US 2016.0347799A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0347799 A1 BARRY et al. (43) Pub. Date: Dec. 1, 2016
(54) INSECTICIDAL PROTEINS FROM PLANTS Related U.S. Application Data AND METHODS FOR THEIR USE (60) Provisional application No. 62/051,720, filed on Sep. (71) Applicants:PIONEER HI-BRED 17, 2014, provisional application No. 61/937.295, ppl1canus: filed on Feb. 7, 2014. INTERNATIONAL, INC., Johnston, IA (US); E. I. DUPONT DE Publication Classification NEMOURS AND COMPANY., (51) Int. Cl Wilmington, DE (US) C07K I4/45 (2006.01) CI2N 5/82 2006.O1 LU LIU, PALO ALTO, CA (US); AMY (52) U.S. Cl. LUM, HAYWARD, CA (US); ERIC CPC ...... C07K 14/415 (2013.01); A0IN 37/18 SCHEPERS, PORT DEPOSIT, MD (2013.01); C12N 15/8286 (2013.01) (US); NASSERYALPANI, JOHNSTON, IA (US); GENHAI ZHU, (57) ABSTRACT SAN JOSE, CA (US) Compositions and methods for controlling pests are pro s vided. The methods involve transforming organisms with a (73) Assignees: PIONEER HI-BRED nucleic acid sequence encoding an insecticidal protein. In INTERNATIONAL INC particular, the nucleic acid sequences are useful for prepar JOHNSTON, IA (US). E i. DU PONT ing plants and microorganisms that possess insecticidal DE NEMOURS AND CoMPANY activity. Thus, transformed bacteria, plants, plant cells, plant s tissues and seeds are provided. Compositions are insecti WILMINGTON, DE (US) cidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors (21) Appl. No.: 15/116,740 for Subsequent transformation into organisms of interest (22) PCT Filed: Feb 6, 2015 including plants, as probes for the isolation of other homolo a vs. gous (or partially homologous) genes. The pesticidal pro (86). PCT No.: PCT/US 15/14824 teins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran S 371 (c)(1), and nematode pest populations and for producing composi 2) Date: Aug.g 4, 2016 tions with insecticidal activitv.y Patent Application Publication Dec. 1, 2016 Sheet 1 of 21 US 2016/0347799 A1
Fig.
Polypodiaceae DaValiaceae Oleancraceae -C Tectariaceae Eupolypodsh.c LOmariopsicaceae mid Dryoptericaceae Eupolypods, OnOceaceae Blechnaceae Polypodiales Eupolypods|FFE Thelyptericaceae C Woodsiaceae Aspleniaceae Polypod Ptericaceae DennStaectaceae Polypodiopsida SaCColomataceae -----C Lindsaeaceae Metaxyaceae DickSOniaceae C Cyatheaceae Cootaceae Cyatheales Core leptospOrangiates Plagiogyriaceae Culcitaceae LOXOmataceae Thyrsopteridaceae Salviniaceae Marsleaceae Salviniaceales Schizaeaceae AnemiaCeae Schizaeaceales Lygodiaceae Matoniaceae Leptosporangiate Diptericaceae Gleicheniales Gleicheniaceae Hymenophyllaceae Hymenophyllales OSMUndaceae Osmundales Fern MarataceaeEquisetaceae MarataesEquisetales -J EquisetopsidaMaratopsida PsilotaceaeOphiogloSSaceae Psilotalesge. PsilotooscaO
Patent Application Publication Dec. 1, 2016 Sheet 3 of 21 US 2016/0347799 A1
Fig.2b
- - - - MOTIF 20 ----- 120 140 Pt.IP-83Aa PTDGRQS---DTELHLTFMKL AI-QREE-NRWEITAADG----MNWGVY : 132 PtP-83Ca PIDGSQ- - - -ATTLRLPFMQL AR-VIEQNVKSEITATDG----MNWGIY : 129 PIP-83C ETDGSE- - - -AAWLELPEMEL T--LIEQ-YRSEIKAADD----AKWGTY : 128 Pt.IP-83CC PIDGSQ- - - -ATTLRLPFMQL. AR-VIEQNVKSEITATDG----MNWGIY : 129 Pt.IP-83Cd PIDGSQ----ATTLRLPFMQL AR-VIEQNVKSEITATDG----MNWGIY : 129 Pt.IP-83Ce PIDGSQ- - - -ATTLRLPFMOL AR-VIEONVKSEITATDG----MNWGIY : 129 PEIP-83CfE PTGGSR----AATLCLPFMKV AT-LIER-FRSEIAAADG----MNWGTY : 128 Pt.IP-83Fa PEVDQRPHMAQCSLDFPFMRL VVGSWHENVGGVMQAFSSDATPSNIGIY : 139 gi-598378568 SSGNGWP--QVALLQGPLVQYNGN------YSPWTG- - - - - NWSAT : 127 PtIP-83Aa SSE : CCCCCCC---CCHE HH 132 PtIP-83Ca SSE : CCCCCC- - - -C 129 PtIP-83Cb SSE : CCCCCC----HHHH 128 PtIP-83Cc SSE : CCCCCC----CCCCCCC 129 PtIP-83Cd SSE : CCCCCC----CCCCCCC 129 PtIP-83Ce SSE : CCCCCC----CCCCCCCH 129 PtIP-83Cf SSE : CCCCCC----CEECCCCC 128 Pt.IP-83Fa SSE : 139 gi-598378568 127
160 180 200 Pt.IP-83Aa IHA EVOVGVLTMSWSSVLRVSALRSVITSGFRA------WSW 169 Pt.IP-83Ca : IYG KVERSPLLPSNA-ILAWWADRCISAR------HNH : 163 Pt.IP-83Cb : WHA EVOLSPLFNGWP-YLVVEAQRCIITAAM------HNT : 162 Pt.IP-83CC YG KWERSPLPSNA-AWWADRCTTSAR------HNH : 163 Pt.IP-83Cd YG EVERSPLLPSNA-LAWWADRCTTSAR------ENE : 163 Pt.IP-83Ce YG KVERSPLLPSNA-ILAWWADRCISAR------HNH : 163 Pt.IP-83Cf IHG EVOVSPLYPNNS-ILGVWADRSIITSAF------HNW : 162 Pt.IP-83Fa : LHADRFIYROATSPASNFVLPLDVRVSFGSSTYSGPTIRPDWONLNVSNI : 189 gi-598378568 : PLL QDTYR ----FFLHGQQLLILSGT------150 PtIP-83Aa SSE : CCC HCCCEE------se 169 PtIP-83Ca SSE : 163 PtIP-83Cb SSE : 162 PtIP-83Cc SSE : 163 PtIP-83Cd SSE : 163 PtIP-83Ce SSE : EccoccCCCCCEEEEEEE : 163 162 PtIP-83Cf SSE : CCCCCCCE-coccaccCCCCCCCc------CCC : PtIP-83Fa SSE : EECCCH CCCCCCCCCCCCCCCCCCCCCC : 189 gi-598378568 : CCCCCCCCE------CCCCCEEECCC------150
Patent Application Publication Dec. 1, 2016 Sheet 10 of 21 US 2016/0347799 A1
Fig. 2.
MOTIF 5 ------(-- 82O 840 PtP-83Aa : TTOTGTVYILL GSTIFHDRRRDQVLPF AAAPLNYHYAYRLDTGDSTLT : BOO PtP-83Ca : STRSGIWYILL, GSTIEHDRRRDEWMTF AADPLNF YAYRLDTGEATLT : 786 PtP-83Cb : ATQTGTIYILL GSTVFHDRRREEVMTF AAVPLNYHYAYRLDTGEATLT : 785 PtP-83CC : STRSGIWYILL, GSTIEHDRRRDEVMT. AADPLNF YAYRLDTGEATLT : 784 PtP-83CC : STRSGIWYILL GSTIEHDRRRDEVMT. AADPLNE YAYRLDTGEATLT : 784 PtP-83Ce : STRSGIWYILL, GSTIEHDRRRDEWMTF AADPLNF YAYRLDTGEATLT : 784 PtIP-83Cf : ATQTGTVYILL, GSTIFHDRRRDEVMTF AAAPLNY YAYRLDTGETTLT : 785 PtP-83Ea : STNTGLVYMLL, GSRFLHDRKHEEVMDY ASILGPWYAYAYDLNTGATTLN : 817 gi-598378568 ATVPAYHYAYNLASETTED : 749 PtIP-83Aa SSE : EEEEECCCCCCCC : BOO PtIP-83Ca SSE : M CCCCCCCC : 786 PtIP-83Cb SSE : , , as CCCCCCCC : 785 PtIP-83cc SSE : CCCCCCCC : 784 PtIP-83Cd SSE : CCCCCCCC : 784 PtIP-83Ce SSE : CCCCCCCC : 784 PtIP-83Cf SSE : CCCCCCCC : 785 PtP-83Fa SSE : CCCCCCCC : 817 gi-598378568 CCCCCCCC : 749
MOTIF 23 ------MOTIF 3 ------860 880 900 PtF-83Aa : NEPSEQFANKFMQMTPFTRWRLRLSASAKENAGLAFPTATALDSTT IVI : 850 PtP-83Ca : NEPSEDFANTFMQMTPFTRWRLRLSASASENAELAFPTATAPDSTT 836 PtP-83Cb : NEPSEQEANTFMQMTPFTHWRLRLSASAAENKGLAFPTATAPDSTT 835 PtP-83CC : NEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAPDSTT 834 PtP-83CC : NEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAPDSTT 834 PtP-83Ce : NEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAPDSTT 834 Pt.IP-83Cf : NOPSEHFANTFMQMTPFTRWRLRLSASAPENAGLAFPTATALDSTT 835 PtP-83Fa : NIPSQQYANTFMQMTPFNAWRLRLSASAVENQGLVFPTATSPDNTT 867 gi-598378568 : NRPDEEFARVFMRMTPFTRWKLRVSTSARENQGLAFPTAITPDATT : 799 PtIP-83Aa SSE : VV V. CCCCCCCCCCCCCCCEEVVVV VVV EE 850 PtIP-83Ca SSE : CCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEE 836 PtIP-83Cb SSE : CCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCCC 835 PtIP-83Cc SSE : CCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCCCC 834 PtIP-83Cd SSE : CCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCCCC 834 PtIP-83Ce SSE : CCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCCCC 834 PtIP-83Cf SSE : cCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCEE 835 Pt.IP-83Fa SSE : CCCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCCCCC 867 gi-598378568 : CCCCCCCCCCCCCCCCCCEEEE CCCCCCCCCCCCCCCCCCCEE : 799 Patent Application Publication Dec. 1, 2016 Sheet 11 of 21 US 2016/0347799 A1
Fig. 2
920 PtP-83Aa : TFHVTAIR IDWRHDEE--- 867 PtP-83Ca : TFHVTAIR WDWRQEEE--- 853 Pt.IP-83Cb : TFHVTAIR IDWRQEEE--- 852 PtP-83CC : TFHVTAIR VDWRQEEE--- 851 PtP-83CC : TFHVTAIR VDWRQEEE--- 851 Pt.IP-83Ce : TFHVTAIR WDWRQEEKEEE : 854 PtP-83Cf : TFHVTAIR IDWRHEEE--- 852 PtP-83Fa : TFYVTAIRRIDHRQEGDVE 886 gi-598378568 : RFYISAIRDFDWNIR------813 PtIP-83Aa SSE : ECCCCCC--- 867 PtIP-83Ca SSE : CCCCCCCC- - - 853 PtIP-83Cb SSE : ECCCCCCC--- 852 PtIP-83Cc SSE : CCCCCCCC- - - 851 PtIP-83Cd SSE : CCCCCCCC- - - 851 PtIP-83Ce SSE : 854 PtIP-83Cf SSE : 852 PtIP-83Fa SSE : 886 gi-598378568 813
Patent Application Publication Dec. 1, 2016 Sheet 13 of 21 US 2016/0347799 A1
Fig. 3b
6O1 W l 650 Pt(P-50Aa (547) QAD-RLSSIKPADLTNYLE MVSEMDMRTTRMLLELIRVLYIONAALOYEYLE. H PtIP-83Aa (597). OADRLAAICPADLTNYLEMATOMDMRTTRMLLGLLNILRIONAALRYEYL
651 7 OO PtIP-50Aa (597) OTPAPLNAWPVTMQTVWGLLVOOETAAINGLLOMGAPSDYTOEYAVRDVP PtIP-83Aa (647) LMPTELTTWPLGMDTVGDLLIAOENAALIGLMOLGPSSDFTSRHVVKDIP
PtIP-50Aa (647) VRLLLGGGDWEFELPVRN--ADFPLTWCRVRIRYVDMRFDAAAE------PtIP-83Aa (697) VNLLLDGEDWEFEIPVOAGMSSFPSSWSRVRIRHLEMHFVKEASGIGGE
751. 8 OO PtIP-50Aa (689) -HLPVTSTGEVYMLLOSSRFFEDRAKRENEFISYEGGMGLOYOYAYRLAT PtIP-83Aa (747) IHOPTTOTGIVYILLOGSTIFHDRRR--DOVLPFOAAAPLNYHYAYRLDT
8O1 85 O PtIP-50Aa (738). GDATVTNVPSEEYANTFMRLAPFTRWRLRLSASAPENKGLAFPTATLADA PtIP-83Aa (795) GDSTILTNEPSEOFANKFMOMTPETRWRLRLSASAKENAGLAFPTATALDS
851 873 PtIP-50Aa (788) TTRIKITFHVSAIRRISTRVAV PtIP-83Aa (845) TTOIVITFHVTAIROIDWRHDEEH
Patent Application Publication Dec. 1, 2016 Sheet 21 of 21 US 2016/0347799 A1
Fig. 5e 801 850 PtIP-83Aa (793) DTGDSTLTNEPSEQFANKFMQMTPFTRWRLRLSASAKENAGLAFPTATAT, PtIP-83ca (779) DTGEATLTNEPSEDFANTFMQMTPFTRWRLRLSASASENAELAFPTATAP PtIP-83Cb (778) DTGEATLTNEPSEQFANTFMQMTPFTHWRLRLSASAAENKGLAFPTATAP PtIP-83cc (777) DTGEATLTNEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAP PtIP-83Cd (777) DTGEATLTNEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAP PtIP-83ce (777) DTGEATLTNEPSEEFANTFMQMTPFTRWRLRLSASASENAELAFPTATAP PtIP-83Cg (778) DTGEATLTNEPSEQFANTFMQMTPFTHWRLRLSASAAENEGLAFPTATAP Pt(P-83Cf (778) TTLTNPSEHFANTFMQMTPFTRWRLRTSASAPENAGLAFPTATAT, PtIP-83Da (777) f:------
PtIP-83Aa (843) Pt.IP-83Ca (829) PtIP-83Ch (828) PtIP-83Cc (827) Pt.IP-83Cd (827) Pt.IP-83Ce wVITFHVTAIRQVDWRQEEKEEE PtIP-83Cg (828) DSTTEIAITFHVMAIRQIDWRQEEE--- PtIP-83Cf (828) DSTTIVITFHVTAIRQIDWRHEEE--- PtIP-83Da (783) ------US 2016/0347799 A1 Dec. 1, 2016
INSECTICIDAL PROTEINS FROM PLANTS economically important insect pests. In some cases, insects AND METHODS FOR THEIR USE can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological CROSS REFERENCE control agents for pest control. 0001. This application claims the benefit of U.S. Provi 10007 Accordingly, there remains a need for new pesti sional Application No. 61/937.295 filed Feb. 7, 2014, and cidal proteins with different ranges of insecticidal activity U.S. Provisional Application No. 62/051,720 filed Sep. 17, against insect pests, e.g., insecticidal proteins which are 2014, which are incorporated herein by reference in its active against a variety of insects in the order Lepidoptera entirety. and/or the order Coleoptera including but not limited to insect pests that have developed resistance to existing insec REFERENCE TO SEQUENCE LISTING ticides. SUBMITTED ELECTRONICALLY SUMMARY 0002 The official copy of the sequence listing is submit ted electronically via EFS-Web as an ASCII formatted 0008 Compositions and methods for conferring pesti sequence listing with a file named “6054PCT Sequence cidal activity to bacteria, plants, plant cells, tissues and seeds Listing” created on Jan. 26, 2015, and having a size of 3,705 are provided. Compositions include nucleic acid molecules kilobytes and is filed concurrently with the specification. encoding sequences for pesticidal and insecticidal polypep The sequence listing contained in this ASCII formatted tides, Vectors comprising those nucleic acid molecules, and document is part of the specification and is herein incorpo host cells comprising the vectors. Compositions also include rated by reference in its entirety. the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in FIELD DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and 0003. This disclosure relates to the field of molecular plants. The nucleotide or amino acid sequences may be biology. Provided are novel genes that encode pesticidal Synthetic sequences that have been designed for expression proteins. These pesticidal proteins and the nucleic acid in an organism including, but not limited to, a microorgan sequences that encode them are useful in preparing pesti ism or a plant. Compositions also comprise transformed cidal formulations and in the production of transgenic pest bacteria, plants, plant cells, tissues and seeds. resistant plants. 0009. In particular, isolated or recombinant nucleic acid molecules are provided encoding Pteridophyta Insecticidal BACKGROUND Protein-83 (PtIP-83) polypeptides including amino acid sub 0004 Biological control of insect pests of agricultural stitutions, deletions, insertions, fragments thereof. Addition significance using a microbial agent, such as fungi, bacteria ally, amino acid sequences corresponding to the PtIP-83 or another species of insect affords an environmentally polypeptides are encompassed. Provided are isolated or friendly and commercially attractive alternative to synthetic recombinant nucleic acid molecules capable of encoding chemical pesticides. Generally speaking, the use of biope PtIP-83 polypeptides of SEQID NO: 1, SEQID NO:3, SEQ sticides presents a lower risk of pollution and environmental ID NO: 5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, hazards and biopesticides provide greater target specificity SEQID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID than is characteristic of traditional broad-spectrum chemical NO: 19, SEQID NO: 21, SEQID NO. 23, SEQID NO: 716, insecticides. In addition, biopesticides often cost less to SEQID NO: 754, SEQID NO: 755, SEQID NO: 756, SEQ produce and thus improve economic yield for a wide variety ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID of crops. NO: 760, SEQ ID NO: 761, SEQID NO: 762, SEQ ID NO: 0005 Certain species of microorganisms of the genus 763, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, Bacillus are known to possess pesticidal activity against a SEQ ID NO: 767, SEQID NO: 768, and SEQID NO: 769, range of insect pests including Lepidoptera, Diptera, Cole as well as amino acid substitutions, deletions, insertions, optera, Hemiptera and others. Bacillus thuringiensis (Bt) fragments thereof, and combinations thereof. Nucleic acid and Bacillus popilliae are among the most successful bio sequences that are complementary to a nucleic acid control agents discovered to date. Insect pathogenicity has sequence of the embodiments or that hybridize to a sequence also been attributed to strains of B. larvae, B. lentimorbus, of the embodiments are also encompassed. Also provided B. Sphaericus and B. cereus. Microbial insecticides, particu are isolated or recombinant PtIP-83 polypeptides of SEQ ID larly those obtained from Bacillus strains, have played an NO: 1, SEQID NO:3, SEQID NO. 5, SEQID NO: 7, SEQ important role in agriculture as alternatives to chemical pest ID NO:9, SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: control. 15, SEQID NO: 17, SEQID NO: 19, SEQID NO: 21, SEQ 0006) Crop plants have been developed with enhanced ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID insect resistance by genetically engineering crop plants to NO: 755, SEQID NO: 756, SEQID NO: 757, SEQ ID NO: produce pesticidal proteins from Bacillus. For example, corn 758, SEQID NO: 759, SEQID NO: 760, SEQID NO:761, and cotton plants have been genetically engineered to pro SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQ duce pesticidal proteins isolated from strains of Bt. These ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID genetically engineered crops are now widely used in agri NO: 768, and SEQ ID NO: 769, as well as amino acid culture and have provided the farmer with an environmen Substitutions, deletions, insertions, fragments thereof and tally friendly alternative to traditional insect-control meth combinations thereof. ods. While they have proven to be very successful I0010 Methods are provided for producing the polypep commercially, these genetically engineered, insect-resistant tides and for using those polypeptides for controlling or crop plants provide resistance to only a narrow range of the killing a Lepidopteran, Coleopteran, nematode, fungi, and/ US 2016/0347799 A1 Dec. 1, 2016
or Dipteran pests. The transgenic plants of the embodiments DETAILED DESCRIPTION express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further 0018. It is to be understood that this disclosure is not comprises one or more additional genes for insect resistance, limited to the particular methodology, protocols, cell lines, for example, one or more additional genes for controlling genera, and reagents described, as Such may vary. It is also Coleopteran, Lepidopteran, Hemipteran or nematode pests. to be understood that the terminology used herein is for the It will be understood by one of skill in the art that the purpose of describing particular embodiments only, and is transgenic plant may comprise any gene imparting an agro not intended to limit the scope of the present disclosure. 0019. As used herein the singular forms “a”, “and”, and nomic trait of interest. “the include plural referents unless the context clearly 0011 Methods for detecting the nucleic acids and poly dictates otherwise. Thus, for example, reference to “a cell peptides of the embodiments in a sample are also included. includes a plurality of such cells and reference to “the A kit for detecting the presence of a PtIP-83 polypeptide or protein’ includes reference to one or more proteins and detecting the presence of a polynucleotide encoding a Pt(P- equivalents thereof known to those skilled in the art, and so 83 polypeptide in a sample is provided. The kit may be forth. All technical and scientific terms used herein have the provided along with all reagents and control samples nec same meaning as commonly understood to one of ordinary essary for carrying out a method for detecting the intended skill in the art to which this disclosure belongs unless clearly agent, as well as instructions for use. indicated otherwise. 0012. The compositions and methods of the embodi 0020. The present disclosure is drawn to compositions ments are useful for the production of organisms with and methods for controlling pests. The methods involve enhanced pest resistance or tolerance. These organisms and transforming organisms with nucleic acid sequences encod compositions comprising the organisms are desirable for ing PtIP-83 polypeptides. In particular, the nucleic acid agricultural purposes. The compositions of the embodiments sequences of the embodiments are useful for preparing are also useful for generating altered or improved proteins plants and microorganisms that possess pesticidal activity. that have pesticidal activity or for detecting the presence of Thus, transformed bacteria, plants, plant cells, plant tissues PtIP-83 polypeptides. and seeds are provided. The compositions are pesticidal nucleic acids and proteins of bacterial species. The nucleic BRIEF DESCRIPTION OF THE FIGURES acid sequences find use in the construction of expression vectors for Subsequent transformation into organisms of 0013 FIG. 1 the phylogeny of ferns based on the clas interest, as probes for the isolation of other homologous (or sification for extant ferns by A. R. Smith et al. TAXON, partially homologous) genes, and for the generation of 55:705-731 (2006). altered PtIP-83 polypeptides by methods known in the art, 0014 FIG. 2a-2i shows an alignment of the amino acid Such as site directed mutagenesis, domain Swapping or DNA sequences of PtIP-83Aa (SEQ ID NO: 1), PtIP-83Ca(SEQ shuffling. The PtIP-83 find use in controlling or killing ID NO. 5), PtIP-83Cb (SEQID NO:7), PtIP-83Cc (SEQ ID Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran NO:9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ ID and nematode pest populations and for producing composi NO: 13), PtIP-83Cf (SEQID NO: 15), and PtIP-83Fa (SEQ tions with pesticidal activity. Insect pests of interest include, ID NO:3); an alignment of the secondary structure predic but are not limited to, Lepidoptera species including but not tion, by the PSIPRED, top ranked secondary structure limited to: Corn Earworm, (CEW) (Helicoverpa zea), Euro prediction method, for PtIP-83Aa (SEQ ID NO: 1) and pean Corn Borer (ECB) (Ostrinia nubilalis), diamond-back PtIP-83Fa (SEQ ID NO:3); and the locations of the amino moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., acid sequence MOTIFs, as predicted by MEME motif analy Pseudoplusia includens Walker; and velvet bean caterpillar sis, relative to PtIP-83Aa (SEQ ID NO: 1). A “H” indicates e.g., Anticarsia gemmatalis Hübner and Coleoptera species a predicted helical structure, an “E” indicates a PtIP-beta including but not limited to Western corn rootworm (Dia strand structure, and a “C” indicates a predicted coil struc brotica virgifera). WCRW, Southern corn rootworm (Dia ture. brotica undecimpunctata howardi)—SCRW, and Northern 0015 FIG. 3a-3b shows a sequence alignment between corn rootworm (Diabrotica barberi)—NCRW. PtIP-83Aa (SEQ ID NO: 1) and PtIP-50Aa (SEQ ID NO: 0021. By “pesticidal toxin' or “pesticidal protein’ is used 34). The crossover points in the PtIP-83 Aa/PtIP-50Aa herein to refer to a toxin that has toxic activity against one chimeras indicated in Table 13 are indicated by an arrow (1) or more pests, including, but not limited to, members of the above the amino acid. Lepidoptera, Diptera, Hemiptera and Coleoptera orders or 0016 FIG. 4a-4d shows an amino acid sequence align the Nematodaphylum or a protein that has homology to Such ment of PtIP-83Aa (SEQ ID NO: 1), PtIP-83Fa (SEQ ID a protein. Pesticidal proteins have been purified from organ NO:3), PtIP-50Aa (SEQ ID NO. 34), PtIP-50Ba (SEQ ID isms including, for example, Bacillus sp., Pseudomonas sp., NO:35), and PtIP-50Bb (SEQ ID NO:36). The conserved Photorhabdus sp., Xenorhabdus sp., Clostridium bifermen sequence motifs identified are indicated and the amino acid tans and Paenibacillus popilliae. Pesticidal proteins include sequence of the motifs in PtIP-83Aa (SEQ ID NO: 1) are but are not limited to: insecticidal proteins from Pseudomo underlined. nas sp. such as PSEEN3174 (Monalysin; (2011) PLOS 0017 FIG. 5a-5e shows an amino acid sequence align Pathogens 7:1-13); from Pseudomonas protegens Strain ment of PtIP-83Aa (SEQ ID NO: 1), PtIP-83Ca(SEQ ID CHAO and Pf-5 (previously fluorescens) (Pechy–Tarr, (2008) NO. 5), Pt|P-83Cb (SEQ ID NO: 7), PtIP-83Cc (SEQ ID Environmental Microbiology 10:2368-2386; GenBank NO:9), PtIP-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ ID Accession No. EU400157); from Pseudomonas Taiwanensis NO: 13), PtIP-83Cf (SEQ ID NO: 15), PtIP-83Cg (SEQ ID (Liu, et al., (2010).J. Agric. Food Chem., 58:12343-12349) NO: 17), and PtIP-83 Da (SEQ ID NO: 19). The sequence and from Pseudomonas pseudoalcligenes (Zhang, et al., diversity is highlighted. (2009) Annals of Microbiology 59:45-50 and Li, et al.,
US 2016/0347799 A1 Dec. 1, 2016
engineered hybrid insecticidal protein (eHIP) created by AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, fusing unique combinations of variable regions and con AXMI100, AXMI101, AXMI102, AXMI 103, AXM1104, served blocks of at least two different Cry proteins (US AXM1107, AXMI108, AXMI109, AXMI110, AXMI111, Patent Application Publication Number 2010/0017914); a AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476, AXMI124, AXM11257, AXM11268, AXM1127, 781, 7,105,332, 7,378.499 and 7.462,760; a Cry9 protein AXM1129, AXMI164, AXMI151, AXMI161, AXM1183, such as such as members of the Cry9A, Cry9B, Cry9C, AXMI132, AXMI138, AXMI137 of US Patent Application Cry9D, Cry9E and Cry9F families; a Cry15 protein of Publication Number 2010/0005543, cry proteins such as Naimov, et al., (2008) Applied and Environmental Micro Cry1A and Cry3A having modified proteolytic sites of U.S. biology, 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. No. 8.319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 protein from Bacillus thuringiensis strain VBTS 2528 of US and cryET34 protein of U.S. Pat. Nos. 6.248,535, 6,326,351, Patent Application Publication Number 2011/0064710. 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 Other Cry proteins are well known to one skilled in the art and CryET34 homologs of US Patent Publication Number (see, Crickmore, et al., “Bacillus thuringiensis toxin nomen 2006/0191034, 2012/0278954, and PCT Publication Num clature' (2011), at lifesci. Sussex.ac.uk/home/Neil Crick ber WO 2012/139004: a Cry35Ab1 protein of U.S. Pat. Nos. more/Bt? which can be accessed on the world-wide web 6,083,499, 6,548,291 and 6,340,593: a Cry46 protein, a Cry using the “www’ prefix). The insecticidal activity of Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; proteins is well known to one skilled in the art (for review, TIC807 of US Patent Application Publication Number 2008/ see, van Frannkenhuyzen, (2009).J. Invert. Path. 101:1-16). 0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, The use of Cry proteins as transgenic plant traits is well TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, known to one skilled in the art and Cry-transgenic plants and AXMI-038 of U.S. Pat. No. 8,236,757: AXMI-031, including but not limited to plants expressing Cry1Ac, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, 602: AXMI-018, AXMI-020 and AXMI-021 of WO 2006/ Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, 083891: AXMI-010 of WO 2005/038032: AXMI-003 of Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt WO 2005/021585; AXMI-008 of US Patent Application have received regulatory approval (see, Sanahuja, (2011) Publication Number 2004/02503 11: AXMI-006 of US Pat Plant Biotech Journal 9:283-300 and the CERA. (2010) GM ent Application Publication Number 2004/0216186: AXMI Crop Database Center for Environmental Risk Assessment 007 of US Patent Application Publication Number 2004/ (CERA), ILSI Research Foundation, Washington D.C. at 0210965; AXMI-009 of US Patent Application Number cera-gmc.org/index.php?action gm crop database which 2004/0210964: AXMI-014 of US Patent Application Publi can be accessed on the world-wide web using the “www.” cation Number 2004/0197917: AXMI-004 of US Patent prefix). More than one pesticidal proteins well known to one Application Publication Number 2004/0197916: AXMI-028 skilled in the art can also be expressed in plants such as and AXMI-029 of WO 2006/119457; AXMI-007, AXMI Vip3Ab & Cry1 Fa (US2012/0317682); Cry1BE & Cry1F 008, AXMI-008Orf2, AXMI-009, AXMI-014 and AXMI (US2012/0311746); Cry1CA & Cry1AB (US2012/ 004 of WO 2004/074462: AXMI-150 of U.S. Pat. No. 0311745); Cry1F & CryCa (US2012/0317681); Cry1DA & 8,084,416: AXMI-205 of US Patent Application Publication Cry 1BE (US2012/0331590); Cry 1DA & Cry1 Fa (US2012/ Number 2011/0023184: AXMI-011, AXMI-012, AXMI 0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1 Fa 013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, & Cry2Aa and Cry1 I & Cry1E (US2012/0324605); AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI Cry34Ab/35Ab and Cry6Aa (US20130167269); Cry34Ab/ 023, AXMI-041, AXMI-063 and AXMI-064 of US Patent VCry35Ab & Cry3Aa (US20130167268); and Cry3A and Application Publication Number 2011/0263488; AXMI-R1 Cry1Ab or Vip3Aa (US201301.16170). Pesticidal proteins and related proteins of US Patent Application Publication also include insecticidal lipases including lipid acyl hydro Number 2010/0197592: AXMI221Z, AXMI222z, lases of U.S. Pat. No. 7,491,869, and cholesterol oxidases AXMI223Z, AXMI224Z and AXMI225Z of WO 2011/ such as from Streptomyces (Purcell et al. (1993) Biochem 103248: AXMI218, AXMI219, AXMI220, AXM1226, Biophy's Res Commun 15:1406-1413). Pesticidal proteins AXM1227, AXM1228, AXM1229, AXMI230 and also include VIP (vegetative insecticidal proteins) toxins of AXMI231 of WO 2011/103247; AXMI-115, AXMI-113, U.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 7,615,686, and 8.237,020 and the like. Other VIP proteins 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 are well known to one skilled in the art (see, lifesci.sussex. and AXMI-045 of US Patent Application Publication Num ac.uk/home/Neil Crickmore/Bt/vip.html which can be ber 2010/0298211: AXMI-066 and AXMI-076 of US Patent accessed on the world-wide web using the “www’ prefix). Application Publication Number 2009/014.4852: AXMI128, Pesticidal proteins also include toxin complex (TC) proteins, AXM1130, AXM1131, AXM1133, AXMI140, AXMI141, obtainable from organisms such as Xenorhabdus, Photo AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, rhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, and 8,084,418). Some TC proteins have “stand alone” AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, insecticidal activity and other TC proteins enhance the AXMI166, AXMI167, AXM1168, AXM1169, AXMI170, activity of the stand-alone toxins produced by the same AXMI171, AXM1172, AXMI173, AXMI174, AXMI175, given organism. The toxicity of a “stand-alone TC protein AXM1176, AXM1177, AXMI178, AXMI179, AXM1180, (from Photorhabdus, Xenorhabdus or Paenibacillus, for AXMI 181, AXM1182, AXM1185, AXMI186, AXM1187, example) can be enhanced by one or more TC protein AXM1188, AXMI 189 of U.S. Pat. No. 8,318,900; “potentiators' derived from a source organism of a different AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, genus. There are three main types of TC proteins. As US 2016/0347799 A1 Dec. 1, 2016
referred to herein, Class A proteins (“Protein A') are stand or genomic nucleic acid sequence. In some embodiments the alone toxins. Class B proteins (“Protein B) and Class C change in the native or genomic nucleic acid sequence proteins (“Protein C) enhance the toxicity of Class A includes but is not limited to: changes in the nucleic acid proteins. Examples of Class A proteins are TcbA, TcdA, sequence due to the degeneracy of the genetic code; changes XptA1 and XptA2. Examples of Class B proteins are TcaG, in the nucleic acid sequence due to the amino acid Substi TcdB, XptB1Xb and XptC1 Wi. Examples of Class C pro tution, insertion, deletion and/or addition compared to the teins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins native or genomic sequence; removal of one or more intron; also include spider, Snake and Scorpion venom proteins. deletion of one or more upstream or downstream regulatory Examples of spider venom peptides include but not limited regions; and deletion of the 5' and/or 3' untranslated region to lycotoxin-1 peptides and mutants thereof (U.S. Pat. No. associated with the genomic nucleic acid sequence. In some 8,334.366). embodiments the nucleic acid molecule encoding a PtIP-83 0023. In some embodiments the PtIP-83 polypeptide polypeptide is a non-genomic sequence. include amino acid sequences deduced from the full-length (0028. A variety of polynucleotides that encode PtIP-83 nucleic acid sequences disclosed herein and amino acid polypeptides or related proteins are contemplated. Such sequences that are shorter than the full-length sequences, polynucleotides are useful for production of PtIP-83 poly either due to the use of an alternate downstream start site or peptides in host cells when operably linked to suitable due to processing that produces a shorter protein having promoter, transcription termination and/or polyadenylation pesticidal activity. Processing may occur in the organism the sequences. Such polynucleotides are also useful as probes protein is expressed in or in the pest after ingestion of the for isolating homologous or Substantially homologous poly protein. nucleotides that encode PtIP-83 polypeptides or related 0024. Thus, provided herein are novel isolated or recom proteins. binant nucleic acid sequences that confer pesticidal activity. Also provided are the amino acid sequences of PtIP-83 Polynucleotides Encoding PtIP-83 Polypeptides polypeptides. The protein resulting from translation of these (0029. One source of polynucleotides that encode PtIP-83 PtIP-83 polypeptide genes allows cells to control or kill polypeptides or related proteins is a fern or other primitive pests that ingest it. plant species which contains a PtIP-83 polynucleotide of Nucleic Acid Molecules, and Variants and Fragments SEQID NO: 2, SEQID NO:4, SEQID NO: 6, SEQID NO: Thereof 8, SEQID NO: 10, SEQID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQID NO: 0025. One aspect pertains to isolated or recombinant 22, SEQ ID NO: 24, SEQ ID NO: 717, SEQ ID NO: 738, nucleic acid molecules comprising nucleic acid sequences SEQID NO: 739, SEQID NO: 740, SEQID NO: 741, SEQ encoding PtIP-83 polypeptides or biologically active por ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID tions thereof, as well as nucleic acid molecules sufficient for NO: 745, SEQID NO: 746, SEQ ID NO: 747, SEQID NO: use as hybridization probes to identify nucleic acid mol 748, SEQID NO: 749, SEQID NO: 750, SEQID NO: 751, ecules encoding proteins with regions of sequence homol SEQID NO: 752 or SEQID NO: 753, encoding a PtIP-83 ogy. As used herein, the term “nucleic acid molecule” refers polypeptide of SEQID NO: 1, SEQID NO:3, SEQID NO: to DNA molecules (e.g., recombinant DNA, cDNA, 5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, SEQ ID genomic DNA, plastid DNA, mitochondrial DNA) and RNA NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, molecules (e.g., mRNA) and analogs of the DNA or RNA SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ generated using nucleotide analogs. The nucleic acid mol ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID ecule can be single-stranded or double-stranded, but pref NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQID NO: erably is double-stranded DNA. 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, 0026. An "isolated nucleic acid molecule (or DNA) is SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ used herein to refer to a nucleic acid sequence (or DNA) that ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. The is no longer in its natural environment, for example in vitro. polynucleotides of SEQID NO: 2, SEQID NO: 4, SEQ ID A “recombinant nucleic acid molecule (or DNA) is used NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, herein to refer to a nucleic acid sequence (or DNA) that is SEQID NO: 14, SEQID NO: 16, SEQID NO: 18, SEQ ID in a recombinant bacterial or plant host cell. In some NO:20, SEQID NO:22, SEQID NO:24, SEQID NO: 717, embodiments, an "isolated' or “recombinant nucleic acid is SEQID NO: 738, SEQID NO: 739, SEQID NO: 740, SEQ free of sequences (preferably protein encoding sequences) ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID that naturally flank the nucleic acid (i.e., sequences located NO: 744, SEQID NO: 745, SEQ ID NO: 746, SEQID NO: at the 5' and 3' ends of the nucleic acid) in the genomic DNA 747, SEQID NO: 748, SEQID NO: 749, SEQID NO: 750, of the organism from which the nucleic acid is derived. For SEQ ID NO: 751, SEQ ID NO: 752 and SEQ ID NO: 753 purposes of the disclosure, "isolated or “recombinant can be used to express Pt|P-83 polypeptides in bacterial when used to refer to nucleic acid molecules excludes hosts that include but are not limited to Agrobacterium, isolated chromosomes. For example, in various embodi Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizo ments, the recombinant nucleic acid molecule encoding bium bacterial host cells. The polynucleotides are also useful PtIP-83 polypeptides can contain less than about 5 kb, 4 kb, as probes for isolating homologous or Substantially homolo 3 kb. 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences gous polynucleotides that encode PtIP-83 polypeptides or that naturally flank the nucleic acid molecule in genomic related proteins. Such probes can be used to identify DNA of the cell from which the nucleic acid is derived. homologous or Substantially homologous polynucleotides 0027. In some embodiments an isolated nucleic acid derived from Pteridophyta species. molecule encoding PtIP-83 polypeptides has one or more 0030 Polynucleotides that encode PtIP-83 polypeptides change in the nucleic acid sequence compared to the native can also be synthesized de novo from a PtIP-83 polypeptide US 2016/0347799 A1 Dec. 1, 2016
sequence. The sequence of the polynucleotide gene can be acid sequence to introduce at least one amino acid Substi deduced from a PtIP-83 polypeptide sequence through use tution, insertion, deletion and/or addition compared to the of the genetic code. Computer programs such as "Back native or genomic sequence; removal of one or more intron Translate” (GCGTM Package. Acclerys, Inc. San Diego, associated with the genomic nucleic acid sequence; insertion Calif.) can be used to convert a peptide sequence to the of one or more heterologous introns; deletion of one or more corresponding nucleotide sequence encoding the peptide. upstream or downstream regulatory regions associated with Examples of PtIP-83 polypeptide sequences that can be used the genomic nucleic acid sequence; insertion of one or more to obtain corresponding nucleotide encoding sequences heterologous upstream or downstream regulatory regions; include, but are not limited to the PtIP-83 polypeptides of deletion of the 5' and/or 3' untranslated region associated SEQID NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: with the genomic nucleic acid sequence; insertion of a 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ heterologous 5' and/or 3' untranslated region; and modifi ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQID NO: cation of a polyadenylation site. In some embodiments the 21, SEQ ID NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, non-genomic nucleic acid molecule is a cDNA. In some SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, SEQ embodiments the non-genomic nucleic acid molecule is a ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID synthetic nucleic acid sequence. NO: 761, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, 0033. In some embodiments the nucleic acid molecule SEQ ID NO: 768, and SEQ ID NO: 769. Furthermore, encoding a Pt(P-83 polypeptide is a the non-genomic poly synthetic PtIP-83 polynucleotide sequences of the disclosure nucleotide having a nucleotide sequence having at least can be designed so that they will be expressed in plants. U.S. 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, Pat. No. 5,500,365 describes a method for synthesizing plant 60%, 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, genes to improve the expression level of the protein encoded 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, by the synthesized gene. This method relates to the modi 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, fication of the structural gene sequences of the exogenous 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% transgene, to cause them to be more efficiently transcribed, identity, to the nucleic acid sequence of SEQID NO: 2, SEQ processed, translated and expressed by the plant. Features of ID NO. 4, SEQID NO: 6, SEQID NO: 8, SEQ ID NO: 10, genes that are expressed well in plants include elimination of SEQID NO: 12, SEQID NO: 14, SEQID NO: 16, SEQ ID sequences that can cause undesired intron splicing or poly NO: 18, SEQID NO: 20, SEQID NO: 22, SEQID NO: 24, adenylation in the coding region of a gene transcript while SEQID NO: 717, SEQID NO: 738, SEQID NO: 739, SEQ retaining Substantially the amino acid sequence of the toxic ID NO: 740, SEQ ID NO: 741, SEQID NO: 742, SEQID portion of the insecticidal protein. A similar method for NO: 743, SEQID NO: 744, SEQ ID NO: 745, SEQID NO: obtaining enhanced expression of transgenes in monocoty 746, SEQID NO: 747, SEQID NO: 748, SEQID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 or ledonous plants is disclosed in U.S. Pat. No. 5.689,052. SEQ ID NO: 753, wherein the PtIP-83 polypeptide has 0031. In some embodiments the nucleic acid molecule insecticidal activity. encoding a PtIP-83 polypeptide is a polynucleotide having the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, 0034. In some embodiments the non-genomic polynucle SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID otide is not the nucleic acid sequence of SEQID NO: 2, SEQ NO: 12, SEQID NO: 14, SEQID NO: 16, SEQID NO: 18, ID NO. 4, SEQID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID SEQID NO: 12, SEQID NO: 14, SEQID NO: 16, SEQ ID NO: 717, SEQID NO: 738, SEQID NO: 739, SEQID NO: NO: 18, SEQID NO: 20, SEQID NO: 22, SEQID NO: 24, 740, SEQID NO: 741, SEQID NO: 742, SEQID NO: 743, SEQID NO: 717, SEQID NO: 738, SEQID NO: 739, SEQ SEQID NO: 744, SEQID NO: 745, SEQID NO: 746, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 743, SEQID NO: 744, SEQ ID NO: 745, SEQID NO: NO: 750, SEQID NO: 751, SEQID NO: 752, SEQID NO: 746, SEQID NO: 747, SEQID NO: 748, SEQID NO: 749, 753, and variants, fragments and complements thereof. SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 or “Complement' is used herein to refer to a nucleic acid SEQ ID NO: 753. sequence that is Sufficiently complementary to a given 0035. In some embodiments the nucleic acid molecule nucleic acid sequence Such that it can hybridize to the given encodes a Pt(P-83 polypeptide comprising an amino acid nucleic acid sequence to thereby form a stable duplex. sequence having at least 40%, 45%, 50%, 51%, 52%. 53%, “Polynucleotide sequence variants' is used herein to refer to 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, a nucleic acid sequence that except for the degeneracy of the 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, genetic code encodes the same polypeptide. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 0032. In some embodiments the nucleic acid molecule 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, encoding the Pt|P-83 polypeptide is a non-genomic nucleic 94%. 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence. As used herein a “non-genomic nucleic acid acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID sequence' or “non-genomic nucleic acid molecule' or “non NO:5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, SEQ genomic polynucleotide' refers to a nucleic acid molecule ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQID NO: that has one or more change in the nucleic acid sequence 19, SEQID NO:21, SEQID NO. 23, SEQID NO: 716, SEQ compared to a native or genomic nucleic acid sequence. In ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID Some embodiments the change to a native or genomic NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQID NO: nucleic acid molecule includes but is not limited to: changes 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, in the nucleic acid sequence due to the degeneracy of the SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ genetic code; codon optimization of the nucleic acid ID NO: 767, SEQID NO: 768 or SEQID NO: 769, wherein sequence for expression in plants; changes in the nucleic the PtIP-83 polypeptide has insecticidal activity. US 2016/0347799 A1 Dec. 1, 2016
