X2- X2 - X4- Y - Y - Yz X - X2- Xz - Y - Y2- Yz- Z -Z2 , Z -Z2 - 23- X7 -X2 - X3- X4 - Y1 Y4- 2 , -22 - 23 - 24 , 27- 22- 23- 24- X , -X2 - Y - Y2- 2 , -22 - 23 - 24 , Y2- Y3- 2 , - Z2, Z - Z2- 23 - X , - X2- Y - Y2- Y3 - Y4- Z -Z2 , Z , -Z2 27- 22 - 23- 24- X -X2 - X3- Y - Y2- 2 , - 22- 23- 24 , Z -Z2 - 23 - 24 Zz- X -X2 - Xz - Y - Y2- Y3- Y4- 27- 22, 27- 22- 23- X -X2 - Xz - X4 X7 -X2 - X3 - X4 - Y , - Y2- 2 , - 22- 23- 24 , Z -Z2 - 23- 24- X -X2 - Y ; Y - Y2- Yz- Y4- 21 - 22 Z -Z2 - 23- 24 - X7 -X2 - Y - Y2- 22 - 22 , Y2- Y3- 21 - 22 - 23 -24 , Z _ -Z2 - 23- 24 -X7 - X , -X3 - Y - Y2- Y3 - 2 , Zj- Z2- 23 - 24 - X , -X7 - X , - Y , - Y2- 27 - Z2, Z , -Z2 - 23 - 24 - X , - X2 Zz- Zz - Z4 , Z -Z2 - 23 - 24- X , - X2- Xz - X4- Y - Y - Y3- 2 , -22 - Zz X3- X4 - Y - Y2- 2 - 22, 2 , - 22- 23- 24 - X , - X2- Y - Y2- Y3- 2 ; - 22, Z4, Z , - 22- 23- 24 - X - X2- Y - Y - Y3- Y4 - 2 , - 22- 23- 24 , 27- 22 Z ; - Z2- 23 - 24 - X , -X2 - Xz- Y - Y - Y3- 2 ; -Z2 , Z - Z2- 23- 24 - X - 23- 24 - X , - X - X3- Y - Y - Y3- Y4 - 2 , - 22- 23- 24, Z , -22 - 23 - 24 X -X3 - X4 - Y , - Y2- Y3- 2 , - 22, Z - Z2- Z2 - 24- X -X2 - Y - Y2- Yz X -X2 - X3 - X4- Y - Y2- Y3- Y4- 2 , -22 - 23 - 24 , Z - Y - Y2- X , Y4- 2 , -22 , 27 - 22- 23- 24- X , -X2 - Xz - Y - Y2- Y3- Y4- 2 , - 22, X2, Z7 - Y , -Y2 - X , -X2 - X3 , Z - Y - Y2- X , - X2- X3- X4, Z / - Y Z - Z2- 23 - 24 - X , -X2 - X3- X4 - Y - Y2- Y3- Y4- 27 -Z2 , X , -X2 - Y - Y2- Y3 - X , -X2 , Z - Y - Y2- Y3- X - X2- X3 , Z - Y - Y2- Y3 - X - Y2- 21 - 22 - 23 , X -X2 - X3 - Y - Y2 -Z -Z2 - Z3 , X7- X2- Xz- X4 X -X3 - X4, Z - Y - Y - Yz- YA- X , -X2 , Z - Y - Y2- Y3 -Y4 - X , Y -Y2 - 2 , -22 - 23 , X -X2 - Y - Y2- Y3- 21 - 22- 23, X , - X , -Xz X -Xz , Z - Y - Y - Yz- Y4 - X , - X - X3- X4 , Z 1 - Z2- Y - Y2- X Y | -Y2 - Y3 - 2 - 2 -23 , X -X2 - X3 - X4 - Y - Y - Y3- 24 - Z2- 23, X2, Z , -Z2 - Y - Y2- X , - X2- X3 , Z , - Z2 - Y - Y2- X , - X2- X3- X4 , X -X2 - Y , - Y2- Yz- Y4- 2 , - 22- 23, X - X2- Xz- Y - Y - Y3- 94 2 , -22 - Y - Y - Y3- X , - X2, Z7 - 22- Y - Y2- Y3- X -X2 - X3 , Z - Z2- 23 , X . -X2 - X3- X4 - Y - Y2- Y3- Y4- 2 , -22 - Z3 , Z1- X - X2 Z -Z2 - Y - Y2- Y3- X - X2- X3 - X4, Z _ -Z2 - Y - Y2- Y3- Y4- X - Y - Y2- 2 , - Z2- Z3 , Z7 - X -X2 - Xz- Y -Y2 - 24 - Z2 -Z3 , Z7 - X , - X2 X2, Z -Z2 - Y - Y - Y3- Y4- X , -X2 - X3 , 2 , - 22- Y - Y2- Y3- Y4 X3- X4- Y - Y2- 21 - 22- 23 , 24 - X -X2 - Y - Y2- Y3- 21 - 22- 23 , X -X2 - X3- X4, Z7 - 22- 23- Y - Y2- X -X2 , Z1- Z2- 23 - Y -Y2 Z7 -X , - X2- X2 - Y - Y2- Y3- 2 , -22 - 23 , 24- X - X2- X3 -X4 - Y - X7 - X2- X3, Z -Z2 - 23 - Y - Y2- X7 - X2- X3 -X4 , Z _ -Z2 - 23 - Y Y2- Yz- Z -Z2 - Z3 , Z , - X , - X2- Y - Y - Y3 -Y4 - 27 -Z2 - Z3 , Z4- X , Y2- Y3- X , - X2, Z -Z2 -23 - Y - Y - Y3- X7 - X , - Xz, Z / -Z2 -Zz X2- X3 - Y - Y - Y3- Y4 - 2 -22 - 23 , 27- X -X2 - X3 - X4 -Y - Y , Y - Y2- Y3 - X , - X2- X3- X4 , Z -Z2 -23 - Y - Y2- Y3- Y4- X - X2, Y3- Y4 - 2 , -22 - 23 , 2 , - 22- X , -X2 - Y - Y2- 2 , - 22- 23 , 2 , -22 - X , Z -Z2 - 23- Y - Y ,- Y3- Y4 - X , - X ,- X3, 2 , - 22- 23- Y - Y2- Y3- Y4 X2- Xz - Y - Y2- 24 -Z2 - Z3, Z - Z2- X -X2 - X3 - X4- Y - Y2- 2 , - 22 X - X2- X3 - X4, Z -Z2 - 23- 24 - Y - Y2- X7 - X2, Z1- Z2 - 23 - 24 - Y - Z3, Z -Z2 - X -X2 - Y - Y2- Y3- 21 - 22 - 23 , Z -Z2 - X , -X2 - X2 - Y - Y2- X -X7 - X3 , 21- 22- 23- 24- Y - Y2- X -X2 - X3 - X4 , 21- 22 - 23 Y2- Y3 - Z -Z2 - Z3 , Z1- Z2- X -X2 - X3 -X4 - Y - Y2- Y3 -21 - 22 - 23 , 24- Y - Y2- Y3- X -X2 , 22- 22- 23- 24 - Y -Y2 - Y3 - X7 - X , -X3 , Zj- Z2 - X , - X2- Y , - Y2- Y3- Y4- 27 - Z2 -Z3 , Z -Z2 - X - X2- Xz - Y Z -Z2 - 23 -24 - Y - Y2- Y3 - X - X2- X3- X4, Z - Z2- 23 -24 - Y - Y2 Y - Yz- Y4- 2 , -22 - Z3, Z . - 22- X -X2 - X3- X4 - Y - Y - Yz- Y4 – Yz - Y4- X , -X2 Z -22 - 23 - 24 - Y - Y2- Y3- Y4- X -X2 - Xz, 2 , -22 - 23 , Z1- Z2 - 23- X , -X2 - Y -Y2 - 21- 22- 23, Z1- 22- 23- X , Z -Z2 - 23 - 24- Y - Y - Yz- Y4- X - X2- X7 - X4, Y - Y - X7- X2- 21 , X2- X3 - Y -Y2 - 2 , - 22- Z3, Z1- Z2 - 23 - X , -X2 - X3 - X4 - Y - Y2- 2 , Y , - Y2- X , -X2 - X3- 21 , Y - Y2 - X , -X2 - X7 -X4 - 21 , Y - Y2- Yz Z2- Z3 , Z1- Z2 - 23 - X -X2 - Y -Y2 - Y3 - 24- Z2 - Z3 , Z -Z2 - 23- X - X7 -X2 - Z1 , Y - Y2- Y3 - X - X2- Xz - Z1, Y - Y2- Y3 - X -X2 - Xz X - Xz - Y - Y2- Y3- Z -Z2 - Z3, Z -Z2 - 23 - X - X2- X3 - X4- Y - Y - X4- 21 , Y - Y2- Y3 - Y4 - X , -X2 - Z1 , Y - Y2- Y3- Y4- X , - X - Xz Y3- 2 - Z2 - Z3, Z - Z2- 23 - X -X2 - Y - Y2- Y3- Y4- 2 -Z2 - 23 , Z1, Y - Y2- Y3- Y4 -X7 - X2 -X3 - X4 -Z1 , Z - Y - Y2- X7 - X2- Z1 , Zj- Z2- 23 - X - X2- X3- Y - Y2- Y3- Y4- 2 - Z2- Z3, Z -Z2 -23 - X - Z - Y - Y2- X -X2 - X3- 21 , Z - Y -Y2 - X7 - X2- X3 - X4- 21 , US 2018 /0362598 A1 Dec. 20 , 2018 33
Z - Y - Y2- Y3 - X , -X2 - Z1 , Z - Y - Y2 - Y3- X - X2- X3 - Z1, Z1- Z2 -23 - Y - Y2- X , - X2- X3 - X4- 24 - Z2- Z3, Z1- Z2 - 23- Y -Y2 Z - Y - Y2- Y3- X - X2- X3- X4 -21 , 21- Y - Y2- Y3- Y4- X -X2 Y3- X , -X2 - 21 - 22 - Z3, Z - Z2- Z2 - Y - Y2 -Y3 - X , - X2- Xz - 2 , -Z2 Z1, Z - Y - Y2- Y3- Y4 - X -X2 - Xz- Z1 , Z - Y - Y2 - Y3- Y4- X Z3, Z1- Z2 -23 - Y - Y2- Y3- X -X2 - X3 -X4 - 21 - 22 - 23, Z -Z2 -23 X2 -X3 - X4 -Z1 , Z -Z2 - Y - Y2- X7 - X2- Z1 , Z -Z2 - Y - Y2- X1 Y - Y2 - Y3 - Y4 - X -X2 - 27- Z2 - Z3 , Z - Z2 -23 - Y - Y2- Y3- Y4- X , X - X , -Z1 , Z ; - Z - Y - Y - X ,- X2- X7- X4 - 21 , Z , -22 - Y - Y , X7 -X , -2 -2 -23 , 2 , - Z2 -22 -Y - Y2- Y2 - YA -X - X , - X ,- X4- 2 , Y3- X - X2- Z1 , Z -Z2 - Y - Y2- Y3 - X , -X2 - Xz- Z1 , Z -Z2 - Y - Zz- Z3 , Z1- Z2 - 23- 24 - Y - Y2- X , - X2- Z -Z2 - Z3 , Z1- Z2 -23 - 24 Y2- Y3- X , -X2 - Xz -X4 - Z1 , Z7 -Z2 - Y - Y2 - Y3- Y4- X7- X2 - 21 , Y - Y2- X7- X2 - X3- 21 - 22 - 23 , Z1 -Z2 - 23 -24 - Y - Y2- X , - X - Xz Z -Z2 - Y - 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23 23- Y - Y2- X ,- X2- X3- Z -Z2 , Z1 - Z2 -23 - Y - Y2- X , -X2 - X3 - X4 24, Z _ -22 - 23 - Y -Y2 - Y3 - Y4 - X7 - X , -X3 - 2 , - 22- 23- 24 , 27 -22 Zj- Z2, Z -Z2 - Zz - Y - Y - Y3- X7- X2 - 2 , -Z2 , Z -Z2 - Zz - Y - Y - Zz- Y - Y - Y3- Y4- X , - X - X7 -X4 - 24 - Z2 - 23 -24 , 27- 22- Zz Y3- X , -X2 - Xz- Z -Z2 , Z -Z2 - 23 - Y - Y2- Y3- X , -X2 - X3- X4 - 2 , 24 - Y - Y2- X , - X2- 21 - 22- 23- 24, 27 - Z2- 23- 24- Y - Y2- X , Z2, Z , - Z2 -23 - Y - Y2- Yz- Y4 - X -X2 - 27- Z2 , Z , - Z2- 23 - Y - Y2 X -X2 - 27 - Z2 - 23 - 24 , - 22- 23- 24- Y - Y2- X -X7 - X , -X4 - 24 Y3 -Y4 - X -X2 - X3 - 2 , - 22 , 22- 22 -23 - Y - Y2- Yz- Y4- X , - Xz 22- 23- 24 , Z _ - Z2- 23- 24- Y - Y2- Y3 -X , -X2 - 2 , -22 - 23 - 24 , X3- X4 - 24 - 22 , 22- 22 - 23- 24 - Y - Y2- X , -X2 - 2 , -22 , Z , -Z2 - 23 Z -Z2 - 23 - 24- Y - Y - Y3- X , - X - X3- 21 - 22- 23 - 24 , 27- 22- 23 24- Y -Y2 - X , - X2- Xz - Z , -Z2 , Z -Z2 - 23 -24 - Y -Y2 - X - X2- Xz Z4- Y - Y2- Y3 - X , -X2 - Xz -X4 - 24 -Z2 - 23- 24 , Z -Z2 - 23- 24 - Y1 X4 -24 - Z2 , Z -Z2 - 23 - 24 - Y - Y - Y3- X -X2 - 2 ,- Z2 , Z , -Z2 - 23 YZ- YZ- YA- X7- X2- 2 ,- 22- 23- 24, Z -Z2 -23 -24 - Y - Y - Y3 - Y4 24- Y - Y - Yz- X ,- X - X2 -2 , - 22, Z , -Z2 - 23- 24- Y - Y2- Y2 -X , X -X2 - X2 - 2 , - 22- 23 - 24, Z - Z2- 23 - 24 - Y - Y2 - Y3- Y4- X , -X2 X - X7 -X4 - 24 - Z2 , Z - Z2- 23 - 24 - Y - Y - Y3- Y4- X7 - X , - 2 -22 , X3- X4 - 2 , - 22- 23- 24 , 27- X , - X ,- Y - Y2, Z - X7 - X , -X3 - Y Zj- Z2- 23 - 24 - Y - Y2 - Y3- Y4- X , - X - Xz- Z - Z2, Z , -Z2 - 23 - 24 Y2, Z7 - X , -X2 - X7- X4 - Y - Y2, Z7- X , -X2 - Y - Y2- Y3, Z7 - X , Y - Y2- Y3 - Y4 - X , -X2 - Xz -X4 - ZA - Z2, Y - Y2- X , -X2 - 27 -Z2 - 23 , X2- Xz - Y - Y2 - Y3, Z4- X - X2- Xz - X4- Y - Y2- Y3 , Z - X , -X2 Y - Y2- X - X2- Xz - Z -Z2 -23 , Y , - Y2- X - X2- X3- X4- 21 - -23 , Y - Y2- Yz- Y4, 21- X -X2 - X3- Y - Y2- Y3- Y4, Z , - X , - X7- X , Y - Y2 - Y3- X - X2- 21 - 22- 23 , Y - Y2- Y3- X - X2- X3 - Z -Z2 - 23, X4- Y - Y - Yz- Y4, Z7- 22 - X7 - X , - Y -Y2 , Z - Z2- X , -X7 - Xz Y - Y2- Y3- X -X2 - X3 -X4 - 21 - 22 - 23 , Y - Y2- Y3 - Y4- X7 - X2 Yi - Y2 , Z , -22 - X - X2- X3 - X4- Y - Y2, 2 , -22 - X , - X - Y - Y2 Z ; -Z2 - Zz , Y - Y - Y3 - Y4 - X , - X2- X3- 27 -Z2 - Zz, Y , - Y2- Yz Y3, Z -Z2 - X , - X2- X2 - Y - Y2- Y3, Z -Z2 - X , - X - X7- X4 - Y Y4- X , - X2- X3- X4 -24 - Z2 - Z3 , Z - Y - Y2- X , -X2 - 27 -Z2 - 23 , Y2- Y3, Z -Z2 - X , -X2 - Y - Y2 - Yz- Y4, Z _ -Z2 - X - X2- Xz - Y1 2 , - Y - Y2- X -X2 - X3 - 2 - 2 -23 , 22- Y - Y ,- X , - X ,- X , -X4 - 2 , Y2- Y3- Y4, Z -Z2 - X - X2- X3- X4 - Y - Y2- Yz - Y4, Z7 -22 -23 Z2- Z3 , Z - Y - Y - Y3- X -X2 - 21- 22 - 23, Z - Y -Y2 - Y3- X , -X2 X -X2 - Y - Y2, Z7- 22 - 23- X -X2 - Xz - Y - Y2, 21- 22 - - X - X3- 2 , -22 - 23 , Z - Y -Y2 - Y3 - X , -X2 - X3 - X4 - 21- 22- 23, 27 - Y , X2 -X3 - X4 - Y - Y2, Z1- Z2 -23 - X - X2- Y - Y - Y3, Z -Z2 -23 Y2- Y3- Y4- X , - X -ZA - Z2 - Z3, Z - Y - Y2- Y3- Y4 - X , -X2 - Xz X - X2- Xz - Y - Y2- Y3, Z1- Z2 - 23- X - X2- X3 - X4- Y - Y2- Y3, Z -Z2 - Z3 , Z - Y - Y2- Y3- Y4- X7 -X2 - X3- X4 - 2 , -22 - Z3 , Z -Z2 Z -Z2 - 23- X -X2 - Y - Y2- Y3- Y4, Z _ -Z2 - 23- X - X2- X2 - Y - Y2 Y - Y2- X , -X2 - 27- Z2- Z3, Z - Z2- Y - Y - X , - X2- X2- 27 - 22- 23, Yz- Y4, 24- Z2 - 23 - X -X2 - X3- X4 - Y - Y2- Y3- Y4, Z , -Z2 - 23- 24 2 , -22 - Y - Y2- X -X7 - X3 - X4 - 2 -22 - 23, Z - Z2- Y -Y2 - Y3- X , X - X2- Y - Y2, Z -Z2 - 23 - 24 -X , - X - X2 - Y - Y2, Z1- Z2 - 23 -24 X2- 2 , -22 - 23 , 27- 22- Y - Y2- Y3- X , -X2 - X3 - 2 , - 22- 23 , 2 , -22 X -X2 - X3 - X4- Y - Y2, 27 - 22- 23- 24- X - X - Y - Y2- Y3, Y - Y - Y3- X -X2 - Xz -X4 - ZA - Z2 - Z3 , Z1- Z2 - Y - Y2- Y3- Y4 Z -Z2 - 23 -24 - X7- X2 - X3- Y - Y2- Y3, Z1 -Z2 - 23 -24 - X , - X2- Xz X - X2- 27 - Z2- Z3 , Z1- Z2- Y - Y2 - Y3- Y4 - X , -X2 - Xz- Z -Z2 - 23 , X4 - Y - Y2- Y3 , Z -Z2 - 23 - 24 - X -X2 - Y - Y2- Y3 - Y4, Z _ -Z2 - 23 21- 22- Y - Y2- Y3- Y4- X -X2 -X3 -X4 -21 - 22 - 23 , Z1- Z2 - 23 - Y1 Z4- X - X2- X3 - Y - Y2- Y3- Y4, 21- 22- 23 -24 - X , -X2 - X3 -X4 Y2- X7 - X2- 21 - 22 -23 , Z -Z2 - 23 - Y - Y2- X - X2- X3 - 2 - 2 - 23, Y - Y2- Y3- Y4 , 22- Y - Y2- X - X2, Z - Y - Y2- X7 - X , -X3 , US 2018 /0362598 A1 Dec. 20 , 2018 34
Z - Y - Y2 - X , -X2 - Xz- X4 , Z - Y - Y2- Y3- X , -X2 , Z - Y - Y2 Y3 -Y4 , Z - Z2- X - X2- X3 - X4- Y -Y2 - Y3 -Y4 , Z -Z2 - 23 - X , Yz- X , -X2 - Xz, Z - Y - Y2- Y3 - X , -X2 - X3- X4 , Z - Y - Y2- Y3 X - Y -Y2 , Z , -Z2 - 23- X , - X2 - X3- Y - Y2, Z -Z2 -23 - X , -X2 Y4- X7 - X2, Z - Y - Y2- Y3- Y4- X - X2- X3 , Z - Y - Y2- Y3- Y4 X3 -X4 - Y - Y2, Z1- Z2- 23 -X -X2 - Y - Y2 - Y3, Z1- Z2 - 23 - X1 X - X2- X3 - X4, Z _ -Z2 - Y - Y2 - X7 -X2 , Z - Z2 - Y - Y2- X7 - X2 X2- X3- Y - Y2- Y3, Z1- Z2 - 23- X -X2 - X3 -X4 - Y -Y2 - Y3, Xz, Z - Z2- Y - Y2- X , - X - X7 - X4, Z -Z2 - Y - Y - Y3- X , -X2 Z - Z2 -Z2 - X , - X2- Y - Y - Y3- Y4, Z _ - Z2- Zz - X , - X2- X3- Y -Y2 Z -Z2 - Y - Y2- Y3- X - X2- X3 , Z1- Z2 - Y - Y2 - Y3- X - X2- Xz Yz- Y4, Z1- Z2- 23- X -X2 - X3 - X4 - Y - Y2- Y3- Y4, 21- 22 - 23 - 24 X4, Z _ -Z2 - Y - Y2- Y3 - Y4- X - X2, Z1- Z2- Y - Y2 - Y3- Y4- X X -X2 - Y - Y2, Z1- Z2- 23- 24- X - X2- X3 - Y - Y2, Zj- Z2- 23- 24 X2 -X3 , Z -Z2 - Y - Y2 - Y3 - Y4 - X , -X2 - X3 -X4 , Z _ - Z2- 23 - Y X - X2- X3- X4- Y - Y2, 21- 22- 23 - 24- X - X2- Y - Y2- Y3, Y2- X , -X2 , Z , - 22- 23- Y - Y2- X , - X2- X3, Z -Z2 - 23 - Y - Y2 Z -Z2 - 23- 24 - X -X2 - X3- Y - Y2- Y3, Z -Z2 - 23 -24 - X - X2 - Xz X -X2 - X3 - X4 , Z - 22- 23- Y , - Y2- Yz- X , - X2, Z7 -22 - 23 - Y - X4- Y -Y2 - Y3 , Z -Z2 - 23 - 24- X -X2 - Y -Y2 - Y3 -Y4 , Z1- Z2- Zz Y2- Y3 -X -X2 - X3 , Z / -Z2 - 23 - Y - Y2- Y3- X - X2- X3- X4, Z4- X - X2- X3- Y - Y2- Y3- Y4, Z - Z2- 23- 24 - X -X2 -X3 -X4 21- 22- 23- Y - Y2- Y3- Y4- X -X2 , 22- 22- 23- Y - Y2- Y3 -Y4 - X , Y - Y2- Y3 -Y4 , X7- X2- Y - Y2- Z1, X7- X2- Xz - Y - Y2- Z1 , X2- X3, Z -Z2 - 23- Y -Y2 - Yz- Y4- X , - X2- Xz - X4, Z _ - Z2- 23 - 24 X -X2 - X3- X4 - Y - Y2- 21 , X7 -X2 - Y - Y2- Y3- 21 , X , - X2- Xz Y - Y2- X -X2 , Z , - Z2- 23- 24- Y - Y2- X , -X2 - X3 , Z -Z2 - 23 - 24 Y - Y - Yz- 21 , X7 - X ,- X3- X4 - Y -Y2 - Y3 - Z1, X -X2 - Y - Y2 Y -Y2 - X , -X2 - X3 - X4 , 21- 22- 23- 24 - Y - Y2- Yz- X , - X2, Yz- Y4 -21 , X , -X2 - X3- Y - Y2- Y3 - Y4- 21 , X , -X2 - X3- X4 - Y - Z - Z2- 23 - 24 - Y - Y2 - Y3- X - X2- X3, Z - Z2 -23 - 24 - Y - Y2- Y3 Y2- Y3- Y4- Z1 , Z , - X , -X2 - Y - Y2- Z1 , Z1- X - X2- Xz - Y - Y2 X - X2- Xz - X4, Z - Z2- 23 - 24- Y -Y2 - Y3 -Y4 - X , -X2 , Z1- Z2 - Zz Z1, Z7- X , - X2- X3- X4 - Y - Y2 - 21, Z1- X - X - Y - Y2- Y3- 21 , 24- Y -Y2 - Y3 - Y2- X , -X2 - Xz, Z - Z2- 23- 24 - Y - Y - Y3- Y4 Z - X , -X2 - X3 - Y - Y - Yz- 21 Z , - X , -X2 - X3 - X4 - Y - Y2- Yz X - X2- X7- X4 , X , - X - Y - Y - Z1, X7 -X7 - X , - Y - Y2- 21 , Z1, Z , - X , - X - Y - Y2 - Yz- Y4 -21 , 21- X - X2- X3 - Y - Y2- Yz X , - X2- X2 - X4 - Y , - Y2- Z1 , X , - X2- Y - Y - Yz- Z1 , X , - X - Xz Y4- 21 , Z , - X , -X2 - X7 -X4 - Y - Y - Y3 -Y4 - 21 , Z7 -22 - X , - X2 Y - Y2- Yz -Z1 , X7- X2- Xz - X4 - Y - Y2- Y3- Z1 , X7 -X2 - Y - Y2 Y , - Y2- Z1 , Z , -Z2 - X , - X2- X3 - Y - Y2- Z1, Z -Z2 - X7- X2 - Xz Yz - Y4- Z1 , X -X2 - Xz- Y - Y2- Y3- Y4- Z1 , X - X2- X3 - X4- Y X4- Y - Y2- Z1 , Z -Z2 - X - X2- Y - Y2- Y3- Z1, Z -Z2 - X7- X2 Y2- Y3 -Y4 - 21 , X7- X2- Y -Y2 - 27 -22 , X -X2 - X3- Y -Y2 - 21 Xz - Y - Y2- Y3 -Z1 , Zi -Z2 - X - X2- Xz - X4 - Y - Y2- Yz- Z1, Z2, X -X2 - X3- X4 - Y - Y2 - 2 , -Z2 , X - X2- Y - Y2- Y3- 2 , -22 , 2 , -22 - X , - X - Y - Y2- Y3- Y4- 21, 22- 22 - X , - X2- X2 - Y - Y2 X - X -Xz - Y - Y - Y3- 24- 22 , X -X2 - X3 - X4 - Y - Y2- Y3- 2 , Yz - Y4- Z1 , Z , -Z2 - X , - X2- X2 - X4- Y - Y - Y3 - 94- 21 , Z7 -Z2 -Zz Z2, X -X2 - Y -Y2 - Y3 - Y4 - 2 , -22 , X -X2 - X3 - Y - Y2- Y3- Y4 X -X2 - Y - Y2 -Z1 , Z1- Z2 - 23 - X - X2- X3 - Y - Y2- 21, 21- 22 - 23 Z ; - Z2, X -X2 - X3- X4 - Y - Y2 - Y3- Y4- 2 , -22 , X , - X2- Y -Y2 X -X2 - X7 -X4 - Y , -Y2 - 21 , 21 - 22- 23 - X - X2- Y - Y2- Y3- Z1 , Z1- Zz - Z3, X -X2 - X3- Y - Y2- 21 - 22 -23 , X - X2- X3 - X4- Y - Z -Z2 - 23- X , - X - X2- Y - Y - Yz- Z1, Z - Z2- 23- X -X2 - X3 - X4 Y2- 27 - - 23, X7 -X2 - Y - Y2 - Y3- 2 , -22 - Zz , X - X2- Xz - Y - Y -Y2 - Yz - Z1, 2 - 22- 23 - X , - X2- Y -Y2 - Y3 - Y4 - 21 , 27- 22 - 23 Y2- Y3- 21 - 22- 23, X7- X2- X3- X4- Y1- Y2- Y3- 21- 22- 23, X - X2- X , - Y - Y -Y3 - Y4 - 21, 27- 22- 23 - X , - X ,- X , -X4 - Y X - X2- Y - Y - Y3- Y4 - 2 , - 22- Z3 , X7 -X2 - X3- Y - Y2- Y3- 94 Y2- Y3- Y4- 21 , 21 - 22- 23- 24 - X , - X2 - Y - Y2- 21, Z - Z2- 23 - 24 27 -22 - 23 , X -X2 - X3 - X4 - Y , - Y2- Yz- Y4- 27- 22- 23, X . -X2 X7- X2 - X3- Y -Y2 - 21 , Z , -22 - 23 -24 - X , - X2- X2 - X4 - Y , - Y2- Z1 , Y - Y2- 2 , - 22- 23 - 24 , X7- X2- X3- Y -Y2 - 21 - 22 - 23 -24 , X - X2 Z -Z2 - 23 -24 - X7- X2 - Y - Y2- Y3- Z1 , Z , -Z2 - 23 -24 - X - X2- Xz Xz- X4- Y - Y2- 2 , - 22- 23 - 24, X7- X2- Y - Y2- Y3- 2 , -22 - 23- 24 , Y - Y2- Y3 - 21 , 21- 22- 23 -24 - X , -X2 - Xz -X4 - Y - Y2- Yz- Z1 , X -X2 - X3 - Y - Y - Y3- 2 , -22 - 23- 24 , X7 -X2 - X3- X4 - Y - Y2 Z -Z2 - 23 - 24- X7 - X2- Y - Y2- Y3 - Y4 -Z1 , Z -Z2 - 23- 24 - X7 - X2 Y3- 2 , -22 - 23 - 24 , X , -X2 - Y - Y2- Y3- 94- 21- 22- 23- 24, X7- X2 X2 - Y - Y2- Y3- Y4 -21 , Z -Z2 - 23 - 24 -X , -X2 - X3- X4 - Y - Y2 X3- Y - Y - Y3 - 94- 21 - 22- 23 - 24 X - X7- X2 - X4- Y - Y - Yz Y3- Y4 -Z1 , X , - X - Y - Y2 - Z / -Z2 , X , - X2- X3- Y - Y2- 27 -Z2 , Y4- 27 - 22- 23- 24 , Y - Y2- X , - X2- 21, Y - Y2- X -X7 - X , -21 , X -X2 - Xz -X4 - Y -Y2 - 24 - Z2 X , -X2 - Y -Y2 - Yz- Z -Z2 , Y - Y2- X , - X2- X3- X4- 21 , Y - Y - Y3- X , -XZ - Z , Y - Y - Y3 X -X7 - X , - Y -Y2 - Y3 - 2 , - Z2, X , -X2 - X ,- X4- Y - Y -Y3 - 2 , X -X2 - Xz- Z1 , Y - Y2- Y3- X7 - X , -X3 - X4 - 21, Y -Y2 - Y3 - Y4 Z2, X , - X2- Y - Y2- Y3 - Y4- 2 , - 22, X - X2- X2 - Y - Y - Y3- Y4 X -X2 - 21 , Y - Y2- Yz- Y4- X , - X - Xz- 21 , Y -Y2 - Y3- Y4- X , Z -Z2 , X , -X2 - X3- 84 - Y , - Y2- Y3- Y4 - 2 , -22 , Z , - X , - X - Y - X - Xz -X4 - 21 , Y - Y2 - X , -X2 - Z2 - Z2 , Y - Y2- X7 - X2- X3 - 21 Y2- 27 - Z2 , Z7 - X , - X2- X3 - Y - Y2 - 2 , -Z2 , Z7- X7- X2 - X3- X4 Z2, Y1- Y2- X -X2 - X3 - X4 - 24 - Z2 , Y - Y2- Y3- X - X2- 27 -22 , Y - Y2- Zi -Z2 , Z , - X , -X2 - Y - Y2- Yz- Z -Z2 , Z7 - X , - X2- Xz Y - Y2- Y3- X , - X2- X3- 2 , - 22, Y - Y2- Y3- X -X2 - X3- X - 2 Y - Y - Y3- 2 -22 27- X , - X2- X , - X4- Y - Y -Y2 - 27 - 22, Z2, Y - Y - Y3- 94- X -X2 - 27- 22 , Y - Y2- Y3- Y4- X , - X - Xz Z - X , - X - Y - Y2- Yz- Y4- 2 , -22 , 2 , - X , - X2- X2 - Y - Y2- Yz Zj- Z2, Y - Y2- Y3- Y4 - X , - X - X7 -X4 - 24 - 22 , Y , - Y2- X , - Xz YA- Z -Z2 , Z7- X , - X - X7- X4 - Y - Y2- Y3- Y4 - 2 , -22 , Z , -Z2 - X , Z -Z2 - Zz , Y - Y2- X -X2 - Xz - Z -Z2 - Z3 , Y - Y2- X , - X2- Xz X2- Y - Y2- Z -Z2 , Z , -Z2 - X7- X2 - X3- Y - Y - Z -Z2 , Z , -Z2 - X - X4- 24 -Z2 - Zz, Y - Y2 - Y3- X - X2- 2 , - 22- 23 , Y , - Y2- Y3- X - X - X3- X4 - Y - Y2- 27 -Z2 , Z - Z2- X - X2- Y - Y2- Y3- 2 , -22 , XZ- X3 - 21 - 22 - 23, Y - Y2- Y3- X , - X - X3- X4- 21 - 22- 23, Z -Z2 - X - X2- Xz - Y - Y2- Y3- 21 - 22 , Z -Z2 - X -X2 - X3- X4 - Y1 Y - Y2- Yz- Y4 - X , -X2 - 2 , -22 - 23, Y -Y2 - Y3 - Y4- X - X2- Xz Y2- Y3- 2 , - Z2, Z -Z2 - X , -X2 - Y - Y2- Y3- Y4- 21 - 22 , Z -Z2 - X , Z ; -Z2 - Z3 , Y - Y - Y3 - Y4- X , - X2- X3 - X4 -24 - Z2 - Z3 , Y - Y - X2- Xz - Y - Y2 - Yz - Y4- Z -Z2 , Z -Z2 - X , - X - X7- X4 - Y - Y2 X -X2 - Z2 - Z2 - 23 -24 , Y -Y2 - X - X2- X3 - 2 , -Z2 - 23 - 24, Y - Y2 Yz - Y4- Z -Z2 , Z1- Z2 - Zz - X7 -X2 - Y - Y2- 2 , -Z2 , Z1- Z2- 23 - X - X - X2- Xz - X4 - 2 , - 22- 23 - 24, Y - Y - Y3 - X , - X2- 21 - 22 - 23- 24 , X2- X2 - Y - Y2- 2 , - Z2, Z , -Z2 - 23 - X , -X2 - X3 -X4 - Y - Y2- 2 , -22 , Y - Y2- Y3- X - X2- Xz - 2 ,- 22 -23 - 24 , Y - Y2- Y3- X -X2 - Xz Z -Z2 - 23 - X -X2 - Y - Y2- Y3- 21 - 22, Z1- Z2 -23 -X7 - X , -X3 - Y , X4- 2 , -22 - 23- 24, Y - Y2- Y3- Y4- X , -X2 - 2 , -22 - 23 - 24 , Y - Y2 Yz- Y3- 2 -22 , 22- 22- 23- X - X2- X3- X4 - Y - Y - Y3- 2 , - 22, Y3- Y4 - X7 -X2 - X3- 21 - 22- 23 - 24 , Y -Y2 - Y3 -Y4 - X - X2- Xz Z - Z2- 23 - X , - X - Y - Y2- Y3- Y4- 24- Z2 , Z , -Z2 - 23 - X -X2 - Xz X4- 21 - 22 -23 - 24 , 27- X , -X2 - Y - Y2, Z , -X7 - X2 - Xz- Y -Y2 , Y - Y2- Y3- Y4- 2 , -22 Z -Z2 - 23 - X , -X2 - X3- X4 - Y - Y2- Y3 21- X , -X2 - Xz - X4 - Y - Y2, Z ,- X7- X2- Y - Y2- Y3, Z ,- X ,- Xz Y4- 27 - Z2 , Z - Z2- 23- 24- X -X2 - Y - Y - Z -Z2 , Z -Z2 - 23 - 24 X3- Y - Y2- Y3, Z ,- X7- X2- Xz- X4- Y - Y2- Y3, Z4 - X -X2 - Y - X -X2 - Xz - Y - Y2- 27 - Z2, Z , -Z2 - 23- 24 -X7 - X2- X3- X4- Y - Y2 Yz- Y3 - Y4 , 27- X , -X2 - Xz - Y - Y2- Y3- Y4, Z7 - X , -X2 - X3 - X4 27- 22, Z , - 22- 23- 24 - X , - X , - Y -Y2 - Y3 - 2 , - 22, Z , -22 - 23 - 24 Y - Y2- Y3- Y4, Z1- Z2 - X7 -X2 - Y - Y2, Z , -Z2 - X , - X2- X3 - Y - X7 -X2 - Xz- Y - Y2- Y3- 27 -Z2 , Z -22 - 23 -24 - X , - X2- X3 - X4- Y Y2, Z -Z2 - X - X2- X3- X4- Y - Y2, Z7 - Z2- X7- X2 - Y - Y2- Y3 , Y2- Y3- 2 - 22 Z1- Z2 -23 - 24- X7 -X2 - Y - Y2- Y3- Y4 - 2 , -22 , Z -Z2 - X -X2 - X3- Y - Y2- Y3, Z1- Z2 - X -X2 - X3- X4 - Y - Y2 Z - Z2 - 23 -24 - X7 -X2 - X3- Y - Y2- Y3 - Y4- 27 - Z2 , Z - Z2- 23 - 24 Y3, Z1 - Z2- X7 - X2- Y -Y2 - Y3- Y4, Z _ -Z2 - X -X2 - X3- Y -Y2 - X -X2 - X3 -X4 — Y - Y2- Y3- Y4- 2 , -22 , X -X2 - Y -Y2 - 2 -22 US 2018 /0362598 A1 Dec. 20 , 2018 35
Z3, X7- X2- Xz - Y - Y - Z -Z2 - Z3 , X - X2- X3 - X4- Y - Y2- 21 X2, Z - Y - Y - Y3- Y4 - X -X2 - X3 , Z - Y - Y2- Y3- Y4- X , - X2 22- 23 , X7 -X2 - Y - Y2- Y3- 21 - 22 - 23 , X7- X2- Xz- Y - Y2- Y3 Xz -X4 , Z - Z2- Y - Y2- X , - X2, Z , -Z2 - Y - Y2- X7 -X2 - Xz, Z -Z2 - 23 , X7 -X2 - X3- X4 - Y - Y2- Y3- 21 - 22- 23 , X - X2- Y - Z - Z2 - Y - Y2 - X -X2 - X3- X4 Z -Z2 - Y - Y2- Y3- X - X2, Y2- Y3- Y4 - 2 , - 22- Z3 , X - X2- X3 - Y - Y2- Y3- Y4- 2 - 2 - 23 , Z - Z2 - Y - Y2 - Y3- X - X2- X3 , Z - Z2- Y - Y2 - Y3 -X7 - X , -X3 X , - X2- Xz- X4- Y - Y2- Y3- 94- 21- 22- Z3, Z7 -X , -X2 - Y , - Y2 X4, Z -Z2 - Y - Y2- Y3- Y4- X , -X2 , Z , -Z2 - Y - Y - Y3- Y4- X , Z -Z2 - Z3 , Z1- X - X2- X3 - Y - Y2- Zi -Z2 -Z3 , Z4- X7- X2- Xz- X4 X2- X3 , Z -Z2 - Y - Y2 - Y3- Y4- X , -X2 - Xz -X4 , Z _ -Z2 - Zz - Y Y - Y2- 2 , - Z2- Z3 , Z7 - X , - X2- Y - Y2- Y3 - 24 - Z2 - Z3 , Z7 - X , - X2 Y2- X , -X2 , Z , -Z2 - Zz - Y - Y2- X , -X2 - Xz, Z / -Z2 - 23 - Y - Y2 X3- Y - Y2- Y3 - 2 - 2 - 23, Z -X7 - X , -X3 - X4- Y - Y2- Y3- 2 / -Z2 X -X2 - X3- X4 , Z _ - Z2- 23 - Y - Y - Y3- X , -X2 , Z -Z2 -23 - Y Z3, Z7 - X , -X2 - Y -Y2 - Y3 - Y4 - 2 , -22 - 23 , Z7- X , - X2- Xz- Y - Y2- Y3 - X -X2 - Xz, Z -Z2 - 23 - Y - Y2- Y3 - X -X2 - X3- X4 , Yz- Yz- Y4- 27 - 22- 23, 27- X , -X2 - X3 - X4 - Y , - Y2- Y3- Y4- 2 , -22 Z ;- Z2- 23- Y - Y2- Y3- Y4- X , -X2 , Z , - Z2- Zz - Y - Y2 - Y3- Y4- X , Z3, Z , -Z2 - X , - X2- Y - Y2- Z -Z2 - Z3 , Z1- Z2- X7 -X2 - Xz- Y - Y - X2- X3, Z -Z2 - Zz- Y -Y2 - Y3- Y4- X , - X2- X3 - X4, Z - Z2- 23 -24 Z - Z2- 23 , Z1- Z2 - X -X2 - X3 - X4- Y -Y2 - 24 - Z2 -Z3 , Z1- Z2 - X Y - Y2- X , -X2 , Z7 - Z2- 23 - 24 - Y - Y2- X , -X2 - Xz, Z -Z2 - Zz X2- Y - Y2- Y3- 21 - 22- Z3, Z -Z2 - X -X2 - X3 - Y - Y - Y3 -2 , -22 24 - Y - Y2- X , - X - X3- X4, Z7- 22 - 23- 24- Y - Y2- Y3- X , -X2 , Z3, Z1- Z2 - X -X2 - X3- X4 - Y - Y2 - Y3- 21 - 22 -Z3 , Z1- Z2 - X7 -X2 Z -Z2 - 23 - 24 - Y -Y2 - Y3- X -X2 - X3 , 27 - Z2- 23- 24- Y - Y2- Yz Y - Y2- Y3 - Y4 - 2 , -22 - 23, Z -Z2 - X , -X2 - X3 - Y - Y - Y3- Y4 -21 X7- X2- Xz- X4, 2 , - 22- 23- 24 - Y - Y2- Y3 - Y4 - X - X2, Z -Z2 - 23 Z2- 23, Z - Z2- X - X2- X3 -84 - Y - Y2- Y3 - Y4 -2 , -22 - 23 , Z1- Z2 24- Y - Y - Y3- Y4- X -X2 - Xz , Z -Z2 - 23 - 24- Y - Y2- Y3- Y4 Zz- X , -X2 - Y - Y2- 2 , -22 - Z3, Z1- Z2 - 23- X - X2- Xz - Y - Y2- 2 , X7- X2- Xz - X4, Y - Y2- X , -X2 - Z1 , Y - Y2 - X , -X2 - Xz- Z1 , Zz- Z3, Z -Z2 - 23 - X -X2 - X3- X4 - Y - Y2- 2 , -22 - Z3 , Z1- Zz - Zz Y - Y2- X , - X2- Xz - X4- 21 , Y , - Y2- Yz- X , -X2 - 21 , Y - Y2- Y3 X7 -X2 - Y -Y2 - Y3 - 2 - 2 - 23, 2 ,- 22 - 23- X , -X2 - X2 - Y - Y2- Yz X -X2 - Xz - Z?, Y - Y2- Y3- X -X2 - X3- X4- 21 , Y - Y2- Y3- Y4 Zj- Z2- Z3 , Z - Z - Zz- X , - X2- Xz - X44Y - Y2- Y3- 21 - 22- 23 , X , -XZ - Z1 , Y - Y2- Y3- Y4- X , - X - Xz -Z1 , Y , - Y2- Y3- Y4- X , Z - Z2- 23 - X , - X - Y - Y2- Y3- Y4- 24- Z2 - Z3 , Z1- Z2 - Zz - X - X2 X2- X3- X4 -Z1 , Z - Y - Y2- X , - X -Z1 , Z - Y - Y2- X - X2- Xz Xz - Y - Y2- Y3 - Y - Z -Z2 - Z3 , Z -Z2 - Zz - X , -X2 - X3- X4 - Y - Y2 Z1, Z , - Y - Y2- X - X2- Xz - X4- Z1 , Z - Y - Y2- Y3 - X , - X2- Z1 , Yz- Y4 - 2 ; -Z2 - Z3, Z -Z2 - 23 - 24 - X , - X2- Y - Y2- 2 , - 22 -23 , Z - Y - Y2- Y3- X -X2 - Xz- 21, Z , - Y - Y2- Y3- X -X2 - Xz - X4 2 , -22 - 23 - 24 - X7- X ,- Xz- Y -Y2 - 27 - Z2- Z3, Z7- 22- 23 - 24 - X - Z1, Z - Y - Y2 - Y3 - Y4 -X - X2 -Z1 , Z ,- Y -Y2 - Y3- Y4- X ,- X2 X2- X3 - X4 - Y - Y2- 2 , -Z2 - Z3 , Z , -Z2 - 23- 24 - X , - X - Y - Y - Yz X3- 21 , Z - Y - Y -Y3 - Y4 - X , -X2 - X3 - X4 - 21, 27 -22 - Y - Y2 Z - Z2- Z3 , Z1 -Z2 - 23- 24 - X - X2- X3 - Y - Y2- Y3- 21 - 22- 23 , X -X2 - Z1 , Z -Z2 - Y - Y2- X , -X2 - Xz- Z1 , Z1 -Z2 - Y - Y2- X - Z ; - Z2- 23 - 24 - X , -X2 - X3- X4 - Y - Y2- Y3- 2 , - 22- Z3 , Z -Z2 - 23 X2- X3- X4 -Z1 , Z -Z2 - Y - Y - Y3- X , -X2 - Z1 , Z -Z2 - Y - Y2 24- X -X2 - Y - Y2- 2 , - 22- , Z - Z2 - 23- 24- X , -X2 - Xz - Y - Y2 Y3 -X7 - X , -X3 - 21 , Z -Z2 - Y - Y2- Y3 - X -X2 - X3 -X4 - 21 , Yz- Y4- 2 , - 22- 23 , 24- Z2 - 23 - 24- X - X2- X3 - X4- Y - Y2- Y3- Y4 Z - Z2- Y - Y2- Y3 - Y4- X - X2- Z1 , Z - Z2- Y - Y2 - Y3- Y4- X , 2 , -22 - 23 , X , - X2- Y -Y2 - 27 - 22- 23- 24, X , - X ,- X , - Y -Y2 - 2 ; X - X , -Z1 , 2 , - 22- Y - Y - Y3- Y4- X , -X2 - X2 - X4 - 21 , 27 -22 - 23 ZA- ZX - Z , X - XL- XX- X - Y - Y - - Z - Z - Z , X - XA- Y - Y Y - Y2- X -X2 - 21, 21 - 22- 23- Y - Y2- X -X2 - Xz- Z1 , Z7- 22 - 23 Y3- 2 , -22 - 23 - 24 , X , -X2 - X3 - Y - Y2- Y3- 2 , - 22- 23 - 24, X -X2 Y - Y2- X , - X2- X2 - X4- Z1 Z -Z2 - Zz- Y - Y2 - Y3- X , - X2- Z1 , Xz- X4- Y - Y2- Y3- 21 - 22 - 23- 24 , X7- X2 - Y - Y2- Y3- Y4- Z -Z2 Z - Z2- 23 - Y - Y - Y3- X , - X2- Xz - Z1, Z -Z2 - Zz - Y - Y2- Y3- X Zz- Z4, X -X2 - X2 - Y - Y - Y3- Y4- 2 , -22 - 23 - 24, X -X2 - Xz X2- X3- X4 - 21, Z -Z2 - 23- Y ; - Y2- Y3- YA- X7 - X , -21 , 21 - 22- 23 X4- Y - Y2- Y3- Y4- 2 ,- 22- 23- 24, Z _ - X -X2 - Y - Y2- 2 , -22 -23 Y -Y2 - Y3 - Y4 - X7 - X ,- Xz- Z1, Z7- 22- 22 - Y - Y -Y3 - Y4 - X , Z4, Z -X7 - X2- X3 - Y - Y2- 22 - 22 -23 - 24 , Z _ - X , -X2 - X3- X4 - Y1 X2- Xz- X4- 21 , 21 - 22- 23- 24 - Y - Y2- X , - X2- 21 , 21- 22- 23- 24 Y2- Z , -Z2 - 23 - 24, Z7 - X , - X2- Y - Y - Y2- 2 , -22 - 23 -24 , Z _ - X - Y - Y2- X , - X - Xz- Z1 , Z -Z2 - 23- 24 - Y - Y2 - X7- X , - X7- X4 - 21, X - Xz- Y - Y2- Y3- 2 , -22 - 23- 24 , 27 - X , - X2- X3 - X4- Y - Y2 21- 22 - 23 - 24 - Y - Y2- Y3- X , - X2- 21 , 21- 22- 23 - 24- Y - Y2- Yz Y3- 2 -22 - 23- 24, 27- X , -X2 - Y - Y2- Y3- 94- 2 , - 22- 23- 24, X -X2 - X2 - 21 , 21- 22- 23- 24 - Y -Y2 - Y3 - X7 - X , - X , -X4 - 21, Z - X - X2- Xz- Y - Y2- Y3 - Y4- Z -Z2 - 23- 24, Z , - X - X2- Xz- X4 2 , -22 - 23 - 24 - Y - Y - Yz- Y4- X7- X , -21 , 21- 22 - 23 - 24- Y -Y2 Y - Y - Y3- Y4- 2 , - 22- 23 - 24, 27 - 22- X , -X2 - 2 , -22 - Y - Y2- 22 Y3- Y4- X - X2- X3 - 21, 22 -22 - 23- 24- Y - Y2- Y3 -Y4 - X , - X2 Zz- Z4 , Z - Z2- X - X2- X3 - Y - Y2- 2 , -22 - 23- 24 , Z _ -Z2 - X7 -X2 X3- 84 -21 , Y - Y2 - X7 -X2 - 27 -22 , Y - Y2 - X7- X2 - Xz- Z -Z2 , X3- X4 - Y - Y2 -27 -Z2 - 23- 24 , Z _ -Z2 - X - X2- Y - Y2- Y3- 2 -Z2 Y - Y2- X7 - X2- X3 - X4 -24 - 22 , Y - Y2- Y3- X , -X2 - Z -Z2 , Zz- Z4, Z -Z2 - X7 -X2 - X3- Y -Y2 - Y3- 2 , -22 - 23 - 24 , Zi -Z2 - X - Y - Y - Yz- X , -X2 - XZ- 2 , -22 , Y - Y - Yz- X ,- X2 - Xz - X4- 2 , X -X3 - X4 - Y -Y2 - Y3- 21- 22- 23- 24 , 27 - Z2- X , -X2 - Y - Y2- Yz Z2, Y -Y2 - Y3 - Y4- X , - X2- 2 , -22 , Y - Y -Y3 - Y4 - X7 - X ,- Xz Y4- 2 , -22 - 23- 24 , Z , -Z2 - X , - X - X3- Y - Y2 - Y3- Y4- Z - Z - Zz Z - Z2, Y - Y2- Y3 - Y4 -X7 - X2 - X7- X4 - 2 , -Z2 , Z - Y - Y2- X , Z4, Z -Z2 - X - X2- X3 - X4- Y , - Y2- Y3- Y4- 2 -Z2 -Zz - Z4 , Z -Z2 X2- Z -Z2 , Z - Y - Y2- X - X2- Xz - Z -Z2 , Z , - Y - Y2- X , -X2 Z3- X - X2- Y -Y2 - 2 - - -24 , Z -Z2 -23 - X - X2- X3 - Y - Y2 X3 -X4 - Z -Z2 , Z - Y - Y2- Y3- X , - X2- Z -Z2 , Z1 - Y - Y2- Y3 Zj- Z2- 23 - 24 , Z _ -Z2 - 23- X - X2- X3 - X4- Y -Y2 - 21 - 22- 23- 24 , X -X2 - Xz - Z ;- Z2, Z , - Y - Y2- Y3- X -X2 - X3 - X4- 2 , -22 , Z -Z2 - 23 - X - X2- Y - Y2- Y3 -24 -Z2 - 23- 24, Z _ - 22- 23- X - X2 Z - Y - Y - Yz- YA -X7 - X2- 27 - 22, Z - Y - Y2 - Yz- Y4 - X - X2 X2 - Y - Y - Yz - Z -Z2 - 23- 24 , Z _ -Z2 - 23 - X , - X - X7- X4 - Y - Y2 Xz - Z , -Z2 , Z - Y - Y2- Y3- Y4- X , - X - X7- X4 - 2 , -22 , Z , -Z2 - Y - Y3- 2 , -22 - 23- Z4 , Z7 -Z2 - 23- X - X2- Y - Y2- Y3- Y4- 21 - 22 - 23 Y2- X , - X2- Z -Z2 , Z , -Z2 - Y - Y2- X -X2 - Xz- Z -Z2 , Z , -Z2 - Y1 Z4, Z - Z2 - 23- X - X2- Xz - Y - Y2- Y3- Y4- 2 , -22 - 23 - 24, Z -22 Y2- X , -X2 - X3 -X4 - 24 -Z2 , Z -Z2 - Y - Y2- Y3- X , -X2 - 27 -22 , Z3- X -X2 - X3 - X4 - Y - Y2- Yz- Y4- 21- 22 - 23- 24, 27 - 22- 23 - 24 Z - Z2- Y - Y2- Y3- X - X2- X3- 24- Z2, Z -Z2 - Y - Y2- Y3- X , -X2 X , -X2 - Y -Y2 - 2 , - 22- 23- 24 , 27- 22- 23- 24- X , -X2 - Xz - Y - Y2 X3 -X4 - 2 , -22 , Z , -22 - Y - Y2 - Y3- Y4- X , -X2 - 2 , - Z2 , Z , - Z2- Y - Z - Z2- 23 - 24 , Z _ -Z2 - 23 - 24- X , - X2- X3- X4- Y - Y2- 21 - 22 - 23 Y2- Y3- Y4- X , - X2- Xz- Z -Z2 , Z1- Z2 - Y - Y2 - Y3- Y4- X , -X2 Z4, Z - Z2 -23 - 24 - X , -X2 - Y - Y2 - Y3- 21 - 22 -23 - 24 , Z _ -Z2 - Zz Xz -X4 - Zi -Z2 , Z1- Z2 - 23 - Y - Y2- X7- X2 - 24 -Z2 , Z -Z2 - Zz - Y - Z4- X -X2 - X3- Y , - Y2- Y3- 2 , -22 - 23- 24 , Z _ -Z2 - 23 - 24 - X , - X2 Y2- X , - X2- Xz- Z2 - Z2 , Z - Z2- Z2 - Y - Y2- X , - X2- X3 - X4- 2 , - 22 , X3- X4 - Y -Y2 - Y3- 2 , - 22- 23- 24 , Z - Z2- 23- 24- X -X2 - Y - Y2 Z -Z2 - 23 - Y - Y2- Y3- X -X2 - 27- Z2 , Z -Z2 -23 - Y - Y2- Y3- X - Y3- 94- 21 - 22 - 23- 24 , 27 - 22- 23- 24- X , - X -Xz - Y , - Y2- Yz- Y4 X2- X2 - 27 -Z2 , Z -Z2 - Zz - Y - Y2 - Y3- X , - X2- Xz - X4- 24 - 22, Zj- Z2 - 23 - 24, Z - Z2- 23 - 24- X , -X2 - Xz -X4 - Y - Y2- Y3 -Y4 - 21 Z -Z2 - 23 - Y - Y2- Y3- Y4- X - X2- Z1 - Z2 , Z -Z2 -23 - Y - Y2- Yz Z2- 23 - 24 , Z - Y - Y2- X , -X2 , Z - Y - Y2- X - X2- X3 , Z - Y - Y4- X , - X2- Xz- Z -Z2 Z -Z2 - Zz - Y - Y2- Y3 - Y4- X - X2- Xz Y2- X , -X2 - X3 -X4 , Z - Y -Y2 - Y3 - X -X2 , Z - Y - Y - Y3- X , XA - 2 - 22, Z -Z2 -23 - 24- Y - Y - X , - X - Z - Z2, Z - Z2 - 23- 24 X2- X3 , Z4 - Y - Y - Y3 - X , -X2 - X3 -X4 , Z - Y - Y2- Y3 - Y4- X , Y - Y2- X -X2 - X3- 2 -Z2 , Z7 - - 23 -24 - Y - Y2- X7 - X , -Xz - X4 US 2018 /0362598 A1 Dec. 20 , 2018 36
Z - Z2, Z , -Z2 -23 - 24 - Y - Y2- Y3 - X - X2- 27 -Z2 , Z , -Z2 - 23 - 24- X2- Xz- Z - Z2- 23 - 24 , Z -Z2 - 23- 24 - Y - Y2- Y3 - Y4 -X7 - X2 - Xz Y - Y2- Y3 - X , -X2 -X3 - 2 , -22 , Z1- Z2 - 23 - 24 - Y - Y2- Y3- X - X2 X4 -24 - Z2- 23- 24, wherein X is selected from the group X3 -X4 -21 - 22 , 21- 22 -23 - 24 - Y | - Y2- Y3- Y4- X - X2- 21 - 22 , consisting of: (all single letter amino acid format ) A , D , E , Z -Z2 - 23 -24 - Y - Y - Y3- Y4 -X7 - X2- X3- Z2 - Z2 , Z - Z2- 23 - 24 G , I, K , L , N , P , Q , R , and V ; X , is selected from the group Y - Y2- Y3 - Y4 - X , -X2 - X3- 84 - 2 , -22 , Y , - Y2- X , - X2- Z -Z2 - 23 , consisting of A , F , G , I , K , L , N , P , Q , R , S , T , V , and Y ; X ; Y - Y2- X , -X2 - Xz - Z -Z2 - Z3 , Y - Y2- X , -X2 - Xz -X4 - 27 -Z2 - 23 , is selected from the group consisting of A , D , E , F , G , I , K , Y - Y2- Y3 - X , - X2- Z -Z2 - Z3 , Y - Y2- Y3- X - X2- Xz- Z -Z2 - 23 , L , N , Q , R , S , T , V , and Y ; X4 is selected from the group Y - Y2- Y3- X -X2 - X3 -X4 - 21 - 22 - 23 , Y - Y2- Y3 - Y4- X - X2 consisting of: R , H , K , D , E , S , T , N , Q , G , A , I, L , M , F , W , 2 , -22 - 23 , Y - Y2- Yz- Y4- X , -X2 - X3- 27 - Z2- Zz, Y - Y2- Y3 Y , and V . In some embodiments , Y , is selected from the Y4- X ,- X2- X2- X4- 2 , - 22- Z3, Z - Y - Y2- X , - X2- 27- 22- 23, group consisting of: ( all single letter amino acid format ) A , Z - Y - Y2 - X , -X2 - Xz- Z - Z2- Z3 , Z1- Y - Y2- X , -X2 - Xz -X4 - 21 D , E , F , G , I , L , N , Q , S , T , V , and Y ; Y , is selected from the Z2- 23, 22- Y -Y2 - Y3- X -X2 - 2 - 2 - 23, 27 - Y , -Y2 - Y3 -X7 -X2 group consisting of F , G , I, L , and Y ; and Y2 is selected from X3- 2 -22 - 23, Z , - Y -Y2 - Y3- X -X2 - X3- X4 - 2 , -22 - Z3 , Z - Y the group consisting of A , D , E , F , G , I, K , L , R , S , T , V, and Y2- Y3- Y4- X , -X2 - 21 - 22 - 23, Z - Y - Y2- Y3- Y4 - X7 -X2 - Xz Y ; and Y . is selected from the group consisting of: R , H , K , Z - Z2- Z3 , Z - Y -Y2 - Y3 - Y4- X7 - X2- X3 - X4- 24 -Z2 - Z3 , Z -Z2 D , E , S , T , N , Q , G , A , I , L , M , F , W , Y, and V . 1 [0243 ] In related embodiments , cleavable binary linker Y -Y2 - X7 -X2 - 2 - 2 - 23, Z1- Z2 - Y -Y2 - X7 -X2 - X3 - X4 - 21 - 22 peptides ( L ) comprising an X region and a Y region are Z3, Z -Z2 - Y -Y2 - Y3- X , -X2 - 2 , -22 - Z3 , Z1- Z2 - Y - Y2- Y3- X described above, and exemplary Z regions ( 1 - 4 amino acid X - X2- 2 , -22 - 23 , Zi - Z2- Y - Y - Y3- X , -X2 - X3 - 84 - Z -Z2 - Zz, sequences , for example , 2 amino acid sequences ) that may 2 , -22 - Y - Y - Y3- Y4- X -X2 - 27- 2 ,- 23, Z7 - 22- Y -Y2 - Y3- Y4 be fused in frame to the X and / or Y regions ( either to the X , - X2- Xz - Z -Z2 - Z3, Z -Z2 - Y - Y2- Y3- Y4- X , - X - X7- X4 - 24 N -terminal sequence of an X or Y region , or the C - terminal Zz- Z3, Z1- Z2 - 23 - Y - Y2- X7 -X2 - 2 , -22 - Z3, Z1- Z2 - Zz - Y - Y2 sequence of an X or Y region ) described above form the X - X2- Xz - Z -Z2 - Z3, Z - Z2- Zz - Y - Y2- X - X2- Xz- X4- 24 - Z2 tertiary cleavable linking peptides. Illustrative Z or spacer Z3, Z , -Z2 - 23 - Y - Y - Y3- X -X2 - 21- 22 - 23, Z - Z2- 23 - Y -Y2 amino acid sequences may include : AA , AF , AM , AN , AQ , Yz- X , -X2 - X3 - 21 - 22 - 23 , 2 ,- 22 - 23- Y - Y2- Y3- X ,- X2- X3- X4 AV , AW , AY , DA , DD , DE , DF, DG , DI, DL , DP , DS , DT, Zj- Z2- Z3 , Z1- Zz - Zz- Y -Y2 - Y3 -Y4 - X , -X2 - 27 -Z2 -Z3 , Z -Z2 DV , DW , DY, EA , ED , EE , EF , EG , EI, EL , EP, ES , ET, EV , Zz- Y - Y2 - Y3- Y4- X , -X2 - Xz- Z - Z2 -Z3 , Z -Z2 - 23 - Y - Y2- Y3 EW , EY , FA , FD , FE , FF, FI, FK , FL , FM , FN , FQ , FR , FS , Y4- X , -X2 - X7 -X4 - 24 - Z2 - Z3, Z - Z2- 23 - 24 - Y - Y2- X , -X2 - 21 FT, FV , FW , FY, GA , GD , GE , GF, GI, GL , GM , GN , GQ , Zz- Z3 , Z - Z2- 23 - 24- Y -Y2 - X , -X2 - X2 - 2 - 22- 23, Z ; -Z2 - Zz GS , GV , GW , GY , HA , HD , HE , HF, HH , HI, HK , HL , HN , Z4- Y - Y2 - X , -X2 - X3- X4 - 27 -Z2 - Z3, Z1- Z2 - 23 -24 - Y - Y2- Yz HP . HO , HR , HS , HT, HV, HY, IA , ID , IE , IG , IH , II, IK , IL , X - X2- 2 , - 22- 23 , 24- Z2 - 23 -24 - Y - Y2- Yz - X , - X2- X3- 21 IM , IN , IP , IQ , IR , IS , IT, IV , IW , KA , KD , KE , KF , KI, KL , Zz- Z3, Zi- Z2- 23 - 24- Y - Y2 - Y3- X - X2- X7- X4- 2 , - 22- 23 , KN , KQ , KV, KY, LA , LD , LE , LG , LH , LI, LK , LL , LM , 27 -22 - 23 - 24 - Y , - Y2- Y - Y4- X , - X - X3- 2 , - 22- 23, 2 , -22 - 23 LN , LP , LQ , LR , LS , LT, LV , LW , MA , MF, MG , MI, MK , 24- Y - Y2- Y3- Y4- X -X2 - Xz - Z - Z2- Z3, Z1- Z2- 23- 24 - Y - Y2 ML , MM , MN , MQ , MR , MS , MT, MV, MY, NA , ND , NE , Yz- Y4- X7 - X2- Xz - X4 -24 - Z2 - Z3 , Y - Y2- X , - X2- 21 - 22 - 23- 24 , NF, NI, NL , NM , NN , NQ , NS , NT, NV, NY, QA , QD , QE , Y - Y2- X , -X2 - X3 - 2 , -22 - 23 - 24, Y -Y2 - X , - X2- X3 - X4- 24 -22 QF, QI, QL , QM , QN , QQ , QS , QT , QV, QY, RA , RD , RE, 23- 24, Y - Y2- Y3- X - X2- 2 , - 22- 23 - 24, Y - Y2- Y3 - X - X2 RF, RI, RL , RN , RO , RV , RY, SA , SF , SG , SK , SN , SP, SQ , X3- 2 ,- 22 - 23 - Z4, Y -Y2 - Y3- X , - X7- X ,- X4- 2 ,- - - ZA , SR , SS , ST , SV, SW , SY, TA , TF, TG, TK , TN, TP, TQ, TR, Y - Y2- Y3- Y4- X7 - X2- Z -Z2 - 23 - 24, Y , - Y2- Y3 - Y4- X , - X2 TS , TT, TV , TW , TY , VA , VF, VM , VN , VQ , VV, VW , VY , X3- 2 , - 22 -23 - 24 , Y - Y - Y3- Y4- X - X - X3- X4 - 2 , - Z2- 23 - 24, WA , WF , WI, WK , WL , WN , WP, WQ, WR , WS, WT, WV, Z - Y - Y2- X -X2 - 2 - 22- 23- 24 , Z - Y - Y2- X -X2 - Xz- 2 ,- 22 :AZETZZETETWW , WY , YA , YD , YE , YF , YI, YK , YL , YM , YN , YQ , YR , 23- 24 , Z - Y - Y2- X -X2 - X3 - X4 - 24- 22- 23 - 24, Z - Y - Y - Yz YS , YT, YV, YW , or YY. In exemplary embodiments , X - X -ZA - Z2 - 23 -24 , Z - Y - Y2- Y3- X - X2- Xz - Z -Z2 - 23 - 24 , individual tertiary peptides of the present invention can Z - Y - Y2 - Y3- X - X2- X3 - X4- 24 - Z2 -23 - 24, Z - Y - Y2- Y3- Y4 include a two amino acid sequence, fused in frame to the X , -X2 - 2 , -22 - 23- 24 , Z - Y - Y - Y - YA- X , - X -X2 - 2 , -22 - 23 N - terminus and / or the C - terminus of an illustrative binary Z4, Z - Y - Y2- Y3- Y4- X7 -X2 - X3- X4 - 2 -22 - 23 - 24 , Z -Z2 - Y - peptide disclosed above , wherein the two amino acid Yz- X , -X2 - 27 - 22- 23- 24 , 27 - 22- Y - Y2- X -XZ - X3 - 2 , -22 - 23 sequence is AM , LH , MN , ES , WQ , or DT. Z4, Z -Z2 - Y -Y2 - X , - X2- X3 - X4- 24 - Z2- 23 - 24 , Z _ -Z2 - Y - Y2N Y3- X - X2- 21 - 22 -23 - 24 , Z _ -Z2 - Y - Y2- Y3- X - X2- X3 - 21 - 22 [0244 ] In exemplary embodiments , individual tertiary Z3- 24 , Z -Z2 - Y -Y2 - Y3 - X7 - X - X2 -X4 - 27 - 22- 23- 24 , 27- 22 peptides of the present invention can include a two amino Y - Y - Y3- Y4- X , - X2- 2 , - 22- 23 - 24, 22- - Y - Y2 - Yz- Y4- X , acid sequence , fused in frame to the N -terminus and / or the X - Xz- Z - Z2- Zz - Z4 , Z -Z2 - Y - Y2- Yz- Y4- X , -X2 - X7 -X4 - 2 , C -terminus of an illustrative binary peptide disclosed above , Z2- 23 - 24, Z - Z2- 23 - Y - Y2- X , - X2- Z1- Z2 - 23 -24 , Z - Z2- Zz wherein the two amino acid sequence is GS , WN , WQ , MA Y - Y2- X - X2- X3 - 2 , -22 - 23- 24 , Z , -Z2 - Zz- Y - Y2 - X , -X2 - Xz or MV. X4- 2 , -22 - 23- 24, Z -Z2 - 23 - Y - Y2- Y3- X , - X2- 2 , -22 - 23- 24 , [ 0245 ] In some embodiments , the spacer portion of the 2 , -22 - 23 - Y - Y2 - Y3- X , -X2 - X3- 2 , -22 - 23- 24, 2 , -22 - 23 - Y - tertiary peptide ( Z region ) comprises the amino acid Y2- Y3- X - X2- Xz - X4 -24 - Z2 - 23 - 24, Z -Z2 - 23 - Y - Y2- Y3- Y4 sequence GS, which may be fused in frame to the N - termi X -X2 - 27 -Z2 - 23- 24 , Z _ -Z2 - Zz - Y - Y2 - Y3- Y4 - X , -X2 - Xz- Z - nus or C - terminus of the binary peptide or both . Z2- 23- 24 , Z7 -Z2 - 23- Y - Y2- Yz- Y4- X , - X2- Xz - X4- 24 -Z2 - 23 [0246 ] In various embodiments , exemplary binary pep Z4, Z - 22- 23 - 24- Y -Y2 - X7 -X2 - 2 , - 22- 23- 24, Z -Z2 - 23 - 24 tides for use in the insecticidal and /or nematicidal polypep Y -Y2 - X , -X2 - X2 - 2 , - 22- 23- 24, 27- 22- 23- 24- Y - Y2- X , - X tides and proteins, DNA constructs and polynucleotides Xz- X4- 24 - Z2- 23 - 24 , Z -Z2 - 23 - 24 - Y - Y2 - Y3- X - X2- 2 , - N encoding an insecticidal and / or nematicidal polypeptide and
Zz- Z4, Z _ -Z2 - 23 - 24 - Y12 -Y3 - X -X2 - Xz- Z -Z2 - 23 - 24, Z - Z2N protein and incorporated into a transgenic plants or part Z3- 24- Y - Y2- Y33 - X3- X4 - 2 , -22 - 23- 24, Z _ -Z2 - 23 - 24 - Y - Y2N thereof , of the present invention , may include one or more Y3- Y4 - X -X2 - 21 - 22 - 23- 24 , Z _ -Z2 - 23 - 24- Y -Y2 - Y3- Y4- X - cleavable tertiary peptides selected from the group of or US 2018 /0362598 A1 Dec. 20 , 2018 37 consisting of: a binary peptide selected from IFVRLR (SEQ NO : 1740 ) , GSLKIFV (SEQ ID NO : 1741 ), GSLKLFV ID NO : 1619 ) , ALKLFV (SEQ ID NO : 1605) , ALKFLV ( SEQ ID NO : 1742 ) , GSLRALF ( SEQ ID NO : 1743 ) , (SEQ ID NO : 1603 ), RRKAFV (SEQ ID NO : 1644 ) , or GSLRFF ( SEO ID NO : 1744 ), GSLRIFV (SEQ ID NO : LFAAPF (SEQ ID NO : 1621) , fused in frame either on the 1745 ) , GSLRLFV (SEQ ID NO : 1746 ) , GSRALF (SEQ ID N - terminus , C - terminus or both termini with a Z region or NO : 1747 ) , GSRIFV (SEQ ID NO : 1748 ) , GSRLFV (SEQ spacer sequence selected from the group of or consisting of ID NO : 1749 ) ,GSRLRALF (SEQ ID NO : 1750 ) , GSRLRFF GS, WN , WQ , MA, MV, AM , LH , MN , ES , WQ , or DT . (SEQ ID NO : 1751 ) , GSRLRIFV ( SEQ ID NO : 1752 ) , [0247 ] In some embodiments , an exemplary DNA con GSRLRLFV (SEQ ID NO : 1753 ), GSRRKAFV (SEQ ID structmay encode an insecticidal and / or nematicidal protein NO : 1754 ) , GSRRKLIF (SEQ ID NO : 1755 ) , GSRRRFFA comprising a cleavable tertiary peptide having one or more ( SEO ID NO : 1756 ) , GSVFGRKG (SEQ ID NO : 1757 ) , and of the following illustrative amino acid sequences (ZXY or GSYFVRK (SEQ ID NO : 1758 ) . XYZ ) : AFVRLFGS ( SEQ ID NO : 1649 ) , AKLFVGS ( SEQ [ 0248 ] In various embodiments , the DNA construct com ID NO : 1650 ) , ALFALKGS (SEQ ID NO : 1651 ) , prises a polynucleotide that encodes a tertiary peptide hav ALFLKGS (SEQ ID NO : 1652 ) , ALFLRGS ( SEQ ID NO : ing the amino acid sequence of a binary peptide with the 1653 ) , ALFRGS (SEQ ID NO : 1654 ) , ALFRLRGS (SEQ ID addition of a spacer to the N - terminus of the binary peptide , NO : 1655 ) , ALKALFGS (SEQ ID NO : 1656 ) , ALKFFGS and /or to the C -terminus of the binary peptide , wherein the (SEO ID NO : 1657 ) , ALKFLVGS (SEO ID NO : 1658 ) , spacer is an amino acid or peptide comprising 1 to 4 amino ALKIFVGS (SEQ ID NO : 1659 ) , ALKLFVGS (SEQ ID acids, preferably two amino acids, for example GS, WN , NO : 1660 ) , FFADIKGS (SEQ ID NO : 1661) , FFALKGS WQ, MA , or MV. Other specific spacer examples and ( SEQ ID NO : 1662 ) , FFLKGS ( SEQ ID NO : 1663 ), FFL optimal amino acids for use as spacers are provided below . RGS (SEQ ID NO : 1664 ) , FFRLRGS (SEQ ID NO : 1665 ) , [0249 ] In some embodiments , an exemplary DNA con FGYRIKGS ( SEQ ID NO : 1666 ) , FLRLFGS ( SEQ ID NO : struct encoding an insecticidal and / or nematicidal protein 1667 ) , FYARRGS ( SEQ ID NO : 1668 ), GGLRKKGS ( SEQ comprises one or more polynucleotides which in combina ID NO : 1669 ), IFVALKGS (SEQ ID NO : 1670 ) , IFVLKGS tion or singly encode one or more binary peptides compris (SEO ID NO : 1671 ), IFVLRGS (SEQ ID NO : 1672 ) , ing the amino acid sequence : IFVRLR ( SEO ID NO : 1619 ) , IFVRGS ( SEQ ID NO : 1673 ) , IFVRLRGS (SEQ ID NO : ALKLFV (SEQ ID NO : 1605 ) , ALKFLV ( SEQ ID NO : 1674 ) , ILFNIKGS ( SEQ ID NO : 1675 ) , LFAAPFGS ( SEQ 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF (SEQ ID ID NO : 1676 ) , LFVALKGS (SEQ ID NO : 1677 ) , LFV NO : 1621) fused in frame at the N -terminal and /or the LKGS (SEQ ID NO : 1678 ), LFVLRGS (SEQ ID NO : C -terminal with one or more spacer sequences selected from 1679 ) , LFVRGS (SEO ID NO : 1680 ) , LFVRLRGS ( SEO ID the amino acid sequences: GS, WN , WQ, MA , MV , AM , NO : 1681) , LFVRVFLGS ( SEQ ID NO : 1682 ) , LGERGS LH , MN , ES , WQ , and DT. In various embodiments, an ( SEQ ID NO : 1683 ) , LKALFGS (SEQ ID NO : 1684 ) , exemplary DNA construct for use in transforming or trans LKFFGS (SEQ ID NO : 1685 ) , LKIFVGS (SEQ ID NO : fecting a eukaryotic cell, for example, a yeast cell , or a plant 1686 ) , LKLFVGS (SEO ID NO : 1687 ) , LRALFGS (SEO ID cell comprises a polynucleotide that encodes an insecticidal NO : 1688 ), LRFFGS ( SEQ ID NO : 1689 ) , LRIFVGS ( SEQ and / or nematicidal protein that includes one or more cleav ID NO : 1690 ) , LRLFVGS (SEO ID NO : 1691) , RALFGS able tertiary peptides selected from the group of or consist (SEO ID NO : 1692 ) , RIFVGS (SEQ ID NO : 1693 ) , ing of: a binary peptide selected from IFVRLR (SEQ ID RLFVGS ( SEQ ID NO : 1694 ) , RLRALFGS ( SEQ ID NO : NO : 1619 ) , ALKLFV (SEQ ID NO : 1605) , ALKFLV (SEQ 1695 ) , RLRFFGS ( SEO ID NO : 1696 ) , RLRIFVGS (SEO ID NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF ID NO : 1697 ) , RLRLFVGS (SEQ ID NO : 1698 ), RRKAF (SEQ ID NO : 1621 ), fused in frame either on the N - termi VGS ( SEQ ID NO : 1699 ) , RRKLIFGS (SEQ ID NO : 1700 ) , nus, C - terminus or both termini with a Z region or spacer RRRFFAGS (SEQ ID NO : 1701) , VFGRKGGS (SEQ ID sequence selected from the group of or consisting of GS , NO : 1702 ) , YFVRKGS (SEQ ID NO : 1703) , GSAFVRLF WN , WQ , MA ,MV , AM , LH , MN , ES , WQ , or DT and one ( SEO ID NO : 1704 ) , GSAKLFV (SEO ID NO : 1705 ) , or more TPs as described herein , for example , a CRIP TP, for GSALFALK (SEQ ID NO : 1706 ) , GSALFLK (SEQ ID NO : example , an ICK TP or a non -ICK CRIP TP , a TMOF TP, or 1707 ) , GSALFLR (SEO ID NO : 1708 ) , GSALFR (SEO ID a Bt TP as exemplified throughout the present disclosure . In NO : 1709 ) ,GSALFRLR (SEQ ID NO : 1710 ), GSALKALF related embodiments, the one or more binary or tertiary (SEQ ID NO : 1711) , GSALKFF (SEQ ID NO : 1712 ), peptides ( designated linker L ) described above are fused in GSALKFLV (SEQ ID NO : 1713 ) , GSALKIFV (SEQ ID frame with a TP , and optionally , an ERSP and /or optionally NO : 1714 ) , GSALKLFV (SEQ ID NO : 1715 ) , GSFFADIK a STA , for example , ( from N -terminus to C - terminus) : ( SEQ ID NO : 1716 ), GSFFALK (SER ID NO : 1717 ) , ( L - TP ) ,, - L , or ( L - TP - L ) , ( ERSP ) - ( STA ) - ( L - TP ) , or GSFFLK (SEQ ID NO : 1718 ) , GSFFLR (SEQ ID NO : (ERSP ) - (STA ) - ( L - TP ). - ( L ) , or (ERSP ) - (STA )- ( TP - L ) n , or 1719 ), GSFFRLR (SEQ ID NO : 1720 ), GSFGYRIK ( SEQ (ERSP ) - (STA ) - ( L ) - ( TP - L )n , or (STA ) -( L - TP ) n , or (STA )- ( L ID NO : 1721) , GSFLRLF (SEQ ID NO : 1722) , GSFYARR TP ) n - (L ) , or ( STA )- ( TP - L )n , or (STA )- (L ) -( TP - L ) n, wherein (SEO ID NO : 1723 ) , GSGGLRKK (SEO ID NO : 1724 ) , nis 1 to 200 , for example , from 1 - 100 , or from 1 - 20 , or from GSIFVALK ( SEQ ID NO : 1725 ) , GSIFVLK ( SEQ ID NO : 1 - 10 . In embodiments related to the above , the insecticidal 1726 ), GSIFVLR ( SEQ ID NO : 1727 ) , GSIFVR ( SEO ID and /or nematicidal protein encoded by the one or more NO : 1728 ) , GSIFVRLR (SEQ ID NO : 1729 ) , GSILFNIK polynucleotides may contain TPs that are the same or ( SEO ID NO : 1730 ) , GSLFAAPF (SEO ID NO : 1731) , different, and binary and / or tertiary peptides that are the GSLFVALK (SEQ ID NO : 1732 ), GSLFVLK (SEQ ID NO : same or different. In related embodiments of the above , 1733 ) , GSLFVLR (SEQ ID NO : 1734 ) , GSLFVR (SEQ ID tertiary peptides (designated above as L ) ,may include the NO : 1735 ) , GSLFVRLR (SEO ID NO : 1736 ) , amino acid sequence of a binary peptide selected from GSLFVRVFL (SEQ ID NO : 1737 ) , GSLGER (SEQ ID NO : IFVRLR (SEQ ID NO : 1619 ), ALKLFV (SEQ ID NO : 1738 ) , GSLKALF (SEQ ID NO : 1739 ) , GSLKFF (SEQ ID 1605 ), ALKFLV (SEQ ID NO : 1603 ) , RRKAFV (SEQ ID US 2018 /0362598 A1 Dec. 20 , 2018 38
