WOLTORIO DEUS LA20170247719A1 UN CAMION AND ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2017/ 0247719 A1 HU et al. (43 ) Pub . Date : Aug . 31 , 2017 ( 54 ) COMPOSITIONS AND METHODS TO Related U . S . Application Data CONTROL INSECT PESTS (60 ) Provisional application No . 62 /051 , 901 , filed on Sep . (71 ) Applicants : PIONEER HI- BRED 17 , 2014 . INTERNATIONAL , INC . , Publication Classification JOHNSTON , IA (US ) ; E I DU PONT (51 ) Int. CI. DE NEMOURS AND COMPANY , C12N 15 /82 (2006 . 01 ) WILMINGTON , DE (US ) AOIN 63 /02 ( 2006 . 01 ) ( 72 ) Inventors : XU HU , JOHNSTON , IA (US ) ; 2) U . S . CI. JAMES KEVIN PRESNAIL , ST CPC .. C12N 15/ 8286 ( 2013. 01 ) ; C12N 15/ 8218 LOUIS , MO (US ) (2013 .01 ) ; AOIN 63/ 02 ( 2013 .01 ) ( 73 ) Assignees : PIONEER HI- BRED (57 ) ABSTRACT INTERNATIONAL , INC . , Methods and compositions are provided which employ a JOHNSTON , IA (US ) ; E I DU PONT silencing element that, when ingested by a plant insect pest , DE NEMOURS AND COMPANY . such as a Coleopteran plant pest or a Diabrotica plant pest , decrease the expression of a target sequence in the pest. WILMINGTON , DE (US ) Disclosed are various target polynucleotides set forth in any one of SEO ID NOS : 1 - 33 disclosed herein , (but not (21 ) Appl. No. : 15 /511 , 298 including the forward and reverse primers . ) or variants and fragments thereof, or complements thereof, wherein a ( 22 ) PCT Filed : Sep . 11 , 2015 decrease in expression of one or more of the sequences in the target pest controls the pest ( i. e ., has insecticidal activity ) . ( 86 ) PCT No .: PCT/ US15/ 49678 Plants , plant parts , bacteria and other host cells comprising $ 371 ( c )( 1 ) , the silencing elements or an active variant or fragment ( 2 ) Date : Mar. 15 , 2017 thereof of the invention are also provided . Patent Application Publication Aug . 31 , 2017 Sheet 1 of 6 US 2017 / 0247719 A1 Loopsequncesrepresentedby"Xand ??? Vvsrpponnlkjihhfeccbb-3' Uppercaseletters=sensetotargetsequence Lowercaseletters=antisensecomplementbase Stemmismatchisdenotedby"* FormationofRNAsecondarystructure Underilae=WCRWsequencesBolditalic+ =SCRWsequences. Bold+underline=NCRWsequences 3-abcdefghijklmnopqrstuvxxXXXXXXXXXVUVSRPPONNLKJIBHFECCBB5'(antisensestrand) Transcription * * * option * 5'-ABCDEFGHIJKLMNOPORSTUVXXXXXXXXXXXVUVsepponnikihhfeccbb31(sensestrand) vuvseppannil kjihhfeccbb ConvergentDesign AECDEFGHIJKLMNOPQRSTUV - ' 5 * * * * * * RNAConstruct 5'-ABCDEFGHIJKLMNOPQRSTUV RNAConstruct DNAConstruct FIG.1A Patent Application Publication Aug . 31 , 2017 Sheet 2 of 6 US 2017 / 0247719 A1 and"x*representedXbyLoopsequnces KEY Uppercaseletters=sensetotargetsequence lowercaseletters=antisensecomplementbase denoted*bymismatchisSiem (vuvsrpponnlkiihhfeccbb-3? FormationofRNAsecondarystructure underlineWCBW=sequences BoldItalic=SCRWsequences Bold+underline=NCRWsequences 57-ABCDEFGHIJKLMNOPQRSTUVXXXXXXXXXXXVUVsIpponnikiihhfeccbb3'(sensestrand) 31-abcdefghijklmnopqrstuvxxXXXXXXXXXVUVSRPPONNLKJIHHFECCBB5:(antisensestrand) Transcription # * * vuvsepponnlkjihhfeccbb - 3 / ConvergentDesign - WABCDEFGHIJKLMNOPQRSTUV.TEL ! T ' 5 * * * * * * RNAConstruct 5!-ABCDEFGHIJKLMNOPQRSTUV RNAConstruct DNAConstruct FIG.1B Patent Application Publication Aug . 31 , 2017 Sheet 3 of 6 US 2017 / 0247719 A1 loopsequncesrepresentedby"Xandx KEY Uppercaseletters=sensetotargetsequence Lovercaseletters=3ntisensecomplementbase by*.denoted"mismaichisStem ABCDEFGHIJKLMNOPQRSTUV-3' Bold+underline=NCRWsequences FormationofRNAsecondarystructure Underline=WCRWsequences Boldftalic+SCRW=sequences 5!-vuvsrpponnlkjihhfeccbbxxxxxxxxXXXABCDEFGEIJKLMNOPQRSTUV3?(sensestrand) 31-VUVSRPPONNLKJIHHFECCBBXXXXXXXXXXXabcdefghijklmnopqrstuv5'(antisensestrand) Transcription ABCDEFGHIJKLMNOPQRSTUV - 3 DivergentDesign Ipponnlkjihhfeccbb VLIVS * ' 5 * * * * RNAConstruct 5!-VUvsrpponnlkiihhfeccbb RNAConstruct DNAConstruct FIG.2A Patent Application Publication Aug . 31 , 2017 Sheet 4 of 6 US 2017 / 0247719 A1 "xrepresentedXandbyLoopseqances KEY Uppercaseletters=sensetotargetsequence Lowercaseletters=antisensecomplementbase Stemmismatchisdenotedby* CABCDEFGHIJKLMNOPQRSTUV-3? FormationofRNAsecondarystructure UnderlineWYCRW=sequences d+ta=5C5q.????7/Z25 Bold+underline=NCRWsequences 51-VUVSIpponnikiihhfeccbbxxxXXXXXXXXABCDEFGHIJKZMNOPQRSTUV3'(sensestrand) 31-VUVSAPPONNLKJIHHFECCBBXXXXXXXXXXXabcdefghijklmnopqrstuv5'(antisensestrand) Transcriptionrense * * * * * * ABCDEFGHIJKLMNOPQRSTUV * 3 ' DivergentDesign qg338yuu ????? :??????? - . S * * * * * * 51-yuvsrpponnlkjinnfeccbb! RNAConstruct RNAConstruct DNAConstruct FIG.2B Patent Application Publication Aug . 31 , 2017 Sheet 5 of 6 US 2017 / 0247719 A1 2 . 2 . : . :: . N CRWNIS on ??? SEQ ID NO . 