0036. In some embodiments the nucleic acid molecule Leu, Met, ASn, Pro, Gln, Arg, Thr, Val or Trp; the amino acid encodes a Pt(P-83 polypeptide comprising an amino acid at position 368 is Tyr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, sequence of SEQID NO: 1, SEQID NO:3, SEQ ID NO: 5, Lys, Leu, Met, ASn, Pro, Gln, Arg, Ser. Thr, Val or Trp; the SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID amino acid at position 369 is Leu, Ala, Cys, Asp, Phe, Gly, NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, Ile, Met, Thr or Val; the amino acid at position 370 is Leu, SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Gln, Arg, ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID Ser. Thr, Val, Trp or Tyr; the amino acid at position 371 is NO: 757, SEQID NO: 758, SEQID NO: 759, SEQID NO: Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, ASn, Arg, 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, Ser. Thr, Val or Trp; the amino acid at position 372 is Gln, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769, having Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Arg, Ser, Val or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, Tyr, the amino acid at position 373 is ASn, Ala, Cys, Asp, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, Phe, Gly. His, Ile, Lys, Gln, Ser, Thr, Val or Trp; the amino 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, acid at position 556 is Trp, Phe, Thr or Tyr; the amino acid 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, at position 557 is Arg, Cys, Asp, Gly. His, Ile, Lys, Leu, Met, 68, 69, 70 or more amino acid substitutions compared to the ASn, Pro, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid at native amino acid at the corresponding position of SEQ ID position 558 is Ala, Cys, Asp, Phe, Gly. His, Ile, Lys, Leu, ASn, Pro, Gln, Arg, Ser, Val, Trp or Tyr; the amino acid at NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: 7, SEQ position 559 is Lys, Ala, Cys, Phe, Gly, His, Ile, Leu, Asn. ID NO:9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: Gln, Arg, Ser, Thr, Val or Tyr; the amino acid at position 560 15, SEQID NO: 17, SEQID NO: 19, SEQID NO: 21, SEQ is Cys, Ala, Phe, Gly, Ile, Met, Asn, Arg, Ser. Thr or Val; the ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID amino acid at position 561 is Lys, Ala, Cys, Asp, Glu, Phe, NO: 755, SEQID NO: 756, SEQID NO: 757, SEQID NO: Gly. His, Ile, Leu, Met, Asn, Arg, Ser. Thr, Val or Tyr; the 758, SEQID NO: 759, SEQID NO: 760, SEQID NO: 761, amino acid at position 562 is ASn, Cys, Asp, Glu, Gly, His, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQ Leu, Met, Arg, Ser. Thr, Val or Tyr; the amino acid at ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID position 563 is Val, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, NO: 768 or SEQ ID NO: 769. ASn, Gln, Thr or Trp; the amino acid at position 564 is Ala, 0037. In some embodiments the nucleic acid molecule Cys, Gly, Met, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid encodes a Pt(P-83 polypeptide comprising an amino acid at position 646 is Leu, Ala, Cys, Gly, Ile, Met, Asn., Gln, Ser, sequence of any one of SEQID NO: 236-299, SEQID NO: Thr or Val; the amino acid at position 647 is Leu, Asp, Gly, 334-367, SEQID NO:398-427, SEQID NO:518-607, SEQ Met, Asn., Gln or Thr; the amino acid at position 648 is Met, ID NO: 640-645, and SEQ ID NO: 728-737. Ala, Cys, Asp, Glu, Phe, Gly. His, Lys, Leu, ASn, Pro, Gln, 0038. In some embodiments the nucleic acid molecule Arg, Ser. Thr, Val, Trp or Tyr; the amino acid at position 649 encoding the PtIP-83 polypeptide is any one of SEQID NO: is Pro, Ala, Cys, Asp, Glu, Phe, Gly. His, Lys, Met, Asn., Gln, 172-235, SEQID NO:300-333, SEQID NO:368-397, SEQ Arg, Ser. Thr, Trp or Tyr; the amino acid at position 650 is ID NO: 428-517, SEQID NO: 634–639, and SEQ ID NO: Thr, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, 718-727. Gln, Arg, Ser, Val or Tyr; the amino acid at position 651 is 0039. In some embodiments the nucleic acid molecule Glu, Ala, Cys, Asp, Gly. His, Ile, Leu, Met, ASn, Pro, Gln, encodes a PtIP-83 polypeptide variant of SEQ ID NO: 1, Arg, Ser. Thr, Val or Tyr; the amino acid at position 652 is wherein the amino acid at position 53 is Val, Ala, Cys or Thr: Leu, Cys, Phe, Ile, Lys, Met, Pro, Arg, Ser, Thr or Val; the the amino acid at position 54 is LyS, Ala, Cys, Asp, Glu, Gly, amino acid at position 653 is Thr, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn., Gln, Arg, Ser or Thr; the amino acid His, Ile, Lys, Leu, Pro, Arg, Ser, Val or Trp; the amino acid at position 55 is Arg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, at position 654 is Thr, Ala, Cys, Phe, Ile, Lys, Leu, Met, Pro, Met, Asn., Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at Arg, Ser, Val, Trp or Tyr; the amino acid at position 655 is position 56 is Leu, Glu, Phe, Ile, Met, Thr or Val; the amino Trp, Phe or Tyr; the amino acid at position 771 is Arg, Ala, acid at position 57 is Tyr, Cys, Ile, Leu, Met, Thr or Val; the Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn. Ser, Thr, Val, Trp amino acid at position 58 is Val, Cys, Ile or Leu; the amino or Tyr, the amino acid at position 772 is Arg, Ala, Cys, Asp, acid at position 59 is Phe, Leu, Met, Val or Tyr; the amino Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Ser. Thr, acid at position 60 is Ala, Cys, Gly, Ser, Thr or Val; the Val, Trp or Tyr; the amino acid at position 773 is Asp, Ala, amino acid at position 61 is Asp, Glu, His or Ser; the amino Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Asn., Gln, Arg, Ser, acid at position 62 is Val, Ala, Cys, Ile, Leu or Thr; the amino Thr, Val, Trp or Tyr; the amino acid at position 774 is Gln, acid at position 63 is Val, Ala, Cys, Ile, Leu, Met or Thr; the Ala, Asp, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Arg, Ser, amino acid at position 64 is Glu, Ala, Cys, Phe, Gly. His, Ile, Thr, Val, Trp or Tyr; the amino acid at position 775 is Val, Leu, Met, Asn., Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino Ala, Cys, Asp, Glu, Gly, His, Ile, ASn, Pro, Gln, Arg, Ser. Thr acid at position 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn. or Tyr, the amino acid at position 776 is Leu, Ala, Cys, Asp, Gln, Thr, Val or Trp; the amino acid at position 66 is Pro, Glu, Phe, Gly. His, Ile, Lys, ASn, Pro, Gln, Arg, Ser. Thr, Val Asp, Gly, Met, Gln or Arg; the amino acid at position 363 is or Tyr; the amino acid at position 777 is Pro, Ala, Cys, Asp, Gln, Ala, Cys, Glu, Phe, Gly, His, LyS, Leu, ASn, Arg, Ser, Glu, Phe, Gly. His, Lys, Leu, Met, Asn., Gln, Ser. Thr, Val, Thr, Val or Trp; the amino acid at position 364 is Ile, Ala, Trp or Tyr; the amino acid at position 778 is Phe, Ala, His, Cys, Glu, Phe, His, Lys, Leu, Met, Asn., Gln, Ser. Thr, Val, Ile, Leu, Met, Asn., Gln, Ser, Val, Trp or Tyr; the amino acid Trp or Tyr; the amino acid at position 365 is Leu, Ala, Glu, at position 779 is Gln, Ala, Cys, Asp, Glu, Gly, His, Lys, Phe, Gly. His, Ile, Lys, Met, Asn, Arg, Val, Trp or Tyr; the Leu, ASn, Pro, Arg, Ser, Thr or Val; the amino acid at amino acid at position 366 is Gly, Ala, Cys, Phe, His, Ile, position 780 is Ala, Cys, ASn, Pro, Gln or Ser; the amino Lys, Leu, Met, Asn. Ser. Thr or Val; the amino acid at acid at position 781 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, position 367 is Ser, Ala, Cys, Asp, Glu, Phe, Gly. His, Ile, ASn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at US 2016/0347799 A1 Dec. 1, 2016
position 782 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, 135 is Ala or Gly; the amino acid at position 137 is Glu or Met, Pro, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino acid Lys; the amino acid at position 139 is Glin or Glu; the amino at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn. acid at position 140 is Val, Arg or Leu; the amino acid at Gln, Arg, Ser. Thr or Val; the amino acid at position 784 is position 141 is Gly or Ser; the amino acid at position 142 is Leu, Ala, Glu, Phe, His, Ile, Lys, Met, ASn, Pro, Gln, Ser, Val or Pro; the amino acid at position 144 is Thr, Leu, Phe Thr, Val or Trp; the amino acid at position 785 is Asn, Ala, or Tyr; the amino acid at position 145 is Met, Pro or Asn; the Cys, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Gln, Arg, Ser, amino acid at position 146 is Ser, Gly or ASn; the amino acid Thr, Val, Trp or Tyr; and the amino acid at position 786 is at position 147 is Trp or Asn; the amino acid at position 148 Tyr, Phe, Ile, Leu or Trp. is Ser, Ala or Pro; the amino acid at position 149 is Ser or 0040. In some embodiments the nucleic acid molecule deleted; the amino acid at position 150 is Val, Ile or Tyr; the encodes a PtIP-83 polypeptide variant of SEQ ID NO: 1, amino acid at position 152 is Arg, Ala, Val or Gly; the amino wherein the amino acid at position 1 is Met or deleted; the acid at position 154 is Ser, Trp or Glu; the amino acid at amino acid at position 2 is Ala or deleted; the amino acid at position 156 is Leu, Asp or Gln; the amino acid at position position 3 is Leu, Val or deleted; the amino acid at position 158 is Ser or Cys; the amino acid at position 159 is Val, Thr 4 is Val, Met or Leu; the amino acid at position 7 is Gly or or Ile; the amino acid at position 162 is Seror Ala; the amino Ser; the amino acid at position 8 is Lys or Thr; the amino acid at position 163 is Gly or deleted; the amino acid at acid at position 10 is Phe or Tyr; the amino acid at position position 164 is Phe or deleted; the amino acid at position 165 11 is Glu or Arg; the amino acid at position 18 is Met or Ile: is Arg or Ala; the amino acid at position 166 is Ala, Arg, Met the amino acid at position 19 is Gly, Pro or Ala; the amino or Phe; the amino acid at position 167 is Val or His; the acid at position 20 is Val or deleted; the amino acid at amino acid at position 168 is Ser or ASn; the amino acid at position 21 is Leu or Val; the amino acid at position 23 is Arg position 169 is Val, His or Thr; the amino acid at position or Gln; the amino acid at position 37 is Val or Leu; the amino 170 is Phe or Val; the amino acid at position 171 is Glu, Asn acid at position 38 is Arg or ASn; the amino acid at position or Asp; the amino acid at position 172 is Val, Ala, Arg or 40 is Ala or Ser; the amino acid at position 43 is Asn or Asp; Glu; the amino acid at position 175 is Ser, Arg or Trp; the the amino acid at position 45 is Gly or Ala; the amino acid amino acid at position 176 is Val or Ile; the amino acid at at position 46 is Gln or Glu; the amino acid at position 48 position 177 is Arg or Ile; the amino acid at position 179 is is Glu, Pro or Val; the amino acid at position 51 is Glu or Thr, Ile, Val or Ser; the amino acid at position 180 is Leu, Gly; the amino acid at position 52 is Lys, Arg or Thr; the Phe or Thr; the amino acid at position 181 is Gly, Thr, Gln amino acid at position 56 is Leu or Val; the amino acid at or Ser; the amino acid at position 182 is Ala, Leu, Phe or Ile: position 59 is Phe or Leu; the amino acid at position 66 is the amino acid at position 183 is Thr or Gly; the amino acid Pro or Ala; the amino acid at position 67 is Val, Pro or Thr: at position 184 is Leu, Thr, Ser or Arg; the amino acid at the amino acid at position 68 is Val, Arg, Phe or Gly; the position 185 is Arg, Gly, Asp or Ala; the amino acid at amino acid at position 69 is Glu, Ala or Lys; the amino acid position 186 is Pro, Val or Gln; the amino acid at position at position 70 is Trp. Thr. His, Tyr or Arg; the amino acid at 187 is Asp, Thr or Ser; the amino acid at position 188 is His, position 71 is Arg, Pro or deleted; the amino acid at position Gly or Ala; the amino acid at position 189 is Ala, Arg, Pro 72 is Trp, Asp, Leu or deleted; the amino acid at position 73 or deleted; the amino acid at position 190 is Leu, Asn or is Pro, Gln, Asn. His or deleted; the amino acid at position deleted; the amino acid at position 191 is Tyr or deleted; the 74 is Pro, Met or Thr; the amino acid at position 75 is Gln, amino acid at position 192 is Ser, Ile, Val or Asn; the amino His or Arg; the amino acid at position 76 is Ile, Met or Leu: acid at position 193 is Thr or Asp; the amino acid at position the amino acid at position 84 is Ile or Val; the amino acid at 194 is Thr or Ser; the amino acid at position 195 is Met or position 91 is Trp or Phe; the amino acid at position 93 is Thr Thr; the amino acid at position 196 is Gln, His, Leu or Ser; or Ile; the amino acid at position 94 is Asp or Gly; the amino the amino acid at position 197 is Ala, Gly or Leu; the amino acid at position 96 is Arg or Ser; the amino acid at position acid at position 198 is Thr, Glu or Ala; the amino acid at 97 is Gln, Phe or Arg; the amino acid at position 98 is Ser position 199 is Pro or Arg; the amino acid at position 200 is or deleted; the amino acid at position 99 is Asp or Ala; the Asn. Ser. Thr or Gly; the amino acid at position 201 is Ala, amino acid at position 100 is Thr or Ala; the amino acid at Leu, Glu or Trp; the amino acid at position 202 is Ser, Asp, position 101 is Glu, Thr or Trp the amino acid at position Phe or Leu; the amino acid at position 203 is His, Pro, Gly 103 is His, Arg, Glu or Gln; the amino acid at position 105 or Ser; the amino acid at position 204 is Ile, Trp. His or Gly: is Thr or Pro; the amino acid at position 108 is Lys, Gln or the amino acid at position 205 is Ser, Asn or Ile; the amino Glu; the amino acid at position 109 is Leu or Val; the amino acid at position 206 is Ala, Gly, Asp, Tyr or Arg; the amino acid at position 111 is Ala or Thr, the amino acid at position acid at position 207 is Phe, Val or Leu; the amino acid at 112 is Ile, Arg, Thr or deleted; the amino acid at position 113 position 208 is Asn. Ser, Pro or Leu; the amino acid at is Gln, Ala, Gly or deleted; the amino acid at position 114 position 210 is Arg, Asp, Glu or Tyr, the amino acid at is Arg, Glu or Ile; the amino acid at position 115 is Glu or position 211 is Ile, Seror Thr; the amino acid at position 212 Gln; the amino acid at position 116 is Glu, ASn, Gln or Arg; is Val, Ala or Asp; the amino acid at position 214 is Pro or the amino acid at position 117 is ASn, Val, Tyr or Phe; the Arg; the amino acid at position 215 is Ser or Thr; the amino amino acid at position 118 is Arg or Lys; the amino acid at acid at position 217 is Tyr or Phe; the amino acid at position position 119 is Trp or Ser; the amino acid at position 122 is 218 is Arg or Ser; the amino acid at position 219 is Val or Thr, Lys or Ala; the amino acid at position 124 is Ala or Thr: Ala; the amino acid at position 220 is Cys, Leu or Ser; the the amino acid at position 126 is Gly or Asp; the amino acid amino acid at position 221 is Pro or His; the amino acid at at position 127 is Met or Ala; the amino acid at position 128 position 222 is Leu, Arg or Ser; the amino acid at position is Asn or Lys; the amino acid at position 131 is Val, Ile or 224 is ASn or Ser; the amino acid at position 225 is Asp, Arg Thr; the amino acid at position 133 is Ile or Val; the amino or Thr; the amino acid at position 226 is Thr or Asn; the acid at position 134 is His or Tyr, the amino acid at position amino acid at position 227 is Asp, Leu or deleted; the amino US 2016/0347799 A1 Dec. 1, 2016
acid at position 228 is Thr or deleted; the amino acid at Arg or Gly; the amino acid at position 423 is Lys or Gln; the position 229 is Tyr or deleted; the amino acid at position 230 amino acid at position 431 is Arg or Lys; the amino acid at is Leu or deleted; the amino acid at position 231 is Gly or position 432 is Glin or Glu; the amino acid at position 436 is deleted; the amino acid at position 232 is Ile or deleted; the Arg or Glu; the amino acid at position 440 is ASnor Arg; the amino acid at position 233 is Pro or deleted; the amino acid amino acid at position 442 is Leu or Val; the amino acid at at position 234 is Ala, Pro or deleted; the amino acid at position 447 is Ser, Lys or Arg; the amino acid at position position 235 is Asp, Ile or Val; the amino acid at position 236 448 is Ala or Ser; the amino acid at position 451 is Glin or is Val, Ser or Glu; the amino acid at position 237 is Ala, Phe Met; the amino acid at position 453 is Gly or Ala; the amino or Tyr; the amino acid at position 238 is Ala or Thr; the acid at position 455 is Ala or Val; the amino acid at position amino acid at position 239 is Val, Ser or Gly; the amino acid 457 is Leu or Val; the amino acid at position 467 is Val or at position 240 is Leu or Ile; the amino acid at position 243 Ala; the amino acid at position 471 is Gly or Ala; the amino is Asp or Glu; the amino acid at position 249 is Asn or Ser; acid at position 475 is Ser or ASn; the amino acid at position the amino acid at position 252 is Leu or Met; the amino acid 483 is Gly or Ala; the amino acid at position 493 is Glin or at position 257 is Thr or Ser; the amino acid at position 259 Gly; the amino acid at position 504 is Val or Ile; the amino is His or Leu; the amino acid at position 266 is Ala or Val; acid at position 506 is Asp or His; the amino acid at position the amino acid at position 267 is Cys or Gly; the amino acid 509 is Asp or ASn; the amino acid at position 510 is Ser or at position 268 is His, Arg or Tyr, the amino acid at position Ala; the amino acid at position 512 is Glu or Asp; the amino 272 is Asp or Glu; the amino acid at position 273 is Val, Met, acid at position 515 is Gly or Ser; the amino acid at position Ile or Leu; the amino acid at position 274 is Val or Met; the 516 is Glin or His; the amino acid at position 517 is Ile or amino acid at position 278 is Gly or Ala; the amino acid at Leu; the amino acid at position 519 is Asp, Gly or Gln; the position 279 is Glu or Val; the amino acid at position 281 is amino acid at position 522 is Val, Glu, Pro or Val; the amino Leu or Ala; the amino acid at position 282 is ASn, Leu or Ile; acid at position 525 is Glu or Asp; the amino acid at position the amino acid at position 285 is Asn or Ser; the amino acid 526 is Leu or Met; the amino acid at position 539 is Val or at position 286 is Lys, Asp or Glu; the amino acid at position Ile; the amino acid at position 555 is Val or Ala; the amino 287 is Leu or Val; the amino acid at position 290 is Pro, Gln acid at position 557 is Arg or Lys; the amino acid at position or Arg; the amino acid at position 291 is Leu or Val; the 563 is Val or Met; the amino acid at position 571 is Ser or amino acid at position 292 is Lys or Val; the amino acid at Cys; the amino acid at position 575 is Val or Glu; the amino position 293 is Glu or Gln; the amino acid at position 294 is acid at position 577 is Met or Ile; the amino acid at position Ser, Asn or Lys; the amino acid at position 295 is Thr or Ser; 579 is Glu or Gln; the amino acid at position 583 is Asp or the amino acid at position 296 is Glin or His; the amino acid Glu; the amino acid at position 589 is Met or Leu; the amino at position 297 is Leu or Met; the amino acid at position 300 acid at position 590 is Met or Leu; the amino acid at position is Ser or Thr; the amino acid at position 301 is Glu or Ala; 593 is Met or Ile; the amino acid at position 595 is Arg or the amino acid at position 302 is Ser, Pro or Ala; the amino Gln; the amino acid at position 596 is Ser or Thr; the amino acid at position 304 is Lys or ASn; the amino acid at position acid at position 597 is Glin or His; the amino acid at position 313 is Val or Ile; the amino acid at position 314 is His, Glu 607 is Ala or Val; the amino acid at position 608 is Asp or or Gln; the amino acid at position 315 is Ala, Cys or Ser; the Asn; the amino acid at position 612 is Tyr, His or Phe; the amino acid at position 316 is Ala or Val; the amino acid at amino acid at position 617 is Thr or Ile; the amino acid at position 317 is Met or Ile; the amino acid at position 319 is position 618 is Glin or His; the amino acid at position 625 is Met or Ile; the amino acid at position 320 is Val or Gly; the Arg or Ser; the amino acid at position 626 is Met or Ile; the amino acid at position 321 is Arg or Pro; the amino acid at amino acid at position 628 is Leu or Ile; the amino acid at position 322 is Ile or Phe; the amino acid at position 323 is position 633 is Ile or Met; the amino acid at position 634 is Gly or Val; the amino acid at position 324 is Leu or Ser; the Leu or Met; the amino acid at position 642 is Arg or Met; the amino acid at position 336 is Ser or ASn; the amino acid at amino acid at position 648 is Met or Thr; the amino acid at position 339 is ASn, Lys or Arg; the amino acid at position position 651 is Glu or Gln; the amino acid at position 654 is 350 is Arg or Gln; the amino acid at position 351 is Glu or Thr, Val or Ala; the amino acid at position 658 is Gly or Arg; Asp; the amino acid at position 353 is Lys or Arg; the amino the amino acid at position 663 is Gly or Ala; the amino acid acid at position 354 is Glin or Arg; the amino acid at position at position 664 is Asp or ASn; the amino acid at position 668 355 is Phe or Leu; the amino acid at position 356 is Lys or is Ala or Thr; the amino acid at position 669 is Glin or His: Arg; the amino acid at position 360 is Ile, Val or Ala; the the amino acid at position 671 is Asn or Ser the amino acid amino acid at position 365 is Leu or Phe; the amino acid at at position 675 is Ile, Val or Ser; the amino acid at position position 371 is or Glu; the amino acid at position 372 is or 678 is Met, Ile, Ala or Thr; the amino acid at position 682 Lys; the amino acid at position 374 is Arg or Lys; the amino is Pro or Gln; the amino acid at position 683 is Ser or Pro; acid at position 376 is Phe or Leu; the amino acid at position the amino acid at position 685 is Asp or ASn; the amino acid 378 is Glu or Asp; the amino acid at position 381 is Leu or at position 694 is Asp or Gly; the amino acid at position 697 Val; the amino acid at position 388 is Ala or Ser; the amino is Asn or Ser; the amino acid at position 704 is Glu or Gly: acid at position 395 is Arg or Lys; the amino acid at position the amino acid at position 714 is Ala or Gly; the amino acid 396 is Glu, Gln or Gly; the amino acid at position 399 is Asp at position 721 is Ser or Phe; the amino acid at position 722 or ASn; the amino acid at position 400 is ASn, Thr or Asp; is Ser or Asn; the amino acid at position 724 is Ser or Thr: the amino acid at position 401 is Thr or Ala; the amino acid the amino acid at position 734 is His or Gln; the amino acid at position 402 is Phe, Ile or Leu; the amino acid at position at position 736 is Val or Ala; the amino acid at position 737 406 is Asp or Glu; the amino acid at position 408 is Leu or is Lys or Gln; the amino acid at position 739 is Ala or Ser; Met; the amino acid at position 410 is Gly or Leu; the amino the amino acid at position 740 is Ser or Met; the amino acid acid at position 414 is Ala or Glu; the amino acid at position at position 741 is Gly or Asn; the amino acid at position 742 416 is Ser, Asin or Asp; the amino acid at position 417 is Ser, is Ile or Gly; the amino acid at position 743 is Gly or deleted; US 2016/0347799 A1 Dec. 1, 2016
the amino acid at position 745 is Gly or Asp; the amino acid at position 94 is Asp, Glu, Ala or Gly; the amino acid at at position 751 is Thr, Ser or Ala; the amino acid at position position 96 is Arg, Lys, Thr or Ser; the amino acid at position 753 is Gln or Arg; the amino acid at position 754 is Thr or 97 is Gln, Phe, Asn. Lys or Arg; the amino acid at position Ser; the amino acid at position 756 is Thr or Ile; the amino 98 is Ser. Thr or deleted; the amino acid at position 99 is acid at position 757 is Val or Ile; the amino acid at position Asp, Glu, Gly or Ala; the amino acid at position 100 is Thr, 766 is Ile or Val; the amino acid at position 773 is Asp or Ser, Gly or Ala; the amino acid at position 101 is Glu, Thr, Glu; the amino acid at position 774 is Gln or Glu; the amino Asp, Ser or Trp the amino acid at position 103 is His, Arg, acid at position 776 is Leu or Met; the amino acid at position Lys, Glu or Gln; the amino acid at position 105 is Thr, Ser 777 is Pro or Thr; the amino acid at position 782 is Ala, Asp or Pro; the amino acid at position 108 is Lys, Arg, ASn, Asp, or Val; the amino acid at position 786 is Tyr or Phe; the Gln or Glu; the amino acid at position 109 is Leu, Ile or Val; amino acid at position 787 is His or Gln; the amino acid at the amino acid at position 111 is Ala, Ser or Thr; the amino position 788 is Tyr or Met; the amino acid at position 789 is acid at position 112 is Ile, Arg, Thr, Leu, Val, Lys, Ser or Ala or Arg; the amino acid at position 790 is Tyr or Thr; the deleted; the amino acid at position 113 is Gln, Ala, Gly, Asn amino acid at position 791 is Arg or Ala; the amino acid at or deleted; the amino acid at position 114 is Arg, Glu, Lys, position 792 is Leu or Ser; the amino acid at position 796 is Asp or Ile; the amino acid at position 115 is Glu, Asp, ASn Asp or Glu; the amino acid at position 797 is Ser, Thr or Ala or Gln; the amino acid at position 116 is Glu, ASn, Gln, Asp, the amino acid at position 802 is Glu or Gln; the amino acid Lys or Arg; the amino acid at position 117 is ASn, Val, Tyr, at position 806 is Gln, Asp, Glu or His; the amino acid at Ile, Leu, Gln, Trp or Phe; the amino acid at position 118 is position 810 is Lys or Thr; the amino acid at position 819 is Arg or Lys; the amino acid at position 119 is Trp, Thr or Ser; Arg or His; the amino acid at position 829 is Lys, Ser, Ala the amino acid at position 122 is Thr, Lys, Ser, Arg or Ala; or Pro; the amino acid at position 832 is Ala, Lys or Glu; the the amino acid at position 124 is Ala, Gly, Ser or Thr; the amino acid at position 833 is Gly or Glu; the amino acid at amino acid at position 126 is Gly, Ala, Glu or Asp; the amino position 842 is Leu or Pro; the amino acid at position 847 is acid at position 127 is Met, Gly or Ala; the amino acid at Gln or Glu; the amino acid at position 848 is Ile or Val; the position 128 is ASn, Gln, Arg or Lys; the amino acid at amino acid at position 849 is Val or Ala; the amino acid at position 131 is Val, Ile, Leu, Ser or Thr; the amino acid at position 855 is Thr or Met; the amino acid at position 860 position 133 is Ile, Leu or Val; the amino acid at position 134 is Ile or Val; and the amino acid at position 864 is His or Gln. is His or Tyr; the amino acid at position 135 is Ala or Gly: 0041. In some embodiments the nucleic acid molecule the amino acid at position 137 is Glu, Asp, Arg or Lys; the encodes a PtIP-83 polypeptide variant of SEQ ID NO: 1, amino acid at position 139 is Gln, Asn, Asp or Glu; the wherein the amino acid at position 1 is Met or deleted; the amino acid at position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 2 is Ala or deleted; the amino acid at amino acid at position 141 is Gly, Ala, Thr or Ser; the amino position 3 is Leu, Val, Ile or deleted; the amino acid at acid at position 142 is Val, Ile, Leu or Pro; the amino acid position 4 is Val, Met, Ile or Leu; the amino acid at position at position 144 is Thr, Leu, Phe, Ile, Val or Tyr; the amino 7 is Gly, Thr or Ser; the amino acid at position 8 is Lys, Arg, acid at position 145 is Met, Pro, Gln or Asn; the amino acid Seror Thr; the amino acid at position 10 is Phe, Trp or Tyr; at position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino the amino acid at position 11 is Glu, Asp, Lys or Arg; the acid at position 147 is Trp, Gln, Tyr or ASn; the amino acid amino acid at position 18 is Met, Val, Leu or Ile; the amino at position 148 is Ser, Ala, Thr, Gly or Pro; the amino acid acid at position 19 is Gly, Pro or Ala; the amino acid at at position 149 is Ser, Thr or deleted; the amino acid at position 20 is Val, Ile, Leu or deleted; the amino acid at position 150 is Val, Ile, Leu or Tyr; the amino acid at position 21 is Leu, Ile or Val; the amino acid at position 23 position 152 is Arg, Ala, Val, Ile, Leu, Lys or Gly; the amino is Arg, Lys, ASn or Gln; the amino acid at position 37 is Val, acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino Ile or Leu; the amino acid at position 38 is Arg, Lys, Gln or acid at position 156 is Leu, Asp, Ile, Val, Asn., Glu or Gln; Asn; the amino acid at position 40 is Ala, Gly, Thr or Ser; the amino acid at position 158 is Ser, Thr or Cys; the amino the amino acid at position 43 is ASn, Gln, Glu or Asp; the acid at position 159 is Val, Thr, Leu or Ile; the amino acid amino acid at position 45 is Gly or Ala; the amino acid at at position 162 is Ser. Thr, Gly or Ala; the amino acid at position 46 is Gln, Asp, ASn or Glu; the amino acid at position 163 is Gly, Ala or deleted; the amino acid at position position 48 is Glu, Asp, Pro, Ile, Leu or Val; the amino acid 164 is Phe or deleted; the amino acid at position 165 is Arg, at position 51 is Glu, Asp, Ala or Gly; the amino acid at LyS, Gly or Ala; the amino acid at position 166 is Ala, Arg, position 52 is Lys, Arg, Ser or Thr, the amino acid at position Met, Lys or Phe; the amino acid at position 167 is Val, Ile, 56 is Leu, Ile or Val; the amino acid at position 59 is Phe, Leu or His; the amino acid at position 168 is Ser. Thr, Gln Ile, Val or Leu; the amino acid at position 66 is Pro, Gly or or Asn; the amino acid at position 169 is Val. His, Ile, Leu, Ala; the amino acid at position 67 is Val, Pro, Ile, Leu, Ser Seror Thr; the amino acid at position 170 is Phe, Ile, Leu or or Thr; the amino acid at position 68 is Val, Arg, Phe, Ile, Val; the amino acid at position 171 is Glu, Asn., Gln or Asp; Leu, Lys or Gly; the amino acid at position 69 is Glu, Ala, the amino acid at position 172 is Val, Ala, Arg, Ile, Leu, Gly, Asp, Gly, Arg or Lys; the amino acid at position 70 is Trp, LyS, Asp or Glu; the amino acid at position 175 is Ser, Arg, Thr, His, Tyr, Lys or Arg; the amino acid at position 71 is Thr, Lys or Trp; the amino acid at position 176 is Val, Leu Arg, Pro, Lys or deleted; the amino acid at position 72 is Trp, or Ile; the amino acid at position 177 is Arg, Lys, Leu, Val Asp, Leu, Ile, Val, Glu or deleted; the amino acid at position or Ile; the amino acid at position 179 is Thr, Ile, Val, Leu or 73 is Pro, Gln, Asn. His or deleted; the amino acid at position Ser; the amino acid at position 180 is Leu, Phe, Ile, Val, Ser 74 is Pro, Met, Ser or Thr; the amino acid at position 75 is or Thr; the amino acid at position 181 is Gly, Thr, Gln, Asn Gln, His, ASn, Lys or Arg; the amino acid at position 76 is or Ser; the amino acid at position 182 is Ala, Leu, Phe, Val Ile, Met, Val or Leu; the amino acid at position 84 is Ile, Leu or Ile; the amino acid at position 183 is Thr, Ser, Ala or Gly: or Val; the amino acid at position 91 is Trp or Phe; the amino the amino acid at position 184 is Leu, Thr, Ser, Ile, Val, Lys acid at position 93 is Thr, Ser, Leu, Val or Ile; the amino acid or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys, US 2016/0347799 A1 Dec. 1, 2016
Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, 285 is Asn., Gln, Thr or Ser; the amino acid at position 286 Leu, Asn or Gln; the amino acid at position 187 is Asp, Thr, is Lys, Asp, Arg or Glu; the amino acid at position 287 is Glu or Ser; the amino acid at position 188 is His, Gly or Ala; Leu, Ile or Val; the amino acid at position 290 is Pro, Gln, the amino acid at position 189 is Ala, Arg, Pro, Lys, Gly or ASn, Lys or Arg; the amino acid at position 291 is Leu, Ile deleted; the amino acid at position 190 is Leu, Asn. Ile, Val, or Val; the amino acid at position 292 is Lys, Arg, Ile, Leu Gln or deleted; the amino acid at position 191 is Tyr or or Val; the amino acid at position 293 is Glu, Asp, Asn or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu, Gln; the amino acid at position 294 is Ser, Asn. Thr, Gln, Arg Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Lys; the amino acid at position 295 is Thr or Ser; the or Asp; the amino acid at position 194 is Thr or Ser; the amino acid at position 296 is Gln, ASnor His; the amino acid amino acid at position 195 is Met or Thr; the amino acid at at position 297 is Leu, Ile, Val or Met; the amino acid at position 196 is Gln, His, Leu, Asn. Ile, Val, Thr or Ser; the position 300 is Ser or Thr; the amino acid at position 301 is amino acid at position 197 is Ala, Gly, Ile, Val or Leu; the Glu, Asp, Gly or Ala; the amino acid at position 302 is Ser, amino acid at position 198 is Thr, Glu, Ser, Asp, Gly or Ala; Pro, Thr, Gly or Ala; the amino acid at position 304 is Lys, the amino acid at position 199 is Pro, Lys or Arg; the amino Arg, Gln or ASn; the amino acid at position 313 is Val, Leu acid at position 200 is Asn. Ser. Thr, Gln, Ala or Gly; the or Ile; the amino acid at position 314 is His, Glu, ASn, Asp amino acid at position 201 is Ala, Leu, Glu, Ile, Asp or Trp; or Gln; the amino acid at position 315 is Ala, Cys, Gly, Thr the amino acid at position 202 is Ser, Asp, Phe, Ile, Val, Thr, or Ser; the amino acid at position 316 is Ala, Ile, Leu or Val; Glu or Leu; the amino acid at position 203 is His, Pro, Gly, the amino acid at position 317 is Met, Leu, Val or Ile; the Ala, Thr or Ser; the amino acid at position 204 is Ile, Trp, amino acid at position 319 is Met, Leu, Val or Ile; the amino His, Leu, Val, Ala or Gly; the amino acid at position 205 is acid at position 320 is Val, Ile, Leu, Ala or Gly; the amino Ser, Asn. Leu, Val, Thr, Gln or Ile; the amino acid at position acid at position 321 is Arg, Lys or Pro; the amino acid at 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid position 322 is Ile, Leu, Val or Phe; the amino acid at at position 207 is Phe, Val, Ile or Leu; the amino acid at position 323 is Gly, Ile, Leu or Val; the amino acid at position 208 is Asn. Ser, Pro, Gln, Thr, Val, Ile or Leu; the position 324 is Leu, Ile, Val, Thr or Ser; the amino acid at amino acid at position 210 is Arg, Asp, Glu, Lys, Ser or Tyr; position 336 is Ser. Thr, Gln or ASn; the amino acid at the amino acid at position 211 is Ile, Ser, Leu, Val or Thr; the position 339 is ASn, Lys, Gln or Arg; the amino acid at amino acid at position 212 is Val, Ala, Ile, Leu, Glu, Gly or position 350 is Arg, Lys, ASn or Gln; the amino acid at Asp; the amino acid at position 214 is Pro, Lys or Arg; the position 351 is Glu or Asp; the amino acid at position 353 amino acid at position 215 is Ser or Thr; the amino acid at is Lys or Arg; the amino acid at position 354 is Gln, Asn., Lys position 217 is Tyr or Phe; the amino acid at position 218 is or Arg; the amino acid at position 355 is Phe, Ile, Leu or Leu: Arg, Lys, Thr or Ser; the amino acid at position 219 is Val, the amino acid at position 356 is Lys or Arg; the amino acid Ile, Leu or Ala; the amino acid at position 220 is Cys, Leu, at position 360 is Ile, Val, Leu, Gly or Ala; the amino acid Ile, Val, Thr or Ser; the amino acid at position 221 is Pro or at position 365 is Leu, Ile, Val or Phe; the amino acid at His; the amino acid at position 222 is Leu, Arg, Lys, Ile, Val, position 371 is or Glu or Asp; the amino acid at position 372 Thr or Ser; the amino acid at position 224 is Asn., Gln, Thr is or Lys or Arg; the amino acid at position 374 is Arg or Lys; or Ser; the amino acid at position 225 is Asp, Arg, Glu, LyS, the amino acid at position 376 is Phe, Ile, Val or Leu; the Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln amino acid at position 378 is Glu or Asp; the amino acid at or ASn; the amino acid at position 227 is Asp, Leu, Glu, Ile, position 381 is Leu, Ile or Val; the amino acid at position 388 Val or deleted; the amino acid at position 228 is Thr, Ser or is Ala, Thr, Gly or Ser; the amino acid at position 395 is Arg deleted; the amino acid at position 229 is Tyr or deleted; the or Lys; the amino acid at position 396 is Glu, Gln, Asp, ASn, amino acid at position 230 is Leu, Ile, Val or deleted; the Ala or Gly; the amino acid at position 399 is Asp, Gln, Glu amino acid at position 231 is Gly, Ala or deleted; the amino or ASn; the amino acid at position 400 is Asn. Thr, Ser, Glu, acid at position 232 is Ile, Leu, Val or deleted; the amino acid Gln or Asp; the amino acid at position 401 is Thr, Ser, Gly at position 233 is Pro or deleted; the amino acid at position or Ala; the amino acid at position 402 is Phe, Ile, Val or Leu: 234 is Ala, Pro, Gly or deleted; the amino acid at position the amino acid at position 406 is Asp or Glu; the amino acid 235 is Asp, Ile, Leu, Glu or Val; the amino acid at position at position 408 is Leu, Ile, Val or Met; the amino acid at 236 is Val, Ser, Ile, Leu, Thr, Asp or Glu; the amino acid at position 410 is Gly, Ile, Val, Ala or Leu; the amino acid at position 237 is Ala, Phe or Tyr; the amino acid at position position 414 is Ala, Gly, Asp or Glu; the amino acid at 238 is Ala, Gly, Ser or Thr; the amino acid at position 239 position 416 is Ser, Asn. Thr, Gln, Glu or Asp; the amino is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acid at acid at position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the position 240 is Leu, Val or Ile; the amino acid at position 243 amino acid at position 423 is Lys, Arg, ASnor Gln; the amino is Asp or Glu; the amino acid at position 249 is Asn., Gln, Thr acid at position 431 is Arg or Lys; the amino acid at position or Ser; the amino acid at position 252 is Leu, Ile, Val or Met; 432 is Gln, Asn, Asp or Glu; the amino acid at position 436 the amino acid at position 257 is Thr or Ser; the amino acid is Arg, Lys, Asp or Glu; the amino acid at position 440 is at position 259 is His, Ile, Val or Leu; the amino acid at ASn, Gln, Lys or Arg; the amino acid at position 442 is Leu, position 266 is Ala, Ile, Leu or Val; the amino acid at Ile or Val; the amino acid at position 447 is Ser, Lys, Thr or position 267 is Cys, Ala or Gly; the amino acid at position Arg; the amino acid at position 448 is Ala, Gly, Thr or Ser; 268 is His, Arg, Lys or Tyr; the amino acid at position 272 the amino acid at position 451 is Gln, Asin or Met; the amino is Asp or Glu; the amino acid at position 273 is Val, Met, Ile acid at position 453 is Gly or Ala; the amino acid at position or Leu; the amino acid at position 274 is Val, Ile, Leu or Met; 455 is Ala, Leu, Ile or Val; the amino acid at position 457 is the amino acid at position 278 is Gly or Ala; the amino acid Leu, Ile or Val; the amino acid at position 467 is Val, Ile, at position 279 is Glu, Asp, Gly or Val; the amino acid at Leu, Gly or Ala; the amino acid at position 471 is Gly or Ala; position 281 is Leu, Ile, Val, Gly or Ala; the amino acid at the amino acid at position 475 is Ser. Thr, Gln or Asn; the position 282 is ASn, Leu or Ile; the amino acid at position amino acid at position 483 is Gly or Ala; the amino acid at US 2016/0347799 A1 Dec. 1, 2016
position 493 is Gln, Asn or Gly; the amino acid at position acid at position 756 is Thr, Ser, Leu, Val or Ile; the amino 504 is Val, Leu or Ile; the amino acid at position 506 is Asp, acid at position 757 is Val, Leu or Ile; the amino acid at Glu or His; the amino acid at position 509 is Asp, Glu, Gln position 766 is Ile, Leu or Val; the amino acid at position 773 or ASn; the amino acid at position 510 is Ser. Thr, Gly or Ala; is Asp or Glu; the amino acid at position 774 is Gln, ASn, the amino acid at position 512 is Glu or Asp; the amino acid Asp or Glu; the amino acid at position 776 is Leu, Ile, Val at position 515 is Gly, Ala, Thr or Ser; the amino acid at or Met; the amino acid at position 777 is Pro, Seror Thr; the position 516 is Gln, Asn or His; the amino acid at position amino acid at position 782 is Ala, Asp, Glu, Ile, Leu or Val; 517 is Ile, Val or Leu; the amino acid at position 519 is Asp, the amino acid at position 786 is Tyr or Phe; the amino acid ASn, Glu, Gly or Gln; the amino acid at position 522 is Val, at position 787 is His, Asn or Gln; the amino acid at position Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 is 788 is Tyr or Met; the amino acid at position 789 is Ala, Lys Glu or Asp; the amino acid at position 526 is Leu, Ile, Val or Arg; the amino acid at position 790 is Tyr or Thr; the or Met; the amino acid at position 539 is Val, Leu or Ile; the amino acid at position 791 is Arg, Lys, Gly or Ala; the amino amino acid at position 555 is Val, Leu, Ile or Ala; the amino acid at position 792 is Leu, Ile, Val, Thr or Ser; the amino acid at position 557 is Arg or Lys; the amino acid at position acid at position 796 is Asp or Glu; the amino acid at position 563 is Val, Leu, Ile or Met; the amino acid at position 571 797 is Ser, Thr or Ala the amino acid at position 802 is Glu, is Ser, Thr or Cys; the amino acid at position 575 is Val, Leu, LyS, Asp, ASn or Gln; the amino acid at position 806 is Gln, Ile, Asp or Glu; the amino acid at position 577 is Met, Leu, Asp, Glu, ASn or His; the amino acid at position 810 is Lys, Val or Ile; the amino acid at position 579 is Glu, Asp, Asn Arg or Thr; the amino acid at position 819 is Arg, Lys or His: or Gln; the amino acid at position 583 is Asp or Glu; the the amino acid at position 829 is Lys, Ser, Ala or Pro; the amino acid at position 589 is Met, Ile, Val or Leu; the amino amino acid at position 832 is Ala, Lys, Arg, Asp or Glu; the acid at position 590 is Met, Ile, Val or Leu; the amino acid amino acid at position 833 is Gly, Ala, Asp or Glu; the amino at position 593 is Met, Leu, Val or Ile; the amino acid at acid at position 842 is Leu, Ile, Val or Pro; the amino acid position 595 is Arg, Lys, ASn or Gln; the amino acid at at position 847 is Gln, ASn, Asp or Glu; the amino acid at position 596 is Ser or Thr; the amino acid at position 597 is position 848 is Ile, Leu or Val; the amino acid at position 849 Gln, Asin or His; the amino acid at position 607 is Ala, Gly, is Val, Leu, Ile, Gly or Ala; the amino acid at position 855 Ile, Leu or Val; the amino acid at position 608 is Asp, Glu, is Thr, Ser or Met; the amino acid at position 860 is Ile, Leu Gln or Asn; the amino acid at position 612 is Tyr, His or Phe: or Val; the amino acid at position 864 is His, Asn or Gln; the amino acid at position 617 is Thr, Ser, Leu, Val or Ile; the 0042. In some embodiments the nucleic acid molecule amino acid at position 618 is Gln, Asn or His; the amino acid encodes a PtIP-83 polypeptide variant of SEQ ID NO: 1, at position 625 is Arg, Lys, Thr or Ser; the amino acid at wherein the amino acid at position 1 is Met or deleted; the position 626 is Met, Leu, Val or Ile; the amino acid at amino acid at position 2 is Ala or deleted; the amino acid at position 628 is Leu, Val or Ile; the amino acid at position 633 position 3 is Leu, Val, Ile or deleted; the amino acid at is Ile, Leu, Val or Met; the amino acid at position 634 is Leu, position 4 is Val, Met, Ile or Leu; the amino acid at position Ile, Val or Met; the amino acid at position 642 is Arg, Lys 7 is Gly, Thr or Ser; the amino acid at position 8 is Lys, Arg, or Met; the amino acid at position 648 is Met, Seror Thr; the Ser or Thr; the amino acid at position 10 is Phe, Trp or Tyr; amino acid at position 651 is Glu, Asp, ASn or Gln; the the amino acid at position 11 is Glu, Asp, Lys or Arg; the amino acid at position 654 is Thr, Val, Ser, Ile, Leu, Gly or amino acid at position 18 is Met, Val, Leu or Ile; the amino Ala; the amino acid at position 658 is Gly, Lys, Ala or Arg; acid at position 19 is Gly, Pro or Ala; the amino acid at the amino acid at position 663 is Gly or Ala; the amino acid position 20 is Val, Ile, Leu or deleted; the amino acid at at position 664 is Asp, Glu, Gln or ASn; the amino acid at position 21 is Leu, Ile or Val; the amino acid at position 23 position 668 is Ala, Gly, Ser or Thr; the amino acid at is Arg, Lys, ASn or Gln; the amino acid at position 37 is Val, position 669 is Gln, Asn or His; the amino acid at position Ile or Leu; the amino acid at position 38 is Arg, Lys, Gln or 671 is Asn., Gln, Thr or Ser the amino acid at position 675 Asn; the amino acid at position 40 is Ala, Gly, Thr or Ser; is Ile, Val, Ile, Thr or Ser; the amino acid at position 678 is the amino acid at position 43 is ASn, Gln, Glu or Asp; the Met, Ile, Ala, Leu, Seror Thr; the amino acid at position 682 amino acid at position 45 is Gly or Ala; the amino acid at is Pro, Asin or Gln; the amino acid at position 683 is Ser. Thr position 46 is Gln, Asp, ASn or Glu; the amino acid at or Pro; the amino acid at position 685 is Asp, Glu, Asp or position 48 is Glu, Asp, Pro, Ile, Leu or Val; the amino acid ASn; the amino acid at position 694 is Asp, Glu, Ala or Gly; at position 51 is Glu, Asp, Ala or Gly; the amino acid at the amino acid at position 697 is ASn, Gln, Thr or Ser; the position 52 is Lys, Arg, Ser or Thr, the amino acid at position amino acid at position 704 is Glu, Asp, Ala or Gly; the amino 53 is Val, Ala, Cys or Thr; the amino acid at position 54 is acid at position 714 is Ala or Gly; the amino acid at position LyS, Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met, ASn, Gln, 721 is Ser, Thr or Phe; the amino acid at position 722 is Ser, Arg, Ser or Thr, the amino acid at position 55 is Arg, Ala, Thr, Gln or ASn; the amino acid at position 724 is Seror Thr: Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn., Gln, Ser. Thr, the amino acid at position 734 is His, Asn or Gln; the amino Val, Trp or Tyr; the amino acid at position 56 is Leu, Glu, acid at position 736 is Val, Leu, Ile or Ala; the amino acid Phe, Ile, Met, Thror Val; the amino acid at position 57 is Tyr, at position 737 is Lys, Arg, ASn or Gln; the amino acid at Cys, Ile, Leu, Met, Thr or Val; the amino acid at position 58 position 739 is Ala, Gly, Thr or Ser; the amino acid at is Val, Cys, Ile or Leu; the amino acid at position 59 is Phe, position 740 is Ser. Thr or Met; the amino acid at position Leu, Met, Val or Tyr; the amino acid at position 60 is Ala, 741 is Gly, Ala, Gln or Asn; the amino acid at position 742 Cys, Gly, Ser, Thr or Val; the amino acid at position 61 is is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 Asp, Glu, His or Ser; the amino acid at position 62 is Val, is Gly or deleted; the amino acid at position 745 is Gly, Ala, Ala, Cys, Ile, Leu or Thr; the amino acid at position 63 is Glu or Asp; the amino acid at position 751 is Thr, Ser, Gly Val, Ala, Cys, Ile, Leu, Met or Thr; the amino acid at or Ala; the amino acid at position 753 is Gln, Asn. Lys or position 64 is Glu, Ala, Cys, Phe, Gly, His, Ile, Leu, Met, Arg; the amino acid at position 754 is Thr or Ser; the amino ASn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at US 2016/0347799 A1 Dec. 1, 2016