NO : 1644 ) , or LFAAPF (SEQ ID NO : 1621 ), fused in frame [ 0252 ] As disclosed herein DNA constructs are also pro either on the N -terminus , C - terminus or both termini of the vided that comprise a polynucleotide that encodes a cleav binary peptide with a Z region or spacer sequence selected able binary and / or tertiary peptide fused in frame to a TP , from the group of or consisting of GS , WN , WQ , MA , MV, which when in the presence of an appropriate gut protease is released from the encoded binary and /or tertiary peptide, AM , LH , MN , ES , WQ , or DT. and provides insecticidal and / or nematicidal activity in the [ 0250 ] In related embodiments of the above , TPs useful in intended target insect or nematode. In various embodiments , the manufacture of a DNA construct or polynucleotide the TP can be a class of TPs commonly referred to as CRIP encoding : ( 1 ) an insecticidal and / or nematicidal protein , peptides , ICK TPs, non - ICK TPs , e . g . sea anemone peptides, which may include one or more ICK TPs , for example , a TP TMOF peptides and Bt proteins . In some embodiments , the selected from an “ ACTX ” or “ ACTX peptide” family of TP is a protein selected from the group consisting of a Pore insecticidal ICK peptides that have been isolated from an Forming Insecticidal Protein (PFIP ) and a Cysteine Rich Australian funnel- web spiders belonging to the Atracinae Insecticidal Protein (CRIP ) . The compositions and methods subfamily , for example , one or more TPs selected from an of the present invention can utilize any amino acid polymer Omega- ACTX TP , a Kappa- ACTX TP and an U - ACTX TP , based TP , including those described in International PCT for example , U + 2 - ACTX -Hvla , U - ACTX -Hvla , Application Serial No. PCT/ US2013 /030042 filed on Mar. 8 , rU - ACTX -Hvla , rU -ACTX -Hvlb or a TP having at least 2013 , the contents of the disclosure are incorporated by 80 % , at least 85 % , at least 90 % , at least 95 % , at least 96 % , reference herein in its entirety . at least 97 % , at least 98 % , at least 99 % , or at least 100 % [0253 ] The present invention also relates to nucleic acid sequence identity or sequence homology to a peptide or constructs comprising a polynucleotide encoding an insec protein having an amino acid sequence as set forth in SEO ticidal and / or nematicidal protein comprising at least one ID NOs : 5 , 6 and 1761 , 1762 , 1767, 1771 - 1773 , and variants cleavable linker and one TP. IN some embodiments , the thereof, (wherein the TPs, if more than one , may be the same nucleic acid construct can be a DNA nucleic acid construct or different) and each insecticidal and / or nematicidal protein or an RNA nucleic acid construct . In some embodiments of further comprising one or more tertiary peptides (designated the present invention , a nucleic acid construct can include at above as L ) , which may include the amino acid sequence of least one coding sequence operable to encode at least one a binary peptide selected from IFVRLR (SEQ ID NO : insecticidal and/ or nematicidal protein operably linked to 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID one or more control sequences that direct the expression of NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF the coding sequence in a suitable host cell under conditions ( SEQ ID NO : 1621) , fused in frame either on the N - termi compatible with the control sequences . The term “ operably nus , C - terminus or both termini of the binary peptide with a linked” means a configuration in which a control sequence Z region or spacer sequence selected from the group of or is placed at an appropriate position relative to the coding consisting of GS , WN , WQ, MA, MV, AM , LH , MN , ES , sequence of a polynucleotide such that the control sequence WO , or DT, wherein the TPs, if more than one , may be the directs expression of the coding sequence . same or different, and each insecticidal and / or nematicidal [ 0254 ] The polynucleotide may be manipulated in a vari protein further comprising one or more tertiary peptides ety of ways to provide for expression of an insecticidal (designated above as L ) , which may include the amino acid and /or nematicidal protein . Manipulation of the polynucle sequence of a binary peptide selected from IFVRLR (SEQ otide prior to its insertion into a vector may be desirable or ID NO : 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV necessary depending on the expression vector. The tech (SEQ ID NO : 1603 ), RRKAFV (SEQ ID NO : 1644 ), or niques for modifying polynucleotides utilizing recombinant LFAAPF ( SEQ ID NO : 1621 ) , fused in frame either on the DNA methods are well known in the art. N - terminus, C - terminus or both termini of the binary peptide [0255 ] The control sequence may be or include a pro with a Z region or spacer sequence selected from the group moter , a polynucleotide which is recognized by a host cell of or consisting of GS , WN , WQ , MA, MV , AM , LH , MN , for expression of the polynucleotide . The promoter contains ES , WQ, or DT. transcriptional control sequences that mediate the expres [ 0251 ] In various embodiments , the DNA construct of the sion of the insecticidal and /or nematicidal protein . The present invention encodes an insecticidal and/ or nematicidal promoter may be any polynucleotide that shows transcrip TP that contains one or more binary and / or tertiary peptides tional activity in the host cell including mutant, truncated , as described herein . The one or more binary and /or tertiary and hybrid promoters , and may be obtained from genes peptides are cleavable linking units that can be cleaved in encoding extracellular or intracellular polypeptides either both an animal gut environment, for example a human gut homologous or heterologous to the host cell. through the action of a human digestive protease , and also [0256 ]. In a yeast host, useful promoters are obtained from in the gut environment of an insect or nematode. The various the genes for Saccharomyces cerevisiae enolase (ENO - 1 ) , proteases available in the gut of an animal, for example a Saccharomyces cerevisiae galactokinase (GALI ) , Saccha human gut, and an insect or nematode are provided herein . romyces cerevisiae alcohol dehydrogenase /glyceraldehyde In some embodiments, the DNA construct of the present 3 - phosphate dehydrogenase (ADH1 , ADH2 /GAP ), Saccha invention encodes an insecticidal and /or nematicidal TP that romyces cerevisiae triose phosphate isomerase ( TPI) , contains one or more binary and / or tertiary peptides which Saccharomyces cerevisiae metallothionein (CUP1 ), and are cleavable in the gut of a Lepidoptera or a Coleoptera Saccharomyces cerevisiae 3 -phosphoglycerate kinase . Other insect species . The same DNA constructs which encode a useful promoters for yeast host cells are described by binary and/ or tertiary peptide which is cleavable in the gut Romanos et al ., 1992 , Yeast 8 :423 -488 . of a Lepidoptera or a Coleoptera insect species is also [0257 ] The control sequence may also be a transcription cleavable in a human gut via the action of a human digestive terminator, which is recognized by a host cell to terminate protease . transcription . The terminator sequence is operably linked to US 2018 /0362598 A1 Dec. 20 , 2018 39
the 3 '- terminus of the polynucleotide encoding the insecti expressed by inserting the polynucleotide or a nucleic acid cidal and /or nematicidal protein . Any terminator that is construct comprising the polynucleotide into an appropriate functional in the host cell may be used . Preferred terminators vector for expression . In creating the expression vector, the for bacterial host cells are obtained from the genes for coding sequence is located in the vector so that the coding Bacillus clausii alkaline protease (aprH ) , Bacillus licheni sequence is operably linked with the appropriate control formis alpha - amylase ( amyL ) , and Escherichia coli ribo sequences for expression . somal RNA ( rrnB ) . Preferred terminators for yeast host cells [0263 ] The recombinant expression vector may be any are obtained from the genes for Saccharomyces cerevisiae vector ( e . g . , a plasmid or virus ) that can be conveniently enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ) , subjected to recombinant DNA procedures and can bring and Saccharomyces cerevisiae glyceraldehyde - 3 - phosphate about expression of the polynucleotide. The choice of the dehydrogenase . Other useful terminators for yeast host cells vector will typically depend on the compatibility of the are described by Romanos et al. , 1992 , supra . The control vector with the host cell into which the vector is to be sequence may also be an mRNA stabilizer region down introduced . The vector may be a linear or closed circular stream of a promoter and upstream of the coding sequence plasmid . The vector may be an autonomously replicating of a gene which increases expression of the gene . vector , i . e . , a vector that exists as an extrachromosomal [0258 ] The control sequence may also include a leader , a entity , the replication of which is independent of chromo nontranslated region of an mRNA that is important for somal replication , e . g ., a plasmid , an extrachromosomal translation by the host cell . The leader sequence is operably element, a minichromosome , or an artificial chromosome. linked to the 5 ' - terminus of the polynucleotide encoding the The vector may contain any means for assuring self- repli insecticidal and / or nematicidal protein . Any leader that is cation . Alternatively, the vector may be one that , when functional in the host cell may be used . Suitable leaders for introduced into the host cell , is integrated into the genome, yeast host cells are obtained from the genes for Saccharo for example , a plant genome, and replicated together with myces cerevisiae enolase (ENO - 1 ) , Saccharomyces cerevi the chromosome( s ) into which it has been integrated . Fur siae 3 -phosphoglycerate kinase, Saccharomyces cerevisiae thermore , a single vector or plasmid or two or more vectors alpha - factor, and Saccharomyces cerevisiae alcohol dehy or plasmids that together contain the total DNA to be drogenase /glyceraldehyde - 3 -phosphate dehydrogenase introduced into the genome of the host cell , or a transposon , ( ADH2/ GAP ) . may be used . [0259 ] The control sequence may also include a polyade 0264 The vector preferably contains one or more select nylation sequence, a sequence operably linked to the 3 ' -ter able markers that permit easy selection of transformed , minus of the insecticidal and / or nematicidal protein - encod transfected , transduced , or the like cells . A selectable marker ing sequence and , when transcribed , is recognized by the is a gene the product of which provides for biocide or viral host cell as a signal to add polyadenosine residues to resistance, resistance to heavy metals , prototrophy to auxo transcribed mRNA . Any polyadenylation sequence that is trophs , and the like . Examples of bacterial selectable mark functional in the host cell may be used . Useful polyade ers are Bacillus licheniformis or Bacillus subtilis dal genes , nylation sequences for yeast host cells are described by Guo or markers that confer antibiotic resistance such as ampicil and Sherman , 1995 , Mol. Cellular Biol. 15 :5983 -5990 . lin , chloramphenicol, kanamycin , neomycin , spectinomycin [ 0260 ] It may also be desirable to add regulatory or tetracycline resistance . Suitable markers for yeast host sequences that regulate expression of the insecticidal and / or cells include , but are not limited to , ADE2 , HIS3 , LEU2 , nematicidal protein relative to the growth of the host cell . LYS2 , MET3, TRP 1 , and URA3 . Selectable markers for use Examples of regulatory systems are those that cause expres in a filamentous fungal host cell include, but are not limited sion of the gene to be turned on or off in response to a to , amds ( acetamidase ) , argB (ornithine carbamoyltrans chemical or physical stimulus, including the presence of a ferase ), bar (phosphinothricin acetyltransferase ), hph (hy regulatory compound . Regulatory systems in prokaryotic gromycin phosphotransferase ) , niaD ( nitrate reductase ) , systems include the lac , tac , and trp operator systems. In pyrG (orotidine - 5 '- phosphate decarboxylase ) , sC ( sulfate yeast , the ADH2 system or GAL1 system may be used . adenyltransferase ) , and trpC (anthranilate synthase ) , as well Other examples of regulatory sequences are those that allow as equivalents thereof. for gene amplification . In eukaryotic systems, these regula [0265 ] The vector preferably contains an element( s ) that tory sequences include the dihydrofolate reductase gene that permits integration of the vector into the host cell' s genome is amplified in the presence of methotrexate , and the met or autonomous replication of the vector in the cell indepen allothionein genes that are amplified with heavy metals . In dent of the genome. For integration into the host cell these cases , the polynucleotide encoding the insecticidal genome, the vector may rely on the polynucleotide' s and / or nematicidal would be operably linked with the regu sequence encoding the insecticidal and /or nematicidal pro latory sequence . tein or any other element of the vector for integration into [ 0261] Expression Vectors the genome by homologous or non -homologous recombi [ 0262] The present invention also relates to recombinant nation . Alternatively , the vector may contain additional expression vectors comprising a polynucleotide encoding an polynucleotides for directing integration by homologous insecticidal and / or nematicidal protein of the present inven recombination into the genome of the host cell at a precise tion , a promoter, and transcriptional and translational stop location ( s) in the chromosome( s ). To increase the likelihood signals . The various nucleotide and control sequences may of integration at a precise location , the integrational ele be joined together to produce a recombinant expression ments should contain a sufficient number of nucleic acids , vector that may include one or more convenient restriction such as 100 to 10 ,000 base pairs , 400 to 10 , 000 base pairs , sites to allow for insertion or substitution of the polynucle and 800 to 10 , 000 base pairs , which have a high degree of otide encoding the insecticidal and / or nematicidal protein at sequence identity to the corresponding target sequence to such sites . Alternatively , the polynucleotide may be enhance the probability of homologous recombination . The US 2018 /0362598 A1 Dec. 20 , 2018 40
integrational elements may be any sequence that is homolo constructs comprising a polynucleotide that encodes an gous with the target sequence in the genome of the host cell. insecticidal and / or nematicidal protein that may comprise Furthermore, the integrational elements may be non - encod one or more binary and /or tertiary peptides and one or more ing or encoding polynucleotides. On the other hand , the TPs. Each of the binary and /or tertiary peptides may be the vector may be integrated into the genome of the host cell by same or different and each of the TPs may be the same or non -homologous recombination . different. In some embodiments , the present invention pro [ 0266 ] For autonomous replication , the vector may further vides insecticidal and / or nematicidal proteins and DNA comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question . The constructs comprising a polynucleotide that encodes an origin of replication may be any plasmid replicator mediat insecticidal and / or nematicidal protein that may comprise ing autonomous replication that functions in a cell . The term two or more binary and / or tertiary peptides and two ormore " origin of replication ” or “ plasmid replicator” means a TPs . Each of the binary and / or tertiary peptides may be the polynucleotide that enables a plasmid or vector to replicate same or different and each of the TPs may be the same or in vivo . Examples of bacterial origins of replication are the different. In one illustrative embodiment, the present inven origins of replication of plasmids PBR322 , PUC 19 , PACYC tion provides a DNA construct comprising at least one 177 , and PACYC 184 permitting replication in E . coli, and polynucleotide operably linked to an operable promoter, for PUB110 , pE194 , pTA1060 , and PAM .beta . 1 permitting rep example , a homologous or heterologous promoter, wherein lication in Bacillus . Examples of origins of replication for the polynucleotide encodes an insecticidal and /or nemati use in a yeast host cell are the 2 micron origin of replication , cidal protein having one or more TPs and two or more ARS1 , ARS4 , the combination of ARS 1 and CEN3, and the cleavable binary or tertiary peptides . See FIG . 2 . combination of ARS4 and CEN6 . More than one copy of a [0271 ] In another related aspect, the present invention polynucleotide of the present invention may be inserted into provides an insecticidal and /or nematicidal protein , wherein a host cell to increase production of an insecticidal and/ or nematicidal protein . An increase in the copy number of the the insecticidal and /or nematicidal protein comprises an polynucleotide can be obtained by integrating at least one ERSP fused in frame with the N - terminus of a binary or additional copy of the sequence into the host cell genome or tertiary peptide , the binary or tertiary peptide is fused in by including an amplifiable selectable marker gene with the frame with a construct comprising (TP - L ) n or the insecti polynucleotide where cells containing amplified copies of cidal and /or nematicidal protein comprises an ERSP fused the selectable marker gene , and thereby additional copies of the N - terminus of a binary or tertiary peptide , the binary or the polynucleotide , can be selected for by cultivating the tertiary peptide is fused in framewith a construct comprising cells in the presence of the appropriate selectable agent. The (L - TP )n . procedures used to ligate the elements described above to [0272 ] In related embodiments , DNA constructs described construct the recombinant expression vectors of the present herein at least one TP is encoded in frame with at least one invention are well known to one skilled in the art (see , e . g ., binary or tertiary peptide , preferably two or more TPs are Sambrook et al ., 1989 , supra ). flanked in frame by at least one or at least two cleavable [ 0267] (4B ) . ERSP Fused to a Binary or Tertiary Peptide binary or tertiary peptides cleavable binary linker peptides Fused in Frame to a Construct ( TP - L ), ( L ) comprising an X region and a Y region are described [ 0268 ] In some embodiments , each insecticidal and / or above, and exemplary Z regions ( 1 - 4 amino acid sequences , nematicidal protein can include an ERSP fused to a binary for example , 2 amino acid sequences ) that may be fused in or tertiary peptide which is fused in frame to a construct frame to the X and /or Y regions (either to the N -terminal comprising : ( TP - L ) , wherein TP is a toxic protein , L is a sequence of an X or Y region , or the C -terminal sequence of binary or tertiary peptide and n is an integer ranging from 1 an X or Y region , described above form the tertiary cleav to 200 . See FIG . 4 . In some of these embodiments , the TPs able linking peptides . Illustrative Z or spacer amino acid in the insecticidal and / or nematicidal protein may be the sequences may include : AA , AF , AM , AN , AQ , AV , AW , AY, same or different. DA , DD , DE , DF, DG , DI, DL , DP, DS , DT, DV, DW , DY , 10269 ] All of these classes of TPs are described herein and EA , ED , EE , EF, EG , EI, EL , EP , ES , ET , EV , EW , EY, FA , all of these classes may be incorporated into the DNA FD , FE , FF , FI, FK , FL , FM , FN , FQ , FR , FS , FT, FV , FW , constructs described herein wherein at least one TP is FY ,GA , GD , GE , GF, GI, GL , GM , GN , GQ , GS ,GV , GW , encoded in frame with at least one binary or tertiary peptide , GY, HA , HD , HE , HF , HH , HI, HK , HL , HN , HP, HQ , HR , preferably two or more TPs are flanked in frame by at least HS , HT, HV, HY , IA . ID . IE . IG , IH , II , IK , IL , IM , IN . IP . one or at least two cleavable binary or tertiary peptides for IQ , IR , IS , IT , IV , IW , KA , KD , KE , KF, KI, KL , KN , KO , example : (ERSP ) - ( L ) - TP - ( L ) - TP, (ERSP ) - ( L ) - TP - ( L ) - TP KV, KY, LA , LD , LE , LG , LH , LI, LK , LL , LM , LN , LP , LO , ( L ), ( L - TP ) , -L , (L - TP - L )n , (ERSP ) -( TP - L ). , ( ERSP )- (L ) LR , LS , LT, LV , LW ,MA , MF, MG , MI, MK , ML , MM ,MN , ( TP - L ) n , ( ERSP ) - ( L - TP ) n , (ERSP ) - ( L - TP ) n - ( L ) , (ERSP ) MQ , MR , MS , MT, MV, MY, NA , ND , NE , NF, NI, NL , ( STA ) - ( L - TP ) , ( ERSP ) - (STA ) - ( L - TP ) - - ( L ), (ERSP ) NM , NN , NQ , NS, NT, NV, NY, QA , QD , QE , QF , QI, QL , (STA ) - ( TP - L ) n , or (ERSP ) - ( STA ) - ( L ) - ( TP - L )m , or ( STA ) - ( L QM , QN , QQ , QS , QT, QV , QY, RA , RD , RE , RF , RI, RL , TP )n , or (STA ) - ( L - TP ) n - ( L ), or (STA ) - ( TP - L ) n , or (STA ) RN , RO , RV , RY , SA , SF , SG , SK , SN , SP , SO , SR , SS , ST , ( L ) - ( TP - L ) n , wherein ( L ) is a binary peptide or a tertiary SV, SW , SY, TA , TF, TG, TK , TN, TP, TQ, TR , TS, TT, TV, peptide , and which each L may be the same or different, TP TW , TY , VA , VF, VM , VN , VO , VV , VW , VY , WA , WF, WI, is a toxic peptide , which may be the same or different, and WK , WL , WN , WP, WQ, WR , WS, WT, WV, WW , WY, YA , n is an integer ranging from 1 to 200 , preferably 1 to 100 , YD , YE , YF, YI, YK , YL , YM , YN , YQ , YR , YS , YT, YV , and most preferably , 1 to 10 . See FIGS. 4 and 5 . YW , or YY . In exemplary embodiments , individual tertiary [ 0270 ] In some embodiments , the present invention pro peptides of the present invention can include a two amino vides insecticidal and /or nematicidal proteins and DNA acid sequence , fused in frame to the N - terminus and / or the US 2018 /0362598 A1 Dec. 20 , 2018
C - terminus of an illustrative binary peptide disclosed above, (ERSP ) - ( TP - L ) n . (ERSP ) - ( L ) - ( TP - L ) n (ERSP ) - ( L - TP )m , wherein the two amino acid sequence is AM , LH , MN , ES , (ERSP ) - ( L - TP ) - ( L ) , (ERSP ) - (STA ) - ( L - TP )n (ERSP ) WQ , or DT. ( STA ) - ( L - TP ) n - ( L ) , ( ERSP ) - ( STA ) - ( TP - L )n , or ( ERSP ) [0273 ] In exemplary embodiments , individual tertiary (STA )- ( L ) - ( TP - L )n , or (STA ) -( L - TP )n , or (STA ) -( L - TP )n peptides of the present invention can include a two amino (L ) , or (STA )- ( TP -L )n , or (STA )- (L ) - ( TP - L )n , wherein ( L ) is acid sequence , fused in frame to the N -terminus and / or the a binary peptide or a tertiary peptide, and which each L may C - terminus of an illustrative binary peptide disclosed above , be the same or different, TP is a toxic peptide, which may be wherein the two amino acid sequence is GS, WN , WQ, MA the same or different, and n is an integer ranging from 1 to or MV. 200 , preferably 1 to 100 , and most preferably , 1 to 10 (See [0274 ] In some embodiments , the spacer portion of the for example exemplary insecticidal and /or nematicidal pro tertiary peptide ( Z region ) comprises the amino acid teins as shown in FIG . 4 , wherein n is an integer ranging sequence GS, which may be fused in frame to the N - termi from 1 to 200 , preferably 1 to 100 , and most preferably , 1 to nus or C - terminus of the binary peptide or both . 10 ) . In related embodiments , the insecticidal and /or nemati [0275 ] In various embodiments , exemplary binary pep cidal protein may contain TPs that are the same or different, tides for use in the insecticidal and /or nematicidal polypep and binary and / or tertiary peptides that are the same or tides and proteins , DNA constructs and polynucleotides different. In related embodiments of the above , tertiary encoding an insecticidal and / or nematicidal polypeptide and peptides (designated above as L ) , may include the amino protein and incorporated into a transgenic plants or part acid sequence of a binary peptide selected from IFVRLR thereof, of the present invention , may include one or more ( SEQ ID NO : 1619 ) , ALKLFV ( SEQ ID NO : 1605 ) , ALK cleavable tertiary peptides selected from the group of or FLV (SEQ ID NO : 1603 ) , RRKAFV ( SEQ ID NO : 1644 ) , or consisting of: a binary peptide selected from IFVRLR ( SEQ LFAAPF (SEQ ID NO : 1621 ) , fused in frame either on the ID NO : 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV N -terminus , C - terminus or both termini of the binary peptide (SEQ ID NO : 1603 ), RRKAFV ( SEQ ID NO : 1644 ) , or with a Z region or spacer sequence selected from the group LFAAPF (SEQ ID NO : 1621) , fused in frame either on the of or consisting of GS , WN , WQ , MA , MV, AM , LH , MN , N - terminus , C - terminus or both termini with a Z region or ES , WQ , or DT. In another embodiment , a plant , or part spacer sequence selected from the group of or consisting of thereof comprises a polynucleotide operably linked to an GS, WN , WQ, MA, MV, AM , LH , MN , ES , WQ , or DT. In operable promoter , for example , a homologous or heterolo embodiments related to the above, the insecticidal and/ or gous promoter, wherein the polynucleotide encodes an nematicidal protein may contain TPs that are the same or insecticidal and /or nematicidal protein comprising an ERSP different, and the binary or tertiary peptides may be the same fused in frame with the N - terminus of a stabilizing domain or different. In related embodiments of the above , tertiary (STA ) . See FIGS. 5 and 6C . peptides ( designated above as L ) , may include the amino 10277 ] The STA can be fused in frame with either the acid sequence of a binary peptide selected from IFVRLR N - terminus of a binary or tertiary peptide , or the N - terminus (SEQ ID NO : 1619 ), ALKLFV (SEQ ID NO : 1605 ) , ALK of a construct comprising : ( L - TP ) n . ( L - TP ), - L , ( TP - L ) n , or FLV (SEQ ID NO : 1603) , RRKAFV (SEQ ID NO : 1644 ), or L - (TP - L )n . LFAAPF (SEQ ID NO : 1621 ), fused in frame either on the [0278 ] In various embodiments , an exemplary insecticidal N - terminus , C - terminus or both termini of the binary peptide and /or nematicidal protein , and /or the DNA construct com with a Z region or spacer sequence selected from the group prising one or more polynucleotides each or in combination of or consisting of GS, WN , WQ , MA , MV , AM , LH , MN , encoding such insecticidal and / or nematicidal proteins can ES , WQ, or DT. See FIG . 4 . In related embodiments of the include the construct: (ERSP ) -( STA )- (L - TP )n , or (ERSP ) above , TPs useful in the manufacture of an insecticidal (STA ) - ( L - TP ) - ( L ) , or ( ERSP ) - (STA ) - ( TP - L ) n , or ( ERSP ) and /or nematicidal protein , which may include one or more (STA ) - ( L ) - ( TP - L ) n , or ( STA ) - ( L - TP ) n , or (STA ) - ( L - TP ) . ICK TPs, for example , a TP selected from an “ ACTX ” or (L ) , or (STA ) - ( TP - L ) . , or (STA )- ( L ) - ( TP - L ) . , wherein TP is “ ACTX peptide” family of insecticidal ICK peptides that toxic protein , L is a binary or tertiary peptide and n is an have been isolated from an Australian funnel- web spiders integer ranging from 1 to 200 , preferably an integer ranging belonging to the Atracinae subfamily , for example , one or from 1 to 100 , and more preferably an integer ranging from more TPs selected from an Omega - ACTX TP , a Kappa 1 to 10 . In some embodiments , the insecticidal and / or ACTX TP and an U - ACTX TP , for example , U + 2 - ACTX nematicidal protein may contain TPs that are the same or Hvla , U - ACTX -Hvla , rU - ACTX -Hvla , rU - ACTX -Hvlb different, and binary and / or tertiary peptides may be the (wherein the TPs , if more than one , may be the same or same or different. In some embodiments, the insecticidal different) and each insecticidal and / or nematicidal protein and /or nematicidal protein has the C - terminal TP fused with further comprising one or more tertiary peptides (designated or unfused at its C -terminus with a binary or tertiary peptide . above as L ) , which may include the amino acid sequence of In related embodiments of the above , TPs useful in the a binary peptide selected from IFVRLR (SEQ ID NO : manufacture of a DNA construct or polynucleotide encod 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID ing: ( 1 ) an insecticidal and /or nematicidal protein , which NO : 1603 ) , RRKAFV (SEO ID NO : 1644 ) , or LFAAPF may include one or more ICK TPs, for example , a TP ( SEQ ID NO : 1621) , fused in frame either on the N - termi selected from an " ACTX ” or “ ACTX peptide ” family of nus, C - terminus or both termini of the binary peptide with a insecticidal ICK peptides that have been isolated from an Z region or spacer sequence selected from the group of or Australian funnel -web spiders belonging to the Atracinae consisting of GS, WN , WQ , MA , MV, AM , LH , MN , ES , subfamily , for example , one or more TPs selected from an WQ, or DT. Omega -ACTX TP, a Kappa - ACTX TP and an U -ACTX TP , [ 0276 In various embodiments , an exemplary insecticidal for example, U + 2 - ACTX -Hvla , U -ACTX -Hvla , and / or nematicidal protein can include: (ERSP ) - ( L ) - TP - ( L ) rU -ACTX -Hvla , rU -ACTX -Hvlb , or a TP having at least TP, (ERSP ) - ( L )- TP - ( L )- TP -( L ) , ( L - TP ) . - L , ( L - TP - L ) n 80 % , at least 85 % , at least 90 % , at least 95 % , at least 96 % , US 2018 /0362598 A1 Dec. 20 , 2018 at least 97 % , at least 98 % , at least 99 % , or at least 100 % binary peptide has 2 different regions and the tertiary peptide sequence identity or sequence homology to a peptide or has 3 different regions ; wherein , the binary and / or the protein having an amino acid sequence as set forth in SEO tertiary peptide can be cleaved by both an animal gut ID NOs: 5 , 6 and 1761 , 1762 , 1767, 1771 - 1773 , and variants protease , ( for example a human gut protease ) and an insect thereof, (wherein the TPs , if more than one , may be the same or nematode gut protease, and in some embodiments , the or different ) and each insecticidal and/ or nematicidal protein binary and / or the tertiary peptide is operably fused to the further comprising one ormore tertiary peptides (designated C - terminus of an Endoplasmic Reticulum Signal Peptide above as L ) , which may include the amino acid sequence of (ERSP ) and operably fused to the N - terminus of a TP . In a binary peptide selected from IFVRLR (SEQ ID NO : some of these embodiments , exemplary tertiary peptides 1619 ), ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEO ID (designated as L ), may include the amino acid sequence of NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ), or LFAAPF a binary peptide selected from IFVRLR (SEQ ID NO : (SEQ ID NO : 1621) , fused in frame either on the N - termi 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID nus, C - terminus or both termini of the binary peptide with a NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF Z region or spacer sequence selected from the group of or (SEQ ID NO : 1621) , fused in frame either on the N - termi consisting of GS , WN , WQ, MA, MV , AM , LH , MN, ES , nus , C -terminus or both termini of the binary peptide with a WQ , or DT. Z region or spacer sequence selected from the group of or [0279 ] DNA constructs encoding a cleavable linking pep consisting of GS , WN , WQ , MA , MV, AM , LH , MN , ES , tide operatively fused to a TP can be constructed with a WQ , or DT. variety of host cell specific regulatory components . In some [0282 ] In some embodiments , the insecticidal and / or nem embodiments , the DNA constructs may be transformed into aticidal compositions comprising cleavable linking peptides a variety of host cells for expression , synthesis of encoded described herein are intended for the dissemination or con proteins or for genetic manipulation . In some embodiments , sumption of TPs into or by insect and /or nematode pests that the host cell can be a bacterial cell, a yeast cell , a plant cell , ingest or otherwise come into contact with the insecticidal or an animal cell, for example , an Agrobacterium cell , a and /or nematicidal compositions. The present invention pro Bacillus cell , an Escherichia cell, a Salmonella cell, a vides a plant, or part thereof, (a “ plant or part thereof” , is Pseudomonas cell , and a Rhizobium cell . In various embodi defined herein as including , without limitation , a plant and ments , the host cell is a plant cell . the plant tissue , a plant cell, or a plant seed of said plant ) [ 0280 ] In some embodiments , the insecticidal and / or nem comprising an insecticidal and /or nematicidal protein or aticidal compositions comprising cleavable linking peptides polypeptide comprising either a binary or a tertiary peptide , described herein are intended for the dissemination of TPS the binary and / or the tertiary peptide containing at least 4 into insect and /or nematode pests that ingest or otherwise amino acids and up to a maximum of 16 amino acids, and come into contact with the insecticidal and / or nematicidal is comprised of 2 or 3 different regions , wherein the binary compositions. The present invention provides a plant , or part peptide has 2 different regions and the tertiary peptide has 3 thereof, ( a " plant or part thereof , is defined herein as different regions; wherein , the binary and / or the tertiary including , without limitation , a plant and the plant ' s tissue peptide can be cleaved by both an animal gut protease and ( for example , a plant' s roots , stem , leaves, flowers or parts an insect or nematode gut protease , and in some embodi thereof) , a plant cell , or a plant seed of said plant) compris ments , the binary and/ or the tertiary peptide is operably ing : an insecticidal and / or nematicidal protein or polypep fused to the C - terminus of an Endoplasmic Reticulum Signal tide comprising either a binary or a tertiary peptide , the Peptide (ERSP ) and operably fused to the N - terminus of a binary and / or the tertiary peptide containing at least 4 amino TP. acids and up to a maximum of 16 amino acids , and is [0283 ] In a related embodiment, the plant or part thereof, comprised of 2 or 3 different regions, wherein the binary comprises a polynucleotide that encodes an insecticidal peptide has 2 different regions and the tertiary peptide has 3 and /or nematicidal protein , wherein the insecticidal and /or different regions ; wherein , the binary and /or the tertiary nematicidal protein comprises an ERSP fused in frame with peptide can be cleaved by both an animal gut protease and the N - terminus of a binary or tertiary peptide , the binary or an insect or nematode gut protease, and in some embodi tertiary peptide is fused in frame with a construct comprising ments , the binary and /or the tertiary peptide is operably ( TP - L ) n or the insecticidal and / or nematicidal protein com fused to the C -terminus of an Endoplasmic Reticulum Signal prises an ERSP fused in frame with a binary peptide and /or Peptide (ERSP ) and operably fused to the N -terminus of a a repeat construct ( L - TP ) , or ( TP - L ) n . See FIG . 4 . In various TP . embodiments , the exemplary insecticidal and / or nematicidal [ 0281 ] In some embodiments , the insecticidal and /or nem protein can include a construct from N - terminus to C - ter aticidal compositions comprising cleavable linking peptides minus: ( ERSP ) - ( L ) - TP - ( L ) - TP , (ERSP ) - ( L ) - TP - ( L ) - TP - ( L ) , described herein are intended for the dissemination or con ( L - TP ) n - L , ( L - TP - L ) n , (ERSP ) - ( TP - L ) n , ( ERSP ) - ( L ) - ( TP - L ) sumption of TPs into or by insect and /or nematode pests that no ( ERSP ) - ( L - TP ) n . ( ERSP ) - ( L - TP ). - ( L ) , (ERSP ) - (STA )- ( L ingest or otherwise come into contact with the insecticidal TP ) n , (ERSP ) -( STA )- (L - TP )n - (L ), (ERSP )- (STA )- ( TP -L ) n , and / or nematicidal compositions. The present invention pro or (ERSP ) - (STA ) - ( L ) - ( TP - L ) , or (STA ) - ( L - TP ) , or (STA ) vides a plant , or part thereof, ( a " plant or part thereof " , is ( L - TP ) n - ( L ) , or ( STA ) - ( TP - L ) n , or (STA ) - ( L ) - ( TP - L ) n , defined herein as including, without limitation , a plant and wherein ( L ) is a binary peptide or a tertiary peptide , and the plant tissue, a plant cell , or a plant seed of said plant) which each L may be the same or different, TP is a toxic comprising an insecticidal and /or nematicidal protein or peptide , which may be the same or different, and n is an polypeptide comprising one or more binary and /or tertiary integer ranging from 1 to 200 , preferably 1 to 100 , and most peptides, the binary and / or the tertiary peptide containing at preferably , 1 to 10 . In some of these embodiments , exem least 4 amino acids and up to a maximum of 16 amino acids, plary tertiary peptides (designated as L ), may include the and is comprised of 2 or 3 different regions, wherein the amino acid sequence of a binary peptide selected from US 2018 /0362598 A1 Dec. 20 , 2018 43
IFVRLR (SEQ ID NO : 1619 ), ALKLFV (SEQ ID NO : each insecticidal and /or nematicidal protein further compris 1605 ) , ALKFLV (SEQ ID NO : 1603 ) , RRKAFV (SEQ ID ing one or more tertiary peptides (designated above as L ) , NO : 1644 ), or LFAAPF ( SEQ ID NO : 1621) , fused in frame which may include the amino acid sequence of a binary either on the N - terminus, C - terminus or both termini of the peptide selected from IFVRLR ( SEQ ID NO : 1619 ) , binary peptide with a Z region or spacer sequence selected ALKLFV ( SEQ ID NO : 1605 ) , ALKFLV ( SEO ID NO : from the group of or consisting ofGS , WN , WQ ,MA , MV, 1603 ) , RRKAFV (SEQ ID NO : 1644 ), or LFAAPF (SEQ ID AM , LH , MN , ES , WQ , or DT. In related embodiments of NO : 1621 ) , fused in frame either on the N - terminus, C - ter the above , TPs useful in the manufacture of a DNA construct minus or both termini of the binary peptide with a Z region or polynucleotide encoding : ( 1 ) an insecticidal and /or nem or spacer sequence selected from the group of or consisting aticidal protein , which may include one or more ICK TPs, of GS , WN , WQ, MA , MV, AM , LH , MN , ES , WQ , or DT. for example , a TP selected from an “ ACTX ” or “ ACTX [0285 ] In some embodiments , the C - terminal TP is fused peptide” family of insecticidal ICK peptides that have been or unfused at its C - terminus with a binary or tertiary peptide . isolated from an Australian funnel- web spiders belonging to See FIGS . 2 , 4 and 5 . In some of these embodiments , the Atracinae subfamily , for example , one or more TPS exemplary tertiary peptides (designated as L ), may include selected from an Omega - ACTX TP, a Kappa -ACTX TP and the amino acid sequence of a binary peptide selected from an U -ACTX TP , for example , U + 2 - ACTX -Hvla , U - ACTX IFVRLR (SEQ ID NO : 1619 ) , ALKLFV (SEQ ID NO : Hvla , rU -ACTX -Hvla , rU - ACTX - Hvlb or a TP having at 1605 ) , ALKFLV (SEQ ID NO : 1603 ) , RRKAFV (SEQ ID least 80 % , at least 85 % , at least 90 % , at least 95 % , at least NO : 1644 ), or LFAAPF ( SEQ ID NO : 1621 ) , fused in frame 96 % , at least 97 % , at least 98 % , at least 99 % , or at least either on the N -terminus , C - terminus or both termini of the 100 % sequence identity or sequence homology to a peptide binary peptide with a Z region or spacer sequence selected or protein having an amino acid sequence as set forth in SEO from the group of or consisting of GS , WN , WQ , MA, MV, ID NOs: 5 , 6 and 1761, 1762 , 1767 , 1771 - 1773 , and variants AM , LH , MN , ES , WQ , or DT. thereof, (wherein the TPs , if more than one, may be the same [0286 ] It should be appreciated by the reader that in or different) and each insecticidal and / or nematicidal protein practice a TP - Linker construct ( the Linker being defined as further comprising one or more tertiary peptides (designated including a cleavable binary or tertiary peptide ) that can be above as L ) , which may include the amino acid sequence of written as ( TP - L ) , or the variations shown in FIG . 2 could a binary peptide selected from IFVRLR (SEQ ID NO : also be written out in long form . For example , when n is 5 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID then ( TP - L ) , written out in long form would appear as : NO : 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF TP - L - TP - L - TP -L - TP -L - TP - L . After a construct like this is ( SEQ ID NO : 1621) , fused in frame either on the N - termi made and produced in a system such as a plant, or part nus , C - terminus or both termini of the binary peptide with a thereof, the L or Linkers will be split apart by insect and / or Z region or spacer sequence selected from the group of or nematode proteases in the gut or hemolymph environment of consisting of GS, WN , WQ , MA , MV , AM , LH , MN , ES , the insect and /or nematode to allow large numbers single WQ , or DT. toxic peptides to be released and exert their toxic effects , [ 0284 ] In another embodiment, a plant, or part thereof killing or immobilizing or slowing the feeding of the insects comprises a polynucleotide operably linked to an operable and /or nematodes . When this happens , depending on various promoter, for example , a homologous or heterologous pro circumstances, the L or Linker can be cleaved between moter , wherein the polynucleotide encodes an insecticidal different amino acids in the linker or between the Toxic and / or nematicidal protein comprising an ERSP fused in Peptide and the linker such that on either side of the TP or frame with the N - terminus of a stabilizing domain (STA ) . Toxic Peptide there can be from 0 to 16 amino acids still See FIG . 5 . The STA is fused in frame with either the attached to the toxic peptide remaining from the Lor Linker N - terminus of a binary or tertiary peptide , or the N - terminus that had an initial length of from 1 - 16 amino acids to start of a construct comprising : ( L - TP ) n , ( L - TP ), - L , ( TP - L ) n , or with . Said another way , post protease cleavage , the TP could L - ( TP - L ) n . In various embodiments , the exemplary insecti either have no amino acids from the Linker attached to it , or cidal and /or nematicidal protein can include : (ERSP )- (STA ) it could have one or two “ stubs ” aka " nubbins ” ( one or both ( L - TP ) n , or (ERSP ) - ( STA ) - ( L - TP ), , - ( L ) , or ( ERSP ) - (STA ) stubs fused to the N - terminus and /or the C - terminus) made ( TP - L )n , or ( ERSP ) -( STA )- (L ) - ( TP - L ) n , or (STA )- ( L - TP ) n, of remnants of the Linker, that vary in size from one to 16 or (STA )- ( L - TP ) n - ( L ) , or (STA ) - ( TP - L ) n , or (STA ) - ( L ) - ( TP amino acids , but more often from 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , L )n , wherein TP is toxic protein as described herein , L is a 11 , 12 , 13 or 14 amino acids . The stubs can be on the binary or tertiary peptide and n is an integer ranging from 1 N - terminal, the C - terminal, or both the N -terminal and the to 200 , preferably an integer ranging from 1 to 100 , and C -terminals . These stubs are not always the same size , it more preferably an integer ranging from 1 to 10 . In some depends on how they are cleaved by the proteases in the embodiments , the insecticidal and / or nematicidal protein insect and / or human gut environment and this can vary having a construct as shown above may contain TPs that are depending on the protease and the Linker used . Normally the same or different, and binary and / or tertiary peptides that there will be stubs attached to both “ sides ” of the Toxic are the same or different. In some eof these embodiments , Peptides with both stubs originating from different parts of the insecticidal and /or nematicidal protein having a con the Linking Peptide. Sometimes there are no stubs or only 1 struct as shown above may contain a TP having at least 80 % , stub attached to the TP after cleavage . at least 85 % , at least 90 % , at least 95 % , at least 96 % , at least [0287 ] The stubs can range in size from 1 - 16 amino acids , 97 % , at least 98 % , at least 99 % , or at least 100 % sequence (and there may be no stubs ) depending on the length of the identity or sequence homology to a peptide or protein having starting Linker, more often the stubs will be from 2 - 14 an amino acid sequence as set forth in SEQ ID NOs: 5 , 6 and amino acids, from 3 - 12 , from 4 - 8 , or from 5 - 7 amino acids 1761, 1762 , 1767 , 1771- 1773 , and variants thereof, (wherein or but they could be any of the following lengths of amino the TPs, if more than one , may be the same or different) and acids: from : 1 - 3 , 1 -4 , 1- 5 , 1 -6 , 1 - 7 , 1- 8 , 1 -9 , 10 , 1 - 11 , 2 -3 , US 2018 /0362598 A1 Dec. 20 , 2018 44