11 SEQ ID NO . 13 SEQD NO . 14 Count 25 24 18 13 Median 1 . 5 0 . 75 0 . 8 ???? TIETO. 1111111 : . ' . ' . ' . ' . ' . ' . ' . ' FIG . 3A Patent Application Publication Aug . 31 , 2017 Sheet 6 of 6 US 2017 / 0247719 A1 . ... CRWNIS " E. ???? SEO ID NO . 10 SEQD NO . 12 Count2 :57 : :: : :: : :: 16 28 10 os : : : : : : : : : : : : : : : : : : : :: : : : : : : : : : : : :: 1 . 8 1 . 35 Outlidis . FIG . 3B US 2017 /0247719 A1 Aug. 31, 2017 COMPOSITIONS AND METHODS TO [0005 ] In specific embodiments , the decrease in expres CONTROL INSECT PESTS sion of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to FIELD a plant . Described herein are various target polynucleotides as set forth in SEQ ID NOs. : 1 -33 , or variants or fragments [0001 ] The present disclosure relates generally to methods thereof, or complements thereof, wherein a decrease in of molecular biology and gene silencing to control pests . expression of one or more of the sequences in the target pest controls the pest ( i . e . , has insecticidal activity ) . Further REFERENCE TO A SEQUENCE LISTING provided are silencing elements , which when ingested by the SUBMITTED AS A TEXT FILE VIA EFS -WEB pest , decrease the level of expression of one or more of the [0002 ] The Sequence Listing created on Aug . 12 , 2015 as target polynucleotides . Plants , plant parts , plant cells , bac a text file named “ 6862 SeqList, ” and having a size of teria and other host cells comprising the silencing elements 34 ,733 bytes is hereby incorporated by reference pursuant to or an active variant or fragment thereof are also provided . 37 C . F . R . $ 1 . 52 ( e ) (5 ) . Sprayable compositions comprising the silencing agents for topical applications to pest insects or substrates are also BACKGROUND contemplated . [0006 ] In another embodiment, a method for controlling a [ 0003 ] Plant insect pests are a serious problem in agricul plant insect pest , such as a Coleopteran plant pest or a ture . They destroy millions of acres of staple crops such as Diabrotica plant pest, is provided . The method comprises corn , soybeans , peas , and cotton . Yearly , plant insect pests feeding to a plant insect pest a composition comprising a cause over $ 100 billion dollars in crop damage in the U . S . silencing element, wherein the silencing element, when alone . In an ongoing seasonal battle , farmers must apply ingested by the pest , reduces the level of a target sequence billions of gallons of synthetic pesticides to combat these in the pest and thereby controls the pest . Further provided pests . Other methods employed in the past delivered insec are methods to protect a plant from a plant insect pest. Such ticidal activity by microorganisms or genes derived from methods comprise introducing into the plant or plant part a microorganisms expressed in transgenic plants . For disclosed silencing element. When the plant expressing the example , certain species of microorganisms of the genus silencing element is ingested by the pest , the level of the Bacillus are known to possess pesticidal activity against a target sequence is decreased and the pest is controlled . broad range of insect pests including Lepidoptera , Diptera , Coleoptera , Hemiptera , and others . In fact , microbial pesti BRIEF DESCRIPTION OF THE DRAWINGS cides , particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to [0007 ] FIG . 1 (FIG . 1A and FIG . 1B ) shows schematic chemical pest control. Agricultural scientists have developed representations of the convergent type of mismatch con crop plants with enhanced insect resistance by genetically struct as disclosed herein . FIG . 1A shows a DNA construct engineering crop plants to produce insecticidal proteins from that when transcribed to RNA comprises a segment 1 in the Bacillus. For example, corn and cotton plants genetically sense orientation to the target RNA , wherein segment 1 is engineered to produce Cry toxins ( see , e . g ., Aronson ( 2002 ) consists of a hypothetical WCRW sequence ; a segment 2 Cell Mol. Life Sci. 59 ( 3 ) :417 -425 ; Schnepf et al. ( 1998 ) that consists of a sequence that is not self -complementary ; Microbiol. Mol. Biol. Rev. 62 ( 3 ) :775 - 806 ) are now widely and a segment 3 in the antisense orientation to the target used in American agriculture and have provided the farmer RNA , wherein segment 3 is complementary to segment 1 with an alternative to traditional insect - control methods . and consists of a hypothetical NCRW sequence . FIG . 1B However , in some instances these Bt insecticidal proteins shows a DNA construct that when transcribed to RNA may only protect plants from a relatively narrow range of comprises a segment 1 in the sense orientation to the target