position 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn., Gln, Phe; the amino acid at position 167 is Val, Ile, Leu or His: Thr, Val or Trp; the amino acid at position 66 is Pro, Asp, the amino acid at position 168 is Ser. Thr, Gln or Asn; the Gly, Met, Gln or Arg; the amino acid at position 67 is Val, amino acid at position 169 is Val, His, Ile, Leu, Ser or Thr: Pro, Ile, Leu, Seror Thr; the amino acid at position 68 is Val, the amino acid at position 170 is Phe, Ile, Leu or Val; the Arg, Phe, Ile, Leu, Lys or Gly; the amino acid at position 69 amino acid at position 171 is Glu, ASn, Gln or Asp; the is Glu, Ala, Asp, Gly, Arg or Lys; the amino acid at position amino acid at position 172 is Val, Ala, Arg, Ile, Leu, Gly, 70 is Trp, Thr, His, Tyr, Lys or Arg; the amino acid at LyS, Asp or Glu; the amino acid at position 175 is Ser, Arg, position 71 is Arg, Pro, Lys or deleted; the amino acid at Thr, Lys or Trp; the amino acid at position 176 is Val, Leu position 72 is Trp, Asp, Leu, Ile, Val, Glu or deleted; the or Ile; the amino acid at position 177 is Arg, Lys, Leu, Val amino acid at position 73 is Pro, Gln, Asn. His or deleted; or Ile; the amino acid at position 179 is Thr, Ile, Val, Leu or the amino acid at position 74 is Pro, Met, Ser or Thr; the Ser; the amino acid at position 180 is Leu, Phe, Ile, Val, Ser amino acid at position 75 is Gln, His, ASn, Lys or Arg; the or Thr; the amino acid at position 181 is Gly, Thr, Gln, Asn amino acid at position 76 is Ile, Met, Val or Leu; the amino or Ser; the amino acid at position 182 is Ala, Leu, Phe, Val acid at position 84 is Ile, Leu or Val; the amino acid at or Ile; the amino acid at position 183 is Thr, Ser, Ala or Gly: position 91 is Trp or Phe; the amino acid at position 93 is the amino acid at position 184 is Leu, Thr, Ser, Ile, Val, Lys Thr, Ser, Leu, Val or Ile; the amino acid at position 94 is Asp, or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys, Glu, Ala or Gly; the amino acid at position 96 is Arg, LyS, Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Thr or Ser; the amino acid at position 97 is Gln, Phe, Asn. Leu, Asn or Gln; the amino acid at position 187 is Asp, Thr, Lys or Arg; the amino acid at position 98 is Ser. Thr or Glu or Ser; the amino acid at position 188 is His, Gly or Ala; deleted; the amino acid at position 99 is Asp, Glu, Gly or the amino acid at position 189 is Ala, Arg, Pro, Lys, Gly or Ala; the amino acid at position 100 is Thr, Ser, Gly or Ala; deleted; the amino acid at position 190 is Leu, Asn. Ile, Val, the amino acid at position 101 is Glu, Thr, Asp, Ser or Trp Gln or deleted; the amino acid at position 191 is Tyr or the amino acid at position 103 is His, Arg, Lys, Glu or Gln; deleted; the amino acid at position 192 is Ser, Ile, Val, Leu, the amino acid at position 105 is Thr, Ser or Pro; the amino Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu acid at position 108 is Lys, Arg, ASn, Asp, Gln or Glu; the or Asp; the amino acid at position 194 is Thr or Ser; the amino acid at position 109 is Leu, Ile or Val; the amino acid amino acid at position 195 is Met or Thr; the amino acid at at position 111 is Ala, Seror Thr; the amino acid at position position 196 is Gln, His, Leu, Asn. Ile, Val, Thr or Ser; the 112 is Ile, Arg, Thr, Leu, Val, Lys, Ser or deleted; the amino amino acid at position 197 is Ala, Gly, Ile, Val or Leu; the acid at position 113 is Gln, Ala, Gly, Asin or deleted; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly or Ala: amino acid at position 114 is Arg, Glu, Lys, Asp or Ile; the the amino acid at position 199 is Pro, Lys or Arg; the amino amino acid at position 115 is Glu, Asp, ASn or Gln; the acid at position 200 is Asn. Ser. Thr, Gln, Ala or Gly; the amino acid at position 116 is Glu, ASn, Gln, Asp, Lys or Arg; amino acid at position 201 is Ala, Leu, Glu, Ile, Asp or Trp; the amino acid at position 117 is Asn. Val, Tyr, Ile, Leu, Gln, the amino acid at position 202 is Ser, Asp, Phe, Ile, Val, Thr, Trp or Phe; the amino acid at position 118 is Arg or Lys; the Glu or Leu; the amino acid at position 203 is His, Pro, Gly, amino acid at position 119 is Trp, Thr or Ser; the amino acid Ala, Thr or Ser; the amino acid at position 204 is Ile, Trp, at position 122 is Thr, Lys, Ser, Arg or Ala; the amino acid His, Leu, Val, Ala or Gly; the amino acid at position 205 is at position 124 is Ala, Gly, Ser or Thr; the amino acid at Ser, Asn. Leu, Val, Thr, Gln or Ile; the amino acid at position position 126 is Gly, Ala, Glu or Asp; the amino acid at 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid position 127 is Met, Gly or Ala; the amino acid at position at position 207 is Phe, Val, Ile or Leu; the amino acid at 128 is ASn, Gln, Arg or Lys; the amino acid at position 131 position 208 is Asn. Ser, Pro, Gln, Thr, Val, Ile or Leu; the is Val, Ile, Leu, Ser or Thr; the amino acid at position 133 amino acid at position 210 is Arg, Asp, Glu, Lys, Ser or Tyr; is Ile, Leu or Val; the amino acid at position 134 is His or the amino acid at position 211 is Ile, Ser, Leu, Val or Thr; the Tyr; the amino acid at position 135 is Ala or Gly; the amino amino acid at position 212 is Val, Ala, Ile, Leu, Glu, Gly or acid at position 137 is Glu, Asp, Arg or Lys; the amino acid Asp; the amino acid at position 214 is Pro, Lys or Arg; the at position 139 is Gln, Asn., Asp or Glu; the amino acid at amino acid at position 215 is Ser or Thr; the amino acid at position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 217 is Tyr or Phe; the amino acid at position 218 is position 141 is Gly, Ala, Thr or Ser; the amino acid at Arg, Lys, Thr or Ser; the amino acid at position 219 is Val, position 142 is Val, Ile, Leu or Pro; the amino acid at Ile, Leu or Ala; the amino acid at position 220 is Cys, Leu, position 144 is Thr, Leu, Phe, Ile, Val or Tyr; the amino acid Ile, Val, Thr or Ser; the amino acid at position 221 is Pro or at position 145 is Met, Pro, Gln or ASn; the amino acid at His; the amino acid at position 222 is Leu, Arg, Lys, Ile, Val, position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acid Thr or Ser; the amino acid at position 224 is Asn., Gln, Thr at position 147 is Trp, Gln, Tyr or Asn; the amino acid at or Ser; the amino acid at position 225 is Asp, Arg, Glu, Lys, position 148 is Ser, Ala, Thr, Gly or Pro; the amino acid at Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln position 149 is Ser. Thr or deleted; the amino acid at position or ASn; the amino acid at position 227 is Asp, Leu, Glu, Ile, 150 is Val, Ile, Leu or Tyr; the amino acid at position 152 is Val or deleted; the amino acid at position 228 is Thr, Ser or Arg, Ala, Val, Ile, Leu, Lys or Gly; the amino acid at position deleted; the amino acid at position 229 is Tyr or deleted; the 154 is Ser, Trp, Thr, Asp or Glu; the amino acid at position amino acid at position 230 is Leu, Ile, Val or deleted; the 156 is Leu, Asp, Ile, Val, Asn., Glu or Gln; the amino acid at amino acid at position 231 is Gly, Ala or deleted; the amino position 158 is Ser. Thr or Cys; the amino acid at position acid at position 232 is Ile, Leu, Val or deleted; the amino acid 159 is Val, Thr, Leu or Ile; the amino acid at position 162 is at position 233 is Pro or deleted; the amino acid at position Ser. Thr, Gly or Ala; the amino acid at position 163 is Gly, 234 is Ala, Pro, Gly or deleted; the amino acid at position Ala or deleted; the amino acid at position 164 is Phe or 235 is Asp, Ile, Leu, Glu or Val; the amino acid at position deleted; the amino acid at position 165 is Arg, Lys, Gly or 236 is Val, Ser, Ile, Leu, Thr, Asp or Glu; the amino acid at Ala; the amino acid at position 166 is Ala, Arg, Met, Lys or position 237 is Ala, Phe or Tyr; the amino acid at position US 2016/0347799 A1 Dec. 1, 2016
238 is Ala, Gly, Ser or Thr; the amino acid at position 239 Leu, ASn, Arg, Ser, Thr, Val or Trp; the amino acid at is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acid at position 372 is Gln, Ala, Cys, Asp, Phe, Gly. His, Ile, Leu, position 240 is Leu, Val or Ile; the amino acid at position 243 Asn, Arg, Ser, Val or Tyr; the amino acid at position 373 is is Asp or Glu; the amino acid at position 249 is Asn., Gln, Thr Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Gln, Ser. Thr, Val or Ser; the amino acid at position 252 is Leu, Ile, Val or Met; or Trp; the amino acid at position 374 is Arg or Lys; the the amino acid at position 257 is Thr or Ser; the amino acid amino acid at position 376 is Phe, Ile, Val or Leu; the amino at position 259 is His, Ile, Val or Leu; the amino acid at acid at position 378 is Glu or Asp; the amino acid at position position 266 is Ala, Ile, Leu or Val; the amino acid at 381 is Leu, Ile or Val; the amino acid at position 388 is Ala, position 267 is Cys, Ala or Gly; the amino acid at position Thr, Gly or Ser; the amino acid at position 395 is Arg or Lys: 268 is His, Arg, Lys or Tyr; the amino acid at position 272 the amino acid at position 396 is Glu, Gln, Asp, Asn, Ala or is Asp or Glu; the amino acid at position 273 is Val, Met, Ile Gly; the amino acid at position 399 is Asp, Gln, Glu or Asn; or Leu; the amino acid at position 274 is Val, Ile, Leu or Met; the amino acid at position 400 is Asn. Thr, Ser, Glu, Gln or the amino acid at position 278 is Gly or Ala; the amino acid Asp; the amino acid at position 401 is Thr, Ser, Gly or Ala; at position 279 is Glu, Asp, Gly or Val; the amino acid at the amino acid at position 402 is Phe, Ile, Val or Leu; the position 281 is Leu, Ile, Val, Gly or Ala; the amino acid at amino acid at position 406 is Asp or Glu; the amino acid at position 282 is ASn, Leu or Ile; the amino acid at position position 408 is Leu, Ile, Val or Met; the amino acid at 285 is Asn., Gln, Thr or Ser; the amino acid at position 286 position 410 is Gly, Ile, Val, Ala or Leu; the amino acid at is Lys, Asp, Arg or Glu; the amino acid at position 287 is position 414 is Ala, Gly, Asp or Glu; the amino acid at Leu, Ile or Val; the amino acid at position 290 is Pro, Gln, position 416 is Ser, Asn. Thr, Gln, Glu or Asp; the amino ASn, Lys or Arg; the amino acid at position 291 is Leu, Ile acid at position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the or Val; the amino acid at position 292 is Lys, Arg, Ile, Leu amino acid at position 423 is Lys, Arg, ASnor Gln; the amino or Val; the amino acid at position 293 is Glu, Asp, Asn or acid at position 431 is Arg or Lys; the amino acid at position Gln; the amino acid at position 294 is Ser, Asn. Thr, Gln, Arg 432 is Gln, Asn, Asp or Glu; the amino acid at position 436 or Lys; the amino acid at position 295 is Thr or Ser; the is Arg, Lys, Asp or Glu; the amino acid at position 440 is amino acid at position 296 is Gln, ASnor His; the amino acid ASn, Gln, Lys or Arg; the amino acid at position 442 is Leu, at position 297 is Leu, Ile, Val or Met; the amino acid at Ile or Val; the amino acid at position 447 is Ser, Lys, Thr or position 300 is Ser or Thr; the amino acid at position 301 is Arg; the amino acid at position 448 is Ala, Gly, Thr or Ser; Glu, Asp, Gly or Ala; the amino acid at position 302 is Ser, the amino acid at position 451 is Gln, Asin or Met; the amino Pro, Thr, Gly or Ala; the amino acid at position 304 is Lys, acid at position 453 is Gly or Ala; the amino acid at position Arg, Gln or ASn; the amino acid at position 313 is Val, Leu 455 is Ala, Leu, Ile or Val; the amino acid at position 457 is or Ile; the amino acid at position 314 is His, Glu, ASn, Asp Leu, Ile or Val; the amino acid at position 467 is Val, Ile, or Gln; the amino acid at position 315 is Ala, Cys, Gly, Thr Leu, Gly or Ala; the amino acid at position 471 is Gly or Ala; or Ser; the amino acid at position 316 is Ala, Ile, Leu or Val; the amino acid at position 475 is Ser. Thr, Gln or Asn; the the amino acid at position 317 is Met, Leu, Val or Ile; the amino acid at position 483 is Gly or Ala; the amino acid at amino acid at position 319 is Met, Leu, Val or Ile; the amino position 493 is Gln, Asn or Gly; the amino acid at position acid at position 320 is Val, Ile, Leu, Ala or Gly; the amino 504 is Val, Leu or Ile; the amino acid at position 506 is Asp, acid at position 321 is Arg, Lys or Pro; the amino acid at Glu or His; the amino acid at position 509 is Asp, Glu, Gln position 322 is Ile, Leu, Val or Phe; the amino acid at or ASn; the amino acid at position 510 is Ser. Thr, Gly or Ala; position 323 is Gly, Ile, Leu or Val; the amino acid at the amino acid at position 512 is Glu or Asp; the amino acid position 324 is Leu, Ile, Val, Thr or Ser; the amino acid at at position 515 is Gly, Ala, Thr or Ser; the amino acid at position 336 is Ser. Thr, Gln or ASn; the amino acid at position 516 is Gln, Asin or His; the amino acid at position position 339 is ASn, Lys, Gln or Arg; the amino acid at 517 is Ile, Val or Leu; the amino acid at position 519 is Asp, position 350 is Arg, Lys, ASn or Gln; the amino acid at ASn, Glu, Gly or Gln; the amino acid at position 522 is Val, position 351 is Glu or Asp; the amino acid at position 353 Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 is is Lys or Arg; the amino acid at position 354 is Gln, ASn, Lys Glu or Asp; the amino acid at position 526 is Leu, Ile, Val or Arg; the amino acid at position 355 is Phe, Ile, Leu or Leu: or Met; the amino acid at position 539 is Val, Leu or Ile; the the amino acid at position 356 is Lys or Arg; the amino acid amino acid at position 555 is Val, Leu, Ile or Ala; the amino at position 360 is Ile, Val, Leu, Gly or Ala; the amino acid acid at position 556 is Trp, Phe, Thr or Tyr; the amino acid at position 363 is Gln, Ala, Cys, Glu, Phe, Gly, His, Lys, at position 557 is Arg, Cys, Asp, Gly. His, Ile, Lys, Leu, Met, Leu, ASn, Arg, Ser, Thr, Val or Trp; the amino acid at ASn, Pro, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid at position 364 is Ile, Ala, Cys, Glu, Phe, His, Lys, Leu, Met, position 558 is Ala, Cys, Asp, Phe, Gly. His, Ile, Lys, Leu, ASn, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid at position ASn, Pro, Gln, Arg, Ser, Val, Trp or Tyr; the amino acid at 365 is Leu, Ala, Glu, Phe, Gly. His, Ile, Lys, Met, Asn, Arg, position 559 is Lys, Ala, Cys, Phe, Gly, His, Ile, Leu, Asn. Val, Trp or Tyr; the amino acid at position 366 is Gly, Ala, Gln, Arg, Ser, Thr, Val or Tyr; the amino acid at position 560 Cys, Phe, His, Ile, Lys, Leu, Met, Asn. Ser, Thr or Val; the is Cys, Ala, Phe, Gly, Ile, Met, Asn, Arg, Ser. Thr or Val; the amino acid at position 367 is Ser, Ala, Cys, Asp, Glu, Phe, amino acid at position 561 is Lys, Ala, Cys, Asp, Glu, Phe, Gly. His, Ile, Leu, Met, Asn. Pro, Gln, Arg, Thr, Val or Trp; Gly. His, Ile, Leu, Met, Asn, Arg, Ser. Thr, Val or Tyr; the the amino acid at position 368 is Tyr, Ala, Cys, Asp, Glu, amino acid at position 562 is ASn, Cys, Asp, Glu, Gly, His, Phe, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Gln, Arg, Ser, Leu, Met, Arg, Ser. Thr, Val or Tyr; the amino acid at Thr, Val or Trp; the amino acid at position 369 is Leu, Ala, position 563 is Val, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Cys, Asp, Phe, Gly, Ile, Met, Thr or Val; the amino acid at ASn, Gln, Thr or Trp; the amino acid at position 564 is Ala, position 370 is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Cys, Gly, Met, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid Lys, Met, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino acid at position 571 is Ser. Thr or Cys; the amino acid at position at position 371 is Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position US 2016/0347799 A1 Dec. 1, 2016
577 is Met, Leu, Val or Ile; the amino acid at position 579 is Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ASn, Ser, is Glu, Asp, Asn or Gln; the amino acid at position 583 is Thr, Val, Trp or Tyr; the amino acid at position 772 is Arg, Asp or Glu; the amino acid at position 589 is Met, Ile, Val Ala, Cys, Asp, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Pro, or Leu; the amino acid at position 590 is Met, Ile, Val or Leu: Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 773 the amino acid at position 593 is Met, Leu, Val or Ile; the is Asp, Ala, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Asn., Gln, amino acid at position 595 is Arg, Lys, ASnor Gln; the amino Arg, Ser. Thr, Val, Trp or Tyr; the amino acid at position 774 acid at position 596 is Seror Thr; the amino acid at position is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Arg, 597 is Gln, Asn or His; the amino acid at position 607 is Ala, Ser. Thr, Val, Trp or Tyr; the amino acid at position 775 is Gly, Ile, Leu or Val; the amino acid at position 608 is Asp, Val, Ala, Cys, Asp, Glu, Gly, His, Ile, Asn. Pro, Gln, Arg, Glu, Gln or ASn; the amino acid at position 612 is Tyr, His Ser. Thr or Tyr; the amino acid at position 776 is Leu, Ala, or Phe; the amino acid at position 617 is Thr, Ser, Leu, Val Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, ASn, Pro, Gln, Arg, or Ile; the amino acid at position 618 is Gln, Asn or His; the Ser. Thr, Val or Tyr; the amino acid at position 777 is Pro, amino acid at position 625 is Arg, Lys, Thr or Ser; the amino Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn., Gln, acid at position 626 is Met, Leu, Val or Ile; the amino acid Ser. Thr, Val, Trp or Tyr; the amino acid at position 778 is at position 628 is Leu, Val or Ile; the amino acid at position Phe, Ala, His, Ile, Leu, Met, Asn., Gln, Ser, Val, Trp or Tyr; 633 is Ile, Leu, Val or Met; the amino acid at position 634 the amino acid at position 779 is Gln, Ala, Cys, Asp, Glu, is Leu, Ile, Val or Met; the amino acid at position 642 is Arg, Gly. His, Lys, Leu, Asn. Pro, Arg, Ser. Thr or Val; the amino Lys or Met; the amino acid at position 646 is Leu, Ala, Cys, acid at position 780 is Ala, Cys, ASn, Pro, Gln or Ser; the Gly, Ile, Met, Asn., Gln, Ser. Thr or Val; the amino acid at amino acid at position 781 is Ala, Cys, Asp, Glu, Phe, Gly, position 647 is Leu, Asp, Gly, Met, Asn., Gln or Thr; the His, Ile, Asn., Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino amino acid at position 648 is Met, Ala, Cys, Asp, Glu, Phe, Gly. His, Lys, Leu, Asn. Pro, Gln, Arg, Ser, Thr, Val, Trp or acid at position 782 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Tyr; the amino acid at position 649 is Pro, Ala, Cys, Asp, Lys, Met, Pro, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino Glu, Phe, Gly, His, Lys, Met, Asn., Gln, Arg, Ser. Thr, Trp or acid at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn. Tyr; the amino acid at position 650 is Thr, Ala, Cys, Asp, Gln, Arg, Ser. Thr or Val; the amino acid at position 784 is Phe, Gly. His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Val or Leu, Ala, Glu, Phe, His, Ile, Lys, Met, ASn, Pro, Gln, Ser, Tyr; the amino acid at position 651 is Glu, Ala, Cys, Asp, Thr, Val or Trp; the amino acid at position 785 is Asn, Ala, Gly. His, Ile, Leu, Met, Asn. Pro, Gln, Arg, Ser. Thr, Val or Cys, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Gln, Arg, Ser, Tyr; the amino acid at position 652 is Leu, Cys, Phe, Ile, Lys, Thr, Val, Trp or Tyr; the amino acid at position 786 is Tyr, Met, Pro, Arg, Ser. Thr or Val; the amino acid at position 653 Phe, Ile, Leu or Trp; the amino acid at position 787 is His, is Thr, Cys, Asp, Glu, Phe, Gly. His, Ile, Lys, Leu, Pro, Arg, Asin or Gln; the amino acid at position 788 is Tyr or Met; the Ser, Val or Trp; the amino acid at position 654 is Thr, Ala, amino acid at position 789 is Ala, Lys or Arg; the amino acid Cys, Phe, Ile, Lys, Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; at position 790 is Tyr or Thr; the amino acid at position 791 the amino acid at position 655 is Trp, Phe or Tyr; the amino is Arg, LyS, Gly or Ala; the amino acid at position 792 is Leu, acid at position 658 is Gly, Lys, Ala or Arg; the amino acid Ile, Val, Thr or Ser; the amino acid at position 796 is Asp or at position 663 is Gly or Ala; the amino acid at position 664 Glu; the amino acid at position 797 is Ser, Thr or Ala the is Asp, Glu, Gln or ASn; the amino acid at position 668 is amino acid at position 802 is Glu, Lys, Asp, ASn or Gln; the Ala, Gly, Ser or Thr; the amino acid at position 669 is Gln, amino acid at position 806 is Gln, Asp, Glu, Asn or His; the Asin or His; the amino acid at position 671 is Asn., Gln, Thr amino acid at position 810 is Lys, Arg or Thr, the amino acid or Ser the amino acid at position 675 is Ile, Val, Ile, Thr or at position 819 is Arg, Lys or His; the amino acid at position Ser; the amino acid at position 678 is Met, Ile, Ala, Leu, Ser 829 is Lys, Ser, Ala or Pro; the amino acid at position 832 or Thr; the amino acid at position 682 is Pro, Asn or Gln; the is Ala, Lys, Arg, Asp or Glu, the amino acid at position 833 amino acid at position 683 is Ser. Thr or Pro; the amino acid is Gly, Ala, Asp or Glu; the amino acid at position 842 is at position 685 is Asp, Glu, Asp or ASn; the amino acid at Leu, Ile, Val or Pro; the amino acid at position 847 is Gln, position 694 is Asp, Glu, Ala or Gly; the amino acid at ASn, Asp or Glu; the amino acid at position 848 is Ile, Leu position 697 is ASn, Gln, Thr or Ser; the amino acid at or Val; the amino acid at position 849 is Val, Leu, Ile, Gly position 704 is Glu, Asp, Ala or Gly; the amino acid at or Ala; the amino acid at position 855 is Thr, Ser or Met; the position 714 is Ala or Gly; the amino acid at position 721 is amino acid at position 860 is Ile, Leu or Val; and the amino Ser. Thr or Phe; the amino acid at position 722 is Ser, Thr, acid at position 864 is His, Asn or Gln. Gln or Asn; the amino acid at position 724 is Ser or Thr; the 0043. In some embodiments the nucleic acid molecule amino acid at position 734 is His, ASnor Gln; the amino acid encoding the PtIP-83 polypeptide is derived from a fern at position 736 is Val, Leu, Ile or Ala; the amino acid at species in the Division Pteridophyta. The phylogeny offerns position 737 is Lys, Arg, ASn or Gln; the amino acid at as used herein is based on the classification for extant ferns position 739 is Ala, Gly, Thr or Ser; the amino acid at by A. R. Smith et al, TAXON, 55:705-731 (2006). The position 740 is Ser. Thr or Met; the amino acid at position consensus phylogeny based on the classification by A. R. 741 is Gly, Ala, Gln or Asn; the amino acid at position 742 Smith is shown in FIG. 1. Other phylogenic classifications is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 of extant ferns are known to one skilled in the art. Additional is Gly or deleted; the amino acid at position 745 is Gly, Ala, information on the phylogeny of ferns can be found at Glu or Asp; the amino acid at position 751 is Thr, Ser, Gly mobot.org/MOBOT/research/APweb? (which can be or Ala; the amino acid at position 753 is Gln, Asn. Lys or accessed using the “www’ prefix) and Schuettpelz. E. and Arg; the amino acid at position 754 is Thr or Ser; the amino Pryer K. M., TAXON 56: 1037-1050 (2007) based on three acid at position 756 is Thr, Ser, Leu, Val or Ile; the amino plastid genes. Additional fern and other primitive plant acid at position 757 is Val, Leu or Ile; the amino acid at species can be found at homepages.caverock.net.nz/-b/fern/ position 766 is Ile, Leu or Val; the amino acid at position 771 list.htm (which can be accessed using the http://prefix). US 2016/0347799 A1 Dec. 1, 2016
0044. In some embodiments the nucleic acid molecule niaceae, Family Thelypteridaceae, Family Woodsiaceae, encoding the PtIP-83 polypeptide is derived from a fern Family Onocleaceae, Family Blechnaceae, Family species in the Class Psilotopsida. In some embodiments the Dryopteridaceae, Family Lomariopsidaceae, Family Tecta nucleic acid molecule encoding the PtIP-83 polypeptide is riaceae, Family Oleandraceae, Family Davalliaceae or Fam derived from a fern species in the Class Psilotopsida, Order ily Polypodiaceae. Psilotales. In some embodiments the nucleic acid molecule 0047. In some embodiments the nucleic acid molecule encoding PtIP-83 polypeptide is derived from a fern species encoding the PtIP-83 polypeptide is derived from a fern in the Class Psilotopsida, Order Ophioglossales. In some species in the Order Polypodiales, Family Pteridaceae, embodiments the nucleic acid molecule encoding the PtIP Genus Adiantaceae selected from but not limited to Adian 83 polypeptide is derived from a fern species in the Class tum aethiopicum, Adiantum aleuticum, Adiantum bonati Psilotopsida, Order Ophioglossales, Family Psilotaceae. In anum, Adiantum cajennense, Adiantum capillus-junionis, Some embodiments the nucleic acid molecule encoding the Adiantum capillus-veneris, Adiantum caudatum, Adiantum PtIP-83 polypeptide is derived from a fern species in the chienii, Adiantum chilense, Adiantum cuneatum, Adiantum Class Psilotopsida, Order Ophioglossales Family Ophio cunninghamii, Adiantum davidii, Adiantum diaphanum, Adi glossaceae. In some embodiments the nucleic acid molecule antum edentulum, Adiantum edgeworthii, Adiantum encoding the PtIP-83 polypeptide is derived from a fern excisum, Adiantum fengianum, Adiantum fimbriatum, Adi species in the Genus Ophioglossum L. Botrychium, Botry antum flabellulatum, Adiantum formosanum, Adiantum for pus, Helminthostachys, Ophioderma, Cheiroglossa, Scep mosum, Adiantum fulvum, Adiantum gravesii, Adiantum tridium or Mankyua. hispidulum, Adiantum induratum, Adiantum jordani, Adi 0045. In some embodiments the nucleic acid molecule antum juxtapositum, Adiantum latifolium, Adiantum leveil encoding the PtIP-83 polypeptide is derived from a species lei, Adiantum lianxianense, Adiantum malesianum, Adian in the Class Polypodiopsida/Pteridopsida. In some embodi tum mariesii, Adiantum monochlamys, Adiantum ments the nucleic acid molecule encoding the PtIP-83 poly myriosorum, Adiantum obliquum, Adiantum Ogasawarense, peptide is derived from a fern species in the Order Osmun Adiantum pedatum, Adiantum pentadactylon, Adiantum dales (royal ferns); Family Osmundaceae. In some peruvianum, Adiantum philippense, Adiantum princeps, embodiments the nucleic acid molecule encoding the PtIP Adiantum pubescens, Adiantum raddianum, Adiantum reni 83 polypeptide is derived from a fern species in the Order forme, Adiantum roborowski, Adiantum serratodentatum, Hymenophyllales; Family Hymenophyllaceae. In some Adiantum sinicum, Adiantum Soboliferum, Adiantum sub embodiments the nucleic acid molecule encoding the Pt|P- cordatum, Adiantum tenerum, Adiantum terminatum, Adi 83 polypeptide is derived from a fern species in the Order antum tetraphyllum, Adiantum trapeziforme, Adiantum Gleicheniales; Family Gleicheniaceae, Family Dipteri venustum, Adiantum viridescens, and Adiantum viridinon daceae or Family Matoniaceae. In some embodiments the tantiin. nucleic acid molecule encoding the PtIP-83 polypeptide is 0048. In some embodiments the nucleic acid molecule derived from a fern species in the Order Schizaeales; Family encoding the PtIP-83 polypeptide is derived from a fern Lygodiaceae, Family Anemiaceae or Family Schizaeaceae. species in the Order Polypodiales, Family Aspleniaceae, In some embodiments the nucleic acid encoding the PtIP-83 Genus Asplenium. polypeptide is derived from a fern species in the Order 0049. In some embodiments the nucleic acid molecule Schizaeales; Family Schizaeaceae, Genus Lygodium encoding the PtIP-83 polypeptide is derived from a fern selected from but not limited to Lygodium articulatum, species in the Order Polypodiales, Family Aspleniaceae, Lygodium circinatum, Lygodium conforme, Lygodium Genus Aspleniuml selected from but not limited to Asple cubense, Lygodium digitatum, Lygodium flexuosum, Lygo nium adiantum, Asplenium adulterinum, Asplenium dium heterodoxum, Lygodium japonicum, Lygodium ker aequibasis, Asplenium aethiopicum, Asplenium africanum, stenii, Lygodium lanceolatum, Lygodium longifolium, Lygo Aspleniumxalternifolium, Asplenium angustum, Asplenium dium merrilii, Lygodium micans, Lygodium microphyllum, antiquum, Asplenium ascensionis, Asplenium attenuatum, Lygodium microstachyum, Lygodium Oligostachyum, Lygo Asplenium aureum, Asplenium auritum, Asplenium austral dium palmatum, Lygodium polystachyum, Lygodium radia asicum, Asplenium azoricum, Asplenium bifrons, Asplenium tum, Lygodium reticulatum, Lygodium salicifolium, Lygo billottii, Asplenium bipinnatifidum, Asplenium brachycar dium scandens, Lygodium Smithianum, Lygodium pum, Asplenium bradleyi, Asplenium bulbiferum, Asplenium subareolatum, Lygodium trifiurcatum, Lygodium venustum, caudatum, Asplenium ceterach, Asplenium compressum, Lygodium versteeghi, Lygodium volubile, and Lygodium Asplenium congestum, Asplenium corderoanum, Asplenium yunnanense. In some embodiments the nucleic acid mol crinicaule, Asplenium cristatum, Asplenium cuneifolium, ecule encoding the PtIP-83 polypeptide is derived from a Asplenium cymbifolium, Asplenium daghestanicum, Asple fern species in the Order Salviniales; Family Marsileaceae nium dalhousiae, Asplenium dareoides, Asplenium daucifo or Family Salviniaceae. In some embodiments the nucleic lium, Asplenium diforme, Asplenium fissum, Asplenium acid molecule encoding the PtIP-83 polypeptide is derived dimorphum, Asplenium divaricatum, Asplenium dregeanum, from a fern species in the Order Cyatheales; Family Thyr Aspleniumxebenoides, Asplenium ecuadorense, Asplenium Sopteridaceae, Family LOXSomataceae, Family Culcitaceae, feei Kunze, Asplenium fissum, Asplenium flabellifolium, Asp Family Plagiogyriaceae, Family Cibotiaceae, Family Cya lenium flaccidum, Asplenium fontanum, Asplenium theaceae, Family Dicksoniaceae or Family Metaxyaceae. forisiense, Asplenium formosum, Asplenium gemmiferum, 0046. In some embodiments the nucleic acid molecule Aspleniumxgermanicum, Asplenium gueinzi, Asplenium encoding the PtIP-83 polypeptide is derived from a fern goudeyi, Asplenium hemionitis, Asplenium hermannii-chris species in the Order Polypodiales; Family Lindsaeaceae, tii, Asplenium hookerianum, Asplenium hybridum, Asple Family Saccolomataceae, Family Cystodiaceae, Family nium incisum, Aspleniumxjacksonii, Aspleniumxkenzoi, Dennstaedtiaceae, Family Pteridaceae, Family Asple Asplenium laciniatum, Asplenium lamprophyllum, Asple US 2016/0347799 A1 Dec. 1, 2016 20 nium laserpitifolium, Asplenium lepidum, Asplenium listeri, chum castaneum, Polystichum chilense, Polystichum christii Asplenium longissimum, Asplenium lucidum, Asplenium Ching, Polystichum chuni Ching, Polystichum craspedo so lunulatum, Asplenium lyallii, Asplenium macedonicum, Asp rum, Polystichum cyclolobum, Polystichum cystostegia, lenium majoricum, Asplenium marinum, Aspleniumxmicro Polystichum deltodon, Polystichum dielsii, Polystichum dis don, Asplenium milnei, Asplenium montanum, Asplenium Cretum, Polystichum drepanum, Polystichum dudleyi, Poly musifolium, Asplenium nidus, Asplenium normale, Asple Stichum duthiei, Polystichum echinatum, Polystichum ero nium obliquum, Asplenium oblongifolium, Asplenium obo sum, Polystichum excellens, Polystichum eximium, vatum, Asplenium obtusatum, Asplenium Oligolepidum, Asp Polystichum falcatipinnum, Polystichum falcinellum, Poly lenium Oligophlebium, Asplenium Onopteris, Asplenium Stichum fallax, Polystichum formosanum, Polystichum pacificum, Asplenium paleaceum, Asplenium palmeri, Asp gongboense, Polystichum grandifrons, Polystichum gymno lenium petrarchae, Asplenium pinnatifidum, Asplenium carpium, Polystichum halleakalense, Polystichum hancockii, planicaule, Asplenium platybasis, Asplenium platyneuron, Polystichum hecatopteron, Polystichum herbaceum, Poly Asplenium polyodon, Asplenium praemorsum, Asplenium Stichum imbricans, Polystichum incongruum, Polystichum prolongatum, Asplenium pteridoides, Asplenium resiliens, Kruckebergii, Polystichum kwakiutlii, Polystichum Asplenium rhizophyllum, Asplenium richardii, Asplenium lachenense, Polystichum lanceolatum, Polystichum lem ruprechtii, Asplenium ruta-muraria, Asplenium rustifolium, monii, Polystichum lentum, Polystichum lonchitis, Polysti Asplenium sagittatum, Asplenium sandersonii, Aspleniumx chum longidens, Polystichum longipaleatum, Polystichum Sarniense, Asplenium schizotrichum, Asplenium Schwein longipes, Polystichum luctuosum, Polystichum macleae, firthii, Asplenium scleroprium, Asplenium scolopendrium Polystichum macrochlaenum, Polystichum makinoi, Polysti (syn. Phyllitis scolopendrium), Asplenium Seelosii, Asple chum martini, Polystichum mavebarae, Polystichum medio nium Septentrionale, Asplenium Septentrionalextricho cre, Polystichum medogense, Polystichum microchlamys, manes, Asplenium serra, Asplenium serratum, Asplenium Polystichum mohrioides, Polystichum mollissimum, Polysti sessilifolium, Asplenium shuttleworthianum, Asplenium sim chum monticola, Polystichum moorei, Polystichum morii, plici?ions, Asplenium splendens, Asplenium surrogatum, Polystichum moupinense, Polystichum muricatum, Polysti Asplenium tenerum, Asplenium terrestre, Asplenium the chum nakenense, Polystichum neolobatum, Polystichum ciferum, Asplenium thunbergii, Asplenium trichomanes, nepalense, Polystichum ningshenense, Polystichum Asplenium tutwilerae, Asplenium vespertinum, Asplenium obliquum, Polystichum Omeiense, Polystichum Ordinatum, vieillardii, Asplenium virens, Asplenium viride, Asplenium Polystichum Orientalitibeticum, Polystichum paramoupin vittiforme, and Asplenium viviparum. ense, Polystichum parvipinnulum, Polystichum piceopalea 0050. In some embodiments the nucleic acid molecule ceum, Polystichum poly blepharum, Polystichum prescot encoding the PtIP-83 polypeptide is derived from a fern tianum, Polystichum prionolepis, Polystichum proliferum, species in the Order Polypodiales, Family Blechnaceae, Polystichum pseudocastaneum, Polystichum pseudomaki Genus Blecnum. noi, Polystichum punctiferum, Polystichum pungens, Poly 0051. In some embodiments the nucleic acid molecule Stichum qandoense, Polystichum retrosopaleaceum, Poly encoding the PtIP-83 polypeptide is derived from a fern Stichum rhombiforme, Polystichum rhomboidea, species in the Order Polypodiales, Family Dryopteridaceae Polystichum richardii, Polystichum rigens, Polystichum Genus Acrophorus, Genus Acrorumohra, Genus Anapausia, rotundilobum, Polystichum Scopulinum, Polystichum semi Genus Arachniodes, Genus Bolbitis, Genus Ctenitis, Genus fertile, Polystichum setiferum, Polystichum setigerum, Poly Cyclodium, Genus Cyrtogonellum, Genus Cyrtomidictyum, Stichum Shensiense, Polystichum Silvaticum, Polystichum Genus Cyrtonium, Genus Diacalpe, Genus Didymochlaena, simplicipinnum, Polystichum sinense, Polystichum squarro Genus Dryopsis, Genus Dryopteris, Genus Elaphoglossum, sum, Polystichum Stenophyllum, Polystichum stimulans, Genus Hypodematium, Genus LaStreopsis, Genus Leptoru Polystichum submite, Polystichum tacticopterum, Polysti mohra, Genus Leucostegia, Genus Lithostegia, Genus chum thomsoni, Polystichum tibeticum, Polystichum trans Lomagramma, Genus Maxonia, Genus Megalastrum, Genus vaalense, Polystichum tripteron, Polystichum tsus-simense, Olfersia, Genus Peranema, Genus Phanerophlebia, Genus Polystichum vestitum, Polystichum wattii, Polystichum Phanerophlebiopsis, Genus Polybotrya, Genus Polystichop whiteleggei, Polystichum xiphophyllum, Polystichum sis, Genus Polystichum, Genus Rumohra, Genus Sorole yadongense, and Polystichum yunnanense. pidium, Genus Stigmatopteris or Genus Teratophyllum. 0053. In some embodiments the nucleic acid molecule 0.052. In some embodiments the nucleic acid molecule encoding the PtIP-83 polypeptide is derived from a fern encoding the PtIP-83 polypeptide is derived from a fern species in the Order Polypodiales, Family Dryopteridaceae, species in the Order Polypodiales, Family Dryopteridaceae, Genus Rumohra. In some embodiments the nucleic acid Genus Polystichum. In some embodiments the nucleic acid molecule encoding the PtIP-83 polypeptide is derived from molecule encoding the PtIP-83 polypeptide is derived from a fern species in the Order Polypodiales, Family Dryopteri a fern species in the Order Polypodiales, Family Dryopteri daceae, Genus Rumohra selected from but not limited to daceae, Genus Polystichum selected from but not limited to Rumohra adiantiformis, Rumohra aristata, Rumohra bar Polystichum acanthophyllum, Polystichum acrostichoides, tonae, Rumohra berteroana, Rumohra capuronii, Rumohra Polystichum aculeatum, Polystichum acutidens, Polysti glandulosa, Rumohra humbertii, Rumohra linearisquamosa, chum acutipinnulum, Polystichum alcicorne, Polystichum Rumohra lokohensis, Rumohra madagascarica, and aleuticum, Polystichum andersonii, Polystichum atkinsonii, Rumohra quadrangularis. Polystichum australiense, Polystichum bakerianum, Poly 0054. In some embodiments the nucleic acid molecule Stichum biaristatum, Polystichum bomiense, Polystichum encoding the PtIP-83 polypeptide is derived from a fern bonseyi, Polystichum brachypterum, Polystichum braunii, species in the Order Polypodiales, Family Polypodiaceae Polystichum brachypterum, Polystichum calderonense, Genus Campyloneurum, Genus Drynaria, Genus Lepisorus, Polystichum californicum, Polystichum capillipes, Polysti Genus Microgramma, Genus Microsorum, Genus Neuro US 2016/0347799 A1 Dec. 1, 2016