2 -4 , 2 -5 , 2 -6 , 2 -7 , 2 - 8 , 2 - 9 , 2 -10 , and 2 - 11, or 3 - 4 , 3 -5 , 3 - 6 , [0292 ] The present invention provides insecticidal and /or 3 - 7 , 3 - 8 , 3 - 9 , 3 - 10 , and 3 - 11, or 4 - 5 , 4 - 6 , 4 - 7 , 4 - 8 , 4 - 9 , 4 - 10 nematicidal proteins, such that when the insecticidal and /or and 4 - 11 ; or 5 - 6 , 5 - 7 , 5 - 8 , 5 - 9 , 5 - 10 and 5 - 11 ; or 6 - 7 , 6 - 8 , nematicidal protein is ingested by an insect and/ or a nema 6 - 9 , 6 - 10 and 6 - 11; or 7 - 8 , 7 - 9 , 7 - 10 , and 7 - 11; and 8 - 9 , tode , the gut environment proteases of the insect and / or 8 - 10 , or 8 - 11, and 9 - 10 or 9 - 11, or 10 or 11 amino acids in nematode cleaves the binary and / or tertiary peptide thereby length . liberating the fused TPs and is killed , immobilized , rendered [0288 ] Thus, when , before cleavage , the construct might unable to function normally or slowing the feeding or appear as TP - L - TP - L - TP - L - TP - L - TP - L , ( our example growth of the insects and/ or nematodes contacted with the above , and shown in equation form in FIG . 2 ) where , for insecticidal and /or nematicidal protein . In certain embodi example, if the Linker has 6 aa (or amino acids ) , then after ments, the selection of TPs to be incorporated into the protease cleavage , the TP - L - TP - L - TP - L - TP - L - TP - L con insecticidal and /or nematicidal protein can be made to target struct is broken up into individual Toxic Peptides ( TPs ) with a specific insect or nematode pest, or for expression in a stubs , which might appear as follows: the first peptide could specific plant or specific conditions experienced by the be TP - 2aa ( 2aa from one side of the 6 aa Linker ) and then plant . In some embodiments , an insecticidal and / or nemati 4aa from the last part of the Linker attached to the other end cidal protein which is produced by a transgenic plant, plant of the toxic peptide as in : 4aa - TP - 2aa , and a third peptide tissue, or plant seed or a composition comprising one or 4aa - TP - 2aa , and a fourth 4aa - TP - 2aa and then the fifth and more insecticidal and /or nematicidal proteins having the last cleaved TP would appear as 4aa - TP -6aa , because the last same or different TPs formulated for direct application to a Linker may not be cleaved . Sometimes cleavage does not plant, has TPs comprised of a combination of ICK TPs and always take place because the last TP of the construct is not Bt TPs . The transgenic plant, plant tissue , or plant seed can attached to another TP . Or it might be cleaved leaving a express an insecticidal protein , or one or more plynucle 4aa - TP - 2aa . These bits of aa fragments attached to the toxic otides encoding an insecticidal protein comprising one or peptides following peptidase cleavage are what we call more ICK TPs derived from any species of Australian " stubs” and they do not typically affect or inhibit the activity Funnel -web spider, or the Australian Funnel- web spiders of of the cleaved toxic peptides produced from cleavage in the genus Atrax or Hadronyche , and the Australian Blue Moun insects ' gut . tains Funnel- web , Hadronyche versuta . In some embodi 10289 ) Stubs may also appear when an ERSP is fused to ments , the present invention provides a transgenic plant, or the ( TP - L ) n or ( L - TP ) , as in FIG . 4 and in such a construct part thereof, (for example , a plant tissue , a plant cell , or a the stub could include pieces of the ERSP , and or in FIG . 5 plant seed ) wherein the transgenic plant or part thereof where the L - TP is further fused to a STA or BAAS , or contains an insecticidal protein comprising a construct ( from Ubiquitin , whatever is used in the construct. The stubs could N - terminal to C -terminal ) (ERSP ) - ( L ) - TP - ( L ) - TP, (ERSP ) be any combination of any length of peptide of 1 amino acid ( L ) - TP - ( L ) - TP - ( L ), ( L - TP ) - L , ( L - TP - L )n , (ERSP ) - ( TP - L )m , or greater up to 16 amino acids in length . They may be (ERSP ) - ( L ) - ( TP - L ) n , (ERSP ) - ( L - TP ) n , ( ERSP ) - ( L - TP ) n irregularly attached to the TP . Stubs may not have the same ( L ), (ERSP )- (STA )- ( L - TP ) n , (ERSP )- ( STA )- ( L - TP )n -( L ), length or number of amino acids before and after the Toxic (ERSP ) - (STA )- ( TP -L ) n , or ( ERSP ) -( STA )- (L ) - ( TP - L ) n , or Peptide. (STA ) - ( L - TP )n , or (STA )- ( L - TP ) n - ( L ) , or (STA ) - ( TP - L ) n , or [0290 ] In various embodiments , a host cell is provided that (STA ) - ( L ) - ( TP - L )n , wherein ( L ) is a binary peptide or a has been transformed with a DNA construct described herein tertiary peptide , and which each L may be the same or that is operable to express an insecticidal and / or nematicidal different, TP is a toxic peptide, which may be the same or protein of the present invention . In various embodiments , different, and n is an integer ranging from 1 to 200 , prefer the host cell may be a eukaryotic cell , for example , a yeast ably 1 to 100 , and most preferably , 1 to 10 . cell, for example , a yeast cell from the genus Saccharomy [0293 ] In related embodiments , the transgenic plant or part ces, Pichia , Hansenula , Kluyveromyces, Schizosaccharomy thereof may express an insecticidal and /or nematicidal pro ces, Schwanniomyces , Yarrowia , Aspergillus ; plant cells ; tein that may contain TPs that are the same or different, and and mammalian cells. binary and / or tertiary peptides that are the same or different . [0291 ] In various embodiments , the intended host cell that In related embodiments, the transgenic plant, or part thereof, is transformed ( transiently , or stably ) with the DNA con ( for example , a plant tissue , a plant cell , or a plant seed ) structs encoding an insecticidal and /or nematicidal protein contains one or more polynucleotides each or in combina comprising a cleavable linking peptide operatively fused to tion , operable to express an insecticidal protein comprising a TP are plant cells . In some embodiments , the plant cells a construct, comprising , selected from the group consisting that may be transformed with the DNA constructs described of, or consisting of : ( from N - terminal to C - terminal) herein may include but not limited to : alfalfa , banana , barley, (ERSP ) - ( L ) - TP - ( L ) - TP , (ERSP )- ( L ) - TP - ( L ) - TP - ( L ), ( L - TP ) bean , broccoli , cabbage , canola , carrot, cassava , castor, n - L , ( L - TP - L ) n (ERSP ) - ( TP - L ) n , ( ERSP ) - ( L ) - ( TP - L )m , cauliflower , celery , chickpea , Chinese cabbage , citrus , coco (ERSP )- (L - TP )n . (ERSP ) -( L - TP )n - (L ) , (ERSP )- (STA )- ( L nut, coffee , corn , clover , cotton , a cucurbit , cucumber, TP ) n , (ERSP ) - (STA ) - ( L - TP ) n - ( L ) , (ERSP ) - ( STA ) - ( TP - L )n , Douglas fir , eggplant , eucalyptus, flax , garlic , grape , hops , (ERSP ) - (STA ) - ( L ) - ( TP - L ) n , ( STA ) -( L - TP )n , (STA ) - ( L - TP ) leek , lettuce , Loblolly pine , millets , melons, nut, oat, olive , n - (L ), (STA )- ( TP -L )n , (STA )- ( L ) -( TP - L )n , wherein (L ) is a onion , ornamental, palm , pasture grass , pea, peanut , pepper, binary peptide or a tertiary peptide, and which each L may pigeon pea , pine , potato , poplar, pumpkin , Radiata pine , be the same or different, TP is a toxic peptide , which may be radish , rapeseed , rice , rootstocks, rye , safflower, shrub , the same or different, and n is an integer ranging from 1 to sorghum , Southern pine , soybean , spinach , squash , straw 200 , preferably 1 to 100 , and most preferably , 1 to 10 . In berry , sugar beet , sugarcane , sunflower, sweet corn , sweet related embodiments of the above , TPs useful in the manu gum , sweet potato , switchgrass, tea , tobacco , tomato , triti facture of a transgenic plant or part thereof, comprising a cale , turf grass , watermelon , and a wheat plant cell . DNA construct or polynucleotide encoding : ( 1 ) an insecti US 2018 /0362598 A1 Dec. 20 , 2018 45 cidal and / or nematicidal protein , which may include one or 90 : 10 , 85 : 15 , 80 :20 , 75 :25 , 70 : 30 , 65 : 35 , 60 :40 , 55 :45 , more ICK TPs , for example , a TP selected from an “ ACTX ” 50 :50 , 45 :55 , 40 :60 , 35 :65 , 30 : 70 , 25 :75 , 20 :80 , 15 :85 , or “ ACTX peptide” family of insecticidal ICK peptides that 10 : 90 , 5 :95 and 1 :99 , or any combination of any two of these have been isolated from an Australian funnel -web spiders values. The ratio of Bt TP to ICK TP , on a dry weight basis belonging to the Atracinae subfamily , for example , one or thus released , can be selected from about the following more TPs selected from an Omega -ACTX TP, a Kappa ratios: 50 : 50 , 45 : 55 , 40 :60 , 35 :65 , 30 : 70 , 25 : 75 , 20 : 80 , ACTX TP and an U - ACTX TP , for example , U + 2 - ACTX 15 : 85 , 10 : 90 , 5 : 95 , 1 : 99 , 0 . 5 : 99 . 5 , 0 . 1 : 99 . 9 and 0 .01 : 99 . 99 Hvla , U -ACTX -Hvla , rU -ACTX -Hvla , rU -ACTX -Hvlb or one or more TPs having at least 80 % , at least 85 % , at least or any combination of any two of these values. The formu 90 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , lation may comprise the insecticidal and /or nematicidal at least 99 % , or at least 100 % sequence identity or sequence protein and appropriate excipients, or the insecticidal and /or homology to a peptide or protein having an amino acid nematicidal protein may be expressed by a culture of one or sequence as set forth in SEQ ID NOs: 5 , 6 and 1761, 1762 , more bacterial strains, that when formulated and applied to 1767 , 1771 - 1773 , and variants thereof, (wherein the TPs, if the plant, the bacterial strains produce the insecticidal and /or more than one, may be the same or different ) and each nematicidal protein which is subsequently ingested by the insecticidal and / or nematicidal protein further comprising insect and / or nematode pest. In various embodiments , the one or more tertiary peptides (designated above as L ) , which total concentration of Bt TP and ICK TP in the composition may include the amino acid sequence of a binary peptide is selected from the following percent concentrations : 1 , 5 , selected from IFVRLR (SEQ ID NO : 1619 ) , ALKLFV (SEQ 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75, 80 , 85 , ID NO : 1605 ) , ALKFLV (SEQ ID NO : 1603 ), RRKAFV 90 , 95 or 99 % , or any range between any two of these (SEQ ID NO : 1644 ), or LFAAPF (SEQ ID NO : 1621 ), fused values , and the remaining percentage of the composition is in frame either on the N - terminus, C - terminus or both comprised of excipients . Any of the insecticidal and /or termini of the binary peptide with a Z region or spacer nematicidal proteins and plants described herein can be used sequence selected from the group of or consisting of GS , to control insects and /or nematodes , their growth and dam WN , WQ, MA, MV , AM , LH , MN , ES , WQ , or DT. age , especially their damage to plants . In some embodi [0294 ] In related embodiments , the one or more poly ments , insecticidal and / or nematicidal proteins comprising nucleotides are provided in an expression vector, operable to the combination of Bt TP and ICK TP can be applied by express an insecticidal and /or nematicidal protein as being sprayed on a plant, or the insect' s locus , or the locus described above and herein , and is under the control of a of a plant in need of protecting . constitutive or inducible promoter , wherein the expression [ 0296 ] In some embodiments , illustrative methods for vector is transiently expressed in a host cell , for example , a controlling a pest infection comprises preparing a formula plant cell, or wherein the polynucleotide operable to encode tion comprising : an insecticidal and /or nematicidal protein the insecticidal protein is stably integrated into the plant ' s , comprising a combination of a Bt TP ; and an ICK TP, which or part thereof nuclear DNA . In related embodiments of the can include any of the compositions described herein or above , TPs useful in the manufacture of a DNA construct or capable of being made by one skilled in the art given this polynucleotide encoding : (1 ) an insecticidal and /or nemati disclosure . Some of the described formulations include the cidal protein , which may include one or more ICK TPs, for use of a polar aprotic solvent, and or water, and or where the example, a TP selected from an “ ACTX ” or “ ACTX pep polar aprotic solvent is present in an amount of 1 - 99 wt % , tide” family of insecticidal ICK peptides that have been the polar protic solvent is present in an amount of 1 - 99 wt isolated from an Australian funnel -web spiders belonging to % , and the water is present in an amount of 0 - 98 wt % . The the Atracinae subfamily , for example , one or more TPs formulations include formulations where the Bt TP is Dipel selected from an Omega - ACTX TP , a Kappa - ACTX TP and and where the ICK TP is a hybrid - ACTX -Hvla peptide, or an U - ACTX TP , for example , U + 2 - ACTX - Hvla , U - ACTX a TP having at least 80 % , at least 85 % , at least 90 % , at least Hvla , rU - ACTX -Hvla , rU - ACTX - Hv1b (wherein the TPs , 95 % , at least 96 % , at least 97 % , at least 98 % , at least 99 % , if more than one , may be the same or different) and each or at least 100 % sequence identity or sequence homology to insecticidal and /or nematicidal protein further comprising a peptide or protein having an amino acid sequence as set one or more tertiary peptides ( designated above as L ), which forth in SEQ ID NOs: 5 , 6 and 1761 , 1762 , 1767 , 1771 - 1773 , may include the amino acid sequence of a binary peptide and variants thereof, (wherein the TPs, if more than one , selected from IFVRLR (SEO ID NO : 1619 ) , ALKLFV (SEO ID NO : 1605 ) , ALKFLV (SEQ ID NO : 1603 ), RRKAFV may be the same or different) . (SEQ ID NO : 1644 ) , or LFAAPF (SEQ ID NO : 1621) , fused [0297 ] The polar aprotic solvent formulations are espe in frame either on the N - terminus , C - terminus or both cially effective when they contain MSO . The examples termini of the binary peptide with a Z region or spacer below are intended to illustrate and not limit the invention in sequence selected from the group of or consisting of GS, any manner. WN , WQ , MA, MV, AM , LH , MN , ES , WQ, or DT. [0298 ] In some embodiments , an insecticidal and /or nem [0295 ] In some embodiments ,methods for controlling an aticidal proteins which are produced by a transgenic plant, insect and / or nematode pest may include applying a formu or part thereof or formulated for direct application to a plant , lation comprising an insecticidal and /or nematicidal protein comprises a combination of a Bt TP and a TMOF TP, that comprising a combination of a Bt ( Bacillus thuringiensis ) when combined in a single insecticidal and/ or nematicidal TP and an ICK (Inhibitor Cystine Knot) TP to said insect protein , may inhibit the growth , impair the movement, or and / or nematode . Upon release of at least 50 % of the TPS even kill an insect when the combination of TPs is appro from the insecticidal and /or nematicidal protein , the ratio of priately delivered to the locus inhabited by the insect . The Bt TP to ICK TP , on a dry weight basis thus released , can be spray - dried powders are can be made using as the primary selected from at least about the following ratios: 99 : 1 , 95 : 5 , active agent an insecticidal and /or nematicidal protein as US 2018 /0362598 A1 Dec. 20 , 2018 46 described herein comprising a combination of TPs as dis - amino acid on the C - terminal of the dipeptide , including cussed above, and one or more various excipients and embodiments where the dipeptide is glycine - serine, includ fermentation by -products . ing embodiments where the TMOF TP includes embodi [0299 ] We describe an insecticidal and /or nematicidal ments where the TMOF TP is at least 50 % , 55 % , 60 % , 65 % , protein composition comprising both a Bt (Bacillus 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % thuringiensis ) protein ; and a TMOF TP . In some embodi or greater sequence identity to any of the TMOF sequences ments , methods for controlling an insect and / or nematode disclosed herein , including embodiments where the Bt Pro pest may include applying a formulation comprising an tein is any Bt TP , including embodiments where the Bt TP insecticidal and/ or nematicidal protein comprising a com is a Cry or Cyt protein , including embodiments where the Bt bination of a Bt ( Bacillus thuringiensis ) TP and a TMOF TP TP is selected from the group consisting of a Cryl , Cry3 , to said insect and / or nematode . Upon release of at least 50 % TIC851, CryET70 , Cry22 , TIC901, TIC201, TIC407 , of the TPs from the insecticidal and /or nematicidal protein , TIC417 , a binary insecticidal protein CryET80 , and the ratio of Bt TP to TMOF TP , on a dry weight basis thus CryET76 , a binary insecticidal protein TIC100 and TIC101 , released , can be selected from about any or all of the a combination of an insecticidal protein ET29 or ET37 with following ratios: 99: 1 , 95 : 5 , 90 : 10 , 85 :15 , 80 : 20 , 75 :25 , an insecticidal protein TIC810 or TIC812 and a binary 70 : 30 , 65 : 35 , 60 :40 , 55 :45 , 50 :50 , 45 :55 , 40 :60 , 35 :65 , insecticidal protein PS149B1, including embodiments 30 :70 , 25 : 75 , 20 : 80 , 15 : 85 , 10 : 90 , 5 : 95 and 1: 99 , or any where the Bt TP is selected from a Cry protein , a Cry1A combination of any two of these values. The formulation protein or a Cry1F protein , including embodiments where may comprise the insecticidal and /or nematicidal protein the Bt TP is a combination Cry1F -Cry1 A protein , including and appropriate excipients , or the insecticidal and / or nem embodiments where the Bt TP comprises an amino acid aticidal protein may be expressed by a culture of one or more sequence at least 90 % identical to SEQ ID NO : 10 , 12 , 14 , bacterial strains, that when formulated and applied to the 26 , 28 , or 34 of U . S . Pat. No . 7 ,304 , 206 , including embodi plant, the bacterial strains produce the insecticidal and /or ments where the Bt Endotoxin is Dipel , including embodi nematicidal protein which is subsequently ingested by the ments where the Bt Protein is Thuricide . insect and / or nematode pest . In various embodiments , the [0301 ] In some embodiments , methods for controlling an total concentration of Bt TP and TMOF TP in the compo insect and / or nematode pest may include applying a formu sition is selected from the following percent concentrations: lation comprising an insecticidal and / or nematicidal protein 1 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55, 60 , 65 , 70 , 75 , 80 , comprising a combination of a Bt (Bacillus thuringiensis ) 85 , 90 , 95 or 99 % , or any range between any two of these TP and a TMOF TP to said insect and / or nematode . Upon values , and the remaining percentage of the composition is release of at least 50 % of the TPs from the insecticidal comprised of excipients . Any of the insecticidal and / or and /or nematicidal protein , the ratio of Bt TP to TMOF TP , nematicidal proteins and plants described herein can be used on a dry weight basis thus released , can be selected from at to control insects and / or nematodes , their growth and dam least about the following ratios: 99 : 1 , 95 : 5 , 90 : 10 , 85 : 15 , age , especially their damage to plants . In some embodi 80 : 20 , 75 : 25 , 70 : 30 , 65 : 35 , 60 :40 , 55 : 45 , 50 : 50 , 45 :55 , ments , insecticidal and / or nematicidal proteins comprising 40 :60 , 35 :65 , 30 :70 , 25 : 75 , 20 :80 , 15 : 85 , 10 : 90 , 5 : 95 and the combination of Bt TP and TMOF TP can be applied by 1 : 99 , or any combination of any two of these values . The being sprayed on a plant, or the insect 's locus, or the locus ratio of Bt TP to TMOF TP , on a dry weight basis thus of a plant in need of protecting . released , can be selected from about the following ratios : [ 0300 ] In some embodiments , illustrative methods for 50 : 50 , 45 : 55 , 40 :60 , 35 : 65 , 30 :70 , 25 : 75 , 20 : 80 , 15 : 85 , controlling a pest infection comprises preparing a formula 10 : 90 , 5 : 95 , 1 : 99 , 0 . 5 : 99 . 5 , 0 . 1 : 99 . 9 and 0 .01 : 99 . 99 or any tion comprising : an insecticidal and /or nematicidal protein combination of any two of these values . The formulation comprising a combination of a Bt TP ; and a TMOF TP , may comprise the insecticidal and / or nematicidal protein which can include any of the compositions described herein and appropriate excipients , or the insecticidal and /or nem or capable of being made by one skilled in the art given this aticidal protein may be expressed by a culture of one or more disclosure . Some of the described formulations include the bacterial strains , that when formulated and applied to the use of a polar aprotic solvent, and or water, and or where the plant, the bacterial strains produce the insecticidal and /or polar aprotic solvent is present in an amount of 1 - 99 wt % , nematicidal protein which is subsequently ingested by the the polar protic solvent is present in an amount of 1 - 99 wt insect and / or nematode pest . In various embodiments , the % , and the water is present in an amount of 0 - 98 wt % . In total concentration of Bt TP and TMOF TP in the compo certain embodiments , as provided herein , the insecticidal sition is selected from the following percent concentrations: and /or nematicidal protein for any combination of TPs can 1 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , include one or more binary and / or tertiary peptides . The 85 , 90 , 95 or 99 % , or any range between any two of these insecticidal and /or nematicidal protein can include one or values , and the remaining percentage of the composition is more TPs from the illustrative combination linked to a comprised of excipients . Any of the insecticidal and /or tertiary peptide , in which the spacer is positioned either to nematicidal proteins and plants described herein can be used the N - terminal side of the binary peptide portion of the to control insects and / or nematodes , their growth and dam tertiary peptide or to the C - terminal side of the binary age, especially their damage to plants . In some embodi peptide portion of the tertiary peptide . In some embodi ments , insecticidal and/ or nematicidal proteins comprising ments , the spacer is a dipeptide, for example ,GS , which may the combination of Bt TP and TMOF TP can be applied by be operably linked to the TMOF TP units within the insec being sprayed on a plant, or the insect ' s locus, or the locus ticidal and /or nematicidal protein . In some embodiments , the of a plant in need of protecting8 . spacer dipeptide is linked at the N - terminal of the TMOF [0302 ] In some embodiments , the method for controlling TP ; and wherein the spacer is comprised of one nonpolar a pest infection includes providing a transformed plant amino acid on the N - terminal of the dipeptide and one polar expressing an insecticidal and /or nematicidal protein US 2018 /0362598 A1 Dec. 20 , 2018 47 expressing the combination of Bt TP and TMOF TPs added to the cell by infection , transfection , microinjection , described herein where the average concentration of each of electroporation , microprojection , or the like. Bt and TMOF TPs as a percentage weight of the total [0309 ] Transformation of plant cells can be accomplished amount of expressed TP in an average leaf of a transformed by one of several techniques known in the art . The DNA plant is about: 1 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , construct of the invention may be modified to obtain or 65 , 70 , 75 , 80 , 85 , 90 , 95 or 99 % , or any range between any enhance expression in plant cells . Typically a construct that two of these values. expresses such a protein would contain a promoter to drive [ 0303] In some embodiments , the insecticidal and /or nem transcription of the gene , as well as a 3 ' untranslated region aticidal protein expressing the combination of Bt TP and to allow transcription termination and polyadenylation . The TMOF TPs described herein is expressed in corn , soybean , organization of such constructs is well known in the art . In cotton , rice, sorghum , switchgrass, sugarcane, alfalfa , pota some instances, it may be useful to engineer the gene such toes or tomatoes. In some embodiments , the insecticidal that the resulting peptide is secreted , or otherwise targeted and / or nematicidal protein is expressed in a transgenic seed within the plant cell . For example, the gene can be engi having an insecticidal and / or nematicidal protein as neered to contain a signal peptide to facilitate transfer of the described above . peptide to the endoplasmic reticulum . It may also be pref [ 0304 ] In some embodiments , a transformed plant erable to engineer the plant expression cassette to contain an expressing properly folded combination of Bt TP and TMOF intron , such that mRNA processing of the intron is required TPs described herein in the transformed plant and to cause for expression . the accumulation of the expressed and properly folded [ 0310 ] In various embodiments , DNA constructs compris combination ofBt TP and TMOF TPs described herein in ing a polynucleotide that encodes an insecticidal and /or said plant and to cause an increase in the plant' s yield or nematicidal protein of the present invention may be tran resistance to insect and /or nematode damage and they con siently or stably transformed into unmodified plant cells or trol insect and / or nematode pests in crops and natural or may be transformed into previously genetically altered plant man -made forests . We describe transgenic plants made by cells . In one embodiment, DNA constructs comprising a any of the products and processes described herein . polynucleotide that encodes an insecticidal and/ or nemati cidal protein of the present invention may be stably trans [ 0305 ] ( 5 ) . Transgenic Plants formed into unmodified plant cells , i. e . plant cells that have [0306 ] Methods of the invention involve introducing a not previously been transformed or transfected with any nucleotide or DNA construct into a host cell, for example , a heterologous DNA or RNA material. Methods for stable plant. By “ introducing ” is intended to present to the plant the integration of foreign or heterologous expression vectors , DNA construct in such a manner that the construct gains are described below . In other embodiments , the genetically access to the interior of a cell of the plant. The methods of unaltered plant cells can be transformed with one DNA the invention do not require that a particular method for construct comprising a polynucleotide operable to encode introducing a DNA construct to a plant is used , only that the one insecticidal and /or nematicidal protein of the present DNA construct gains access to the interior of at least one cell invention . In this embodiment , the insecticidal and / or nem of the plant. Methods for introducing DNA constructs into aticidal protein contains only one type of TP, for example , an plants are known in the art including , but not limited to , ICK motif TP . In other embodiments , the genetically unal stable transformation methods , transient transformation tered plant cells can be transformed with two different DNA methods , and virus -mediated methods . Methods for trans constructs , each construct comprising a polynucleotide oper forming polynucleotides including cloning and expression able to encode one insecticidal and / or nematicidal protein of vectors containing insecticidal proteins are known in the art . the present invention , wherein the insecticidal and / or nem For example , methods for cloning and transforming plants aticidal protein of each construct comprises different TPs , by introducing recombinant insecticidal proteins fused to for example , in one DNA construct, the insecticidal and / or signal peptides and molecular markers can be found in nematicidal protein comprises one or more copies of an ICK International PCT Application Serial No. PCT/ US2013 / motif TP and the other DNA construct comprises a poly 030042 filed on Mar . 8 , 2013 , the contents of the disclosure nucleotide that encodes a different TP , for example a Bt TP . are incorporated by reference herein in its entirety. In some Expression of both types of insecticidal and / or nematicidal embodiments , proteins can be manipulated by using the same promoter or [ 0307 ] By " plant" is intended whole plants , plant organs different promoters and other control sequences to cause the ( e . g . , leaves , stems, roots , etc . ) , seeds , plant cells , propa differential expression of either insecticidal and / or nemati gules, embryos and progeny of the same. Plant cells can be cidal protein , or both insecticidal and / or nematicidal pro differentiated or undifferentiated ( e. g . callus , suspension teins together. culture cells , protoplasts , leaf cells , root cells , phloem cells , [0311 ] In some embodiments , a transgenic plant or part pollen ) . thereof, can produce or express one or more insecticidal [ 0308 ] “ Transgenic plants ” or “ transformed plants ” or and / or nematicidal proteins containing one or more TPs . In “ stably transformed ” plants or cells or tissues refers to plants one embodiment, the TP is an ICK TP , for example , a that have incorporated or integrated exogenous nucleic acid Hybrid - ACTX -Hvla , for example , a TP having an amino sequences or DNA fragments into the plant cell . These acid sequence of SEQ ID NO : 5 or 6 ( or a variant thereof) nucleic acid sequences include those that are exogenous, or and /or the ICK TP expressed may contain 20 - 100 amino not present in the untransformed plant cell , as well as those acids and 2 - 4 disulfide bonds and or the ICK TP is any that may be endogenous , or present in the untransformed insecticidal peptide with at least 50 % , 55 % , 60 % , 65 % , plant cell . “ Heterologous” generally refers to the nucleic 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % acid sequences that are not endogenous to the cell or part of or greater sequence identity to any of the ICK TPs described the native genome in which they are present, and have been herein and/ or a Bt TP and specifically exemplified in any TP US 2018 /0362598 A1 Dec. 20 , 2018 48 having an amino acid sequence as set forth in SEQ ID NOs: each of the insecticidal and /or nematicidal protein and 5 - 1593 and 1761 - 1775 , or a variant thereof. previously introduced TP can be controlled using different [0312 ] In various embodiments , a transgenic plant may promoters or expression conditions to facilitate differential express and produce one or more ICK TPs, which may be expression of both . It is to be understood , that transforma the same or they may be different, ( for example , an Omega tion of two different Bt TPs cannot occur, whether the DNA ACTX TP , a Kappa -ACTX TP or a U - ACTX TP , for construct comprises a polynucleotide operable to encode an example , a Hybrid - ACTX -Hvla TP ) , or a TP having at least insecticidal and/ or nematicidal protein having multiple TPs 80 % , at least 85 % , at least 90 % , at least 95 % , at least 96 % , or transformation of multiple DNA constructs containing at least 97 % , at least 98 % , at least 99 % , or at least 100 % single TPs. In either case, only a single species of Bt TP can sequence identity or sequence homology to a peptide or be transformed into the plant, whether expressed as a single protein having an amino acid sequence as set forth in SEO TP or in insecticidal and/ or nematicidal proteins containing ID NOs: 5 , 6 and 1761, 1762, 1767 , 1771 - 1773 , and variants multiple copies of the same species of Bt TP . thereof, (wherein the TPs, if more than one , may be the same [0317 ] In some embodiments , the present invention pro or different) . vides a transgenic plant comprising a combination of a Bt [0313 ] In another embodiment, a transgenic plant or part (Bacillus thuringiensis ) TP and an ICK ( Inhibitor Cystine thereof, may express and produce one or more Bt TPs , which Knot) TP . This transgenic plant can be made from indepen may be the same or different. It is to be understood , that dent and separate stable genetic insertions of DNA con although a transgenic plant , or part thereof may express and structs as provided herein that create a first flowering plant produce the same type of TPs or different TPs , a transgenic expressing a Bt TP and a second flowering plant expressing plantwill not have a single DNA construct which is operable an ICK TP. The pollen of one of those plants will be crossed to encode and express an ICK TP and a Bt TP from the same onto the female flower organs of the other plant to form a DNA construct. These two types of TPs may be expressed genetic hybridization that contains both an insecticidal and / and produced in a plant or part thereof, but these two types or nematicidal proteins comprising a Bt TP and a different of TPs originate from separate DNA constructs as described insecticidal and /or nematicidal protein that comprises an herein . The transgenic plants disclosed herein can contain , ICK TP genetic traits . The progeny of that sexual cross and express , and produce an insecticidal and /or nematicidal further crossing that results in the inheritance of both traits protein containing a known Bt TP , including a Cry or Cyt will result in a transgenic plant comprising a combination of protein TP , and /or a Bt TP selected from the group consisting a Bt (Bacillus thuringiensis ) TP and an ICK ( Inhibitor of a Cry1, Cry3 , TIC851, CryET70 , Cry22 , TIC901 , Cystine Knot) TP. TIC201, TIC407 , TIC417 , a CryET80 , and CryET76 , [0318 ] In some embodiments , transgenic plants or parts TIC100 TIC101 , ET29 , ET37 , TIC810 , TIC812 , PS149B1 , thereof, are operable to express an insecticidal and / or nem and combinations thereof. The Bt TP can be selected from a aticidal protein encoded by a DNA construct encoding an Cry protein , a Cry1A protein or a Cry1F protein , or a insecticidal and / or nematicidal protein , wherein the insecti combination Cry1F - Cry1A protein , or it comprises an amino cidal and/ or nematicidal protein comprises an ERSP fused in acid sequence at least 90 % identical to SEO ID NO : 10 , 12 , framewith the N - terminus of a binary or tertiary peptide , the 14 , 26 , 28 , or 34 of U . S . Pat . No . 7 , 304 , 206 . In some binary or tertiary peptide is fused in frame with a construct embodiments , a transgenic plant can produce an insecticidal comprising ( TP - L )n or the insecticidal and /or nematicidal and / or nematicidal protein comprising a Bt TP, for example , protein comprises an ERSP fused in frame with a binary Dipel and /or Thuricide . peptide and / or a repeat construct ( L - TP ) , or ( TP - L ) n . In [ 03141 In some embodiments , a transgenic plant express various embodiments, the exemplary insecticidal and /or ing the TPs described herein where the average concentra nematicidal protein can include the construct: (ERSP ) - ( TP tion of each of Bt and ICK TPs as a percentage weight of the L )n , or (ERSP )- (L )- ( TP -L )n , or ( ERSP ) -( L - TP )n , or (ERSP ) total amount of expressed TP in an average leaf of a ( L - TP ) . - ( L ) . In another embodiment, a transgenic plant, or transformed plant is about: 1 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , part thereof comprises a polynucleotide operably linked to 45 , 50 , 55, 60 , 65 , 70 , 75 , 80 , 85, 90 , 95 or 99 % , or any range an operable promoter, wherein the polynucleotide encodes between any two of these values . an insecticidal and/ or nematicidal protein comprising an [0315 ] In some embodiments , the insecticidal and / or nem ERSP fused in frame with the N - terminus of a stabilizing aticidal protein described herein is expressed in corn , soy domain (STA ) . The STA is fused in frame with either the bean , cotton , rice , sorghum , switchgrass , sugarcane , alfalfa , N -terminus of a binary or tertiary peptide , or the N -terminus potatoes or tomatoes. In some embodiments , the insecticidal of a construct comprising: ( L - TP )n , ( L - TP ), - L , ( TP -L ) n , or and/ or nematicidal protein is expressed in a transgenic seed L -( TP - L )n . In various embodiments , an exemplary insecti having an insecticidal and / or nematicidal protein as cidal and/ or nematicidal protein can include a construct from described above. N - terminus to C - terminus : ( ERSP ) - ( L ) - TP - ( L ) - TP , ( ERSP ) [ 0316 ] In other embodiments , a DNA construct compris (L )- TP -( L )- TP - (L ), ( L - TP ), , - L , ( L - TP -L ) n. (ERSP )- ( TP -L )n , ing a polynucleotide operable to encode an insecticidal (ERSP ) - ( L ) - ( TP - L ) n , (ERSP ) - ( L - TP ) n , ( ERSP ) - ( L - TP ) n and / or nematicidal protein of the present invention can be ( L ) , (ERSP ) - (STA ) - ( L - TP ) n , (ERSP ) - (STA ) - ( L - TP ) n - ( L ) , transiently or stably expressed in a plant cell that has been (ERSP ) - (STA ) - ( TP - L ) n , or (ERSP ) - (STA ) - ( L ) - ( TP - L ) n , or previously genetically modified , for example , a plant cell (STA ) - ( L - TP ) n , or ( STA ) - ( L - TP ). - ( L ) , or (STA ) - ( TP - L ) n , or that has been previously transformed with another TP, for ( STA ) - ( L ) - ( TP - L ) n , wherein ( L ) is a binary peptide or a example , an ICK motif TP or a Bt TP as described herein . In tertiary peptide, and which each L may be the same or this embodiment, the DNA construct of the present inven different, TP is a toxic peptide , which may be the same or tion can be transformed such that the expression of either the different, and n is an integer ranging from 1 to 200 , prefer insecticidal and / or nematicidal protein or the previously ably 1 to 100 , and most preferably , 1 to 10 . In some introduced TP can be controlled by the same promoters or embodiments , the insecticidal and /or nematicidal protein US 2018 /0362598 A1 Dec. 20 , 2018 49 may contain TPs that are the same or different, and binary has stable expression of a Bt TP and an insecticidal and /or and / or tertiary peptides that are the same or different. In nematicidal protein containing one or more ICK motif TPs . some embodiments , the C - terminal TP is fused or unfused at [ 0320 ] In various embodiments , insecticidal and / or nem its C - terminus with a binary or tertiary peptide . In related aticidal proteins can also be integrated into plant genome embodiments of the above, transgenic plants or parts thereof using stable plant transformation technology , and therefore may comprise one or more TPs useful in the manufacture of insecticidal and / or nematicidal proteins can be stably an insecticidal and / or nematicidal protein . Transgenic plants expressed in plants and protect the transformed plants from or parts thereof may comprise an insecticidal and / or nem generation to generation . For the stable transformation of aticidal protein , which may include one or more ICK TPs, plants , an expression vector comprising the DNA construct for example , a TP selected from an “ ACTX ” or “ ACTX can be circular or linear. A few critical components must be peptide ” family of insecticidal ICK peptides that have been included in the vector DNA . The insecticidal and / or nem isolated from an Australian funnel -web spiders belonging to aticidal insecticidal and / or nematicidal protein ORF for the Atracinae subfamily , for example , one or more TPS stable plant transformation should be carefully designed for selected from an Omega -ACTX TP, a Kappa -ACTX TP and optimal expression in plants based on the study in the an U -ACTX TP, for example , U + 2 - ACTX - Hvla , U - ACTX transient plant expression as described above . The expres Hvla , rU - ACTX -Hvla , rU - ACTX -Hvlb , or a hybrid sion of insecticidal and / or nematicidal insecticidal and / or ACTX -Hvla peptide , or a TP having at least 80 % , at least nematicidal protein is usually controlled by a promoter that 85 % , at least 90 % , at least 95 % , at least 96 % , at least 97 % , drives transcription in some of all cells of the transgenic at least 98 % , at least 99 % , or at least 100 % sequence identity plant. The promoter can be a strong plant viral promoter , for or sequence homology to a peptide or protein having an example , the constitutive 35S promoter from Cauliflower amino acid sequence as set forth in SEQ ID NOs: 5 , 6 and Mosaic Virus (CaMV ) ; it also can be a strong plant pro 1761, 1762 , 1767 , 1771 - 1773 , and variants thereof , ( wherein moter, for example , the hydroperoxide lyase promoter the TPs , if more than one, may be the same or different ) and (PHPL ) from Arabidopsis thaliana ; the Glycine max polyu each insecticidal and /or nematicidal protein further compris biquitin (Gmubi ) promoter from soybean ; the ubiquitin ing one or more tertiary peptides (designated above as L ), promoters from different plant species ( rice, corn , potato , which may include the amino acid sequence of a binary etc . ) , etc . A plant transcriptional terminator often occurs peptide selected from IFVRLR (SEQ ID NO : 1619 ) , after the stop codon of the ORF to halt the RNA polymerase ALKLFV ( SEO ID NO : 1605 ) , ALKFLV (SEQ ID NO : and transcription of the mRNA . To evaluate the insecticidal 1603 ) , RRKAFV (SEQ ID NO : 1644 ) , or LFAAPF (SEQ ID and / or nematicidal insecticidal and / or nematicidal protein NO : 1621 ) , fused in frame either on the N - terminus, C - ter expression , a reporter gene can be included in the expression minus or both termini of the binary peptide with a Z region vector , for example , beta -glucuronidase gene (GUS ) for or spacer sequence selected from the group of or consisting GUS straining assay, green fluorescent protein (GFP ) gene of GS, WN , WQ, MA , MV, AM , LH , MN , ES , WQ , or DT . for green fluorescence detection under UV light , etc . For [ 0319 ] In various embodiments , the plant or part thereof, selection of transformed plants , a selection marker gene is for example a plant cell , has been previously transformed usually included in the expression vector. The marker gene with a Bt TP and the plant or part thereof, for example a plant expression product can provide the transformed plant with cell containing a Bt TP is subsequently transformed with a resistance to specific antibiotics, for example , kanamycin , DNA construct operable to encode an insecticidal and / or hygromycin , etc . , or specific herbicide , for example , gly nematicidal protein , which may include one or more ICK phosate etc . If agroinfection technology is adopted for plant TPs, for example , a TP selected from an “ ACTX ” or “ ACTX transformation , T- DNA left border and right border peptide ” family of insecticidal ICK peptides that have been sequences are also included in the expression vector to isolated from an Australian funnel -web spiders belonging to transport the T -DNA portion into the plant. The constructed the Atracinae subfamily , for example , one or more TPS expression vector can be transformed into plant cells or selected from an Omega - ACTX TP , a Kappa -ACTX TP and tissues using any number of established and well known an U - ACTX TP , for example , U + 2 -ACTX -Hvla , U - ACTX transformation technologies . Agroinfection is a very popular Hvla , rU -ACTX -Hvla , rU -ACTX -Hvlb , or a hybrid way to transform a plant using an Agrobacterium tumefa ACTX -Hvla peptide , or a TP having at least 80 % , at least ciens strain or an Agrobacterium rhizogenes strain . Particle 85 % , at least 90 % , at least 95 % , at least 96 % , at least 97 % , bombardment ( also called Gene Gun , or Biolistics ) technol at least 98 % , at least 99 % , or at least 100 % sequence identity ogy is also very commonly used for plant transformation . or sequence homology to a peptide or protein having an Other less commonly used transformation methods include amino acid sequence as set forth in SEQ ID NOs: 5 , 6 and tissue electroportation , silicon carbide whiskers , direct 1761, 1762 , 1767, 1771 - 1773 , and variants thereof, (wherein injection of DNA , etc . After transformation , the transformed the TPs , if more than one , may be the same or different) and plant cells or tissues placed on plant regeneration media to each insecticidal and/ or nematicidal protein further compris regenerate successfully transformed plant cells or tissues ing one or more tertiary peptides (designated above as L ) , into transgenic plants . The evaluation of the integration and which may include the amino acid sequence of a binary expression of the insecticidal and /or nematicidal insecticidal peptide selected from IFVRLR ( SEQ ID NO : 1619 ) , and /or nematicidal protein ORF in the transformed plant can ALKLFV ( SEQ ID NO : 1605 ) , ALKFLV (SEQ ID NO : be performed . 