dium, Genus Niphidium, Genus Pecluma M.G., Genus Phle var. curtii, Polypodium allosuroides, Polypodium alsophili bodium, Genus Phymatosorus, Genus Platycerium, Genus cola, Polypodium amamianum, Polypodium amoenum, Pleopeltis, Genus Polypodium. Polypodium amorphum, Polypodium anetioides, Polypo 0055. In some embodiments the PtIP-83 polypeptide is dium anfractuosum, Polypodium anguinum, Polypodium derived from a fern species in the Order Polypodiales, angustifolium f. remotifolia, Polypodium angustifolium var. Family Polypodiaceae, Genus Microsorum. amphostenon, Polypodium angustifolium var. heterolepis, 0056. In some embodiments the nucleic acid molecule Polypodium angustifolium var. monstrosa, Polypodium encoding the PtIP-83 polypeptide is derived from a fern angustipaleatum, Polypodium angustissimum, Polypodium species in the Order Polypodiales, Family Polypodiaceae, anisomeron var. pectinatum, Polypodium antioquianum, Genus Microsorum. selected from but not limited to Polypodium aoristisorum, Polypodium apagolepis, Polypo Microsorum alatum, Microsorum angustifolium, Microso dium apicidens, Polypodium apiculatum, Polypodium rum aurantiacum, Microsorum australiense, Microsorum apoense, Polypodium appalachianum, Polypodium appres baithoense, Microsorum basicordatum, Microsorum bise sum, Polypodium arenarium, Polypodium argentinum, Poly riatum, Microsorum brassii, Microsorum buergerianum, podium argutum, Polypodium armatum, Polypodium aro Microsorum chapaense, Microsorum cinctum, Microsorum maticum, Polypodium aspersum, Polypodium assurgens, commutatum, Microsorum congregatifolium, Microsorum Polypodium atrum, Polypodium auriculatum, Polypodium cuneatum, Microsorum cuspidatum, Microsorum dengii, balaonense, Polypodium balliviani, Polypodium bamleri, Microsorum egregium, Microsorum emeiensis, Microsorum Polypodium bangii, Polypodium bartlettii, Polypodium ensatum, Microsorum ensiforme, Microsorum excelsum, basale, Polypodium bernoullii, Polypodium biauritum, Microsorum fortunei, Microsorum griseorhizoma, Microso Polypodium bifrons, Polypodium blepharodes, Polypodium rum grossum, Microsorum hemionitideum, Microsorum hen bolivari, Polypodium bolivianum, Polypodium bolobense, rvi, Microsorum heterocarpum, Microsorum heterolobum, Polypodium bombycinum, Polypodium bombycinum var. Microsorum howense, Microsorum insigne, Microsorum insularum, Polypodium bradeorum, Polypodium bryophi intermedium, Microsorum kongtingense, Microsorum lum, Polypodium bryopodum, Polypodium buchtienii, Poly kravanense, Microsorum lanceolatum, Microsorum lancifo podium buesi, Polypodium bulbotrichum, Polypodium cac lium, Microsorum lastii, Microsorum latilobatum, Microso eresii, Polypodium californicum f. brauscombii, rum leandrianum, Microsorum lineare, Microsorum lingui Polypodium Californicum f. parsonsiae, Polypodium cali forme, Microsorum longissimum, Microsorum fornicum, Polypodium calophlebium, Polypodium calvum, longshengense, Microsorum maculosum, Microsorum maxi Polypodium camptophyllarium var. abbreviatum, Polypo mum, Microsorum membranaceum, Microsorum membrani dium capitellatum, Polypodium carpinterae, Polypodium folium, Microsorum microsorioides, Microsorum minor, chachapoyense, Polypodium chartaceum, Polypodium chi Microsorum monstrosum, Microsorum muliense, Microso mantense, Polypodium chiricanum, Polypodium choquetan rum mutense, Microsorum manchuanense, Microsorum gense, Polypodium christensenii, Polypodium christii, Poly ningpoense, Microsorum normale, Microsorum novae-zea podium chrysotrichum, Polypodium ciliolepis, Polypodium landiae, Microsorum ovalifolium, Microsorum ovatum, cinerascens, Polypodium collinsii, Polypodium colysoides, Microsorum palmatopedatum, Microsorum pappei, Polypodium confluens, Polypodium conforme, Polypodium Microsorum papuanum, Microsorum parksii, Microsorum confilsum, Polypodium congregatifolium, Polypodium con pentaphyllum, Microsorum piliferum, Microsorum pit nellii, Polypodium consimile var. bourgaeanum, Polypo cairnense, Microsorum powelli, Microsorum pteropodum, dium consimile var. minor; Polypodium conterminans, Poly Microsorum pteropus, Microsorum punctatum, Microsorum podium contiguum, Polypodium cookii, Polypodium pustulatum, Microsorun rampans, Microsorum revolutum, coriaceum, Polypodium coronans, Polypodium costari Microsorum rubidum, Microsorum Samarense, Microsorum cense, Polypodium costatum, Polypodium Crassifolium f. Sapaense, Microsorum Sarawakense, Microsorum Scandens, angustissimum, Polypodium crassifolium var. longipes, Microsorum scolopendria, Microsorum sibOmense, Polypodium crassulum, Polypodium craterisorum, Polypo Microsorum sinense, Microsorum Sopuense, Microsorum dium cryptum, Polypodium crystalloneuron, Polypodium spectrum, Microsorum steerei, Microsorum subhemioniti cucullatum var. planum, Polypodium cuencanum, Polypo deum, Microsorum submarginale, Microsorum subnudum, dium cumingianum, Polypodium cupreolepis, Polypodium Microsorum superficiale, Microsorum takhtajani, Microso curranii, Polypodium curvans, Polypodium cyathicola, rum tenuipes, Microsorum tibeticum, Microsorum triglos Polypodium cyathisorum, Polypodium cyclocolpon, Polypo sum, Microsorum truncatum, Microsorum tsai, Microsorum dium daguense, Polypodium damunense, Polypodium dare varians, Microsorum venosum, Microsorum vieillardii, iformioides, Polypodium dasypleura, Polypodium decipiens, Microsorumxinaequibasis, Microsorum viliangensis, and Polypodium decorum, Polypodium delicatulum, Polypo Microsorum zippelii. dium deltoideum, Polypodium demeraranum, Polypodium 0057. In some embodiments the nucleic acid molecule denticulatum, Polypodium diaphanum, Polypodium dilata encoding the PtIP-83 polypeptide is derived from a fern tum, Polypodium dispersum, Polypodium dissectum, Poly species in the Order Polypodiales, Family Polypodiaceae, podium dissimulans, Polypodium dolichosorum, Polypo Genus Polypodium L. In some embodiments the nucleic acid dium dolorense, Polypodium donnell-Smithii, Polypodium molecule encoding the PtIP-83 polypeptide is derived from drymoglossoides, Polypodium ebeninum, Polypodium egg a fern species in the Order Polypodiales, Family Polypodi ersii, Polypodium elmeri, Polypodium elongatum, Polypo aceae, Genus Polypodium L. selected from but not limited to dium enterosoroides, Polypodium erubescens, Polypodium Polypodium absidatum, Polypodium acutifolium, Polypo erythrolepis, Polypodium erythrotrichum, Polypodium eury dium adiantiforme, Polypodium aequale, Polypodium affine, basis, Polypodium eury basis var. villosum, Polypodium Polypodium albidopaleatum, Polypodium alcicorne, Poly exormans, Polypodium falcoideum, Polypodium fallacissi podium alfari, Polypodium alfredii, Polypodium alfredii mum, Polypodium farinosum, Polypodium faucium, Polypo US 2016/0347799 A1 Dec. 1, 2016 22 dium feei, Polypodium ferrugineum, Polypodium feuillei, Polypodium Oligosorum, Polypodium Oligosorum, Polypo Polypodium firmulum, Polypodium firmum, Polypodium dium Olivaceum, Polypodium Olivaceum var. elatum, Poly flaccidum, Polypodium flagellare, Polypodium flexuosum, podium Oodes, Polypodium oosphaerum, Polypodium Oreo Polypodium flexuosum var. ekmanii, Polypodium forbesii, philum, Polypodium ornatissimum, Polypodium ornatum, Polypodium formosanum, Polypodium fraxinifolium Subsp. Polypodium ovatum, Polypodium oxylobum, Polypodium articulatum, Polypodium fraxinifolium Subsp. luridum, Oxypholis, Polypodium pakkaense, Polypodium pallidum, Polypodium fructuosum, Polypodium fucoides, Polypodium Polypodium palmatopedatum, Polypodium palmeri, Poly filvescens, Polypodium galeottii, Polypodium glaucum, podium panamense, Polypodium parvum, Polypodium Polypodium glycyrrhiza, Polypodium gracillimum, Polypo patagonicum, Polypodium paucisorum, Polypodium pavo dium gramineum, Polypodium grandifolium, Polypodium nianum, Polypodium pectinatum var. Caliense, Polypodium gratum, Polypodium graveolens, Polypodium griseo-ni pectinatum var. hispidum, Polypodium pellucidum, Polypo grum, Polypodium griseum, Polypodium guttatum, Polypo dium pendulum var. boliviense, Polypodium percrassum, dium haalilioanum, Polypodium hammatisorum, Polypo Polypodium perpusillum, Polypodium peruvianum var. Sub dium hancockii, Polypodium haplophlebicum, Polypodium gibbosum, Polypodium phyllitidis var. elongatum, Polypo harrisii, Polypodium hastatum var. Simplex, Polypodium dium pichinchense, Polypodium pilosissimum, Polypodium hawaiiense, Polypodium heanophyllum, Polypodium helleri, pilosissimum var. glabriusculum, Polypodium pilossimum Polypodium hemionitidium, Polypodium henryi, Polypo var. tunguraquensis, Polypodium pity rolepis, Polypodium dium herzogii, Polypodium hesperium, Polypodium hessii, platyphyllum, Polypodium playfairii, Polypodium plebeium Polypodium hombersleyi, Polypodium hostmannii, Polypo var. cooperi, Polypodium plectolepidioides, Polypodium dium humile, Polypodium hyalinum, Polypodium iboense, pleolepis, Polypodium plesiosorum var. i. Polypodium podo Polypodium induens var. Subdentatum, Polypodium insidi basis, Polypodium podocarpum, Polypodium poloense, Osum, Polypodium insigne, Polypodium intermedium Subsp. Polypodium polydatylon, Polypodium polypodioides var. masafiteranum var. Obtuseserratum, Polypodium intramar aciculare, Polypodium polypodioides var. michauxianum, ginale, Polypodium involutum, Polypodium itatiavense, Polypodium praetermissium, Polypodium preslianum var. Polypodium javanicum, Polypodium juglandifolium, Poly immersum, Polypodium procerum, Polypodium procerum, podium kamiense, Polypodium knowltoniorum, Polypodium Polypodium productum, Polypodium productum, Polypo Kyimbilense, Polypodium lherminieri var. costaricense, dium prolongilobum, Polypodium propinguum, Polypodium Polypodium lachniferum f. incurvata, Polypodium lach proteus, Polypodium pruinatum, Polypodium pseudocapil niferum var. glabrescens, Polypodium lachnopus, Polypo lare, Polypodium pseudofraternum, Polypodium pseudo dium lanceolatum var. complanatum, Polypodium lanceo nutans, Polypodium pseudoserratum, Polypodium pulcher latum var. trichophorum, Polypodium latevagans, rimum, Polypodium pulogense, Polypodium pungens, Polypodium laxifrons, Polypodium laxifrons var. lividum, Polypodium purpusii, Polypodium radicale, Polypodium Polypodium lehmannianum, Polypodium leiorhizum, Poly randallii, Polypodium 25 ratiborii, Polypodium reclinatum, podium leptopodon, Polypodium leuconeuron var. angusti Polypodium recreense, Polypodium repens var. abruptum, folia, Polypodium leuconeuron var. latifolium, Polypodium Polypodium revolvens, Polypodium rhachipterygium, Poly leucosticta, Polypodium limulum, Polypodium lindigii, podium rhomboideum, Polypodium rigens, Polypodium Polypodium lineatum, Polypodium lomarioides, Polypo robustum, Polypodium roraimense, Polypodium roraimense, dium longifrons, Polypodium loretense, Polypodium loric Polypodium rosei, Polypodium rosenstockii, Polypodium eum var. umbraticum, Polypodium loriforme, Polypodium rubidum, Polypodium rudimentum, Polypodium rusbyi, loxogramme f. gigas, Polypodium ludens, Polypodium Polypodium sablanianum, Polypodium sarmentosum, Poly luzonicum, Polypodium lycopodioides f. Obtusum, Polypo podium saxicola, Polypodium schenckii, Polypodium dium lycopodioides L., Polypodium macrolepis, Polypodium Schlechteri, Polypodium scolopendria, Polypodium scolo macrophyllum, Polypodium macrosorum, Polypodium mac pendria, Polypodium scolopendrium, Polypodium scouleri, rosphaerum, Polypodium maculosum, Polypodium Polypodium scutulatum, Polypodium segregatum, Polypo madrense, Polypodium manmeiense, Polypodium margari dium semihirsutum, Polypodium semihirsutum var. fiascose tiferum, Polypodium maritimum, Polypodium martensii, tosum, Polypodium senile var. minor, Polypodium sericeo Polypodium mayoris, Polypodium megalolepis, Polypodium lanatum, Polypodium serraeforme, Polypodium serricula, melanotrichum, Polypodium menisciifolium var. pubescens, Polypodium sesquipedala, Polypodium sessilifolium, Poly Polypodium meniscioides, Polypodium merrillii, Polypo podium setosum var. Calvum, Polypodium setulosum, Poly dium mettenii, Polypodium mexiae, Polypodium microso podium shaferi, Polypodium sibOmense, Polypodium sic rum, Polypodium militare, Polypodium minimum, Polypo cum, Polypodium simacense, Polypodium simulans, dium minusculum, Polypodium mixtum, Polypodium Polypodium singeri, Polypodium sinicum, Polypodium sin mollendense, Polypodium mollissimum, Polypodium monili tenisii, Polypodium Skutchii, Polypodium Sloanei, Polypo forme var. minus, Polypodium monoides, Polypodium mon dium sodiroi, Polypodium Sordidulum, Polypodium Sordi ticola, Polypodium montigenium, Polypodium moritzianum, dum, Polypodium sphaeropteroides, Polypodium sphenodes, Polypodium moultonii, Polypodium multicaudatum, Poly Polypodium sprucei, Polypodium spruceivar. furcativenosa, podium multilineatum, Polypodium multisorum, Polypo Polypodium Steirolepis, Polypodium Stenobasis, Polypo dium munchi, Polypodium muscoides, Polypodium myriole dium Stenolepis, Polypodium Stenopterum, Polypodium sub pis, Polypodium myriophyllum, Polypodium myriotrichum, capillare, Polypodium subflabelliforme, Polypodium subhe Polypodium nematorhizon, Polypodium memorale, Polypo mionitidium, Polypodium subinaequale, Polypodium dium nesioticum, Polypodium nigrescentium, Polypodium subintegrum, Polypodium subspathulatum, Polypodium nigripes, Polypodium nigrocinctum, Polypodium nimbatum, subtile, Polypodium subvestitum, Polypodium subviride, Polypodium nitidissimum, Polypodium nitidissimum var. Polypodium superficiale var. attenuatum, Polypodium lation; Polypodium nubrigemum, Polypodium Oligolepis, superficiale var. Chinensis, Polypodium Sursuincurrens, US 2016/0347799 A1 Dec. 1, 2016
Polypodium tablazianum, Polypodium taenifolium, Polypo tachys, Lycopodiastrum, Lycopodiella, Lycopodium, Palhin dium tamandarei, Polypodium tatei, Polypodium tenuiculum haea, Pseudodiphasium, Pseudolycopodiella, var. acrosora, Polypodium tenuiculum var. brasiliense, Pseudolycopodium or Spinulum. In some embodiments the Polypodium tenuilore, Polypodium tenuinerve, Polypodium nucleic acid molecule encoding the PtIP-83 polypeptide is tepuiense, Polypodium teresae, Polypodium tetragonum var. derived from a species in the Genus Lycopodium. incompletum, Polypodium thysanolepis var. bipinnatifidum, Polypodium thyssanolepis, var. thyssanolepis, Polypodium 0063. In some embodiments the nucleic acid molecule thyssanolepsi, Polypodium tobagense, Polypodium encodes a PtIP-83 polypeptide comprises an amino acid trichophyllum, Polypodium tridactylum, Polypodium triden sequence MOTIF selected from: an amino acid sequence tatum, Polypodium trifurcatum var. brevipes, Polypodium MOTIF 1 as represented by an amino acid sequence of the triglossum, Polypodium truncatulum, Polypodium trunci formula MPIDEMPSEADWSIFVNEIVEAVAEGMPT colla var. major; Polypodium truncicola var. minor, Polypo EVSEVPLAVIWKAKCKNMVIAALGREMSCI (SEQ dium tuberosum, Polypodium tunguraguae, Polypodium ID NO: 646); an amino acid sequence MOTIF 2 as repre turquinum, Polypodium turrialbae, Polypodium ursipes, sented by an amino acid sequence of the formula Polypodium vagans, Polypodium valdealatum, Polypodium PQLQYRMYGNSLIKNIQMAQVAQNYDOEDIFKQ versteegii, Polypodium villagranii, Polypodium virginianum |FLIKLFIIAQNQILFIG.SYLLQQNKRIA F (SEQ ID f. Cambroideum, Polypodium virginianum f. peraferens, NO: 647); an amino acid sequence MOTIF 3 as represented Polypodium vittarioides, Polypodium vulgare, Polypodium by an amino acid sequence of the formula NTFMQMTPF vulgare L., Polypodium vulgare subsp. Oreophilum, Polypo TRWRLRLSASASENAEGLAFPTATAPLDSTTEQ dium vulgare var. acuminatum, Polypodium vulpinum, Poly IVIVITFHVTAIR (SEQ ID NO: 648); an amino acid podium williamsii, Polypodium wobbense, Polypodiumxfal sequence MOTIF 4 as represented by an amino acid lacissimum-guttatum, Polypodium xantholepis, Polypodium sequence of the formula DNFTSRHVVKGDIPVSLLL xiphopteris, Polypodium varumalense, Polypodium yun DGEDWEFEIPVQAGIGMSSFP (SEQ ID NO: 649); an gense, and Polypodium Zosteriforme. amino acid sequence MOTIF 5 as represented by an amino acid sequence of the formula IIHQPSAITIRQISTIGIT 0.058. In some embodiments the nucleic acid molecule VYILLQGSTIFHDRRRDEEVMTFQAADAPLNFY encoding the PtIP-83 polypeptide is derived from a fern QHIYAYRLDT. G. (SEQ ID NO: 650); an amino acid species in the Order Polypodiales, Family Polypodiaceae, sequence MOTIF 6 as represented by an amino acid Genus Platycerium. sequence of the formula SHOADRLAAIQPAVIDLTN 0059. In some embodiments the nucleic acid molecule HYLEMATHQMDMRTTRSMILILIGLLNMI encoding the PtIP-83 polypeptide is derived from a species LRIONAALMY EY (SEQ ID NO: 651); an amino acid in the Division Lycophyta. The phylogeny of extant Lyco sequence MOTIF 7 as represented by an amino acid pods as used herein is based on the classification by N. sequence of the formula VLDRVEFSEVMVIHRMYVRL Wikstrom, American Fern Journal, 91:150-156 (2001). SADLINDIVGELPEIGAEGRKTVKRVLIYVFL Other phylogenic classifications of extant Lycopods are ADWE (SEQID NO: 652); an amino acid sequence MOTIF known to one skilled in the art. Additional information on 8 as represented by an amino acid sequence of the formula the phylogeny of ferns can be found at mobot.org/MOBOT/ ADERELQMESFHSAVISQRRQELINDITALIFAKM research/APweb? (which can be accessed using the “www. DERLMISLQMEEENSRAMEQAQKE M (SEQ ID prefix) and Schuettpelz. E. and Pryer K. M., TAXON 56: NO: 653); an amino acid sequence MOTIF 9 as represented 1037-1050 (2007) based on three plastid genes. Additional by an amino acid sequence of the formula FVTAGATAPGA Lycopod species can be found at homepages.caverock.net. |AVIASAGQAVSIAGQAAQAGLRRWEILEGQ nz/-b/fern/list.htm (which can be accessed using the http:// LEAVMEWAAVIK (SEQ ID NO: 654); an amino acid prefix). sequence MOTIF 10 as represented by an amino acid 0060. In some embodiments the nucleic acid molecule sequence of the formula DGMNWGITIYIYHIGELKETV encoding the PtIP-83 polypeptide is derived from a species EQRSPLLPSNAILAVWADRCTIITSARHNHIVF in the Class Isoetopsida or Class Lycopodiopsida. NAPGRIVII (SEQ ID NO: 655); an amino acid sequence 0061. In some embodiments the nucleic acid molecule MOTIF 11 as represented by an amino acid sequence of the encoding the PtIP-83 polypeptide is derived from a species formula KVVKCARPPSPDMMVISAVAEHAL in the Class Isoetopsida, Order Selaginales. In some embodi WLNDVLLQWQKNESQLMIQGTAEPYNECLAL ments the nucleic acid molecule encoding the PtIP-83 poly LGR (SEQID NO: 656); an amino acid sequence MOTIF 12 peptide is derived from a fern species in the Class Isoetop as represented by an amino acid sequence of the formula sida, Order Selaginales, Family Selaginellaceae. In some PTELTIVAWPLGMDTVLAGNLLIAQENAALVL embodiments the nucleic acid molecule encoding the PtIP GLIQLGPSS (SEQ ID NO: 657); an amino acid sequence 83 polypeptide is derived from a species in the Genus MOTIF 13 as represented by an amino acid sequence of the Selaginella. formula RDQIMTHQIMPGSVTVILIVILCRLLQFPIT 0062. In some embodiments the nucleic acid molecule DGSQAITAT (SEQ ID NO: 658); an amino acid sequence encoding the PtIP-83 polypeptide is derived from a species MOTIF 14 as represented by an amino acid sequence of the in the Class Lycopodiopsida, Order Lycopodiales. In some formula TSIPVEVVTDPSNILLGMQTTVILHIAEL embodiments the nucleic acid molecule encoding the PtIP (SEQ ID NO: 659); an amino acid sequence MOTIF 15 as 83 polypeptide is derived from a fern species in the Class represented by an amino acid sequence of the formula Lycopodiopsida, Order Lycopodiales Family Lycopodiaceae EGLREQIFQNRQVARAVLFAVLKAVAMQILAG) or Family HuperZiaceae. In some embodiments the nucleic (SEQ ID NO: 660); an amino acid sequence MOTIF 16 as acid molecule encoding the PtIP-83 polypeptide is derived represented by an amino acid sequence of the formula from a species in the Genus Austrolycopodium, Dendroly WITSIRVRIRHLEMIQHIFLAVIQEASG (SEQ ID NO: copodium, Diphasiastrum, Diphasium, Huperzia, Lateris 661); an amino acid sequence MOTIF 17 as represented by US 2016/0347799 A1 Dec. 1, 2016 24 an amino acid sequence of the formula QISELQYED sequence as represented by the formula DGMNWGITIYI IWVOGLMMLRDIA (SEQ ID NO: 662); an amino acid YHIGEIKEIVEQRSPLLPSNAILAVWADRCITIIT sequence MOTIF 18 as represented by an amino acid SARHNHIVFINAPGRIVII (SEQ ID NO: 655); an amino sequence of the formula TFTLGSGVTGITSMHGEPSLD acid sequence MOTIF 11 having at least 90% sequence PWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acid identity to the amino acid sequence as represented by the sequence MOTIF 19 as represented by an amino acid formula KVVKCARPPSPDMMVISAVAEHAL sequence of the formula MDYSTLYRDLNOIS (SEQ ID WLNDVLLQWQKNESQLMIQGTAEPYNECLAL NO: 664); an amino acid sequence MOTIF 20 as represented LGR (SEQID NO: 656); an amino acid sequence MOTIF 12 by an amino acid sequence of the formula LRLPFMQK having at least 90% sequence identity to the amino acid LHARVIEQNVRKSE (SEQID NO: 665); an amino acid sequence as represented by the formula PTELTIVAWPL sequence MOTIF 21 as represented by an amino acid GMDTVLAGNLLIAOENAALIVLIGLIQLGPSS (SEQID sequence of the formula VDSLEQVGQHILIVIGDAP NO: 657); an amino acid sequence MOTIF 13 having at least (SEQ ID NO: 666); an amino acid sequence MOTIF 22 as 90% sequence identity to the amino acid sequence as represented by an amino acid sequence of the formula represented by the formula RDQIMTHQMPGSVTVI IVEQCAVMKIIMIGRFVGSLIW (SEQ ID NO: IVILCRLLQFPITIDGSQAITAT (SEQ ID NO: 658); an 667); an amino acid sequence MOTIF 23 as represented by amino acid sequence MOTIF 14 having at least 90% an amino acid sequence of the formula TLTNEPSEEQF sequence identity to the amino acid sequence as represented (SEQ ID NO: 668); and an amino acid sequence MOTIF 24 by the formula TSIPVEWTDPSNILLGMQTTVILH as represented by an amino acid sequence of the formula IAEL (SEQ ID NO: 659); an amino acid sequence MOTIF LPRQSRNISF (SEQ ID NO: 669). 15 having at least 90% sequence identity to the amino acid 0064. In some embodiments the nucleic acid molecule sequence as represented by the formula EGLREQFQN encodes a PtIP-83 polypeptide comprises an amino acid RQVARAVLFAVLKAVAMQILAG) (SEQID NO: 660); sequence MOTIF selected from: an amino acid sequence an amino acid sequence MOTIF 16 having at least 90% MOTIF 1 having at least 90% sequence identity to the amino sequence identity to the amino acid sequence as represented acid sequence as represented by the formula MPIDE by the formula WITSRVRIRHLEMIQHIFLAVIQEASG MPSEADWSIFVNEIVEAVAEGMPTEVSEVPAV (SEQ ID NO: 661); an amino acid sequence MOTIF 17 WKAKCKNMVIAALGREMSCI (SEQID NO: 646); an having at least 90% sequence identity to the amino acid amino acid sequence MOTIF 2 having at least 90% sequence as represented by the formula QISELQYED sequence identity to the amino acid sequence as represented IWVOGLMMLRDIA (SEQID NO: 662); an amino acid by the formula PQLQYRMYGINSLIKN sequence MOTIF 18 having at least 90% sequence identity QMAQVAQNYDQEDIFKQFLIKLFILIAQNQILF to the amino acid sequence as represented by the formula GSYLLQQNKRAF (SEQ ID NO: 647); an amino acid TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF sequence MOTIF3 having at least 90% sequence identity to (SEQ ID NO: 663); an amino acid sequence MOTIF 19 the amino acid sequence as represented by the formula having at least 90% sequence identity to the amino acid NTFMQMTPFTRWRLRLSASASENAEGLAFPTATA sequence as represented by the formula MDYSTLYRDLN PLDSTTEQIVIVITFHVTAIR (SEQ ID NO: 648); an QIS (SEQID NO: 664); an amino acid sequence MOTIF 20 amino acid sequence MOTIF 4 having at least 90% having at least 90% sequence identity to the amino acid sequence identity to the amino acid sequence as represented sequence as represented by the formula LRLPFMQK LHARVIEQNVRKSE (SEQID NO: 665); an amino acid by the formula DNFTSRHVVKGDIPVSLLLDGED sequence MOTIF 21 having at least 90% sequence identity WEFEIPVQAGIGMSSFP (SEQ ID NO: 649); an amino to the amino acid sequence as represented by the formula acid sequence MOTIF 5 having at least 90% sequence VDSLEQVGQHILIVIGDAP (SEQ ID NO: 666); an identity to the amino acid sequence as represented by the amino acid sequence MOTIF 22 having at least 90% formula IIHQPSAITIRQISTIGITIVYILLQGSTIFH sequence identity to the amino acid sequence as represented DRRRDEEVMTFQAADAPLNFYIQHIYAYRLDT G (SEQ ID NO: 650); an amino acid sequence MOTIF 6 by the formula IVIEQCAVMKIIMGRFIVGSLIVV having at least 90% sequence identity to the amino acid (SEQ ID NO: 667): an amino acid sequence MOTIF 23 sequence as represented by the formula SHOADRLAAI having at least 90% sequence identity to the amino acid QPAVIDLTNIHYLEMATHQMDMRTTRSMILIL sequence as represented by the formula TLTNEPSEEQF GLLNMILRIQNAALMY EY (SEQ ID NO: 651); an (SEQ ID NO: 668); and an amino acid sequence MOTIF 24 amino acid sequence MOTIF 7 having at least 90% having at least 90% sequence identity to the amino acid sequence identity to the amino acid sequence as represented sequence as represented by the formula LPROSRNISF by the formula VLDRVEFSEVMVIHRMYVRLISADL (SEQ ID NO: 669). NDTVGELIPEIGAEGIRKIVKRVLIYVFLADWE 0065. In some embodiments the nucleic acid molecule (SEQ ID NO: 652); an amino acid sequence MOTIF 8 encodes a PtIP-83 polypeptide comprises an amino acid having at least 90% sequence identity to the amino acid sequence MOTIF selected from: an amino acid sequence sequence as represented by the formula AIDERELQMES MOTIF 1 as represented by an amino acid sequence of the FHSAVISQRRQELNDITAIFAKMIDERLM formula MPIDEMPSTEDADWSIFVNEIVLEAVAE SLQMEEENSRAMEQAQKEM (SEQ ID NO: 653); an GMPTEVSEVPAVIWKRAKCKNMVIAALGRE amino acid sequence MOTIF 9 having at least 90% MISC1 (SEQID NO: 670); an amino acid sequence MOTIF sequence identity to the amino acid sequence as represented 2 as represented by an amino acid sequence of the formula by the formula FVTAGATAPGAAVIASAGQAV PQLQYRMYGNSLIKRNIQMAQVAQNYDIQRED SIAGQAAQAGLRRWEILEGQLEAVMEWAAVIK FKQRFLIKRLFILIAVLQNQILFIG.SYL LQEQN (SEQ ID NO: 654); an amino acid sequence MOTIF 10 KRAF (SEQ ID NO: 671); an amino acid sequence having at least 90% sequence identity to the amino acid MOTIF 3 as represented by an amino acid sequence of the US 2016/0347799 A1 Dec. 1, 2016
formula NITKFMQMTPFTRHIWRLRLSASASPKAJE QERNFVRKWSIISE (SEQ ID NO: 688): an amino NAKEGLAFPTATAPLDSTTEQIVIVAITF acid sequence MOTIF 21 as represented by an amino acid HVTAIR (SEQ ID NO: 672); an amino acid sequence sequence of the formula VIDNISALEDIQVGSIQH MOTIF 4 as represented by an amino acid sequence of the ILIVIGDAP (SEQ ID NO: 689); an amino acid sequence formula DNFTSRHVVKGDIPVSNLLLDGEG MOTIF 22 as represented by an amino acid sequence of the DWEFEIPVQAGIGMSSFP (SEQID NO: 673); an amino formula IVEOHCASIVAMIKIMIGVIRPFI) acid sequence MOTIF 5 as represented by an amino acid VGSLIW (SEQ ID NO: 690); an amino acid sequence sequence of the formula IIHQPSAITIRQISTIGITIVII MOTIF23 as represented by an amino acid sequence of the YILLOGSTIVFHDRRRDEEQIVIMLITIFPIQAA formula TLTNEQIPSEEQDHF (SEQ ID NO: 691); and DAVIPLNFYIQHIYAYRLDTG (SEQ ID NO: 674); an an amino acid sequence MOTIF 24 as represented by an amino acid sequence MOTIF 6 as represented by an amino amino acid sequence of the formula LPRSIQSRTINIV acid sequence of the formula SHOADRLAAIQPAV SF (SEQ ID NO: 692). DNLTNIHYFILEMATHQMDMRTTRSIMILIL) 0066. In some embodiments the nucleic acid molecule GLLNMILMRIQNAALMRIYEY (SEQ ID NO: 675); encodes a PtIP-83 polypeptide comprises an amino acid an amino acid sequence MOTIF 7 as represented by an sequence MOTIF selected from: an amino acid sequence amino acid sequence of the formula VLDRQIVEF MOTIF 1 having at least 90% sequence identity to the amino SEVMVIHRMYVINRLISADLINDIVIGAEQLIPE acid sequence as represented by the formula MPIDEMP GAEGIRKIVKRVLIYVFLADVVE (SEQ ID NO: STEDADWSIFVNEIVLEAVAEGMPTEVSEVPAV 676); an amino acid sequence MOTIF 8 as represented by an WKRAKCKNMVIAALGRE MISCII (SEQ ID NO: amino acid sequence of the formula AIDERELQMESFH 670); an amino acid sequence MOTIF2 having at least 90% SAAVISQRKRQGEELNDTDATIFAKMIDE sequence identity to the amino acid sequence as represented RILMISLQMEEENS DIRGAMEQAQRIKEM (SEQID by the formula PQLQYRMYGINSLIKRN NO: 677); an amino acid sequence MOTIF 9 as represented QMAQVAQNYDIQRIEDIFKQRFLKRLFILAVL) by an amino acid sequence of the formula FVLITAGAT QNQILFIG.SYL LQEQNKRAF (SEQ ID NO: 671); an APGAAVIASAGQAVSNIAGQAAQAGLRRVVEILE amino acid sequence MOTIF 3 having at least 90% GQLEAVMEVVAAVIIK (SEQ ID NO: 678); an amino sequence identity to the amino acid sequence as represented acid sequence MOTIF 10 as represented by an amino acid by the formula NITKFMQMTPFTRHIWRLRLSASASP sequence of the formula DIGDIMAINKIWGITIYIV KAENAKEGLAFPTATAPLDSTTEQIVIVAITF YHIGAEKEVTEQIRVLISPLLYFIPNISNGINW HVTAIR (SEQ ID NO: 672); an amino acid sequence ASPIYLAG VIVIWEADQRICSTIITISAA MOTIF4 having at least 90% sequence identity to the amino RFMHNIHVTIVFINDIAERPG|RWILIVIIIR (SEQ acid sequence as represented by the formula DNFTSRHV ID NO: 679); an amino acid sequence MOTIF 11 as repre VKGDIPVSNLLLDGEGDWEFEIPVQAG) sented by an amino acid sequence of the formula KVVK GMSSFP (SEQ ID NO: 673); an amino acid sequence |CARGCPHYPSPIDEMILIMVISAVAGEVHA MOTIF5 having at least 90% sequence identity to the amino LI NWLNSDKVLLIQRIWOK NESQHILLMIQGT acid sequence as represented by the formula IIHQPISAT AEPSAYNECLALLGR (SEQ ID NO: 680); an amino RQISTIGITIVIIYILLQGSTIVFHDRRRDEEQIV acid sequence MOTIF 12 as represented by an amino acid MLITIFPIQAADAVIPLNFYIQHIYAYRLDTG (SEQ sequence of the formula (PNITEQLTIVATIWPLGRM ID NO: 674); an amino acid sequence MOTIF 6 having at DTVAGINDILLIATIQHEINSAALIVLSIGLITMA least 90% sequence identity to the amino acid sequence as QLGPQIS PS (SEQ ID NO: 681); an amino acid represented by the formula SHOADRLAAIQPLAVIDN sequence MOTIF 13 as represented by an amino acid LTNIHYFILEMATHQMDMRTTIRSMILILIGLLN sequence of the formula RLCIDLWKQNPRIMTP MILMRIQN AALMRYEY (SEQ ID NO: 675); an HQRIMILPGSVTVILIVILCRLLQFPITIDGIGSR) amino acid sequence MOTIF 7 having at least 90% QFRASTADITW (SEQ ID NO: 682); an amino acid sequence identity to the amino acid sequence as represented sequence MOTIF 14 as represented by an amino acid by the formula VLDRQIVEFSEVMVIHRMYVINRL sequence of the formula (TAISGVIILPVIEDIWTDPSN) SADLINDIVIGAEQLLPEIGAEGIRKIVKRIVLIYV ILLMIGMOTITSVILHIAEL (SEQ ID NO: 683); an (FLADVVE (SEQ ID NO: 676); an amino acid sequence amino acid sequence MOTIF 15 as represented by an amino MOTIF8 having at least 90% sequence identity to the amino acid sequence of the formula EGLREQIFQNREQVA acid sequence as represented by the formula ADER RNAIVLFAVLKSASIVAMQILAG) (SEQ ID NO: ELQMESFHSAAVISQRKRQGEELNDITDAT 684); an amino acid sequence MOTIF 16 as represented by IFAKMIDERLMISLQMEEENS DIRGAMEQAQR an amino acid sequence of the formula WITSRVRIRHLEM KEM (SEQ ID NO: 677); an amino acid sequence MOTIF QHIFLAVIQKEASISMIGN (SEQ ID NO: 685); an 9 having at least 90% sequence identity to the amino acid amino acid sequence MOTIF 17 as represented by an amino sequence as represented by the formula FIVLITAGATAP acid sequence of the formula QIMSEQLQYED GAAVIASAGQAVISNIAGQAAQAGLRRVVEILE IWVOGLMMLRDIMA (SEQ ID NO: 686); an amino GQLEAVMEVVAAVIIK (SEQ ID NO: 678); an amino acid sequence MOTIF 18 as represented by an amino acid acid sequence MOTIF 10 having at least 90% sequence sequence of the formula TFTLGSGVTGITSMHGEPSLD identity to the amino acid sequence as represented by the PWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acid formula DIGDIMANKWGITYIVYHIGAEKEIV sequence MOTIF 19 as represented by an amino acid EQIRVLISPLLYFIPNSNGINWASPIIIYLAG sequence of the formula MLVDYISKITSKILIYFIRE VIVIWEADQRICSTIITISAARFMHNIHVTIVF DLNOIS (SEQ ID NO: 687); an amino acid sequence NDIAERPG|RWIVIIR (SEQ ID NO: 679); an amino MOTIF 20 as represented by an amino acid sequence of the acid sequence MOTIF 11 having at least 90% sequence formula LRHQLPTFMQKLHARITIVQLIRE identity to the amino acid sequence as represented by the US 2016/0347799 A1 Dec. 1, 2016 26 formula KVVKCARGCPHYPSPIDEMILIMV acid sequence of the formula NITKSRFMQMTPFTRHK SAVAGEVHALI NIWLNSDKVLLIQRIWOK WRLRLSASASPKATRENAKREGLAFPTATA NESQHILMIQGTAEIPSAYNECLALLGR (SEQ ID PLIVIDSTTEQ NDIIIVLIVAILITFHVTAIR (SEQ ID NO: 680); an amino acid sequence MOTIF 12 having at least NO: 695); an amino acid sequence MOTIF 4 as represented 90% sequence identity to the amino acid sequence as by an amino acid sequence of the formula DNQEFTSRH represented by the formula (PNITEQLTIVATIWPLGR) WKGDEIPVISNTQLLLDGEGDIDWEFEIPVQAG) MDTVAGINDILLIATIQHIENSIAALIVLSIGL GMSSFP (SEQ ID NO: 696); an amino acid sequence ITMAQLGPQIS PIS (SEQ ID NO: 681); an amino acid MOTIF 5 as represented by an amino acid sequence of the sequence MOTIF 13 having at least 90% sequence identity formula IIHQPSATITIRQKNISTIGITLVSIVIL) to the amino acid sequence as represented by the formula YILLQGSTIVLFHDRRRDEEQDNIVIMLIVITIFP RLCIDLWKIQNPRIMTPHQRIMILPGSVTVIIIV LCRLLQFPITIDGIGSRQFRASTADITW (SEQ QA AIDAVEILIPLNIFYIQHNIYAYRLDTG (SEQ ID ID NO: 682); an amino acid sequence MOTIF 14 having at NO: 697); an amino acid sequence MOTIF 6 as represented least 90% sequence identity to the amino acid sequence as by an amino acid sequence of the formula SHONADR represented by the formula TAISGVIILPVLEDVVTDP LAAIQPAVILIDNILTNIHYFILEMATHQNMDMRTT SNILLMIGMOTITSVILHIAEL (SEQID NO: 683); an RSKTIMILVILLILVIGLLNLM ILVIILMIVIRIQNAAL amino acid sequence MOTIF 15 having at least 90% MRILVKYEY (SEQ ID NO: 698); an amino acid sequence identity to the amino acid sequence as represented sequence MOTIF 7 as represented by an amino acid by the formula EGLREQIFQNREQVARNAVLFAVL sequence of the formula VLIDRQKNIVEFSEVM KSASIVAMQIAGI(SEQ ID NO: 684); an amino acid VIHRMYVINRLISATDLNDQEIVIGAEQNDL sequence MOTIF 16 having at least 90% sequence identity PEDIGAEGDIRKIVKRIVLIYVFLIVADWE (SEQ to the amino acid sequence as represented by the formula ID NO: 699); an amino acid sequence MOTIF 8 as repre WITSRVRIRHLEMIQHIFLAVIQKEASISMIGN sented by an amino acid sequence of the formula ADE (SEQ ID NO: 685); an amino acid sequence MOTIF 17 RELQMESFHSAT|AVISQRKRQGENDELNDQE) having at least 90% sequence identity to the amino acid TDSEATSIFLVAKMDERLMI VISLQMEEE sequence as represented by the formula QIMSEQLQY NSDQETRGKAMEQAQRNKKEM (SEQ ID NO: EDIWVOGLMMLRDIMA (SEQ ID NO: 686); an 700); an amino acid sequence MOTIF 9 as represented by an amino acid sequence MOTIF 18 having at least 90% amino acid sequence of the formula FIVLITAGATAPGA sequence identity to the amino acid sequence as represented |AVILIASAGQAVISNTQIAGQAAQAGLRRVVEILE by the formula TFTLGSGVTGITSMHGEPSLDP GQNILEAVMEVVA AVILIK (SEQ ID NO: 701); an WNGVSLDSASPTAF (SEQ ID NO: 663); an amino acid amino acid sequence MOTIF 10 as represented by an amino sequence MOTIF 19 having at least 90% sequence identity acid sequence of the formula DIGDEMANKOKIWG to the amino acid sequence as represented by the formula ITLVSIYIVLYHIGAEKERDIVIEQNDIRVLKI MLVDYSKITSKILIYFIREDLNOIS (SEQ ID NO: SPLLYFIVPNQS NGTQNWQIASPTIYLVL 687); an amino acid sequence MOTIF 20 having at least AGVILIVIWEDADQNERICSTITISLVITISATA 90% sequence identity to the amino acid sequence as |RFMKHNIHVTILSVFILNDQEAERDKPG|RWK) represented by the formula LRHQLPTIFMIQKLHAR IVLIRLVK) (SEQID NO: 702); an amino acid sequence ITVQLIREQERNFVRKWSIISE (SEQ ID NO: MOTIF 11 as represented by an amino acid sequence of the 688); an amino acid sequence MOTIF 21 having at least formula KVRILIVKRILCARGCKPHYPSPIDE 90% sequence identity to the amino acid sequence as MILVI MVILISAVAGEVDILHALINVQWLNSQ TDKERVLLQRNKVVQKNRQESQHNLMIV represented by the formula VDNISALEDIQVGSIQH QGTAEDIPSATYNECLALLGR (SEQ ID NO: 703); an ILIVIGDAP (SEQ ID NO: 689); an amino acid sequence amino acid sequence MOTIF 12 as represented by an amino MOTIF 22 having at least 90% sequence identity to the acid sequence of the formula PNQITEQDNLTIVATILS amino acid sequence as represented by the formula IV WPLGRKMDTVAGINDQELLIATSQHNIE EQHICASIVAMIKIMIGVIRPFIVGSLIW NSQTAALIVLSITIGLITMALVSIQLGPQNSPTIS (SEQ ID NO: 690); an amino acid sequence MOTIF 23 (SEQ ID NO: 704); an amino acid sequence MOTIF 13 as having at least 90% sequence identity to the amino acid represented by an amino acid sequence of the formula sequence as represented by the formula TLTNEQIPSE RLCKIVIDLWKEIVRQNPRKMTPHQRIMILV EQDHF (SEQ ID NO: 691); and an amino acid sequence PGSVTVIIIVLLCRLLQFPITLVSIDGEIGSRTK) MOTIF 24 having at least 90% sequence identity to the QFRNKASTITADESITWS) (SEQ ID NO: 705); an amino acid sequence as represented by the formula LPRS amino acid sequence MOTIF 14 as represented by an amino QSIRTINIVISF (SEQ ID NO: 692). acid sequence of the formula TASGVTILILVIPVIED 0067. In some embodiments the nucleic acid molecule WTDPISNTQILLMIVIGMQTITSVILHIVIAEL (SEQ encodes a PtIP-83 polypeptide comprises an amino acid ID NO: 706); an amino acid sequence MOTIF 15 as repre sequence MOTIF selected from: an amino acid sequence sented by an amino acid sequence of the formula EGLR MOTIF 1 as represented by an amino acid sequence of the EQNDIFQNREKDIQVARNKQAVLIFAVLKSRT formula MPIDEMPSTEDADWSIFVNEIVLEAVAE ASTIVAMQNILAG) (SEQ ID NO: 707); an amino acid GMPTEVSEVPAVILIWKRAKCKNMVILIAAL sequence MOTIF 16 as represented by an amino acid GREMISCTI (SEQ ID NO: 693); an amino acid sequence sequence of the formula WITSRVRIRHLEMIQHN.F MOTIF 2 as represented by an amino acid sequence of the |AVILIQKNRELASTISMTIGNQ (SEQ ID NO: 708); formula PQLQYRMYGNSLIKRNQQMAQVAQNYD an amino acid sequence MOTIF 17 as represented by an QRNKEDIFKQRNKFLIKRLFILAVLQNQIL amino acid sequence of the formula QIMLVISEQND FIVIGSYLLIQENDIQNKRAF (SEQ ID NO: 694); an LQYEDIWVOGLMIVIMLIVIRDIMLVA (SEQ ID amino acid sequence MOTIF 3 as represented by an amino NO: 709); an amino acid sequence MOTIF 18 as represented US 2016/0347799 A1 Dec. 1, 2016 27 by an amino acid sequence of the formula TFTLGSGVT having at least 90% sequence identity to the amino acid GITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: sequence as represented by the formula FVLITAGATAP 663); an amino acid sequence MOTIF 19 as represented by GAAVILIASAGQAVISNTQIAGQAAQAGLRRV an amino acid sequence of the formula MLVIDYISKTR VEILEIGQNILEAVMEVVA AVILIK (SEQ ID NO: 701): ITSKRLYFIREKDDLNQIS (SEQ ID NO: 710); an an amino acid sequence MOTIF 10 having at least 90% amino acid sequence MOTIF 20 as represented by an amino sequence identity to the amino acid sequence as represented acid sequence of the formula LRHQKNLPTSFMQKN by the formula DIGDEMANKOKIWGITLVSIYIVL) RILHARITKLVSIVQLINIIRLVKJEQERNDKINFO YHIGAEKERDIVIEQNDIRVLKIISPLLYFIV VRILKKWSRTSETD (SEQ ID NO: 711); an amino PNQS NGTQNWQIASPTIIIYLVLAGVILIV acid sequence MOTIF 21 as represented by an amino acid WEDADQNERICSTITISLVIITISATARFMKHN sequence of the formula VDNQESATLEDIQVGST HV TILSVFILNDQEAERDKPG|RWKIVL) QHNILVIVIGDEAP (SEQ ID NO: 712); an amino acid IRLVK) (SEQ ID NO: 702); an amino acid sequence sequence MOTIF 22 as represented by an amino acid MOTIF 11 having at least 90% sequence identity to the sequence of the formula IVLEQHNDICASTIVAILIM amino acid sequence as represented by the formula KVRIL ILVKIMLVGVILRPKFILVVGILISLTIVVV VKRILICAIRGCKPHYPSPIDEMILVMVILISAV (SEQ ID NO: 713); an amino acid sequence MOTIF 23 as AGIEVDILHALLINVQWLNSQ TDKERVLL represented by an amino acid sequence of the formula QRNKVVQKNRQESQHNLMIVIQGTAED TLTNEQDNPSEEQDHNIF (SEQ ID NO: 714); and an PSATYNECLALLGR (SEQ ID NO: 703); an amino acid amino acid sequence MOTIF 24 as represented by an amino sequence MOTIF 12 having at least 90% sequence identity acid sequence of the formula LPRSKTIQSRTKSNIVL) to the amino acid sequence as represented by the formula SF (SEQ ID NO: 715). PNQITEQDNLTIVATILSWPLGRKIMDTVLAG) 0068. In some embodiments the nucleic acid molecule NDQELLIATSIOHNIENSQTAALIVLSITIGLIT encodes a PtIP-83 polypeptide comprises an amino acid MALVSIQLGPQNSPTIS (SEQ ID NO: 704); an amino sequence MOTIF selected from: an amino acid sequence acid sequence MOTIF 13 having at least 90% sequence MOTIF 1 having at least 90% sequence identity to the amino identity to the amino acid sequence as represented by the acid sequence as represented by the formula MPIDEMP formula RLCKIVIDLWKEIVRQNPRKMTPHQR STEDADWSIFVNEIVLEAVAEGMPTEVSEVP MILVIPGSVTVIIVLLCRLLQFPITLVSIDGEIG |AVILIWKRAKCKNMVILIAAL GREMSCTI (SEQ SRTKQFRNKASTITADESITWS) (SEQ ID NO: ID NO: 693); an amino acid sequence MOTIF 2 having at 705); an amino acid sequence MOTIF 14 having at least least 90% sequence identity to the amino acid sequence as 90% sequence identity to the amino acid sequence as represented by the formula PQLQYRMYGNSLIKRNQ represented by the formula TASGVTILILVIPVIED QMAQVAQNYDIQRNKEDIFKQRNKFLKRLFI WTDPISNTQILLMIVIGMQTITSVILHIVIAEL (SEQ (IAVLQNQIL FIVIGSYLLIQENDIQNKRAF (SEQ ID ID NO: 706); an amino acid sequence MOTIF 15 having at NO: 694); an amino acid sequence MOTIF 3 having at least least 90% sequence identity to the amino acid sequence as 90% sequence identity to the amino acid sequence as represented by the formula EGLREQNDFQNREKDIQ represented by the formula NITKSRFMQMTPFTRHK VARNKQAVLIFAVLKSRTASTIVAMQNILAG) WRLRLSASASPKATRENAKREGLAFPTATA (SEQ ID NO: 707); an amino acid sequence MOTIF 16 PLIVIDSTTEQ NDIVLIVAILITFHVTAIR (SEQ ID having at least 90% sequence identity to the amino acid NO: 695); an amino acid sequence MOTIF 4 having at least sequence as represented by the formula WITS 90% sequence identity to the amino acid sequence as RVRIRHLEMIQHNIFLAVILIOKNRELASTISMT represented by the formula DNQEFTSRHWKGDEIPV IGNQ (SEQ ID NO: 708); an amino acid sequence MOTIF ISNTQLLLDGIEGDIDWEFEIPVQAGIGMSSFP (SEQ 17 having at least 90% sequence identity to the amino acid ID NO: 696); an amino acid sequence MOTIF 5 having at sequence as represented by the formula QIMLVISEQND least 90% sequence identity to the amino acid sequence as LQYEDIWVOGLMIVIMLIVIRDIMLVA (SEQ ID represented by the formula IIHQPSATITIRQKNISTIG NO: 709); an amino acid sequence MOTIF 18 having at least ITLVSIVILIYILLQGSTIVLFHDRRRDEEQDNIV 90% sequence identity to the amino acid sequence as MLIVITIFPIQA ADAVEILPLNFYIQHNIYAY represented by the formula TFTLGSGVTGITSMH RLDTG (SEQ ID NO: 697); an amino acid sequence GEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an MOTIF6 having at least 90% sequence identity to the amino amino acid sequence MOTIF 19 having at least 90% acid sequence as represented by the formula SHONADR sequence identity to the amino acid sequence as represented LAAIQPAVILIDNILTNIHYFILEMATHQNMDMRTT by the formula MLVIDYSKTRITSKRLYFIREKD RSKTIMILVILILVIGLLNLM ILVILMIVIRIQNAAL DLNOIS (SEQ ID NO: 710); an amino acid sequence MRILVKYEY (SEQ ID NO: 698); an amino acid MOTIF 20 having at least 90% sequence identity to the sequence MOTIF 7 having at least 90% sequence identity to amino acid sequence as represented by the formula the amino acid sequence as represented by the formula LRHQKNILPTSFMQKNRLHARITKLVSIVQLIN VLIDIRQKNIVEFSEVMVIHRMYVINRLISATDL IRLVKEQERNDKINFOIVRILKKWSR TISETD NDQEVGAEQNDLIPEDIGAEGDRKTVKRIVLI (SEQ ID NO: 711); an amino acid sequence MOTIF 21 YVFLIVADWE (SEQ ID NO: 699); an amino acid having at least 90% sequence identity to the amino acid sequence MOTIF8 having at least 90% sequence identity to sequence as represented by the formula VDNQESATL the amino acid sequence as represented by the formula EDIQVGSTIQHNILVIVIGDEAP (SEQID NO: 712); AIDERELQMESFHSAT|AVISQRKRQGENDEL an amino acid sequence MOTIF 22 having at least 90% NDQEITDSEATSIFLVAKMDERLMI sequence identity to the amino acid sequence as represented VISLQMEEENSDQETIRGKAMEQAQRNKKEM by the formula IVLEQHNDICASTIVAILIMILVK (SEQ ID NO: 700); an amino acid sequence MOTIF 9 IMLVIGVILIRPKFILVVGILISLTIVIW (SEQ ID US 2016/0347799 A1 Dec. 1, 2016 28
NO: 713); an amino acid sequence MOTIF23 having at least 668, SEQ ID NO: 691 or SEQID NO: 714), and MOTIF 3 90% sequence identity to the amino acid sequence as (SEQ ID NO: 648, SEQ ID NO: 672 or SEQID NO: 695). represented by the formula TLTNEQDNPSEEQDHNIF (0071. In some embodiments the nucleic acid molecule (SEQ ID NO: 714); and an amino acid sequence MOTIF 24 encodes a Pt(P-83 polypeptide comprising, sequentially having at least 90% sequence identity to the amino acid from the N-terminus to the C-terminus, the amino acid sequence as represented by the formula LPRSKTIQSRTK sequence motifs: MOTIF 19 (SEQ ID NO: 664, SEQ ID SNIVLISF (SEQ ID NO: 715). NO: 687 or SEQID NO: 710), MOTIF 7 (SEQID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF 13 (SEQ ID 0069. In some embodiments the nucleic acid molecule NO: 658, SEQID NO: 682 or SEQID NO: 705), MOTIF 20 encodes a Pt(P-83 polypeptide comprising, sequentially (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO: 711), from the N-terminus to the C-terminus, an amino acid MOTIF 10 (SEQID NO: 655, SEQID NO: 679 or SEQ ID sequence MOTIF selected from: MOTIF 19 (SEQ ID NO: NO: 702), MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ 664, SEQID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQ ID NO: 669, SEQID NO: 692 or SEQID NO: 715), MOTIF ID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 683 or SEQ ID NO: 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQ ID NO: 706), MOTIF 11 (SEQ ID NO: 656, SEQ ID NO: 680 or 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO: 703), MOTIF 22 (SEQ ID NO: 667, SEQ ID SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID NO: 690 or SEQID NO: 713), MOTIF 2 (SEQID NO: 647, NO: 679 or SEQ ID NO: 702), MOTIF 18 (SEQ ID NO: SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 or NO: 653, SEQID NO: 677 or SEQID NO: 700), MOTIF 15 SEQ ID NO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ ID (SEQ ID NO: 660, SEQ ID NO: 684 or SEQID NO: 707), NO: 683 or SEQ ID NO: 706), MOTIF 11 (SEQ ID NO: MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID 656, SEQ ID NO: 680 or SEQ ID NO: 703), MOTIF 22 NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQID NO: 689 (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), or SEQID NO: 712), MOTIF 1 (SEQID NO: 646, SEQID MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID NO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ ID NO: NO: 694), MOTIF8 (SEQID NO: 653, SEQID NO: 677 or 662, SEQID NO: 686 or SEQID NO: 709), MOTIF 6 (SEQ SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID ID NO: 651, SEQID NO: 675 or SEQID NO: 698), MOTIF NO: 684 or SEQID NO: 707), MOTIF9 (SEQID NO: 654, 12 (SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: SEQID NO: 678 or SEQID NO: 701), MOTIF 21 (SEQ ID 704), MOTIF4 (SEQID NO: 649, SEQID NO: 673 or SEQ NO: 666, SEQID NO: 689 or SEQID NO: 712), MOTIF 1 ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: (SEQ ID NO: 646, SEQID NO: 670 or SEQ ID NO: 693), 685 or SEQID NO: 708), MOTIF5 (SEQID NO: 650, SEQ MOTIF 17 (SEQID NO: 662, SEQID NO: 686 or SEQ ID ID NO: 674 or SEQID NO: 697), MOTIF 23 (SEQ ID NO: NO: 709), MOTIF 6 (SEQID NO: 651, SEQID NO: 675 or 668, SEQ ID NO: 691 or SEQID NO: 714), and MOTIF 3 SEQ ID NO: 698), MOTIF 12 (SEQ ID NO: 657, SEQ ID (SEQ ID NO: 648, SEQ ID NO: 672 or SEQID NO: 695). NO: 681 or SEQID NO: 704), MOTIF4 (SEQID NO: 649, 0072. In some embodiments the nucleic acid molecule SEQID NO: 673 or SEQID NO: 696), MOTIF 16 (SEQ ID encodes a Pt(P-83 polypeptide comprising, sequentially NO: 661, SEQID NO: 685 or SEQID NO: 708), MOTIF5 from the N-terminus to the C-terminus, the amino acid (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO: 697), sequence motifs: MOTIF 19 (SEQ ID NO: 664, SEQ ID MOTIF 23 (SEQID NO: 668, SEQID NO: 691 or SEQ ID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQID NO: 652, NO: 714), and MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: SEQID NO: 676 or SEQID NO: 699), MOTIF 13 (SEQ ID 672 or SEQ ID NO: 695). NO: 658, SEQID NO: 682 or SEQID NO: 705), MOTIF 20 0070. In some embodiments the nucleic acid molecule (SEQ ID NO: 665, SEQ ID NO: 688 or SEQ ID NO: 711), encodes a Pt(P-83 polypeptide comprising, sequentially MOTIF 14 (SEQID NO: 659, SEQID NO: 683 or SEQ ID from the N-terminus to the C-terminus, an amino acid NO:706), MOTIF 2 (SEQID NO: 647, SEQID NO: 671 or sequence MOTIF selected from: MOTIF 19 (SEQ ID NO: SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ ID 664, SEQID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQ NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: ID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF 660, SEQID NO: 684 or SEQID NO: 707), MOTIF 9 (SEQ 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQ ID NO: ID NO: 654, SEQID NO: 678 or SEQID NO: 701), MOTIF 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: SEQ ID NO: 711), MOTIF 14 (SEQ ID NO: 659, SEQ ID 712), MOTIF 1 (SEQID NO: 646, SEQID NO: 670 or SEQ NO: 683 or SEQID NO: 706), MOTIF 2 (SEQID NO: 647, ID NO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID 686 or SEQID NO: 709), MOTIF 6 (SEQID NO: 651, SEQ NO: 653, SEQID NO: 677 or SEQID NO: 700), MOTIF 15 ID NO: 675 or SEQID NO: 698), MOTIF 12 (SEQ ID NO: (SEQ ID NO: 660, SEQ ID NO: 684 or SEQ ID NO: 707), 657, SEQID NO: 681 or SEQID NO: 704), MOTIF4 (SEQ MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID ID NO: 649, SEQID NO: 673 or SEQID NO: 696), MOTIF NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID NO: 689 16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO: or SEQID NO: 712), MOTIF 1 (SEQID NO: 646, SEQID 708), MOTIF5 (SEQID NO: 650, SEQID NO: 674 or SEQ NO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ ID NO: ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 662, SEQID NO: 686 or SEQID NO: 709), MOTIF 6 (SEQ 691 or SEQID NO: 714), and MOTIF 3 (SEQID NO: 648, ID NO: 651, SEQID NO: 675 or SEQID NO: 698), MOTIF SEQ ID NO: 672 or SEQ ID NO: 695). 12 (SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: 0073. In some embodiments the nucleic acid molecule 704), MOTIF4 (SEQ ID NO: 649, SEQID NO: 673 or SEQ encodes a PtIP-83 polypeptide comprising sequentially from ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: the N-terminus to the C-terminus: a Region A of between 685 or SEQID NO: 708), MOTIF5 (SEQID NO: 650, SEQ about 200 to about 300 amino acids in length comprising an ID NO: 674 or SEQID NO: 697), MOTIF 23 (SEQ ID NO: amino acid sequence MOTIF of MOTIF 19 (SEQ ID NO: US 2016/0347799 A1 Dec. 1, 2016 29
664, SEQID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQ predominantly nonconserved secondary structure; a Region ID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF B of between about 380 to about 465 amino acids in length 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQ ID NO: having a consensus secondary structure comprising nine 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or segments of predominately alpha helical structure; and a SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID Region C of between about 150 to about 180 amino acids in NO: 679 or SEQ ID NO: 702), MOTIF 18 (SEQ ID NO: length having a consensus secondary structure comprising 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 or seven segments of predominately beta Strand structure. SEQ ID NO: 715), and/or MOTIF 14 having a predomi 0077. In some embodiments the nucleic acid molecule nantly nonconserved secondary structure; a Region B of encodes a PtIP-83 polypeptide comprising sequentially from between about 380 to about 465 amino acids in length the N-terminus to the C-terminus: a Region A of between comprising an amino acid sequence MOTIF of MOTIF 22 about 200 to about 300 amino acids in length having a (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), predominantly nonconserved secondary structure, wherein MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID the Region. A comprises a conserved beta Strand 1 (B1a) of NO: 694), MOTIF8 (SEQID NO: 653, SEQID NO: 677 or between about 4 and about 12 amino acids in length within SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID about amino acid residue 30 to about amino acid residue 130 NO: 684 or SEQID NO: 707), MOTIF9 (SEQID NO: 654, from the N-terminus of the PtIP-83 polypeptide; a Region B SEQID NO: 678 or SEQID NO: 701), MOTIF 21 (SEQ ID of between about 380 to about 465 amino acids in length NO: 666, SEQID NO: 689 or SEQID NO: 712), MOTIF 1 having a consensus secondary structure comprising nine (SEQ ID NO: 646, SEQID NO: 670 or SEQ ID NO: 693), segments of predominately alpha helical structure; and a MOTIF 17 (SEQID NO: 662, SEQID NO: 686 or SEQ ID Region C of between about 150 to about 180 amino acids in NO: 709), MOTIF 6 (SEQID NO: 651, SEQID NO: 675 or length having a consensus secondary structure comprising SEQ ID NO: 698), and/or MOTIF 12 and having a pre seven segments of predominately beta Strand structure. dominately alpha helical structure; and a Region C of 0078. In some embodiments the nucleic acid molecule between about 150 to about 180 amino acids in length encodes a PtIP-83 polypeptide comprising sequentially from comprising an amino acid sequence MOTIF of MOTIF 16 the N-terminus to the C-terminus: a Region A of between (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO: 708), about 200 to about 300 amino acids in length having a MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID flexible consensus secondary structure, wherein the Region NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: 691 A comprises a conserved beta strand 1 (B1a) of between or SEQID NO: 714), and/or MOTIF 3 (SEQ ID NO: 648, about 4 and about 12 amino acids in length, a coil of between SEQ ID NO: 672 or SEQ ID NO: 695), having a consensus about 3 and about 18 amino acids in length and a beta Strand secondary structure comprising predominately beta Strand 2 (B1b) of between about 4 and about 32 amino acids in Structure. length, within about amino acid residue 50 to about amino 0074. In some embodiments the PtIP-83 polypeptide acid residue 165 from the N-terminus of the PtIP-83 poly comprises an amino acid sequence MOTIF at the positions peptide; a Region B of between about 380 to about 465 as shown in Table 2. amino acids in length having a consensus secondary struc 0075. In some embodiments the nucleic acid molecule ture comprising nine segments of predominately alpha heli encodes a PtIP-83 polypeptide comprising sequentially from cal structure; and a Region C of between about 150 to about the N-terminus to the C-terminus: a Region A of between 180 amino acids in length having a consensus secondary about 200 to about 300 amino acids in length having a structure comprising seven segments of predominately beta predominantly nonconserved secondary structure; a Region Strand structure. B of between about 380 to about 465 amino acids in length 0079. In some embodiments the nucleic acid molecule having a consensus secondary structure comprising 8 to 10 encodes a PtIP-83 polypeptide comprising sequentially from segments of predominately alpha helical structure; and a the N-terminus to the C-terminus: a Region A of between Region C of between about 150 to about 180 amino acids in about 200 to about 300 amino acids in length having a length having a consensus secondary structure comprising 6 predominantly nonconserved secondary structure; a Region to 8 segments of predominately beta Strand structure. As B of between about 380 to about 465 amino acids in length used herein "predominantly nonconserved secondary struc having a consensus secondary structure comprising sequen ture” means that the regions of secondary structure don't tially: i) an alpha helix-1 of between about 10 and about 26 consistently align within the family of PtIP polypeptides. As amino acids in length; ii) a coil-1 of between about 2 and used herein “predominately alpha helical structure” means about 8 amino acids in length flanked by alpha helix-1 and that secondary structure prediction may have one or more alpha helix-2; iii) an alpha helix-2 of between about 15 and gap of between 1 to 6 amino acids of coil and/or beta Strand about 24 amino acids in length; iv) a coil-2 of between about structure intervening in the alpha helix structure. As used 4 and about 14 amino acids in length flanked by alpha herein “predominately beta strand structure” means that helix-2 and alpha helix-3; v) an alpha helix 3 of between secondary structure prediction may have one or more gap of about 15 and about 27 amino acids in length; vi) a coil-3 of between 1 to 6 amino acids of coil and/or alpha helix between about 11 and about 13 amino acids in length flanked structure intervening in the beta Strand structure. In some by alpha helix-3 and alpha helix-4; vii) an alpha helix-4 of embodiments the secondary structure is generated by the about 24 180 amino acids in length; viii) a coil-4 of between PSIPRED, top ranked secondary structure prediction about 4 and about 5 amino acids in length flanked by alpha method (Jones D T. (1999).J. Mol. Biol. 292: 195-202). helix-4 and alpha helix-5; ix) an alpha helix-5 of between 0076. In some embodiments the nucleic acid molecule about 50 and about 54 amino acids in length; x) a coil-5 of encodes a PtIP-83 polypeptide comprising sequentially from between about 11 and about 17 amino acids in length flanked the N-terminus to the C-terminus: a Region A of between by alpha helix-5 and alpha helix-6; xi) an alpha helix-6 of about 200 to about 300 amino acids in length having a between about 15 and about 16 amino acids in length; xii) US 2016/0347799 A1 Dec. 1, 2016 30 a coil-6 of between about 6 and about 9 amino acids in helix-4 and alpha helix-5; ix) an alpha helix-5 of between length flanked by alpha helix-6 and alpha helix-7: xiii) an about 50 and about 54 amino acids in length; x) a coil-5 of alpha helix-7 of between about 49 and about 55 amino acids between about 11 and about 17 amino acids in length flanked in length; xiv) a coil-7 of between about 3 and about 8 amino by alpha helix-5 and alpha helix-6; xi) an alpha helix-6 of acids in length flanked by alpha helix-7 and alpha helix-8; between about 15 and about 16 amino acids in length; xii) XV) an alpha helix-8 of between about 33 and about 36 a coil-6 of between about 6 and about 9 amino acids in amino acids in length; xvi) a coil-8 of between about 14 and length flanked by alpha helix-6 and alpha helix-7: xiii) an about 16 amino acids in length flanked by alpha helix-8 and alpha helix-7 of between about 49 and about 55 amino acids alpha helix-9; xvii) an alpha helix-9 of between about 16 and in length; xiv) a coil-7 of between about 3 and about 8 amino about 23 amino acids in length; xviii) a coil-9 of between acids in length flanked by alpha helix-7 and alpha helix-8; about 21 and about 28 amino acids in length flanked by XV) an alpha helix-8 of between about 33 and about 36 alpha helix-9 and Region C; and a Region C of between amino acids in length; xvi) a coil-8 of between about 14 and about 150 to about 180 amino acids in length having a about 16 amino acids in length flanked by alpha helix-8 and consensus secondary structure comprising seven segments alpha helix-9; xvii) an alpha helix-9 of between about 16 and of predominately beta Strand structure. about 23 amino acids in length; xviii) a coil-9 of between 0080. In some embodiments the nucleic acid molecule about 21 and about 28 amino acids in length flanked by encodes a PtIP-83 polypeptide comprising sequentially from alpha helix-9 and Region C; and a Region C of between the N-terminus to the C-terminus: a Region A of between about 150 to about 180 amino acids in length having a about 200 to about 300 amino acids in length having a consensus secondary structure comprising sequentially: i) a predominantly nonconserved secondary structure; a Region beta strand-1 (B1) of between about 3 amino acids and about B of between about 380 to about 465 amino acids in length 5 amino acids in length; ii) a coil of between about 13 amino having a consensus secondary structure comprising nine acids and about 17 amino acids in length; iii) a beta Strand-2 segments of predominately alpha helical structure; and a (B2) of between about 7 amino acids and about 11 amino Region C of between about 150 to about 180 amino acids in acids in length; iv) a coil of between about 17 amino acids length having a consensus secondary structure comprising and about 23 amino acids in length; v) a beta strand-3 (B3) sequentially: i) a beta strand-1 (B1) of between about 3 of between about 5 amino acids and about 7 amino acids in amino acids and about 5 amino acids in length; ii) a coil of length; vi) a coil of between about 12 amino acids and about between about 13 amino acids and about 17 amino acids in 14 amino acids in length; vii) a beta Strand-4 (B4) of between length; iii) a beta strand-2 (32) of between about 7 amino about 5 amino acids and about 6 amino acids in length; viii) acids and about 11 amino acids in length; iv) a coil of a coil of between about 2 amino acids and about 7 amino between about 17 amino acids and about 23 amino acids in acids in length; ix) a beta strand-5 (B5) of between about 5 length; v) a beta strand-3 (B3) of between about 5 amino amino acids and about 7 amino acids in length; X) a coil of acids and about 7 amino acids in length; vi) a coil of between between about 26 amino acids and about 28 amino acids in about 12 amino acids and about 14 amino acids in length; length; xi) a beta strand-6 (B6) of between about 5 amino vii) a beta strand-4 (B4) of between about 5 amino acids and acids and about 7 amino acids in length; xii) a coil of about 6 amino acids in length; viii) a coil of between about between about 16 amino acids and about 20 amino acids in 2 amino acids and about 7 amino acids in length; ix) a beta length; and xiii) a beta strand-1 (37) of between about 13 strand-5 (B5) of between about 5 amino acids and about 7 amino acids and about 17 amino acids in length. amino acids in length: X) a coil of between about 26 amino 0082 In some embodiments the nucleic acid molecule acids and about 28 amino acids in length; xi) a beta Strand-6 encodes a PtIP-83 polypeptide comprising sequentially from (B6) of between about 5 amino acids and about 7 amino the N-terminus to the C-terminus: a Region A of between acids in length; xii) a coil of between about 16 amino acids about 200 to about 300 amino acids in length having a and about 20 amino acids in length; and xiii) a beta Strand-1 flexible consensus secondary structure, wherein the Region (B7) of between about 13 amino acids and about 17 amino A comprises a conserved beta strand 1 (B1a) of between acids in length. about 4 and about 12 amino acids in length within about 0081. In some embodiments the nucleic acid molecule amino acid residue 30 to about amino acid residue 130 from encodes a PtIP-83 polypeptide comprising sequentially from the N-terminus of the PtIP-83 polypeptide; a Region B of the N-terminus to the C-terminus: a Region A of between between about 380 to about 465 amino acids in length about 200 to about 300 amino acids in length having a having a consensus secondary structure comprising sequen predominantly nonconserved secondary structure; a Region tially: i) an alpha helix-1 of between about 10 and about 26 B of between about 380 to about 465 amino acids in length amino acids in length; ii) a coil-1 of between about 2 and having a consensus secondary structure comprising sequen about 8 amino acids in length flanked by alpha helix-1 and tially: i) an alpha helix-1 of between about 10 and about 26 alpha helix-2; iii) an alpha helix-2 of between about 15 and amino acids in length; ii) a coil-1 of between about 2 and about 24 amino acids in length; iv) a coil-2 of between about about 8 amino acids in length flanked by alpha helix-1 and 4 and about 14 amino acids in length flanked by alpha alpha helix-2; iii) an alpha helix-2 of between about 15 and helix-2 and alpha helix-3; v) an alpha helix 3 of between about 24 amino acids in length; iv) a coil-2 of between about about 15 and about 27 amino acids in length; vi) a coil-3 of 4 and about 14 amino acids in length flanked by alpha between about 11 and about 13 amino acids in length flanked helix-2 and alpha helix-3; v) an alpha helix 3 of between by alpha helix-3 and alpha helix-4; vii) an alpha helix-4 of about 15 and about 27 amino acids in length; vi) a coil-3 of about 24 180 amino acids in length; viii) a coil-4 of between between about 11 and about 13 amino acids in length flanked about 4 and about 5 amino acids in length flanked by alpha by alpha helix-3 and alpha helix-4; vii) an alpha helix-4 of helix-4 and alpha helix-5; ix) an alpha helix-5 of between about 24 180 amino acids in length; viii) a coil-4 of between about 50 and about 54 amino acids in length; x) a coil-5 of about 4 and about 5 amino acids in length flanked by alpha between about 11 and about 17 amino acids in length flanked US 2016/0347799 A1 Dec. 1, 2016
by alpha helix-5 and alpha helix-6; xi) an alpha helix-6 of another example, introduction of one or more insertion between about 15 and about 16 amino acids in length; xii) sequences into a polynucleotide can facilitate recombination a coil-6 of between about 6 and about 9 amino acids in with a low homology polynucleotide; use of an intron or length flanked by alpha helix-6 and alpha helix-7: xiii) an intein for the insertion sequence facilitates the removal of alpha helix-7 of between about 49 and about 55 amino acids the intervening sequence, thereby restoring function of the in length; xiv) a coil-7 of between about 3 and about 8 amino encoded variant. acids in length flanked by alpha helix-7 and alpha helix-8; I0085 Nucleic acid molecules that are fragments of these XV) an alpha helix-8 of between about 33 and about 36 nucleic acid sequences encoding Pt|P-83 polypeptides are amino acids in length; xvi) a coil-8 of between about 14 and also encompassed by the embodiments. "Fragment as used about 16 amino acids in length flanked by alpha helix-8 and herein refers to a portion of the nucleic acid sequence alpha helix-9; xvii) an alpha helix-9 of between about 16 and encoding a PtIP-83 polypeptide. A fragment of a nucleic acid about 23 amino acids in length; xviii) a coil-9 of between sequence may encode a biologically active portion of a about 21 and about 28 amino acids in length flanked by PtIP-83 polypeptide or it may be a fragment that can be used alpha helix-9 and Region C; and a Region C of between as a hybridization probe or PCR primer using methods about 150 to about 180 amino acids in length having a disclosed below. Nucleic acid molecules that are fragments consensus secondary comprising sequentially: i) a beta of a nucleic acid sequence encoding a Pt(P-83 polypeptide strand-1 (B1) of between about 3 amino acids and about 5 comprise at least about 150, 180,210, 240,270, 300,330 or amino acids in length; ii) a coil of between about 13 amino 360, contiguous nucleotides or up to the number of nucleo acids and about 17 amino acids in length; iii) a beta Strand-2 tides present in a full-length nucleic acid sequence encoding (B2) of between about 7 amino acids and about 11 amino a PtIP-83 polypeptide disclosed herein, depending upon the acids in length; iv) a coil of between about 17 amino acids intended use. “Contiguous nucleotides’ is used herein to and about 23 amino acids in length; v) a beta strand-3 (B3) refer to nucleotide residues that are immediately adjacent to of between about 5 amino acids and about 7 amino acids in one another. Fragments of the nucleic acid sequences of the length; vi) a coil of between about 12 amino acids and about embodiments will encode protein fragments that retain the 14 amino acids in length; vii) a beta Strand-4 (B4) of between biological activity of the PtIP-83 polypeptide and, hence, about 5 amino acids and about 6 amino acids in length; viii) retain insecticidal activity. “Retains insecticidal activity” is a coil of between about 2 amino acids and about 7 amino used herein to refer to a polypeptide having at least about acids in length; ix) a beta strand-5 (B5) of between about 5 10%, at least about 30%, at least about 50%, at least about amino acids and about 7 amino acids in length; X) a coil of 70%, 80%, 90%, 95% or higher of the insecticidal activity between about 26 amino acids and about 28 amino acids in of the full-length PtIP-83Aa polypeptide (SEQID NO: 1). In length; xi) a beta strand-6 (B6) of between about 5 amino Some embodiments, the insecticidal activity is Lepidoptera acids and about 7 amino acids in length; xii) a coil of activity. In one embodiment, the insecticidal activity is between about 16 amino acids and about 20 amino acids in against a Coleopteran species. In some embodiments, the length; and xiii) a beta strand-1 (37) of between about 13 insecticidal activity is against one or more insect pests of the amino acids and about 17 amino acids in length. corn rootworm complex: western corn rootworm, Dia 0083. Also provided are nucleic acid molecules that brotica virgifera; northern corn rootworm, D. barberi: encode transcription and/or translation products that are Southern corn rootworm or spotted cucumber beetle; Dia subsequently spliced to ultimately produce functional PtIP brotica undecimpunctata howardi, and the Mexican corn 83 polypeptides. Splicing can be accomplished in vitro or in rootworm, D. virgifera zeae. In one embodiment, the insec Vivo, and can involve cis- or trans-splicing. The Substrate for ticidal activity is against a Diabrotica species. splicing can be polynucleotides (e.g., RNA transcripts) or I0086. In some embodiments a fragment of a nucleic acid polypeptides. An example of cis-splicing of a polynucleotide sequence encoding a Pt(P-83 polypeptide encoding a bio is where an intron inserted into a coding sequence is logically active portion of a protein will encode at least removed and the two flanking exon regions are spliced to about 15, 20, 30, 50, 75, 100, 125, contiguous amino acids generate a Pt(P-83 polypeptide encoding sequence. An or up to the total number of amino acids present in a example of trans splicing would be where a polynucleotide full-length PtIP-83 polypeptide of the embodiments. In is encrypted by separating the coding sequence into two or Some embodiments, the fragment is an N-terminal and/or a more fragments that can be separately transcribed and then C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, spliced to form the full-length pesticidal encoding sequence. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 0084. The use of a splicing enhancer sequence, which can 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids be introduced into a construct, can facilitate splicing either from the N-terminus and/or C-terminus relative to SEQ ID in cis or trans-splicing of polypeptides (U.S. Pat. Nos. NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: 7, SEQ 6,365.377 and 6,531,316). Thus, in some embodiments the ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: polynucleotides do not directly encode a full-length PtIP-83 15, SEQID NO: 17, SEQID NO: 19, SEQID NO: 21, SEQ polypeptide, but rather encode a fragment or fragments of a ID NO. 23 or SEQID NO: 716, SEQID NO: 754, SEQ ID PtIP-83 polypeptide. These polynucleotides can be used to NO: 755, SEQID NO: 756, SEQ ID NO: 757, SEQID NO: express a functional PtIP-83 polypeptide through a mecha 758, SEQID NO: 759, SEQID NO: 760, SEQID NO: 761, nism involving splicing, where splicing can occur at the SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQ level of polynucleotide (e.g., intron/exon) and/or polypep ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID tide (e.g., intein/extein). This can be useful, for example, in NO: 768 or SEQ ID NO: 769 variants thereof, e.g., by controlling expression of pesticidal activity, since a func proteolysis, insertion of a start codon, deletion of the codons tional pesticidal polypeptide will only be expressed if all encoding the deleted amino acids with the concomitant required fragments are expressed in an environment that insertion of a stop codon or by insertion of a stop codon in permits splicing processes to generate functional product. In the coding sequence. In some embodiments, the fragments US 2016/0347799 A1 Dec. 1, 2016 32 encompassed herein result from the removal of the N-ter ID NO:368-397, any one of SEQID NO: 428-517, SEQID minal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, NO: 717, any one of SEQID NO: 718–727, and any one of 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids from the SEQ ID NO: 738-753. N-terminus relative to SEQID NO: 1, SEQID NO:3, SEQ 0091. In some embodiments the nucleic acid encodes a ID NO. 5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, PtIP-83 polypeptide having at least about 50%, 55%, 60%, SEQID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, NO: 19, SEQID NO:21, SEQID NO. 23, SEQID NO: 716, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, SEQID NO: 754, SEQID NO: 755, SEQID NO: 756, SEQ 97%, 98%, 99% or greater sequence identity compared to ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: SEQID NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 or ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQID NO: variants thereof, e.g., by proteolysis or by insertion of a start 21, SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, codon in the coding sequence. SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID 0087. In some embodiments, the fragments encompassed NO: 761, SEQID NO: 762, SEQ ID NO: 763, SEQID NO: herein result from the removal of the N-terminal 1, 2, 3, 4, 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acids relative to SEQ SEQID NO: 768 or SEQID NO: 769. In some embodiments ID NO: 1, SEQ ID NO:3, SEQ ID NO. 5, SEQ ID NO: 7, the sequence identity is calculated using ClustalW algorithm SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID in the ALIGNX(R) module of the Vector NTIR) Program Suite NO: 15, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, (Invitrogen Corporation, Carlsbad, Calif.) with all default SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ parameters. In some embodiments the sequence identity is ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID across the entire length of polypeptide calculated using NO: 758, SEQID NO: 759, SEQID NO: 760, SEQID NO: ClustalW algorithm in the ALIGNX module of the Vector 761, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, NTI Program Suite (Invitrogen Corporation, Carlsbad, SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, SEQ ID NO: 768, SEQ ID NO: 769 or variants thereof, e.g., by Calif.) with all default parameters. proteolysis or by insertion of a start codon in the coding 0092. To determine the percent identity of two amino Sequence. acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. The I0088. In some embodiments the PtIP-83 polypeptide is percent identity between the two sequences is a function of encoded by a nucleic acid sequence Sufficiently homologous the number of identical positions shared by the sequences to the nucleic acid sequence of SEQID NO: 2, SEQID NO: (i.e., percent identity=number of identical positions/total 4, SEQID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQID number of positions (e.g., overlapping positions)x100). In NO: 12, SEQID NO: 14, SEQID NO: 16, SEQID NO: 18, one embodiment, the two sequences are the same length. In SEQID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID another embodiment, the comparison is across the entirety NO: 717, SEQID NO: 738, SEQID NO: 739, SEQID NO: of the reference sequence (e.g., across the entirety of SEQ 740, SEQID NO: 741, SEQID NO: 742, SEQID NO: 743, ID NO: 1). The percent identity between two sequences can SEQID NO: 744, SEQID NO: 745, SEQID NO: 746, SEQ be determined using techniques similar to those described ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID below, with or without allowing gaps. In calculating percent NO: 750, SEQ ID NO: 751, SEQ ID NO: 752 or SEQ ID NO: 753. “Sufficiently homologous” is used herein to refer identity, typically exact matches are counted. to an amino acid or nucleic acid sequence that has at least 0093. The determination of percent identity between two about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, sequences can be accomplished using a mathematical algo 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, rithm. A non-limiting example of a mathematical algorithm 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence utilized for the comparison of two sequences is the algo homology compared to a reference sequence using one of rithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. the alignment programs described herein using standard USA 87:2264, modified as in Karlin and Altschul, (1993) parameters. One of skill in the art will recognize that these Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm values can be appropriately adjusted to determine corre is incorporated into the BLASTN and BLASTX programs of sponding homology of proteins encoded by two nucleic acid Altschul, et al., (1990) J. Mol. Biol. 215:403. BLAST sequences by taking into account codon degeneracy, amino nucleotide searches can be performed with the BLASTN acid similarity, reading frame positioning, and the like. program, score=100, wordlength=12, to obtain nucleic acid sequences homologous to pesticidal nucleic acid molecules 0089. In some embodiments the sequence homology is of the embodiments. BLAST protein searches can be per against the full length sequence of the polynucleotide encod formed with the BLASTX program, score=50, word ing a PtIP-83 polypeptide or against the full length sequence length 3, to obtain amino acid sequences homologous to of a PtIP-83 polypeptide. pesticidal protein molecules of the embodiments. To obtain 0090. In some embodiments the nucleic acid encoding a gapped alignments for comparison purposes, Gapped PtIP-83 polypeptide is selected from SEQID NO: 2, SEQID BLAST (in BLAST 2.0) can be utilized as described in NO:4, SEQID NO: 6, SEQID NO: 8, SEQID NO: 10, SEQ Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alter ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQID NO: natively, PSI-Blast can be used to perform an iterated search 18, SEQID NO: 20, SEQID NO: 22, SEQID NO: 24, SEQ that detects distant relationships between molecules. See, ID NO: 28, SEQ ID NO: 29, any one of SEQ ID NO: Altschul, et al., (1997) supra. When utilizing BLAST, 172–235, any one of SEQID NO: 300-333, any one of SEQ Gapped BLAST, and PSI-Blast programs, the default param US 2016/0347799 A1 Dec. 1, 2016 eters of the respective programs (e.g., BLASTX and art will readily appreciate that due to the degeneracy of the BLASTN) can be used. Alignment may also be performed genetic code, a multitude of nucleotide sequences encoding manually by inspection. PtIP-83 polypeptides of the present disclosure exist. Table 1 0094. Another non-limiting example of a mathematical is a codon table that provides the synonymous codons for algorithm utilized for the comparison of sequences is the each amino acid. For example, the codons AGA, AGG, ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids CGA, CGC, CGG, and CGU all encode the amino acid Res. 22:4673-4680). ClustalW compares sequences and arginine. Thus, at every position in the nucleic acids of the aligns the entirety of the amino acid or DNA sequence, and disclosure where an arginine is specified by a codon, the thus can provide data about the sequence conservation of the codon can be altered to any of the corresponding codons entire amino acid sequence. The ClustalW algorithm is used described above without altering the encoded polypeptide. It in several commercially available DNA/amino acid analysis is understood that U in an RNA sequence corresponds to T software packages, such as the ALIGNX(R) module of the in a DNA sequence. Vector NTIR) Program Suite (Invitrogen Corporation, Carls bad, Calif.). After alignment of amino acid sequences with TABLE 1. ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for Alanine Ala A. GCA GCC GCG GC analysis of ClustalW alignments is GENEDOCTM GENE Cystine Cys UGC UGU DOCTM (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Aspartic acid Asp D GAC GAU Another non-limiting example of a mathematical algorithm Glutamic acid Glu E GAA GAG utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algo Phenylalanine Phe F UUC UUU rithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Glycine Gly G GGA. GGC GGG GGU Package, Version 10 (available from Accelrys, Inc., 9685 Histidine His CAC CAU Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a Isoleucine II I AUA AUC AUU PAM120 weight residue table, a gap length penalty of 12, Lysine Llys K AAA AAG and a gap penalty of 4 can be used. 0095. Another non-limiting example of a mathematical Leucine Leu L UUA UUG. CUA CUC CUG C U algorithm utilized for the comparison of sequences is the Methionine Me M AUG algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software to determine Asparagine ASn N AAC AAU sequence identity or similarity using the following default parameters: % identity and % similarity for a nucleic acid Proline Pro P CCA CCC CCG CCU sequence using GAP Weight of 50 and Length Weight of 3, Glutamine Glin Q CAA CAG and the nwsgapdna.cmpii scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of Arginine Arg R AGA AGG CGA CGC CGG CGU 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. “Equiva Serine Ser S AGC AGU UCA, UCC UCG UCC lent program' is used herein to refer to any sequence Threonine Thir T ACA ACC ACG ACU comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue Waline Wall W. GUA GUC GUG GUU matches and an identical percent sequence identity when Tryptophan Trp W TGG compared to the corresponding alignment generated by GAP Version 10. Tyrosine Tyr Y UAC UAU 0096. The embodiments also encompass nucleic acid molecules encoding PtIP-83 polypeptide variants. “Vari 0098. The skilled artisan will further appreciate that ants of the PtIP-83 polypeptide encoding nucleic acid changes can be introduced by mutation of the nucleic acid sequences include those sequences that encode the PtIP-83 sequences thereby leading to changes in the amino acid polypeptides disclosed herein but that differ conservatively sequence of the encoded PtIP-83 polypeptides, without because of the degeneracy of the genetic code as well as altering the biological activity of the proteins. Thus, variant those that are sufficiently identical as discussed above. nucleic acid molecules can be created by introducing one or Naturally occurring allelic variants can be identified with the more nucleotide Substitutions, additions and/or deletions use of well-known molecular biology techniques, such as into the corresponding nucleic acid sequence disclosed polymerase chain reaction (PCR) and hybridization tech herein, such that one or more amino acid Substitutions, niques as outlined below. Variant nucleic acid sequences additions or deletions are introduced into the encoded pro also include synthetically derived nucleic acid sequences tein. Mutations can be introduced by Standard techniques, that have been generated, for example, by using site-directed Such as site-directed mutagenesis and PCR-mediated muta mutagenesis but which still encode the PtIP-83 polypeptides genesis. Such variant nucleic acid sequences are also encom disclosed as discussed below. passed by the present disclosure. 0097. The present disclosure provides isolated or recom 0099. Alternatively, variant nucleic acid sequences can binant polynucleotides that encode any of the PtIP-83 poly be made by introducing mutations randomly along all or part peptides disclosed herein. Those having ordinary skill in the of the coding sequence. Such as by Saturation mutagenesis, US 2016/0347799 A1 Dec. 1, 2016 34 and the resultant mutants can be screened for ability to geting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; confer pesticidal activity to identify mutants that retain Chang, et al., (1999) Nat Biotechnol 17:793-797; Minshull activity. Following mutagenesis, the encoded protein can be and Stemmer, (1999) Curr Opin Chem Biol 3:284-290; expressed recombinantly, and the activity of the protein can Christians, et al., (1999) Nat Biotechnol 17:259-264; Cra be determined using standard assay techniques. meri, et al., (1998) Nature 391:288-291; Crameri, et al., 0100. The polynucleotides of the disclosure and frag (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) ments thereof are optionally used as Substrates for a variety PNAS USA 94:4504-4509: Patten, et al., (1997) Curr Opin of recombination and recursive recombination reactions, in Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med addition to standard cloning methods as set forth in, e.g., 2:100-103: Crameri, et al., (1996) Nat Biotechnol 14:315 Ausubel, Berger and Sambrook, i.e., to produce additional 319; Gates, et al., (1996).J Mol Biol 255:373-386; Stemmer, pesticidal polypeptide homologues and fragments thereof (1996) “Sexual PCR and Assembly PCR” In: The Encyclo with desired properties. A variety of such reactions are pedia of Molecular Biology. VCH Publishers, New York. pp. known, including those developed by the inventors and their 447-457; Crameri and Stemmer, (1995) BioTechniques co-workers. Methods for producing a variant of any nucleic 18:194-195; Stemmer, et al., (1995) Gene, 164:49-53: Stem acid listed herein comprising recursively recombining Such mer, (1995) Science 270: 1510; Stemmer, (1995) Bio/Tech polynucleotide with a second (or more) polynucleotide, thus nology 13:549-553; Stemmer, (1994) Nature 370:389-391 forming a library of variant polynucleotides are also and Stemmer, (1994) PNAS USA 91: 10747-10751. embodiments of the disclosure, as are the libraries produced, 0105 Mutational methods of generating diversity the cells comprising the libraries and any recombinant include, for example, site-directed mutagenesis (Ling, et al., polynucleotide produces by Such methods. Additionally, (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Such methods optionally comprise selecting a variant poly Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev nucleotide from such libraries based on pesticidal activity, as Genet 19:423-462: Botstein and Shortle, (1985) Science is wherein such recursive recombination is done in vitro or 229:1193-1201; Carter, (1986) Biochem J237:1-7 and Kun in vivo. kel, (1987) “The efficiency of oligonucleotide directed 0101. A variety of diversity generating protocols, includ mutagenesis' in Nucleic Acids & Molecular Biology (Eck ing nucleic acid recursive recombination protocols are avail Stein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis able and fully described in the art. The procedures can be using uracil containing templates (Kunkel, (1985) PNAS used separately, and/or in combination to produce one or USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol more variants of a nucleic acid or set of nucleic acids, as well 154:367-382 and Bass, et al., (1988) Science 242:240-245): as variants of encoded proteins. Individually and collec oligonucleotide-directed mutagenesis (Zoller and Smith, tively, these procedures provide robust, widely applicable (1983) Methods Enzymol 100:468-500; Zoller and Smith, ways of generating diversified nucleic acids and sets of (1987) Methods Enzymol 154:329-350 (1987); Zoller and nucleic acids (including, e.g., nucleic acid libraries) useful, Smith, (1982) Nucleic Acids Res 10:6487-6500), phospho e.g., for the engineering or rapid evolution of nucleic acids, rothioate-modified DNA mutagenesis (Taylor, et al., (1985) proteins, pathways, cells and/or organisms with new and/or Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl improved characteristics. Acids Res 13:8765-8787 (1985): Nakamaye and Eckstein, 0102. While distinctions and classifications are made in (1986) Nucl Acids Res 14:9679–9698; Sayers, et al., (1988) the course of the ensuing discussion for clarity, it will be Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl appreciated that the techniques are often not mutually exclu Acids Res 16:803-814); mutagenesis using gapped duplex sive. Indeed, the various methods can be used singly or in DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456: combination, in parallel or in series, to access diverse Kramer and Fritz, (1987) Methods Enzymol 154:350-367: sequence variants. Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et 0103) The result of any of the diversity generating pro al., (1988) Nucl Acids Res 16:6987-6999). cedures described herein can be the generation of one or 010.6 Additional suitable methods include point mis more nucleic acids, which can be selected or screened for match repair (Kramer, et al., (1984) Cell 38:879-887), nucleic acids with or which confer desirable properties or mutagenesis using repair-deficient host strains (Carter, et al., that encode proteins with or which confer desirable proper (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) ties. Following diversification by one or more of the meth Methods in Enzymol 154:382-403), deletion mutagenesis ods herein or otherwise available to one of skill, any nucleic (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res acids that are produced can be selected for a desired activity 14:5115), restriction-selection and restriction-purification or property, e.g. pesticidal activity or, such activity at a (Wells, et al., (1986) Phil Trans R Soc Lond A317:415-423), desired pH, etc. This can include identifying any activity that mutagenesis by total gene synthesis (Nambiar, et al., (1984) can be detected, for example, in an automated or automat Science 223:1299-1301: Sakamar and Khorana, (1988) Nucl able format, by any of the assays in the art, see, e.g., Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315 discussion of Screening of insecticidal activity, infra. A 323 and Grundström, et al., (1985) Nucl Acids Res 13:3305 variety of related (or even unrelated) properties can be 3316), double-strand break repair (Mandecki, (1986) PNAS evaluated, in serial or in parallel, at the discretion of the USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech practitioner. 4:450-455). Additional details on many of the above meth 0104. Descriptions of a variety of diversity generating ods can be found in Methods Enzymol Volume 154, which procedures for generating modified nucleic acid sequences, also describes useful controls for trouble-shooting problems e.g., those coding for polypeptides having pesticidal activity with various mutagenesis methods. or fragments thereof, are found in the following publications 0107 Additional details regarding various diversity gen and the references cited therein: Soong, et al., (2000) Nat erating methods can be found in the following US patents, Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Tar PCT Publications and applications and EPO publications: US 2016/0347799 A1 Dec. 1, 2016