1603 ) , RRKAFV (SEO ID NO : 1644 ) , or LFAAPF (SEO ID [ 0321 ] In some embodiments , a “ plant expression cas NO : 1621 ) , fused in frame either on the N - terminus, C - ter sette ” can be inserted into a “ plant transformation vector” . minus or both termini of the binary peptide with a Z region The illustrative plant transformation vector may be com or spacer sequence selected from the group of or consisting prised of one or more DNA vectors needed for achieving of GS , WN , WQ , MA , MV, AM , LH , MN , ES , WQ , or DT. plant transformation . For example , it is a common practice The resulting plant or part thereof, for example , a plant cell, in the art to utilize plant transformation vectors that are US 2018 /0362598 A1 Dec. 20 , 2018 50 comprised of more than one contiguous DNA segment. [ 0323 ] Transformation protocols as well as protocols for These vectors are often referred to in the art as “ binary introducing nucleotide sequences into plants may vary vectors” . Binary vectors as well as vectors with helper depending on the type of plant or plant cell , i . e ., monocot or plasmids are most often used for Agrobacterium -mediated dicot, targeted for transformation . Generation of transgenic transformation , where the size and complexity of DNA plants may be performed by one of several methods, includ segments needed to achieve efficient transformation is quite ing , but not limited to , microinjection , electroporation , direct large , and it is advantageous to separate functions onto gene transfer, introduction of heterologous DNA by Agro bacterium into plant cells ( Agrobacterium -mediated trans separate DNA molecules. Binary vectors typically contain a formation ) , bombardment of plant cells with heterologous plasmid vector that contains the cis - acting sequences foreign DNA adhered to particles, ballistic particle accel required for T -DNA transfer ( such as left border and right eration , aerosol beam transformation ( U . S . Published Appli border ), a selectable marker that is engineered to be capable cation No . 20010026941 ; U . S . Pat. No. 4 , 945 ,050 ; Interna of expression in a plant cell, and a " gene of interest” ( a gene tional Publication No. WO 91/ 00915 ; U . S . Published engineered to be capable of expression in a plant cell for Application No . 2002015066 ) , Lecl transformation , and which generation of transgenic plants is desired ) . Also various other non - particle direct -mediated methods to trans present on this plasmid vector are sequences required for fer DNA . bacterial replication . The cis -acting sequences are arranged 0324 ] Methods for transformation of chloroplasts are in a fashion to allow efficient transfer into plant cells and known in the art . See , for example , Svab et al. ( 1990 ) Proc . expression therein . For example , the selectable marker gene Natl . Acad . Sci. USA 87 : 8526 - 8530 ; Svab and Maliga and the insecticidal and / or nematicidal protein described ( 1993 ) Proc . Natl. Acad . Sci. USA 90 : 913 - 917 ; Svab and herein are located between the left and right borders . Often Maliga ( 1993 ) EMBO J . 12 :601 -606 . The method relies on a second plasmid vector contains the trans- acting factors that particle gun delivery of DNA containing a selectable marker mediate T -DNA transfer from Agrobacterium to plant cells . and targeting of the DNA to the plastid genome through This plasmid often contains the virulence functions ( Vir homologous recombination . Additionally , plastid transfor genes ) that allow infection of plant cells by Agrobacterium , mation can be accomplished by transactivation of a silent and transfer of DNA by cleavage at border sequences and plastid -borne transgene by tissue - preferred expression of a vir -mediated DNA transfer, as is understood in the art nuclear - encoded and plastid - directed RNA polymerase . (Hellens and Mullineaux ( 2000 ) Trends in Plant Science Such a system has been reported in McBride et al . (1994 ) 5 :446 - 451 ) . Several types of Agrobacterium strains ( e . g ., Proc . Natl. Acad . Sci. USA 91: 7301- 7305 . LBA4404 , GV3101, EHA101, EHA105 , etc . ) can be used [ 0325 ] Following integration of heterologous foreign for plant transformation . The second plasmid vector is not DNA into plant cells , one then applies a maximum threshold necessary for transforming the plants by other methods such level of appropriate selection in the medium to kill the as microprojection ,microinjection , electroporation , polyeth untransformed cells and separate and proliferate the puta ylene glycol, etc . tively transformed cells that survive from this selection [0322 ] In general , plant transformation methods involve treatment by transferring regularly to a fresh medium . By transferring heterologous DNA into target plant cells ( e . g ., continuous passage and challenge with appropriate selec immature or mature embryos , suspension cultures , undiffer tion , one identifies and proliferates the cells that are trans entiated callus, protoplasts , etc. ), followed by applying a formed with the plasmid vector. Molecular and biochemical maximum threshold level of appropriate selection (depend methods can then be used to confirm the presence of the ing on the selectable marker gene ) to recover the trans integrated heterologous gene of interest into the genome of formed plant cells from a group of untransformed cell mass . the transgenic plant. Explants are typically transferred to a fresh supply of the [0326 . The cells that have been transformed may be grown same medium and cultured routinely . Subsequently , the into plants in accordance with conventional ways . See , for transformed cells are differentiated into shoots after placing example , McCormick et al. ( 1986 ) Plant Cell Reports 5 :81 on regeneration medium supplemented with a maximum 84 . These plants may then be grown, and either pollinated threshold level of selecting agent. The shoots are then with the same transformed strain or different strains , and the transferred to a selective rooting medium for recovering resulting hybrid having constitutive expression of the rooted shoot or plantlet . The transgenic plantlet then grows desired phenotypic characteristic identified . Two or more into a mature plant and produces fertile seeds ( e . g . Hiei et al. generations may be grown to ensure that expression of the ( 1994 ) The Plant Journal 6 :271 - 282 ; Ishida et al. ( 1996 ) desired phenotypic characteristic is stably maintained and Nature Biotechnology 14 :745 -750 ) . Explants are typically inherited and then seeds harvested to ensure expression of transferred to a fresh supply of the same medium and the desired phenotypic characteristic has been achieved . In cultured routinely . A general description of the techniques this manner , the present invention provides transformed seed and methods for generating transgenic plants are found in (also referred to as “ transgenic seed ” ) having a nucleotide Ayres and Park (1994 ) Critical Reviews in Plant Science construct of the invention , for example, an expression cas 13 : 219 - 239 and Bommineni and Jauhar ( 1997 ) Maydica sette of the invention , stably incorporated into their genome. 42 : 107 - 120 . Since the transformed material contains many [0327 ] (5A ). Evaluation of Plant Transformations cells ; both transformed and non - transformed cells are pres [0328 ] Following introduction of heterologous foreign ent in any piece of subjected target callus or tissue or group DNA into plant cells , the transformation or integration of of cells . The ability to kill non - transformed cells and allow heterologous gene in the plant genome is confirmed by transformed cells to proliferate results in transformed plant various methods such as analysis of nucleic acids, proteins cultures . Often , the ability to remove non - transformed cells and metabolites associated with the integrated gene . is a limitation to rapid recovery of transformed plant cells 0 329 ) PCR analysis is a rapid method to screen trans and successful generation of transgenic plants . formed cells , tissue or shoots for the presence of incorpo US 2018 /0362598 A1 Dec. 20 , 2018 51 rated gene at the earlier stage before transplanting into the nematicidal protein , i . e . at its C - terminus , may be fused or soil (Sambrook and Russell ( 2001) Molecular Cloning : A unfused at its C - terminus with a binary or tertiary peptide . Laboratory Manual. Cold Spring Harbor Laboratory Press , In various embodiments , an exemplary insecticidal and / or Cold Spring Harbor , N . Y .) . PCR is carried out using oligo nematicidal protein can include the structure : ( ERSP ) -( TP nucleotide primers specific to the gene of interest or Agro L )n , or (ERSP ) - ( L ) - ( TP - L ) n , or (ERSP ) - ( L - TP ) n , or (ERSP ) bacterium vector background , etc . ( L - TP ) . - ( L ) . In another embodiment, a plant, or part thereof [ 0330 ] Plant transformation may be confirmed by South comprises a polynucleotide operably linked to an operable ern blot analysis of genomic DNA ( Sambrook and Russell , promoter, for example , a homologous or heterologous pro 2001, supra ) . In general, total DNA is extracted from the moter , wherein the polynucleotide encodes an insecticidal transformant, digested with appropriate restriction enzymes, and /or nematicidal protein comprising an ERSP fused in fractionated in an agarose gel and transferred to a nitrocel frame with the N - terminus of a stabilizing domain (STA ) . lulose or nylon membrane. The membrane or " blot ” is then The STA is fused in frame with either the N - terminus of a probed with , for example, radiolabeled .sup . 32P target DNA binary or tertiary peptide , or the N - terminus of a construct fragment to confirm the integration of introduced gene into comprising: (L - TP ) , ( L - TP ) .- L , ( TP - L )n , or L -( TP - L ) n. In the plant genome according to standard techniques (Sam various embodiments , an exemplary insecticidal and / or brook and Russell, 2001, supra ) . nematicidal protein can comprise , or selected from the group [0331 ] In Northern blot analysis , RNA is isolated from consisting of, or consisting of a construct from N -terminus specific tissues of transformant, fractionated in a formalde to C - terminus : (ERSP )- ( L ) - TP - ( L ) - TP , (ERSP ) - ( L ) - TP - ( L ) hyde agarose gel , and blotted onto a nylon filter according TP - ( L ) , ( L - TP ) n - L , ( L - TP - L ) n , (ERSP ) - ( TP - L ) n . (ERSP ) to standard procedures that are routinely used in the art ( L ) -( TP - L )n (ERSP ) -( L - TP )n . ( ERSP )- (L - TP )n -( L ), (Sambrook and Russell , 2001, supra ) . Expression of RNA (ERSP ) - ( STA ) - ( L - TP ) n . (ERSP ) - ( STA ) - ( L - TP ) n - ( L ) , encoded by the Bt- protein is then tested by hybridizing the ( ERSP ) - (STA ) - ( TP - L ) , or ( ERSP ) - (STA ) - ( L ) - ( TP - L ) n , or filter to a radioactive probe derived from a Bt- protein , by (STA ) - (L - TP )n , or (STA )- ( L - TP )n -( L ), or (STA )- ( TP -L ) n , or methods known in the art (Sambrook and Russell, 2001 , (STA ) - ( L ) - ( TP - L )n , wherein ( L ) is a binary peptide or a supra ). tertiary peptide, and which each L may be the same or [0332 ] Western blot, biochemical assays and the like may different, TP is a toxic peptide , which may be the same or be carried out on the transgenic plants to confirm the different, and n is an integer ranging from 1 to 200 , prefer presence of the transformed TP by standard procedures ably 1 to 100 , and most preferably , 1 to 10 . In some (Sambrook and Russell, 2001 , supra) using antibodies that embodiments , the insecticidal and / or nematicidal protein bind to one or more epitopes present on the TP . may contain TPs that are the same or different, and binary [ 0333] Accordingly , the present invention provides a and/ or tertiary peptides that are the same or different. In transgenic plant or part thereof, which comprises a poly some embodiments , the C - terminal TP is fused or unfused at nucleotide operably linked to an operable promoter, for its C -terminus with a binary or tertiary peptide . example , a homologous or heterologous promoter , wherein [0334 ] In some embodiments , transgenic plants or parts the polynucleotide encodes : a peptide comprising either a thereof, that may be receptive to the expression of insecti binary or a tertiary peptide as described herein . In some cidal and /or nematicidal peptide compositions comprising a embodiments , the binary or tertiary peptide can include an cleavable linking peptide described herein , can include: amino acid sequence of at least 4 amino acids and up to a alfalfa , banana , barley , bean , broccoli , cabbage , canola , maximum of 16 amino acids. The binary peptide has two carrot, cassava , castor, cauliflower , celery, chickpea , Chi regions and the tertiary has the same two regions and one nese cabbage , citrus , coconut, coffee , corn , clover, cotton , a additional region called a spacer. Each of the binary and /or cucurbit , cucumber, Douglas fir , eggplant, eucalyptus, flax , tertiary peptides can be cleaved by both an animal gut garlic , grape, hops, leek , lettuce, Loblolly pine , millets , protease, for example a human gastrointestinal protease and melons, nut, oat , olive , onion , ornamental, palm , pasture an insect or nematode gut protease . In various embodiments , grass , pea , peanut, pepper , pigeonpea , pine , potato , poplar, the polynucleotide also encodes an Endoplasmic Reticulum pumpkin , Radiata pine , radish , rapeseed , rice , rootstocks , Signal Peptide (ERSP ) which is fused at its C - terminus with rye , safflower, shrub , sorghum , Southern pine , soybean , the N -terminus of a binary and /or tertiary peptide . In these spinach , squash , strawberry , sugar beet, sugarcane, sun embodiments , the binary and / or tertiary peptides are also flower, sweet corn , sweet gum , sweet potato , switchgrass , operably fused to the N - terminus of a TP. In still other tea , tobacco , tomato , triticale , turf grass , watermelon , and a embodiments , the transgenic plant may contain a cell or a wheat plant. In some embodiments the transgenic plant may plurality of cells transformed with a DNA construct com be grown from cells that were initially transformed with the prising at least one polynucleotide operably linked to an DNA constructs described herein . In other embodiments , the operable promoter, for example , a homologous or heterolo transgenic plantmay express the encoded insecticidal and /or gous promoter, wherein the polynucleotide encodes an nematicidal peptide compositions in a specific tissue, or insecticidal and /or nematicidal protein having two or more plant part, for example , a leaf, a stem a flower, a sepal, a cleavable binary or tertiary peptides . The insecticidal and / or fruit, a root, or a seed or combinations thereof. nematicidal protein comprises an ERSP fused in frame at its [0335 ] In another aspect of the invention , one may gen C - terminus with the N - terminus of the binary or tertiary erate transgenic plants expressing an insecticidal and /or peptide , and the binary or tertiary peptide is fused in frame nematicidal insecticidal and / or nematicidal protein that at its C - terminus with a construct comprising : ( TP - L ) when cleaved in the presence of an appropriate protease has wherein TP is toxic peptide or protein , L is a binary or pesticidal activity . Methods described above by way of tertiary peptide and n is an integer ranging from 1 to 200 , or example may be utilized to generate transgenic plants , but from 1 to 100 , or from 1 to 10 . In these insecticidal and / or the manner in which the transgenic plant cells are generated nematicidal proteins, the last TP of the insecticidal and / or is not critical to this invention . Methods known or described US 2018 /0362598 A1 Dec. 20 , 2018 in the art such as Agrobacterium -mediated transformation , tertiary peptide, and the binary or tertiary peptide is fused in biolistic transformation ( See for example , Kikkert JR1, frame with a construct comprising ( TP - L ) , wherein TP is Vidal JR , Reisch B I ., “ Stable transformation of plant cells toxic protein , L is a binary or tertiary peptide and n is an by particle bombardment/ biolistics . " , Methods Mol Biol. integer ranging from 1 to 200 and wherein the C - terminal TP 2005 ; 286 :61 - 78 , the disclosure of which is incorporated is fused or unfused at its C -terminus with a binary or tertiary herein by reference in its entirety ) , and non - particle -medi peptide. In some embodiments , the present invention pro ated methods may be used . Plants expressing an insecticidal vides a transgenic plant wherein the insecticidal and /or and / or nematicidal insecticidal and / or nematicidal protein nematicidal protein that is expressed in the transgenic plant, may be isolated by common methods described in the art , for or part thereof, is derived from a DNA construct that example by transformation of callus, selection of trans encodes an ERSP fused in frame with the N - terminus of the formed callus, and regeneration of fertile plants from such binary or tertiary peptide , and the binary or tertiary peptide transgenic callus. In such process, one may use any gene as is fused in frame with a construct comprising ( TP - L ) , a selectable marker so long as its expression in plant cells wherein TP is toxic protein , L is a binary or tertiary peptide confers ability to identify or select for transformed cells . and n is an integer ranging from 1 to 200 and wherein the [0336 ] A number of markers have been developed for use C -terminal TP is fused or unfused at its C - terminus with a with plant cells , such as resistance to chloramphenicol, the binary or tertiary peptide . In various embodiments , an aminoglycoside G418 , hygromycin , or the like . Other genes exemplary insecticidal and / or nematicidal protein can that encode a product involved in chloroplast metabolism include the structure : ( ERSP ) - ( TP - L ) n , or (ERSP ) - ( L ) - ( TP may also be used as selectable markers. For example , genes L ) n , or (ERSP ) - ( L - TP ) , or ( ERSP ) - ( L - TP ) , ( L ) . In another that provide resistance to plant herbicides such as glypho embodiment, a plant, or part thereof comprises a polynucle sate , bromoxynil , or imidazolinone may find particular use . otide operably linked to an operable promoter , for example , Such genes have been reported (Stalker et al. ( 1985 ) J . Biol. a homologous or heterologous promoter , wherein the poly Chem . 263: 6310 - 6314 (bromoxynil resistance nitrilase nucleotide encodes an insecticidal and / or nematicidal pro gene ); and Sathasivan et al. ( 1990 ) Nucl. Acids Res. 18 :2188 tein comprising an ERSP fused in frame with the N - terminus ( AHAS imidazolinone resistance gene ). Additionally , the of a stabilizing domain ( STA ). The STA is fused in frame genes disclosed herein are useful as markers to assess with either the N - terminus of a binary or tertiary peptide , or transformation of bacterial or plant cells . Methods for the N -terminus of a construct comprising : ( L - TP ) , ( L - TP ) detecting the presence of a transgene in a plant, plant organ „ - L , ( TP - L ) , , or L -( TP - L ) . . In various embodiments , an ( e . g . , leaves , stems, roots , etc . ) , seed , plant cell , propagule , exemplary insecticidal and /or nematicidal protein can embryo or progeny of the same are well known in the art . In include a construct from N - terminus to C - terminus compris one embodiment, the presence of the transgene is detected ing : (ERSP ) - (L ) - TP - ( L ) - TP , (ERSP ) - ( L ) - TP - (L )- TP - ( L ) , by testing for pesticidal activity . ( L - TP ) ,, - L , ( L - TP - L ). , (ERSP ) - ( TP - L ) . , ( ERSP ) - ( L ) - ( TP - L ) [0337 ] Fertile plants expressing an insecticidal and /or n ( ERSP ) - (L - TP )n , ( ERSP ) -( L - TP )n -( L ), (ERSP ) -( STA )- ( L nematicidal protein may be tested for pesticidal activity , and TP )n , ( ERSP ) -( STA )- (L - TP )n - (L ), (ERSP )- (STA )- ( TP -L ) n , the plants showing optimal activity selected for further or (ERSP ) - (STA ) - ( L ) - ( TP - L ) n , or (STA ) - ( L - TP ) n , or ( STA ) breeding . Methods are available in the art to assay for pest ( L - TP ) - ( L ) , or (STA ) - ( TP - L ) n , or (STA ) - ( L ) - ( TP - L ) n , activity . Generally, the cleaved TP is mixed and used in wherein (L ) is a binary peptide or a tertiary peptide, and feeding assays . See , for example Marrone et al. ( 1985 ) J . of which each L may be the same or different, TP is a toxic Economic Entomology 78 : 290 -293 . peptide , which may be the same or different, and n is an [ 0338 ] Accordingly , the present invention provides a integer ranging from 1 to 200 , preferably 1 to 100 , and most transgenic plant, meaning a plant, or part thereof that has preferably , 1 to 10 . In some embodiments , the insecticidal been transformed with a DNA construct comprising a poly and / or nematicidal protein may contain TPs that are the nucleotide operably linked to an operable promoter, for same or different, and binary and /or tertiary peptides that are example , a homologous or heterologous promoter that is the same or different. In some embodiments , the C - terminal operable in the plant cell thus transformed . In some embodi TP is fused or unfused at its C -terminus with a binary or ments , the transformed polynucleotide encodes: a peptide tertiary peptide . In related embodiments of the above , TPs comprising either a binary or a tertiary peptide , wherein the useful in the manufacture of a DNA construct or polynucle peptide contains at least 4 amino acids and up to a maximum otide encoding: ( 1 ) an insecticidal and / or nematicidal pro of 16 amino acids, and is comprised of 2 or 3 different tein , which may include one or more ICK TPs , for example , regions. The binary peptide has 2 different regions and the a TP selected from an " ACTX ” or “ ACTX peptide ” family tertiary peptide has 3 different regions. The binary and of insecticidal ICK peptides that have been isolated from an tertiary peptide can be cleaved by both an animal , for Australian funnel- web spiders belonging to the Atracinae example a human , gut protease and an insect or nematode subfamily , for example , one or more TPs for example , gut protease. The binary or tertiary peptide is operably fused selected from an Omega - ACTX TP, a Kappa -ACTX TP and to the C - terminus of an Endoplasmic Reticulum Signal an U -ACTX TP, for example , U + 2 - ACTX -Hvla , U - ACTX Peptide (ERSP ) and operably fused to the N - terminus of a Hvla , rU - ACTX -Hvla , rU - ACTX -Hvlb , or a hybrid TP . In various embodiments , the plant cell is transformed ACTX -Hvla peptide , or a TP having at least 80 % , at least with a DNA construct comprising at least one polynucle 85 % , at least 90 % , at least 95 % , at least 96 % , at least 97 % , otide operably linked to an operable promoter, for example , at least 98 % , at least 99 % , or at least 100 % sequence identity a homologous or heterologous promoter, wherein the poly or sequence homology to a peptide or protein having an nucleotide encodes an insecticidal and / or nematicidal pro - amino acid sequence as set forth in SEO ID NOs : 5 , 6 and tein having one or more cleavable binary or tertiary peptides. 1761 , 1762, 1767 , 1771 - 1773 , and variants thereof, (wherein The insecticidal and / or nematicidal protein comprises an the TPs, if more than one , may be the same or different ) and ERSP fused in frame with the N -terminus of the binary or each insecticidal and /or nematicidal protein further compris US 2018 /0362598 A1 Dec. 20 , 2018 53 ing one or more tertiary peptides ( designated above as L ) , (ERSP )- (L )- ( TP - L ) n , or (ERSP ) - ( L - TP )n , or (ERSP ) -( L - TP ) which may include the amino acid sequence of a binary n - ( L ) . In another embodiment, a plant, or part thereof peptide selected from IFVRLR (SEQ ID NO : 1619 ), comprises a polynucleotide operably linked to an operable ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID NO : promoter , for example , a homologous or heterologous pro 1603 ), RRKAFV ( SEQ ID NO : 1644 ) , or LFAAPF ( SEQ ID moter, wherein the polynucleotide encodes an insecticidal NO : 1621 ), fused in frame either on the N -terminus , C - ter and /or nematicidal protein described herein , the insecticidal minus or both termini of the binary peptide with a Z region and / or nematicidal protein comprising an ERSP fused in or spacer sequence selected from the group of or consisting frame with the N -terminus of a stabilizing domain (STA ) . of GS, WN , WQ, MA , MV, AM , LH ,MN , ES , WQ , or DT. The STA is fused in frame with either the N - terminus of a [ 0339] In related embodiments , the transgenic plant binary or tertiary peptide , or the N - terminus of a construct expresses and produces an insecticidal and /or nematicidal comprising : ( L - TP )n , ( L - TP ) , - L , ( TP - L ) n , or L - ( TP - L ) n . In protein containing one or more copies of a CRIP TP which various embodiments , an exemplary insecticidal and / or may be derived from an Australian Funnel -web spider or sea nematicidal protein can comprise , or is selected from the anemone. In some embodiments , real or notional examples group consisting of, or consists of: a construct from N -ter of transformed plants described and exemplified herein , are minus to C - terminus : (ERSP ) -( L )- TP - ( L )- TP, (ERSP ) -( L ) transformed with a CRIP TP from a spider selected from the TP - ( L ) - TP - ( L ) , ( L - TP ) n - L , ( L - TP - L )n , (ERSP ) - ( TP - L )n , Australian Funnel- web spiders of genus Atrax or Hadro (ERSP ) - ( L ) - ( TP - L ) , (ERSP ) - ( L - TP ). , (ERSP ) - ( L - TP ) . nyche or a sea anemone selected from Anemonia viridis . The ( L ) , (ERSP ) - (STA ) - ( L - TP ) n . (ERSP ) - ( STA ) - ( L - TP ). - ( L ), transgenic plant can produce an insecticidal and / or nemati ( ERSP ) - (STA ) - ( TP - L ) , or ( ERSP ) - ( STA ) - ( L ) - ( TP - L ) n , or cidal protein comprising one or more ICK TPs , for example , ( STA ) -( L - TP ) n, or ( STA ) -( L - TP )n -( L ), or (STA ) - ( TP - L ) n , or Hybrid - ACTX - Hvla . The CRIP TP can be an ICK TP or (STA )- ( L ) -( TP - L ) n, wherein ( L ) is a binary peptide or a Non -ICK TP that when expressed contains 20 - 100 amino tertiary peptide, and which each L may be the same or acids and 2 - 4 disulfide bonds . The TPs useful in the trans different, TP is a toxic peptide, which may be the same or genic peptides disclosed herein can have at least 50 % , 55 % , different, and n is an integer ranging from 1 to 200, prefer 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , ably 1 to 100 , and most preferably , 1 to 10 . In some 98 % , or 99 % or greater sequence identity to SEQ ID NOs: embodiments , the insecticidal and / or nematicidal protein 5 or 6 , SEQ ID NO : 33 - 1032 and SEQ ID NO : 1761 - 1775 may contain TPs that are the same or different, and binary and variants thereof. In various embodiments , one or more and /or tertiary peptides that are the same or different. In of the CRIP TPs become active when the insecticidal and/ or some embodiments , the C -terminal TP is fused or unfused at nematicidal protein 's binary and /or tertiary peptides are its C - terminus with a binary or tertiary peptide . The TPs cleaved by an appropriate protease found in the insect and/ or useful in the above constructs disclosed above and herein nematode gut environment, thereby releasing the one or can have at least 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , more TPs from the insecticidal and / or nematicidal protein to 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % or greater exert its insecticidal and / or nematicidal activity in the insect sequence identity to SEQ ID NOs: 5 or 6 , SEQ ID NO : 33 or nematode . and /or a TP selected from SEQ ID NOs: 33 - 1032 and [ 0340 ] ( 6 ) . Methods for Controlling a Pest Infection variants thereof. In related embodiments of the above, TPs [0341 ] In various embodiments of the present invention , useful in the manufacture of a DNA construct or polynucle methods are provided for controlling a pest infection or otide encoding : ( 1 ) an insecticidal and /or nematicidal pro infestation ( used interchangeably ) , wherein the pest may be tein , which may include one or more ICK TPs, for example , an insect, a nematode or both . In some embodiments , the a TP selected from an “ ACTX ” or “ ACTX peptide” family method for controlling such a pest infection includes pro of insecticidal ICK peptides that have been isolated from an viding in a diet of the pest , a plant, or part thereof, wherein Australian funnel -web spiders belonging to the Atracinae the plant or part thereof, contains and expresses a polynucle subfamily , for example , one or more TPs selected from an otide which encodes an insecticidal and / or nematicidal TP as Omega - ACTX TP , a Kappa - ACTX TP and an U -ACTX TP , described herein . In other embodiments , a pesticidally effec for example , U + 2 - ACTX -Hvla , U - ACTX - Hvla , tive amount of a composition comprising an insecticidal rU -ACTX -Hvla , rU - ACTX -Hv1b (wherein the TPs , if more and / or nematicidal protein is applied to a plant, prior to the than one, may be the same or different) , or a TP having at exposure to the pest , or during the pest infection or infes least 80 % , at least 85 % , at least 90 % , at least 95 % , at least tation , such that, the application of the insecticidal and /or 96 % , at least 97 % , at least 98 % , at least 99 % , or at least nematicidal protein containing composition reduces the 100 % sequence identity or sequence homology to a peptide number of pests affecting the plant, or the severity of the pest or protein having an amino acid sequence as set forth in SEO infection on the plant thus contacted with the composition , ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771 - 1773 , and variants and optionally enables the plant to survive and / or grow thereof , (wherein the TPs, if more than one , may be the same compared to similar plants exposed to the same pests in the or different ) and each insecticidal and / or nematicidal protein absence of the composition . further comprising one or more tertiary peptides ( designated [0342 ] In some embodiments, the plant expresses a poly above as L ), which may include the amino acid sequence of nucleotide which is operable to express an insecticidal a binary peptide selected from IFVRLR (SEQ ID NO : and / or nematicidal protein constitutively, or inducibly . The 1619 ) , ALKLFV (SEQ ID NO : 1605 ) , ALKFLV (SEQ ID polynucleotide may be operably linked to an operable pro NO : 1603 ), RRKAFV (SEQ ID NO : 1644 ), or LFAAPF moter, for example , a homologous or heterologous pro (SEQ ID NO : 1621 ), fused in frame either on the N - termi moter, wherein the polynucleotide encodes an insecticidal nus , C -terminus or both termini of the binary peptide with a and / or nematicidal protein having one or more cleavable Z region or spacer sequence selected from the group of or binary or tertiary peptides . The insecticidal and / or nemati consisting of GS , WN , WQ , MA , MV, AM , LH , MN , ES , cidal protein can include the structure : ( ERSP ) - ( TP - L ) , , or WQ , or DT. In related embodiments, the method for con US 2018 /0362598 A1 Dec. 20 , 2018 54 trolling a pest infection provides expression and synthesis of [ 0344 ] In various embodiments , an illustrative insecticidal an insecticidal and / or nematicidal protein in a plant or part and /or nematicidal protein comprising a cleavable tertiary thereof, for example , a plant tissue , a plant cell , and /or a peptide having the following amino acid sequence construct : plant seed . The insecticidal and /or nematicidal protein com Z - X7 -X2 - Y - Y2, Z , - X -XZ - X3 - Y , - Y2, Z7- X , - X2 - X3- X4 prises one or more cleavable linker peptides fused in frame Y - Y2, Z7 - X , - X - Y - Y - Y3, 27 - X , -X2 - Xz - Y - Y2- Y3, to one or more TPs. In some embodiments , the cleavable linker peptide is a binary peptide comprising an amino acid 27- X , - X2- X3- X4 - Y - Y2- Y3 , Z7 - X , - X2- Y - Y2- Y3- Y4, sequence of: X -X2 - Y - Y2, X - X2-Xz - Y - Y2, X ,- X2- Xz Z7- X -X2 - X3 - Y - Y - Y3- Y4, 27- X , -X2 - Xz- X4 - Y - Y2- Yz X4- Y ,- Y2, X , -X2 - Y/ -Y2 - Y3, X ,- X7- X ,- Y - Y - Y3, X7- X , Y4, Z / -Z2 - X -X2 - Y - Y2, Z - Z2- X - X2- X3 - Y - Y2 , Z -Z2 X3- X4 - Y - Y2 - Y3, X7 -X2 - Y - Y2 - Y3- Y4, X - X2- Xz - Y - Y2 X -X2 - X3 - X4- Y -Y2 , Z -Z2 - X -X2 - Y - Y2 - Y3, Z1- Z2 - X / Y3 - Y4, X -X2 - X3- X4 - Y - Y2- Y3 - Y4, Y1- Y2- X -X2 , Y1- Y2 X -Xz - Y - Y - Y3, Z7- 22- X , -X2 - X3 - X4 - Y - Y2- Y3, 27- 22 X - X2- X3 , Y - Y2- X7- X , -X3 - X4, Y - Y2- Y3 - X -X2 , Y - Y2 X -X2 - Y - Y2 - Y3 - Y4 Z -Z2 - X -X2 - Xz- Y -Y2 - Y3 -Y4 , Y3- X7 - X , -X3 , Y - Y2- Y3- X7 -X2 - X3- X4 , Y - Y2- Y3- Y4- X - Z -Z2 - X7 -X2 - X3- X4 - Y - Y2- Y3- Y4, Z -Z2 -Zz - X - X2- Y1 X2, Y - Y2- Y3- Y4- X - X - X3, Y - Y2 -Y3 - Y4 - X -X2 - X3- X4 , Y2, Z - 22- 23 - X - X2- Xz - Y - Y2, Z -Z2 - 23 - X , -X2 - X3 -X4 wherein each Xn and each Y ,, is an amino acid , and wherein Y - Y2, Z -Z2 - 23 - X - X2- Y - Y2- Y3, Z1- Z2 - 23 - X -X2 - Xz each Xn and each Yn is an amino acid , and wherein X is Y - Y2- Y3, Z1- Z2 -23 - X - X2 -X3 - X4 - Y - Y - Y3, Z -Z2 -23 selected from the group consisting of: ( all single letter amino X -X2 - Y - Y2 - Y3 -Y4 , Z _ -Z2 - Zz - X , -X2 - Xz- Y - Y2- Y3- Y4, acid format) A , D , E , G , I, K , L , N , P , Q , R , and V ; X , is Z -Z2 - 23- X - XZ- X3 - X4- Y - Y2- Y3- Y4, Z - Z2 -23 - 24 - X , -X2 selected from the group consisting of A , F , G , I , K , L , N , P , Y - Y2, Z -Z2 -23 - 24 - X7 -X2 - Xz- Y -Y2 , Z , -Z2 - 23- 24 - X7 - X2 Q , R , S , T, V , and Y ; X2 is selected from the group consisting X3- X4 - Y - Y2 , Z -Z2 - 23 - 24- X , -X2 - Y - Y2 - Y3 , Z1- Z2 - 23 - 24 of A , D , E , F , G , I , K , L , N , Q , R , S , T , V , and Y ; X4 is X , -XZ - X3 - Y , - Y2- Y3, 21- 22- 23 - 24 - X , - X - X7- X4 - Y - Y2 selected from the group consisting of: R , H , K , D , E , S , T , Y3, Z , -22 - 23 -24 - X - X2- Y -Y2 - Y3- Y4, Z , -Z2 - 23- 24 - X - X2'N X3 - Y - Y2- Y3 - Y4, 27 -Z2 -23 - 24 - X , - X2- Xz - X4- Y - Y2- Yzw N , Q , G , A , I , L , M , F , W , Y , and V . In some embodiments , Y4, X - X - Y - Y2- 21 , X , - X2- Xz - Y - Y2- Z1, X , - X - X7 -X4 Y is selected from the group consisting of: (all single letter Y - Y2- Z1 , X - X2- Y - Y - Y3- 21 , X - X2 -Xz - Y - Y2- Y3- Z1 , amino acid format ) A , D , E , F , G , I , L , N , R , S , T , V , and Y ; X -X2 - X3 - X4 - Y - Y2- Y3- 21 , X - X2 - Y , -Y2 - Yz - Y4 - 21 , Y , is selected from the group consisting of F , G , I , L , and Y ; X -X2 - Xz- Y - Y2- Y3- Y4- Z1 , X -X2 - X3- X4 - Y - Y2- Y3 - Y4 and Yz is selected from the group consisting of A , D , E , F , Z1, Z , - X , -X2 - Y , - Y - Z1, Z , - X , - X - X , - Y - Y ,- 21, Z , - X , G , I , K , L , R , S , T , V , and Y ; and Y4 is selected from the X -X3 - X4 - Y - Y2- 21 , Z7 - X , -X2 - Y -Y2 - Yz- Z1, Z , - X - X2 group consisting of: R , H , K , D , E , S , T , N , Q , G , A , I , L , Xz- Y - Y2- Yz- Z1 , Z , - X , -X2 - Xz -X4 - Y - Y2- Y3 - 21, 21- X , M , F , W , Y , and V . X2- Y - Y2- Y3 - 94 -21 , 21- X , - X2- Xz - Y - Y2- Y3- Y4- 21 , [ 0343] In some embodiments , an illustrative insecticidal Z - X -X7 - X3 - X4 - Y - Y2- Y3- Y4- 21 , 2 ; - 22- X -X2 - Y - Y - and / or nematicidal protein for use in a method to control a Z1, Z1- Z2- X , -X2 - X3 - Y -Y2 - 21 , 21- 22- X -X2 - X3 - X4- Y , pest comprises one ormore illustrative binary peptides (XY Y2- 21 , 21- 22 - X7- X2- Y -Y2 - Y3- 21, 21- 22- X -X7 - X3 - Y - or YX ) . In some embodiments , the insecticidal and / or nem Y2- Y3- 21 , 21- 22 - X , - X2- X3- X4- Y -Y2 - Yz- 21, 21- 22- X , aticidal protein comprises a cleavable binary peptide having X2- Y - Y2- Y3- Y4- 21, 21- 22- X7- X - Xz- Y -Y2 - Y3 - Y4 - 21, the following amino acid sequence : AFVRLF (SEQ ID NO : Z -Z2 - X - X2- X3 - X4- Y - Y2- Y3 - 94- 21 , 21 -22 -23 - X -X2 - Y1 1594 ) , AKLFV ( SEQ ID NO : 1595 ) , ALFALK (SEQ ID Y2- Z1 , Z , -Z2 - 23 - X7- X , - X3- Y -Y2 - Z1 , Z / -Z2 -Z2 - X , - X2- Xz NO : 1596 ) , ALFLK ( SEQ ID NO : 1597 ) , ALFLR (SEQ ID X4 - Y - Y2- Z1 , Z1- Z2 - 23 - X -X2 - Y - Y2 -Y3 - 21 , Z - Z2- Zz - X - NO : 1598 ), ALFR (SEO ID NO : 1599 ) , ALFRLR (SEO ID X2- X2 - Y - Y2- Yz- Z1 , Z -Z2 - 23 - X -X2 - X3- 84 - Y - Y2- Y3 NO : 1600 ) , ALKALF (SEQ ID NO : 1601) , ALKFF (SEQ ID Z1, Z1- Z2 - 23 - X - X2- Y - Y2- Y3- 94- 21 , 21 -22 - 23 - X -X2 - Xz NO : 1602 ) , ALKFLV (SEO ID NO : 1603 ) , ALKIFV (SEO Y - Y - Y3- Y4- 21 , 2 ; - -23 - X - X2- X3 - X4- Y - Y - Y3 - Y4 ID NO : 1604 ) , ALKLFV (SEO ID NO : 1605 ) , FFADIK Z1, Z -Z2 - 23 - 24 - X -X2 - Y - Y2- Z1 , Z , -Z2 -23 -24 - X - X2- Xz (SEQ ID NO : 1606 ) , FFALK (SEQ ID NO : 1607 ) , FFLK Y - Y2- 21 , Z ,- Z2- 23 - 24 - X -X2 - Xz- X4 - Y - Y2- 21 , Z -Z2 - 23 ( SEO ID NO : 1608 ) , FFLR (SEO ID NO : 1609 ) , FFRLR Z4 - X - X2- Y - Y - Yz- Z1, Z -Z2 - 23- 24 -X7 - X2- Xz - Y - Y2- Yz ( SEQ ID NO : 1610 ) , FGYRIK (SEQ ID NO : 1611) , FLRLF Z1, Z -Z2 -23 - 24- X7 - X2- X3 - X4- Y - Y2- Y3 - Z1, Z -Z2 - 23- 24 ( SEO ID NO : 1612 ) , FYARR (SEO ID NO : 1613 ) , GGL X , -X2 - Y - Y2 - Y3 -Y4 - 21 , Z7 -Z2 - 23- 24 - X , - X2- X2 - Y - Y2 RKK ( SEQ ID NO : 1614 ) , IFVALK (SEO ID NO : 1615 ) , Yz - Y4- Z1 , Z - Z2- 23 -24 - X , -X2 - Xz -X4 - Y - Y2- Y3 - Y4- Z1 , IFVLK (SEQ ID NO : 1616 ) , IFVLR (SEQ ID NO : 1617 ) , X - X - Y - Y2- 2 , -Z2 , X -X2 - Xz- Y - Y2- 27 - Z2, X7 -X2 - Xz IFVR (SEO ID NO : 1618 ) , IFVRLR (SEO ID NO : 1619 ) , X4- Y -Y2 - 2 , - Z2, X , -X2 - Y - Y2- Yz- Z - Z2, X -X2 - Xz - Y - ILFNIK (SEQ ID NO : 1620 ), LFAAPF (SEQ ID NO : 1621 ), Yz- Y3- 2 -22 , X , -X2 - X3- X4 - Y -Y2 - Y3 - 2 , - 22, X , -X2 - Y , LFVALK (SEQ ID NO : 1622 ) , LFVLK ( SEQ ID NO : 1623 ) , Y2- Y3 -Y4 - 24 -Z2 , X , - X2- X3- Y -Y2 - Y3 -Y4 - 27 - Z2, X , - X2 LFVLR (SEQ ID NO : 1624 ) , LFVR (SEQ ID NO : 1625 ) , X3- 84 - Y - Y2- Y3 - Y4 - 24- Z2 , Z , - X , -X2 - Y - Y2- 27 -22 , LFVRLR (SEQ ID NO : 1626 ) , LFVRVFL (SEQ ID NO : Z7 - X , - X2- Xz- Y - Y - Z -Z2 , Z , - X , -X2 - X3- X4 - Y - Y2- 2 , -22 , 1627 ) , LGER (SEQ ID NO : 1628 ), LKALF (SEQ ID NO : 24- X ,- X2- Y - Y2- Y32, - Z2, Z7 -X , - X2- X3 - Y - Y2- Y3- 21 1629 ) , LKFF (SEQ ID NO : 1630 ) , LKIFV (SEQ ID NO : Z2, Z , - X , - X - Xz - X4 - Y - Y2- Yz- Z -Z2 , Z , - X - X2 - Y - Y2 1631 ) , LKLFV ( SEO ID NO : 1632 ) , LRALF (SEO ID NO : Y3- Y4- 27 - 22 , Z - X -X2 - X3 - Y -Y2 - Y3 - Y4 - 2 , - 22, Z7- X 1633 ) , LRFF (SEQ ID NO : 1634 ) , LRIFV ( SEO ID NO : X2- Xz - X4- Y -Y2 - Y3 -Y4 - Z -Z2 , Z - Z2 - X - X2- Y - Y - Z -Z2 , 1635 ), LRLFV (SEQ ID NO : 1636 ), RALF (SEQ ID NO : Z -Z2 - X , - X2- Xz - Y - Y2- 27 -Z2 , Z , -Z2 - X - X2- X3- X4 - Y - Y2 1637 ) , RIFV ( SEO ID NO : 1638 ) , RLFV (SEO ID NO : Z -Z2 , Z1 - Z2- X , -X2 - Y -Y2 - Y3 -24 - Z2 , Z , -Z2 - X -X2 - X3- Y - 1639 ), RLRALF (SEQ ID NO : 1640 ), RLRFF (SEQ ID NO : Yz- Y3- 2 -22 , 2 , - 22- X - X -X3 - X4 - Y - Y - Y3- 27 - 22, 22- 22 1641) , RLRIFV (SEQ ID NO : 1642 ) , RLRLFV (SEQ ID X - X2- Y -Y2 - Y3 -Y4 - Z -Z2 , Z - Z2- X , -X2 - X3 - Y - Y2- Y3 NO : 1643 ) , RRKAFV (SEQ ID NO : 1644 ) , RRKLIF (SEQ Y4- Z -Z2 , Z , -Z2 - X7- X2 - X3- X4 - Y - Y2 -Y3 - Y4- 2 , -Z2 , Z -Z2 ID NO : 1645 ) , RRRFFA ( SEQ ID NO : 1646 ) , VFGRKG Z3- X - X2- Y - Y2- 21- 22, Z1- Z2- 23- X - X2- Xz- Y - Y2- 2 - 22, (SEQ ID NO : 1647 ), and YFVRK (SEQ ID NO : 1648 ). Z - Z2- 23 - X - X2- X3- X4 - Y - Y - Z -Z2 , Z -Z2 - 23 - X7 -X2 - Y US 2018 /0362598 A1 Dec. 20 , 2018 55
Y2- Y3- 2 ; - Z2, Z - Z2- 23 - X , - X2- Xz- Y -Y2 - Y3 -24 - Z2 , Z1- Zz Y - Y2- Y3- 94- 21- 22- 23- 24, 27- 22 - 23- 24 - X -X2 - Y - Y2- 21 Zz- X -X2 - X3- X4 - Y - Y2- Y3- 2 , - Z2 , Z1- Z2 - Z2 - X , - X - Y - Y , Zz- Zz - Z4 , -22 - 23 - 24- X - X2- Xz - Y - Y2- 2 , -22 - 23 -24 , Y3- Y4 -21 - 22 , 22 -22 - 23 - X - X2- Xz - Y - Y2- Y3- Y4- 21 - 22 , Z -Z2 - 23 -24 - X -X2 - X3- X4 - Y - Y - 2 , -22 - 23- 24, Z -Z2 -23 Zj- Z2- 23 - X - X2- X3- X4 - Y - Y - Y3 - Y - Z -Z2 , Z -Z2 - 23 - 24 24- X , - X2- Y - Y2- Y3- 21- 22- 23- 24, Z , - 22- 23- 24- X , - X2- Xz X , -X2 - Y - Y - Z -Z2 , Z - Z2 -23 - 24- X , -X7 - X , - Y , - Y - Z - Z2 , Y - Y2- Y3 - Z -Z2 - 23 - 24 , Z _- Z2 - 23 - 24 - X , - X2- X7- X4- Y - Y2 Zj- Z2 - 23 - 24- X , - X - X3- X4- Y - Y2 -Zi - Z2, Z - Z2- Zz -Z4 - X1 Y3- 21- 22- 23- 24, Zj- Z2- 23- 24 -X -X2 - Y - Y2 - Y3 - Y4 -2 , -22 X - Y - Y2- Yz- Z -Z2 , Z - Z2- 23 -24 - X , -X2 - X3 - Y - Y2- Y3- 21 Zz- Z4 , Z -Z2 - 23 - 24 - X7- X2 - Xz- Y - Y2- Y3- Y4- 24 - Z2- 23 - 24 , Z2, Z - Z2 - 23- 24 - X -X2 - X3- X4 - Y - Y2 -Y3 - 2 -Z2 , Z -Z2 - Zz 21 -22 - 23 - 24 - X -X2 - X3 - X4- Y |- Y2- Y3- Y4- 21 - 22 - 23 - 24 24- X7- X , - Y , -Y2 - Y3 - Y4 - 27 - 22, Z , - 22- 23- 24- X , - X2- X3 - Y , Z - Y -Y2 - X , -X2 , Z / - Y -Y2 - X , -X2 - X3 , Z / - Y -Y2 - X , -X2 Yz- Y3 - Y4 - 2 , - 22, Z - Z2 - 23- 24- X , -X2 - X3 - X4 - Y - Y2- Y3- 94 X3- X4, 27 - Y , - Y2- Y3- X -X2 , Z - Y , - Y2- Y3- X , - X2- X3, Z1- Z2 , X -X2 - Y - Y2- 21 - 22 - 23 , X -X2 - X3 - Y - Y2- 21 - 22 - 23 , Z ; - Y - Y2- Y3- X - X2- X3- X4, Z - Y - Y2 - Y3 - Y4 -X -X2 , X - X2- Xz - X4- Y - Y2- Z -Z2 - 23 , X , -X2 - Y - Y2- Yz- Z - Z2- 23 , Z - Y - Y2- Y3- Y4- X , - X2- Xz, Z - Y - Y2- Y3- Y4- X , - X - Xz X - X - X2 - Y - Y - Yz- Z -Z2 - Z3, X ,- X - X2 -X4 - Y - Y2 - Yz X4, Z - Z2- Y -Y2 - X , - X2, Z , -Z2 - Y - Y2- X7 -X2 - Xz, Z -Z2 Z -Z2 - 23 , X7 -X2 - Y - Y2- Y3 - Y - Z -Z2 - Z3, X7 -X2 - X3 - Y Y - Y2- X -X2 - X3 - X4 , Z7- 22 - Y -Y2 - Y3 - X , -X2 , Z , -Z2 - Y - Y2 - Yz- Y4 - 2 , - -23 , X -X2 - X3- X4- Y - Y2- Yz- Y4 - 2 , - 22 -23 , Y2- Yz - X7 - X ,- Xz, 2 , -22 - Y - Y - Y3- X -X7 - X3 - X4 , Z _ -22 27 - X , - X2- Y -Y2 - 27 - Z2- Z3 , Z7 - X , - X2- X3- Y - Y2- 27 -22 - 23, Y - Y2- Y3- Y4- X -X2 , Z7 - 22- Y - Y2- Y3- Y4- X -X2 - X3 , Z ; - X7 -X2 - X3- X4 - Y - Y2- 27 -Z2 - Z3, Z7 - X , - X2- Y - Y2- Y3- 21 Z -Z2 - Y - Y2- Y3- Y4- X7 -X2 - X3- X4 , Z -Z2 -Zz - Y - Y2- X - Zz- Z3 , Z ,- X , - X - X , - Y - Y -Y3 - 27 - 22- 23, 27- X , -X7 - X , -X4 X2, Z7- 22 - 23 - Y - Y2- X , -X2 - X3 , Z - 22- 22 - Y -Y2 - X , -X2 Y -Y2 - Y3 - 2 - 2 -23 , 24- X -X2 - Y - Y - Y3- 94- 21 - 22 - 23, X3- X4 , 27- 22 - 23- Y - Y2- Y3- X -X2 , Z - Z2- 23 - Y -Y2 - Yz 2 , - X ,- X2- X - Y - Y2 - Y3 - Y4- 2 , -Z2 - Z3, Z7 -X , - X - X2 -X4 X - X - Xz, Z -Z2 - 23 - Y - Y - Y3 - X , - X - X7- X4 , Z -Z2 - Zz Y ; -Y2 - Y3 - Y4 - 2 , - 22- 23, 21- 22- X , -X2 - Y - Y2- 2 - 2 - 23, Y , - Y2- Y3- Y4- X , - X2, Z7 -Z2 - Zz- Y - Y2 - Yz- Y4- X , -X2 - Xz, Z1- Z2 - X7- X2- X3- Y - Y2- Z1 - Z2 - Z3, Z1- Z2- X7- X2- X3- X4- Y1 Z -Z2 - 23 - Y - Y2- Y3- Y4- X - X2- Xz - X4, Z - Z2- 23 - 24 - Y - Y2 Y2- 21 - 22 - 23, Z1- Z2 - X -X2 - Y - Y2 - Y3- 21 - 22 - 23 , Z -Z2 - X1 X -X2 , Z - Z2- 23- 24- Y - Y2- X -X2 - X3, Z - Z2 -23 - 24 - Y1 X2- X3- Y - Y2- Y3- 2 ; -Z2 - Z3 , Z -Z2 - X -X2 - Xz - X4- Y - Y2 Yz- X -XZ - X3 - X4 , 27- 22- 23 - 24- Y -Y2 - Y3- X , -X2 , 2 , -22 - 23 Y3- 2 -22 - Z3, Z7 -Z2 - X , - X2- Y , - Y2- Y3 - Y4 - 2 , -Z2 - Z3 , Z -Z2 24- Y , - Yz- Y3- X - X2- Xz, 27 - 22- 23- 24- Y - Y - Y3- X -X2 X - X2- Xz - Y - Y2- Yz- Y4- Z -Z2 -Z3 , Z1- Z2 - X - X2- X3 - X4 Xz- X4, Z _ -Z2 - 23- 24 - Y - Y2- Y3 - Y4 -X7 - X2 , Z - Z2- 23 - 24 - Y - Y -Y2 - Y3 - Y4 - 2 , - 22- 23, Z7 - 22- 23- X -X2 - Y - Y - Z - Z2- 23, Y2- Y3- Y4- X , -X2 - Xz, 2 -22 - 23 - 24 - Y , - Y2- Yz - Y4- X , - X2 2 , -22 - 23 - X , - X2- X3- Y - Y2- 2 , - 22- 23, Z - Z2- 23- X -X2 - Xz X3- X4 , Y , - Y2- X -X2 - 21, Y - Y2- X -X2 - X3 - 21 , Y - Y2- X X - Y - Y2- 2 - 22- 23, 2 , - 22- 23 - X , - X2- Y - Y - Y3- 2 , -22 - 23 , X2- X3- X4 - 21, Y - Y2- Y3- X , - X2- 21 , Y - Y2- Y3- X -X2 - Xz 2 , - 22- 22 - X , -X2 - X2- Y - Y2- Y2- 2 , -22 - 23 , 27 -22 - 23 - X - X2 Zi, Y , - Y2- Y3- X -X7 - X -X4 - 21 , Y - Y2- Y3- YA- X , - X2- 21 , X7- X4 - Y - Y2- Y3 - 2 , -22 - 23, Z -Z2 - 23- X , - X - Y - Y2- Yz Y - Y2- Y3- Y4- X , - X2- Xz- 21 , Y - Y2- Y3 - Y4- X , - X - X3- X4 Y4- 2 , -22 - Z3, Z -Z2 - 23- X , -X7 - X , - Y - Y - Y3- Y4- 2 , -22 - 23 , Z1, Z , - Y -Y2 - X , - XZ- Z1 , Z - Y - Y - X , - X - Xz- Z1 , Z , - Y Z -Z2 - 23 - X - X2- Xz - X4- Y - Y2- Y3 - Y4- 24 - Z2 - Z3 , Z1- Z2 - Zz Y2- X - X2- X3 - X4- 21 , Z - Y - Y2 - Y3- X - X2- Z1 , Z - Y - Y2 24- X - X2- Y - Y - 2 - 2 -23 , Z1 -Z2 - 23- 24 - X , - X2- X2 - Y - Y2 Y3 - X , - X2- Xz- Z1 , Z / - Y - Y2- Y3- X -X2 - Xz -X4 - Z1 , Z - Y1 Z ; - Z2- 23, 21- 22- 23- 24 - X7- X ,- X3- X4 - Y -Y2 - 27 -22 - 23 , Y2- Y3 -Y4 - X -X2 - 21 , 21- Y - Y2- Y3- Y4- X -X2 - X3 -Z1 , 21- 22 - 23- 24 - X -X2 - Y - Y2- Y3- 2 , - 22- 23, Z4- 22 - 23 - 24 - X , Z - Y - Y2- Y3- Y4- X , - X2- X3 -X4 - Z1 , Z -Z2 - Y - Y2- X7 - X2 X2- X2 - Y - Y2 - Y3- 21 - 22- Z3 , Z -Z2 - 23 - 24- X , - XZ- X3 - X4- Y , Z1, Z , -Z2 - Y - Y2- X , - X2- Xz - Z1 , Zi -Z2 - Y - Y2- X , - X - Xz Y2- Y3- 2 , -22 - 23 , 2 - 22- 23 - 24- X , - X - Y - Y2- Y3- Y4- 2 -22 X4- 21 , 21- 22- Y - Y2- Y3- X7 -X2 - 21 , 2 , - 22- Y - Y2- Y3- X , Z3, Z - Z2 - 23- 24 - X , - X - Xz- Y - Y2 - Y3 - Y4 - 2 , -22 - 23, Z7 -22 X -Xz - Z1 , Z , -22 - Y - Y - Y3 - X7 - X -X3 - X4 - 21 , Z -Z2 - Y - 23- 24 - X , -X2 - X3- X4 - Y - Y2 - Yz- Y4 - 2 , -22 - 23, X - X2- Y - Y2- Y3- Y - X , -X2 - Z1, Z -Z2 - Y - Y - Y3 - Y4- X - X2- X2 - 21 , Y2- 2 , -22 - 23 - 24 , X , -X2 - X3 - Y , -Y2 - 2 - 22- 23- 24 , X . -X2 - Xz 2 , -Z2 - Y - Y - Y3- Y4- X -XZ - X3 - X4 - 21 , 21- 22 - 23- Y - Y2- X X4- Y - Y2- 2 , -22 - 23- 24 , X7- X - Y - Y2- Y3- 21 - 22 - 23- 24 , X2- Z1 , Z - Z2- 23 - Y - Y2- X , - X2- Xz- Z1 , Z / -Z2 - Zz - Y -Y2 - X , X -X2 - X3- Y - Y2- Y3- 2 , - 22- 23- 24 , X , -X2 - X3 - X4 - Y - Y2 X2- Xz - X4- 21 , Z , -Z2 - Z2 - Y - Y2- Y3 - X - X2- 21 , Z / -Z2 - Zz - Y Y3- 2 , -22 - 23 - 24 , X , -X2 - Y - Y2- Y3- Y4- 2 , - 22- 23 - 24, X , -X2 Y2- Y3- X - X2- Xz - Z1 , Z -Z2 - 23 - Y - Y2- Y3- X , - X2- Xz - X4 X2- Y - Y2 - Y3 - Y4- 2 , -22 - 23 - 24 , X ,- X2- X3 -X4 - Y - Y2 - Yz Z1, Z7 -22 - 23 - Y - Y2- Y3- Y4- X -X2 - 21, 21- 22- 23- Y - Y - Yz Y4- 21 - 22 -23 - 24 , Z , - X , -X2 - Y - Y - Z - Z2- 23- 24 , 27- X , - X , Y4- X7 - X , - Xz- Z1 Z -Z2 - Zz- Y -Y2 - Y3 -Y4 - X , -X7 - X7 -X4 Xz- Y1- Y2- 21- 22- 23- 24, Z _ -X -X2 - X3 -X4 - Y - Y2- Z1- Zz -Zz Z1, Z1- Z2 - 23- 24 - Y - Y2- X , - X2- Z1 , Z1- Z2 - 23 -24 - Y , - Y2- X - 24, Z7- X -X2 - Y - Y2- Y3- Z -Z2 - 23 - 24 , Z , - X - X2- X2 - Y - Y2 X -X3 - 21 , 22- 22- 23 - 24- Y - Y2- X -X2 - X3 - 84 - 21 , 21- 22 - 23 Y3- 21 - 22 - 23- 24 , Z - X - X2- X3- X4 - Y - Y2- Y3- 21 - 22 - 23- 24 , 24- Y -Y2 - Y3 - X -X2 - 21, 21- 22- 23- 24 - Y -Y2 - Y3 - X , - X - Xz Z - X -X2 - Y - Y2- Yz- Y4- 21 - 22 - 23 - 24, 27- X ,- X2- X3- Y - Y2 Z1, Z , - 22- 23- 24 - Y -Y2 - Y3 - X , -X2 - X3- X4 - 21 , 21- 22 - 23 - 24 Yz- Y4- 2 , - 22- 23 - 24 , Z7 - X , - X2- X3- X4- Y - Y - Y3- Y4- 2 , - 22 Y - Y2- Yz - Y4 - X , -XZ - Z1 , Z7 -Z2 - 23- 24 - Y - Y - Y3- Y4- X , Zz- Z4 , Z - Z2- X - X2- Y -Y2 - 2 , - -23 - 24 , Z _ -Z2 - X - X2- Xz X2- Xz- Z1 , Z - Z2- 23 - 24 - Y - Y2 - Y3- Y4 - X , -X2 - Xz -X4 - Z1 , Y -Y2 - 2 , -22 - 23 - 24 , Z - Z2- X , - X2- X3- X4- Y - Y2- 21 - 22- 23 Y - Y2- X , -X2 - 2 , -22 , Y - Y2- X , - X - X3- 2 , -22 , Y - Y2- X , Z4, Z - Z2 - X - X2- Y - Y2- Y3- 22- 22 -23 - 24, Z -Z2 - X - X2- Xz X2 -X3 - X4- 2 - 22 , Y - Y2- Y3- X - X2- 27 - 22, Y - Y2- Y3- X - Y - Y2- Y3- 21 - 22 - 23 - 24, Z _ - Z2- X - X2- X3- X4- Y - Y2- Y3- 2 ; X2 -X3 - 2 , -22 , Y - Y - Y3- X , -X2 - X3 -X4 - ZA - Z2 , Y - Y2- Y3 Z2- 23- 24, 27- 22- X -X2 - Y - Y2- Y3- 94- 21- 22- 23- 24, 27- 22 Y4- X -X2 - 27- 22 , Y -Y2 - Y3 - Y4 - X -X2 - X3 - 2 , - 22, Y - Y2 X - X2- Xz - Y - Y2- Yz- Y4- Z2 - Z2 - 23 -24 , Z _ -Z2 - X - X2- X3 - X4 Yz- Y4- X7 - X2- X3 - X4 -24 - Z2 , Z - Y - Y2- X , -X2 - Z -Z2 , Y -Y2 - Y3 - Y4 - 2 , - 22- 23- 24, 27- 22 - 23- X , -X2 - Y - Y2- 2 , 2 , - Y - Y2- X , - X - Xz- Z -Z2 , Z ,- Y - Y2 - X , -X2 - X2 -X4 - 2 ,- 22 , Z2- 23- 24 , 27- 22- 23 - X , - X2- X3 - Y - Y2- 27 - 22- 23- 24, 27- 22 Z - Y - Y - Y3- X -X2 - 2 , -22 , Z - Y - Y - Y3 - X - X - X3- 27 - 22, Zz- X , - X2- X2 - X4- Y - Y - Z -Z2 - Zz - Z4 , Z -Z2 - Z2 - X , - X2- Y Z - Y - Y - Y3 - X , - X - X7 -X4 - 24 - Z2 , Z - Y - Y - Y3- Y4- X , Y2- Y3- 2 ; - Z2- 23 - 24 , Z - Z2- Zz - X , - X2- Xz- Y - Y2- Y3- 2 , - 22 X2 - 2 , -Z2 , Z - Y - Y2- Y3 - Y4 -X7 - X2 - Xz- Z -Z2 , Z - Y -Y2 Zz- Z4 Z -Z2 - 23- X - X2- Xz- X4- Y - Y2- Y3 -24 - Z2 - 23 - 24 , Yz - Y4- X , -X2 - Xz -X4 - Z1 - Z2 Z -Z2 - Y - Y2- X - X2- 27 - 22 , 21- 22- 23- X - X2- Y - Y2- Y3- Y4- 21 - 22 -23 -24 , Zi -Z2 -23 - X - Zi -Z2 - Y1- Y2- X -X2 - X3 - 21 - 22 , Z1- Z2 - Y - Y2- X - X2- X3- X4 X2 -X3 - Y - Y2 - Y3 - Y4- 2 -22 - 23- 24 , Z _ -Z2 -23 - X -X2 - X3 -X4 Z -Z2 , Z -Z2 - Y - Y2- Y3- X - X2- 27- Z2, Z -Z2 - Y - Y - Y3- X , US 2018 /0362598 A1 Dec. 20 , 2018 56
X - Xz- Z - Z2, Z7 -22 - Y - Y2- Y3 - X - X2- X3 - X4- 24 -Z2 , Z1- Zz Zj- Z2 - Zz - Y - Y2 - Y3- X - X2- 21 - 22- 23 - 24 , Z - Z2- Zz - Y - Y2 Y - Y2- Y3 - Y4 - X , -X2 - 2 , -Z2 , Z -Z2 - Y - Y2 - Y3- Y4- X , -X2 Y3- X - X2- X3- 21 - 22- 23- 24, Z -Z2 -23 - Y - Y2- Y3- X -X2 - Xz X3- 21 - 22 , Z1- Z2 - Y - Y2 - Y3 - Y4 -X -X2 - X3- X4- 24 -Z2 , Z1- Z2 X4 -21 -22 - 23 - 24 , 21- 22 - 23 - Y |- Y2- Y3- Y4- X - X2- 21 - 22 - 23 Zz- Y - Y2- X7 - X2- Z - Z2, Z -Z2 - 23- Y - Y2 - X -X2 - X3- 2 - 22 , Z4, Z -Z2 - 23 - Y - Y2- Y3- Y4- X7 - X2- X3 - 21 -22 -23 - 24 , Z _ -Z2 Z - Z2 -22 - Y - Y - X ,- X7 - X ,- X4- 2 ,- 22, Z7 -22 -22 - Y , -Y2 - Yz Zz- Y - Y - Y3- Y4- X , - X2- X2 - X4- 21 - 22- 23 - 24 , Z - Z2- 23 - 24 X - X -ZA - Z2 , Z1- Z2 - 23- Y -Y2 - Y3 -X7 - X2 - Xz- Z -Z2 , Z1- Z2 Y -Y2 - X - X2- 27 - Z2- 23 - 24 , Z _ - Z2- 23- 24 - Y - Y2- X7 - X2- Xz Zz- Y -Y2 - Yz- X , -X2 - X3- X4 - 2 , -Z2 , Z - Z2- 23 - Y - Y2- Y3 - Y4 Z1- Z2 - 23- 24, Z -Z2 - 23- 24- Y - Y2- X - X2- Xz- X4- 21- 22- 23 X - X2- 21 - 22, Z1- Z2 - 23- Y - Y2- Y3- Y4- X - X2- X3- 21- 22, Z4, Z - 22 -23 -24 - Y - Y2- Y3- X -X2 - 21 - 22 - 23 - 24 , Z - Z2 -23 21- 22 - 23- Y -Y2 - Y3 - Y4 - X , -X2 - X3 - X4 - 2 , - 22, Z , -Z2 - 23 - 24 24- Y - Y - Y3- X -X2 - X3 - 21- 22- 23- 24, Z7 -22 - 23 - 24 - Y - Y2 Y -Y2 - X , -X2 - 2 , - 22, Z7 - 22- 23- 24- Y , - Y2- X , -X2 - X3 - 2 , - 22, Yz - X , -X2 - X3- X4 -24 -Z2 - 23 -24 , Z - Z2- 23- 24 - Y - Y2- Y3- Y4 Z1- Z2- 23- 24 - Y - Y2- X -X2 - X3- X4- 24 -Z2 , Zi- Z2- 23 -24 - Y , X - X2- 21- 22- 23- 24, Z ; - Z2 -23 -24 - Y - Y2- Y3 - Y4- X -X2 - Xz Y2- Y3- X - X2- Z - Z2, Z - Z2- 23 -24 - Y - Y2- Yz -X7 - X2- X3- 21 Z1- Z2 -23 - 24 , Z _ -Z2 - 23 - 24 - Y - Y2- Y3 - Y4 - X , -X2 - X3- X4 - 2 , Z2, Z , -Z2 - 23 -24 - Y - Y2- Y3 -X , - X - X2- X4 -2 , - 22, Z , - Z2- Zz Z2- 23 - 24 , 27- X , -X2 - Y - Y2, Z , - X , - X2- Xz - Y - Y2, Z , - X - Z4- Y - Y2 - Y3- Y4- X7 -X2 - 2 -22 , Z -Z2 - 23 - 24 - Y - Y2- Y3- Y4 X2 -X3 - X4 - Y - Y2, Z1- X - X2- Y - Y2- Y3, Z1 - X -X2 - X3- Y - X -X2 - X3 - 2 , - 22, Z - Z2- 23 - 24 - Y - Y2 - Y3- Y4 - X -X2 - X3 -X4 Yz- Y3, Z7 - X , - X2- X3 - X4 - Y -Y2 - Y3 , 27- X , - X2- Y - Y2- Yz Z - Z2, Y - Y2- X7 - X2- 2 , -22 - 23 , Y - Y2 - X , -X2 - Xz- Z - Z2- 23 , Y4, Z _ - X , - X2- Xz- Y - Y2- Y3- Y4, Z _ - X -X2 - X3- X4 - Y - Y2 Y - Y2- X , -X2 - Xz -X4 - 21 - 22 - 23, Y - Y2- Y3- X , - X2- Z1 - Z2 - 23 , Yz- Y4, Z _ -Z2 - X , -X2 - Y - Y2 Z -Z2 - X - X2- Xz - Y - Y2, Y - Y - Y3- X , -X2 - X2 - 2 , - 22- 23 , Y - Y2- Y3- X , - X2- Xz - X4 Z -Z2 - X , - X2- X3 - X4- Y , - Y2, Z -Z2 - X , - X2- Y , - Y2- Y3 , 21- 22 - 23, Y - Y2- Y3- Y4- X , -X2 - 2 , -22 - 23, Y - Y - Y3- Y4 Z -Z2 - X7 - X , -X3 - Y - Y2- Y3, Z -Z2 - X - X2 -X3 - X4 - Y - Y2 X , - X2- X2 - 2 , -22 - 23, Y - Y - Y3 - Y4- X , - X2- X3 - X4- 21 - 22- 23 , Y3, Z -Z2 - X , - X2- Y - Y - Y3- Y4, Z -Z2 - X , -X7 - X , Y , - Y2 Z - Y - Y2- X -X2 - 2 , - 22- 23 , Z - Y - Y2- X , - X -X3 - 21 - 22 - 23, Y3- Y4, Z _ -Z2 - X , -X2 - X3- X4 - Y - Y2- Y3- Y4, Z _ -Z2 - 23- X , Z - Y - Y2 - X7 -X2 - X3- X4 - 2 , -22 - Z3, Z - Y - Y2- Y3- X , -X2 - 27 X2- Y -Y2 , Z / -Z2 -23 - X , -X2 - X3 - Y - Y2, Z7- Z2- 23 - X - X2 Z2- 23 , 24- Y - Y2- Y3- X -X2 - X3 - 2 ,- 22 - 23, 2 ,- Y - Y2- Y3- X , X3- X4 - Y -Y2 , 2 , - 22- 23- X -X2 - Y - Y2- Y3; Z7 -22 - 23 - X , X2- X3 -X4 - 2 , - 22- 23 , Z - Y - Y2- Y3- Y4- X -X2 - 27- - 23 , X2- X3 - Y - Y - Y3, Z -Z2 - 23- X -X2 - X3 -X4 - Y - Y2 - Y3, Z - Y - Y - Y3- Y4- X , - X2- X2 - 27 -Z2 - Z3 , Z - Y - Y2- Y3- Y4 Zi -Z2 - 23 - X , - X2- Y - Y - Y3- Y4, Z _ - Z2- 23 - X , - X2- X2 - Y - Y2 X - X2- Xz - X4- 24 - Z2- Z3 , Z1- Z2 - Y - Y2- X -X2 - X3- 24 - Z2 - 3 , Yz - Y4, Z _ -Z2 - 23- X - X2- X3 - X4- Y - Y2- Y3 - Y4, Z - Z2- 23 -24 Z -Z2 - Y - Y2- X -X2 - Xz- X4 - 24- Z2 - Z3 , Z -Z2 - Y - Y2- Y3- X , X -X2 - Y - Y2 , Z , -Z2 - 23 - 24- X , - X2- Xz - Y - Y2, 21- 22- 23 -24 X2- 2 , -22 - 23 , Z ; - Z2- Y - Y2- Y3- X -X2 - Xz - Z - Z2- 23 , Z1 -Z2 X -X2 - X3 -X4 - Y - Y2 21- 22- 23 - 24 - X -X2 - Y - Y2- Y3, Y - Y - Yz- X -X2 - Xz -X4 - 2 , - -23 , Z - Z2 - Y -Y2 - Y3- Y4 Z -Z2 - 23 -24 - X -X2 - Xz- Y - Y2- Y3, Z , -Z2 - 23 -24 - X7 - X2- Xz X -XZ - ZA - Z2 - Z3 , Z -Z2 - Y - Y - Y3- Y4- X7 - X - X3- 2 , -22 - 23 , X4- Y / -Y2 - Y3 , ?7 - Z2- 23- 24- X , - X2- Y - Y - Y3 -Y4 , Z7 - Z2- 23 2 , -22 - Y - Y - Y3- Y4- X -X7 - X3 - X4 - 27- 22 - 23, 2 ,- 2 ,- 23- Y , 24- X -X2 - Xz - Y - Y - Y3- Y4, 27- 22- 23 - 24- X -X2 - Xz - X4 Y2- X , - X -Z2 - Z2 - Z3, Z -Z2 - Zz - Y - Y2 - X , - X - Xz- Z -Z2 - 23 , Y - Y2 - Y3- Y4 , Z - Y - Y2- X , -X2 , Z - Y - Y2 - X , -X2 - X3, Z -Z2 - 23 - Y - Y2- X - X2- Xz - X4- 24 - Z2 - Z3, Z1- Z2 - Zz - Y - Y2 Z - Y - Y2 - X -X2 - X3- X4 , Z - Y -Y2 - Y3- X7- X2 , Z , - Y -Y2 Y3- X - X2- 27 - Z2 -Z3 , Z -Z2 - Zz - Y - Y2- Y3- X - X2- X3 - 22 - 22 Y3- X - X2- X3 , Z - Y - Y2- Y3- X - X2- Xz - X4, Z - Y - Y2- Yz Z3, Z1 - 22 -23 - Y - Y2 -Y3 - X -X2 - X3 - X4- 24 -Z2 -23 , Z1- Z2 - 23 Y4- X -X2 , Z - Y - Y2- Y3- Y4- X - X2- X3, 2 , - Y - Y2- Y3- Y4 Y - Y2 - Y3- Y4 - X , -X2 - 22 - 22 - 23, Z -Z2 -23 - Y - Y2- Y3- Y4- X - X7 - X ,- Xz -X4 , 2 , - 22- Y - Y2- X -X2 , Z7- 22- Y -Y2 - X , -X2 X2- X2 - 27 -Z2 - Z3, Z -Z2 - Zz - Y - Y - Y3 - Y4- X , - X2- X2 - X4 -24 Xz, Z -Z2 - Y - Y2- X , - X ,- X3- X4, Z -Z2 - Y - Y2- Y3- X , - X2, 22- 23 , 2 , - 22- 23- 24- Y - Y2- X -X2 - 27- 22- 23, 21- 22- 23 - 24 Z -Z2 - Y - Y2- Y3- X , -X2 - Xz, Z -Z2 - Y - Y2- Y3- X , - X - Xz Y -Y2 - X , - X -X2 - 27- 22- 23, Z - Z2- 23 - 24- Y - Y , - X , -X7 - Xz X4, Z -Z2 - Y - Y2- Y3- Y4- X , -X2 , Z - Z2 - Y -Y2 - Y3 - Y4 - X , X4 - 2 , - 22- 23 , Z1- Z2 - 23 - 24- Y - Y2- Y3- X - X2- 2 , -22 - 23 , XZ- X3 , 27 - 22- Y - Y - Y3- Y4- X -XZ - X3 - X4 , Z -Z2 - 23 - Y - 2 , -22 - 23 -24 - Y - Y - Y3 - X - X - X3- 2 , - 22- 23 , 2 , -22 - 23 Y2- X , -X2 , Z , -22 - 23 - Y - Y2- X , - X2- X3, Z1 -Z2 - 23 - Y - Y2 Z4- Y - Y2 - Y3- X - X2- X3 - X4- 24 - Z2 -23 , Z , -Z2 - 23- 24 - Y - Y - X -X2 - X3 - X4 , Z _ - 22- 23- Y - Y2- Y3- X - X2, Z7 -22 - 23 - Y - Yz- YA- X , -X2 - X3 - 21 - 22 - 23 , 2 , - 22- 23 - 24- Y - Y2- Y3- Y4- X , Y2- Y3- X , -X2 - Xz, Z -Z2 - 23 - Y - Y2- Y3- X , - X2- X3 - X4, X -X3 - 2 ,- 22- 23, Z , - Z2- 23- 24- Y - Y2- Y3- Y4- X , - X2- X3- X4 2 , -22 - 22 - Y - Y - Yz- Y4- X -X2 , Z , - Z2 -23 - Y - Y - Y3- Y4- X , 21- 22 - 23, Y - Y2- X -X2 N - 27- 22 - 23- 24, Y - Y2- X -X2 - X3 - 2 ; X -X3 , 2 , -22 - 23 - Y - Y2- Y3- Y4 - X7 - X - X3- X4 , Z -Z2 - -24 Z2- 23- 24, Y -Y2 - X , -X2 N - X3 - X4 - 2 , - 22- 23- 24, Y , -Y2 - Yz - X , Y , - Y2- X , -X2 , Z7- 22- -24 - Y - Y2- X7 - X , - X3, Z7 -22 - 23- 24 X2- Z -Z2 - 23 - 24, Y - Y2N - Y3 - X - X2- Xz - Z -Z2 - 23 - 24, Y - Y2 Y , - Y2- X - X2- X3 - X4 Z - Z2 -23 - 24 - Y - Y2- Y3 - X , - X2, Yz- X , - X2- X3- X4- 24 - Z2- 23- 24, Y - Y2- Yz- Y4- X , -X2 - 27 - 22 Z1- Z2 -23 - 24 - Y - Y2- Y3- X - X2- X3 , Z -Z2 - 23- 24 - Y - Y2- Yz Zz- Z4, Y - Y2- Y3- Y4 - X , -X2 - X3 - Z - Z2- 23 -24 , Y - Y2- Y3 X -X2 - X3- X4 , Z _ -Z2 - 23 - 24- Y -Y2 - Y3 -Y4 - X7 - X2, Z1- Z2- 23 Y4- X ,- X2 - X2 - X4 - 2 , -22 - 23 - 24 , Z , - Y - Y2- X -X2 - 2 , - 22 -23 24- Y - Y2- Y3- Y4- X - X2- X3 , Z - Z2- 23 -24 - Y - Y2 - Y3- Y4 Z4, Z - Y - Y2 - X , - X - Xz - Z - Z2- 23 -24 , Z - Y - Y2- X , - X2- Xz X , - X2- X3- X4 , X , - X - Y - Y -Z1 , X , - X - X2 - Y - Y2- Z1 , X4- 27 - 22 - 23 - 24 , Z - Y - Y2- Y3 - X -X2 - 27 -22 - 23 - 24 , Z -Y - X7- X2- X3- 84 - Y - Y2- Z1, X , - X2- Y - Y2- Y3 -Z1 , X7 -X2 - Xz Y2- Y3 - X , -X2 - X3 - 2 , - 22- 23- 24 , Z - Y -Y2 - Y3 - X , - X -Xz Y - Y -Y3 - Z1, X , -X2 - X3 - X4 - Y - Y - Yz- 21 , X , -X2 - Y -Y2 X4 - 2 , - 22- 23 - 24 , Z - Y - Y2 - Y3- Y4- X , - X2- 2 , -22 - 23 - 24 , Y3- 94- 21 , X ,- X2- Xz - Y -Y2 - Y3- Y4 -21 , X , -X2 - X3- X4- Y , Z ;- Y - Y2- Y3- Y4- X ,- X2- X3- 2 , - 22- 23- 24, Z - Y - Y2- Y3 - Y4 Y2- Y3 - Y4- 21 , X ,- X2 - Y - Y2- 2 , -Z2 , X -X2 - X3- Y - Y2- 2 , X -X2 - X3 - X4 - 21- 22 - 23- 24, Z _- 22- Y - Y2- X -X2 - 2 -22 - 23 Z2, X - X2- X3 - X4- Y - Y2- Z - Z2, X , -X2 - Y -Y2 - Y3- 2 -22 , Z4, Z -Z2 - Y -Y2 - X - X2- Xz - Z , -Z2 - 23 - 24, Z - Z2 - Y -Y2 - X - X -X2 - X3 - Y - Y2- Y3 - 24 - Z2 , X , -X2 - Xz -X4 - Y -Y2 - Y3 -21 X2- Xz - X4- 2 , - 22 -23 - 24 , Z - Z2- Y - Y2- Y3- X - X2- 2 , - 22- 23 Z2, X , - X - Y - Y2 - Yz - YA - 2 , - 22, X , - X -X2 - Y - Y - Y - YA Z4, Z -Z2 - Y - Y - Y3- X -X2 - X2 - 27- - 23- 24, 27- 22- Y - Y2 Z -Z2 , X -X2 - X3 - X4 - Y - Y - Y3- Y4- 2 -22 , X7 - X , - Y -Y2 Y3- X , - X2- X2 - X4- 21 - 22- 23 - 24 , Z _ -Z2 - Y - Y2 -Y3 - Y4- X7- X , 27- 22- 23 , X -X2 - Xz - Y - Y2- 2 , -22 - 23, X -X2 - X3 -X4 - Y - Z - Z2- 23 - 24 , Z -Z2 - Y - Y - Y3- Y4- X , -X2 - X3- 21 - 22 - 23 - 24 , Y2- 2 , -Z2 - Z3 , X7- X2- Y - Y2- Y3 - 21 - 22 - 23 , X -X2 - Xz - Y - Zj- Z2 - Y - Y2- Y3- Y4- X - X2- Xz - X4- 24 - Z2 -23 - 24 , Z - Z2- Zz Y2- Y3- Z -Z2 - Z3 , X7- X2- Xz- X4- Y1- Y2- Y3- 2 , -22 - 23 , Y -Y2 - X7 -X2 - 27 - 22- 23- 24, Z - Z2- 22- Y -Y2 - X7 - X ,- X3- 2 , X -X2 - Y - Y2 - Y3- Y4 - 2 -Z2 - Z3 , X7 -X2 - X3- Y - Y2- Y3- 94 Z2- 23- 24 , Z _ -Z2 - 23- Y - Y2- X - X2- X3 - X4- 21 - 22 - 23- 24 , Z - Z2- Zz , X -X2 - X3 -X4 - Y -Y2 - Y3 -Y4 - 27 -Z2 - Zz , X -X2 US 2018 /0362598 A1 Dec. 20 , 2018 57
Y - Y2- 2 -Z2 - Zz - Z4 , X7- X2 - X3- Y -Y2 - Z -Z2 -Zz - Z4 , X7- X2 Z -Z2 - 23 - 24- X - X2- Y - Y2- Yz- Z1, Z - Z2 -23 -24 - X , - X - Xz X3 -X4 - Y - Y2- 21 - 22 - 23 - 24 , X -X2 - Y - Y2- Y3 -21 - 22 - 23 - 24 , Y - Y2- Y3 -21 , 21- 22- 23 -24 - X , -X2 - X3 -X4 - Y - Y2- Y3- 21 , X - X2- Xz- Y - Y2- Y3- 21 - 22- 23- 24, X -X2 - X3 -X4 - Y - Y2 21 - 22- 23 - 24 - X , -X2 - Y - Y2- Y3- Y4- Z1, Z -Z2 - 23 - 24- X7 - X2 Y3 - 2 , - 22 -23 - 24, X -X2 - Y - Y2- Y3- Y4- 2 , -22 - 23 - 24, X -X2 Xz- Y - Y - Y3- Y4- 21 , 21- 22- 23- 24- X , - X2- X3- X4 - Y , -Y2 Xz- Y - Y - Y3- 94- 21- 22 - 23- 24, X , - X -X3 - X4 - Y , - Y2- Yz Y3- Y4 -Z1 , X , - X - Y - Y - Z , -Z2 , X , - X2- X3 - Y - Y2- 2 , -22 , Y4 -21 - 22 - 23 - 24, Y - Y2- X - X2- 21 , Y - Y2- X - X2- Xz- Z1 , X - X2- X3 - X4- Y - Y2- 27 - 22 X - X2- Y - Y2- Y3 - 2 , -22 , Y - Y2- X7 - X2- X3 - X4 -Z1 , Y - Y - Y3- X , - X -Z1 , Y - Y2- Yz X7- X2 - Xz- Y -Y2 - Y3 - 2 , -Z2 , X7- X2 - X3- X4 - Y - Y2- Y3 - 21 X - X2 -X3 - 21 , Y - Y2 - Y3- X7 - X , -Xz - X4- 21 , Y - Y2- Y3- Y4 Z2, X , -X2 - Y -Y2 - Y3 -Y4 - 24 -Z2 , X -X2 - X3 - Y - Y - Y3- Y4 X -X2 - 21 , Y -Y2 - Y3 - Y4 - X , -X2 - Xz - Z1, Y - Y2- Y3 -Y4 - X , 2 -22 , X -XZ - X3 - X4 - Y - Y - Y3- Y4- 27- 22, 27- X , - X2 - Y , X2- X3 - X4 -21 , Y , - Y2- X , - X2- 27 -22 , Y , - Y2- X , - X2- Xz- Z , Y2- 27 -22 , 27- X , -X2 - Xz - Y - Y2- 2 , -22 , Z7 - X , - X - X3 -X4 Z2, Y - Y2- X - X2- X3- X4- 24- Z2, Y - Y2- Y3- X - X2- Z - Z2, Y - Y2 -27 -Z2 , Z , - X7 -X2 - Y - Y2- Y3- Z -Z2 , Z7 - X , - X2- Xz Y - Y2- Y3 - X , - X2- Xz- Z -Z2 , Y - Y2- Y3 - X , - X2- X3- X4 -24 Y - Y2- Yz - Z -Z2 , Z , - X , -X2 - Xz - X4- Y -Y2 - Yz- Z -Z2 , Z2, Y - Y - Yz- YA- X , -X2 - 2 ,- 22 , Y - Y2- Y3- YA- X ,- X2- Xz Z ; - X -X2 - Y - Y2- Y3- Y4- 27 -Z2 Z - X , - X2- Xz - Y - Y2- Yz 2 , -22 , Y - Y2- Yz- Y4- X - X2- X3- X4- 2 , - 22, Y - Y2- X7 - X , Y4- 27 - Z2 , Z - X - X ,- X3- X4- Y -Y2 - Y3 - Y4 - 2 , -22 , 27- 22 - X , Z -Z2 - 23, Y - Y2- X -X2 - X3- 21 -22 -23 , Y -Y2 - X7 -X2 - Xz X - Y - Y2- Z - Z2 , Z -Z2 - X -X2 - X3- Y -Y2 - 2 , -Z2 , Z -Z2 - X1 X4- 24 -Z2 - 23, Y - Y2- Y3- X -X2 - 27 - Z2- 23 , Y - Y2- Y3- X , X2- X3- X4 - Y - Y2- Z -Z2 , Z1- Z2- X , -X2 - Y - Y2- Y3- 2 -22 , X - Xz- Z - Z2- 23 , Y - Y2 - Y3- X - X2- X3- X4- 21 - -23 , Z - Z2- X - X2- Xz - Y - Y2- Y3- 27 - Z2, Z1- Z2- X , -X2 - X3- X4 - Y - Y -Y2 - Y3 - Y4 - X , -X2 - 2 , - 22- 23, Y - Y -Y3 - Y4 - X , - X , -Xz Y2- Y3- 2 , - Z2, Z , -Z2 - X7 -X2 - Y - Y2 - Y3- Y4 - 2 , -22 , Z -Z2 - X , 21- 22- 23, Y , - Y2- Y3- 94- X -X2 - X3 - X4 - 21- 22- 23, Y - Y , X -Xz - Y - Y - Y3- Y4- 24- 22 , 27- 22- X -X7 - X3 - X4 - Y - Y2 X , - X2- 2 , - -23 - 24 , Y - Y2- X7- X , - X2- 27 -Z2 -23 - 24 , Y - Y - Yz- Y4- Z -Z2 , Z , -Z2 - Z2 - X7- X , - Y - Y2- 2 , -Z2 , Z -Z2 - 23 - X - X - X2- Xz - X4 -24 - Z2 - 23 - 24, Y - Y - Y3 - X , - X2- 21 - - 23- 24 X2- Xz- Y - Y2 - Z -Z2 , Z - Z2- Z2 - X , - X2- X3- X4- Y , - Y2- 24 - 22 , Y - Y2 - Y3- X - X2- Xz - Z -Z2 - 23- 24 , Y - Y2- Y3- X - X2- Xz Z - Z2- 23 - X , - X - Y - Y2- Y3- 24 - Z2 , Z -Z2 - 23 - X - X2- Xz - Y1 X4- 2 ,- 22- 23- 24 , Y - Y2- Y3- Y4- X - X2- 27 - Z2- 23- 24, Y - Y , Y2- Y3 - 2 -22 , 21- 22- 23 - X -X2 - X3- X4 - Y - Y - Y3- 2 , -22 , Yz- Y4- X , - X2- X3- 2 , - 22- 23- 24, Y - Y2- Y3 - Y4- X , - X2- Xz Z - Z2- 23- X -X2 - Y - Y2- Y3- Y4- 2 , - 22, 21- 22 - 23 - X , - X2- Xz X4- 2 , -22 - 23- 24 , Z7 - X , - X2- Y - Y2, Z7 - X , - X2- X2 - Y - Y2, Y - Y - Y3- Y4- 27- 22, 27 - 22- 23- X - X2- Xz- X4- Y - Y - Yz Z1- X -X2 - X3 - X4 - Y1- Y2, Z1- X - X2- Y - Y2- Y3, Z1- X7- X2 Y4- 27 - Z2 , Z / -Z2 - 23 - 24 -X7 - X2 - Y - Y2- 2 , -Z2 , Z , - Z2 -23 - 24 Xz- Y - Y2- Y3, Z - X , - X2- Xz - X4- Y - Y - Y3, Z7 - X , -X2 - Y - X -X2 - XZ- Y , -Y2 - 27 - Z2, Z -Z2 - 23 -24 - X , - X2- X3- X4 - Y - Y2 Y2- Y3 - Y4 , 27- X , -X7 - X3 - Y - Y2- Yz- Y4, 27 - X , -X2 - X3 - X4 Z -Z2 , Z7- 22 - 23- 24- X -X2 - Y - Y2- Y3- 27- Z2, Z7 - 22- 23- 24 Y - Y - Yz- Y4 , Z -Z2 - X , -X2 - Y - Y2, 24- Z2 - X - X2- Xz - Y - X -X2 - Xz- Y -Y2 - Y3 - 24- Z2 , Z - Z2- 23 -24 - X -X2 - Xz -X4 - Y , Y , Z -Z2 - X , - X - X7- X4- Y - Y Z - Z2- X , - X2- Y -Y2 - Y3, Y2- Y3 - 2 , - 22 21- 22 - 23- 24- X -X2 - Y - Y2- Y3- 94- 2 ; -22 , 2 , -22 - X -X2 - Xz - Y - Y - Yz, Z - Z2- X , -X2 - X3 - X4 - Y - Y2 2 , -22 - 23 -24 - X7- X , - X2- Y - Y2- Yz- Y4- 2 , - 22, 27- 22- 23 - 24 Y3, Z -Z2 - X , - X2- Y - Y - Y3 - Y4, Z - Z2- X , - X - X2- Y - Y - X -X2 - X3 - X4- Y - Y - Y3 -Y4 - 27 - 22, X , - X - Y - Y2- 21 - 22 Yz- Y4, Z _ -Z2 - X7 - X2- Xz - X4 - Y - Y2- Y3- Y4, Z _ -Z2 - Zz - X - Zz, X7- X2 - Xz- Y -Y2 - 24 - Z2 - Zz, X7- X2- Xz - X4- Y - Y2- 21 X - Y - Y2, Z - Z2- 23- X -X7 - X3 - Y - Y2, 21- 22- 23- X , -X2 Zz- Z3 , X7 -X2 - Y - Y2 - Yz- Z -Z2 - Z3 , X7 -X2 - Xz- Y - Y2- Yz X3 -X4 - Y - Y2 , Z -Z2 -23 - X -X2 - Y - Y2- Y3, Z - Z2- 23 - X - Z1- Zz - Z3, X -X2 - X3- X4 - Y - Y2 - Y3- 21 - 22- 23 , X - X2- Y - X2- X3 - Y - Y - Y3, Z -Z2 - 23- X - X2- X3 - X4- Y - Y2- Y3, Y2- Y3 - Y4 - 2 , - 22- 23, X -XZ - X3 - Y - Y - Y3- Y4- 2 - 22- 23, Zj- Z2- Zz - X , - X - Y , - Y2- Y3- Y4, Z _ -22 - 23- X , -X7 - X , - Y - Y X , - X2- X3- 84 - Y , - Y2- Y3 - Y4 - 2 , -Z2 - Z3 , Z7 - X , - X2- Y - Y2 Y3- Y4, 21- 22- 23- X -X7 - X7 - X4 - Y - Y2- Y3- Y4, Z - Z2- 23- 24 2 , -22 - 23 , 2 ,- X -X2 - X3- Y , - Y2- 2 , - 22- 23, 2 , - X , -X2 - X3 - X4 X7 - X , - Y -Y2 , Z , - Z2- 23 - 24- X , -X7 - X2 - Y - Y2, Z - Z2- 23- 24 Y -Y2 - 27 - 22- Z3, Z7- X , -X2 - Y - Y -Y3 - 2 , - 22- 23, 27- X , -X2 X - X2- X7- X4 - Y - Y Z - Z2- 23- 24- X , - X2- Y - Y2- Y3, X3- Y - Y2- Y3- 2 , -22 - 23 , 27 -X , - X - X3- X4 - Y - Y2- Y3- 2 ,- 22 2 , -22 - 23 -24 - X , - X2- XZ- Y - Y2- Y3, 2 , -22 - 23 -24 - X , - X2- Xz Zz, Z7 - X , -X2 - Y - Y2- Y3 - Y4 - 2 , -22 - 23 , Z , - X , -X7 - X - Y X4- Y - Y2- Y3 , Z7 -Z2 - 23- 24 - X7 - X2- Y - Y2- Y3 - Y4, Z _ -Z2 - Zz Y2- Y3- Y4- Z -Z2 - Z3, Z -X7 - X2 - X3- X4 - Y - Y2 -Y3 - Y4- 2 , -22 24- X , - X2- X - Y - Y2 - Y3 - Y4, 2 , - 22- 23- 24- X , - X2- X3- X4 Z3, Z , - Z2- X ,- X2 - Y -Y2 - 2 , -22 - 23, 2 ,- 22 - X , - X - Xz- Y - Y2 Y - Y2- Y3- Y4, X , - X - Y - Y - Z1, X -X7 - X - Y - Y2- 21 , 2 , -Z2 - 23 , Z - Z2- X , - X , -X7 - X4 - Y - Y2- 2 , - 22- 23 , Z1 -Z2 - X , X -X2 - X3- X4 - Y - Y2- Z1 , X7 -X2 - Y - Y2- Y3- Z1 , X - X2- Xz X2- Y - Y2- Y3- 2 ; - Z2- Z3, Z7 - Z2- X -X2 - Xz - Y - Y2- Y3- 21 - 22 Y , - Y2- Y3- 21 , X - X2- X3- X4 - Y , - Y2- Yz- 21, X , - X2- Y - Y2 Z3, Z ,- 22- X , - X2- X3- X4 - Y - Y2- Y3- 2 -Z2 -Z3 , Z , - Z2- X ,- X2 Yz - Y4- Z1 , X7 -X2 - X3 - Y - Y2- Y3- Y4- Z1 , X - X2- Xz - X4 - Y1 Y - Y2- Y3 - Y4 -Zi -Z2 - Z3, Z -Z2 - X , -X2 - Xz - Y - Y2- Y3- Y4- 21 Y2- Y3- Y4- 21 , Z7 - X , - X2- Y - Y2- 21 , 21- X7 -X2 - Xz- Y - Y2 Z2 -Z3 , Z , -Z2 - X7 - X2- X3- X4 - Y - Y2- Y3 - Y4 -24 - Z2 - Z3 , Z1- Z2 Z1, Z , - X7- X ,- X2- X4- Y -Y2 - Z1 , Z , - X -X2 - Y - Y - Y3- 21 , Zz- X - X2- Y - Y2- 21 - 22- 23 , Z -Z2 - 23- X -X2 - X3 - Y - Y2- 21 21 - X -X2 - X3 - Y - Y2- Y3 -21 , Z - X -X2 - Xz - X4- Y - Y2 - Y3 Zz - Z3, Z1- Z2 - 23- X - X - X3 -X4 - Y - Y2- 2 , -Z2 - Z3, Z -Z2 - Zz Z1, Z7 - X , - X2- Y - Y - Y3- Y4- Z1 , Z , - X , -X7 - X - Y - Y - Yz X , -X2 - Y - Y2 - Yz- Z -Z2 - Z3 , Z , -Z2 - 23 - X , - X - XZ- Y - Y - Yz Y4- 21 , Z , - X , -X2 - X3 - X4- Y -Y2 - Y3 -Y4 - 21 , Z -Z2 - X , - X2 21- 22- 23, 27- 22- 23- X , -X2 - X3- X4 - Y - Y2- Y3- 2 , - 22- 23, Y - Y2- Z1 , Z -Z2 - X , - X2- Xz - Y - Y2- 21 , Z -Z2 - X -X2 - Xz Z -Z2 - 23 - X - X2- Y - Y2- Y3- Y4- 21 - 22 -23 , 27 -22 - 23 - X -X2 X4- Y -Y2 - 21, 21- 22- X , -X2 - Y - Y2- Yz- 21 , 21 -22 - X7 - X , X3 - Y - Y - Y3 - Y - Z - Z2- Z3 , Z -Z2 - 23 - X -X2 - X3- X4 - Y - Y2 X3- Y -Y2 - Y3 - 21, 21- 22- X , -X2 - X3 - X4- Y - Y - Yz- 21, Y3- Y4- 2 , -22 - 23, 2 , - 22- 23- 24- X7- X ,- Y , - Y2- 27 - 22- 23, 21- 22 - X , - X2- Y - Y - Y3- Y4 - 21, 21- 22- X -X2 - X3 - Y - Y2 21- 22- 23- 24 - X -X2 - X3 - Y - Y2- 21- 22- 23 , 21- 22 - 23 - 24 - X , Yz - Y4- Z1 , Z , -Z2 - X , - X2- X3 - X4- Y - Y2- Y3 - 94- 21 , Z , -Z2 -Zz X2- X3- X4 - Y - Y2- Z -Z2 - Z3 , Z7 -Z2 - 23- 24 - X - X2- Y - Y2- Yz X , -X2 - Y -Y2 - 21, 21- 22 - 23- X7 - X - Y - Y , - Z1, Z , -Z2 - 23 - X , Z -Z2 - 23 , Z - Z2- 23- 24- X , -X2 - X3 - Y - Y2- Y3- 2 , -22 - 23 , X - X7- X4 - Y - Y2- 21 , 21- 22- 23 - X -X2 - Y - Y2 - Yz- 21 , 2 , -22 Z -Z2 - 23 - 24 - X , -X2 - X3- X4- Y3- 27 - 22- 23, 21- 22 - 23- 24 - X , Zz- X ,- X2- X - Y - Y2 - Y3 -21 , 22 -22 - 23- X -X2 - Xz- X4 - Y , X - Y - Y - Y3- 94- 21- 22 - 23, 24 - 22- 23- 24- X , - X -X3 - Y , - Y2 Y2- Yz- Z1 , Z -Z2 - Zz -X7 - X2 - Y - Y2- Y3 - 94 -21 , Zj- Z2 - Zz - X Yz - Y4- Z -Z2 - Z3 , Z1- Z2- 23 - 24 - X , -X2 - X3- X4 - Y - Y2- Y3- Y4 X2- Xz- Y - Y2 - Y3 -Y4 - 21 , 21- 22 - 23 - X -X2 - Xz -X4 - Y - Y2 Z -Z2 - Z3 , X -X2 - Y - Y2- 27 -22 - 23- 24 , X , -X2 - X3 - Y - Y2- Z1 Y3 - Y4- Z1 , Z -Z2 - 23 - 24 - X -X2 - Y - Y2- Z1 , Z -Z2 - 23- 24 - X Z2 -23 - 24 , X1- X2 - X3 - X4 - Y - Y2- 21 - 22 - 23 - 24 , X - X2- Y - Y2 X2- X3 - Y - Y2 -21 , Z -Z2 - 23 -24 - X7 - X2 -X3 - X4- Y - Y2- 21 , Y3- 21 - 22 - 23- 24 , X -X2 - X3 - Y - Y2 -Y3 - 2 - Z2- 23 - 24 , X - X2 US 2018 /0362598 A1 Dec. 20 , 2018 58
X3 -X4 - Y - Y2 - Y3 -24 - Z2 - 23 - 24, X - X2- Y - Y2- Y3 - Y4- Z -Z2 - Z7 -Z2 - Zz- Y - Y2 - Y3- X - X2- Xz- Z1 , Z -Z2 - 23 - Y - Y2 - Y3- X - Zz -Z4 , X , -X2 - X2 - Y - Y2- Yz- Y - 2 , -22 - 23- 24, X , - X2- Xz X2 -X3 -X4 -21 , 21- 22 - 23 - Y - Y2- Y3 -Y4 - X - X2- 21, 21- 22 - 23 X4- Y - Y2- Y3- Y4- 21 - 22 - 23- 24 , Z _ - X , - X2- Y - Y2- 21 - 22- Zz Y - Y2- Y3- Y4- X - X2- Xz- Z1, Z1 -Z2 -23 - Y - Y2- Y3- Y4 -X1 Z4, Z -X7 - X2- Xz - Y - Y2- 21 - 22 -23 - 24 , Z _ - X7 -X2 - X3- X4 - Y X2 -X3 - X4 -Z1 , Z -Z2 - 23 - 24- Y -Y2 - X7 - X2- 21 , 21- 22- 23 - 24 Y2- 21- - - 24, Z - X - X2- Y - Y - Y3- 2 ,- Z2- 23- 24, Z , -X , Y - Y2- X , -X2 - Xz -Z1 , Z - Z2- Zz - Z4 - Y - Y2 - X , - X - X7 -X4 - Z1 , X2- Xz - Y - Y2 - Y3- 21 - 22- 23 - 24 , 27- X7- X2 - X3- X4 - Y - Y2 Z -Z2 - 23 - 24- Y -Y2 - Y3- X , - X2- Z1, Z - Z2 -23 -24 - Y - Y2- Yz Yz- Z -Z2 - 23 - 24, 27- X , - X - Y - Y - Y3- Y4 -24 - Z2 - 23 - 24 X -X3 - 21 , 21- 22- 23- 24- Y - Y2- Y3- X - X2- X3- X4- 21 , 21- 22 Z1- X - X2 -Xz - Y - Y2- Y3- Y4- 21 - 22 - 23- 24 , 27 - X , -X2 - X3 -X4 Z3- 24 - Y - Y2- Y3- Y4- X - X2- Z1 , Z / -Z2 - 23 -24 - Y - Y - Y3- Y4 Y - Y2- Y3 - Y4 - 2 , -22 - 23- 24 , Z _ - Z2- X -X2 - Y - Y - Z -Z2 - Zz X7- X - Xz- 21, 21- 22- 23- 24- Y - Y2- Y3- Y4- X7 - X2 - X3 - X4 - 21, Z4, Z - Z2 -X - X2- X3 - Y , - Y2- 2 , -Z2 - Zz - Z4, Z -Z2 - X ,- X2 - Xz Y , - Y2- X , -X2 - 27 -22 , Y , - Y2- X , -X2 - Xz - Z -Z2 , Y - Y2- X , X4- Y - Y2- 21- 22- 23- 24, Z - Z2- X -X2 - Y - Y2- Y3 -21 - 22 - 23 X2- X3 -X4 - Z -Z2 , Y - Y2- Y3- X - X2- 27 - Z2, Y - Y2- Y3- X - Z4, Z -Z2 - X - X2- Xz - Y - Y2- Y3- Z - Z2- 23 - 24 , Z _ -Z2 - X , -X2 X -X3 - 27 - 22 , Y -Y2 - Y3 - X , -X2 - X3 - X4 - 2 - 22, Y - Y2- Yz Xz- X4- Y - Y2- Y3 -21 -22 - 23- 24, Z - Z2- X - X2- Y - Y - Y3 - Y4 Y4- X , - X2- 2 , - 22 , Y - Y2- Y3- Y4 - X , -X2 - X3- 2 , - 22 , Y - Y2 Zj- Z2- 23 - 24 , Z _ -Z2 - X - X2- X3- Y - Y2 - Y3 - Y4- Z2 - Z2 - 23- 24 , Y3 - Y4- X , -X2 - X3 -X4 - 2 , -22 Z - Y - Y2- X - X2- 2 -22 , 27- Z2- X7 - X ,- X3- X4 - Y -Y2 - Y3- Y4- 2 , - 22- 23- 24 , Z -Z2 - Zz 21 - Y - Y2- X - X2- Xz- Z ; -Z2 , Z , - Y - Y2- X - X2- X3 - X4- 2 , - 22, X - X - Y - Y2- 2 , -Z2 - 23 - 24 , Z - Z2- 23 - X -X2 - Xz- Y - Y2- 21 Z - Y - Y2- Y3 - X , -X2 - 27 -Z2 , Z - Y - Y2- Y3 - X , -X2 - Xz- Z -Z2 , Z2- 23- 24 , Z7 -Z2 - 23- X -X2 - Xz -X4 - Y - Y2- 2 , -22 - 23- 24 , Z - Y - Y2- Y3 - X , -X2 - X3- X4 - 27 -Z2 , Z - Y - Y2- Y3- Y4- X - 2 , -22 - 23 - X , - X2- Y - Y2- Y3- 2 , - 22- 23- 24, 2 ,- 22- 23- X - X , X - Z - Z2, Z - Y - Y2- Y3- 94- X , -X2 - X2 - 27- 22 Z - Y - Y - X3- Y -Y2 - Y3- 21- 22- 23- 24 , 27- 22 - 23- X -X2 - X3 - X4 - Y - Y2 Yz- Y4- X - X -X3 - X4 - 24- 22 21- 22 - Y -Y2 - X , -X2 - 27 - 22, Yz- Z , -Z2 - 23 - 24, Z -ZZ - Zz - X , - X - Y , - Y2- Y3- Y4- 2 , -Z2 - Zz Z -Z2 - Y - Y2- X , - X2- X2 - 27 -Z2 , Z , -Z2 - Y - Y2- X , - X2- X3- X4 Z4, Z - Z2- 23 - X , -X2 - Xz- Y - Y2- Y3- 94- 21 - 22 -23 - 24 , Z _ -Z2 Z -Z2 , Z , - Z2- Y -Y2 - Y3- X -X2 - 2 , -Z2 , Z1- Z2 - Y - Y2- Y3- X - Zz- X7- X2 - X3- X4 - Y - Y2- Y3 - Y4 - 2 , -Z2 - 23- 24, Z _ -Z2 - 23 - 24 X2- Xz- Z -Z2 , Z7 -Z2 - Y - Y2- Y3 - X - X2- X7 - X4- 24 -Z2 , Z , -Z2 X - X2- Y1- Y2- 21- 22- 23 -24 , Z1- 22 -23 - 24 - X -X2 - X3 - Y - Y2 Y - Y2- Y3 - Y4- X -X2 - 27 -Z2 , Zi -Z2 - Y - Y2- Yz- Y4- X - X2 Z - Z2- Zz- Z4 , Z ,- Z2- 23- 24 - X - X2- Xz - X4- Y - Y2- 2 , - Z2- Zz X3- 2 ,- Z2, Z ,- Z2 - Y - Y2- Y3- Y4- X7- X2- Xz- X4- 2 ,- 22, Z -22 24, 2 , - 22- 23 - 24- X , - X - Y - Y -Y3 - 2 , - 22- 23- 24 , 27 -22 - 23 Zz- Y , - Y2- X , - X2- Z - Z2, Z , -Z2 - Zz- Y -Y2 - X , -XZ - X3- 2 , -22 , Z4- X - X2- X3 - Y - Y2- Y3- 2 ; -Z2 - 23 -24 , Z -Z2 - 23 -24 - X -X2 Z -Z2 - Zz - Y - Y2 - X -X2 - X3- X4 - 24 -Z2 , Z , -Z2 - Zz - Y - Y2- Yz Xz- X4- Y - Y - Y3- 2 , -22 - 23- 24 , Z _ -Z2 - 23 - 24 - X , - X2- Y - Y2 X -XZ - Z -Z2 , Z , -Z2 - 23- Y , -Y2 - Y3 - X -X2 - X2- 27 -Z2 , Z -NZ2 Y3- Y4 - 2 , -22 - 23 - 24 , 27- Z2- 23 - 24- X -X2 - Xz - Y - Y2- Y3- Y4 Zz- Y - Y - Y3 - X -X2 - X3- X4 - 24 - Z2 , Z -Z2 -23 - Y - Y2- Y3- Y4 2 , -22 - 23 - 24 , Z _ - 22- 23- 24 - X , - X2- X3- X4- Y -Y2 - Y3 - Y4 - 2 , X , - X2- 2 , - 22 27- Z2 - 23- Y - Y - Y3- Y4- X -X2 - X3 - 2 , -22 , Z2- 23 - 24 , Z - Y - Y2- X , - X2, Z - Y - Y ,- X , - X ,- X3, Z , - Y , Z -Z2 - Z2- Y - Y -Y3 - Y4 - X , - X ,- X7- X4 - 2 , - 22, Z ; -Z2 - 23 - 24 Y2- X - X - X3- X4 , Z - Y - Y2 - Y3- X - X2, Z - Y -Y2 - Yz- X , Y -Y2 - X , -X2 - 24- Z2, Z7 - 22- 23- 24- Y - Y2- X , - X2- X3- 2 -22 , X - Xz, Z / - Y , - Y2- Y3 - X , - X - X7 -X4 , Z - Y - Y2 - Y3- Y4- X , 2 , -22 - 23- 24- Y -Y2 - X7 - X , -X3 - 84 - 24- 22 , 20- 22 - 23 - 24 X2, Z7 - Y , - Y2- Y3- Y4- X , - X2- X3 , Z - Y - Y2 -Y3 - Y4- X7 - X , Y - Y2- Y3 - X , - X2- Z -Z2 , Z - Z2- 23 -24 - Y - Y2 - Y3- X - X2- Xz X3- X4, 27- 22- Y - Y2- X , -X2 2 - 22- Y - Y2- X - X -X3 , Zj- Z2 , Z -Z2 -23 - 24 - Y - Y2- Y3- X , -X2 - Xz - X4- 24 - Z2 , Z , -Z2 Zj- Z2- Y - Y2- X7 - X2- X3 - X4, 27- Z2- Y -Y2 - Y3- X -X2 , Z3- 24- Y - Y2- Y3 - Y4- X7 - X2- 27 - 22, Z - Z2 -23 - 24 - Y - Y2- Y3 21- 22 - Y - Y2- Y3- X , - X - Xz, Z - Z2- Y - Y2- Y3- X , - X2- Xz Y4 - X -X2 - X3- 24 - 22, Z / -Z2 - 23- 24 - Y - Y - Y3 - Y4 - X -X2 - Xz X4, Z _ -Z2 - Y - Y - Y3 - Y4- X , - X2, Z - Z2- Y - Y2 - Y3- Y4- X , X4- 24 - Z2 , Y - Y2- X , -X2 - 2 , -22 - 23 , Y -Y2 - X , - X7- X2 - 2 , -Z2 X2- X3, Z -Z2 - Y - Y2 - Y3 -Y4 - X , -X2 - X3 - X4, Z - Z2- 23 - Y1 Zz, Y - Y2- X -X2 - X3 - X4 - 2 , -22 - 23, Y - Y2- Y3- X - X2- 27 Y2- X , -X2 , Z , -Z2 - Zz - Y - Y2 - X , -X2 - Xz, Z -Z2 - 23- Y , - Y , Zz- Zz , Y - Y2- Y3- X , - X2- X2 - 27 - - 23, Y , - Y2- Y3- X -X2 X , - X2- Xz- X4, Z -22 - 23 - Y - Y2- Y3- X7 - X2 , Z -Z2 -Zz - Y X3- 84 - 24 - Z2 - Zz , Y - Y -Y3 - Y4 - X7 -X2 - 2 , - 22- 23, Y - Y2 Yz- Y3- X -X7 - X3 , 21- 22 - 23- Y -Y2 - Y3- X -X2 - X3 - X4 , Y3- Y4- X , - X2- X3 - 2 , - 22- 23, Y - Y2- Y3 -Y4 - X , - X2- Xz - X4 Z -Z2 - 23 - Y - Y2- Y3- Y4- X - X2, Z / - Z2- Zz - Y - Y2- Y3- Y4- X - Zj- Z2 - Z3 , Z - Y - Y2- X7 -X2 - 2 , -22 - Z3 , Z - Y - Y2- X , -X2 - Xz X2- X3, Z -Z2 - Zz - Y - Y2- Y3- Y4 - X7 -X2 - Xz- X4 , Z -Z2 - 23- 24 Z -Z2 - Z3 , Z - Y -Y2 - X - X2- X7 - X4- Z -Z2 - Z3 , Z - Y - Y2- Yz Y -Y2 - X , -X2 , Z , - 22- 23- 24 - Y - Y ,- X , -X2 - X3 , 27- 22 - 23- 24 X -X2 - 27 - Z2 - Z3, Z - Y - Y - Y3- X -X7 - X , - 21- 22 - 23, Z - Y - Y - Y2- X -X7 - X3 - X4 , 27- 22- 23- 24- Y - Y2- Y3- X -X2 , Y2- Y3- X -X7 - X3 - X4 - 24- 22- 23, 2 , - Y - Y - Y3- Y4- X -X2 Zj- Z2- 23 - 24 - Y - Y2 - Y3- X , - X2- X3 , Z -Z2 - 23 -24 - Y - Y2- Yz Z -Z2 - Z3 , Z - Y - Y2- Y3- Y4- X7 -XZ - X3- 21 - 22 -Z3 , Z - Y , - Y2 X -X2 - Xz -X4 , Z _ -Z2 - 23 - 24 - Y - Y2- Y3 - Y4 -X7 - X2 , Z -Z2 - Zz Yz - Y4- X , -X2 - Xz -X4 - Z1 - Zz - Z3, Z1- Z2- Y -Y2 - X - X2- 27 -Z2 24- Y -Y2 - Y3- Y2 - X , - X2- Xz, 2 , - 22- 23- 24 - Y - Y2- Y3- Y4 23, 2 , - 22- Y - Y2- X -X7 - X3 - 2 ; - 22- 23, 2 ; -22 - Y - Y - X , - X2 X -X2 - X3- X4 , Y - Y2- X -X2 - Z , Y -Y2 - X7 - X2- X3 - 21, X3- X4 - 27 - Z2 - Z3, Z - Z2- Y - Y2- Y3- X - X2- 27 -Z2 - Z3 , Z7- 22 Y - Y2- X , - X - X2 -X4 - 21 , Y - Y2 - Y3- X ,- X2- 21, Y - Y - Yz Y , - Y2- Y3- X , -XZ - X3- 2 , -22 - 23 , 2 ,- 22 - Y - Y2 - Y3- X -X2 X , - X - Xz -Z1 , Y - Y - Y3- X , - X - X7 -X4 - 21 , Y - Y2- Y3- Y4 X3- X4 -24 - Z2 - Z3 , Z -Z2 - Y - Y - Y3 - Y4- X , - X2- Z -Z2 - 23 , X -X2 - 21 , Y - Y2- Y3- Y4- X7 -X2 - Xz- 21 , Y -Y2 - Y3 -Y4 - X - Zj- Z2 - Y - Y2- Y3- Y4- X - X2- Xz- Z - Z2- Z3, Z -Z2 - Y - Y2- Yz X2- X3 -X4 - 21 , Z - Y - Y2- X , - X2- 21 , Z - Y - Y2 - X7 -X2 - Xz Y4- X , - X2- X3- X4 - 2 , -Z2 - Z3 , Z - Z2- 23- Y -Y2 - X , - X2- 27 -22 Z1, Z ,- Y - Y2- X - X2- X3- X4 -Z1 , Z - Y - Y2 - Y3- X - X2- 21, Z3, Z1- Z2 - 23 - Y , -Y2 - X , -X2 - Xz - 2 - -23 , 2 , - 22- 23- Y - Y2 27 - Y , -Y2 - Y3 - X , - X - Xz- 21, 21 - Y -Y2 - Y3- X7- X ,- Xz -X4 X7 -X2 - X3 - X4 - 24- 22- 23, 2 , - 22- 23 - Y - Y - Y3- X , - X2- 2 , -22 Z1, Z7 - Y , - Y2- Y3- Y4- X - X2- Z1 , Z - Y - Y2- Y3- Y4- X , -X2 Z3, Z -Z2 - 23 - Y -Y2 - Y3 - X , -X2 - X3 - 21- 22- 23, 27 -22 - 23 - Y - Xz- Z1 , Z - Y - Y2- Y3- Y4- X , -X2 - Xz -X4 - Z1 , Z -Z2 - Y - Y2 Y2- Y3 - X , -X2 - Xz -X4 - 24 - Z2 - Z3, Z -Z2 -23 - Y - Y2- Y3- Y4- X - X - X2- Z1 , Z -Z2 - Y - Y2- X , - X2- Xz - 21 , Z -Z2 - Y - Y2- X X2- 2 , - Z2 -Z3 , Z - Z2- Z2 - Y - Y - Y3- Y4- X - X - X3- 2 , -22 - 23 , X - X7- X4 - 21 , 2 ; -Z2 - Y - Y2 - Y3- X - X2- 21 , 21- 22 - Y -Y2 Z - Z2- 23 - Y - Y2- Y3- Y4- X - X2- X3 -XA - Z -Z2 - Z3, Z - Z2- Zz Y3 - X -X2 -X3 - 21 , 21- 22 - Y - Y2- Y3 - X7 -X2 - X3- X4 - 21 , 24- Y , - Y2- X , -X2 - 2 , - 22 - Z3, Z -Z2 - 23- 24- Y - Y2 - X , - X2- Xz Z -Z2 - Y - Y2- Y3- Y4- X7 -X2 - Z1 Z1- Z2 - Y - Y2- Y3- Y4- X , 21- 22 - 23 ; 2 - 22- 23- 24- Y - Y2- X , -X2 - X3- X4 - 2 , - 22- 23, X2- Xz -Z1 , Z1- Z2 - Y - Y2- Y3- Y4 - X , -X2 - X3- X4 - 21 , 21- Z2 - Zz Z -Z2 - 23 -24 - Y - Y2- Y3 - X , - X - Z -Z2 - Z3 , Z , -Z2 -23 - 24 - Y1 Y - Y2- X -X2 -21 , Z -Z2 - 23 - Y - Y2- X -X2 - X3- 21 , 21- 22- 23 Y2- Y3 - X -X2 - X3- 2 - Z2- Z3, Z1 - Z2- 23 -24 - Y - Y2- Y3 - X - X2 Y - Y2- X7 - X , -X3 - X4 -Z1 , Z - Z2- 23- Y - Y2 - Y3- X - X2- 21 , X3- X4- 21 - 22- 23 , Z -Z2 - 23 - 24 - Y - Y2- Y3 - Y4- X7 - X2- 2 , - 22 US 2018 /0362598 A1 Dec. 20 , 2018 59
ID NO : 1650 ) ALFALKGS (SEQ ID NO : 1651) , Z3- 24 - Y - Y2- Y3- Y4- X -X2 - X3- X4 - 24- Z2 - Z3 , Y - Y2- X - ALFLKGS (SEQ ID NO : 1652 ), ALFLRGS (SEQ ID NO : X2- 22 - 22 - 23- 24 , Y -Y2 - X -X2 - X3 - 2 , -Z2 - 23 - 24 , Y - Y2- X , 1653 ) , ALFRGS (SEQ ID NO : 1654 ) , ALFRLRGS (SEQ ID X2- X3 - X - Z -Z2 - 23 - 24; Y , - Y2 - Yz - X , - X2- Z -Z2 - 23 - 24 , NO: : 16551655) ), , ALS ALKALFGS( SEQ ID NO (SEQ : 165ALFLRGS ID NO : 1656( SEÇ), ALKFFGS Y - Y2- Y3- X7- X2- X3- 2 ,- 22- 23- 24, Y - Y2- Y3 -X , -X2 - Xz (SEQ ID NO : 1657 ), ALKFLVGS (SEQ ID NO : 1658 ), X4- 21 - 22 - 23- 24 , Y , - Y2- Yz- Y4- X , - X2- 2 , - 22 -23 - 24 , Y - Y2 ALKIFVGS ( SEQ ID NO : 1659 ), ALKLFVGS ( SEO ID Y3- Y4 - X - X2 -X3 - 21 - 22 - 23- 24 , Y - Y2- Y3- Y4 - X7 -X2 - Xz NO : 1660 ) , FFADIKGS ( SEO ID NO : 1661) , FFALKGS X4- 24 - Z2 - 23- 24 , Z - Y - Y2- X -X2 - 27- Z2 - 23 - 24 , Z - Y - Y2 ( SEO ID NO : 1662 ) , FFLKGS (SEO ID NO : 1663 ) , FFL X -X2 - Xz - Z1 - Z2 - Zz - Z4 , Z , - Y - Y2- X7- X2- Xz- X4- 24 - Z2- Zz Z4, Z - Y - Y2 - Y3- X - X2- 21 - 22- 23 - 24 , Z _ - Y , - Y2- Y3- X , -X2 RGS (SEQ ID NO : 1664) , FFRLRGS (SEQ ID NO : 1665 ) , X2- 27 - - 23- 24 , Z - Y - Y - Y3- X , - X2- X7 -X4 - 21 - 22 - 23- 24 , FGYRIKGS (SER ID NO : 1666 ), FLRLFGS (SEQ ID NO : Z - Y - Y2 - Y3- Y4- X7 -X2 - Z -Z2 - 23- 24, Z - Y - Y2 - Y3- Y4- X / 1667) , FYARRGS ( SEQ ID NO : 1668 ) , GGLRKKGS (SEQ X2- X2 - 27 -22 - 23- 24 , Z - Y - Y2- Yz- Y4- X7 - X , - X7- X4 - 24 -Z2 ID NO : 1669 ) , IFVALKGS (SEQ ID NO : 1670 ) , IFVLKGS Zz- Z4, Z _ -Z2 - Y - Y2- X , -X2 - 2 , -22 - 23 -24 , Z _ -Z2 - Y - Y2- X1 ( SEQ ID NO : 1671 ) , IFVLRGS (SEQ ID NO : 1672 ), X2- X3 - 27 -Z2 - 23- 24 , Z _ -Z2 - Y - Y2- X7- X2- Xz- X4 - 2 , -22 - 23 IFVRGS (SEQ ID NO : 1673 ) , IFVRLRGS (SEQ ID NO : Z4, Z -Z2 - Y - Y - Y3- X -X2 - 27- 22 - 23- 24, Z - Z2- Y - Y2- Yz 1674 ) , ILFNIKGS (SEQ ID NO : 1675 ) , LFAAPFGS (SEQ X -X7 - X , - 2 - Z2 -23 - 24, 2 , -22 - Y - Y2 - Y3- X , - X2- X7- X4 - 2 ; ID NO : 1676 ) , LFVALKGS (SEQ ID NO : 1677 ) , LFV Zz- Zz - Z4, Z - Z2 - Y - Y - Y3 -Y4 - X , - X2- 27 -Z2 -23 - 24 , Z -Z2 LKGS ( SEO ID NO : 1678 ) , LFVLRGS (SEO ID NO : Y - Y2- Y3- Y4- X7 - X2- Xz - ZA - Z2 - 23 -24 , Z _ -Z2 - Y - Y2- Y3- Y4 1679 ) , LFVRGS ( SEO ID NO : 1680 ) , LFVRLRGS (SEO ID X - X2- Xz - X4- 24 - Z2- 23 - 24, Z - Z2- Zz - Y - Y2- X - X2- 2 , - 22 NO : 1681) , LFVRVFLGS (SEQ ID NO : 1682 ), LGERGS Zz- Z4, Z _ -Z2 - 23- Y -Y2 - X7 - X2- X3 - 2 , -22 - 23 - 24 , Z _ -Z2 - 23 (SEQ ID NO : 1683 ) , LKALFGS (SEQ ID NO : 1684 ) , Y? - Y2- X -X2 - X3 -X4 -21 - 22 - 23 - 24 , Z1- Z2- 23 - Y1- Y2- Y3- X1 LKFFGS (SEQ ID NO : 1685 ) , LKIFVGS (SEQ ID NO : X2- 27 -Y2 - 23- 24 , Z7 - 22- 23- Y - Y2- Y3- X - X -X3 - 21 - 22 - 23 1686 ) , LKLFVGS (SEQ ID NO : 1687 ) , LRALFGS (SEQ ID Z4, Z - Z2 -Zz - Y - Y2- Y3 - X - X2- X3 - X4- 24 -Z2 -23 - 24 , Z _ -Z2 NO : 1688 ) , LRFFGS (SEQ ID NO : 1689 ) , LRIFVGS (SEQ Z - Y - Y -Y3 - Y2- X , -X2 - 2 , - 22- 23- 24, Z - Z2- 23 - Y - Y -Yz ID NO : 1690 ) , LRLFVGS (SEQ ID NO : 1691 ) , RALFGS Y4- X7 - X , -X2 - 2 , - 22- 23- 24 , 27- 22 - 23- Y - Y2- Y3- Y4- X7 - X , (SEQ ID NO : 1692 ) , RIFVGS (SEQ ID NO : 1693 ) , X3- X4- 24 -Z2 - 23 - 24 , 27- 22 - 23- 24- Y - Y2- X -X2 - 27 - 22 - 23 RLFVGS (SEQ ID NO : 1694 ) , RLRALFGS (SEQ ID NO : 24, Z _ - 22- 23- 24- Y - Y2- X -X2 - X3 - 21- 22- 23- 24, 27- 22 - 23 1695 ) , RLRFFGS (SEQ ID NO : 1696 ) , RLRIFVGS (SEQ 24- Y - Y2- X - X2- X3 -X4 - 21- 22- 23- 24, Z -Z2 - 23 - 24 - Y - Y2 ID NO : 1697 ) , RLRLFVGS (SEQ ID NO : 1698 ) , RRKAF Y3- X , - X2- 27 - Z2 -23 - 24, Z -Z2 - 23 - 24 - Y - Y2- Y3- X , - X2- Xz VGS (SEQ ID NO : 1699 ), RRKLIFGS (SEQ ID NO : 1700 ), Z - Z2- 23 - 24 , Z - Z2- 23 - 24- Y -Y2 - Y3- X - X2- Xz- X4- 2 , - 22 RRRFFAGS ( SEQ ID NO : 1701 ) , VFGRKGGS (SEQ ID Zz- Z4, Z - Z2- 23 - 24- Y - Y2- Yz - Y4 - X , - X2- , - - 23- 24 , NO : 1702 ), YFVRKGS (SEO ID NO : 1703 ) , GSAFVRLF Z - Z2- 23 - 24 - Y - Y2 - Y3- Y4- X , -X2 - X3 - Z - Z2- 23 - 24 , Z - Z2 ( SEQ ID NO : 1704 ) , GSAKLFV (SEQ ID NO : 1705) , Z3- 24- Y -Y2 - Y3 - Y4- X -X2 - X3 - X4 - Z1- Z2- 23- 24, wherein GSALFALK (SEO ID NO : 1706 ) , GSALFLK (SEO ID NO : X , is selected from the group consisting of: (all single letter 1707) ,GSALFLR ( SEQ ID NO : 1708 ), GSALFR (SEQ ID amino acid format ) A , D , E , G , I , K , L , N , P , Q , R , and V ; NO : 1709 ) , GSALFRLR ( SEQ ID NO : 1710 ) , GSALKALF X , is selected from the group consisting of A , F , G , I, K , L , ( SEO ID NO : 1711 ) , GSALKFF ( SEO ID NO : 1712 ) , N , P , Q , R , S , T , V , and Y ; X2 is selected from the group GSALKFLV (SEQ ID NO : 1713 ) , GSALKIFV (SEQ ID consisting of A , D , E , F , G , I , K , L , N , Q , R , S , T , V , and Y ; NO : 1714 ) , GSALKLFV ( SEO ID NO : 1715 ) , GSFFADIK X4 is selected from the group consisting of: R , H , K , D , E , ( SEQ ID NO : 1716 ) , GSFFALK (SEQ ID NO : 1717) , S , T , N , Q , G , A , I, L , M , F , W , Y , and V . In some GSFFLK (SEO ID NO : 1718 ) , GSFFLR (SEO ID NO : embodiments , Y1 is selected from the group consisting of: 1719 ) , GSFFRLR ( SEO ID NO : 1720 ) , GSFGYRIK (SEO (all single letter amino acid format ) A , D , E , F , G , I, L , N , ID NO : 1721) , GSFLRLF (SEQ ID NO : 1722 ) , GSFYARR Q , S , T , V, and Y ; Y , is selected from the group consisting (SEQ ID NO : 1723 ) , GSGGLRKK (SEQ ID NO : 1724 ) , of F , G , I, L , and Y ; and Yz is selected from the group GSIFVALK (SEQ ID NO : 1725 ) , GSIFVLK (SEQ ID NO : consisting of A , D , E , F , G , I, K , L , R , S , T , V , and Y ; and 1726 ) , GSIFVLR (SEQ ID NO : 1727 ) , GSIFVR ( SEQ ID YA is selected from the group consisting of: R , H , K , D , E , NO : 1728 ) , GSIFVRLR (SEO ID NO : 1729 ) , GSILFNIK S , T , N , Q , G , A , I , L , M , F , W , Y , and V . (SEQ ID NO : 1730 ), GSLFAAPF ( SEQ ID NO : 1731 ), [ 0345 ] In related embodiments , themethod for controlling GSLFVALK ( SEQ ID NO : 1732 ) , GSLFVLK ( SEQ ID NO : a pest infection provides expression and synthesis of an 1733 ) , GSLFVLR (SEQ ID NO : 1734 ), GSLFVR (SEQ ID insecticidal and /or nematicidal protein in a plant or part NO : 1735 ), GSLFVRLR ( SEQ ID NO : 1736 ), thereof, for example , a plant tissue , a plant cell , and /or a GSLFVRVFL (SEQ ID NO : 1737 ) , GSLGER (SEQ ID NO : plant seed . The insecticidal and /or nematicidal protein com 1738 ) , GSLKALF (SEQ ID NO : 1739 ) , GSLKFF (SEQ ID prises a binary peptide as described above and can include NO : 1740 ), GSLKIFV (SEQ ID NO : 1741) , GSLKLFV a two amino acid spacer sequence ( a Z region ) fused in (SEQ ID NO : 1742 ) , GSLRALF (SEQ ID NO : 1743 ) , frame to the N - terminus and / or the C - terminus of an illus GSLRFF (SEQ ID NO : 1744 ) , GSLRIFV (SEQ ID NO : trative binary peptide disclosed above , wherein the two 1745 ) , GSLRLFV (SEQ ID NO : 1746 ) , GSRALF (SEQ ID amino acid spacer sequence ZZ, sequence can be one or NO : 1747 ) , GSRIFV ( SEQ ID NO : 1748 ) , GSRLFV (SEQ more of GS, WN , WQ, MA, and MV. ID NO : 1749 ) , GSRLRALF (SEQ ID NO : 1750 ) , GSRLRFF [ 0346 ] In some embodiments , the insecticidal and / or nem (SEO ID NO : 1751) , GSRLRIFV (SEO ID NO : 1752 ) , aticidal protein for use in methods described above can GSRLRLFV (SEQ ID NO : 1753 ), GSRRKAFV (SEQ ID comprise a cleavable tertiary peptide having one or more of NO : 1754 ), GSRRKLIF (SEQ ID NO : 1755 ) , GSRRRFFA the following illustrative amino acid sequences (ZXY or (SEQ ID NO : 1756 ) , GSVFGRKG (SEQ ID NO : 1757 ) , and XYZ ): AFVRLFGS (SEQ ID NO : 1649 ) , AKLFVGS (SE G SYFVRK (SEQ ID NO : 1758 ) . US 2018 /0362598 A1 Dec. 20 , 2018 60