U.S. Pat. No. 5,723,323, U.S. Pat. No. 5,763,192, U.S. Pat. lar Biology published by John Wiley & Son Inc). Specifi No. 5,814,476, U.S. Pat. No. 5,817,483, U.S. Pat. No. cally, LC-MS/MS based protein identification method is 5,824,514, U.S. Pat. No. 5,976,862, U.S. Pat. No. 5,605,793, used to associate the MS data of given cell lysate or desired U.S. Pat. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. molecular weight enriched samples (excised from SDS No. 5,834,252, U.S. Pat. No. 5,837,458, WO 1995/22625, PAGE gel of relevant molecular weight bands to PtIP-83 WO 1996/33207, WO 1997/20078, WO 1997/35966, WO polypeptides) with sequence information of PtIP-83 poly 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/ peptides SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, 41368, EP 752008, EP 0932670, WO 1999/23107, WO SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID 1999/21979, WO 1998/31837, WO 1998/27230, WO 1998/ NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, 27230, WO 2000/00632, WO 2000/09679, WO 1998/42832, SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ WO 1999/29902, WO 1998/41653, WO 1998/41622, WO ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID 1998/.42727, WO 2000/18906, WO 2000/04190, WO 2000/ NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQID NO: 42561, WO 2000/42559, WO 2000/42560, WO 2001/23401 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, and PCTFUSO1/O6775. SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ 0108. The nucleotide sequences of the embodiments can ID NO: 767, SEQID NO: 768 or SEQID NO: 769 and their also be used to isolate corresponding sequences from ferns homologs. Any match in peptide sequences indicates the or other primitive plant, particularly a Asplenium, Polypo potential of having the homologous proteins in the samples. dium, Adiantum, Platycerium, Nephrolepis, Ophioglossum, Additional techniques (protein purification and molecular Colysis, Bolbitis, Blechnum, Selaginella, Lycopodium, and biology) can be used to isolate the protein and identify the Huperzia species. In this manner, methods such as PCR, sequences of the homologs. hybridization, and the like can be used to identify such 0111. In hybridization methods, all or part of the pesti sequences based on their sequence homology to the cidal nucleic acid sequence can be used to screen cDNA or sequences set forth herein. Sequences that are selected based genomic libraries. Methods for construction of such cDNA on their sequence identity to the entire sequences set forth and genomic libraries are generally known in the art and are herein or to fragments thereof are encompassed by the disclosed in Sambrook and Russell. (2001), supra. The embodiments. Such sequences include sequences that are so-called hybridization probes may be genomic DNA frag orthologs of the disclosed sequences. The term “orthologs' ments, cDNA fragments, RNA fragments or other oligo refers to genes derived from a common ancestral gene and nucleotides and may be labeled with a detectable group such which are found in different species as a result of speciation. as 32P or any other detectable marker, such as other radio Genes found in different species are considered orthologs isotopes, a fluorescent compound, an enzyme or an enzyme when their nucleotide sequences and/or their encoded pro co-factor. Probes for hybridization can be made by labeling tein sequences share substantial identity as defined else synthetic oligonucleotides based on the known PtIP-83 where herein. Functions of orthologs are often highly con polypeptide-encoding nucleic acid sequence disclosed served among species. herein. Degenerate primers designed on the basis of con 0109. In a PCR approach, oligonucleotide primers can be served nucleotides or amino acid residues in the nucleic acid designed for use in PCR reactions to amplify corresponding sequence or encoded amino acid sequence can additionally DNA sequences from cDNA or genomic DNA extracted be used. The probe typically comprises a region of nucleic from any organism of interest. Methods for designing PCR acid sequence that hybridizes under stringent conditions to primers and PCR cloning are generally known in the art and at least about 12, at least about 25, at least about 50, 75, 100, are disclosed in Sambrook, et al., (1989) Molecular Clon 125, 150, 175 or 200 consecutive nucleotides of nucleic acid ing: A Laboratory Manual (2d ed., Cold Spring Harbor sequence encoding a PtIP-83 polypeptide of the disclosure Laboratory Press, Plainview, N.Y.), hereinafter “Sambrook”. or a fragment or variant thereof. Methods for the preparation See also, Innis, et al., eds. (1990) PCR Protocols: A Guide of probes for hybridization are generally known in the art to Methods and Applications (Academic Press, New York); and are disclosed in Sambrook and Russell. (2001), supra, Innis and Gelfand, eds. (1995) PCR Strategies (Academic herein incorporated by reference. Press, New York); and Innis and Gelfand, eds. (1999) PCR 0112 For example, an entire nucleic acid sequence, Methods Manual (Academic Press, New York). Known encoding a PtIP-83 polypeptide, disclosed herein or one or methods of PCR include, but are not limited to, methods more portions thereof may be used as a probe capable of using paired primers, nested primers, single specific primers, specifically hybridizing to corresponding nucleic acid degenerate primers, gene-specific primers, vector-specific sequences encoding Pt|P-83 polypeptide-like sequences and primers, partially-mismatched primers, and the like. To messenger RNAs. To achieve specific hybridization under a identify potential Pt(P-83 polypeptides from fern or moss variety of conditions, such probes include sequences that are collections, the fern or moss cell lysates can be screened unique and are preferably at least about 10 nucleotides in with antibodies generated against a PtIP-83 polypeptides length or at least about 20 nucleotides in length. Such probes and/or PtIP-83 polypeptides using Western blotting and/or may be used to amplify corresponding pesticidal sequences ELISA methods. This type of assays can be performed in a from a chosen organism by PCR. This technique may be high throughput fashion. Positive samples can be further used to isolate additional coding sequences from a desired analyzed by various techniques such as antibody based organism or as a diagnostic assay to determine the presence protein purification and identification. Methods of generat of coding sequences in an organism. Hybridization tech ing antibodies are well known in the art as discussed infra. niques include hybridization screening of plated DNA librar 0110. Alternatively, mass spectrometry based protein ies (either plaques or colonies; see, for example, Sambrook, identification method can be used to identify homologs of et al., (1989) Molecular Cloning: A Laboratory Manual (2d PtIP-83 polypeptides using protocols in the literatures (Scott ed., Cold Spring Harbor Laboratory Press, Cold Spring Patterson, (1998), 10.22, 1-24, Current Protocol in Molecu Harbor, N.Y.). Hybridization of such sequences may be US 2016/0347799 A1 Dec. 1, 2016 36 carried out under stringent conditions. “Stringent condi ization and wash compositions, and desired Tm, those of tions” or “stringent hybridization conditions” is used herein ordinary skill will understand that variations in the strin to refer to conditions under which a probe will hybridize to gency of hybridization and/or wash Solutions are inherently its target sequence to a detectably greater degree than to described. If the desired degree of mismatching results in a other sequences (e.g., at least 2-fold over background). Tm of less than 45° C. (aqueous solution) or 32° C. Stringent conditions are sequence-dependent and will be (formamide solution), it is preferred to increase the SSC different in different circumstances. By controlling the strin concentration so that a higher temperature can be used. An gency of the hybridization and/or washing conditions, target extensive guide to the hybridization of nucleic acids is found sequences that are 100% complementary to the probe can be in Tijssen, (1993) Laboratory Techniques in Biochemistry identified (homologous probing). Alternatively, stringency and Molecular Biology-Hybridization with Nucleic Acid conditions can be adjusted to allow some mismatching in Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al., sequences so that lower degrees of similarity are detected eds. (1995) Current Protocols in Molecular Biology, Chapter (heterologous probing). Generally, a probe is less than about 2 (Greene Publishing and Wiley-Interscience, New York). 1000 nucleotides in length, preferably less than 500 nucleo See, Sambrook, et al., (1989) Molecular Cloning: A Labo tides in length. ratory Manual (2d ed., Cold Spring Harbor Laboratory 0113 Typically, stringent conditions will be those in Press, Cold Spring Harbor, N.Y.). which the salt concentration is less than about 1.5 MNaion, 0.115. In some embodiments polynucleotides are pro typically about 0.01 to 1.0 M Na ion concentration (or other vided encoding a Pt(P-83 polypeptide comprising an amino salts) at pH 7.0 to 8.3 and the temperature is at least about acid sequence having at least 80%, at least 85%, at least 30° C. for short probes (e.g., 10 to 50 nucleotides) and at 90%, at least 95%, at least 96%, at least 97%, at least 98% least about 60° C. for long probes (e.g., greater than 50 or at least 99% sequence identity to any one of SEQID NO: nucleotides). Stringent conditions may also be achieved with 786-888. the addition of destabilizing agents such as formamide. 0116. In some embodiments polynucleotides are pro Exemplary low stringency conditions include hybridization vided encoding a PtIP-83 polypeptide comprising the amino with a buffer solution of 30 to 35% formamide, 1 M NaCl, acid sequence of any one of SEQ ID NO: 786-888. 1% SDS (sodium dodecyl sulphate) at 37°C., and a wash in 0117. In some embodiments the polynucleotide encoding 1x to 2xSSC (20xSSC=3.0 MNaC1/0.3 M trisodium citrate) the PtIP-83 polypeptide comprising the amino acid sequence at 50 to 55° C. Exemplary moderate stringency conditions of any one of SEQ ID NO: 786-888 is a non-genomic include hybridization in 40 to 45% formamide, 1.0 M NaCl, Sequence. 1% SDS at 37° C., and a wash in 0.5x to 1XSSC at 55 to 60° 0118. In some embodiments the polynucleotide encoding C. Exemplary high Stringency conditions include hybridiza the PtIP-83 polypeptide comprising the amino acid sequence tion in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and of any one of SEQ ID NO: 786-888 is a cDNA. a wash in 0.1xSSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of Proteins and Variants and Fragments Thereof hybridization is generally less than about 24 hours, usually 0119 PtIP-83 polypeptides are also encompassed by the about 4 to about 12 hours. disclosure. “Pteridophyta Insecticidal Protein-83” “PtlP-83 0114 Specificity is typically the function of post-hybrid polypeptide', and “Pt|P-83 protein’ as used herein inter ization washes, the critical factors being the ionic strength changeably refers to a polypeptide having insecticidal activ and temperature of the final wash solution. For DNA-DNA ity including but not limited to insecticidal activity against hybrids, the Tm can be approximated from the equation of one or more insect pests of the Lepidoptera and/or Coleop Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: tera orders, and is sufficiently homologous to the protein of Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)- SEQ ID NO: 1. A variety of PtIP-83 polypeptides are 500/L: where M is the molarity of monovalent cations, '% contemplated. Sources of PtIP-83 polypeptides or related GC is the percentage of guanosine and cytosine nucleotides proteins are fern species selected from but not limited to in the DNA,% form is the percentage of formamide in the Polypodium punctatum, Lygodium flexuosum, Microsorum hybridization solution, and L is the length of the hybrid in musifolium, Adiantum peruvianum, Adiantum trapeziforme base pairs. The Tm is the temperature (under defined ionic and Adiantum pedatum. strength and pH) at which 50% of a complementary target I0120 "Sufficiently homologous is used herein to refer to sequence hybridizes to a perfectly matched probe. Tm is an amino acid sequence that has at least about 40%, 45%, reduced by about 1° C. for each 1% of mismatching; thus, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, Tm, hybridization, and/or wash conditions can be adjusted 60%, 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, to hybridize to sequences of the desired identity. For 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, example, if sequences with 90% identity are sought, the Tm 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, can be decreased 10° C. Generally, stringent conditions are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or selected to be about 5° C. lower than the thermal melting greater sequence homology compared to a reference point (Tm) for the specific sequence and its complement at sequence using one of the alignment programs described a defined ionic strength and pH. However, severely stringent herein using standard parameters. In some embodiments the conditions can utilize a hybridization and/or wash at 1, 2, 3 sequence homology is against the full length sequence of a or 4°C. lower than the thermal melting point (Tm); mod PtIP-83 polypeptide. In some embodiments the PtIP-83 erately stringent conditions can utilize a hybridization and/or polypeptide has at least about 40%, 45%, 50%, 51%, 52%, wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, point (Tm); low stringency conditions can utilize a hybrid 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, ization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, the thermal melting point (Tm). Using the equation, hybrid 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, US 2016/0347799 A1 Dec. 1, 2016 37
93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, identity compared to SEQID NO: 1. One of skill in the art SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ will recognize that these values can be appropriately ID NO: 767, SEQID NO: 768 or SEQID NO: 769, e.g., by adjusted to determine corresponding homology of proteins proteolysis, by insertion of a start codon, by deletion of the taking into account amino acid similarity and the like. In codons encoding the deleted amino acids and concomitant Some embodiments the sequence identity is calculated using insertion of a start codon, and/or insertion of a stop codon. ClustalW algorithm in the ALIGNX(R) module of the Vector 0.124. In some embodiments, the PtP-83 polypeptide NTIR) Program Suite (Invitrogen Corporation, Carlsbad, fragments encompassed herein result from the removal of Calif.) with all default parameters. In some embodiments the the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino sequence identity is across the entire length of polypeptide acids relative to SEQID NO: 1, SEQID NO:3, SEQID NO: calculated using ClustalW algorithm in the ALIGNX(R) mod 5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, SEQ ID ule of the Vector NTIR Program Suite (Invitrogen Corpo NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, ration, Carlsbad, Calif.) with all default parameters. SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ 0121. As used herein, the terms “protein,”99 “peptide&g mol ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID ecule,” or “polypeptide' includes any molecule that com NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQID NO: prises five or more amino acids. It is well known in the art 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, that protein, peptide or polypeptide molecules may undergo SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ modification, including post-translational modifications, ID NO: 767, SEQID NO: 768, SEQID NO: 769 or variants Such as, but not limited to, disulfide bond formation, gly thereof, e.g., by proteolysis or by insertion of a start codon, cosylation, phosphorylation or oligomerization. Thus, as by deletion of the codons encoding the deleted amino acids used herein, the terms “protein,” “peptide molecule' or and concomitant insertion of a start codon. "polypeptide' includes any protein that is modified by any 0.125 “Variants' as used herein refers to proteins or biological or non-biological process. The terms "amino polypeptides having an amino acid sequence that is at least acid and “amino acids’ refer to all naturally occurring about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, L-amino acids. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 0122. A “recombinant protein’ or “recombinant polypep 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the tide' is used herein to refer to a protein that is no longer in parental amino acid sequence. its natural environment, for example in vitro or in a recom I0126. In some embodiments a PtIP-83 polypeptide com binant bacterial or plant host cell. A 'purified protein’ or prises an amino acid sequence having at least 40%, 45%, “purified polypeptide' is used herein to refer to a protein that 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, is substantially free of cellular material. A PtIP-83 polypep 60%, 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, tide that is substantially free of cellular material includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, preparations of protein having less than about 30%. 20%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 10% or 5% (by dry weight) of non-pesticidal protein (also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% referred to herein as a “contaminating protein'). identity to the amino acid sequence of SEQID NO: 1, SEQ 0123 “Fragments’ or “biologically active portions' ID NO:3, SEQ ID NO. 5, SEQ ID NO: 7, SEQ ID NO:9, include polypeptide fragments comprising amino acid SEQID NO: 11, SEQID NO: 13, SEQID NO: 15, SEQ ID sequences sufficiently identical to a PtIP-83 polypeptide and NO: 17, SEQID NO: 19, SEQID NO: 21, SEQID NO. 23, that exhibit insecticidal activity. "Fragments' or “biologi SEQID NO: 716, SEQID NO: 754, SEQID NO: 755, SEQ cally active portions of PtIP-83 polypeptides includes frag ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID ments comprising amino acid sequences sufficiently identi NO: 759, SEQID NO: 760, SEQ ID NO: 761, SEQID NO: cal to the amino acid sequence set forth in SEQ ID NO: 1. 762, SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQID NO:3, SEQID NO:5, SEQID NO: 7, SEQID NO: SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or 9, SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ SEQ ID NO: 769, wherein the PtIP-83 polypeptide has ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQID NO: insecticidal activity. 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, I0127. In some embodiments a PtIP-83 polypeptide com SEQID NO: 756, SEQID NO: 757, SEQID NO: 758, SEQ prises an amino acid sequence having at least about 80%, ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, NO: 762, SEQID NO: 763, SEQID NO: 764, SEQID NO: 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 765, SEQ ID NO: 766, SEQID NO: 767, SEQID NO: 768 identity across the entire length of the amino acid sequence or SEQ ID NO: 769, wherein the PtIP-83 polypeptide has of SEQID NO: 1, SEQ ID NO:3, SEQID NO. 5, SEQ ID insecticidal activity. Such biologically active portions can be NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, prepared by recombinant techniques and evaluated for insec SEQID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQ ID ticidal activity. In some embodiments, the PtIP-83 polypep NO: 21, SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: tide fragment is an N-terminal and/or a C-terminal trunca 754, SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, tion of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, SEQID NO: 758, SEQID NO: 759, SEQID NO: 760, SEQ 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID 34 or more amino acids from the N-terminus and/or C-ter NO: 764, SEQID NO: 765, SEQ ID NO: 766, SEQID NO: minus relative to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID 767, SEQ ID NO: 768 or SEQ ID NO: 769. NO:5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, SEQ I0128. In some embodiments a PtIP-83 polypeptide com ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQID NO: prises an amino acid sequence having at least about 80%, 19, SEQID NO:21, SEQID NO. 23, SEQID NO: 716, SEQ 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% NO: 757, SEQID NO: 758, SEQID NO: 759, SEQID NO: identity across the entire length of the amino acid sequence US 2016/0347799 A1 Dec. 1, 2016
of SEQID NO: 1, SEQ ID NO:3, SEQID NO. 5, SEQ ID SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ NO: 7, SEQ ID NO:9, SEQID NO: 11, SEQ ID NO: 13, ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. SEQID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQ ID 0.132. In some embodiments a PtIP-83 polypeptide com NO: 21, SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: prises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 754, SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, SEQID NO: 758, SEQID NO: 759, SEQID NO: 760, SEQ 25, 26, 27, 28 or 29 amino acid substitutions, in any ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID combination, compared to the native amino acid at the NO: 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: corresponding position of SEQ ID NO: 1, SEQ ID NO: 3, 767, SEQ ID NO: 768 or SEQID NO: 769, and has at least SEQID NO:5, SEQID NO: 7, SEQID NO:9, SEQID NO: one amino acid Substitution, deletion, insertion, and/or addi 11, SEQID NO: 13, SEQID NO: 15, SEQID NO: 17, SEQ tion at the N-terminus or C-terminus compared to the native ID NO: 19, SEQ ID NO: 21, SEQID NO. 23, SEQID NO: sequence of SEQID NO: 1, SEQID NO:3, SEQ ID NO: 5, 716, SEQID NO: 754, SEQID NO: 755, SEQID NO: 756, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID SEQID NO: 757, SEQID NO: 758, SEQID NO: 759, SEQ NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ NO: 763, SEQID NO: 764, SEQ ID NO: 765, SEQID NO: ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: NO: 757, SEQID NO: 758, SEQID NO: 759, SEQID NO: 769. 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ I0133. In some embodiments the PtIP-83 polypeptide ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. comprises an amino acid sequence of SEQ ID NO: 1, SEQ 0129. In some embodiments a PtIP-83 polypeptide com ID NO:3, SEQ ID NO. 5, SEQ ID NO: 7, SEQ ID NO:9, prises an amino acid sequence of SEQ ID NO: 1, SEQ ID SEQID NO: 11, SEQID NO: 13, SEQID NO: 15, SEQ ID NO:3, SEQID NO. 5, SEQID NO:7, SEQID NO:9, SEQ NO: 17, SEQID NO: 19, SEQID NO: 21, SEQID NO. 23, ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQID NO: SEQID NO: 716, SEQID NO: 754, SEQID NO: 755, SEQ 17, SEQID NO: 19, SEQID NO: 21, SEQID NO. 23, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 759, SEQID NO: 760, SEQ ID NO: 761, SEQID NO: NO: 756, SEQID NO: 757, SEQID NO: 758, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, 759, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQ SEQ ID NO: 769. ID NO: 766, SEQID NO: 767, SEQID NO: 768 or SEQID I0134. In some embodiments the PtIP-83 polypeptide NO: 769 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, comprises a non-naturally occurring amino acid sequence. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, As used herein the term “non-naturally occurring amino acid 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, sequence” means an amino acid sequence not found in 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, nature. 64, 65, 66, 67, 68, 69, 70 or more amino acid substitutions I0135) In some embodiments the PtIP-83 polypeptide is compared to the native amino acid at the corresponding not the polypeptide of SEQID NO: 1, SEQID NO:3, SEQ position of SEQID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, ID NO: 5, SEQID NO: 7, SEQID NO:9, SEQID NO: 11, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID SEQID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, NO: 19, SEQID NO: 21, SEQID NO. 23, SEQID NO: 716, SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ SEQID NO: 754, SEQID NO: 755, SEQID NO: 756, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID ID NO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 757, SEQID NO: 758, SEQID NO: 759, SEQID NO: NO: 760, SEQID NO: 761, SEQ ID NO: 762, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, 763, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. 0.136. In some embodiments the PtIP-83 polypeptide is a 0130. In some embodiments the sequence identity is variant of the polypeptide of SEQ ID NO: 1, SEQ ID NO: across the entire length of the polypeptide calculated using 3, SEQ ID NO. 5, SEQID NO: 7, SEQ ID NO:9, SEQ ID ClustalW algorithm in the ALIGNX(R) module of the Vector NO: 11, SEQID NO: 13, SEQID NO: 15, SEQID NO: 17, NTIR) Program Suite (Invitrogen Corporation, Carlsbad, SEQID NO: 19, SEQ ID NO: 21, SEQID NO. 23, SEQ ID Calif.) with all default parameters. NO: 716, SEQID NO: 754, SEQ ID NO: 755, SEQID NO: 0131. In some embodiments a PtIP-83 polypeptide com 756, SEQID NO: 757, SEQID NO: 758, SEQID NO: 759, prises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, SEQ 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, NO: 766, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, NO: 769, wherein the PtIP-83 polypeptide variant has at 57, 58, 59 or 60 amino acid substitutions, in any combina least one amino acid Substitution, deletion, insertion, and/or tion, compared to the native amino acid at the corresponding addition at the N-terminus or C-terminus compared to SEQ position of SEQID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, ID NO: 1, SEQ ID NO:3, SEQ ID NO. 5, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, NO: 15, SEQID NO: 17, SEQID NO: 19, SEQID NO: 21, SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ SEQ ID NO. 23, SEQ ID NO: 716, SEQID NO: 754, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID ID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 757, SEQID NO: 758, SEQID NO: 759, SEQID NO: NO: 758, SEQID NO: 759, SEQ ID NO: 760, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, 761, SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, US 2016/0347799 A1 Dec. 1, 2016 39
SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, SEQ at position 648 is Met, Ala, Cys, Asp, Glu, Phe, Gly. His, ID NO: 768 or SEQ ID NO: 769. Lys, Leu, ASn, Pro, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the 0.137 In some embodiments PtIP-83 polypeptide com amino acid at position 649 is Pro, Ala, Cys, Asp, Glu, Phe, prising an amino acid sequence of any one of SEQ ID NO: Gly. His, Lys, Met, Asn., Gln, Arg, Ser. Thr, Trp or Tyr; the 236-299, SEQID NO:334-367, SEQID NO:398-427, SEQ amino acid at position 650 is Thr, Ala, Cys, Asp, Phe, Gly, ID NO. 518-607, SEQID NO: 640-645, and SEQ ID NO: His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Val or Tyr; the 728-737. amino acid at position 651 is Glu, Ala, Cys, Asp, Gly, His, 0.138. In some embodiments the PtIP-83 polypeptide is a Ile, Leu, Met, ASn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; the variant of SEQID NO: 1, wherein the amino acid at position amino acid at position 652 is Leu, Cys, Phe, Ile, Lys, Met, 53 is Val, Ala, Cys or Thr; the amino acid at position 54 is Pro, Arg, Ser, Thr or Val; the amino acid at position 653 is Lys, Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met, ASn, Gln, Thr, Cys, Asp, Glu, Phe, Gly. His, Ile, Lys, Leu, Pro, Arg, Arg, Ser or Thr; the amino acid at position 55 is Arg, Ala, Ser, Val or Trp; the amino acid at position 654 is Thr, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn., Gln, Ser. Thr, Cys, Phe, Ile, Lys, Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; Val, Trp or Tyr; the amino acid at position 56 is Leu, Glu, the amino acid at position 655 is Trp, Phe or Tyr; the amino Phe, Ile, Met, Thror Val; the amino acid at position 57 is Tyr, acid at position 771 is Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Cys, Ile, Leu, Met, Thr or Val; the amino acid at position 58 Lys, Leu, Asn. Ser. Thr, Val, Trp or Tyr; the amino acid at is Val, Cys, Ile or Leu; the amino acid at position 59 is Phe, position 772 is Arg, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Val or Tyr; the amino acid at position 60 is Ala, Lys, Leu, Met, Pro, Gln, Ser. Thr, Val, Trp or Tyr; the amino Cys, Gly, Ser, Thr or Val; the amino acid at position 61 is acid at position 773 is Asp, Ala, Glu, Phe, Gly, His, Ile, Lys, Asp, Glu, His or Ser; the amino acid at position 62 is Val, Leu, Met, Asn., Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino Ala, Cys, Ile, Leu or Thr; the amino acid at position 63 is acid at position 774 is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Val, Ala, Cys, Ile, Leu, Met or Thr; the amino acid at Met, ASn, Pro, Arg, Ser. Thr, Val, Trp or Tyr; the amino acid position 64 is Glu, Ala, Cys, Phe, Gly, His, Ile, Leu, Met, at position 775 is Val, Ala, Cys, Asp, Glu, Gly. His, Ile, Asn. ASn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino acid at Pro, Gln, Arg, Ser. Thror Tyr; the amino acid at position 776 position 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn., Gln, is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, ASn, Pro, Thr, Val or Trp; the amino acid at position 66 is Pro, Asp, Gln, Arg, Ser, Thr, Val or Tyr; the amino acid at position 777 Gly, Met, Gln or Arg; the amino acid at position 363 is Gln, is Pro, Ala, Cys, Asp, Glu, Phe, Gly. His, Lys, Leu, Met, Asn. Ala, Cys, Glu, Phe, Gly. His, Lys, Leu, Asn, Arg, Ser. Thr, Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 778 Val or Trp; the amino acid at position 364 is Ile, Ala, Cys, is Phe, Ala, His, Ile, Leu, Met, ASn, Gln, Ser, Val, Trp or Tyr; Glu, Phe, His, Lys, Leu, Met, Asn., Gln, Ser. Thr, Val, Trp or the amino acid at position 779 is Gln, Ala, Cys, Asp, Glu, Tyr; the amino acid at position 365 is Leu, Ala, Glu, Phe, Gly. His, Lys, Leu, Asn. Pro, Arg, Ser. Thr or Val; the amino Gly. His, Ile, Lys, Met, Asn, Arg, Val, Trp or Tyr; the amino acid at position 780 is Ala, Cys, ASn, Pro, Gln or Ser; the acid at position 366 is Gly, Ala, Cys, Phe, His, Ile, Lys, Leu, amino acid at position 781 is Ala, Cys, Asp, Glu, Phe, Gly, Met, Asn. Ser. Thr or Val; the amino acid at position 367 is His, Ile, Asn., Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, ASn, acid at position 782 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Pro, Gln, Arg, Thr, Val or Trp; the amino acid at position 368 Lys, Met, Pro, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino is Tyr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, acid at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn. ASn, Pro, Gln, Arg, Ser, Thr, Val or Trp; the amino acid at Gln, Arg, Ser. Thr or Val; the amino acid at position 784 is position 369 is Leu, Ala, Cys, Asp, Phe, Gly, Ile, Met, Thr Leu, Ala, Glu, Phe, His, Ile, Lys, Met, ASn, Pro, Gln, Ser, or Val; the amino acid at position 370 is Leu, Ala, Cys, Asp, Thr, Val or Trp; the amino acid at position 785 is Asn, Ala, Glu, Phe, Gly. His, Ile, Lys, Met, Gln, Arg, Ser. Thr, Val, Trp Cys, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Gln, Arg, Ser, or Tyr, the amino acid at position 371 is Gln, Ala, Cys, Asp, Thr, Val, Trp or Tyr; and the amino acid at position 786 is Glu, Phe, Gly, Ile, Lys, Leu, Asn, Arg, Ser. Thr, Val or Trp; Tyr, Phe, Ile, Leu or Trp. the amino acid at position 372 is Gln, Ala, Cys, Asp, Phe, (0.139. In some embodiments the PtIP-83 polypeptide is a Gly. His, Ile, Leu, Asn, Arg, Ser, Val or Tyr; the amino acid variant of SEQID NO: 1, wherein the amino acid at position at position 373 is Asn, Ala, Cys, Asp, Phe, Gly. His, Ile, Lys, 1 is Met or deleted; the amino acid at position 2 is Ala or Gln, Ser. Thr, Val or Trp; the amino acid at position 556 is deleted; the amino acid at position 3 is Leu, Val or deleted; Trp, Phe, Thr or Tyr; the amino acid at position 557 is Arg, the amino acid at position 4 is Val, Met or Leu; the amino Cys, Asp, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Gln, Ser, acid at position 7 is Gly or Ser; the amino acid at position Thr, Val, Trp or Tyr; the amino acid at position 558 is Ala, 8 is Lys or Thr; the amino acid at position 10 is Phe or Tyr; Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, ASn, Pro, Gln, Arg, the amino acid at position 11 is Glu or Arg; the amino acid Ser, Val, Trp or Tyr; the amino acid at position 559 is Lys, at position 18 is Met or Ile; the amino acid at position 19 is Ala, Cys, Phe, Gly. His, Ile, Leu, Asn., Gln, Arg, Ser. Thr, Val Gly, Pro or Ala; the amino acid at position 20 is Val or or Tyr; the amino acid at position 560 is Cys, Ala, Phe, Gly, deleted; the amino acid at position 21 is Leu or Val; the Ile, Met, Asn, Arg, Ser. Thr or Val; the amino acid at position amino acid at position 23 is Arg or Gln; the amino acid at 561 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, position 37 is Val or Leu; the amino acid at position 38 is Arg Asn, Arg, Ser. Thr, Val or Tyr; the amino acid at position 562 or ASn; the amino acid at position 40 is Ala or Ser; the amino is Asn. Cys, Asp, Glu, Gly, His, Leu, Met, Arg, Ser. Thr, Val acid at position 43 is ASn or Asp; the amino acid at position or Tyr; the amino acid at position 563 is Val, Ala, Cys, Asp, 45 is Gly or Ala; the amino acid at position 46 is Glin or Glu; Phe, His, Ile, Leu, Met, Asn., Gln, Thr or Trp; the amino acid the amino acid at position 48 is Glu, Pro or Val; the amino at position 564 is Ala, Cys, Gly, Met, Gln, Ser. Thr, Val, Trp acid at position 51 is Glu or Gly; the amino acid at position or Tyr; the amino acid at position 646 is Leu, Ala, Cys, Gly, 52 is Lys, Arg or Thr; the amino acid at position 56 is Leu Ile, Met, Asn., Gln, Ser. Thr or Val; the amino acid at position or Val; the amino acid at position 59 is Phe or Leu; the amino 647 is Leu, Asp, Gly, Met, Asn., Gln or Thr; the amino acid acid at position 66 is Pro or Ala; the amino acid at position US 2016/0347799 A1 Dec. 1, 2016 40
67 is Val, Pro or Thr; the amino acid at position 68 is Val, is Leu, Thr, Seror Arg; the amino acid at position 185 is Arg, Arg, Phe or Gly; the amino acid at position 69 is Glu, Ala Gly, Asp or Ala; the amino acid at position 186 is Pro, Val or Lys; the amino acid at position 70 is Trp, Thr. His, Tyr or or Gln; the amino acid at position 187 is Asp, Thr or Ser; the Arg; the amino acid at position 71 is Arg, Pro or deleted; the amino acid at position 188 is His, Gly or Ala; the amino acid amino acid at position 72 is Trp, Asp, Leu or deleted; the at position 189 is Ala, Arg, Pro or deleted; the amino acid at amino acid at position 73 is Pro, Gln, Asn. His or deleted; position 190 is Leu, Asn or deleted; the amino acid at the amino acid at position 74 is Pro, Met or Thr; the amino position 191 is Tyr or deleted; the amino acid at position 192 acid at position 75 is Gln, His or Arg; the amino acid at is Ser, Ile, Val or Asn; the amino acid at position 193 is Thr position 76 is Ile, Met or Leu; the amino acid at position 84 or Asp; the amino acid at position 194 is Thr or Ser; the is Ile or Val; the amino acid at position 91 is Trp or Phe; the amino acid at position 195 is Met or Thr; the amino acid at amino acid at position 93 is Thr or Ile; the amino acid at position 196 is Gln, His, Leu or Ser; the amino acid at position 94 is Asp or Gly; the amino acid at position 96 is position 197 is Ala, Gly or Leu; the amino acid at position Arg or Ser; the amino acid at position 97 is Gln, Phe or Arg; 198 is Thr, Glu or Ala; the amino acid at position 199 is Pro the amino acid at position 98 is Ser or deleted; the amino or Arg; the amino acid at position 200 is Asn. Ser. Thr or acid at position 99 is Asp or Ala; the amino acid at position Gly; the amino acid at position 201 is Ala, Leu, Glu or Trp; 100 is Thr or Ala; the amino acid at position 101 is Glu, Thr the amino acid at position 202 is Ser, Asp, Phe or Leu; the or Trp the amino acid at position 103 is His, Arg, Glu or Gln; amino acid at position 203 is His, Pro, Gly or Ser; the amino the amino acid at position 105 is Thr or Pro; the amino acid acid at position 204 is Ile, Trp. His or Gly; the amino acid at position 108 is Lys, Gln or Glu; the amino acid at position at position 205 is Ser, Asn or Ile; the amino acid at position 109 is Leu or Val; the amino acid at position 111 is Ala or 206 is Ala, Gly, Asp, Tyr or Arg; the amino acid at position Thr; the amino acid at position 112 is Ile, Arg, Thr or 207 is Phe, Val or Leu; the amino acid at position 208 is Asn. deleted; the amino acid at position 113 is Gln, Ala, Gly or Ser, Pro or Leu; the amino acid at position 210 is Arg, Asp, deleted; the amino acid at position 114 is Arg, Glu or Ile; the Glu or Tyr; the amino acid at position 211 is Ile, Ser or Thr: amino acid at position 115 is Glu or Gln; the amino acid at the amino acid at position 212 is Val, Ala or Asp; the amino position 116 is Glu, ASn, Gln or Arg; the amino acid at acid at position 214 is Pro or Arg; the amino acid at position position 117 is Asn. Val, Tyr or Phe; the amino acid at 215 is Ser or Thr; the amino acid at position 217 is Tyr or position 118 is Arg or Lys; the amino acid at position 119 is Phe; the amino acid at position 218 is Arg or Ser; the amino Trp or Ser; the amino acid at position 122 is Thr, Lys or Ala; acid at position 219 is Val or Ala; the amino acid at position the amino acid at position 124 is Ala or Thr; the amino acid 220 is Cys, Leu or Ser; the amino acid at position 221 is Pro at position 126 is Gly or Asp; the amino acid at position 127 or His; the amino acid at position 222 is Leu, Arg or Ser; the is Met or Ala; the amino acid at position 128 is Asn or Lys: amino acid at position 224 is ASn or Ser; the amino acid at the amino acid at position 131 is Val, Ile or Thr; the amino position 225 is Asp, Arg or Thr; the amino acid at position acid at position 133 is Ile or Val; the amino acid at position 226 is Thr or ASn; the amino acid at position 227 is Asp, Leu 134 is His or Tyr; the amino acid at position 135 is Ala or or deleted; the amino acid at position 228 is Thr or deleted; Gly; the amino acid at position 137 is Glu or Lys; the amino the amino acid at position 229 is Tyr or deleted; the amino acid at position 139 is Glin or Glu; the amino acid at position acid at position 230 is Leu or deleted; the amino acid at 140 is Val, Arg or Leu; the amino acid at position 141 is Gly position 231 is Gly or deleted; the amino acid at position 232 or Ser; the amino acid at position 142 is Val or Pro; the is Ile or deleted; the amino acid at position 233 is Pro or amino acid at position 144 is Thr, Leu, Phe or Tyr; the amino deleted; the amino acid at position 234 is Ala, Pro or deleted; acid at position 145 is Met, Pro or ASn; the amino acid at the amino acid at position 235 is Asp, Ile or Val; the amino position 146 is Ser, Gly or ASn; the amino acid at position acid at position 236 is Val, Ser or Glu; the amino acid at 147 is Trp or ASn; the amino acid at position 148 is Ser, Ala position 237 is Ala, Phe or Tyr; the amino acid at position or Pro; the amino acid at position 149 is Ser or deleted; the 238 is Ala or Thr; the amino acid at position 239 is Val, Ser amino acid at position 150 is Val, Ile or Tyr; the amino acid or Gly; the amino acid at position 240 is Leu or Ile; the at position 152 is Arg, Ala, Val or Gly; the amino acid at amino acid at position 243 is Asp or Glu; the amino acid at position 154 is Ser, Trp or Glu; the amino acid at position position 249 is Asn or Ser; the amino acid at position 252 is 156 is Leu, Asp or Gln; the amino acid at position 158 is Ser Leu or Met; the amino acid at position 257 is Thr or Ser; the or Cys; the amino acid at position 159 is Val, Thr or Ile; the amino acid at position 259 is His or Leu; the amino acid at amino acid at position 162 is Ser or Ala; the amino acid at position 266 is Ala or Val; the amino acid at position 267 is position 163 is Gly or deleted; the amino acid at position 164 Cys or Gly; the amino acid at position 268 is His, Arg or Tyr; is Phe or deleted; the amino acid at position 165 is Arg or the amino acid at position 272 is Asp or Glu; the amino acid Ala; the amino acid at position 166 is Ala, Arg, Met or Phe: at position 273 is Val, Met, Ile or Leu; the amino acid at the amino acid at position 167 is Val or His; the amino acid position 274 is Val or Met; the amino acid at position 278 is at position 168 is Ser or Asn; the amino acid at position 169 Gly or Ala; the amino acid at position 279 is Glu or Val; the is Val, His or Thr; the amino acid at position 170 is Phe or amino acid at position 281 is Leu or Ala; the amino acid at Val; the amino acid at position 171 is Glu, Asn or Asp; the position 282 is ASn, Leu or Ile; the amino acid at position amino acid at position 172 is Val, Ala, Arg or Glu, the amino 285 is Asn or Ser; the amino acid at position 286 is Lys, Asp acid at position 175 is Ser, Arg or Trp; the amino acid at or Glu; the amino acid at position 287 is Leu or Val; the position 176 is Val or Ile; the amino acid at position 177 is amino acid at position 290 is Pro, Gln or Arg; the amino acid Arg or Ile; the amino acid at position 179 is Thr, Ile, Val or at position 291 is Leu or Val; the amino acid at position 292 Ser; the amino acid at position 180 is Leu, Phe or Thr; the is Lys or Val; the amino acid at position 293 is Glu or Gln; amino acid at position 181 is Gly, Thr, Gln or Ser; the amino the amino acid at position 294 is Ser, ASn or Lys; the amino acid at position 182 is Ala, Leu, Phe or Ile; the amino acid acid at position 295 is Thr or Ser; the amino acid at position at position 183 is Thr or Gly; the amino acid at position 184 296 is Gln or His; the amino acid at position 297 is Leu or US 2016/0347799 A1 Dec. 1, 2016