[ 0347 ] In various embodiments, the insecticidal and /or method for controlling a pest infection can include an nematicidal protein for use in methods described above may Inhibitor Cysteine Knot ( ICK ) motif protein derived from , be encoded by a polynucleotide that encodes a cleavable or originates from , Hadronyche versuta , Atrax robustus , tertiary peptide having the amino acid sequence of a binary Atrax formidabilis , Atrax infensus , including TPs known as peptide as described above and herein , with the addition of U - ACTX polypetides , U - ACTX - Hvla , rU - ACTX -Hvla , a spacer to the N -terminus of the binary peptide , and /or to rU - ACTX -Hvlb , selected from an Omega -ACTX TP, a the C - terminus of the binary peptide, wherein the spacer is Kappa - ACTX TP and an U - ACTX TP , for example , U + 2 an amino acid or peptide comprising 1 to 4 amino acids , ACTX -Hvla , U - ACTX -Hvla , rU -ACTX -Hvla , rU -ACTX preferably two amino acids, for example GS, WN ,WQ , MA , Hvlb , or a hybrid - ACTX - Hvla peptide , or a TP having at or MV. Other specific spacer examples and optimal amino least 80 % , at least 85 % , at least 90 % , at least 95 % , at least acids for use as spacers are provided below . In related 96 % , at least 97 % , at least 98 % , at least 99 % , or at least embodiments of the above, TPs useful in the manufacture of 100 % sequence identity or sequence homology to a peptide a DNA construct or polynucleotide encoding : ( 1 ) an insec or protein having an amino acid sequence as set forth in SEQ ticidal and / or nematicidal protein , which may include one or ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771 - 1773 , and mutants more ICK TPs, for example , a TP selected from an “ ACTX ” or variants thereof. or “ ACTX peptide” family of insecticidal ICK peptides that [03491 . In some embodiments , the method of controlling a have been isolated from an Australian funnel- web spiders pest infection of a plant provides a pesticidally effective belonging to the Atracinae subfamily , for example , one or amount of one or more TPs synthesized by the plant or part more TPs selected from an Omega -ACTX TP , a Kappa thereof, in the form of an insecticidal and / or nematicidal ACTX TP and an U - ACTX TP , for example , U + 2 - ACTX protein . In some embodiments , an insecticidal and / or nem Hvla , U -ACTX -Hvla , rU -ACTX -Hvla , rU - ACTX -Hvlb aticidal protein comprising one or more TPs that are cleav (wherein the TPs , if more than one , may be the same or able upon exposure to the gut environment of an insect different ) , or a TP having at least 80 % , at least 85 % , at least and /or nematode can be produced from a DNA construct 90 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , transformed into a plant or part thereof. In some embodi at least 99 % , or at least 100 % sequence identity or sequence ments, an exemplary DNA construct encodes one or more homology to a peptide or protein having an amino acid insecticidal and / or nematicidal proteins, each insecticidal sequence as set forth in SEQ ID NOs: 5 , 6 and 1761, 1762, and / or nematicidal protein comprises an ERSP fused in 1767 , 1771 - 1773 , and variants thereof, (wherein the TPs , if frame with the N - terminus of a binary or tertiary peptide , more than one , may be the same or different) and each and the binary or tertiary peptide is fused in frame with a insecticidal and /or nematicidal protein further comprising construct comprising ( TP - L ) , or ( L - TP ) , wherein each TP one or more tertiary peptides ( designated above as L ) , which is toxic protein , L is a binary or tertiary peptide and n is an may include the amino acid sequence of a binary peptide integer ranging from 1 to 200 and wherein the C -terminal TP selected from IFVRLR (SEQ ID NO : 1619 ) , ALKLFV (SEQ is fused or unfused at its C -terminus with a binary or tertiary ID NO : 1605 ) , ALKFLV (SEQ ID NO : 1603 ) , RRKAFV peptide . When n is greater than 1 , each TP and each L may ( SEQ ID NO : 1644 ) , or LFAAPF (SEQ ID NO : 1621 ) , fused be the same or different. in frame either on the N - terminus , C - terminus or both [0350 ] In other embodiments , an exemplary method for termini of the binary peptide with a Z region or spacer controlling such a pest infection includes providing in a diet sequence selected from the group of or consisting of GS , of the pest , a plant, or part thereof, wherein the plant or part WN , WQ , MA , MV, AM , LH , MN , ES, WQ , or DT. thereof, contains and expresses a polynucleotide which [ 0348 ] Exemplary binary and tertiary peptides, for encodes an insecticidal and /or nematicidal insecticidal and / example , ALKLFV ( SEQ ID NO : 1605 ) , ALKFLV (SEQ ID or nematicidal protein comprising one or more TPs as NO : 1603 ) or IFVRLR (SEQ ID NO : 1619 ), fused to one or described herein . In some embodiments , the plant expresses more TPs as described herein may be used to produce an a polynucleotide constitutively , or inducibly . The polynucle insecticidal and /or nematicidal protein which when otide may be operably linked to an operable promoter, for expressed and synthesized by a transgenic plant can fight a example , a homologous or heterologous promoter, wherein pest infection . In various embodiments, the method for the polynucleotide encodes an insecticidal and /or nemati controlling a pest infection of a plant includes planting a cidal protein having one or more cleavable binary or tertiary seed or a plant in a field , wherein the plant or seed is peptides . The insecticidal and /or nematicidal protein can operable to express an insecticidal and / or nematicidal pro include the structure : ( ERSP ) -( TP - L ) , or ( ERSP ) - ( L ) - ( TP tein in the plant or the application of a composition con L ) , or (ERSP ) - ( L - TP ) , or (ERSP ) - ( L - TP ) , ( L ) . In another taining an insecticidal and /or nematicidal protein compris embodiment, a plant, or part thereof comprises a polynucle ing one or more TP proteins selected from the group otide operably linked to an operable promoter, for example , consisting of a Pore Forming Insecticidal Protein ( PFIP ) and a homologous or heterologous promoter , wherein the poly a Cysteine Rich Insecticidal Protein (CRIP ) , for example , nucleotide encodes an insecticidal and / or nematicidal pro the TP may be an Inhibitor Cysteine Knot ( ICK ) motif tein comprising an ERSP fused in frame with the N -terminus protein , a non -ICK protein , a Bt protein or any combination of a stabilizing domain (STA ). The STA is fused in frame of these , and growing the plant or seed in the field which in with either the N -terminus of a binary or tertiary peptide, or the presence of the pest , is able to continue to survive and / or the N - terminus of a construct comprising : ( L - TP ) , ( L - TP ) grow in the field in the presence of the pest . In such ,, - L , ( TP - L )n , or L - ( TP -L ) , . In various embodiments , an embodiments , the pest infection is reduced , by the death of exemplary insecticidal and / or nematicidal protein can at least a portion of the pests, after consuming the plant include a construct from N - terminus to C - terminus : ( ERSP ) expressing the insecticidal and /or nematicidal protein or ( L ) - TP - ( L ) - TP , (ERSP ) - ( L ) - TP -( L ) - TP - ( L ), ( L - TP ), - L , coated at least in part, with the insecticidal and /or nemati ( L - TP - L ) n , ( ERSP ) - ( TP - L ) n , (ERSP ) - ( L ) - ( TP - L ) n , (ERSP ) cidal protein . In one embodiment, the TP useful in the ( L - TP ) , (ERSP ) - ( L - TP ). - ( L ), (ERSP )- ( STA ) - ( L - TP )m , US 2018 /0362598 A1 Dec. 20 , 2018 61
( ERSP ) - (STA ) - ( L - TP ) n - ( L ) , (ERSP ) - ( STA ) - ( TP - L ) n , or a Bt (Bacillus thuringiensis ) TP and an ICK ( Inhibitor ( ERSP ) - ( STA ) - ( L ) - ( TP - L ) n , or (STA ) - ( L - TP ) n , or (STA ) - ( L Cystine Knot) TP to said insect and /or nematode . TP ) n - ( L ) , or (STA ) - ( TP - L )n , or (STA ) - ( L ) - ( TP - L ) n , wherein [ 0352 ] In some embodiments , the DNA constructs ( L ) is a binary peptide or a tertiary peptide , and which each described herein may encode insecticidal and/ or nematicidal L may be the same or different , TP is a toxic peptide , which proteins that contain specific TPs that are suited to treat a may be the same or different, and n is an integer ranging specific pest infestation in a specific plant or groups of from 1 to 200 , preferably 1 to 100 , and most preferably , 1 to plants , for example , a particular type of crop . In one embodi 10 . In some embodiments , the insecticidal and / or nemati ment, insecticidal and /or nematicidal proteins of the present cidal protein may contain TPs that are the same or different, invention may comprise a TP selected from one or more of: and binary and / or tertiary peptides that are the same or Cry 1A . 105 , Cry2Ab2, Cry1F , Cry3Bb1, Cry34Ab1 and different. The TPs useful in the transgenic plants or parts Cry35Ab1. These TPs may be expressed singly or in com thereof disclosed herein , can have at least 50 % , 55 % , 60 % , binations to treat a specific crop , for example , corn . In some 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or embodiments , insecticidal and / or nematicidal proteins hav 99 % or greater sequence identity to SEQ ID NOs: 5 or 6 , ing the structure from N -terminus to C -terminus : ( ERSP ) SEQ ID NO : 33 and / or a peptide selected from SEQ ID ( L ) - TP - ( L ) - TP ( ERSP ) - ( L ) - TP - ( L ) - TP - ( L ) , ( L - TP ) , - L , NOs: 33 - 1032 and variants thereof. In some embodiments , ( L - TP - L )n , ( ERSP ) -( TP - L )no (ERSP )- ( L ) -( TP - L )n . (ERSP ) the C -terminal TP is fused or unfused at its C - terminus with ( L - TP )n . (ERSP )- (L - TP ) -( L ), ( ERSP )- (STA )- ( L - TP ) , a binary or tertiary peptide . In related embodiments of the (ERSP )- (STA )- ( L - TP ) n -( L ), (ERSP ) -( STA )- ( TP -L )n , or above , TPs useful in the manufacture of a DNA construct or (ERSP ) - (STA ) - ( L ) - ( TP - L ) n , or (STA ) - ( L - TP ) n , or ( STA ) - ( L polynucleotide encoding : (1 ) an insecticidal and /or nemati TP ) n - (L ) , or (STA )- ( TP - L )n , or (STA )- (L ) -( TP - L ) n , wherein cidal protein , which may include one or more ICK TPs, for ( L ) is a binary peptide or a tertiary peptide, and which each example , a TP selected from an “ ACTX ” or “ ACTX pep L may be the same or different, TP is a toxic peptide , which tide ” family of insecticidal ICK peptides that have been may be the same or different, and n is an integer ranging isolated from an Australian funnel- web spiders belonging to from 1 to 200 , preferably 1 to 100 , and most preferably , 1 to the Atracinae subfamily , for example , one or more TPs 10 . In some embodiments , the insecticidal and / or nemati selected from an Omega - ACTX TP , a Kappa -ACTX TP and cidal protein may contain TPs that are the same or different, an U - ACTX TP , for example , U + 2 -ACTX -Hvla , U - ACTX and binary and / or tertiary peptides that are the same or Hvla , rU - ACTX - Hvla , rU - ACTX - Hvlb , or a TP having at different, wherein TP is toxic protein selected from Cry least 80 % , at least 85 % , at least 90 % , at least 95 % , at least 1A . 105 , Cry2Ab2, Cry1F , Cry3Bb1 , Cry34Ab1 and 96 % , at least 97 % , at least 98 % , at least 99 % , or at least Cry35Abi , or combinations thereof, and L is a binary or 100 % sequence identity or sequence homology to a peptide tertiary peptide and n is an integer ranging from 1 to 200 , or protein having an amino acid sequence as set forth in SEQ preferably an integer ranging from 1 to 100 , and more ID NOs: 5 , 6 and 1761, 1762 , 1767, 1771 - 1773 , and variants preferably an integer ranging from 1 to 10 can be used to thereof, (wherein the TPs , if more than one , may be the same treat the following corn pests : European corn borer ( ECB ) or different) and wherein the DNA construct or polynucle Southwestern corn borer (SWCB ) Southern cornstalk borer otide further encodes ( 2 ) one or more tertiary peptides (SCSB ) , Corn earworm (CEW ) , Fall armyworm ( FAW ) , ( designated above as L ) fused in frame to at least one binary Stalk borer , Lesser corn stalk borer , Sugarcane borer ( SCB ) , or tertiary peptide , which may include the amino acid Western bean cutworm (WBC ), Black cutworm , Western sequence of a binary peptide selected from IFVRLR (SEQ com rootworm (WCRW ) , Northern corn rootworm ID NO : 1619 ) , ALKLFV (SEO ID NO : 1605 ) , ALKFLV (NCRW ) , or Mexican corn rootworm . (MCRW ) . ( SEQ ID NO : 1603 ) , RRKAFV ( SEQ ID NO : 1644 ) , or [0353 ] In other embodiments , insecticidal and /or nemati LFAAPF (SEQ ID NO : 1621) , fused in frame either on the cidal proteins having the structure from N -terminus to N - terminus , C - terminus or both termini of the binary peptide C - terminus : ( ERSP ) - ( L ) - TP - ( L ) - TP, (ERSP ) - ( L ) - TP - ( L ) with a Z region or spacer sequence selected from the group TP - ( L ) , ( L - TP - L , ( L - TP - L ) , ( ERSP ) - ( TP - L ) , (ERSP ) of or consisting of GS , WN , WQ , MA , MV, AM , LH , MN , ( L ) -( TP - L )n , (ERSP ) -( L - TP ) , ( ERSP )- (L - TP )n -( L ), ES , WQ , or DT. (ERSP ) - (STA )- ( L - TP ) ( ERSP ) - (STA ) - ( L - TP ) . - ( L ) , [ 0351 ] In some embodiments , methods for controlling an (ERSP ) - (STA ) - ( TP - L ) n , or (ERSP ) - ( STA ) - ( L ) - ( TP - L )n , or insect and / or a nematode pest infection or infestation , may (STA ) - ( L - TP )n , or (STA )- ( L - TP ) n - ( L ) , or (STA ) - ( TP - L ) n , or include the creation of a transgenic plant comprising a (STA )- ( L ) -( TP - L ). , wherein ( L ) is a binary peptide or a combination of insecticidal and /or nematicidal proteins, tertiary peptide, and which each L may be the same or each insecticidal and /or nematicidal protein including a Bt different, TP is a toxic peptide, which may be the same or (Bacillus thuringiensis ) TP or an ICK (Inhibitor Cystine different, and n is an integer ranging from 1 to 200 , prefer Knot ) TP which when predated upon by said insect and / or ably 1 to 100 , and most preferably , 1 to 10 . In some nematode , will exert insecticidal and / or nematicidal activity embodiments , the insecticidal and / or nematicidal protein and control or reduce or eliminate the pest infection and / or may contain TPs that are the same or different, and binary infestation . This plant can be made from independent stable and / or tertiary peptides that are the same or different, genetic insertions of DNA that create a flowering plant wherein TP is cotton Cry 1AC , can be used to treat the expressing a Bt TP and a second flowering plant expressing following cotton PIP (Bollgard I and II — first one gene , then a ICK TP . The pollen of one of those plants will be crossed two genes ) pests: cotton bollworm (CBW ) Helicoverpa zea onto the female flower organs of the other plant to form a (Boddie ), tobacco budworm ( TBW ) Heliothis virescens , and genetic hybridization that contains both the Bt TP and the pink bollworm ( PBW ) Pectinophora gossypiella ( Sanders ) . ICK TP genetic traits . The progeny of that sexual cross and In a related embodiment, insecticidal and /or nematicidal further crossing that results in the inheritance of both traits proteins having the structure from N - terminus to C - termi will result in a transgenic plant comprising a combination of nus : (ERSP )- (L ) - TP -( L ) - TP , (ERSP ) -( L )- TP -( L ) - TP - ( L ), US 2018 /0362598 A1 Dec. 20 , 2018 62
( L - TP ) . - L , ( L - TP - L ) n , ( ERSP ) - ( TP - L ) n (ERSP ) - ( L ) - ( TP - L ) ticidal and /or nematicidal compositions containing the ( ERSP ) - ( L - TP ) , ( ERSP ) - ( L - TP ) ,, - ( L ) , ( ERSP ) - (STA ) - ( L insecticidal and / or nematicidal proteins can be applied TP ) n , (ERSP ) -( STA )- (L - TP )n - ( L ), ( ERSP )- (STA )- ( TP - L ) n, directly to the surface of the plants . or (ERSP ) - (STA ) -( L ) - ( TP - L ) n, or (STA )- ( L - TP )n , or (STA ) [0356 As used herein , pesticidally effective amounts of ( L - TP )n - (L ), or (STA ) -( TP - L )n , or (STA )- ( L )- ( TP -L ) n , the insecticidal and / or nematicidal proteins described herein wherein ( L ) is a binary peptide or a tertiary peptide , and includes any amount that may inhibit the growth , impair the which each L may be the same or different, TP is a toxic movement, or even kill an insect and / or nematode when the peptide , which may be the same or different, and n is an TP cleaved from the insecticidal and /or nematicidal protein integer ranging from 1 to 200 , preferably 1 to 100 , and most is appropriately delivered to the locus inhabited by the insect preferably , 1 to 10 . In some embodiments , the insecticidal and /or nematode . In various embodiments , the insecticidal and / or nematicidal protein may contain TPs that are the and /or nematicidal protein containing compositions can be same or different, and binary and /or tertiary peptides that are formulated as powders , emulsions, solutions, granules , the same or different, wherein TP is Cotton Cry1Ac and /or micronized particles , and the like and other formulations and Cry2Ab , can be used to treat the following cotton pests : forms of compositions used in the art for applying insecti cotton bollworm (CBW ) Helicoverpa zea (Boddie ), tobacco cidal proteins such as Bt proteins that can be applied to the budworm ( TBW ) Heliothis virescens, pink bollworm surface of the plants for which pest control is desired . (PBW ) Pectinophora gossypiella ( Sanders ), Fall armyworm Amounts of TPs thus distributed assuming at least 50 % ( FAW ), Beet armyworm (BAW ), and Soybean looper ( SBL ). cleavage and release of the TPs from the insecticidal and /or [0354 ] In other embodiments , insecticidal and /or nemati nematicidal protein can be quantified using Cig rpHPLC cidal proteins having the structure from N - terminus to methods known by those skilled in the art . C - terminus : (ERSP ) - ( L ) - TP - ( L ) - TP, ( ERSP ) - ( L ) - TP - ( L ) [0357 ] In the embodiments described herein , the present TP - ( L ), (L - TP )n - L , (L - TP - L )n , ( ERSP )- ( TP -L )n , (ERSP ) insecticidal and /or nematicidal proteins comprising one or ( L ) -( TP - L )n (ERSP )- ( L - TP )n (ERSP )- ( L - TP )n -( L ), more TPs and one or more cleavable linkers are effective (ERSP ) - (STA ) -( L - TP ) 77. (ERSP ) - (STA ) - ( L - TP ) ,, - ( L ) , against a variety of insects and nematodes . The methods ( ERSP ) - (STA ) - ( TP - L ) , , or (ERSP ) - ( STA ) - ( L ) - ( TP - L ) . , or provided herein can be used to control, or prevent, or reduce (STA ) - ( L - TP ) , or (STA )- ( L - TP ) . - ( L ) , or (STA ) - ( TP - L ) n , or the severity of a pest infection , to kill insects and /or nema (STA ) - ( L ) - ( TP - L ) n , wherein ( L ) is a binary peptide or a todes which consume plants or parts thereof in their diets , to tertiary peptide , and which each L may be the same or optimize the growth potential of a plant, to increase the yield different, TP is a toxic peptide , which may be the same or of a crop susceptible to a pest infection , and various other different, and n is an integer ranging from 1 to 200 , prefer methods requiring the control and / or elimination of insects ably 1 to 100 , and most preferably , 1 to 10 . In some and / or nematodes which predate on plant tissue. Various embodiments , the insecticidal and / or nematicidal protein types of insects and nematodes susceptible to the methods , may contain TPs that are the same or different, and binary compositions and procedures described herein are as fol and / or tertiary peptides that are the same or different, lows . wherein TP is Soybean Cry1Ac, can be used to treat the [0358 ] Insect and nematodes: Insects may be selected following soyabean PIP pests : velvetbean caterpillar (Anti from the orders Coleoptera , Diptera, Hymenoptera, Lepi carcia gemmatalis ), soybean looper (Pseudoplusia doptera , Mallophaga , Homoptera , Hemiptera , Orthroptera , includens ) , soybean anxil borer ( Epinotia aporema ) , and Thysanoptera , Dermaptera , Isoptera , Anoplura , Siphonap sunflower looper (Rachiplusia nu ). tera , Trichoptera , and the like .More particularly , insect pests [ 0355 ] In some embodiments , the method provides trans include Coleoptera, Lepidoptera, and Diptera . forming a plant cell or a plurality of plant cells with a DNA [0359 ] Insects of suitable agricultural, household and /or construct which encodes insecticidal and /or nematicidal medical/ veterinary importance for treatment with the insec insecticidal and /or nematicidal protein containing composi ticidal polypeptides include , but are not limited to , members tions . The transformed plant may be a recombinant plant, a of the following classes and orders . part of the plant, or a product of the plant or the plant part. [0360 ] The order Coleoptera includes the suborders While transformation of a plant or plant part may be Adephaga and Polyphaga. Suborder Adephaga includes the pursued , the insecticidal and / or nematicidal compositions superfamilies Caraboidea and Gyrinoidea . Suborder may be produced recombinantly in host cells including a Polyphaga includes the superfamilies Hydrophiloidea , bacterial host cell , a yeast host cell , a plant host cell or an Staphylinoidea , Cantharoidea , Cleroidea , Elateroidea , Das animal host cell. In some embodiments , the insecticidal cilloidea , Dryopoidea, Byrrhoidea , Cucujoidea, Meloidea , and/ or nematicidal protein comprising an ERSP, one or more Mordelloidea , Tenebrionoidea , Bostrichoidea , Scarabaeoi cleavable linking peptides and one or more TPs can be dea , Cerambycoidea , Chrysomeloidea , and Curculionoidea . synthesized in a yeast production strain or in a bacterial Superfamily Caraboidea includes the families Cicindelidae , production strain to produce milligram , gram or kilogram Carabidae , and Dytiscidae . Superfamily Gyrinoidea quantities of the insecticidal and/ or nematicidal protein for includes the family Gyrinidae . Superfamily Hydrophiloidea application to a plurality of plants or a field of plants , i. e ., includes the family Hydrophilidae . Superfamily Staphyli crops to control or prevent a pest infection . In some embodi noidea includes the families Silphidae and Staphylinidae . ments , a pesticidally effective amount of the insecticidal Superfamily Cantharoidea includes the families Cantharidae and/ or nematicidal protein can be provided in one or more and Lampyridae. Superfamily Cleroidea includes the fami formulations topically applied on the plant or a part of the lies Cleridae and Dermestidae . Superfamily Elateroidea plant, wherein the insecticidal and /or nematicidal protein is includes the families Elateridae and Buprestidae. Superfam formulated in bacterial cells , bacterial spores , fungal cells , ily Cucujoidea includes the family Coccinellidae . Superfam fungal spores , or parasporal crystals that comprise the insec ily Meloidea includes the family Meloidae . Superfamily ticidal and /or nematicidal protein . Alternatively , the insec - Tenebrionoidea includes the family Tenebrionidae. Super US 2018 /0362598 A1 Dec. 20 , 2018
family Scarabaeoidea includes the families Passalidae and dae, Bombyliidae , and Dolichopodidae . Suborder Cyclor Scarabaeidae . Superfamily Cerambycoidea includes the rhapha includes the Divisions Aschiza and Aschiza . Division family Cerambycidae . Superfamily Chrysomeloidea Aschiza includes the families Phoridae , Syrphidae, and includes the family Chrysomelidae . Superfamily Curculion Conopidae . Division Aschiza includes the Sections Acalyp oidea includes the families Curculionidae and Scolytidae . tratae and Calyptratae. Section Acalyptratae includes the [ 0361 ] Examples of Coleoptera include , but are not lim families Otitidae , Tephritidae , Agromyzidae , and Droso ited to : the American bean weevil Acanthoscelides obtectus, philidae . Section Calyptratae includes the families Hippo the leaf beetle Agelastica alni, click beetles (Agriotes lin boscidae, Oestridae , Tachinidae, Anthomyiidae , Muscidae , eatus , Agriotes obscurus , Agriotes bicolor ), the grain beetle Calliphoridae , and Sarcophagidae . Ahasverus advena , the summer schafer Amphimallon solsti [ 0366 ] Examples of Diptera include, but are not limited to : tialis , the furniture beetle Anobium punctatum , Anthonomus the house fly (Musca domestica ), the African tumbu fly spp . (weevils ) , the Pygmy mangold beetle Atomaria lin (Cordylobia anthropophaga ), biting midges (Culicoides earis , carpet beetles ( Anthrenus spp ., Attagenus spp . ), the spp . ) , bee louse (Braula spp . ), the beet fly Pegomyia betae , cowpea weevil Callosobruchus maculates , the fried fruit black flies ( Cnephia spp . , Eusimulium spp . , Simulium spp . ) , beetle Carpophilus hemipterus, the cabbage seedpod weevil bot flies ( Cuterebra spp ., Gastrophilus spp. , Oestrus spp . ), Ceutorhynchus assimilis , the rape winter stem weevil Ceu craneflies (Tipula spp .) , eye gnats (Hippelates spp .) , filth torhynchus picitarsis , the wireworms Conoderus vesperti breeding flies ( Calliphora spp ., Fannia spp ., Hermetia spp ., nus and Conoderus falli, the banana weevil Cosmopolites Lucilia spp ., Musca spp ., Muscina spp ., Phaenicia spp ., sordidus, the New Zealand grass grub Costelytra zea Phormia spp . ), flesh flies ( Sarcophaga spp ., Wohlfahrtia landica , the June beetle Cotinis nitida , the sunflower stem spp .) ; the flit fly Oscinella frit , fruitflies (Dacus spp ., Droso weevil Cylindrocopturus adspersus, the larder beetle Der phila spp .) , head and canon flies (Hydrotea spp . ) , the hessian mestes lardarius, the corn rootworms Diabrotica virgifera , fly Mayetiola destructor , horn and buffalo flies (Haematobia Diabrotica virgifera virgifera , and Diabrotica barberi , the spp . ), horse and deer flies (Chrysops spp . , Haematopota Mexican bean beetle Epilachna varivestis , the old house spp . , Tabanus spp . ), louse flies ( Lipoptena spp . , Lynchia borer Hylotropes bajulus, the lucerne weevil Hypera pos spp ., and Pseudolynchia spp . ), medflies ( Ceratitus spp .) , tica , the shiny spider beetle Gibbium psylloides, the cigarette mosquitoes (Aedes spp . , Anopheles spp ., Culex spp . , Pso beetle Lasioderma serricorne, the Colorado potato beetle rophora spp . ) , sandflies ( Phlebotomus spp . , Lutzomyia spp . ) , Leptinotarsa decemlineata , Lyctus beetles' (Lyctus spp . ) , screw -worm flies (Chtysomya bezziana and Cochliomyia the pollen beetle Meligethes aeneus , the common cockshafer hominivorax ), sheep keds (Melophagus spp .) ; stable flies Melolontha melolontha , the American spider beetle Mezium (Stomoxys spp .) , tsetse flies (Glossina spp .) , and warble flies americanum , the golden spider beetle Niptus hololeucus, the (Hypoderma spp .) . grain beetles Oryzaephilus surinamensis and Oryzaephilus [0367 ) Examples of Isontera ( termites) include, but are not mercator, the black vine weevil Otiorhynchus sulcatus , the limited to : species from the familes Hodotennitidae , Kalo mustard beetle Phaedon cochleariae , the crucifer flea beetle termitidae , Mastotermitidae , Rhinotennitidae , Serritermiti Phyllotreta cruciferae , the striped flea beetle Phyllotreta dae , Termitidae, Termopsidae . striolata , the cabbage steam flea beetle Psylliodes chryso [0368 ] Examples of Heteroptera include, but are not lim cephala , Ptinus spp . ( spider beetles ), the lesser grain borer ited to : the bed bug Cimex lectularius, the cotton stainer Rhizopertha dominica , the pea and been weevil Sitona Dysdercus intermedius , the Sunn pest Eurygaster integri lineatus, the rice and granary beetles Sitophilus oryzae and ceps , the tarnished plant bug Lygus lineolaris , the green Sitophilus granaries , the red sunflower seed weevil Smicro stink bug Nezara antennata , the southern green stink bug nyx fulvus, the drugstore beetle Stegobium paniceum , the Nezara viridula , and the triatomid bugs Panstrogylus megis yellow mealworm beetle Tenebrio molitor , the flour beetles tus , Rhodnius ecuadoriensis , Rhodnius pallescans, Rhod Tribolium castaneum and Tribolium confusum , warehouse nius prolixus, Rhodnius robustus, Triatoma dimidiata , Tria and cabinet beetles ( Trogoderma spp . ) , and the sunflower toma infestans, and Triatoma sordida . beetle Zygogramma exclamation ' s . [0369 ] Examples of Homoptera include , but are not lim [ 0362 ] Examples of Dermaptera ( earwigs) include, but are ited to : the California red scale Aonidiella aurantii , the black not limited to : the European earwig Forficula auricularia , bean aphid Aphisfabae , the cotton or melon aphid Aphis and the striped earwig Labidura riparia . gossypii , the green apple aphid Aphis pomi, the citrus spiny 10363 ) Examples of Dictvontera include, but are not lim whitefly Aleurocanthus spiniferus, the oleander scale Aspi ited to : the oriental cockroach Blatta orientalis, the German diotus hederae , the sweet potato whitefly Bemesia tabaci , cockroach Blatella germanica , the Madeira cockroach Leu the cabbage aphid Brevicoryne brassicae , the pear psylla cophaea maderae , the American cockroach Periplaneta Cacopsylla pyricola , the currant aphid Cryptomyzus ribis , americana , and the smokybrown cockroach Periplaneta the grape phylloxera Daktulosphaira vitifoliae , the citrus fuliginosa . psylla Diaphorina citri, the potato leafhopper Empoascafa [0364 ] Examples of Diplonoda include , but are not limited bae, the bean leafhopper Empoasca solana , the vine leaf to : the spotted snake millipede Blaniulus guttulatus, the hopper Empoasca vitis , the woolly aphid Eriosoma lanige flat -back millipede Brachydesmus superus, and the green rum , the European fruit scale Eulecanium corni, the mealy house millipede Oxidus gracilis. plum aphid Hyalopterus arundinis, the small brown plan [ 0365 ] The order Diptera includes the Suborders Nemato thopper Laodelphax striatellus , the potato aphid Macro cera , Brachycera, and Cyclorrhapha . Suborder Nematocera siphum euphorbiae , the green peach aphid Myzus persicae , includes the families Tipulidae , Psychodidae, Culicidae , the green rice leafhopper Nephotettix cinticeps, the brown Ceratopogonidae , Chironomidae , Simuliidae , Bibionidae , planthopper Nilaparvata lugens , gall - forming aphids ( Pem and Cecidomyiidae . Suborder Brachycera includes the fami phigus spp . ), the hop aphid Phorodon humuli , the bird lies Stratiomyidae, Tabanidae , Therevidae , Asilidae , Mydi cherry aphid Rhopalosiphum padi, the black scale Saissetia US 2018 /0362598 A1 Dec. 20 , 2018 64 oleae, the greenbug Schizaphis graminum , the grain aphid shortnosed cattle louse Haematopinus eloysternus , the tail Sitobion avenae, and the greenhouse whitefly Trialeurodes switch louse Haematopinus quadriperiussus, the hog louse vaporariorum . Haematopinus suis , the face louse Linognathus ovillus, the [0370 ] Examples of Isopoda include, but are not limited foot louse Linognathus pedalis , the dog sucking louse to : the common pillbug Armadillidium vulgare and the Linognathus setosus, the long -nosed cattle louse Linogna common woodlouse Oniscus asellus. thus vituli, the chicken body louse Menacanthus stramineus , [ 0371] The order Lepidoptera includes the families Pap the poultry shaft louse Menopon gallinae , the human body ilionidae , Pieridae, Lycaenidae, Nymphalidae, Danaidae , louse Pediculus humanus , the pubic louse Phthirus pubis , Satyridae , Hesperiidae, Sphingidae, Saturniidae , Geometri the little blue cattle louse Solenopotes capillatus, and the dae, Arctiidae , Noctuidae , Lymantriidae , Sesiidae , and dog biting louse Trichodectes canis. Tineidae . 10375 ] Examples of Psocoptera include , but are not lim [ 0372 ] Examples of Lepidoptera include, but are not lim ited to : the booklice Liposcelis bostrychophila , Liposcelis ited to : Adoxophyes orana ( summer fruit tortrix moth ) , decolor, Liposcelis entomophila , and Trogium pulsatorium . Agrotis ipsolon (black cutworm ) , Archips podana ( fruit tree [0376 ] Examples of Siphonaptera include , but are not tortrix moth ) , Bucculatrix pyrivorella (pear leafminer ), Buc limited to : the bird flea Ceratophyllus gallinae , the dog flea culatrix thurberiella (cotton leaf perforator ), Bupalus pini Ctenocephalides canis , the cat flea Ctenocephalides fells , arius (pine looper ), Carpocapsa pomonella ( codling moth ) , the human flea Pulex irritans , and the oriental rat flea Chilo suppressalis ( striped rice borer ), Choristoneura Xenopsylla cheopis . fumiferana (eastern spruce budworm ), Cochylis hospes [ 0377 ] Examples of Symphyla include, but are not limited (banded sunflower moth ), Diatraea grandiosella ( south to : the garden symphylan Scutigerella immaculate . western corn borer ) , Earls insulana (Egyptian bollworm ), (0378 ] Examples of Thysanura include , but are not limited Euphestia kuehniella (Mediterranean flour moth ), Eupoe to : the gray silverfish Ctenolepisma longicaudata , the four cilia ambiguella ( European grape berry moth ) , Euproctis lined silverfish Ctenolepisma quadriseriata , the common chrysorrhoea (brown - tail moth ), Euproctis subflava (orien silverfish Lepisma saccharina , and the firebrat Thennobia tal tussock moth ), Galleria mellonella (greater wax moth ), domestica . Helicoverpa armigera (cotton bollworm ) , Helicoverpa zea [0379 ] Examples of Thysanoptera include , but are not ( cotton bollworm ) , Heliothis virescens ( tobacco budworm ), limited to : the tobacco thrips Frankliniella fusca , the flower Hofmannophila pseudopretella (brown house moth ) , thrips Frankliniella intonsa , the western flower thrips Homeosoma electellum ( sunflower moth ), Homona magn Frankliniella occidentalis , the cotton bud thrips Franklin anima (oriental tea tree tortrix moth ) , Lithocolletis blan iella schultzei, the banded greenhouse thrips Hercinothrips cardella (spotted tentiform leafminer) , Lymantria dispar femoralis , the soybean thrips Neohydatothrips variabilis , ( gypsy moth ) , Malacosoma neustria ( tent caterpillar ) , Kelly ' s citrus thrips Pezothrips kellyanus, the avocado Mamestra brassicae (cabbage armyworm ) , Mamestra con thrips Scirtothrips perseae , the melon thrips Thrips palmi, figurata (Bertha armyworm ) , the hornwormsManduca sexta and the onion thrips Thrips tabaci . and Manuduca quinquemaculata , Operophtera brumata [0380 ] Examples of Nematodes include, but are not lim (winter moth ), Ostrinia nubilalis (European corn borer ), ited to : parasitic nematodes such as root- knot , cyst , and Panolis flammea ( pine beauty moth ) , Pectinophora gossyp lesion nematodes, including Heterodera spp . , Meloidogyne iella (pink bollworm ) , Phyllocnistis citrella ( citrus leaf spp . , and Globodera spp . ; particularly members of the cyst miner ), Pieris brassicae (cabbage white butterfly ) , Plutella nematodes , including, but not limited to : Heterodera glv xylostella ( diamondback moth ) , Rachiplusia ni ( soybean cines ( soybean cyst nematode ); Heterodera schachtii (beet looper ), Spilosoma virginica (yellow bear moth ) , Spodop cyst nematode ) ; Heterodera avenae ( cereal cyst nematode ) ; tera exigua (beet armyworm ), Spodoptera frugiperda ( fall and Globodera rostochiensis and Globodera pailida (potato armyworm ) , Spodoptera littoralis (cotton leafworin ), cyst nematodes ) . Lesion nematodes include , but are not Spodoptera litura ( common cutworm ), Spodoptera praefica limited to : Pratylenchus spp . ( yellowstriped armyworm ) , Sylepta derogata ( cotton leaf [0381 ] In one embodiment, the insecticidal and nemati roller ) , Tineola bisselliella (webbing clothes moth ) , Tineola cidal compositions comprising the polypeptides, polynucle pellionella ( case- making clothes moth ), Tortrix viridana otides, cells , vectors, etc . described herein , can be employed (European oak leafroller ), Trichoplusia ni ( cabbage looper ), to treat ectoparasites . Ectoparasites include , but are not and Yponomeuta padella (small ermine moth ) . limited to : fleas , ticks, mange, mites , mosquitoes , nuisance [ 0373] Examples of Orthoptera include, but are not limited and biting flies , lice , and combinations comprising one or to : the common cricket Acheta domesticus, tree locusts more of the foregoing ectoparasites . The term “ fleas ” ( Anacridium spp . ) , the migratory locust Locusta migratoria , includes the usual or accidental species of parasitic flea of the twostriped grasshopper Melanoplus bivittatus, the dif the order Siphonaptera , and in particular the species Cteno ferential grasshopper Melanoplus dfferentialis , the cephalides , in particular C . fells and C . cams, rat fleas redlegged grasshopper Melanoplus femurrubrum , the migra ( Xenopsylla cheopis ) and human fleas (Pulex irritans ) . tory grasshopper Melanoplus sanguinipes , the northern mole [0382 ] Insect pests of the invention for the major crops cricket Neocurtilla hexadectyla , the red locust Nomadacris include , but are not limited to : Maize : Ostrinia nubilalis , septemfasciata , the shortwinged mole cricket Scapteriscus European corn borer ; Agrotis ipsilon , black cutworm ; Heli abbreviatus, the southern mole cricket Scapteriscus borellii , coverpa zea , corn earworm ; Spodoptera frugiperda, fall the tawny mole cricket Scapteriscus vicinus , and the desert armyworm ; Diatraea grandiosella , southwestern corn locust Schistocerca gregaria . borer ; Elasmopalpus lignosellus , lesser cornstalk borer ; Dia 0374 ] Examples of Phthiraptera include , but are not lim traea saccharalis , surgarcane borer; Diabrotica virgifera , ited to : the cattle biting louse Bovicola bovis , biting lice western corn rootworm ; Diabrotica longicornis barberi , (Damalinia spp . ) , the cat louse Felicola subrostrata , the northern corn rootworm ; Diabrotica undecimpunctata how US 2018 /0362598 A1 Dec. 20 , 2018 65
ardi, southern corn rootworm ; Melanotus spp ., wireworms ; armyworm ; Heliothis virescens, cotton budworm ; Helicov Cyclocephala borealis , northern masked chafer (white erpa zea , cotton bollworm ; Epilachna varivestis , Mexican grub ) ; Cyclocephala immaculata , southern masked chafer bean beetle ; Myzus persicae , green peach aphid ; Empoasca ( white grub ); Popillia japonica , Japanese beetle ; Chaetoc fabae , potato leafhopper; Acrosternum hilare , green stink nema pulicaria , corn flea beetle ; Sphenophorus maidis , bug ; Melanoplus femurrubrum , redlegged grasshopper ; maize billbug ; Rhopalosiphum maidis , corn leaf aphid ; Melanoplus differentialis , differential grasshopper; Hylemya platura , seedcorn maggot; Sericothrips variabilis , soybean Anuraphis maidiradicis , corn root aphid ; Blissus leucop thrips; Thrips tabaci, onion thrips ; Tetranychus turkestani, terus leucopterus, chinch bug ; Melanoplus femurrubrum , strawberry spider mite ; Tetranychus urticae , twospotted redlegged grasshopper; Melanoplus sanguinipes , migratory spider mite ; Barley : Ostrinia nubilalis , European corn borer; grasshopper ; Hylemya platura , seedcorn maggot; Agromyza Agrotis ipsilon , black cutworm ; Schizaphis graminum , parvicornis , corn blot leafminer ; Anaphothrips obscrurus, greenbug ; Blissus leucopterus leucopterus, chinch bug ; grass thrips ; Solenopsis milesta , thief ant; Tetranychus urti Acrosternum hilare , green stink bug ; Euschistus servus , cae , twospotted spider mite ; Sorghum : Chilo partellus, brown stink bug ; Delia platura , seedcorn maggot; Mayetiola sorghum borer ; Spodoptera frugiperda , fall armyworm ; destructor, Hessian fly ; Petrobia latens, brown wheat mite ; Helicoverpa zea , corn earworm ; Elasmopalpus lignosellus , Oil Seed Rape : Brevicoryne brassicae , cabbage aphid ; Phyl lesser cornstalk borer; Feltia subterranea , granulate cut worm ; Phyllophaga crinita , white grub ; Eleodes, lotreta cruciferae , Flea beetle ; Mamestra configurata , Ber Conoderus , and Aeolus spp . , wireworms; Oulema melano tha armyworm ; Plutella xylostella , Diamond - back moth ; pus, cereal leaf beetle ; Chaetocnema pulicaria , corn flea Delia ssp ., Root maggots . beetle ; Sphenophorus maidis , maize billbug ; Rhopalosi [0383 ] In some embodiments , the insecticidal composi phum maidis , corn leaf aphid ; Sipha flava , yellow sugarcane tions can be employed to treat combinations comprising one aphid ; Blissus leucopterus leucopterus , chinch bug ; Con or more of the foregoing insects . tarinia sorghicola , sorghum midge ; Tetranychus cinnabari [0384 ] The insects that are susceptible to the peptides of nus, carmine spider mite; Tetranychus urticae , twospotted this invention include but are not limited to the following : spider mite ; Wheat: Pseudaletia unipunctata , army worm ; Blattaria , Coleoptera , Collembola , Diptera, Echinostomida , Spodoptera frugiperda , fall armyworm ; Elasmopalpus Hemiptera , Hymenoptera , Isoptera , Lepidoptera , Neurop lignosellus, lesser cornstalk borer; Agrotis orthogonia , west tera , Orthoptera, Rhabditida, Siphonoptera , Thysanoptera . ern cutworm ; Elasmopalpus lignosellus, lesser cornstalk Genus -Species are indicated as follows: Actebia - fennica , borer; Oulema melanopus, cereal leaf beetle ; Hypera punc Agrotis - ipsilon , A .- segetum , Anticarsia -gemmatalis , Argyro tata , clover leaf weevil; Diabrotica undecimpunctata how taenia - citrana , Artogeia - rapae , Bombyx mori, Busseola ardi , southern corn rootworm ; Russian wheat aphid ; Schiza fusca , Cacyreus -marshall , Chilo - suppressalis , Chris phis graminum , greenbug; Macrosiphum avenae , English toneura - fumiferana , C . -occidentalis , C . - pinus pinus , grain aphid ; Melanoplus femurrubrum , redlegged grasshop C .- rosacena , Cnaphalocrocis -medinalis , Conopomorpha per ;Melanoplus differentialis , differential grasshopper ; Mel cramerella , Ctenopsuestis - obliquana , Cydia - pomonella , anoplus sanguinipes , migratory grasshopper; Mayetiola Danaus- plexippus, Diabrotica undecimpunctata howardi, destructor, Hessian fly ; Sitodiplosis mosellana , wheat Diatraea - saccharallis , D . - grandiosella , Earias -vittella , midge ; Meromyza americana , wheat stem maggot; Hylemya Elasmolpalpus- lignoselius, Eldana -saccharina , Ephestia coarctata , wheat bulb fly ; Frankliniella fusca , tobacco kuehniella , Epinotia -aporema , Epiphyas -postvittana , Galle thrips; Cephus cinctus, wheat stem sawfly ; Aceria tulipae , ria -mellonella , Helicoverpa - zea, H . - punctigera , H . - armig wheat curl mite ; Sunflower : Suleima helianthana , sunflower era , Heliothis - virescens, Hyphantria - cunea , Lambdina bud moth ; Homoeosoma electellum , sunflower moth ; Zygo fiscellaria , Leguminivora - glycinivorella , Lobesia - botrana , gramma exclamationis , sunflower beetle ; Bothyrus gibbo Lymantria -dispar , Malacosoma -disstria , Mamestra -brassi sus, carrot beetle ; Neolasioptera murtfeldtiana , sunflower cae , M . configurata , Manduca - sexta , Marasmia - patnalis , seed midge ; Cotton : Heliothis virescens , cotton budworm ; Maruca - vitrata , Orgyia - leucostigma , Ostrinia -nubilalis , Helicoverpa zea , cotton bollworm ; Spodoptera exigua , beet 0 .- furnacalis , Pandemis -pyrusana , Pectinophora - gossyp armyworm ; Pectinophora gossypiella , pink bollworm ; iella , Perileucoptera -coffeella , Phthorimaea - opercullela , Anthonomus grandis, boll weevil; Aphis gossypii, cotton Pianotortrix -octo , Piatynota -stultana , Pieris -brassicae , aphid ; Pseudatomoscelis seriatus, cotton fleahopper; Tri Plodia - interpunctala , Plutella - xylostella , Pseudoplusia - in aleurodes abutilonea , bandedwinged whitefly ; Lygus line cludens, Rachiplusia - nu , Sciropophaga - incertulas, Sesa olaris , tarnished plant bug ; Melanoplus femurrubrum , mia - calamistis, Spilosoma - virginica , Spodoptera -exigua , redlegged grasshopper ; Melanoplus differentialis , differen S .- Jfrugiperda , S .- littoralis , S .- exempta, S .- litura , Tecia -so tial grasshopper ; Thrips tabaci, onion thrips; Franklinkiella lanivora , Thaumetopoea -pityocampa , Trichoplusia -ni , fusca , tobacco thrips; Tetranychus cinnabarinus, carmine Wiseana - cervinata , Wiseana - copularis, Wiseana - jocosa , spider mite ; Tetranychus urticae , twospotted spider mite ; Blattaria - Blattella , Collembola - Xenylla , C .- Folsomia , Echi Rice : Diatraea saccharalis , sugarcane borer ; Spodoptera nostomida - Fasciola , Hemiptera - Oncopeltrus, He. -Bemisia , frugiperda , fall armyworm ; Helicoverpa zea , corn earworm ; He. -Macrosiphum , He. - Rhopalosiphum , He. -Myzus , Hyme Colaspis brunnea , grape colaspis; Lissorhoptrus oryzophi noptera - Diprion , Hy .- Apis , Hy. - Macrocentrus , Hy .- Meteo lus, rice water weevil; Sitophilus oryzae , rice weevil; Nepho rus, Hy .- Nasonia , Hy. - Solenopsis , Isopoda - Porcellio , Isop tettix nigropictus , rice leafhopper; Blissus leucopterus leu tera -Reticulitermes , Orthoptera - Achta , Prostigmata copterus, chinch bug; Acrosternum hilare , green stink bug ; Tetranychus, Rhabitida - Acrobeloides , R .- Caenorhabditis , Soybean : Pseudoplusia includens, soybean looper, Anticar R .- Distolabrellus, R .- Panagrellus , R . -Pristionchus , R . -Pra sia gemmatalis , velvetbean caterpillar; Plathypena scabra , tylenchus, R . - Ancylostoma, R . - Nippostrongylus, R . -Pa green cloverworm ; Ostrinia nubilalis , European corn borer ; nagrellus , R . - Haemonchus, R . -Meloidogyne , and Siphonap Agrotis ipsilon , black cutworm ; Spodoptera exigua , beet tera - Ctenocephalides. As used herein , insect pests and US 2018 /0362598 A1 Dec. 20 , 2018 nematode pests are those species of insects and nematodes then the plate is read once a minute for detectable fluores that are susceptible to the insecticidal and /or nematicidal cence . The output of fluorescence detection is recorded for transgenic protein compositions and polypeptides that each reaction . encode them . [0390 ] FRET samples were prepared immediately prior to [0385 ). Select list of highly susceptible insects and nema assay as recommended by the manufacturer (add 5 uL 50 % todes. The list below contains both insects and nematodes . acetonitrile in water to each well and agitate plate on shaker Nematodes are also susceptible to the compositions and for 5 minutes . Then add 45 uL of assay buffer and agitate treatments identified in the application and claims. The again for 5 minutes ) . At this point, the FRET pools were insects and nematodes below are considered especially ready of the addition of 50 uL of the working enzyme stock suitable and susceptible to the compositions and treatments to start the reactions . Using a multichannel , repeat pipetter, identified in the specification and claims. 50 uL of the working enzyme stock ( for each of the enzyme [0386 ] Stalk borer - Busseola fusca , Southern Cornstalk types tested ) was added to each well and then the plate was Borer — Diatraea cramnbidoides , True armyworm — Pseu immediately placed into a plate reader (SpectraMax plate daletia unipuncta , Western bean cutworm - Striacosta albi reader with SoftMax Pro 6 .0 software , Molecular Devices, costa , Black cutworm - Agrotis ipsilon , Corn earworm Sunnyvale Calif. ) and emissions were read using the setting Helicoverpa zea , European corn borer — Ostrinia nubilalis , of Excitation 320 nm , Emission 420 nm , with a cut- off of Fall armyworm - Spodoptera frugiperda , Western corn root 420 nm . Readings occurred every 1 minute for 15 minutes . worm - Diabrotica virgifera virgifera , Southern Corn Root Once the plates were completed , they were sealed with an worm - Diabrotica undecimpunctata howardi, Northern aluminum sealer and stored at - 80° C . Corn Rootworm - Diabrotica barberi, Western tarnished [0391 ] To test the FRET pool cleavage by pepsin or plant bug - Lygus Hesperus, Tarnished plant bug - Lygus simulated human gastric fluid (SGF ) , the assay buffer was 35 lineolaris , Soybean cyst nematode - Heterodera glycines, mM NaCl, pH 1 . 2 . The commercially sourced enzyme Root- Knot Nematode - Meloidogyne hapla , Lesion nema (Sigma P6887 ) enzyme was prepared by dissolving 1 mg/ ml tode — Pratylenchus penetrans, Pink bollworm - Pectino pepsin in the assay buffer. Pepsin stock was then diluted phora gossypiella , Cotton bollworm — Helicoverpa armig 1 :200 by adding 250 uL of stock enzyme to 50 mL of assay era , Corn earworm - Helicoverpa zea , Sugarcane stalk buffer . This working solution was then used for all plate borer - Diatraea saccharalis, Asian corn borer - Ostrinia screenings . furnacalis, Native budworm Helicoverpa punctigera , [0392 ] To test the FRET pool cleavage by gut or intestinal Tobacco budworm Heliothis virescens, Beet armyworm enzymes of a coleopteran pest, gut enzymes (CELOP ) were Spodoptera exigua , Soybean Looper — Chrysodeixis isolated from Southern Corn Rootworm ( Diabrotica undec includens, Velvetbean caterpillar - Anticarcia gemmatalis , impunctata ) . Southern Corn Rootworm were commercially Soybean looper — Pseudoplusia includes , Soybean anxil obtained from Crop Characteristics (Farmington , MN ) as borer - Epinotia aporema, and Sunflower looper - Rachip 3rd instar larvae . Insects were anesthetized by placing them lusia nu . on a cold pack for several minutes before the dissection began . To isolate the gut, the posterior end was pinned to a EXAMPLES dissection plate and another pin was used to nick the soft tissue behind the head . The pin was inserted behind the head Example 1 . Identification and Isolation of Binary capsule and then pulled away from pinned insect , pulling the and Tertiary Cleavable Linking Peptides digestive tract out of the body with the head . The gut could [ 0387] 1 . ) The FRET Research and Discovery of Cleav then be collected . About 20 guts were added to a pre able Linking Peptides weighed tube with 100 uL solution of 100 mM PBS pH 5 .7 [0388 ] To find a Linker with insect and /or nematode and weighed after to determine the mass of guts added . Gut protease and human gastrointestinal cleavable properties, contents were extracted by vortexing the solution for 1 - 2 the inventors used a peptide library coupled to FRET minutes . Tubes were then spun at 15 ,000 rpm for 1 minute . molecules ( a dye and a quencher ) for the identification of The liquid layer was then transferred to a 0 . 2 um mico - spin peptides that cleave in either insect gut environment or in filter and spun at 15 , 000 rcf for 1 minute . The filter was simulated human gastrointestinal environment as reporter washed with 400 uL of 100 mM PBS buffer pH 5 . 7 and the assays . As shown in FIG . 1 , a FRET kit contains pools of permeate from both spins were combined . The sterile filtered FRET molecules with a stretch of 3 variable amino acid gut contents were then diluted to 10 mg/ mL in buffer and sequences ( grid pattern circle ) bracketed by a series of stored at - 20 C . To make a working stock , the gut contents glycine amino acids (horizontal line pattern ) attached to where then diluted 10x by mixing 700 uL stock to 7 mL with either a dye (solid black circle with a protruding square ) or assay buffer for each plate . Using a multichannel , repeat a quencher ( solid black circle with a square- shaped indent) . pipetter, 50 uL of 1x gut extract was added to each well and FIG . 1 illustrates that each FRET molecule gives no signal read immediately as described above . if the variable region is not cleaved but can be excited if it [0393 ] To test the FRET pool cleavage by gut enzymes of is cleaved . The speed at which the cleavage occurs ( i . e . the a lepidopteran pest (LEP ) , gut enzymes were isolated from specificity of the sequence for the proteases ) can be ranked Corn Earworm (Helicoverpa zea ) . Corn Earworm insects by the rate at which the fluorescent signal occurs over time, were obtained commercially from Benzon Research (Carl i. e . , its slope. isle , Pa . ) as eggs. Hatched larvae were raised on artificial [ 0389 ] The FRET kit used (Cat . No . PSREPLI005 , Mimo diet until 4/ 5th instar ( 20 mm long ) before guts were isolated . topes, Victoria , Australia ) contains 512 pools of up to 8 Before gut extract , larvae were anesthetized using CO , . The different FRET molecules per pool. The FRET reaction is larva was then pinned on the dissection plate at both the head very high - throughput, with the enzymes added to the plate and the tail . Using dissection scissors, the cuticle was of pooled material with a multichannel loading pipet and nicked . The dissection scissors were then inserted into the US 2018 /0362598 A1 Dec. 20 , 2018 67 nick and the cuticle was lengthwise along the insect. The TABLE 4 - continued cuticle was then carefully pulled back and pinned open to reveal the digestive track . Using DI water, the insect was Representative cleavable sequences from pooled FRET screening thoroughly rinsed to remove hemolymph . The gut was then for each of the digestive conditions; SGF, COLEOP, and LEP excised with tweezers and placed in a solution of 500 uL of 200 mM Tris -HCl PH 8 . 1 150 mM NaCl (assay buffer ) . The tube was pre - weighed and post -weighed to calculate the SGF COLEOP LEP total amount of gut added . CEW gut was diluted to a stock % of Max % of Max % of Max concentration of 7 mg/ ml ( 10x ) in 200 mM Tris -HCl pH 8 . 1 Sequence Slope Sequence Slope Sequence Slope 150 mM NaCl. This was diluted fresh each plate by 20x with ALY 78 QLK 74 KKY 92 the assay buffer . Using a multichannel, repeat pipetter, 50 uL VIF QLR KRF of 1x gut extract was added to each well and read immedi VLF AIK KRY ately as described above . VLY AIR RKF 92 [0394 ] 1 . 1 ) Protease Cleavage Analysis of FRET Peptide DFF 66 ALK RKY 92 DFY 66 ALR RRF 92 Pools DYF 66 VIK RTR 90 [ 0395 ] The FRET kits ( Cat . No. PSREPLI005, Mimo EFF 66 VIR 70 KSK 90 topes , Victoria , Australia ) were tested against LEP , EFY 66 VLK 70 KSR 90 EYF 66 VLR KTK 90 COLEOP and SGF gut simulated protease environments and IFA 65 IKS 68 KTR data was recovered . There were two types of data ( slope of IFV IKT RSK cleavage over time ( which is presented as either a raw slope, LFA 65 IRS RSR 90 or as an indexed value where the steepest slope is 100 % and LFV 65 IRT RTK 90 LYA LKS VRT 90 each slope is represented as a percentage of the highest LYV 65 LKT AKS O slope ) and cleaved components) were generated . The first AFF 3 LRS AKT C type of data was the slope of fluorescence detection over 10 AFY LRT ARS 90 minutes . The slope of the reaction allowed the ranking of the AYF INK ART 90 VFF INR VKS 90 two or three amino acid sequences for their speed to cleave VFY IOK 56 VKT 90 in the various protease environments . Table 4 shows the top VYF IQR 56 VRS 90 50 sequences that cleaved for each of the digestive condi IIF LNK LTR tions (SGF represents human gastrointestinal environment; ILF LNR ISK ILY LOK ISR COLEOP is the gut environment of Coleoptera insects; and LIF LOR ITK LEP is the gut environment ofLepidopteran insects ) . Shown LIY 63 IKA 56 ITR in Table 5 are sample sequences that did not cleave in the LLF 63 IKV 5 LSK digestive conditions , and represent an example of sequences outside of the Markush that are not cleavable in the X and Y conditions . TABLE 5 TABLE 4 Example of 5 failed cleavable sequences from pooled FRET screening for each of the digestive conditions; SGF, COLEOP, and LEP Representative cleavable sequences from pooled FRET screening for each of the digestive conditions: SGF. COLEOP , and LEP Y SGF COLEOP LEP SGF COLEOP _ _ LEP % of Max % of Max % of Max Sequence Slope Sequence Slope Sequence Slope % of Max % of Max % of Max Sequence Slope Sequence Slope Sequence Slope IFF ? ??? PKK IFY IKQ PKR IFF 100 IKN 100 PKK 100 IYF IRN PRK IFY 100 IKO 100 PKR 100 LFF - 1 IRO PRR IYF 100 IRN 100 PRK 100 LFY LKN VRY LFF 100 PRR 100 ooooo IRQ 100 NOOOO LFY 100 LKN 100 VRY LYF 100 LKO 100 AKF LYY 100 LRN 100 AKY [0396 ] The amino acids represented in the top 5 % of wells FFA 84 LRQ 100 ARF from each digestive condition were used to create the FFV KIK 83 ARY FYA KIR 83 VKF Markush group that constitutes a binary peptide that com FYV t KLK 83 VKY prises of an amino acid sequence selected from the group YFA t KLR VRF consisting of: X7- X2- Y - Y2, X - X2- X3 - Y - Y2, X - X2 -Xz YFV RIK RRT FFI RIR 83 KKS X4- Y - Y2, X -X2 - Y -Y2 - Y3 , X -X2 - X3 - Y - Y2- Y3, X -X2 FFL O RLK 83 KKT X3- X4 - Y - Y2 - Y3, X7 -X2 - Y - Y2 - Y3- Y4, X - X2- X3 - Y - Y2 FYI O RLR 83 KRS Yz- Y4 , X , - X2- Xz - X4- Y - Y - Yz - Y4, Y , -Y2 - X , -X2 , Y - Y2 FYL O NIK 74 KRT X - X2- X3, Y - Y2- X - X2- X3 -X4 , Y , - Y2- Y3- X , -X2 , Y , - Y2 YFI O NIR 74 RKS YFL O NLK 74 RKT Y3- X , - X - Xz, Y - Y - Y3- X , - X2- X2 - X4, Y - Y2 - Y3- Y4- X , YYL O NLR 74 RRS X2, Y - Y2- Y3- Y4- X , -X2 - Xz, and Y - Y2- Y3- Y4 - X -X2 - Xz AIF O QIK 74 RRY X4 , wherein each Xn and each Yn is an amino acid , and ALF 78O QIR 74 KKF wherein X , is selected from the group consisting of: (all single letter amino acid format ) A , D , E , G , I , K , L , N , P , Q , US 2018 /0362598 A1 Dec. 20 , 2018
R , and V ; X , is selected from the group consisting of A , F, AKL , AKN , AKQ , AKR , AKS, AKT, AKY, ALG , ALK , G , I , K , L , N , P , Q , R , S , T , V , and Y ; X2 is selected from the ALN , ALQ , ALR , APF, APG , APK , APR , ARF, ARG , ARI, group consisting of A , D , E , F, G , I, K , L , N , Q , R , S , T , V, ARK , ARL , ARN , ARQ , ARR , ARS , ART, ARY, ASG , ASK , and Y ; X , is selected from the group consisting of: R , H , K , ASR , ATG , ATK , ATR , AVG , AVK , AVR , AYG , AYK , AYR , D , E , S , T, N , Q , G , A , I, L , M , F , W , Y , and V . In some DGK , DGR , DIG , DIK , DIR , DLG , DLK , DLR , EGK , embodiments , Y , is selected from the group consisting of : EGR , EIG , EIK , EIR , ELG , ELK , ELR , ER , FVR , GAF , (all single letter amino acid format ) A , D , E , F , G , I , L , N , GAI, GAK , GAL , GAR , GAY, GFK , GFR , GIK , GIN , GIO , Q , S , T , V , and Y ; Y2 is selected from the group consisting GIR , GKA , GKD , GKE , GKF, GKI, GKK , GKL , GKN , of F , G , I , L , and Y ; and Y , is selected from the group GKO , GKR , GKS , GKT, GKV, GKY, GLK , GLN , GLO , consisting of A , D , E , F , G , I, K , L , R , S , T , V , and Y ; and GLR , GNA , GNK , GNR , GNV , GPK , GPR , GQA , GOK , YA is selected from the group consisting of: R , H , K , D , E , GQR , GQV, GRA , GRD , GRE , GRF, GRI, GRK , GRL , S , T, N , Q , G , A , I, L , M , F , W , Y , and V . GRN , GRQ , GRR , GRS , GRT, GRV , GRY, GSI, GSK , GSL , [ 0397 ] One embodiment of the sequences discovered from GSR ,GTI , GTK , GTL ,GTR , GVF, GVI , GVK , GVL , GVR , SGF ( simulated human gut environment ) screening , in the GVY, GYK , GYR , IGA , IGF, IGI, IGK , IGL , IGN , IGQ , XY or YX format, the human gut protease cleavable sub - IGR , IGS , IGT, IGV, IGY, IIG , IIK , IIR , IKA , IKF, IKG , IKI, strate may include one or more of the following peptides : IKK , IKL , IKN , IKO , IKR , IKS, IKT, IKV, IKY, ILG , ILK , AFF , AFG , AFI, AFL , AFV, AFY, AGF , AGL , AIF , AIL , ILR , INA , ING , INK , INR , INV, IPG , IPK , IPR , IQA , IQG , ALF , ALG , ALI, ALL , ALY , AYF , AYL , DFF , DFG , DFI, IQK , IQR , IQV, IRA , IRF, IRG , IRI, IRK , IRL , IRN , IRO , DFL , DFY , DGF , DGL , DIF , DLF , DLG , DLY , DYF , DYL , IRR , IRS , IRT, IRV , IRY, ISG , ISK , ISR , ITG , ITK , ITR , EFF , EFG , EFI, EFL , EFY , EGF, EGL , EIF , ELF, ELG , ELY, KAF, KAG , KAI, KAK , KAL , KAR , KAY , KFF , KFG , EYF , EYL , FF , FFA , FFD , FFE , FFF, FFG , FFI, FFK , FFL , KFK , KFR , KGA , KGD , KGE, KGF, KGI, KGK , KGL , FFR , FFS , FFT , FFV , FFY , FGA , FGD , FGE , FGF, FGI, KGN , KGQ , KGR , KGS , KGT, KGV, KGY, KIG , KIK , FGK , FGL, FGR , FGS, FGT, FGV , FGY, FLV, FLR , FYA , KIN , KIQ , KIR , KKA, KKD , KKE , KKF, KKG , KKN , FYD , FYE, FYF , FYG , FYI, FYK , FYL , FYR , FYS , FYT, KKQ , KKS, KKT, KKV, KKY , KLG , KLK , KLN , KLQ , FYV , FYY, GFA , GFD , GFE , GFF , GFI, GFK , GFL , GFR . KLR , KNG , KNK , KNR , KPG , KPK , KPR , KOG , KOK , GFS , GFT, GFV , GFY , GGL , GIF , GIL , GLF , GLI, GLL , KOR , KRA , KRD , KRE , KRF, KRG , KRN , KRQ , KRS , GLY, GYF ,GYL , IF , IFA , IFF , IFG , IFI, IFL , IFV , IFY , IGF, KRT, KRV , KRY, KSG , KSI, KSK , KSL , KSR , KTG , KTI, IGL , IIF , IIL , ILF, ILG , ILI, ILL , ILY, IYF, IYL , LFA , LFF , KTK , KTL , KTR , KVF , KVG , KVI, KVK , KVL , KVR , LFG , LFI, LFL , LFV, LFY , LG , LGA , LGE , LGF, LGI, KVY, KYG , KYK , KYR , LFR , LGA, LGF, LGI, LGK , NIELLGL, , LLYLGV ,, LGY LGY,, LIF , , LIGYLIG , ,LII LG , ,LIL LGAYT , LIY , LLFLYL ,, LEALLG , , IGELLI , LGL , LGN , LGQ , LGR , LGS , LGT, LGV , LGY, LIG , LIK , LLL , LLY, LYA , LYF, LYG , LYI, LYL , LYV , LYY , NFF , LIR , LK , LKA , LKF, LKG , LKI, LKK , LKL , LKN , LKQ , NFG , NFI, NFL , NFY , NGF, NGL , NIL , NLG , NLI, NLL , LKR , LKS, LKT, LKV , LKY, LLG , LLK , LLR , LNA , LNG , NYF , NYL , QFF , QFG , QFI, QFL , QFY, QGF, QGL , QIL , LNK , LNR , LNV , LPG , LPK , LPR , LQA , LOG , LOK , QLG , QLI, QLL , QYF, QYL , SFF , SFG , SFI, SFL , SFY , LOR , LOV , LRA , LRF , LRG , LRI, LRK , LRL , LRN , LRO , SGF, SGL , SIF , SIL , SLF , SLG , SLI, SLL , SLY , SYF , SYL , LRR , LRS, LRT, LRV, LRY, LSG , LSK , LSR , LTG , LTK . TFF , TFG , TFI, TFL , TFY, TGF, TGL , TIF , TIL , TLF , TLG , LTR , NGK , NGR , NIG , NIK , NIR , NLG , NLK , NLR , PGK , TLI, TLL , TLY, TYF , TYL , VFF , VFG , VFI, VFL , VFY , PGR , PIG , PIK , PIR , PKG , PKK , PKR , PLG , PLK , PLR , VGF, VGL , VIF , VIL , VLF, VLG , VLI, VLL , VLY, VYF , PRG , PRK , PRR , QGK , OGR , QIG , QIK , QIR , QLG , QLK , VYL , YFA , YFD , YFE , YFF , YFG , YFI, YFK , YFL , YFR , QLR , RAF , RAG , RAI, RAK , RAL , RAR , RAY, RFF , RFG , YFS , YFT, YFV , YFY , YGF, YGL , YYF , YYL . RFK , RFR , RGA , RGD , RGE , RGF, RGI, RGK , RGL , [ 0398 ]. Another embodiment of the sequences discovered RGN , RGO , RGR , RGS , RGT, RGV, RGY, RIG , RIK , RIN , from SGF screening , Y SGF cleavable peptides may include RIQ , RIR , RKA , RKD , RKE , RKF, RKG , RKK , RKN , one or more of: AFF , AFI, AFL , AFY , AIF , AIL , ALF, ALI, RKQ , RKS , RKT, RKV , RKY, RLF , RLFL , RLG , RLK , ALL , ALY , AYF , AYL , DFF , DFI, DFL , DFY, DIF , DLF, RLN , RLQ , RLR , RNG , RNK , RNR , RPG , RPK , RPR , DLY , DYF , DYL , YFL , EFF , EFI, EFL , 0 , EFY, EIF , ELF , ROG , ROK , ROR , RRA , RRD , RRE , RRF, RRG , RRK , ELY , EYF, EYL , FFA , FFD , FFE , FFF , FFI, FFK , FFL , FFR , RRN , RRQ , RRR , RRS , RRT, RRV , RRY , RSG , RSI, RSK , FFS , FFT, FFV , FFY , FYA , FYD , FYE , FYF, FYI , FYK , RSL , RSR , RTG , RTI, RTK , RTL , RTR , RVF , RVG , RVI, FYL , FYR , FYS , FYT, FYV , FYY , IFA , IFF, IFI, IFL , IFV , RVK , RVL , RVR , RVY, RYG , RYK , RYR , VAG , VAK , IFY, IIF , IIL , ILF, ILI, ILL , ILY, IYF, IYL , LFA , LFF , LFI, VAR , VFG , VFK , VFR , VGF, VGI, VGK , VGL , VGN , LFL , LFV, LFY , LIF , LII , LIL , LIY , LLF, LLI, LLL , LLY , VGO , VGR , VGS , VGT, VGY, VIG , VIK , VIN , VIQ , VIR , LYA , LYF , LYI, LYL , LYV, LYY, NFF , NFI, NFL , NFY , VKF, VKG , VKI, VKK , VKL , VKN , VKQ, VKR , VKS , NIL , NLI, NLL , NYF, NYL , QFF , QFI, QFL , QFY , QIL , VKT, VKY, VLG , VLK , VLN , VLQ , VLR , VPG , VPK , QLI, QLL , QYF , QYL , SFF , SFI, SFL , SFY, SIF , SIL , SLF , VPR , VR , VRF , VRG , VRI, VRK , VRL , VRN , VRQ , VRR , SLI, SLL , SLY , SYF , SYL , TFF , TFI, TFL , TFY , TIF , TIL , VRS , VRT, VRY, VSG , VSK , VSR , VTG , VTK , VTR , TLF , TLI, TLL , TLY , TYF, TYL , VFF , VFI, VFL , VFY , VIF , VVG , VVK , VVR , VYG , VYK , VYR . VIL , VLF, VLI, VLL , VLY , VYF, VYL , YFA , YFD , YFE , [0400 . In another embodiment of the sequences discov YFF , YFI, YFK , YFL , YFR , YFS , YFT, YFV , YFY , YYF , ered from insect cleavability , the X region may include one YYL . or more insect and /or nematode gut protease cleavable [0399 ] In some embodiments , exemplary sequences dis peptide sequences : AAK , AAR , AFK , AFR , AIK , AIN , AIQ , covered from insect cleavability , the X region may include AIR , AKF, AKI, AKK , AKL , AKN , AKO , AKR , AKS , AKT, one or more insect and / or nematode gut protease cleavable AKY, ALK , ALN , ALQ , ALR , APK , APR , ARF, ARI, ARK , peptide sequences : AAG , AAK , AAR , AFG , AFK , AFR , ARL , ARN , ARQ , ARR , ARS , ART, ARY, ASK , ASR , ATK , AGF, AGI, AGK , AGL , AGN , AGQ , AGR , AGS , AGT, ATR , AVK , AVR , AYK , AYR , DIK , DIR , DLK , DLR , EIK , AGY, AIG , AIK , AIN , AIQ , AIR , AKF, AKG , AKI, AKK , EIR , ELK , ELR , IIK , IIR , IKA , IKF , IKI, IKK , IKL , IKN , US 2018 /0362598 A1 Dec. 20 , 2018 69
IKQ, IKR , IKS , IKT, IKV, IKY, ?LK , ILR , INA, INK , INR , INV , IPK , IPR , IQA , IQK , IQR , IQV , IRA , IRF, IRI, IRK , ( SEQ ID NO : 1649 ) IRL , IRN , IRQ , IRR , IRS , IRT , IRV , IRY, ISK , ISR , ITK , AFVRLFGS , ITR , KAF, KAI, KAK , KAL , KAR , KAY, KFK , KFR , KIK , ( SEQ ID NO : 1650 ) KIN , KIQ , KIR , KKA , KKD , KKE , KKF, KKN , KKQ , AKLFVGS , KKS , KKT, KKV , KKY, KLK , KLN , KLQ , KLR , KNK , ( SEQ ID NO : 1651 ) KNR , KPK , KPR , KOK , KOR , KRA , KRD , KRE , KRF , ALFALKGS , KRN , KRO , KRS , KRT, KRV , KRY, KSI, KSK , KSL , KSR , KTI, KTK , KTL , KTR , KVF, KVI, KVK , KVL , KVR , ( SEO ID NO : 1652 ) KVY, KYK , KYR , LIK , LIR , LKA , LKF , LKI, LKK , LKL , ALFLKGS , LKN , LKQ , LKR , LKS , LKT, LKV , LKY, LLK , LLR , LNA , ( SEO ID NO : 1653 ) LNKLKN , LKOLNR, , LKRLNV , ,IK LPK , LPR , LQA , LQK , LQR , LOV , ALFLRGS , LRA , LRF, LRI, LRK , LRL , LRN , LRQ , LRR , LRS, LRT, ( SEO ID NO : 1654 ) LRV , LRY , LSK , LSR , LTK , LTR , NIK , NIR , NLK , NLR , ALFRGS , PIK , PIR , PKK , PKR , PLK , PLR , PRK , PRR , QIK , QIR , ( SEO ID NO : 1655 ) QLK , QLR , RAF, RAI, RAK , RAL , RAR , RAY , RFK , RFR , ALFRLRGS , RIK , RIN , RIO , RIR , RKA , RKD , RKE , RKF, RKN , RKO , RKS , RKT, RKV , RKY , RLK , RLN , RLQ , RLR , RNK , ( SEO ID NO : 1656 ) RNR , RPK , RPR , ROK , ROR , RRA , RRD , RRE , RRF, ALKALFGS , RRN , RRQ, RRS , RRT, RRV , RRY, RSI, RSK , RSL , RSR , ( SEO ID NO : 1657 ) RTI, RTK , RTL , RTR , RVF, RVI, RVK , RVL , RVR , RVY , ALKFFGS, RYK , RYR , VAK , VAR , VFK , VFR , VIK , VIN , VIQ , VIR , ( SEO ID NO : 1658 ) VKF, VKI, VKK , VKL , VKN , VKQ , VKR , VKS , VKT, ALKFLVGS , VKY, VLK , VLN , VLQ , VLR , VPK , VPR , VRF, VRI, VRK , VRL , VRN , VRQ , VRR , VRS , VRT, VRY, VSK , VSR , ( SEO ID NO : 1659 ) VTK , VTR , VVK , VVR , VYK , VYR . ALKIFVGS , [ 0401] The lack of numerous common sequences that ( SEQ ID NO : 1660 ) meet the both high ranked X and Y cleavability conditions ALKLFVGS , led to the merging of the sequences discovered in the X and ( SEO ID NO : 1661 ) Y categories to build binary and tertiary peptides. This FFADIKGS , research was called Round 2 and involved combinations ( SEO ID NO : 1662 ) from the Round 1 data , above . One way this was tested FFALKGS , involved several combinations of insect cleavable and SGF ( SEO ID NO : 1663 ) cleavable sequences ( X and Y regions respecdesigned and FFLKGS, ordered to be synthesized as FRET molecules. Another way this was tested involved the combinations of insect cleavable ( SEQ ID NO : 1664 ) and SGF cleavable sequences designed and ordered to be FFLRGS , synthesized as DNA encoding the ERSP -STA - ( TP - L ) n pro ( SEQ ID NO : 1665 ) tein for transient plant expression . Both FRET and transient FFRLRGS , expressed proteins were used to exemplify the insect cleav able and SGF cleavable nature of the binary and tertiary ( SEQ ID NO : 1666 ) peptides. FGYRIKGS , ( SEO ID NO : 1667 ) [0402 ] Round 2 data from FRET molecules that repre FLRLFGS , sented a subset of binary combinations based on the observed top hits in Round 1 provided a hierarchical ranking ( SEO ID NO : 1668 ) of the various binary combinations . Round 2 FRET mol FYARRGS , ecules were synthesized , resuspended , and tested in cleavage ( SEO ID NO : 1669 ) enzyme assays as described above in Round 1. Many GGLRKKGS , combinations of binary cleavable linking peptides were ( SEQ ID NO : 1670 ) tested against LEP , COLEOP and SGF conditions. IFVALKGS , [0403 ] Transiently expressed protein from DNA encoding variation of these the ERSP - STA - ( TP - L ) n protein where Lis ( SEQ ID NO : 1671 ) binary and tertiary peptide based on Round 2 data , and was IFVLKGS , also treated with the same cleavage enzymes (LEP , ( SEQ ID NO : 1672 ) COLEOP and SGF ) . IFVLRGS , [0404 ] Data that embodies Round 2 FRET experiment is ( SEQ ID NO : 1673 ) listed below in section 1 . 1 . Data that embodies Round 2 IFVRGS , plant expression system in listed in section 1 . 2 . The amino ( SEO ID NO : 1674 ) acid sequences listed in SEQ ID NOs: 1594 - 1758 were IFVRLRGS , synthesized as either/ or FRET and transient plant expres sion . US 2018 /0362598 A1 Dec. 20 , 2018
- continued - continued ( SEQ ID NO : 1675 ) ( SEO ID NO : 1701 ) ILFNIKGS , RRRFFAGS , ( SEO ID NO : 1676 ) ( SEO ID NO : 1702 ) LFAAPFGS , VFGRKGGS , ( SEO ID NO : 1677 ) ( SEQ ID NO : 1703 ) LFVALKGS , YFVRKGS . ( SEQ ID NO : 1678 ) [0405 ] Data from the Round 2 FRET that exemplifies the LFVLKGS , combination of both X and Y cleavability in a binary or ( SEO ID NO : 1679 ) tertiary peptide are presented in Table 6 . LFVLRGS , ( SEQ ID NO : 1680 ) TABLE 6 LFVRGS , Representative sequences for X - Y combinations showing cleavability in ( SEQ ID NO : 1681) both X and Y cleavage conditions LFVRLRGS , % of Max ( SEQ ID NO : 1682 ) Slope LFVRVFLGS ( SEQ ID NO : 1683 ) Sequence LGERGS , IFVALKGS ( SEQ ID NO : 1670 ) 69 ( SEO ID NO : 1684 ) ALKLFVGS ( SEQ ID NO : 1660 ) 100 LKALFGS , LFVALKGS (SEQ ID NO : 1677 ) 92 ( SEQ ID NO : 1685 ) LKFFGS , LRLFVGS ( SEQ ID NO : 1691 ) 74 ( SEO ID NO : 1686 ) LFVRLRGS ( SEQ ID NO : 1681 ) 94 LKIFVGS ( SEQ ID NO : 1687 ) LKLFVGS ( SEQ ID NO : 1687 ) 98 LKLFVGS , ( SEQ ID NO : 1688 ) Example 3 . Plant Protein Expression Testing of the LRALFGS , Candidate Binary / Tertiary Cleavable Linking ( SEQ ID NO : 1689 ) Peptides LRFFGS , [0406 ] The cloning and expression of a proteins contain ( SEQ ID NO : 1690 ) ing multiple copies of TP intervened by binary or tertiary LRIFVGS , cleavable linking peptides was performed using a tobacco ( SEQ ID NO : 1691 ) transient expression system technology referred to as FECT LRLFVGS , (Liu Z & Kearney CM , BMC Biotechnology, 2010 , 10 :88 , ( SEQ ID NO : 1692 ) the disclosure of which is incorporated herein by reference RALFGS , in its entirety ) . The FECT vector contains a T -DNA region for agroinfection , which contains a CaMV 35S promoter that ( SEQ ID NO : 1693 ) drives the expression of the foxtail mosaic virus RNA RIFVGS , without the genes encoding the viral coating protein and the ( SEQ ID NO : 1694 ) triple gene block . In the place of the coating protein and RLFVGS , triple block are a pair of subcloning sites (Pac ) and AvrII ) that allow an ORF to be subcloned N ' to C ' following the ( SEQ ID NO : 1695 ) Pacl site for high levels of transient viral expression . This RLRALFGS , “ disarmed ” virus genome prevents plant to plant transmis ( SEQ ID NO : 1696 ) sion . In addition to the FECT vector subcloned to express the RLRFFGS , various proteins with TP intervened by binary or tertiary ( SEO ID NO : 1697 ) cleavable linking peptides, a second FECT vector is co RLRIFVGS , expressed that encodes P19 , a RNA silencing suppressor protein from tomato bushy stunt virus, to prevent the post ( SEQ ID NO : 1698 ) transcriptional gene silencing (PTGS ) of the introduced RLRLFVGS , T -DNA . Agrobacterium containing the transient plant ( SEQ ID NO : 1699 ) expression system are injected into the leaves of tobacco RRKAFVGS , (Nicotiana benthamiana ) as described below . ( SEQ ID NO : 1700 ) [ 0407 ] With reference to FIGS. 2 - 5 , various designs of the RRKLIFGS , insecticidal and / or nematicidal proteins and DNA construct encoding insecticidal and / or nematicidal proteins are illus trated . In some examples , a TP repeat with intervening US 2018 /0362598 A1 Dec. 20 , 2018 binary or tertiary cleavable linking peptides is shown . An vectors DNA. The cell - DNA mixture was then kept on ice illustrative ERSP motif which can be used in accordance for 5 minutes , then transferred to - 80° C . for 5 minutes, and with the exemplary embodiments provided here is the Bar then incubated in a 37° C . water bath for 5 minutes . The ley Alpha -Amylase Signal peptide (BAAS ), which is com freeze - thaw treated cells were then diluted into 1 mL LB prised of 24 Amino acids as shown below ( N ' to C ' , one letter medium and shaken on a rocking table for 2 - 4 hours at room code ): temperature . Cell - LB mixture was then spun down at 5 ,000 rcf for 2 minutes to pellet cells and then 800 uL of LB ( SEO ID NO : 4 ) supernatant is removed . The cells are then resuspended in MANKHLSLSLFLVLLGLSASLASG . the remaining liquid and the entire volume, approximately 200 uL , of the transformed cell -LB mixture was then spread [0408 ] The Zea mays ubiquitin monomer used in some of onto LB agar plates with the appropriate antibiotics ( 10 the proteins was comprised of 75 Amino acids as shown ug /mL rifampicin , 25 ug /mL gentamycin , and 50 ug /mL below ( N ' to C ', one letter code) : kanamycin ) and incubated at 28° C . for two days. Resulting transformed colonies were then picked and culture in 6 mL QIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDOORLIFAGKOLE aliquots of LB medium with the appropriate antibiotics for DGRTLADYNIQKESTLHLVLRLRGG ( Accession Number XP transformed DNA analysis and making glycerol stocks of 020404049 . 1 , SEQ ID NO : 1759 ) . the transformed GV3 101 cells . [0409 ] An exemplary TP useful in the example described [0412 ] The transformed GV3101 cells were streaked onto here can include: U - ACTX -Hvla , which has the following an LB plate with the appropriate antibiotics (as described amino acid sequence ( N ' to C ', one letter code ): above ) from their glycerol stock and incubated at 28° C . for two days . A colony of transformed GV3 101 cells was used to inoculate 5 mL of LB -MESA medium ( LB media supple ( SEQ ID NO : 6 ) mented with 10 mM MES , 20 ?M acetosyringone ) and the QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA same antibiotics described above, and grown overnight at 28° C . The cells of the overnight culture were collected by or U + 2 -ACTX -Hvla , which has the following amino acid centrifugation at 5000 rpm for 10 minutes and resuspended sequence ( N ' to C ' , one letter code ): in the induction medium ( 10 mM MES , 10 mM MgC12 , 100 uM acetosyringone ) at a final OD600 of 1 . 0 . The cells were ( SEQ ID NO : 5 ) then incubated in the induction medium for 2 hours to GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA . overnight at room temperature and were then ready for transient transformation of tobacco leaves. Because FECT [0410 ] In separate examples, three different intervening uses a mixture of P19 expression and the gene of interest binary or tertiary cleavable linking peptides (denoted by the expression , cultures of cells for the pFECT- P19 transformed short hand form L1 -L4 ) were used (N ' to C ', one letter code ): GV3 101 cells and the gene of interest cultures were mixed together in equal amounts for infiltration of tobacco leaves before injection into the plant leaves. The treated cells were (L1 ) (SEQ ID NO : 1 ) infiltrated into the underside of attached leaves of Nicotiana IGER benthamiana plants by injection , using a 3 mL syringe without a needle attached . Protein expression in tobacco ( L2 ) ( SEQ ID NO : 1660 ) leaves was evaluated at 6 - 8 days post - infiltration . ALKLFVGS [ 0413 ] Full length gene- of - interest proteins were purified from the tobacco by using a syringe vacuum extraction ( L3 ) technique . The leaf tissue was rolled up and placed inside the IFVRLRGS ( SEQ ID NO : 1674 ) barrel of a 60 mL syringe with the needle port capped along with 10 mL of buffer (35 mM NaC1, 5 mM EDTA , 1 Roche (L4 ) ( SEO ID NO : 1658 ) cOmplete Tab protease inhibitor tab , pH 1 .2 using HCl) . The ALKFLVGS . plunger was then inserted into the barrel, inverted so the needle port was up , the cap was removed and excess air was [ 0411 ] In addition , a histidine tag, which has the following removed by forcing the plunger in . Once the majority of the amino acid sequence ( N ' to C ' , one letter code ) : HHHHH air had been removed , the syringe was capped and the HHHHH (SEQ ID NO : 1760 ) is used as part of the con plunger was pulled back as far as possible , creating a structed insecticidal and / or nematicidal protein . The follow vacuum , and held for 30 seconds . The vacuum was then ing insecticidal and / or nematicidal proteins: BAAS :UBI : U + slowly released by allowing the plunger to go back to the 2 - ACTX -Hvla :( L : U -ACTX - Hvla )z :His10 and BAAS : point in which there is no vacuum . This was then repeated ( U -ACTX -Hvla : L ) z : U - ACTX -Hvla :H10 ORF DNAs three more times . After the third time, the liquid was then (where BAAS is the ERSP ; and L is a binary or tertiary expelled out of the syringe by applying light pressure to the cleavable linking peptide ) as schematically shown in FIGS . leaves , the liquid was saved , and the process was repeated 6A and 6B , were cloned into the Pac I and Avr II restriction again before the leaves were discarded . The extracted liquid sites of the FECT expression vector to create the transient was then sterile filtered using a Pall 0 . 2 um Nylon syringe vectors which were then transformed into Agrobacterium filter and then concentrated in a GE Healthcare Viva Spin tumefaciens strain , GV3101 cells using a freeze - thaw 500 , 10K mwco rinsing samples with an additional 20 mL method as follows: the stored competent GV3101 cells were of the buffer and spinning the samples down until they were thawed on ice and then mixed with 1 - 5 ug pure transient concentrated about 30x. US 2018 /0362598 A1 Dec. 20 , 2018
[0414 ] The samples were then analyzed using standard Pro used as control software. Serially diluted known con Western Blotting techniques. Samples were prepared for a centrations of pure U - ACTX -Hvla samples were treated in protein gel by mixing 10 uL of protein sample with 9 uL the same manner as described above in the iELISA assay to Invitrogen 2xSDS loading buffer and 2 uL Novex 10x generate a mass - absorbance standard curve for quantities Reducing agent and heating the sample at 85° C . for 5 analysis . minutes. The samples were then loaded and ran on a Novex [ 0416 ] To determine pepsin cleavability of the Linkers, the Precast, 16 % Tricine gel in 1x Invitrogen Tricine running peptides are digested with simulated gastric fluid ( SGF, 35 buffer with 0 . 1 % sodium thioglycolate in the top tank . The mM NaCl, pH 1 . 2 with 1 mg/ ml pepsin from porcine gastric gel was run at 150V for 75 minutes . The gel was then mucosa ) . Purified protein samples were mixed with 1 : 3 , transferred to a Novel PVDF membrane using a 7 -minute SGF: protein ( v : v ) and were incubated shaking at 37° C . A transfer program on the iBLOT system . Once the transfer sample is taken at 0 , 2 and 10 minutes immediately prepared was complete , the blot membrane was then moved to a for a protein gel (see above ). The samples were then run on container and washed with Buffer A ( 1xTBS made from a Western Blot using the technique described above. See Quality Biological' s 10xTBS ( 0 . 25M tris base , 1 . 37M NaCl, FIG . 6 . 0 .03M KCL , PH 7. 4 )) , for five minutes by rocking gently at [0417 ] To determine insect cleavability of the binary / room temperature. This was then followed with a blocking tertiary peptides , the proteins were digested with gut extract step using Buffer B (Buffer A with 1 % BSA ) for 1 hour. The isolated from Southern Corn Rootworm (Diabrotica undec blot was then rinsed three times with 5 minute washes of impunctata ) . Isolation of the gut contents is explained in Buffer C (Buffer B with 0 .05 % Tween 20 ) . This was Example A . 1 . Prior to peptide digestion , the proteins were followed with a 1 : 10000 dilution of Maine Biotech Anti -His buffer exchanged into 100 mM Phosphate buffer , 150 mM antibody in Buffer C for 1 hour . The blot was then rinsed NaCl, pH 5 .83 using a GE Healthcare Viva Spin 20 returning three times with Buffer C for 5 minutes each . This was the final volume to the same as the starting volume. Digests followed with a 1 :3000 dilution of BioRad goat anti- mouse were carried out using SCR gut extract at a 1 : 3 dilution of AP conjugated antibody ( secondary antibody ) in Buffer C SCR gut extract stock :protein ( v : v ) shaking at 500 rpm , 25° for 1 hour. The blot was then rinsed with two times with C . Samples were taken at 0 , 30 and 210 minutes . Samples Buffer C for 5 minutes each and once with Buffer A for 5 were then prepared for SDS - PAGE and western blot as minutes . The blot is then developed with BioRad AP devel described above. oper and stopped by rinsing with water. The bands for the [0418 ] The binary / tertiary peptides were also tested for concentrated samples were then verified and diluted to make cleavability against CEW gut extract (described in Example the bands similar in intensities/ concentrations . The concen A . 1 ) . The peptides were buffer exchanged , just like they tration of the samples was then also validated via iELISA to were for the SCR assay above , into 200 mM Tris -HCl pH 8 . 1 be between 10 - 20 ng / ul. 150 mM NaCl. The assay conditions are repeated as listed [0415 ] Indirect ELISA ( ELISA ) assay was performed as above for SCR gut extract digest . follows to quantitatively evaluate the ICK motif TP from the [0419 ] These protocols resulted in the creation and vali tobacco leaves transiently transformed with the FECT dation of candidate tertiary cleavable linking peptides inter expression systems. 1 uL of the leaf extracted protein was vening copies of a TP expressed in plant. Additional spacers diluted into 99 UL CB2 solution ( Immunochemistry Tech to enhance cleavability were desired and they are detailed in nologies ) in the well of an Immulon 2HD 96 -well plate , with section 1 .3 . serial dilutions performed as necessary . Leaf proteins were Example 4 . FRET Research and Spacers that Make from the extract samples were then allowed to coat the well Multiple Candidate Tertiary Cleavable Linking walls for 3 hours in the dark at room temperature , and then Peptides the CB2 solution was removed , and each well was washed twice with 200 UL PBS (Gibco ) . 150 UL blocking solution [0420 ] From the experiments performed and described ( Block BSA in PBS with 5 % non - fat dry milk ) was then above two exemplary effective binary peptides, ALKLFV added into each well and incubated for 1 hour, in the dark , (SEO ID NO : 1605 ) and IFVRLR (SEO ID NO : 1619 ) , gave at room temperature . After the removal of the blocking rapid SGF cleavage and rapid LEP and COLEOP cleavage solution and a PBS wash off the wells , 100 L of rabbit of the peptides in human ( SGF ) and insect (LEP ) and anti - U -ACTX -Hvla antibody (primary antibody ) ( 1 : 250 ( COLEOP ) simulated gut environments . dilution in blocking solution ) was added to each well and [0421 ] In order for the binary cleavable linking peptide to incubated for 1 hour in the dark at room temperature . The cleave , a proteolytic enzyme has to be able to recognize and primary antibody was then removed and each well was land on the recognized sequence . If the space is tight or too washed with PBS 4 times. Then 100 uL of HRP - conjugated small, it may cause slower or lower digesting efficiency. By goat anti- rabbit antibody ( secondary antibody , used at making the binary peptide a tertiary peptide using the most 1 : 1000 dilution in the blocking solution ) was added into favorable amino acids for the conditions , the speed in which each well and incubated for 1 hour in the dark at room the peptides cleave should increase . The use of “ spacers ” temperature. After removal of the secondary antibody and allows for such increased peptidase cleavage and increased wash of the wells with PBS , 100 uL substrate solution (a 1 : 1 insecticidal activity . mixture of ABTS peroxidase substrate solution A and solu [0422 ] Spacers tion B , KPL ) was added to each well , and the chromogenic [0423 ] To screen for potential spacers, FRET peptides reaction was allowed to go until sufficient color develop were ordered as the binary peptide ALKLFVZ1Z , and ment was apparent. Then 100 uL of peroxidase stop solution varied amino acids in positions Z and Z2. Some of the was added to each well to stop the reaction . The light amino acids were screened as individuals and some were absorbance of each reaction mixture in the plate was read at screened as pools . The peptides were re - suspended using 405 nm using a Spectro Max -M2 plate reader , with SoftMax 50 % acetonitrile in water at a concentration of 2 uM of each US 2018 /0362598 A1 Dec. 20 , 2018 73 peptide . For the one containing more than one FRET pep tissue culture media , and an expression cassette for the tide , they were re - suspended so that each peptide was as 2 stable expression of the transgenic promoter. T - DNA borders UM ( assuming an equal portion of each one ). These were are repeat sequences (25 bp ) that flank the T- DNA in direct then re -suspended and screened as described above for orientation and delineate the region that the virulence genes Round 1 . The data does confirm that some amino acids in the will replicate and package for genetic transformation . In this Z position are more favorable than others . Table 7 shows example the backbone and the T -DNA sequences are pub representative amino acids for Z , and Z2 based on the FRET lished by Coutu et al. , in , Transgenic Res ( 2007 ) 16 : 771 peptide speed to cleave data . The greater the value of the 781 , the disclosure of which is incorporated herein by slope in in Table 7 the greater the activity of the amino acid reference in its entirety . In this example , the expression when part of a spacer. The column in Table 7 labeled “ Slope” cassettes for both the selectable marker and the gene of indicates the speed of cleavage of the Linker . interest ( an insecticidal or nematicidal insecticidal and /or nematicidal protein containing binary and tertiary cleavable TABLE 7 linking peptides and a TP ) are similar in design and differ only in the region encoding the open reading frame (ORF ) Representative spacer sequences for increasing speed to cleave for both X and Y conditions for their respective proteins . with binary peptide ALKLFV (SEQ ID NO : 1605 ) [0427 ] A good example of this expression cassette is the corn Ubi - 1 promoter and expression cassette defined by Spacer sequence Y Slope X Slope Christensen and Quail in Transgenic Res. 1996 May ; 5 ( 3 ) : AM 23461 13328 213 - 8 , the disclosure of which is incorporated herein by LH 36648 6278 reference in its entirety . The promoter region from a con MN 27088 3981 stitutively expressing gene cassette from a plant can be ES 24393 7394 WO 26735 6880 subcloned and the gene removed and replaced by a multi DT 24393 7394 cloning site (MCS ) to drop in the other genes of interest . In Sequences that decreased speed to cleave the case of the Ubi- 1 example the 906 bp promoter region (UBI - 1 prom ) promotes the transcription of an 81 bp 5' PG 1925 - 79 untranslated region ( 5 'UTR ) and a Ubi- 1 1025 bp intron IF - 5662 - 3064 (UBI - 1 intron ) which is followed by a MCS . The cassette is terminated by the insertion of a terminator sequence , in this [ 0424 ] An example of the gene construction for the tran example the terminator sequence is about 250 bp of nopaline sient Tobacco expression system can be seen in FIGS. 6A synthase (NOS ) 3' untranslated sequence and polyade and 6B . For example shown in FIG . 6A , the Linker nylation signals . For the selectable marker cassette , the sequences ALKLFVGS (SEQ ID NO : 1660 ) and IFVRL insertion of a gene which confers plant resistance to an RGS (SEQ ID NO : 1674 ) are represented in portions of FIG . herbicidal chemical can be used such that cells expressing 6A as (L ). In gels , FIG . 7, these show as 2 top bands for that cassette will be resistant to the inclusion of the herbi intact 4x ( w & w / o Ubiquitin ) . Example Linker sequence cidal chemical in the tissue culture media . In this example , IGER is represented by portions of FIG . 6B . In gels it shows the selectable marker gene can be a gene that encodes as 1 top band for intact 4x . phosphinothricin acetyltransferase activity ( see U . S . Pat . No . 5 ,648 , 477 , the disclosure of which is incorporated Example 5 . Cloning and Expression of an herein by reference in its entirety ) . Insecticidal Transgenic Protein Containing Binary [ 0428 ] The expression of an insecticidal transgenic protein and Tertiary Cleavable Linking Peptides containing binary and tertiary cleavable linking peptide and [0425 ] This example details an example of methods to a TP is exemplified herein , specifically in Example A , where recover a transgenic corn plant with the stable expression of such a gene is transiently expressed in tobacco . The linear an insecticidal and / or nematicidal protein containing binary composition of the entire labeled exemplary corn transfor and tertiary cleavable linking peptides and a TP . There are mation vector example can be seen in FIG . 8 . three components to making in this example: ( 1 ) vector [ 0429 ] The second component to this example is corn design , ( 2 ) plant transformation and regeneration , and ( 3 ) plant transformation by standard binary vectors and subse validation of expression of the intact protein . quence transgenic plant regeneration which is well known in [ 0426 ] The first component is the standard binary ) vector the art , and is defined and documented by Frame et al ., Plant design required for corn transformation , which involves the Physiology , May 2002 , Vol. 129 , pp . 13 - 22 and Vega et al. , creation of a vector which has two regions : a backbone ( FIG . Plant Cell Rep (2008 ) 27 : 297 - 305 , the disclosures of which 8 : white arrows ) and a t - DNA region (FIG . 8 : black arrows) . are incorporated herein by reference in their entireties. The backbone has two components : an origin of replication Following these protocols will result in the recovery of a (Ori or ori ) and a selectable marker. The backbone described transgenic plant with the t -DNA described above . in this example has two Oris so it can be replicated in both [0430 ] The third component to this section is the valida E . coli for subcloning purposes ( for example the ColEl a tion of expression of the intact protein . Transgenic plant high copy Ori ) and Agrobacterium for propagation of the tissue can be prepared to isolate the total soluble protein and plasmid ( for example the OriV a low copy ori and its trfA this can be quantified on an iELISA using a primary anti initiation protein ) . The backbone described in this example body specifically binds the TP recombinantly generated ( as has a bacterial neomycin phosphotransferase II (nptll ) gene defined in Example A as provided herein ) . The intact nature for resistance to kanamycin . This gene allows the selection of the protein can be validated using a similar total soluble for E . coli or Agrobacterium transformed with the plasmid . protein isolation followed by a protein gel and western The t - DNA region contains three components : borders , an blotting procedures (also as defined and exemplified in expression cassette for selection of transgenic plant cells on Example A herein ). US 2018 /0362598 A1 Dec. 20 , 2018 74