Met; the amino acid at position 300 is Ser or Thr; the amino is Met or Leu; the amino acid at position 593 is Met or Ile: acid at position 301 is Glu or Ala; the amino acid at position the amino acid at position 595 is Arg or Gln; the amino acid 302 is Ser, Pro or Ala; the amino acid at position 304 is Lys at position 596 is Ser or Thr; the amino acid at position 597 or ASn; the amino acid at position 313 is Val or Ile; the amino is Glin or His; the amino acid at position 607 is Ala or Val; acid at position 314 is His, Glu or Gln; the amino acid at the amino acid at position 608 is Asp or ASn; the amino acid position 315 is Ala, Cys or Ser; the amino acid at position at position 612 is Tyr, His or Phe; the amino acid at position 316 is Ala or Val; the amino acid at position 317 is Met or 617 is Thr or Ile; the amino acid at position 618 is Gln or Ile; the amino acid at position 319 is Met or Ile; the amino His; the amino acid at position 625 is Arg or Ser; the amino acid at position 320 is Val or Gly; the amino acid at position acid at position 626 is Met or Ile; the amino acid at position 321 is Arg or Pro; the amino acid at position 322 is Ile or 628 is Leu or Ile; the amino acid at position 633 is Ile or Met; Phe, the amino acid at position 323 is Gly or Val; the amino the amino acid at position 634 is Leu or Met; the amino acid acid at position 324 is Leu or Ser; the amino acid at position at position 642 is Arg or Met; the amino acid at position 648 336 is Ser or ASn; the amino acid at position 339 is ASn, Lys is Met or Thr; the amino acid at position 651 is Glu or Gln; or Arg; the amino acid at position 350 is Arg or Gln; the the amino acid at position 654 is Thr, Val or Ala; the amino amino acid at position 351 is Glu or Asp; the amino acid at acid at position 658 is Gly or Arg; the amino acid at position position 353 is Lys or Arg; the amino acid at position 354 is 663 is Gly or Ala; the amino acid at position 664 is Asp or Gln or Arg; the amino acid at position 355 is Phe or Leu; the Asn; the amino acid at position 668 is Ala or Thr; the amino amino acid at position 356 is Lys or Arg; the amino acid at acid at position 669 is Glin or His; the amino acid at position position 360 is Ile, Val or Ala; the amino acid at position 365 671 is Asn or Ser the amino acid at position 675 is Ile, Val is Leu or Phe; the amino acid at position 371 is or Glu; the or Ser; the amino acid at position 678 is Met, Ile, Ala or Thr: amino acid at position 372 is or Lys; the amino acid at the amino acid at position 682 is Pro or Gln; the amino acid position 374 is Arg or Lys; the amino acid at position 376 is at position 683 is Ser or Pro; the amino acid at position 685 Phe or Leu; the amino acid at position 378 is Glu or Asp; the is Asp or ASn; the amino acid at position 694 is Asp or Gly; amino acid at position 381 is Leu or Val; the amino acid at the amino acid at position 697 is Asn or Ser; the amino acid position 388 is Ala or Ser; the amino acid at position 395 is at position 704 is Glu or Gly; the amino acid at position 714 Arg or Lys; the amino acid at position 396 is Glu, Gln or is Ala or Gly; the amino acid at position 721 is Ser or Phe: Gly; the amino acid at position 399 is Asp or Asn; the amino the amino acid at position 722 is Ser or ASn; the amino acid acid at position 400 is ASn, Thr or Asp; the amino acid at at position 724 is Ser or Thr; the amino acid at position 734 position 401 is Thr or Ala; the amino acid at position 402 is is His or Gln; the amino acid at position 736 is Val or Ala; Phe, Ile or Leu; the amino acid at position 406 is Asp or Glu; the amino acid at position 737 is Lys or Gln; the amino acid the amino acid at position 408 is Leu or Met; the amino acid at position 739 is Ala or Ser; the amino acid at position 740 at position 410 is Gly or Leu; the amino acid at position 414 is Ser or Met; the amino acid at position 741 is Gly or Asn; is Ala or Glu; the amino acid at position 416 is Ser, Asn or the amino acid at position 742 is Ile or Gly; the amino acid Asp; the amino acid at position 417 is Ser, Arg or Gly; the at position 743 is Gly or deleted; the amino acid at position amino acid at position 423 is Lys or Gln; the amino acid at 745 is Gly or Asp; the amino acid at position 751 is Thr, Ser position 431 is Arg or Lys; the amino acid at position 432 is or Ala; the amino acid at position 753 is Glin or Arg; the Gln or Glu; the amino acid at position 436 is Arg or Glu; the amino acid at position 754 is Thr or Ser; the amino acid at amino acid at position 440 is ASn or Arg; the amino acid at position 756 is Thr or Ile; the amino acid at position 757 is position 442 is Leu or Val; the amino acid at position 447 is Val or Ile; the amino acid at position 766 is Ile or Val; the Ser, Lys or Arg; the amino acid at position 448 is Ala or Ser; amino acid at position 773 is Asp or Glu; the amino acid at the amino acid at position 451 is Gln or Met; the amino acid position 774 is Glin or Glu; the amino acid at position 776 is at position 453 is Gly or Ala; the amino acid at position 455 Leu or Met; the amino acid at position 777 is Pro or Thr; the is Ala or Val; the amino acid at position 457 is Leu or Val; amino acid at position 782 is Ala, Asp or Val; the amino acid the amino acid at position 467 is Val or Ala; the amino acid at position 786 is Tyr or Phe; the amino acid at position 787 at position 471 is Gly or Ala; the amino acid at position 475 is His or Gln; the amino acid at position 788 is Tyr or Met; is Ser or Asn; the amino acid at position 483 is Gly or Ala; the amino acid at position 789 is Ala or Arg; the amino acid the amino acid at position 493 is Glin or Gly; the amino acid at position 790 is Tyr or Thr; the amino acid at position 791 at position 504 is Val or Ile; the amino acid at position 506 is Arg or Ala; the amino acid at position 792 is Leu or Ser; is Asp or His; the amino acid at position 509 is Asp or ASn; the amino acid at position 796 is Asp or Glu; the amino acid the amino acid at position 510 is Ser or Ala; the amino acid at position 797 is Ser, Thr or Ala the amino acid at position at position 512 is Glu or Asp; the amino acid at position 515 802 is Glu or Gln; the amino acid at position 806 is Gln, Asp, is Gly or Ser; the amino acid at position 516 is Glin or His: Glu or His; the amino acid at position 810 is Lys or Thr; the the amino acid at position 517 is Ile or Leu; the amino acid amino acid at position 819 is Arg or His; the amino acid at at position 519 is Asp, Gly or Gln; the amino acid at position position 829 is Lys, Ser, Ala or Pro; the amino acid at 522 is Val, Glu, Pro or Val; the amino acid at position 525 position 832 is Ala, Lys or Glu; the amino acid at position is Glu or Asp; the amino acid at position 526 is Leu or Met; 833 is Gly or Glu; the amino acid at position 842 is Leu or the amino acid at position 539 is Val or Ile; the amino acid Pro; the amino acid at position 847 is Glin or Glu; the amino at position 555 is Val or Ala; the amino acid at position 557 acid at position 848 is Ile or Val; the amino acid at position is Arg or Lys; the amino acid at position 563 is Val or Met; 849 is Val or Ala; the amino acid at position 855 is Thr or the amino acid at position 571 is Ser or Cys; the amino acid Met; the amino acid at position 860 is Ile or Val; and the at position 575 is Val or Glu; the amino acid at position 577 amino acid at position 864 is His or Gln. is Met or Ile; the amino acid at position 579 is Glu or Gln; 0140. In some embodiments the PtIP-83 polypeptide is a the amino acid at position 583 is Asp or Glu; the amino acid variant of SEQID NO: 1, wherein the amino acid at position at position 589 is Met or Leu; the amino acid at position 590 1 is Met or deleted; the amino acid at position 2 is Ala or US 2016/0347799 A1 Dec. 1, 2016 42 deleted; the amino acid at position 3 is Leu, Val, Ile or position 141 is Gly, Ala, Thr or Ser; the amino acid at deleted; the amino acid at position 4 is Val, Met, Ile or Leu: position 142 is Val, Ile, Leu or Pro; the amino acid at the amino acid at position 7 is Gly, Thr or Ser; the amino position 144 is Thr, Leu, Phe, Ile, Val or Tyr; the amino acid acid at position 8 is Lys, Arg, Ser or Thr, the amino acid at at position 145 is Met, Pro, Gln or Asn; the amino acid at position 10 is Phe, Trp or Tyr; the amino acid at position 11 position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acid is Glu, Asp, Lys or Arg; the amino acid at position 18 is Met, at position 147 is Trp, Gln, Tyr or Asn; the amino acid at Val, Leu or Ile; the amino acid at position 19 is Gly, Pro or position 148 is Ser, Ala, Thr, Gly or Pro; the amino acid at Ala; the amino acid at position 20 is Val, Ile, Leu or deleted; position 149 is Ser. Thr or deleted; the amino acid at position the amino acid at position 21 is Leu, Ile or Val; the amino 150 is Val, Ile, Leu or Tyr; the amino acid at position 152 is acid at position 23 is Arg, Lys, ASn or Gln; the amino acid Arg, Ala, Val, Ile, Leu, Lys or Gly; the amino acid at position at position 37 is Val, Ile or Leu; the amino acid at position 154 is Ser, Trp, Thr, Asp or Glu; the amino acid at position 38 is Arg, Lys, Gln or ASn; the amino acid at position 40 is 156 is Leu, Asp, Ile, Val, Asn., Glu or Gln; the amino acid at Ala, Gly, Thr or Ser; the amino acid at position 43 is Asn. position 158 is Ser. Thr or Cys; the amino acid at position Gln, Glu or Asp; the amino acid at position 45 is Gly or Ala; 159 is Val, Thr, Leu or Ile; the amino acid at position 162 is the amino acid at position 46 is Gln, Asp, ASn or Glu; the Ser. Thr, Gly or Ala; the amino acid at position 163 is Gly, amino acid at position 48 is Glu, Asp, Pro, Ile, Leu or Val; Ala or deleted; the amino acid at position 164 is Phe or the amino acid at position 51 is Glu, Asp, Ala or Gly; the deleted; the amino acid at position 165 is Arg, Lys, Gly or amino acid at position 52 is Lys, Arg, Ser or Thr, the amino Ala; the amino acid at position 166 is Ala, Arg, Met, Lys or acid at position 56 is Leu, Ile or Val; the amino acid at Phe; the amino acid at position 167 is Val, Ile, Leu or His: position 59 is Phe, Ile, Val or Leu; the amino acid at position the amino acid at position 168 is Ser. Thr, Gln or Asn; the 66 is Pro, Gly or Ala; the amino acid at position 67 is Val, amino acid at position 169 is Val, His, Ile, Leu, Ser or Thr: Pro, Ile, Leu, Seror Thr; the amino acid at position 68 is Val, the amino acid at position 170 is Phe, Ile, Leu or Val; the Arg, Phe, Ile, Leu, Lys or Gly; the amino acid at position 69 amino acid at position 171 is Glu, ASn, Gln or Asp; the is Glu, Ala, Asp, Gly, Arg or Lys; the amino acid at position amino acid at position 172 is Val, Ala, Arg, Ile, Leu, Gly, 70 is Trp, Thr, His, Tyr, Lys or Arg; the amino acid at LyS, Asp or Glu; the amino acid at position 175 is Ser, Arg, position 71 is Arg, Pro, Lys or deleted; the amino acid at Thr, Lys or Trp; the amino acid at position 176 is Val, Leu position 72 is Trp, Asp, Leu, Ile, Val, Glu or deleted; the or Ile; the amino acid at position 177 is Arg, Lys, Leu, Val amino acid at position 73 is Pro, Gln, Asn. His or deleted; or Ile; the amino acid at position 179 is Thr, Ile, Val, Leu or the amino acid at position 74 is Pro, Met, Ser or Thr: the Ser; the amino acid at position 180 is Leu, Phe, Ile, Val, Ser amino acid at position 75 is Gln, His, ASn, Lys or Arg; the or Thr; the amino acid at position 181 is Gly, Thr, Gln, Asn amino acid at position 76 is Ile, Met, Val or Leu; the amino or Ser; the amino acid at position 182 is Ala, Leu, Phe, Val acid at position 84 is Ile, Leu or Val; the amino acid at or Ile; the amino acid at position 183 is Thr, Ser, Ala or Gly: position 91 is Trp or Phe; the amino acid at position 93 is the amino acid at position 184 is Leu, Thr, Ser, Ile, Val, Lys Thr, Ser, Leu, Val or Ile; the amino acid at position 94 is Asp, or Arg; the amino acid at position 185 is Arg, Gly, Asp, Lys, Glu, Ala or Gly; the amino acid at position 96 is Arg, LyS, Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, Thr or Ser; the amino acid at position 97 is Gln, Phe, Asn. Leu, Asn or Gln; the amino acid at position 187 is Asp, Thr, Lys or Arg; the amino acid at position 98 is Ser. Thr or Glu or Ser; the amino acid at position 188 is His, Gly or Ala; deleted; the amino acid at position 99 is Asp, Glu, Gly or the amino acid at position 189 is Ala, Arg, Pro, Lys, Gly or Ala; the amino acid at position 100 is Thr, Ser, Gly or Ala; deleted; the amino acid at position 190 is Leu, Asn. Ile, Val, the amino acid at position 101 is Glu, Thr, Asp, Ser or Trp Gln or deleted; the amino acid at position 191 is Tyr or the amino acid at position 103 is His, Arg, Lys, Glu or Gln; deleted; the amino acid at position 192 is Ser, Ile, Val, Leu, the amino acid at position 105 is Thr, Ser or Pro; the amino Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu acid at position 108 is Lys, Arg, ASn, Asp, Gln or Glu; the or Asp; the amino acid at position 194 is Thr or Ser; the amino acid at position 109 is Leu, Ile or Val; the amino acid amino acid at position 195 is Met or Thr; the amino acid at at position 111 is Ala, Seror Thr; the amino acid at position position 196 is Gln, His, Leu, Asn. Ile, Val, Thr or Ser; the 112 is Ile, Arg, Thr, Leu, Val, Lys, Ser or deleted; the amino amino acid at position 197 is Ala, Gly, Ile, Val or Leu; the acid at position 113 is Gln, Ala, Gly, Asn or deleted; the amino acid at position 198 is Thr, Glu, Ser, Asp, Gly or Ala; amino acid at position 114 is Arg, Glu, Lys, Asp or Ile; the the amino acid at position 199 is Pro, Lys or Arg; the amino amino acid at position 115 is Glu, Asp, ASn or Gln; the acid at position 200 is Asn. Ser. Thr, Gln, Ala or Gly; the amino acid at position 116 is Glu, ASn, Gln, Asp, Lys or Arg; amino acid at position 201 is Ala, Leu, Glu, Ile, Asp or Trp; the amino acid at position 117 is Asn. Val, Tyr, Ile, Leu, Gln, the amino acid at position 202 is Ser, Asp, Phe, Ile, Val, Thr, Trp or Phe; the amino acid at position 118 is Arg or Lys; the Glu or Leu; the amino acid at position 203 is His, Pro, Gly, amino acid at position 119 is Trp, Thr or Ser; the amino acid Ala, Thr or Ser; the amino acid at position 204 is Ile, Trp, at position 122 is Thr, Lys, Ser, Arg or Ala; the amino acid His, Leu, Val, Ala or Gly; the amino acid at position 205 is at position 124 is Ala, Gly, Ser or Thr; the amino acid at Ser, Asn. Leu, Val, Thr, Gln or Ile; the amino acid at position position 126 is Gly, Ala, Glu or Asp; the amino acid at 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid position 127 is Met, Gly or Ala; the amino acid at position at position 207 is Phe, Val, Ile or Leu; the amino acid at 128 is ASn, Gln, Arg or Lys; the amino acid at position 131 position 208 is Asn. Ser, Pro, Gln, Thr, Val, Ile or Leu; the is Val, Ile, Leu, Ser or Thr; the amino acid at position 133 amino acid at position 210 is Arg, Asp, Glu, Lys, Ser or Tyr; is Ile, Leu or Val; the amino acid at position 134 is His or the amino acid at position 211 is Ile, Ser, Leu, Val or Thr; the Tyr; the amino acid at position 135 is Ala or Gly; the amino amino acid at position 212 is Val, Ala, Ile, Leu, Glu, Gly or acid at position 137 is Glu, Asp, Arg or Lys; the amino acid Asp; the amino acid at position 214 is Pro, Lys or Arg; the at position 139 is Gln, Asn., Asp or Glu; the amino acid at amino acid at position 215 is Ser or Thr; the amino acid at position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 217 is Tyr or Phe; the amino acid at position 218 is US 2016/0347799 A1 Dec. 1, 2016
Arg, Lys, Thr or Ser; the amino acid at position 219 is Val, the amino acid at position 356 is Lys or Arg; the amino acid Ile, Leu or Ala; the amino acid at position 220 is Cys, Leu, at position 360 is Ile, Val, Leu, Gly or Ala; the amino acid Ile, Val, Thr or Ser; the amino acid at position 221 is Pro or at position 365 is Leu, Ile, Val or Phe; the amino acid at His; the amino acid at position 222 is Leu, Arg, Lys, Ile, Val, position 371 is or Glu or Asp; the amino acid at position 372 Thr or Ser; the amino acid at position 224 is Asn., Gln, Thr is or Lys or Arg; the amino acid at position 374 is Arg or Lys; or Ser; the amino acid at position 225 is Asp, Arg, Glu, LyS, the amino acid at position 376 is Phe, Ile, Val or Leu; the Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln amino acid at position 378 is Glu or Asp; the amino acid at or ASn; the amino acid at position 227 is Asp, Leu, Glu, Ile, position 381 is Leu, Ile or Val; the amino acid at position 388 Val or deleted; the amino acid at position 228 is Thr, Ser or is Ala, Thr, Gly or Ser; the amino acid at position 395 is Arg deleted; the amino acid at position 229 is Tyr or deleted; the or Lys; the amino acid at position 396 is Glu, Gln, Asp, ASn, amino acid at position 230 is Leu, Ile, Val or deleted; the Ala or Gly; the amino acid at position 399 is Asp, Gln, Glu amino acid at position 231 is Gly, Ala or deleted; the amino or ASn; the amino acid at position 400 is Asn. Thr, Ser, Glu, acid at position 232 is Ile, Leu, Val or deleted; the amino acid Gln or Asp; the amino acid at position 401 is Thr, Ser, Gly at position 233 is Pro or deleted; the amino acid at position or Ala; the amino acid at position 402 is Phe, Ile, Val or Leu: 234 is Ala, Pro, Gly or deleted; the amino acid at position the amino acid at position 406 is Asp or Glu; the amino acid 235 is Asp, Ile, Leu, Glu or Val; the amino acid at position at position 408 is Leu, Ile, Val or Met; the amino acid at 236 is Val, Ser, Ile, Leu, Thr, Asp or Glu; the amino acid at position 410 is Gly, Ile, Val, Ala or Leu; the amino acid at position 237 is Ala, Phe or Tyr; the amino acid at position position 414 is Ala, Gly, Asp or Glu; the amino acid at 238 is Ala, Gly, Ser or Thr; the amino acid at position 239 position 416 is Ser, Asn. Thr, Gln, Glu or Asp; the amino is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acid at acid at position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the position 240 is Leu, Val or Ile; the amino acid at position 243 amino acid at position 423 is Lys, Arg, ASnor Gln; the amino is Asp or Glu; the amino acid at position 249 is Asn., Gln, Thr acid at position 431 is Arg or Lys; the amino acid at position or Ser; the amino acid at position 252 is Leu, Ile, Val or Met; 432 is Gln, Asn, Asp or Glu; the amino acid at position 436 the amino acid at position 257 is Thr or Ser; the amino acid is Arg, Lys, Asp or Glu; the amino acid at position 440 is at position 259 is His, Ile, Val or Leu; the amino acid at ASn, Gln, Lys or Arg; the amino acid at position 442 is Leu, position 266 is Ala, Ile, Leu or Val; the amino acid at Ile or Val; the amino acid at position 447 is Ser, Lys, Thr or position 267 is Cys, Ala or Gly; the amino acid at position Arg; the amino acid at position 448 is Ala, Gly, Thr or Ser; 268 is His, Arg, Lys or Tyr; the amino acid at position 272 the amino acid at position 451 is Gln, Asin or Met; the amino is Asp or Glu; the amino acid at position 273 is Val, Met, Ile acid at position 453 is Gly or Ala; the amino acid at position or Leu; the amino acid at position 274 is Val, Ile, Leu or Met; 455 is Ala, Leu, Ile or Val; the amino acid at position 457 is the amino acid at position 278 is Gly or Ala; the amino acid Leu, Ile or Val; the amino acid at position 467 is Val, Ile, at position 279 is Glu, Asp, Gly or Val; the amino acid at Leu, Gly or Ala; the amino acid at position 471 is Gly or Ala; position 281 is Leu, Ile, Val, Gly or Ala; the amino acid at the amino acid at position 475 is Ser. Thr, Gln or Asn; the position 282 is ASn, Leu or Ile; the amino acid at position amino acid at position 483 is Gly or Ala; the amino acid at 285 is Asn., Gln, Thr or Ser; the amino acid at position 286 position 493 is Gln, Asn or Gly; the amino acid at position is Lys, Asp, Arg or Glu; the amino acid at position 287 is 504 is Val, Leu or Ile; the amino acid at position 506 is Asp, Leu, Ile or Val; the amino acid at position 290 is Pro, Gln, Glu or His; the amino acid at position 509 is Asp, Glu, Gln ASn, Lys or Arg; the amino acid at position 291 is Leu, Ile or ASn; the amino acid at position 510 is Ser. Thr, Gly or Ala; or Val; the amino acid at position 292 is Lys, Arg, Ile, Leu the amino acid at position 512 is Glu or Asp; the amino acid or Val; the amino acid at position 293 is Glu, Asp, Asn or at position 515 is Gly, Ala, Thr or Ser; the amino acid at Gln; the amino acid at position 294 is Ser, Asn. Thr, Gln, Arg position 516 is Gln, Asin or His; the amino acid at position or Lys; the amino acid at position 295 is Thr or Ser; the 517 is Ile, Val or Leu; the amino acid at position 519 is Asp, amino acid at position 296 is Gln, ASnor His; the amino acid ASn, Glu, Gly or Gln; the amino acid at position 522 is Val, at position 297 is Leu, Ile, Val or Met; the amino acid at Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 is position 300 is Ser or Thr; the amino acid at position 301 is Glu or Asp; the amino acid at position 526 is Leu, Ile, Val Glu, Asp, Gly or Ala; the amino acid at position 302 is Ser, or Met; the amino acid at position 539 is Val, Leu or Ile; the Pro, Thr, Gly or Ala; the amino acid at position 304 is Lys, amino acid at position 555 is Val, Leu, Ile or Ala; the amino Arg, Gln or ASn; the amino acid at position 313 is Val, Leu acid at position 557 is Arg or Lys; the amino acid at position or Ile; the amino acid at position 314 is His, Glu, ASn, Asp 563 is Val, Leu, Ile or Met; the amino acid at position 571 or Gln; the amino acid at position 315 is Ala, Cys, Gly, Thr is Ser. Thror Cys; the amino acid at position 575 is Val, Leu, or Ser; the amino acid at position 316 is Ala, Ile, Leu or Val; Ile, Asp or Glu; the amino acid at position 577 is Met, Leu, the amino acid at position 317 is Met, Leu, Val or Ile; the Val or Ile; the amino acid at position 579 is Glu, Asp, Asn amino acid at position 319 is Met, Leu, Val or Ile; the amino or Gln; the amino acid at position 583 is Asp or Glu; the acid at position 320 is Val, Ile, Leu, Ala or Gly; the amino amino acid at position 589 is Met, Ile, Val or Leu; the amino acid at position 321 is Arg, Lys or Pro; the amino acid at acid at position 590 is Met, Ile, Val or Leu; the amino acid position 322 is Ile, Leu, Val or Phe; the amino acid at at position 593 is Met, Leu, Val or Ile; the amino acid at position 323 is Gly, Ile, Leu or Val; the amino acid at position 595 is Arg, Lys, ASn or Gln; the amino acid at position 324 is Leu, Ile, Val, Thr or Ser; the amino acid at position 596 is Ser or Thr; the amino acid at position 597 is position 336 is Ser. Thr, Gln or ASn; the amino acid at Gln, Asn or His; the amino acid at position 607 is Ala, Gly, position 339 is ASn, Lys, Gln or Arg; the amino acid at Ile, Leu or Val; the amino acid at position 608 is Asp, Glu, position 350 is Arg, Lys, ASn or Gln; the amino acid at Gln or Asn; the amino acid at position 612 is Tyr, His or Phe: position 351 is Glu or Asp; the amino acid at position 353 the amino acid at position 617 is Thr, Ser, Leu, Val or Ile; the is Lys or Arg; the amino acid at position 354 is Gln, ASn, Lys amino acid at position 618 is Gln, ASnor His; the amino acid or Arg; the amino acid at position 355 is Phe, Ile, Leu or Leu: at position 625 is Arg, Lys, Thr or Ser; the amino acid at US 2016/0347799 A1 Dec. 1, 2016 44 position 626 is Met, Leu, Val or Ile; the amino acid at deleted; the amino acid at position 3 is Leu, Val, Ile or position 628 is Leu, Val or Ile; the amino acid at position 633 deleted; the amino acid at position 4 is Val, Met, Ile or Leu: is Ile, Leu, Val or Met; the amino acid at position 634 is Leu, the amino acid at position 7 is Gly, Thr or Ser; the amino Ile, Val or Met; the amino acid at position 642 is Arg, Lys acid at position 8 is Lys, Arg, Ser or Thr, the amino acid at or Met; the amino acid at position 648 is Met, Seror Thr; the position 10 is Phe, Trp or Tyr; the amino acid at position 11 amino acid at position 651 is Glu, Asp, ASn or Gln; the is Glu, Asp, Lys or Arg; the amino acid at position 18 is Met, amino acid at position 654 is Thr, Val, Ser, Ile, Leu, Gly or Val, Leu or Ile; the amino acid at position 19 is Gly, Pro or Ala; the amino acid at position 658 is Gly, Lys, Ala or Arg; Ala; the amino acid at position 20 is Val, Ile, Leu or deleted; the amino acid at position 663 is Gly or Ala; the amino acid the amino acid at position 21 is Leu, Ile or Val; the amino at position 664 is Asp, Glu, Gln or ASn; the amino acid at acid at position 23 is Arg, Lys, ASn or Gln; the amino acid position 668 is Ala, Gly, Ser or Thr; the amino acid at at position 37 is Val, Ile or Leu; the amino acid at position position 669 is Gln, Asn or His; the amino acid at position 38 is Arg, Lys, Gln or ASn; the amino acid at position 40 is 671 is Asn., Gln, Thr or Ser the amino acid at position 675 Ala, Gly, Thr or Ser; the amino acid at position 43 is Asn. is Ile, Val, Ile, Thr or Ser; the amino acid at position 678 is Gln, Glu or Asp; the amino acid at position 45 is Gly or Ala; Met, Ile, Ala, Leu, Seror Thr; the amino acid at position 682 the amino acid at position 46 is Gln, Asp, ASn or Glu; the is Pro, Asin or Gln; the amino acid at position 683 is Ser. Thr amino acid at position 48 is Glu, Asp, Pro, Ile, Leu or Val; or Pro; the amino acid at position 685 is Asp, Glu, Asp or the amino acid at position 51 is Glu, Asp, Ala or Gly; the ASn; the amino acid at position 694 is Asp, Glu, Ala or Gly; amino acid at position 52 is Lys, Arg, Ser or Thr, the amino the amino acid at position 697 is ASn, Gln, Thr or Ser; the acid at position 53 is Val, Ala, Cys or Thr; the amino acid at amino acid at position 704 is Glu, Asp, Ala or Gly; the amino position 54 is Lys, Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, acid at position 714 is Ala or Gly; the amino acid at position Met, ASn, Gln, Arg, Ser or Thr, the amino acid at position 721 is Ser, Thr or Phe; the amino acid at position 722 is Ser, 55 is Arg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn. Thr, Gln or ASn; the amino acid at position 724 is Seror Thr: Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 56 the amino acid at position 734 is His, Asn or Gln; the amino is Leu, Glu, Phe, Ile, Met, Thr or Val; the amino acid at acid at position 736 is Val, Leu, Ile or Ala; the amino acid position 57 is Tyr, Cys, Ile, Leu, Met, Thr or Val; the amino at position 737 is Lys, Arg, ASn or Gln; the amino acid at acid at position 58 is Val, Cys, Ile or Leu; the amino acid at position 739 is Ala, Gly, Thr or Ser; the amino acid at position 59 is Phe, Leu, Met, Val or Tyr; the amino acid at position 740 is Ser. Thr or Met; the amino acid at position position 60 is Ala, Cys, Gly, Ser. Thr or Val; the amino acid 741 is Gly, Ala, Gln or ASn; the amino acid at position 742 at position 61 is Asp, Glu, His or Ser; the amino acid at is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 position 62 is Val, Ala, Cys, Ile, Leu or Thr; the amino acid is Gly or deleted; the amino acid at position 745 is Gly, Ala, at position 63 is Val, Ala, Cys, Ile, Leu, Met or Thr; the Glu or Asp; the amino acid at position 751 is Thr, Ser, Gly amino acid at position 64 is Glu, Ala, Cys, Phe, Gly. His, Ile, or Ala; the amino acid at position 753 is Gln, Asn. Lys or Leu, Met, Asn., Gln, Arg, Ser, Thr, Val, Trp or Tyr; the amino Arg; the amino acid at position 754 is Thr or Ser; the amino acid at position 65 is Leu, Ala, Cys, Phe, His, Ile, Met, Asn. acid at position 756 is Thr, Ser, Leu, Val or Ile; the amino Gln, Thr, Val or Trp; the amino acid at position 66 is Pro, acid at position 757 is Val, Leu or Ile; the amino acid at Asp, Gly, Met, Gln or Arg; the amino acid at position 67 is position 766 is Ile, Leu or Val; the amino acid at position 773 Val, Pro, Ile, Leu, Ser or Thr; the amino acid at position 68 is Asp or Glu; the amino acid at position 774 is Gln, ASn, is Val, Arg, Phe, Ile, Leu, Lys or Gly; the amino acid at Asp or Glu; the amino acid at position 776 is Leu, Ile, Val position 69 is Glu, Ala, Asp, Gly, Arg or Lys; the amino acid or Met; the amino acid at position 777 is Pro, Seror Thr; the at position 70 is Trp, Thr. His, Tyr, Lys or Arg; the amino amino acid at position 782 is Ala, Asp, Glu, Ile, Leu or Val; acid at position 71 is Arg, Pro, Lys or deleted; the amino acid the amino acid at position 786 is Tyr or Phe; the amino acid at position 72 is Trp, Asp, Leu, Ile, Val, Glu or deleted; the at position 787 is His, Asin or Gln; the amino acid at position amino acid at position 73 is Pro, Gln, Asn. His or deleted; 788 is Tyr or Met; the amino acid at position 789 is Ala, Lys the amino acid at position 74 is Pro, Met, Ser or Thr; the or Arg; the amino acid at position 790 is Tyr or Thr; the amino acid at position 75 is Gln, His, ASn, Lys or Arg; the amino acid at position 791 is Arg, Lys, Gly or Ala; the amino amino acid at position 76 is Ile, Met, Val or Leu; the amino acid at position 792 is Leu, Ile, Val, Thr or Ser; the amino acid at position 84 is Ile, Leu or Val; the amino acid at acid at position 796 is Asp or Glu; the amino acid at position position 91 is Trp or Phe; the amino acid at position 93 is 797 is Ser, Thr or Ala the amino acid at position 802 is Glu, Thr, Ser, Leu, Val or Ile; the amino acid at position 94 is Asp, LyS, Asp, ASn or Gln; the amino acid at position 806 is Gln, Glu, Ala or Gly; the amino acid at position 96 is Arg, Lys, Asp, Glu, ASn or His; the amino acid at position 810 is LyS, Thr or Ser; the amino acid at position 97 is Gln, Phe, Asn. Arg or Thr; the amino acid at position 819 is Arg, Lys or His: Lys or Arg; the amino acid at position 98 is Ser. Thr or the amino acid at position 829 is Lys, Ser, Ala or Pro; the deleted; the amino acid at position 99 is Asp, Glu, Gly or amino acid at position 832 is Ala, Lys, Arg, Asp or Glu; the Ala; the amino acid at position 100 is Thr, Ser, Gly or Ala; amino acid at position 833 is Gly, Ala, Asp or Glu; the amino the amino acid at position 101 is Glu, Thr, Asp, Ser or Trp acid at position 842 is Leu, Ile, Val or Pro; the amino acid the amino acid at position 103 is His, Arg, Lys, Glu or Gln; at position 847 is Gln, ASn, Asp or Glu, the amino acid at the amino acid at position 105 is Thr, Ser or Pro; the amino position 848 is Ile, Leu or Val; the amino acid at position 849 acid at position 108 is Lys, Arg, ASn, Asp, Gln or Glu; the is Val, Leu, Ile, Gly or Ala; the amino acid at position 855 amino acid at position 109 is Leu, Ile or Val; the amino acid is Thr, Ser or Met; the amino acid at position 860 is Ile, Leu at position 111 is Ala, Seror Thr; the amino acid at position or Val; the amino acid at position 864 is His, Asin or Gln; 112 is Ile, Arg, Thr, Leu, Val, Lys, Ser or deleted; the amino 0141. In some embodiments the PtIP-83 polypeptide is a acid at position 113 is Gln, Ala, Gly, Asn or deleted; the variant of SEQID NO: 1, wherein the amino acid at position amino acid at position 114 is Arg, Glu, Lys, Asp or Ile; the 1 is Met or deleted; the amino acid at position 2 is Ala or amino acid at position 115 is Glu, Asp, ASn or Gln; the US 2016/0347799 A1 Dec. 1, 2016 amino acid at position 116 is Glu, ASn, Gln, Asp, Lys or Arg; amino acid at position 201 is Ala, Leu, Glu, Ile, Asp or Trp; the amino acid at position 117 is Asn. Val, Tyr, Ile, Leu, Gln, the amino acid at position 202 is Ser, Asp, Phe, Ile, Val, Thr, Trp or Phe; the amino acid at position 118 is Arg or Lys; the Glu or Leu; the amino acid at position 203 is His, Pro, Gly, amino acid at position 119 is Trp, Thr or Ser; the amino acid Ala, Thr or Ser; the amino acid at position 204 is Ile, Trp, at position 122 is Thr, Lys, Ser, Arg or Ala; the amino acid His, Leu, Val, Ala or Gly; the amino acid at position 205 is at position 124 is Ala, Gly, Ser or Thr; the amino acid at Ser, Asn. Leu, Val, Thr, Gln or Ile; the amino acid at position position 126 is Gly, Ala, Glu or Asp; the amino acid at 206 is Ala, Gly, Asp, Tyr, Glu, Lys or Arg; the amino acid position 127 is Met, Gly or Ala; the amino acid at position at position 207 is Phe, Val, Ile or Leu; the amino acid at 128 is ASn, Gln, Arg or Lys; the amino acid at position 131 position 208 is Asn. Ser, Pro, Gln, Thr, Val, Ile or Leu; the is Val, Ile, Leu, Ser or Thr; the amino acid at position 133 amino acid at position 210 is Arg, Asp, Glu, Lys, Ser or Tyr; is Ile, Leu or Val; the amino acid at position 134 is His or the amino acid at position 211 is Ile, Ser, Leu, Val or Thr; the Tyr; the amino acid at position 135 is Ala or Gly; the amino amino acid at position 212 is Val, Ala, Ile, Leu, Glu, Gly or acid at position 137 is Glu, Asp, Arg or Lys; the amino acid Asp; the amino acid at position 214 is Pro, Lys or Arg; the at position 139 is Gln, Asn., Asp or Glu; the amino acid at amino acid at position 215 is Ser or Thr; the amino acid at position 140 is Val, Arg, Ile, Lys or Leu; the amino acid at position 217 is Tyr or Phe; the amino acid at position 218 is position 141 is Gly, Ala, Thr or Ser; the amino acid at Arg, Lys, Thr or Ser; the amino acid at position 219 is Val, position 142 is Val, Ile, Leu or Pro; the amino acid at Ile, Leu or Ala; the amino acid at position 220 is Cys, Leu, position 144 is Thr, Leu, Phe, Ile, Val or Tyr; the amino acid Ile, Val, Thr or Ser; the amino acid at position 221 is Pro or at position 145 is Met, Pro, Gln or ASn; the amino acid at His; the amino acid at position 222 is Leu, Arg, Lys, Ile, Val, position 146 is Ser, Gly, Thr, Ala, Gln or Asn; the amino acid Thr or Ser; the amino acid at position 224 is Asn., Gln, Thr at position 147 is Trp, Gln, Tyr or Asn; the amino acid at or Ser; the amino acid at position 225 is Asp, Arg, Glu, Lys, position 148 is Ser, Ala, Thr, Gly or Pro; the amino acid at Ser or Thr; the amino acid at position 226 is Thr, Ser, Gln position 149 is Ser. Thr or deleted; the amino acid at position or ASn; the amino acid at position 227 is Asp, Leu, Glu, Ile, 150 is Val, Ile, Leu or Tyr; the amino acid at position 152 is Val or deleted; the amino acid at position 228 is Thr, Ser or Arg, Ala, Val, Ile, Leu, Lys or Gly; the amino acid at position deleted; the amino acid at position 229 is Tyr or deleted; the 154 is Ser, Trp, Thr, Asp or Glu; the amino acid at position amino acid at position 230 is Leu, Ile, Val or deleted; the 156 is Leu, Asp, Ile, Val, Asn., Glu or Gln; the amino acid at amino acid at position 231 is Gly, Ala or deleted; the amino position 158 is Ser. Thr or Cys; the amino acid at position acid at position 232 is Ile, Leu, Val or deleted; the amino acid 159 is Val, Thr, Leu or Ile; the amino acid at position 162 is at position 233 is Pro or deleted; the amino acid at position Ser. Thr, Gly or Ala; the amino acid at position 163 is Gly, 234 is Ala, Pro, Gly or deleted; the amino acid at position Ala or deleted; the amino acid at position 164 is Phe or 235 is Asp, Ile, Leu, Glu or Val; the amino acid at position deleted; the amino acid at position 165 is Arg, Lys, Gly or 236 is Val, Ser, Ile, Leu, Thr, Asp or Glu; the amino acid at Ala; the amino acid at position 166 is Ala, Arg, Met, Lys or position 237 is Ala, Phe or Tyr; the amino acid at position Phe, the amino acid at position 167 is Val, Ile, Leu or His: 238 is Ala, Gly, Ser or Thr; the amino acid at position 239 the amino acid at position 168 is Ser. Thr, Gln or ASn; the is Val, Ser, Ile, Leu, Thr, Ala or Gly; the amino acid at amino acid at position 169 is Val, His, Ile, Leu, Ser or Thr: position 240 is Leu, Val or Ile; the amino acid at position 243 the amino acid at position 170 is Phe, Ile, Leu or Val; the is Asp or Glu; the amino acid at position 249 is Asn., Gln, Thr amino acid at position 171 is Glu, ASn, Gln or Asp; the or Ser; the amino acid at position 252 is Leu, Ile, Val or Met; amino acid at position 172 is Val, Ala, Arg, Ile, Leu, Gly, the amino acid at position 257 is Thr or Ser; the amino acid LyS, Asp or Glu; the amino acid at position 175 is Ser, Arg, at position 259 is His, Ile, Val or Leu; the amino acid at Thr, Lys or Trp; the amino acid at position 176 is Val, Leu position 266 is Ala, Ile, Leu or Val; the amino acid at or Ile; the amino acid at position 177 is Arg, Lys, Leu, Val position 267 is Cys, Ala or Gly; the amino acid at position or Ile; the amino acid at position 179 is Thr, Ile, Val, Leu or 268 is His, Arg, Lys or Tyr; the amino acid at position 272 Ser; the amino acid at position 180 is Leu, Phe, Ile, Val, Ser is Asp or Glu; the amino acid at position 273 is Val, Met, Ile or Thr; the amino acid at position 181 is Gly, Thr, Gln, Asn or Leu; the amino acid at position 274 is Val, Ile, Leu or Met; or Ser; the amino acid at position 182 is Ala, Leu, Phe, Val the amino acid at position 278 is Gly or Ala; the amino acid or Ile; the amino acid at position 183 is Thr, Ser, Ala or Gly: at position 279 is Glu, Asp, Gly or Val; the amino acid at the amino acid at position 184 is Leu, Thr, Ser, Ile, Val, Lys position 281 is Leu, Ile, Val, Gly or Ala; the amino acid at or Arg; the amino acid at position 185 is Arg, Gly, Asp, LyS, position 282 is ASn, Leu or Ile; the amino acid at position Glu or Ala; the amino acid at position 186 is Pro, Val, Ile, 285 is Asn., Gln, Thr or Ser; the amino acid at position 286 Leu, Asn or Gln; the amino acid at position 187 is Asp, Thr, is Lys, Asp, Arg or Glu; the amino acid at position 287 is Glu or Ser; the amino acid at position 188 is His, Gly or Ala; Leu, Ile or Val; the amino acid at position 290 is Pro, Gln, the amino acid at position 189 is Ala, Arg, Pro, Lys, Gly or ASn, Lys or Arg; the amino acid at position 291 is Leu, Ile deleted; the amino acid at position 190 is Leu, Asn. Ile, Val, or Val; the amino acid at position 292 is Lys, Arg, Ile, Leu Gln or deleted; the amino acid at position 191 is Tyr or or Val; the amino acid at position 293 is Glu, Asp, Asn or deleted; the amino acid at position 192 is Ser, Ile, Val, Leu, Gln; the amino acid at position 294 is Ser, Asn. Thr, Gln, Arg Thr or Asn; the amino acid at position 193 is Thr, Ser, Glu or Lys; the amino acid at position 295 is Thr or Ser; the or Asp; the amino acid at position 194 is Thr or Ser; the amino acid at position 296 is Gln, ASnor His; the amino acid amino acid at position 195 is Met or Thr; the amino acid at at position 297 is Leu, Ile, Val or Met; the amino acid at position 196 is Gln, His, Leu, Asn. Ile, Val, Thr or Ser; the position 300 is Ser or Thr; the amino acid at position 301 is amino acid at position 197 is Ala, Gly, Ile, Val or Leu; the Glu, Asp, Gly or Ala; the amino acid at position 302 is Ser, amino acid at position 198 is Thr, Glu, Ser, Asp, Gly or Ala; Pro, Thr, Gly or Ala; the amino acid at position 304 is Lys, the amino acid at position 199 is Pro, Lys or Arg; the amino Arg, Gln or ASn; the amino acid at position 313 is Val, Leu acid at position 200 is Asn. Ser. Thr, Gln, Ala or Gly; the or Ile; the amino acid at position 314 is His, Glu, ASn, Asp US 2016/0347799 A1 Dec. 1, 2016 46 or Gln; the amino acid at position 315 is Ala, Cys, Gly, Thr Leu, Gly or Ala; the amino acid at position 471 is Gly or Ala; or Ser; the amino acid at position 316 is Ala, Ile, Leu or Val; the amino acid at position 475 is Ser. Thr, Gln or Asn; the the amino acid at position 317 is Met, Leu, Val or Ile; the amino acid at position 483 is Gly or Ala; the amino acid at amino acid at position 319 is Met, Leu, Val or Ile; the amino position 493 is Gln, Asn or Gly; the amino acid at position acid at position 320 is Val, Ile, Leu, Ala or Gly; the amino 504 is Val, Leu or Ile; the amino acid at position 506 is Asp, acid at position 321 is Arg, Lys or Pro; the amino acid at Glu or His; the amino acid at position 509 is Asp, Glu, Gln position 322 is Ile, Leu, Val or Phe; the amino acid at or ASn; the amino acid at position 510 is Ser. Thr, Gly or Ala; position 323 is Gly, Ile, Leu or Val; the amino acid at the amino acid at position 512 is Glu or Asp; the amino acid position 324 is Leu, Ile, Val, Thr or Ser; the amino acid at at position 515 is Gly, Ala, Thr or Ser; the amino acid at position 336 is Ser. Thr, Gln or ASn; the amino acid at position 516 is Gln, Asin or His; the amino acid at position position 339 is ASn, Lys, Gln or Arg; the amino acid at 517 is Ile, Val or Leu; the amino acid at position 519 is Asp, position 350 is Arg, Lys, ASn or Gln; the amino acid at ASn, Glu, Gly or Gln; the amino acid at position 522 is Val, position 351 is Glu or Asp; the amino acid at position 353 Glu, Pro, Ile, Leu or Asp; the amino acid at position 525 is is Lys or Arg; the amino acid at position 354 is Gln, ASn, Lys Glu or Asp; the amino acid at position 526 is Leu, Ile, Val or Arg; the amino acid at position 355 is Phe, Ile, Leu or Leu: or Met; the amino acid at position 539 is Val, Leu or Ile; the the amino acid at position 356 is Lys or Arg; the amino acid amino acid at position 555 is Val, Leu, Ile or Ala; the amino at position 360 is Ile, Val, Leu, Gly or Ala; the amino acid acid at position 556 is Trp, Phe, Thr or Tyr; the amino acid at position 363 is Gln, Ala, Cys, Glu, Phe, Gly, His, Lys, at position 557 is Arg, Cys, Asp, Gly. His, Ile, Lys, Leu, Met, Leu, ASn, Arg, Ser, Thr, Val or Trp; the amino acid at ASn, Pro, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid at position 364 is Ile, Ala, Cys, Glu, Phe, His, Lys, Leu, Met, position 558 is Ala, Cys, Asp, Phe, Gly. His, Ile, Lys, Leu, ASn, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid at position ASn, Pro, Gln, Arg, Ser, Val, Trp or Tyr; the amino acid at 365 is Leu, Ala, Glu, Phe, Gly. His, Ile, Lys, Met, Asn, Arg, position 559 is Lys, Ala, Cys, Phe, Gly, His, Ile, Leu, Asn. Val, Trp or Tyr; the amino acid at position 366 is Gly, Ala, Gln, Arg, Ser, Thr, Val or Tyr; the amino acid at position 560 Cys, Phe, His, Ile, Lys, Leu, Met, Asn. Ser, Thr or Val; the is Cys, Ala, Phe, Gly, Ile, Met, Asn, Arg, Ser. Thr or Val; the amino acid at position 367 is Ser, Ala, Cys, Asp, Glu, Phe, amino acid at position 561 is Lys, Ala, Cys, Asp, Glu, Phe, Gly. His, Ile, Leu, Met, Asn. Pro, Gln, Arg, Thr, Val or Trp; Gly. His, Ile, Leu, Met, Asn, Arg, Ser. Thr, Val or Tyr; the the amino acid at position 368 is Tyr, Ala, Cys, Asp, Glu, amino acid at position 562 is ASn, Cys, Asp, Glu, Gly, His, Phe, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Gln, Arg, Ser, Leu, Met, Arg, Ser. Thr, Val or Tyr; the amino acid at Thr, Val or Trp; the amino acid at position 369 is Leu, Ala, position 563 is Val, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Cys, Asp, Phe, Gly, Ile, Met, Thr or Val; the amino acid at ASn, Gln, Thr or Trp; the amino acid at position 564 is Ala, position 370 is Leu, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Cys, Gly, Met, Gln, Ser. Thr, Val, Trp or Tyr; the amino acid Lys, Met, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino acid at position 571 is Ser. Thr or Cys; the amino acid at position at position 371 is Gln, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, 575 is Val, Leu, Ile, Asp or Glu; the amino acid at position Leu, ASn, Arg, Ser, Thr, Val or Trp; the amino acid at 577 is Met, Leu, Val or Ile; the amino acid at position 579 position 372 is Gln, Ala, Cys, Asp, Phe, Gly. His, Ile, Leu, is Glu, Asp, Asin or Gln; the amino acid at position 583 is Asn, Arg, Ser, Val or Tyr; the amino acid at position 373 is Asp or Glu; the amino acid at position 589 is Met, Ile, Val Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Gln, Ser. Thr, Val or Leu; the amino acid at position 590 is Met, Ile, Val or Leu: or Trp; the amino acid at position 374 is Arg or Lys; the the amino acid at position 593 is Met, Leu, Val or Ile; the amino acid at position 376 is Phe, Ile, Val or Leu; the amino amino acid at position 595 is Arg, Lys, ASnor Gln; the amino acid at position 378 is Glu or Asp; the amino acid at position acid at position 596 is Seror Thr; the amino acid at position 381 is Leu, Ile or Val; the amino acid at position 388 is Ala, 597 is Gln, Asin or His; the amino acid at position 607 is Ala, Thr, Gly or Ser; the amino acid at position 395 is Arg or Lys: Gly, Ile, Leu or Val; the amino acid at position 608 is Asp, the amino acid at position 396 is Glu, Gln, Asp, Asn, Ala or Glu, Gln or ASn; the amino acid at position 612 is Tyr, His Gly; the amino acid at position 399 is Asp, Gln, Glu or ASn; or Phe; the amino acid at position 617 is Thr, Ser, Leu, Val the amino acid at position 400 is Asn. Thr, Ser, Glu, Gln or or Ile; the amino acid at position 618 is Gln, Asn or His; the Asp; the amino acid at position 401 is Thr, Ser, Gly or Ala; amino acid at position 625 is Arg, Lys, Thr or Ser; the amino the amino acid at position 402 is Phe, Ile, Val or Leu; the acid at position 626 is Met, Leu, Val or Ile; the amino acid amino acid at position 406 is Asp or Glu; the amino acid at at position 628 is Leu, Val or Ile; the amino acid at position position 408 is Leu, Ile, Val or Met; the amino acid at 633 is Ile, Leu, Val or Met; the amino acid at position 634 position 410 is Gly, Ile, Val, Ala or Leu; the amino acid at is Leu, Ile, Val or Met; the amino acid at position 642 is Arg, position 414 is Ala, Gly, Asp or Glu; the amino acid at Lys or Met; the amino acid at position 646 is Leu, Ala, Cys, position 416 is Ser, Asn. Thr, Gln, Glu or Asp; the amino Gly, Ile, Met, Asn., Gln, Ser. Thr or Val; the amino acid at acid at position 417 is Ser, Arg, Lys, Thr, Ala or Gly; the position 647 is Leu, Asp, Gly, Met, Asn., Gln or Thr; the amino acid at position 423 is LyS, Arg, ASnor Gln; the amino amino acid at position 648 is Met, Ala, Cys, Asp, Glu, Phe, acid at position 431 is Arg or Lys; the amino acid at position Gly. His, Lys, Leu, ASn, Pro, Gln, Arg, Ser, Thr, Val, Trp or 432 is Gln, Asn., Asp or Glu; the amino acid at position 436 Tyr; the amino acid at position 649 is Pro, Ala, Cys, Asp, is Arg, Lys, Asp or Glu; the amino acid at position 440 is Glu, Phe, Gly, His, Lys, Met, Asn., Gln, Arg, Ser. Thr, Trp or ASn, Gln, Lys or Arg; the amino acid at position 442 is Leu, Tyr; the amino acid at position 650 is Thr, Ala, Cys, Asp, Ile or Val; the amino acid at position 447 is Ser, Lys, Thr or Phe, Gly. His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Val or Arg; the amino acid at position 448 is Ala, Gly, Thr or Ser; Tyr, the amino acid at position 651 is Glu, Ala, Cys, Asp, the amino acid at position 451 is Gln, ASnor Met; the amino Gly. His, Ile, Leu, Met, ASn, Pro, Gln, Arg, Ser. Thr, Val or acid at position 453 is Gly or Ala; the amino acid at position Tyr; the amino acid at position 652 is Leu, Cys, Phe, Ile, Lys, 455 is Ala, Leu, Ile or Val; the amino acid at position 457 is Met, Pro, Arg, Ser. Thr or Val; the amino acid at position 653 Leu, Ile or Val; the amino acid at position 467 is Val, Ile, is Thr, Cys, Asp, Glu, Phe, Gly. His, Ile, Lys, Leu, Pro, Arg, US 2016/0347799 A1 Dec. 1, 2016 47