Example 6 . Subcloning and Expression of a Bt Cry the tertiary cleavable linking peptide ALKLFVGS (SEQ ID Insecticidal Protein to Contain Binary and Tertiary NO : 1660 ). This would insert the cleavable binary or tertiary Cleavable Linking Peptides peptide between the propeptide (Btinactive ) and the Bt active [0431 ] In some representative examples, protocols for the toxin (Btatud ) as illustrated in FIG . 3B where the cleavable expression and synthesis of modified TPs are provided binary or tertiary peptide is placed in frame between the herein . In one example , further detailed below , modified Bt inactive portion of the Bt TP (Btwactive inactive ) and the active proteins are described that when exposed to the contents of portion of the Bt TP (Btactive ) . insect and nematode and human gut environments , the Bt [0436 ] For the expression of a Bt Cry TP to contain a protein is more efficiently cleaved by the constituent insect binary and / or a tertiary cleavable linking peptide , the DNA or nematode proteases. The presence of the binary or tertiary described above would be subcloned into the multi - cloning cleavable peptide results in a more complete dissociation of site within the insecticidal gene expression cassette , and the stabilizing or inactive region of the Bt protein resulting transformed in corn as previously described by Example B . in the enhanced production of insecticidal and nematicidal TPs. Example 7. Exemplary Binary Peptides [0432 ] This example details how to subclone an exem [0437 ] In some embodiments , the binary peptide of the plary Bt cry TP to contain binary and tertiary cleavable present invention is the binary peptide that has an amino acid linking peptide (L ). There are two steps to this example : The sequence that can be assembled by combining a sequence conversion of a Bt cry TP gene sequence to contain the from the SGF screening Y set and the insect cleavability X binary or tertiary cleavable linking peptide ( L ) between a set, in X - Y or Y - X orientation from the X sequences and Y nonon - insecticidal domain (Btinactive ) and an insecticidal sequences . domain (Btactive ) gene , and the creation of a plant expressing [0438 ]. One embodiment of the sequences discovered from that gene . SGF screening , Y , are: AFF , AFG , AFI, AFL , AFV, AFY , [ 0433] The simplest method to insert a binary or tertiary AGF, AGL , AIF , AIL , ALF, ALG , ALI, ALL , ALY, AYF, cleavable peptide ( L ) into a cry gene is to order the synthesis AYL , DFF , DFG , DFI, DFL , DFY , DGF, DGL , DIF , DLF, of the DNA encoding the entire gene with the inserted DLG , DLY , DYF, DYL , EFF , EFG , EFI, EFL , EFY , EGF. sequence . EGL , EIF , ELF , ELG , ELY , EYF , EYL , FF , FFA , FFD , FFE , [ 0434 ] The Bt cry TP gene can be any number of genes; a FFF , FFG , FFI, FFK , FFL , FFR , FFS , FFT, FFV , FFY , FGA , good example of a Bt TP gene is the Cry3a gene . A variant FGD , FGE , FGF, FGI, FGK , FGL , FGR , FGS , FGT, FGV , of Cry3a gene can be found in Genbank as accession no . FGY, FLV , FLR , FYA , FYD , FYE , FYF , FYG , FYI, FYK , AX712174 : Sequence 8 from Patent WO03018810 , (acces FYL , FYR , FYS , FYT, FYV, FYY , GFA , GFD , GFE , GFF, sible at blast .ncbi . nlm .nih . gov / Blast . GFI, GFK , GFL , GFR , GFS , GFT, GFV , GFY, GGL , GIF , cgi ? PROGRAM = blastn & PAGE _ GIL , GLF , GLI, GLL , GLY , GYF , GYL , IF , IFA , IFF , IFG , TYPE = BlastSearch & LINK _ LOC = blasthome) which is IFI, IFL , IFV, IFY , IGF, IGL, IIF, IIL , ILF , ILG , ILI, ILL , incorporated herein by reference in its entirety . This ILY , IYF , IYL , LFA , LFF , LFG , LFI, LFL , LFV, LFY, LG , sequence is derived from a publication Walters et al . , Appl. LGA , LGE , LGF, LGI, LGL , LGV, LGY, LIF , LIG , LII , LIL , Environ . Microbiol. January 2008 vol. 74 no . 2 367 -374 , the LIY , LLF , LLG , LLI, LLL , LLY , LYA , LYF , LYG , LYI , LYL , disclosure of which is incorporated herein by reference in its LYV , LYY , NFF , NFG , NFI, NFL , NFY , NGF, NGL , NIL , entirety. In this publication , the authors replace a “ VSS” NLG , NLI, NLL , NYF , NYL , QFF , QFG , QFI, QFL , QFY, wild - type three amino acid sequence with a “ AAPF ” four QGF, QGL, QIL , QLG , QLI, QLL , QYF , QYL , SFF , SFG , amino acid sequence in the domain I loop between alpha SFI, SFL , SFY, SGF, SGL , SIF , SIL , SLF , SLG , SLI, SLL , helix 3 and alpha -helix 4 , to increase the cleavage of the SLY , SYF , SYL , TFF , TFG , TFI, TFL , TFY, TGF , TGL , TIF , non - active 12 kDa domain I from the insecticidal or active TIL , TLF, TLG , TLI, TLL , TLY, TYF , TYL , VFF , VFG , VFI, 55 kDa remainder . The authors hypothesize this AAPF is VFL , VFY , VGF, VGL , VIF, VIL , VLF, VLG , VLI, VLL , recognized by the Chymotrypsin /Cathepsin Genzyme in gut VLY, VYF, VYL , YFA , YFD , YFE , YFF , YFG , YFI, YFK , of Diabrotica and leads to more rapid conversation of the YFL , YFR , YFS , YFT , YFV , YFY , YGF, YGL , YYF, YYL . full length 67 kDa protein to a cleavage and the 55 kDa form [0439 ] Another embodiment of the sequences discovered which is the insecticidal mature protein and exhibits specific from SGF screening, Y , are : AFF , AFI, AFL , AFY , AIF , AIL , binding to western corn rootworm brush border membrane ALF, ALI, ALL , ALY , AYF, AYL , DFF, DFI, DFL , DFY , compared to the 67 kDa form . This AAPF amino acid DIF , DLF , DLY , DYF, DYL , YFL , EFF , EFI, EFL , 0 , EFY, sequence was analyzed via FRET and yeast -produced EIF , ELF , ELY , EYF , EYL , FFA , FFD , FFE , FFF , FFI, FFK , dimers of the TP “ U + 2 - ACTX - Hvla ” separated by AAPF FFL , FFR , FFS , FFT, FFV , FFY, FYA , FYD , FYE , FYF , amino acids in our hands and neither FRET nor protein FYI, FYK , FYL , FYR , FYS , FYT, FYV , FYY , IFA , IFF , IFI , showed any cleavability by human gastrointestinal proteases IFL , IFV , IFY, IIF , IIL , ILF , ILI, ILL , ILY, IYF, IYL , LFA , (data not shown ) . Thus, the peptide amino acid sequence LFF, LFI, LFL , LFV , LFY , LIF , LII, LIL , LIY , LLF , LLI , AAPF used in the Walters et al . reference cited above is not LLL , LLY, LYA , LYF , LYI, LYL , LYV , LYY , NFF , NFI, a binary or tertiary cleavable linking peptide as described NFL , NFY, NIL , NLI, NLL , NYF, NYL , OFF , QFI, QFL , herein . QFY , QIL , QLI, QLL , QYF , QYL , SFF , SFI, SFL , SFY , SIF , [0435 ] For the example of converting the Cry3a gene into SIL , SLF , SLI, SLL , SLY , SYF , SYL , TFF , TFI, TFL , TFY , one with an intervening binary or tertiary cleavable linking TIF , TIL , TLF, TLI, TLL , TLY , TYF , TYL , VFF , VFI, VFL , peptide , the cloning of the DNA sequence described in VFY , VIF , VIL , VLF, VLI, VLL , VLY , VYF, VYL , YFA , Walters et al. is performed such that the base pairs encoding YFD , YFE , YFF , YFI, YFK , YFL , YFR , YFS , YFT, YFV , the AAPF amino acids are removed and in their place (in YFY , YYF, YYL . frame) are the codons encoding the amino acids for a binary 0440 ] One embodiment of the sequences discovered from cleavable linking peptide ALKLFV (SEQ ID NO : 1605 ) or insect cleavability , X , are : AAG , AAK , AAR , AFG , AFK , US 2018 /0362598 A1 Dec. 20 , 2018 75
AFR , AGF, AGI, AGK , AGL , AGN , AGQ , AGR , AGS , IKI, IKK , IKL , IKN , IKQ , IKR , IKS, IKT, IKV, IKY, ILK , AGT, AGY, AIG , AIK , AIN , AIQ , AIR , AKF, AKG , AKI, ILR , INA , INK , INR , INV, IPK , IPR , IQA , IQK , IQR , IQV , AKK , AKL , AKN , AKQ , AKR , AKS , AKT, AKY, ALG , ALK , ALN , ALQ , ALR , APF , APG , APK , APR , ARF, ARG , IRA , IRF , IRI, IRK , IRL , IRN , IRQ, IRR , IRS , IRT, IRV , ARI, ARK , ARL , ARN , ARO , ARR , ARS , ART, ARY , ASG , IRY, ISK , ISR , ITK , ITR , KAF , KAI, KAK , KAL , KAR , ASK , ASR , ATG , ATK , ATR , AVG , AVK , AVR , AYG , AYK , KAY, KFK , KFR , KIK , KIN , KIQ , KIR , KKA , KKD , KKE, AYR , DGK , DGR , DIG , DIK , DIR , DLG , DLK , DLR , KKF, KKN , KKQ , KKS, KKT, KKV , KKY, KLK , KLN , EGK , EGR, EIG , EIK , EIR , ELG , ELK , ELR , ER , FVR , KLQ , KLR , KNK , KNR , KPK , KPR , KQK , KQR , KRA , GAF , GAI, GAK , GAL , GAR , GAY, GFK , GFR , GIK , GIN , KRD , KRE , KRF, KRN , KRQ , KRS, KRT, KRV, KRY, KSI, GIO , GIR , GKA , GKD , GKE , GKF, GKI, GKK , GKL , KSK , KSL , KSR , KTI, KTK , KTL , KTR , KVF, KVI, KVK , GKN , GKQ , GKR , GKS, GKT, GKV, GKY, GLK , GLN , GLQ , GLR , GNA , GNK , GNR , GNV , GPK , GPR , GQA , KVL , KVR , KVY, KYK , KYR , LIK , LIR , LKA , LKF, LKI, GOK , GOR , GOV, GRA , GRD , GRE , GRF, GRI, GRK , LKK , LKL , LKN , LKQ , LKR , LKS, LKT, LKV , LKY, LLK , GRL , GRN , GRQ , GRR , GRS , GRT ,GRV , GRY, GSI, GSK , LLR , LNA , LNK , LNR , LNV, LPK , LPR , LQA , LQK , GSL , GSR , GTI, GTK , GTL , GTR , GVF, GVI, GVK , GVL , LQR , LOV, LRA , LRF , LRI, LRK , LRL , LRN , LRQ , LRR , GVR , GVY , GYK , GYR , IGA , IGF, IGI, IGK , IGL, IGN , LRS, LRT, LRV , LRY, LSK , LSR , LTK , LTR , NIK , NIR , IGQ , IGR , IGS, IGT, IGV, IGY, IIG , IIK , IIR , IKA , IKF, NLK , NLR , PIK , PIR , PKK , PKR , PLK , PLR , PRK , PRR , IKG , IKI, IKK , IKL , IKN , IKO , IKR , IKS , IKT, IKV , IKY, QIK , QIR , QLK , QLR , RAF, RAI, RAK , RAL , RAR , RAY , ILG , ILK , ILR , INA , ING , INK , INR , INV, IPG , IPK , IPR , IQA , IQG , IQK , IQR , IQV, IRA , IRF, IRG , IRI, IRK , IRL , RFK , RFR , RIK , RIN , RIQ , RIR , RKA , RKD , RKE , RKF, IRN , IRQ , IRR , IRS , IRT, IRV, IRY, ISG , ISK , ISR , ITG , RKN , RKQ , RKS , RKT, RKV , RKY, RLK , RLN , RLQ , ITK , ITR , KAF, KAG , KAI, KAK , KAL , KAR , KAY , KFF , RLR , RNK , RNR , RPK , RPR , ROK , ROR , RRA , RRD , KFG , KFK , KFR , KGA, KGD , KGE , KGF, KGI, KGK , RRE , RRF , RRN , RRQ, RRS, RRT, RRV, RRY, RSI, RSK , KGL , KGN , KGQ, KGR , KGS, KGT, KGV, KGY, KIG , RSL , RSR , RTI, RTK , RTL , RTR , RVF , RVI, RVK , RVL , KIK , KIN , KIQ ,KIR , KKA , KKD , KKE , KKF, KKG , KKN , RVR , RVY, RYK , RYR , VAK , VAR , VFK , VFR , VIK , VIN , KKQ , KKS, KKT, KKV, KKY, KLG , KLK , KLN , KLQ , VIQ , VIR , VKF , VKI, VKK , VKL , VKN , VKQ , VKR , VKS, KLR , KNG , KNK , KNR , KPG , KPK , KPR , KOG , KOK , KOR , KRA , KRD , KRE , KRF, KRG , KRN , KRO , KRS , VKT, VKY, VLK , VLN , VLQ , VLR , VPK , VPR , VRF, VRI, KRT, KRV, KRY, KSG , KSI, KSK , KSL , KSR , KTG , KTI, VRK , VRL , VRN , VRQ , VRR , VRS , VRT, VRY, VSK , KTK , KTL , KTR , KVF, KVG , KVI, KVK , KVL , KVR , VSR , VTK , VTR , VVK , VVR , VYK , VYR . KVY , KYG , KYK , KYR , LFR , LGA , LGF, LGI, LGK , LGL , LGN , LGQ , LGR , LGS , LGT, LGV, LGY , LIG , LIK , Example 8 . The Insecticidal Efficacy of a LIR , LK , LKA , LKF , LKG , LKI, LKK , LKL , LKN , LKQ , Transgenic Plant Expressing the Insecticidal and/ or LKR , LKS , LKT, LKV, LKY, LLG , LLK , LLR , LNA , LNG , LNK , LNR , LNV , LPG , LPK , LPR , LQA , LQG , LOK , Nematicidal Proteins of the Present Invention LOR , LOV, LRA , LRF, LRG , LRI, LRK , LRL , LRN , LRQ , LRR , LRS , LRT, LRV , LRY, LSG , LSK , LSR , LTG , LTK , [0442 ] Transgenic corn plants were made using the vector LTR , NGK , NGR , NIG , NIK , NIR , NLG , NLK , NLR , PGK , described in FIG . 8 . The ORF comprising the components PGR , PIG , PIK , PIR , PKG , PKK , PKR , PLG , PLK , PLR , STA , L , and TP was defined by FIG . 6B and prepared as a PRG , PRK , PRR , QGK , OGR , QIG , QIK , QIR , OLG , OLK , polynucleotide operable for transformation into corn plant QLR , RAF, RAG , RAI, RAK , RAL , RAR , RAY, RFF , RFG , cells . A field trial was performed by planting seeds at 6 inch RFK , RFR , RGA , RGD , RGE , RGF, RGI, RGK , RGL , spacing, with 125 seeds per row with 6 reps. The UTC corn RGN , RGQ , RGR , RGS , RGT, RGV, RGY, RIG , RIK , RIN , line was the parent line used as an explant for corn trans RIQ , RIR , RKA , RKD , RKE , RKF, RKG , RKK , RKN , formation and the transgenic corn line was the resulting RKQ , RKS , RKT, RKV , RKY, RLF , RLFL , RLG , RLK , transgenic plant expressing the transgene of FIG . 6B . Each RLN , RLQ , RLR , RNG , RNK , RNR , RPG , RPK , RPR , plant was infested with 600 western corn rootworm eggs and ROG , ROK , ROR , RRA , RRD , RRE , RRF, RRG , RRK , then scored once the crop reached maturity . Scoring was RRN , RRO , RRR , RRS , RRT, RRV , RRY, RSG , RSI, RSK , performed on a 0 - 3 scale where the three whorls of roots RSL , RSR , RTG , RTI, RTK , RTL , RTR , RVF, RVG , RVI, underground were scored for damage using (Oleson , J . D . , RVK , RVL , RVR , RVY , RYG , RYK , RYR , VAG , VAK , Y . Park , T . M . Nowatzki, and J . J . Tollefson . 2005 . J . Econ VAR , VFG , VFK , VFR , VGF, VGI, VGK , VGL , VGN , Entomol. 98 ( 1 ): 1 -8 ). A 0 score represents no damage . A 3 VGQ , VGR , VGS, VGT, VGY, VIG , VIK , VIN , VIQ , VIR , score represents that all 3 root whorls were destroyed. The VKF, VKG , VKI, VKK , VKL , VKN , VKQ , VKR , VKS , Field trial data of non - transgenic and transgenic corn plants VKT, VKY, VLG , VLK , VLN , VLQ , VLR , VPG , VPK , where transgenic plants displayed reduced damage by west VPR , VR , VRF , VRG , VRI, VRK , VRL , VRN , VRQ, VRR , ern corn rootworm shown in FIG . 9 is the average score of VRS , VRT, VRY , VSG , VSK , VSR , VTG , VTK , VTR , 30 plants, 5 from each of the 6 reps picked at random . The VVG , VVK , VVR , VYG , VYK , VYR . transgenic plant comprising the insecticidal and / or nemati [0441 ] Another embodiment of the sequences discovered cidal protein shown in FIG . 6B turned in a score of 1. 55 from insect cleavability , X , are : AAK , AAR , AFK , AFR , damage compared to the 1 . 90 damage in the untransformed AIK , AIN , AIQ , AIR , AKF , AKI, AKK , AKL , AKN , AKQ , corn line that was used as an explant to generate the AKR , AKS, AKT, AKY, ALK , ALN , ALQ , ALR , APK , transgenic corn line . The use of the insecticidal and / or APR , ARF, ARI, ARK , ARL , ARN , ARQ , ARR , ARS , ART, nematicidal protein shown in FIG . 6B when transformed ARY, ASK , ASR , ATK , ATR , AVK , AVR , AYK , AYR , DIK , into corn demonstrates the efficacy of the transgene in DIR , DLK , DLR , EIK , EIR , ELK , ELR , IIK , IIR , IKA , IKF, controlling a pest of corn , western corn rootworm . US 2018 /0362598 A1 Dec. 20 , 2018 76
SEQUENCE LISTING The patent application contains a lengthy “ Sequence Listing ” section . A copy of the “ Sequence Listing” is available in electronic form from the USPTO web site ( http :/ / seqdata .uspto . gov / ? pageRequest = docDetail & DocID = US20180362598A1) . An electronic copy of the “ Sequence Listing ” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1 . 19 ( b ) ( 3 ).
1 . A peptide comprised of either a binary or a tertiary ( SEQ ID NO : 1600 ), ALKALF ( SEQ ID NO : 1601) , ALKFF peptide, that contains at least 4 amino acids and up to a (SEQ ID NO : 1602 ), ALKFLV (SER ID NO : 1603 ) , maximum of 16 amino acids, comprised of 2 or 3 different ALKIFV (SEQ ID NO : 1604 ) , ALKLFV (SEQ ID NO : regions, wherein the binary peptides have 2 different regions 1605 ) , FFADIK (SEO ID NO : 1606 ) , FFALK (SEO ID NO : and the tertiary peptides have 3 different regions ; wherein , 1607 ) , FFLK (SEQ ID NO : 1608 ) , FFLR (SEQ ID NO : the binary and tertiary peptide can be cleaved by both an 1609 ) , FFRLR ( SEQ ID NO : 1610 ) , FGYRIK (SEQ ID NO : animal gut protease and an insect or nematode gut protease . 1611 ) , FLRLF (SEQ ID NO : 1612 ) , FYARR (SEQ ID NO : 2 . The peptide of claim 1 , wherein the binary peptide 1613 ) , GGLRKK ( SEQ ID NO : 1614 ) , IFVALK (SEQ ID contains at least 4 amino acids and up to a maximum of 8 NO : 1615 ) , IFVLK (SEO ID NO : 1616 ) , IFVLR (SEO ID amino acids; and the peptide comprises 2 different regions, NO : 1617 ) , IFVR (SEQ ID NO : 1618 ), IFVRLR (SEQ ID wherein one of the regions can be cleaved by an animal gut NO : 1619 ) , ILFNIK (SEO ID NO : 1620 ) , LFAAPF (SEQ ID protease and the other region can be cleaved by an insect or NO : 1621 ) , LFVALK (SEQ ID NO : 1622 ) , LFVLK (SEQ ID nematode gut protease . NO : 1623 ), LFVLR (SEQ ID NO : 1624 ), LFVR (SEQ ID NO : 1625 ) , LFVRLR (SEQ ID NO : 1626 ) , LFVRVFL (SEQ 3 . The peptide of claim 1 , wherein the tertiary peptide ID NO : 1627 ) , LGER ( SEQ ID NO : 1628 ) , LKALF (SEQ contains at least 5 amino acids up to a maximum of 16 amino ID NO : 1629 ) , LKFF (SEO ID NO : 1630 ) , LKIFV (SEO ID acids ; and the tertiary peptide comprises a binary peptide NO : 1631) , LKLFV (SEQ ID NO : 1632 ) , LRALF (SEQ ID and a spacer of 1 to 4 amino acids. NO : 1633 ) , LRFF ( SEQ ID NO : 1634 ), LRIFV (SEQ ID 4 . The peptide of claim 3 , wherein the spacer comprises NO : 1635 ) , LRLFV (SEQ ID NO : 1636 ), RALF (SEQ ID the amino acid sequence GS , WN , WQ, MA , MV, AM , LH , NO : 1637 ) , RIFV (SEQ ID NO : 1638 ) , RLFV (SEQ ID NO : MN , ES , WQ, or DT. 1639 ) , RLRALF (SEO ID NO : 1640 ) , RLRFF (SEO ID NO : 5 . The binary peptide of any one of claims 1 - 4 , wherein 1641) , RLRIFV (SEQ ID NO : 1642 ) , RLRLFV (SEQ ID the binary peptide comprises an amino acid sequence NO : 1643 ) , RRKAFV (SEQ ID NO : 1644 ) , RRKLIF (SEQ selected from the group consisting of: X - X2- Y - Y2, X -X2 ID NO : 1645 ) , RRRFFA (SEQ ID NO : 1646 ), VFGRKG X2- Y - Y2, X -X2 - X2 - X4- Y - Y2, X , - X2- Y - Y2- Y3, X7 - X (SEQ ID NO : 1647 ) , and YFVRK (SEQ ID NO : 1648 ) . X3 - Y - Y2- Y3, X , - X2- X3 -X4 - Y - Y2- Y3, X -X2 - Y - Y2- Yz 7 . The peptide of claim 6 , wherein the binary peptide has Y4, X , -X7 - X , - Y - Y - Y3- Y4, X7- X2 - X3- 84 - Y - Y2 -Y3 - Y4; an amino acid sequence : IFVALK ( SEQ ID NO : 1615 ) , Y - Y2- X7 - X2, Y - Y2- X7 -X2 - Xz, Y - Y2 - X , -X2 - X3- X4 , IFVRLR (SEQ ID NO : 1619 ) , ALKLFV ( SEQ ID NO : Y - Y - Y3- X , -X2 , Y - Y2- Y3 - X , -X2 - Xz, Y - Y2 - Y3- X - X2 1605 ) , ALKFLV (SEQ ID NO : 1603 ) , RRKAFV (SEQ ID Xz- X4, Y - Y2 - Yz- Y4- X7 -X2 , Y -Y2 - Y3 -Y4 - X , - X2- X3 , NO : 1644 ) , LFAAPF (SEO ID NO : 1621 ) , LFVALK (SEO Yi - Y2 - Yz- Y4 - X - X2- X3 - X4 , wherein each X , and each Y , is ID NO : 1622 ) , LRLFV ( SEQ ID NO : 1636 ) , LFVRLR (SEQ an amino acid , and wherein each X , and each Yn is an amino ID NO : 1626 ) , or LKLFV (SEQ ID NO : 1632 ) . acid , and wherein X , is selected from the group consisting 8 . The peptide of claim 1 , wherein the tertiary peptide of: (all single letter amino acid format ) A , D , E , G , I, K , L , comprises an amino acid sequence of IFVALKGS (SEQ ID N , P , Q , R , and V ; X , is selected from the group consisting NO : 1670 ) , IFVRLRGS (SEQ ID NO : 1674 ) , ALKLFVGS of A , F , G , I , K , L , N , P , Q , R , S , T , V , and Y ; X , is selected (SEQ ID NO : 1660 ) , ALKFLVGS (SEQ ID NO : 1658 ) , from the group consisting of A , D , E , F , G , I, K , L , N , Q , R , RRKAFVGS ( SEQ ID NO : 1699 ) , LFAAPFGS (SEQ ID S , T , V , and Y ; X4 is selected from the group consisting of: NO : 1676 ), LFVALKGS (SEQ ID NO : 1677 ) , LRLFVGS R , H , K , D , E , S , T, N , Q , G , A , I, L , M , F , W , Y , and V . In ( SEO ID NO : 1691 ) , LFVRLRGS ( SEQ ID NO : 1681 ) , or some embodiments , Y , is selected from the group consisting LKLFVGS (SEQ ID NO : 1687 ). of: ( all single letter amino acid format ) A , D , E , F , G , I, L , 9 . The peptide of any one of claims 1 - 7 , wherein the N , Q , S , T, V, and Y ; Y2 is selected from the group consisting binary peptide has an amino acid sequence IFVRLR (SEQ of F, G , I, L , and Y; and Y3 is selected from the group ID NO : 1619 ), ALKLFV (SEQ ID NO : 1605 ), ALKFLV consisting of A , D , E , F , G , I , K , L , R , S , T , V , and Y ; and (SEQ ID NO : 1603 ), RRKAFV (SEQ ID NO : 1644 ), or Y4 is selected from the group consisting of: R , H , K , D , E , LFAAPF (SEQ ID NO : 1621 ) . S , T, N , Q , G , A , I, L , M , F , W , Y , and V . 10 . The peptide of claims 1 - 9 , wherein the tertiary peptide 6 . The peptide of any one of claims 1 - 5 , wherein the comprises an amino acid sequence of: IFVRLRGS (SEQ ID binary peptide has an amino acid sequence : AFVRLF ( SEQ NO : 1674 ) , ALKLFVGS (SEQ ID NO : 1660 ) , ALKFLVGS ID NO : 1594 ) , AKLFV ( SEQ ID NO : 1595 ), ALFALK (SEQ ID NO : 1658 ) , RRKAFVGS (SEQ ID NO : 1699 ) , (SEQ ID NO : 1596 ) , ALFLK (SEQ ID NO : 1597 ), ALFLR LFAAPFGS ( SEQ ID NO : 1676 ) , GSIFVRLR (SEO ID NO : (SEQ ID NO : 1598 ) , ALFR (SEQ ID NO : 1599 ) , ALFRLR 1729 ), GSALKLFV (SEQ ID NO : 1715 ) , GSALKFLV US 2018 /0362598 A1 Dec. 20 , 2018
(SEO ID NO : 1713 ), GSRRKAFV (SEQ ID NO : 1754 ) , or nematode gut protease , and the polynucleotide further GSLFAAPF (SEQ ID NO : 1731 ) . encodes a toxic peptide fused in frame with said cleavable 11 . The peptide of any one of claims 1 - 10 , wherein the peptide. binary or tertiary peptide is linked to a toxic protein ( TP ). 25 . The DNA construct of claim 24 , wherein the tertiary 12 . The peptide of claim 11, further comprising a second peptide contains at least 5 amino acids up to a maximum of binary or tertiary peptide linked in frame to the C - terminus 16 amino acids, and the tertiary peptide comprises a binary of the TP. peptide and a spacer of 1 to 4 amino acids. 13 . The peptide of any one of claims 1 - 12 , wherein the binary or tertiary peptide is linked to the C - terminus of an 26 . The DNA construct of claim 25 , wherein the spacer Endoplasmic Reticulum Signal Peptide (ERSP ) . comprises the amino acid sequence GS , WN ,WQ , MA , MV , 14 . An insecticidal and/ or nematicidal protein having one AM , LH , MN , ES , WQ , or DT. or more cleavable linking peptides , the insecticidal and /or 27. The DNA construct of any one of claims 24 - 26 , nematicidal protein comprising an ERSP fused in frame with wherein the binary peptide comprises an amino acid the N -terminus of a binary or tertiary peptide , the binary or sequence of: X -X2 - Y , - Y2, X7- X2- Xz- Y - Y2, X , -X2 - Xz tertiary peptide is fused in frame with a construct compris X4 - Y - Y2, X , - X - Y - Y2- Y3, X - X - X2 - Y - Y - Y3, X -X2 ing : ( L - TP ) n , ( L - TP ) . - L , ( TP - L ) n , or L - ( TP - L )n , wherein TP X3- 84 - Y - Y2- Y3 , X7- X2- Y - Y2- Y3- Y4, X , -X2 - Xz- Y - Y2 is a toxic protein , L is a binary or tertiary peptide and n is Y3- Y4, X -X2 - X3 -X4 - Y - Y2 - Y3 - Y4, Y - Y2- X -X2 , Y - Y2 an integer ranging from 1 to 200 . X -X2 - X3 , Y -Y2 - X - X2- X3 - X4, Y - Y2- Y3- X - X2, Y - Y2 15 . The peptide or insecticidal and /or nematicidal protein Yz- X , - X2- X3, Y - Y2- Y3- X - X2- Xz- X4, Y - Y - Y3- Y4- X , of any one of claims 1 - 14 , wherein the insect protease is a X2, Y - Y2- Y3 - Y4- X - X2- X3, Y - Y2- Y3- Y4- X - X2- X3- X4, protease derived from a Lepidoptera or a Coleoptera insect wherein each X , and each Y , is an amino acid , and wherein species . each Xn and each Yn is an amino acid , and wherein X is selected from the group consisting of: (all single letter amino 16 . The peptide or insecticidal and /or nematicidal protein acid format) A , D , E , G , I , K , L , N , P , Q , R , and V ; X , is of any one of claims 1 - 15 , wherein the animal gut protease selected from the group consisting of A , F , G , I , K , L , N , P , is a human gastrointestinal protease . Q , R , S , T , V , and Y ; X , is selected from the group consisting 17 . The peptide or insecticidal and /or nematicidal protein of A , D , E , F , G , I , K , L , N , Q , R , S , T , V , and Y ; X4 is of any one of claims 1 - 16 , wherein the TP is a protein selected from the group consisting of: R , H , K , D , E , S , T, selected from the group consisting of a Pore Forming N , Q , G , A , I , L , M , F , W , Y , and V . In some embodiments , Insecticidal Protein ( PFIP ) and a Cysteine Rich Insecticidal Y is selected from the group consisting of: (all single letter Protein (CRIP ) . amino acid format ) A , D , E , F , G , I , L , N , O , S , T , V , and Y ; 18 . The peptide or insecticidal and / or nematicidal protein Y , is selected from the group consisting of F , G , I , L , and Y ; of any one of claims 1 - 17 , wherein the TP is a peptide or protein having at least 80 % , at least 85 % , at least 90 % , at and Y? is selected from the group consisting of A , D , E , F , least 95 % , at least 96 % , at least 97 % , at least 98 % , at least G , I , K , L , R , S , T , V , and Y ; and Y . is selected from the 99 % , or at least 100 % sequence identity or sequence homol group consisting of: R , H , K , D , E , S , T , N , Q , G , A , I, L , ogy to a peptide or protein having an amino acid sequence M , F , W , Y , and V . as set forth in SEQ ID NOs: 5 - 1593 and 1761- 1775 . 28 . The DNA construct of claim 27 , wherein the binary 19 . The peptide or insecticidal and /or nematicidal protein peptide comprises the amino acid sequence : AFVRLF (SEO of any one of claims 1 - 18 , wherein the TP is a peptide or ID NO : 1594 ) , AKLFV (SEQ ID NO : 1595 ) , ALFALK protein having at least 80 % , at least 85 % , at least 90 % , at (SEQ ID NO : 1596 ) , ALFLK (SEQ ID NO : 1597 ), ALFLR least 95 % , at least 96 % , at least 97 % , at least 98 % , at least (SEQ ID NO : 1598 ) , ALFR (SEQ ID NO : 1599 ), ALFRLR 99 % , or at least 100 % sequence identity or sequence homol ( SEO ID NO : 1600 ) , ALKALF (SEO ID NO : 1601 ) , ALKFF ogy to a peptide or protein having an amino acid sequence (SEQ ID NO : 1602 ) , ALKFLV ( SEQ ID NO : 1603 ) , as set forth in SEQ ID NOs: 5 , 6 and 1761 , 1762, 1767 , ALKIFV (SEO ID NO : 1604 ) , ALKLFV (SEO ID NO : 1771 - 1773 , and variants thereof. 1605 ) , FFADIK (SEQ ID NO : 1606 ) , FFALK (SEQ ID NO : 1607 ) , FFLK (SEQ ID NO : 1608 ) , FFLR (SEO ID NO : 20 . The peptide or insecticidal and /or nematicidal protein 1609 ) , FFRLR (SEQ ID NO : 1610 ), FGYRIK ( SEQ ID NO : of claim 19 , wherein the TP is a peptide having an amino 1611 ) , FLRLF (SEQ ID NO : 1612 ) , FYARR (SEQ ID NO : acid sequence of any one of SEQ ID NOs : 5 , 6 , 1761, 1762 , 1613 ) , GGLRKK (SEQ ID NO : 1614 ) , IFVALK (SER ID 1767 , 1771 - 1773 , or variants thereof. NO : 1615 ) , IFVLK (SEQ ID NO : 1616 ) , IFVLR (SEQ ID 21. A polynucleotide encoding a peptide or insecticidal NO : 1617 ) , IFVR (SEQ ID NO : 1618 ) , IFVRLR (SEQ ID and / or nematicidal protein of any one of claims 1 - 20 . NO : 1619 ) , ILFNIK (SEO ID NO : 1620 ) , LFAAPF (SEO ID 22 . A vector comprising a polynucleotide of claim 21. NO : 1621 ), LFVALK (SEQ ID NO : 1622 ), LFVLK (SEQ ID 23 . A bacterial host cell , a yeast host cell , a plant host cell NO : 1623 ) , LFVLR (SEQ ID NO : 1624 ) , LFVR (SEQ ID or an animal host cell comprising a vector of claim 22 . NO : 1625) , LFVRLR (SEQ ID NO : 1626 ) , LFVRVFL (SEQ 24 . A DNA construct comprising a polynucleotide linked ID NO : 1627 ) , LGER (SEQ ID NO : 1628 ) , LKALF ( SEO to an operable promoter, wherein said polynucleotide ID NO : 1629 ) , LKFF (SEQ ID NO : 1630 ) , LKIFV ( SEO ID encodes : a cleavable peptide comprising either a binary or a NO : 1631 ) , LKLFV (SEQ ID NO : 1632 ) , LRALF (SEQ ID tertiary peptide , said cleavable peptide containing at least 4 NO : 1633 ) , LRFF ( SEO ID NO : 1634 ) , LRIFV (SEO ID amino acids and up to a maximum of 16 amino acids, and NO : 1635 ) , LRLFV (SEO ID NO : 1636 ) , RALF (SEO ID is comprised of 2 or 3 different regions, wherein the binary NO : 1637 ) , RIFV (SEQ ID NO : 1638 ), RLFV (SEQ ID NO : peptide has 2 different regions and the tertiary peptide has 3 1639) , RLRALF ( SEQ ID NO : 1640 ) , RLRFF (SEQ ID NO : different regions; wherein the cleavable peptide can be 1641) , RLRIFV (SEQ ID NO : 1642 ) , RLRLFV (SEQ ID cleaved by both an animal gut protease and an insect or NO : 1643 ), RRKAFV (SEQ ID NO : 1644 ), RRKLIF (SEQ US 2018 /0362598 A1 Dec . 20 , 2018
ID NO : 1645 ), RRRFFA (SEQ ID NO : 1646 ) , VFGRKG - continued ( SEQ ID NO : 1670 ) ( SEQ ID NO : 1647 ) , and YFVRK (SEQ ID NO : 1648 ) . IFVALKGS , 29 . The DNA construct of claim 28 , wherein the binary peptide comprises an amino acid sequence : IFVRLR (SEQ ( SEQ ID NO : 1671 ) ID NO : 1619 ) , ALKLFV (SEQ ID NO : 1605 , ALKFLV IFVLKGS , (SEQ ID NO : 1603 ), RRKAFV ( SEQ ID NO : 1644 ), or ( SEQ ID NO : 1672 ) LFAAPF (SEQ ID NO : 1621) . IFVLRGS , 30 . The DNA construct of claim 24, wherein said poly ( SEO ID NO : 1673 ) nucleotide encodes a tertiary peptide comprising the amino IFVRGS , acid sequence : ( SEO ID NO : 1674 ) IFVRLRGS , ( SEQ ID NO : 1649 ) ( SEO ID NO : 1675 ) AFVRLFGS , ILFNIKGS , ( SEQ ID NO : 1650 ) ( SEO ID NO : 1676 ) AKLFVGS , LFAAPFGS , ( SEQ ID NO : 1651 ) ( SEQ ID NO : 1677 ) ALFALKGS , LFVALKGS , ( SEQ ID NO : 1652 ) ( SEO ID NO : 1678 ) ALFLKGS , LFVLKGS , ( SEQ ID NO : 1653 ) ( SEO ID NO : 1679 ) ALFLRGS , LFVLRGS , ( SEO ID NO : 1654 ) ( SEO ID NO : 1680 ) ALFRGS, LFVRGS , ( SEO ID NO : 1655 ) ( SEQ ID NO : 1681 ) ALFRLRGS , LFVRLRGS , ( SEQ ID NO : 1656 ) ( SEO ID NO : 1682 ) ALKALFGS , LFVRVFLGS , ( SEQ ID NO : 1657 ) ( SEO ID NO : 1683 ) ALKFFGS , LGERGS , ( SEQ ID NO : 1658 ) ( SEO ID NO : 1684 ) ALKFLVGS , LKALFGS , ( SEO ID NO : 1659 ) ( SEQ ID NO : 1685 ) ALKIFVGS , LKFFGS , ( SEQ ID NO : 1660 ) ( SEQ ID NO : 1686 ) ALKLFVGS , LKIFVGS , ( SEQ ID NO : 1661 ) ( SEO ID NO : 1687 ) FFADIKGS , LKLFVGS , ( SEQ ID NO : 1662 ) ( SEO ID NO : 1688 ) FFALKGS , LRALFGS , ( SEQ ID NO : 1663 ) ( SEO ID NO : 1689 ) FFLKGS , LRFFGS , ( SEQ ID NO : 1664 ) ( SEO ID NO : 1690 ) FFLRGS , LRIFVGS , ( SEQ ID NO : 1665 ) ( SEQ ID NO : 1691 ) FFRLRGS , LRLFVGS , ( SEQ ID NO : 1666 ) ( SEQ ID NO : 1692 ) FGYRIKGS , RALFGS , ( SEQ ID NO : 1667) ( SEQ ID NO : 1693 ) FLRLFGS , RIFVGS , ( SEQ ID NO : 1668 ) ( SEQ ID NO : 1694 ) FYARRGS , RLFVGS , ( SEO ID NO : 1669 ) ( SEO ID NO : 1695 ) GGLRKKGS , RLRALFGS , US 2018 /0362598 A1 Dec. 20 , 2018
- continued - continued ( SEQ ID NO : 1696 ) ( SEQ ID NO : 1722 ) RLRFFGS , GSFLRLF , ( SEO ID NO : 1697 ) ( SEO ID NO : 1723 ) RLRIFVGS , GSFYARR , ( SEO ID NO : 1698 ) ( SEQ ID NO : 1724 ) RLRLFVGS , GSGGLRKK , ( SEQ ID NO : 1699 ) ( SEO ID NO : 1725 ) RRKAFVGS , GSIFVALK , ( SEQ ID NO : 1700 ) ( SEO ID NO : 1726 ) RRKLIFGS , GSIFVLK , ( SEQ ID NO : 1701 ) ( SEO ID NO : 1727 ) RRRFFAGS , GSIFVLR , ( SEQ ID NO : 1702 ) ( SEO ID NO : 1728 ) VFGRKGGS , GSIFVR , ( SEQ ID NO : 1703 ) ( SEO ID NO : 1729 ) YFVRKGS . GSIFVRLR , ( SEQ ID NO : 1704 ) ( SEO ID NO : 1730 ) GSAFVRLF , GSILFNIK , ( SEO ID NO : 1705 ) ( SEO ID NO : 1731 ) GSAKLFV , GSLFAAPF , ( SEQ ID NO : 1706 ) ( SEO ID NO : 1732 ) GSALFALK , GSLFVALK , ( SEO ID NO : 1707 ) ( SEQ ID NO : 1733 ) GSALFLK , GSLFVLK , ( SEQ ID NO : 1708 ) ( SEO ID NO : 1734 ) GSALFLR GSLFVLR , ( SEQ ID NO : 1709 ) ( SEO ID NO : 1735 ) GSALFR , GSLFVR , ( SEQ ID NO : 1710 ) ( SEO ID NO : 1736 ) GSALFRLR , GSLFVRLR , ( SEQ ID NO : 1711 ) ( SEQ ID NO : 1737 ) GSALKALF , GSLFVRVFL , ( SEQ ID NO : 1712 ) ( SEO ID NO : 1738 ) GSALKFF , GSLGER , ( SEQ ID NO : 1713 ) ( SEQ ID NO : 1739 ) GSALKFLV GSLKALF , ( SEQ ID NO : 1714 ) ( SEO ID NO : 1740 ) GSALKIFV , GSLKFF , ( SEQ ID NO : 1715 ) ( SEO ID NO : 1741 ) GSALKLFV , GSLKIFV ( SEQ ID NO : 1716 ) ( SEO ID NO : 1742 ) GSFFADIK , GSLKLFV , ( SEQ ID NO : 1717 ) ( SEQ ID NO : 1743 ) GSFFALK , GSLRALF , ( SEO ID NO : 1718 ) ( SEQ ID NO : 1744 ) GSFFLK , GSLRFF , ( SEQ ID NO : 1719 ) ( SEQ ID NO : 1745 ) GSFFLR , GSLRIFV , ( SEQ ID NO : 1720 ) ( SEQ ID NO : 1746 ) GSFFRLR , GSLRLFV , ( SEQ ID NO : 1721 ) ( SEQ ID NO : 1747 ) GSFGYRIK , GSRALF , US 2018 /0362598 A1 Dec. 20 , 2018
- continued 40 . The host cell of claim 39 , wherein said bacterial cell ( SEO ID NO : 1748 ) is selected from the group consisting of an Agrobacterium , GSRIFV , a Bacillus, an Escherichia , a Salmonella , a Pseudomonas , ( SEO ID NO : 1749 ) and a Rhizobium cell. GSRLFV , 41. The host cell of claim 39 , wherein said plant cell is selected from the group consisting of: alfalfa , banana , bar ( SEO ID NO : 1750 ) ley , bean , broccoli , cabbage, canola , carrot, cassava , castor, GSRLRALF , cauliflower, celery , chickpea , Chinese cabbage , citrus , coco ( SEO ID NO : 1751 ) nut, coffee , corn , clover , cotton , a cucurbit , cucumber , GSRLRFF , Douglas fir, eggplant, eucalyptus , flax , garlic , grape , hops , ( SEQ ID NO : 1752 ) leek , lettuce, Loblolly pine , millets, melons , nut, oat, olive , GSRLRIFV , onion , ornamental, palm , pasture grass , pea , peanut, pepper, pigeonpea , pine, potato , poplar , pumpkin , Radiata pine , ( SEQ ID NO : 1753 ) radish , rapeseed , rice , rootstocks, rye , safflower, shrub , GSRLRLFV , sorghum , Southern pine , soybean , spinach , squash , straw ( SEQ ID NO : 1754 ) berry , sugar beet, sugarcane , sunflower , sweet corn , sweet GSRRKAFV , gum , sweet potato , switchgrass , tea , tobacco , tomato , triti cale , turf grass, watermelon , and a wheat plant cell. ( SEQ ID NO : 1755 ) 42 . A plant, or part thereof comprising a polynucleotide GSRRKLIF , linked to an operable promoter, wherein said polynucleotide ( SEQ ID NO : 1756 ) encodes: a cleavable peptide comprising either a binary or a GSRRRFFA , tertiary peptide , said cleavable peptide containing at least 4 ( SEQ ID NO : 1757 ) amino acids and up to a maximum of 16 amino acids, and GSVFGRKG , is comprised of 2 or 3 different regions, wherein the binary and peptide has 2 different regions and the tertiary peptide has 3 different regions ; wherein the cleavable peptide can be ( SEQ ID NO : 1758 ) cleaved by both an animal gut protease and an insect or GSYFVRK . nematode gut protease , and wherein the polynucleotide 31 . The DNA construct of claim 24 , wherein said insect further encodes a toxic peptide fused in frame with said protease is a protease derived from a Lepidoptera or a cleavable peptide . Coleoptera insect species . 43 . The plant, or part thereof, of claim 42 , wherein said plant is elected from the group consisting of a alfalfa , 32 . The DNA construct of claim 24 , wherein said animal banana , barley , bean , broccoli , cabbage , canola , carrot, cas gut protease is a human gastrointestinal protease . sava , castor , cauliflower , celery , chickpea , Chinese cabbage , 33 . The DNA construct of any one of claims 24 -32 , citrus , coconut, coffee , corn , clover, cotton , a cucurbit , wherein the TP is a peptide or protein selected from the cucumber, Douglas fir, eggplant, eucalyptus, flax , garlic , group consisting of a Pore Forming Insecticidal Protein grape , hops, leek , lettuce , Loblolly pine , millets , melons, (PFIP ) and a Cysteine Rich Insecticidal Protein (CRIP ). nut, oat, olive , onion , ornamental, palm , pasture grass , pea , 34 . The DNA construct of claim 33 , wherein the TP is a peanut, pepper, pigeonpea , pine , potato , poplar, pumpkin , peptide or protein having at least 80 % , at least 85 % , at least Radiata pine , radish , rapeseed , rice , rootstocks, rye , saf 90 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , flower, shrub , sorghum , Southern pine , soybean , spinach , at least 99 % , or at least 100 % sequence identity or sequence squash , strawberry , sugar beet , sugarcane, sunflower, sweet homology to a peptide or protein having an amino acid corn , sweet gum , sweet potato , switchgrass, tea , tobacco , sequence as set forth in SEQ ID NOs: 5 - 1593 and 1761 tomato , triticale , turf grass, watermelon , and a wheat plant, 1775 . and wherein said part is selected from the group consisting of a leaf, a stem a flower , a sepal, a fruit , a root, or a seed . 35 . The DNA construct of claim 34 , wherein the TP is a 44 . A method of controlling a pest infection of a plant , peptide or protein having at least 80 % , at least 85 % , at least said method comprising providing in a diet of the pest , a 90 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , plant, or part thereof, the plant or part thereof expressing an at least 99 % , or at least 100 % sequence identity or sequence insecticidal and/ or nematicidal protein , or coated at least on homology to a peptide or protein having an amino acid a portion of the plant or part thereof, a composition com sequence as set forth in SEQ ID NOs: 5 , 6 and 1761 , 1762 , prising an insecticidal and / or nematicidal protein , wherein 1767 , 1771 - 1773 , and variants thereof. the insecticidal and /or nematicidal protein comprises one or 36 . The DNA construct of claim 35 , wherein the TP is a more cleavable binary or tertiary peptides, and one or more peptide or protein having an amino acid sequence of any one TPs , wherein the binary and tertiary peptide can be cleaved of SEQ ID NOs: 5 , 6 , 1761 , 1762 , 1767 , 1771 - 1773 , or by both an animal gut protease and an insect or nematode gut variants thereof. protease . 37 . A host cell comprising a DNA construct of any one of 45 . The method of controlling a pest infection of a plant of claim 44 , wherein said cleavable binary peptide com claims 24 - 36 . prises an amino acid sequence : X7 -X2 - Y - Y2, X , -X2 - Xz 38 . The host cell of claim 37 , comprising a DNA construct Y - Y2, X , - X2- X3- X4 - Y - Y2, X -X2 - Y - Y - Y3, X -X7 - X , of claim 35 . Y - Y2- Y , X7- X2- X3- X4 - Y - Y - Y3, X - X2- Y -Y2 - Y3- Y4, 39 . The host cell of claim 37, wherein said host cell is a X7- X , -X3 - Y - Y2- Y3 - Y4) X -X2 - X3- X4 - Y - Y2- Y3- Y4, bacterial cell , a yeast cell, a plant cell , or an animal cell. Y - Y2- X -X2 , Y - Y2- X7 -X2 - Xz, Y - Y2 - X -X2 - X3- X4 , US 2018 /0362598 A1 Dec. 20 , 2018
Y - Y2 - Y3- X - X2, Y - Y2- Y3- X - X2- X3 , Y , - Y2- Y3- X , -X2 - continued X3 -X4 , Y - Y2- Y3- Y4- X - X2, Y - Y2 -Yz - Y4 - X -X2 - X3, Y - Y2- Yz- Y4 - X - X2- X3 - X4 , wherein each X , and each Y , is ( SEQ ID NO : 1611 ) an amino acid , and wherein each X , and each Yn is an amino FGYRIK , acid , and wherein X is selected from the group consisting ( SEO ID NO : 1612 ) of: (all single letter amino acid format ) A , D , E , G , I, K , L , FLRLF , N , P , Q , R , and V ; X , is selected from the group consisting ( SEO ID NO : 1613 ) of A , F , G , I , K , L , N , P , Q , R , S , T , V , and Y ; X3 is selected FYARR , from the group consisting of A , D , E , F , G , I , K , L , N , Q , R , S , T , V , and Y ; X4 is selected from the group consisting of: ( SEO ID NO : 1614 ) R , H , K , D , E , S , T, N , Q , G , A , I, L , M , F, W , Y , and V . In GGLRKK , some embodiments , Y is selected from the group consisting ( SEQ ID NO : 1615 ) of: ( all single letter amino acid format ) A , D , E , F , G , I , L , IFVALK , N , Q , S , T , V , and Y ; Y , is selected from the group consisting of F , G , I , L , and Y ; and Y3 is selected from the group ( SEQ ID NO : 1616 ) consisting of A , D , E , F , G , I , K , L , R , S , T , V , and Y ; and IFVLK , Y is selected from the group consisting of: R , H , K , D , E , ( SEQ ID NO : 1617 ) S , T , N , Q , G , A , I , L , M , F , W , Y , and V . IFVLR 46 . The method of controlling a pest infection of a plant ( SEQ ID NO : 1618 ) of claim 44 , wherein the binary peptide comprises an amino IFVR , acid sequence of: ( SEO ID NO : 1619 ) IFVRLR , ( SEQ ID NO : 1594 ) ( SEO ID NO : 1620 ) AFVRLF , ILFNIK , ( SEQ ID NO : 1595 ) ( SEO ID NO : 1621 ) AKLFV , LFAAPF , ( SEQ ID NO : 1596 ) ( SEQ ID NO : 1622 ) ALFALK , LFVALK , ( SEQ ID NO : 1597 ) ( SEQ ID NO : 1623 ) ALFLK , LFVLK , ( SEQ ID NO : 1598 ) ( SEO ID NO : 1624 ) ALFLR , LFVLR , ( SEQ ID NO : 1599 ) ( SEO ID NO : 1625 ) ALFR , LFVR , ( SEQ ID NO : 1600 ) ( SEO ID NO : 1626 ) ALFRLR , LFVRLR , ( SEQ ID NO : 1601 ) ( SEQ ID NO : 1627 ) ALKALF , LFVRVFL , ( SEQ ID NO : 1602 ) ( SEO ID NO : 1628 ) ALKFF , LGER , ( SEO ID NO : 1603 ) ( SEO ID NO : 1629 ) ALKFLV , LKALF , ( SEQ ID NO : 1604 ) ( SEQ ID NO : 1630 ) ALKIFV , LKFF , (SEQ ID NO : 1605 ) ( SEO ID NO : 1631 ) ALKLFV , LKIFV , ( SEQ ID NO : 1606 ) ( SEQ ID NO : 1632 ) FFADIK , LICLFV , ( SEQ ID NO : 1607 ) ( SEO ID NO : 1633 ) FFALK , LRALF , ( SEQ ID NO : 1608 ) ( SEQ ID NO : 1634 ) FFLK , LRFF , ( SEQ ID NO : 1609 ) ( SEQ ID NO : 1635 ) FFLR , LRIFV , ( SEQ ID NO : 1610 ) ( SEO ID NO : 1636 ) FFRLR , LRLFV , US 2018 /0362598 A1 Dec. 20 , 2018 82
- continued 51 . The method of controlling a pest infection of a plant of claim 44 , wherein the animal gut protease is a human ( SEQ ID NO : 1637 ) RALF , gastrointestinal protease . 52 . The method of controlling a pest infection of a plant ( SEQ ID NO : 1638 ) of any one of claims 44 -51 , wherein the TP is a protein RIFV , selected from the group consisting of a Pore Forming ( SEQ ID NO : 1639 ) Insecticidal Protein (PFIP ) and a Cysteine Rich Insecticidal RLFV , Protein (CRIP ). ( SEQ ID NO : 1640 ) 53 . The method of controlling a pest infection of a plant RLRALF , of claim 44 , wherein said pest is an insect or a nematode . 54 . The method of controlling a pest infection of a plant ( SEQ ID NO : 1641 ) of claim 53, wherein said pest is an insect, from the insect RLRFF , order selected from the group consisting of Coleoptera and ( SEO ID NO : 1642 ) Lepidoptera . RLRIFV , 55 . The method of controlling a pest infection of a plant ( SEQ ID NO : 1643 ) of 44 , wherein said TP is a peptide or protein selected from RLRLFV , the group consisting of: a Pore Forming Insecticidal Protein ( SEQ ID NO : 1644 ) (PFIP ) and a Cysteine Rich Insecticidal Peptide (CRIP ). RRKAFV , 56 . Themethod of controlling a pest infection of a plant ( SEO ID NO : 1645 ) of 55 , wherein the TP is an Inhibitor Cysteine Knot (ICK ) RRKLIF , motif protein or a non -ICK protein . ( SEQ ID NO : 1646 ) 57. The method of controlling a pest infection of a plant RRRFFA , of claim 56 , wherein the TP is a peptide or protein having at least 80 % , at least 85 % , at least 90 % , at least 95 % , at least ( SEQ ID NO : 1647 ) 96 % , at least 97 % , at least 98 % , at least 99 % , or at least VFGRKG , 100 % sequence identity or sequence homology to a peptide and or protein having an amino acid sequence as set forth in SEQ ( SEQ ID NO : 1648 ) ID NOs: 5 , 6 and 1761, 1762, 1767 , 1771 -1773 , or variants YFVRK . thereof. 47 . The method of controlling a pest infection of a plant 58 . The method of controlling a pest infection of a plant of claim 46 , wherein the binary peptide comprises an amino of claim 57 , wherein the TP is a peptide or protein having an sequence IFVRLR (SEO ID NO : 1619 ) , ALKLFV (SEO ID amino acid sequence of any one of SEQ ID NOs: 5 , 6 , 1761, NO : 1605 ), ALKFLV (SEO ID NO : 1603 ) , RRKAFV (SEO 1762 , 1767, 1771 - 1773 , or variants thereof. ID NO : 1644 ), or LFAAPF (SEQ ID NO : 1621) . 59. The method of controlling a pest infection of a plant 48 . The method of controlling a pest infection of a plant of claim 44 , wherein a pesticidally effective amount of said of claim 44 , wherein the cleavable tertiary peptide at least 5 TP is provided by the plant, which is a recombinant plant, a amino acids up to a maximum of 16 amino acids; and the tertiary peptide comprises a binary peptide and a spacer of part of the plant, or a product of the plant or the plant part . 1 to 4 amino acids . 60 . The method of controlling a pest infection of a plant 49 . The method of controlling a pest infection of a plant of claim 44 , wherein a pesticidally effective amount of said of claim 48 , wherein the tertiary peptide comprises a binary insecticidal and/ or nematicidal protein is provided in one or peptide fused at its C - terminus to a di- peptide comprising more formulations topically applied on the plant or a part of the amino acid sequence GS, WN , WQ ,MA , MV, AM , LH , the plant , said one or more formulations comprising bacte MN , ES , WQ, or DT. rial cells , bacterial spores, fungal cells , fungal spores, or 50 . The method of controlling a pest infection of a plant parasporal crystals that comprise said insecticidal and/ or of claim 44 , wherein the insect protease is a protease derived nematicidal protein . from a Lepidoptera or a Coleoptera insect species .