Ser, Val or Trp; the amino acid at position 654 is Thr, Ala, at position 790 is Tyr or Thr; the amino acid at position 791 Cys, Phe, Ile, Lys, Leu, Met, Pro, Arg, Ser, Val, Trp or Tyr; is Arg, LyS, Gly or Ala; the amino acid at position 792 is Leu, the amino acid at position 655 is Trp, Phe or Tyr; the amino Ile, Val, Thr or Ser; the amino acid at position 796 is Asp or acid at position 658 is Gly, Lys, Ala or Arg; the amino acid Glu; the amino acid at position 797 is Ser, Thr or Ala the at position 663 is Gly or Ala; the amino acid at position 664 amino acid at position 802 is Glu, Lys, Asp, ASn or Gln; the is Asp, Glu, Gln or ASn; the amino acid at position 668 is amino acid at position 806 is Gln, Asp, Glu, Asn or His; the Ala, Gly, Ser or Thr; the amino acid at position 669 is Gln, amino acid at position 810 is Lys, Arg or Thr, the amino acid Asin or His; the amino acid at position 671 is Asn., Gln, Thr at position 819 is Arg, Lys or His; the amino acid at position or Ser the amino acid at position 675 is Ile, Val, Ile, Thr or 829 is Lys, Ser, Ala or Pro; the amino acid at position 832 Ser; the amino acid at position 678 is Met, Ile, Ala, Leu, Ser is Ala, Lys, Arg, Asp or Glu, the amino acid at position 833 or Thr; the amino acid at position 682 is Pro, Asn or Gln; the is Gly, Ala, Asp or Glu; the amino acid at position 842 is amino acid at position 683 is Ser. Thr or Pro; the amino acid Leu, Ile, Val or Pro; the amino acid at position 847 is Gln, at position 685 is Asp, Glu, Asp or ASn; the amino acid at ASn, Asp or Glu; the amino acid at position 848 is Ile, Leu position 694 is Asp, Glu, Ala or Gly; the amino acid at or Val; the amino acid at position 849 is Val, Leu, Ile, Gly position 697 is ASn, Gln, Thr or Ser; the amino acid at or Ala; the amino acid at position 855 is Thr, Ser or Met; the position 704 is Glu, Asp, Ala or Gly; the amino acid at amino acid at position 860 is Ile, Leu or Val; and the amino position 714 is Ala or Gly; the amino acid at position 721 is acid at position 864 is His, Asn or Gln. Ser. Thr or Phe; the amino acid at position 722 is Ser, Thr, 0142. In some embodiments the nucleic acid molecule Gln or Asn; the amino acid at position 724 is Ser or Thr; the encoding the PtIP-83 polypeptide is derived from a fern amino acid at position 734 is His, ASnor Gln; the amino acid species in the Division Pteridophyta. In some embodiments at position 736 is Val, Leu, Ile or Ala; the amino acid at the PtIP-83 polypeptide is derived from a fern species in the position 737 is Lys, Arg, ASn or Gln; the amino acid at Class Psilotopsida. In some embodiments the PtIP-83 poly position 739 is Ala, Gly, Thr or Ser; the amino acid at peptide is derived from a fern species in the Class Psilo position 740 is Ser. Thr or Met; the amino acid at position topsida, Order Psilotales. In some embodiments the PtIP-83 741 is Gly, Ala, Gln or Asn; the amino acid at position 742 polypeptide is derived from a fern species in the Class is Ile, Leu, Val, Ala or Gly; the amino acid at position 743 Psilotopsida, Order Ophioglossales. In some embodiments is Gly or deleted; the amino acid at position 745 is Gly, Ala, the PtIP-83 polypeptide is derived from a fern species in the Glu or Asp; the amino acid at position 751 is Thr, Ser, Gly Class Psilotopsida, Order Ophioglossales, Family Psilota or Ala; the amino acid at position 753 is Gln, Asn. Lys or ceae. In some embodiments the Pt|P-83 polypeptide is Arg; the amino acid at position 754 is Thr or Ser; the amino derived from a fern species in the Class Psilotopsida, Order acid at position 756 is Thr, Ser, Leu, Val or Ile; the amino Ophioglossales Family Ophioglossaceae. In some embodi acid at position 757 is Val, Leu or Ile; the amino acid at ments the PtIP-83 polypeptide is derived from a fern species position 766 is Ile, Leu or Val; the amino acid at position 771 in the Genus Ophioglossum L. Botrychium, Botrypus, Hel is Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, ASn, Ser, minthostachys, Ophioderma, Cheiroglossa, Sceptridium or Thr, Val, Trp or Tyr; the amino acid at position 772 is Arg, Mankyua. In some embodiments the PtIP-83 polypeptide is Ala, Cys, Asp, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Pro, derived from a species in the Class Polypodiopsida/Pteri Gln, Ser, Thr, Val, Trp or Tyr; the amino acid at position 773 dopsida. In some embodiments the Pt|P-83 polypeptide is is Asp, Ala, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Asn., Gln, derived from a fern species in the Order Osmundales (royal Arg, Ser. Thr, Val, Trp or Tyr; the amino acid at position 774 ferns); Family Osmundaceae. In some embodiments the is Gln, Ala, Asp, Gly, His, Ile, Lys, Leu, Met, ASn, Pro, Arg, PtIP-83 polypeptide is derived from a fern species in the Ser. Thr, Val, Trp or Tyr; the amino acid at position 775 is Order Hymenophyllales; Family Hymenophyllaceae. In Val, Ala, Cys, Asp, Glu, Gly, His, Ile, ASn, Pro, Gln, Arg, some embodiments the PtIP-83 polypeptide is derived from Ser. Thr or Tyr; the amino acid at position 776 is Leu, Ala, a fern species in the Order Gleicheniales; Family Gle Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, ASn, Pro, Gln, Arg, icheniaceae, Family Dipteridaceae or Family Matoniaceae. Ser. Thr, Val or Tyr; the amino acid at position 777 is Pro, In some embodiments the PtIP-83 polypeptide is derived Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn., Gln, from a fern species in the Order Schizaeales; Family Lygo Ser. Thr, Val, Trp or Tyr; the amino acid at position 778 is diaceae, Family Anemiaceae or Family Schizaeaceae. In Phe, Ala, His, Ile, Leu, Met, Asn., Gln, Ser, Val, Trp or Tyr; some embodiments the PtIP-83 polypeptide is derived from the amino acid at position 779 is Gln, Ala, Cys, Asp, Glu, a fern species in the Order Schizaeales; Family Schizae Gly. His, Lys, Leu, Asn. Pro, Arg, Ser. Thr or Val; the amino aceae, Genus Lygodium selected from but not limited to acid at position 780 is Ala, Cys, ASn, Pro, Gln or Ser; the Lygodium articulatum, Lygodium circinatum, Lygodium amino acid at position 781 is Ala, Cys, Asp, Glu, Phe, Gly, conforme, Lygodium cubense, Lygodium digitatum, Lygo His, Ile, Asn., Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino dium flexuosum, Lygodium heterodoxum, Lygodium japoni acid at position 782 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, cum, Lygodium kerstenii, Lygodium lanceolatum, Lygodium Lys, Met, Pro, Gln, Arg, Ser. Thr, Val, Trp or Tyr; the amino longifolium, Lygodium merrilii, Lygodium micans, Lygo acid at position 783 is Pro, Ala, Cys, Asp, Glu, Gly, His, Asn. dium microphyllum, Lygodium microstachyum, Lygodium Gln, Arg, Ser. Thr or Val; the amino acid at position 784 is Oligostachyum, Lygodium palmatum, Lygodium polys Leu, Ala, Glu, Phe, His, Ile, Lys, Met, ASn, Pro, Gln, Ser, tachyum, Lygodium radiatum, Lygodium reticulatum, Lygo Thr, Val or Trp; the amino acid at position 785 is Asn, Ala, dium salicifolium, Lygodium scandens, Lygodium Smithi Cys, Glu, Phe, Gly. His, Ile, Lys, Leu, Met, Gln, Arg, Ser, anum, Lygodium subareolatum, Lygodium trifiurcatum, Thr, Val, Trp or Tyr; the amino acid at position 786 is Tyr, Lygodium venustum, Lygodium versteeghi, Lygodium volu Phe, Ile, Leu or Trp; the amino acid at position 787 is His, bile, and Lygodium yunnanense. Asin or Gln; the amino acid at position 788 is Tyr or Met; the 0143. In some embodiments the PtIP-83 polypeptide is amino acid at position 789 is Ala, Lys or Arg; the amino acid derived from a fern species in the Order Salviniales; Family US 2016/0347799 A1 Dec. 1, 2016 48
Marsileaceae or Family Salviniaceae. In some embodiments Asplenium flabellifolium, Asplenium flaccidum, Asplenium the PtIP-83 polypeptide is derived from a fern species in the fontanum, Asplenium forisiense, Asplenium formosum, Asp Order Cyatheales; Family Thyrsopteridaceae, Family LOX lenium gemmiferum, Aspleniumxgermanicum, Asplenium Somataceae, Family Culcitaceae, Family Plagiogyriaceae, gueinzi, Asplenium goudevi, Asplenium hemionitis, Asple Family Cibotiaceae, Family Cyatheaceae, Family Dickso nium hermannii-christii, Asplenium hookerianum, Asple niaceae or Family Metaxyaceae. nium hybridum, Asplenium incisum, Aspleniumxjacksonii, 0144. In some embodiments the PtIP-83 polypeptide is Aspleniumxkenzoi, Asplenium laciniatum, Asplenium lam derived from a fern species in the Order Polypodiales: prophyllum, Asplenium laserpitifolium, Asplenium lepidum, Family Lindsaeaceae, Family Saccolomataceae, Family Asplenium listeri, Asplenium longissimum, Asplenium luci Cystodiaceae, Family Dennstaedtiaceae, Family Pteri dum, Asplenium lunulatum, Asplenium lyallii, Asplenium daceae, Family Aspleniaceae, Family Thelypteridaceae, macedonicum, Asplenium majoricum, Asplenium marinum, Family Woodsiaceae, Family Onocleaceae, Family Blech Aspleniumxmicrodon, Asplenium milnei Carruth, Asplenium naceae, Family Dryopteridaceae, Family Lomariopsidaceae, montanum, Asplenium musifolium, Asplenium nidus, Asple Family Tectariaceae, Family Oleandraceae, Family Daval nium normale, Asplenium obliquum, Asplenium oblongifo liaceae or Family Polypodiaceae. lium, Asplenium obovatum, Asplenium obtusatum, Asple 0145. In some embodiments the PtIP-83 polypeptide is nium Oligolepidum, Asplenium Oligophlebium, Asplenium derived from a fern species in the Order Polypodiales, Onopteris, Asplenium pacificum, Asplenium paleaceum, Asp Family Pteridaceae, Genus Adiantaceae selected from but lenium palmeri, Asplenium petrarchae, Asplenium pinnati not limited to Adiantum aethiopicum, Adiantum aleuticum, fidum, Asplenium planicaule, Asplenium platybasis, Asple Adiantum bonatianum, Adiantum cajennense, Adiantum nium platyneuron, Asplenium polyodon, Asplenium capillus-junionis, Adiantum capillus-veneris, Adiantum cau praemorsum, Asplenium prolongatum, Asplenium pteri datum, Adiantum chienii, Adiantum chilense, Adiantum doides, Asplenium resiliens, Asplenium rhizophyllum, Asp cuneatum, Adiantum cunninghamii, Adiantum davidii, Adi lenium richardii, Asplenium ruprechtii, Asplenium ruta antum diaphanum, Adiantum edentulum, Adiantum edge muraria, Asplenium rustifolium, Asplenium Sagittatum, worthii, Adiantum excisum, Adiantum fengianum, Adiantum Asplenium sandersonii, Aspleniumxsarmiense, Asplenium fimbriatum, Adiantum flabellulatum, Adiantum formosanum, schizotrichum, Asplenium Schweinfurthii, Asplenium Sclero Adiantum formosum, Adiantum fulvum, Adiantum gravesii, prium, Asplenium scolopendrium (syn. Phyllitis scolopen Adiantum hispidulum, Adiantum induratum, Adiantum jor drium), Asplenium Seelosii, Asplenium Septentrionale, Asp dani, Adiantum iuxtapositum, Adiantum latifolium, Adian lenium Septentrionalextrichomanes, Asplenium serra, tum leveillei, Adiantum lianxianense, Adiantum male Asplenium serratum, Asplenium sessilifolium, Asplenium sianum, Adiantum mariesii, Adiantum monochlamys, shuttleworthianum, Asplenium simplici?ions, Asplenium Adiantum myriosorum, Adiantum obliquum, Adiantum splendens, Asplenium surrogatum, Asplenium tenerum, Asp Ogasawarense, Adiantum pedatum, Adiantum pentadacty lenium terrestre, Asplenium theciferum, Asplenium thunber lon, Adiantum peruvianum, Adiantum philippense, Adian gii, Asplenium trichomanes, Asplenium tutwilerae, Asple tum princeps, Adiantum pubescens, Adiantum raddianum, nium vespertinum, Asplenium vieillardii, Asplenium virens, Adiantum reniforme, Adiantum roborowski, Adiantum ser Asplenium viride, Asplenium vittiforme, and Asplenium ratodentatum, Adiantum sinicum, Adiantum Soboliferum, viviparum. Adiantum subcordatum, Adiantum tenerum, Adiantum ter 0.148. In some embodiments the PtIP-83 polypeptide is minatum, Adiantum tetraphyllum, Adiantum trapeziforme, derived from a fern species in the Order Polypodiales, Adiantum venustum, Adiantum viridescens, and Adiantum Family Blechnaceae, Genus Blecnum. viridimontanum. 0149. In some embodiments the PtIP-83 polypeptide is 0146 In some embodiments the PtIP-83 polypeptide is derived from a fern species in the Order Polypodiales, derived from a fern species in the Order Polypodiales, Family Dryopteridaceae Genus Acrophorus, Genus Acroru Family Aspleniaceae, Genus Asplenium. mohra, Genus Anapausia, Genus Arachniodes, Genus Bol 0147 In some embodiments the PtIP-83 polypeptide is bitis, Genus Ctenitis, Genus Cyclodium, Genus Cyrtogonel derived from a fern species in the Order Polypodiales, lum, Genus Cyrtomidictyum, Genus Cyrtonium, Genus Family Aspleniaceae, Genus Aspleniuml selected from but Diacalpe, Genus Didymochlaena, Genus Dryopsis, Genus not limited to Asplenium adiantum, Asplenium adulterinum, Dryopteris, Genus Elaphoglossum, Genus Hypodematium, Asplenium aequibasis, Asplenium aethiopicum, Asplenium Genus Lastreopsis, Genus Leptorumohra, Genus Leucoste africanum, Aspleniumxalternifolium, Asplenium angustum, gia, Genus Lithostegia, Genus Lomagramma, Genus Maxo Asplenium antiquum, Asplenium ascensionis, Asplenium nia, Genus Megalastrum, Genus Olfersia, Genus Peranema, attenuatum, Asplenium aureum, Asplenium auritum, Asple Genus Phanerophlebia, Genus Phanerophlebiopsis, Genus nium australasicum, Asplenium azoricum, Asplenium Polybotrya, Genus Polystichopsis, Genus Polystichum, bifrons, Asplenium billottii, Asplenium bipinnatifidum, Asp Genus Rumohra, Genus Sorolepidium, Genus Stigmatop lenium brachycarpum, Asplenium bradleyi, Asplenium bul teris or Genus Teratophyllum. biferum, Asplenium caudatum, Asplenium ceterach, Asple 0150. In some embodiments the PtIP-83 polypeptide is nium compressum, Asplenium congestum, Asplenium derived from a fern species in the Order Polypodiales, corderoanum, Asplenium crinicaule, Asplenium Cristatum, Family Dryopteridaceae, Genus Polystichum. In some Asplenium cuneifolium, Asplenium cymbifolium, Asplenium embodiments the nucleic acid molecule encoding the PtIP daghestanicum, Asplenium dalhousiae, Asplenium 83 polypeptide is derived from a fern species in the Order dareoides, Asplenium daucifolium, Asplenium diforme, Asp Polypodiales, Family Dryopteridaceae, Genus Polystichum lenium fissum, Asplenium dimorphum, Asplenium divarica selected from but not limited to Polystichum acanthophyl tum, Asplenium dregeanum, Aspleniumxebenoides, Asple lum, Polystichum acrostichoides, Polystichum aculeatum, nium ecuadorense, Asplenium feei Kunze, Asplenium fissum, Polystichum acutidens, Polystichum acutipinnulum, Polysti US 2016/0347799 A1 Dec. 1, 2016 49 chum alcicorne, Polystichum aleuticum, Polystichum ander bertii, Rumohra linearisquamosa, Rumohra lokohensis, sonii, Polystichum atkinsonii, Polystichum australiense, Rumohra madagascarica, and Rumohra quadrangularis. Polystichum bakerianum, Polystichum biaristatum, Polysti chum bomiense, Polystichum bonseyi, Polystichum brac 0152. In some embodiments the PtIP-83 polypeptide is hypterum, Polystichum braunii, Polystichum brachypterum, derived from a fern species in the Order Polypodiales, Polystichum calderonense, Polystichum californicum, Poly Family Lomariopsidaceae, Genus Nephrolepis. Stichum capillipes, Polystichum castaneum, Polystichum 0153. In some embodiments the PtIP-83 polypeptide is chilense, Polystichum christi Ching, Polystichum chunii derived from a fern species in the Order Polypodiales, Ching, Polystichum Craspedosorum, Polystichum Family Polypodiaceae, Genus Campyloneurum, Genus Dry cyclolobum, Polystichum cystostegia, Polystichum deltodon, naria, Genus Lepisorus, Genus Microgramma, Genus Polystichum dielsii, Polystichum discretum, Polystichum Microsorum, Genus Neurodium, Genus Niphidium, Genus drepanum, Polystichum dudleyi, Polystichum duthiei, Poly Pecluma M.G., Genus Phlebodium, Genus Phymatosorus, Stichum echinatum, Polystichum erosum, Polystichum excel Genus Platycerium, Genus Pleopeltis, Genus Polypodium. lens, Polystichum eximium, Polystichum falcatipinnum, 0154) In some embodiments the PtIP-83 polypeptide is Polystichum falcinellum, Polystichum fallax, Polystichum derived from a fern species in the Order Polypodiales, formosanum, Polystichum gongboense, Polystichum gran Family Polypodiaceae, Genus Microsorum. difrons, Polystichum gymnocarpium, Polystichum halleaka lense, Polystichum hancockii, Polystichum hecatopteron, (O155 In some embodiments the PtIP-83 polypeptide is Polystichum herbaceum, Polystichum imbricans, Polysti derived from a fern species in the Order Polypodiales, chum incongruum, Polystichum kruckebergii, Polystichum Family Polypodiaceae, Genus Microsorum selected from kwakiutlii, Polystichum lachenense, Polystichum lanceola but not limited to Microsorum alatum, Microsorum angus tum, Polystichum lemmonii, Polystichum lentum, Polysti tifolium, Microsorum aurantiacum, Microsorum aus chum lonchitis, Polystichum longidens, Polystichum longi traliense, Microsorum baithoense, Microsorum basicorda paleatum, Polystichum longipes, Polystichum luctuosum, tum, Microsorum biseriatum, Microsorum brassii, Polystichum macleae, Polystichum macrochlaenum, Poly Microsorum buergerianum, Microsorum chapaense, Stichum makinoi, Polystichum martini, Polystichum Microsorum cinctum, Microsorum commutatum, Microso mayebarae, Polystichum mediocre, Polystichum medo rum congregatifolium, Microsorum cuneatum, Microsorum gense, Polystichum microchlamys, Polystichum mohrioides, cuspidatum, Microsorum dengii, Microsorum egregium, Polystichum mollissimum, Polystichum monticola, Polysti Microsorum emeiensis, Microsorum ensatum, Microsorum chum moorei, Polystichum morii, Polystichum moupinense, ensiforme, Microsorum excelsum, Microsorum fortunei, Polystichum muricatum, Polystichum nakenense, PolySti Microsorum griseorhizoma, Microsorum grossum, Microso chum neolobatum, Polystichum nepalense, Polystichum rum hemionitideum, Microsorum henryi, Microsorum het ningshenense, Polystichum obliquum, Polystichum erocarpum, Microsorum heterolobum, Microsorum how Omeiense, Polystichum Ordinatum, Polystichum Orientaliti ense, Microsorum insigne, Microsorum intermedium, beticum, Polystichum paramoupinense, Polystichum parvip Microsorum kongtingense, Microsorum kravanense, innulum, Polystichum piceopaleaceum, Polystichum poly Microsorum lanceolatum, Microsorum lancifolium, blepharum, Polystichum prescottianum, Polystichum Microsorum lastii, Microsorum latilobatum, Microsorum prionolepis, Polystichum proliferum, Polystichum pseudo leandrianum, Microsorum lineare, Microsorum linguiforme, castaneum, Polystichum pseudomakinoi, Polystichum Microsorum longissimum, Microsorum longshengense, punctiferum, Polystichum pungens, Polystichum qam Microsorum maculosum, Microsorum maximum, Microso doense, Polystichum retrosopaleaceum, Polystichum rhom rum membranaceum, Microsorum membranifolium, biforme, Polystichum rhomboidea, Polystichum richardii, Microsorum microsorioides, Microsorum minor; Microso Polystichum rigens, Polystichum rotundilobum, Polysti rum monstrosum, Microsorum muliense, Microsorum chum Scopulinum, Polystichum semifertile, Polystichum mutense, Microsorum manchuanense, Microsorum ning setiferum, Polystichum setigerum, Polystichum Shensiense, poense, Microsorum normale, Microsorum novae-zealan Polystichum Silvaticum, Polystichum simplicipinnum, Poly diae, Microsorum ovalifolium, Microsorum ovatum, Stichum sinense, Polystichum squarrosum, Polystichum Microsorum palmatopedatum, Microsorum pappei, Stenophyllum, Polystichum stimulans, Polystichum submite, Microsorum papuanum, Microsorum parksii, Microsorum Polystichum tacticopterum, Polystichum thomsoni, Polysti pentaphyllum, Microsorum piliferum, Microsorum pit chum tibeticum, Polystichum transvaalense, Polystichum cairnense, Microsorum powelli, Microsorum pteropodum, tripteron, Polystichum tsus-simense, Polystichum vestitum, Microsorum pteropus, Microsorum punctatum, Microsorum Polystichum wattii, Polystichum whiteleggei, Polystichum pustulatum, Microsorum rampans, Microsorum revolutum, xiphophyllum, Polystichum vadongense, and Polystichum Microsorum rubidum, Microsorum Samarense, Microsorum Sapaense, Microsorum Sarawakense, Microsorum Scandens, vunnanense. Microsorum scolopendria, Microsorum sibOmense, 0151. In some embodiments the PtIP-83 polypeptide is Microsorum sinense, Microsorum Sopuense, Microsorum derived from a fern species in the Order Polypodiales, spectrum, Microsorum steerei, Microsorum subhemioniti Family Dryopteridaceae, Genus Rumohra. In some embodi deum, Microsorum submarginale, Microsorum subnudum, ments the nucleic acid molecule encoding the PtIP-83 poly Microsorum superficiale, Microsorum takhtajani, Microso peptide is derived from a fern species in the Order Polypo rum tenuipes, Microsorum tibeticum, Microsorum triglos diales, Family Dryopteridaceae, Genus Rumohra selected sum, Microsorum truncatum, Microsorum tsai, Microsorum from but not limited to Rumohra adiantiformis, Rumohra varians, Microsorum venosum, Microsorum vieillardii, aristata, Rumohra bartonae, Rumohra berteroana, Microsorumxinaequibasis, Microsorum viliangensis, and Rumohra capuronii, Rumohra glandulosa, Rumohra hum Microsorum zippelii. US 2016/0347799 A1 Dec. 1, 2016 50
0156. In some embodiments the PtIP-83 polypeptide is tum, Polypodium dispersum, Polypodium dissectum, Poly derived from a fern species in the Order Polypodiales, podium dissimulans, Polypodium dolichosorum, Polypo Family Polypodiaceae, Genus Polypodium L. dium dolorense, Polypodium donnell-Smithii, Polypodium (O157. In some embodiments the PtIP-83 polypeptide is drymoglossoides, Polypodium ebeninum, Polypodium egg derived from a fern species in the Order Polypodiales, ersii, Polypodium elmeri, Polypodium elongatum, Polypo Family Polypodiaceae, Genus Polypodium L. selected from dium enterosoroides, Polypodium erubescens, Polypodium but not limited to Polypodium absidatum, Polypodium acuti erythrolepis, Polypodium erythrotrichum, Polypodium eury folium, Polypodium adiantiforme, Polypodium aequale, basis, Polypodium eury basis var. villosum, Polypodium Polypodium affine, Polypodium albidopaleatum, Polypo exormans, Polypodium falcoideum, Polypodium fallacissi dium alcicorne, Polypodium alfarii, Polypodium alfredii, mum, Polypodium farinosum, Polypodium faucium, Polypo Polypodium alfredii var. Curtii, Polypodium allosuroides, dium feei, Polypodium ferrugineum, Polypodium feuillei, Polypodium alsophilicola, Polypodium amamianum, Poly Polypodium firmulum, Polypodium firmum, Polypodium podium amoenum, Polypodium amorphum, Polypodium flaccidum, Polypodium flagellare, Polypodium flexuosum, anetioides, Polypodium anfractuosum, Polypodium angui Polypodium flexuosum var. ekmanii, Polypodium forbesii, num, Polypodium angustifolium f. remotifolia, Polypodium Polypodium formosanum, Polypodium fraxinifolium Subsp. angustifolium var. amphostenon, Polypodium angustifolium articulatum, Polypodium fraxinifolium Subsp. luridum, var. heterolepis, Polypodium angustifolium var. monstrosa, Polypodium fructuosum, Polypodium fucoides, Polypodium Polypodium angustipaleatum, Polypodium angustissimum, filvescens, Polypodium galeottii, Polypodium glaucum, Polypodium aniisomeron var. pectinatum, Polypodium antio Polypodium glycyrrhiza, Polypodium gracillimum, Polypo quianum, Polypodium aoristisorum, Polypodium apagole dium gramineum, Polypodium grandifolium, Polypodium pis, Polypodium apicidens, Polypodium apiculatum, Poly gratum, Polypodium graveolens, Polypodium griseo-ni podium apoense, Polypodium appalachianum, Polypodium grum, Polypodium griseum, Polypodium guttatum, Polypo appressum, Polypodium arenarium, Polypodium argenti dium haalilioanum, Polypodium hammatisorum, Polypo num, Polypodium argutum, Polypodium armatum, Polypo dium hancockii, Polypodium haplophlebicum, Polypodium dium aromaticum, Polypodium aspersum, Polypodium harrisii, Polypodium hastatum var. Simplex, Polypodium assurgens, Polypodium atrum, Polypodium auriculatum, hawaiiense, Polypodium heanophyllum, Polypodium helleri, Polypodium balaonense, Polypodium balliviani, Polypo Polypodium hemionitidium, Polypodium henryi, Polypo dium bamleri, Polypodium bangii, Polypodium bartlettii, dium herzogii, Polypodium hesperium, Polypodium hessii, Polypodium basale, Polypodium bernoullii, Polypodium Polypodium hombersleyi, Polypodium hostmannii, Polypo biauritum, Polypodium bifrons, Polypodium blepharodes, dium humile, Polypodium hyalinum, Polypodium iboense, Polypodium bolivari, Polypodium bolivianum, Polypodium Polypodium induens var. Subdentatum, Polypodium insidi bolobense, Polypodium bombycinum, Polypodium bombyci Osum, Polypodium insigne, Polypodium intermedium Subsp. num var. insularum, Polypodium bradeorum, Polypodium masafiteranum var. Obtuseserratum, Polypodium intramar bryophilum, Polypodium bryopodum, Polypodium ginale, Polypodium involutum, Polypodium itatiavense, buchtienii, Polypodium buesii, Polypodium bulbotrichum, Polypodium javanicum, Polypodium juglandifolium, Poly Polypodium caceresii, Polypodium californicum f. braus podium kamiense, Polypodium knowltoniorum, Polypodium combii, Polypodium Californicum f. parsonsiae, Polypodium Kyimbilense, Polypodium lherminieri var. Costaricense, Californicum, Polypodium calophlebium, Polypodium cal Polypodium lachniferum f. incurvata, Polypodium lach vum, Polypodium camptophyllarium var. abbreviatum, Poly niferum var. glabrescens, Polypodium lachnopus, Polypo podium capitellatum, Polypodium carpinterae, Polypodium dium lanceolatum var. complanatum, Polypodium lanceo chachapoyense, Polypodium chartaceum, Polypodium chi latum var. trichophorum, Polypodium latevagans, mantense, Polypodium chiricanum, Polypodium choquetan Polypodium laxifrons, Polypodium laxifrons var. lividum, gense, Polypodium christensenii, Polypodium christii, Poly Polypodium lehmannianum, Polypodium leiorhizum, Poly podium chrysotrichum, Polypodium ciliolepis, Polypodium podium leptopodon, Polypodium leuconeuron var. angusti cinerascens, Polypodium collinsii, Polypodium colysoides, folia, Polypodium leuconeuron var. latifolium, Polypodium Polypodium confluens, Polypodium conforme, Polypodium leucosticta, Polypodium limulum, Polypodium lindigii, confusum, Polypodium congregatifolium, Polypodium con Polypodium lineatum, Polypodium lomarioides, Polypo nellii, Polypodium consimile var. bourgaeanum, Polypo dium longifrons, Polypodium loretense, Polypodium loric dium consimile var. minor; Polypodium conterminans, Poly eum var. umbraticum, Polypodium loriforme, Polypodium podium contiguum, Polypodium cookii, Polypodium loxogramme f. gigas, Polypodium ludens, Polypodium coriaceum, Polypodium coronans, Polypodium costari luzonicum, Polypodium lycopodioides f. Obtusum, Polypo cense, Polypodium costatum, Polypodium Crassifolium f. dium lycopodioides L., Polypodium macrolepis, Polypodium angustissimum, Polypodium crassifolium var. longipes, macrophyllum, Polypodium macrosorum, Polypodium mac Polypodium crassulum, Polypodium Craterisorum, Polypo rosphaerum, Polypodium maculosum, Polypodium dium cryptum, Polypodium crystalloneuron, Polypodium madrense, Polypodium manmeiense, Polypodium margari cucullatum var. planum, Polypodium cuencanum, Polypo tiferum, Polypodium maritimum, Polypodium martensii, dium cumingianum, Polypodium cupreolepis, Polypodium Polypodium mayoris, Polypodium megalolepis, Polypodium curranii, Polypodium curvans, Polypodium cyathicola, melanotrichum, Polypodium menisciifolium var. pubescens, Polypodium cyathisorum, Polypodium cyclocolpon, Polypo Polypodium meniscioides, Polypodium merrillii, Polypo dium daguense, Polypodium damunense, Polypodium dare dium mettenii, Polypodium mexiae, Polypodium microso iformioides, Polypodium dasypleura, Polypodium decipiens, rum, Polypodium militare, Polypodium minimum, Polypo Polypodium decorum, Polypodium delicatulum, Polypo dium minusculum, Polypodium mixtum, Polypodium dium deltoideum, Polypodium demeraranum, Polypodium mollendense, Polypodium mollissimum, Polypodium monili denticulatum, Polypodium diaphanum, Polypodium dilata forme var. minus, Polypodium monoides, Polypodium mon US 2016/0347799 A1 Dec. 1, 2016 51 ticola, Polypodium montigenium, Polypodium moritzianum, dum, Polypodium sphaeropteroides, Polypodium sphenodes, Polypodium moultonii, Polypodium multicaudatum, Poly Polypodium sprucei, Polypodium spruceivar. furcativenosa, podium multilineatum, Polypodium multisorum, Polypo Polypodium Steirolepis, Polypodium Stenobasis, Polypo dium munchi, Polypodium muscoides, Polypodium myriole dium Stenolepis, Polypodium Stenopterum, Polypodium sub pis, Polypodium myriophyllum, Polypodium myriotrichum, capillare, Polypodium subflabelliforme, Polypodium subhe Polypodium nematorhizon, Polypodium memorale, Polypo mionitidium, Polypodium subinaequale, Polypodium dium nesioticum, Polypodium nigrescentium, Polypodium subintegrum, Polypodium subspathulatum, Polypodium nigripes, Polypodium nigrocinctum, Polypodium nimbatum, subtile, Polypodium subvestitum, Polypodium subviride, Polypodium superficiale var. attenuatum, Polypodium Polypodium nitidissimum, Polypodium nitidissimum var. superficiale var. Chinensis, Polypodium Sursuincurrens, lation; Polypodium nubrigemum, Polypodium Oligolepis, Polypodium tablazianum, Polypodium taenifolium, Polypo Polypodium Oligosorum, Polypodium Oligosorum, Polypo dium tamandarei, Polypodium tatei, Polypodium tenuiculum dium Olivaceum, Polypodium Olivaceum var. elatum, Poly var. acrosora, Polypodium tenuiculum var. brasiliense, podium Oodes, Polypodium oosphaerum, Polypodium Oreo Polypodium tenuilore, Polypodium tenuinerve, Polypodium philum, Polypodium ornatissimum, Polypodium ornatum, tepuiense, Polypodium teresae, Polypodium tetragonum var. Polypodium ovatum, Polypodium oxylobum, Polypodium incompletum, Polypodium thysanolepis var. bipinnatifidum, Oxypholis, Polypodium pakkaense, Polypodium pallidum, Polypodium thyssanolepis, var. thyssanolepis, Polypodium Polypodium palmatopedatum, Polypodium palmeri, Poly thyssanolepsi, Polypodium tobagense, Polypodium podium panamense, Polypodium parvum, Polypodium trichophyllum, Polypodium tridactylum, Polypodium triden patagonicum, Polypodium paucisorum, Polypodium pavo tatum, Polypodium trifurcatum var. brevipes, Polypodium nianum, Polypodium pectinatum var. Caliense, Polypodium triglossum, Polypodium truncatulum, Polypodium trunci pectinatum var. hispidum, Polypodium pellucidum, Polypo colla var. major; Polypodium truncicola var. minor, Polypo dium pendulum var. boliviense, Polypodium percrassum, dium tuberosum, Polypodium tunguraguae, Polypodium Polypodium perpusillum, Polypodium peruvianum var. Sub turquinum, Polypodium turrialbae, Polypodium ursipes, gibbosum, Polypodium phyllitidis var. elongatum, Polypo Polypodium vagans, Polypodium valdealatum, Polypodium dium pichinchense, Polypodium pilosissimum, Polypodium versteegii, Polypodium villagranii, Polypodium virginianum pilosissimum var. glabriusculum, Polypodium pilossinum f. Cambroideum, Polypodium virginianum f. peraferens, var. tunguraquensis, Polypodium pitvrolepis, Polypodium Polypodium vittarioides, Polypodium vulgare, Polypodium platyphyllum, Polypodium playfairii, Polypodium plebeium vulgare L., Polypodium vulgare subsp. Oreophilum, Polypo var. cooperi, Polypodium plectolepidioides, Polypodium dium vulgare var. acuminatum, Polypodium vulpinum, Poly pleolepis, Polypodium plesiosorum var. i. Polypodium podo podium williamsii, Polypodium wobbense, Polypodiumxfal basis, Polypodium podocarpum, Polypodium poloense, lacissimum-guttatum, Polypodium xantholepis, Polypodium Polypodium polydatylon, Polypodium polypodioides var. xiphopteris, Polypodium varumalense, Polypodium yun aciculare, Polypodium polypodioides var. michauxianum, Polypodium praetermissium, Polypodium preslianum var. gense, and Polypodium Zosteriforme. immersum, Polypodium procerum, Polypodium procerum, 0158. In some embodiments the PtIP-83 polypeptide is Polypodium productum, Polypodium productum, Polypo derived from a fern species in the Order Polypodiales, dium prolongilobum, Polypodium propinguum, Polypodium Family Polypodiaceae, Genus Platycerium. proteus, Polypodium pruinatum, Polypodium pseudocapil 0159. In some embodiments the PtIP-83 polypeptide is lare, Polypodium pseudofraternum, Polypodium pseudo derived from a species in the Division Lycophyta. nutans, Polypodium pseudoserratum, Polypodium pulcher 0160. In some embodiments the PtIP-83 polypeptide is rimum, Polypodium pulogense, Polypodium pungens, derived from a species in the Class Isoetopsida or Class Polypodium purpusii, Polypodium radicale, Polypodium Lycopodiopsida. randallii, Polypodium ratiborii, Polypodium reclimatum, 0.161. In some embodiments the PtIP-83 polypeptide is Polypodium recreense, Polypodium repens var. abruptum, derived from a species in the Class Isoetopsida Order Polypodium revolvens, Polypodium rhachipterygium, Poly Selaginales. In some embodiments the PtIP-83 polypeptide podium rhomboideum, Polypodium rigens, Polypodium is derived from a fern species in the Class Isoetopsida, Order robustum, Polypodium roraimense, Polypodium roraimense, Selaginales, Family Selaginellaceae. In some embodiments Polypodium rosei, Polypodium rosenstockii, Polypodium the PtIP-83 polypeptide is derived from a species in the rubidum, Polypodium rudimentum, Polypodium rusbyi, Genus Selaginella. In some embodiments the PtIP-83 poly Polypodium sablanianum, Polypodium sarmentosum, Poly peptide is derived from a species in the Class Lycopodiop podium saxicola, Polypodium schenckii, Polypodium sida, Order Lycopodiales. Schlechteri, Polypodium scolopendria, Polypodium scolo 0162. In some embodiments the PtIP-83 polypeptide is pendria, Polypodium scolopendrium, Polypodium scouleri, derived from a fern species in the Class Lycopodiopsida, Polypodium scutulatum, Polypodium segregatum, Polypo Order Lycopodiales Family Lycopodiaceae or Family dium semihirsutum, Polypodium semihirsutum var. fiascose tosum, Polypodium senile var. minor, Polypodium sericeo HuperZiaceae. lanatum, Polypodium serraeforme, Polypodium serricula, 0163. In some embodiments the PtIP-83 polypeptide is Polypodium sesquipedala, Polypodium sessilifolium, Poly derived from a species in the Genus Austrolycopodium, podium setosum var. calvum, Polypodium setulosum, Poly Dendrolycopodium, Diphasiastrum, Diphasium, Huperzia, podium shaferi, Polypodium sibOmense, Polypodium sic Lateristachys, Lycopodiastrum, Lycopodiella, Lycopodium, cum, Polypodium simacense, Polypodium simulans, Palhinhaea, Pseudodiphasium, Pseudolycopodiella, Polypodium singeri, Polypodium sinicum, Polypodium sin Pseudolycopodium or Spinulum. tenisii, Polypodium Skutchii, Polypodium Sloanei, Polypo 0164. In some embodiments the PtIP-83 polypeptide is dium sodiroi, Polypodium Sordidulum, Polypodium Sordi derived from a species in the Genus Lycopodium. US 2016/0347799 A1 Dec. 1, 2016 52
0.165. In some embodiments the PtIP-83 polypeptide is 1994) for identification of key sequence motifs. MEME was derived from a species in the Genus Huperzia. setup as follows: Minimum number of sites 2, Minimum motif width 5, and Maximum number of motifs 30. Phylogenetic, Sequence Motif, and Structural Analyses for Sequence motifs unique to each Sub-family were identified Insecticidal Protein Families by visual observation. The distribution of MOTIFs across 0166 The sequence and structure analysis method the entire gene family could be visualized in HTML web employed is composed of four components: phylogenetic page. The MOTIFs are numbered relative to the ranking of tree construction, protein sequence motifs finding, second the E-value for each MOTIF. The amino acid sequence ary structure prediction, and alignment of protein sequences MOTIFs identified for each of the PtIP-83 polypeptides and and secondary structures. Details about each component are the residue ranges defining the MOTIFs relative to each of illustrated below. the corresponding sequence identifier (SEQ ID NO:) are shown in Table 2. FIG. 2 shows an alignment of the PtIP-83 Phylogenetic Tree Construction polypeptides PtIP-83Aa (SEQ ID NO: 1), PtIP-83Ca(SEQ ID NO. 5), PtIP-83Cb (SEQID NO:7), PtIP-83Cc (SEQ ID 0167. The phylogenetic analysis was performed using the NO:9), Pt|P-83Cd (SEQ ID NO: 11), PtIP-83Ce (SEQ ID software MEGA5. Protein sequences were subjected to NO: 13), PtIP-83Cf (SEQID NO: 15), and PtIP-83Fa (SEQ ClustalW version 2 analysis (Larkin M. A et al (2007) ID NO:3), and the location relative to PtIP-83Aa (SEQ ID Bioinformatics 23 (21): 2947-2948) for multiple sequence NO: 1) of the amino acid sequence MOTIFs present in alignment. The evolutionary history was then inferred by the PtIP-83Aa (SEQ ID NO: 1). Maximum Likelihood method based on the JTT matrix based model. The tree with the highest log likelihood was Secondary Structure Prediction obtained, exported in Newick format, and further processed to extract the sequence IDs in the same order as they (0169 PSIPRED, top ranked secondary structure predic appeared in the tree. A few clades representing Sub-families tion method (Jones D.T. (1999).J. Mol. Biol. 292: 195-202), were manually identified for each insecticidal protein fam was installed in local Linux server, and used for protein ily. secondary structure prediction. The tool provides accurate structure prediction using two feed-forward neural networks based on the PSI-BLAST output. The PSI-BLAST database Protein Sequence Motifs Finding was created by removing low-complexity, transmembrane, 0168 Protein sequences were re-ordered according to the and coiled-coil regions in Uniref100. The PSIPRED results phylogenetic tree built previously, and fed to the MOTIF contain the PtIP-secondary structures (Alpha helix: H, Beta analysis tool MEME (Multiple EM for MOTIF Elicitation) Strand: E, and Coil: C) and the corresponding confidence (Bailey T. L., and Elkan C. Proceedings of the Second scores for each amino acid in a given protein sequence. FIG. International Conference on Intelligent Systems for Molecu 2 shows the PtIP-83 polypeptide amino acid sequence lar Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., alignments and the conserved secondary structural regions. TABLE 2 SEQ ID MOTIF MOTIF MOTIF MOTIF MOTIF MOTIF NO: 19 7 13 2O 10 18
PtP-83Aa 1 4-17 21-64 71-1OO 102-120 Il.p. Il.p. PtP-83Ca 5 1-14 17-60 68-97 98-116 122-171 173-208 PIP-83Cb 7 1-14 17-60 69-98 Il.p. 121-170 m.p. PtP-83CC 9 1-14 17-60 68-97 98-116 122-171 173-208 PtP-83Cd 11 1-14 17-60 68-97 98-116 122-171 173-208 PtP-83Ce 13 1-14 17-60 68-97 98-116 121-17O 173-208 PIP-83Cf 15 1-14 21-64 68-97 98-116 122-171 m.p. PtP-83Fa 3 S-18 17-60 68-97 Il.p. Il.p. Il.p. SEQ ID MOTIF MOTIF MOTIF MOTIF MOTIF MOTIF NO: 24 14 11 22 2 8
PtP-83Aa 1 Il.p. 238-263 Il.p. 313-326 327-376 376-425 PtP-83Ca 5 211-220 221-246 248-297 298-311 312-361 362-411 PIP-83Cb 7 Il.p. 223-248 249-298. 299-312 313-362 363-412 PtP-83CC 9 211-220 221-246 248-297 298-311 312-361 362-411 PtP-83Cd 11 211-220 221-246 248-297 298-311 312-361 362-411 PtP-83Ce 13 211-220 221-246 248-297 298-311 312-361 362-411 PIP-83Cf 15 211-220 221-246 248-297 298-311 312-361 362-411 PtP-83Fa 3 247-256 262-287 Il.p. 329-342 344-393 394-443
SEQ ID MOTIF MOTIF MOTIF MOTIF MOTIF MOTIF NO: 21 15 9 1 17 6
PtP-83Aa 1 SO8-521 428-453 455-504 S23-572 S76-594 S96-645 PtP-83Ca 5 493-506 413-438 441-490 SO8-SS7 S61-579 S81-630 PIP-83Cb 7 494-507 414-439 442-491 SO9-558 S62-580 S82-631 PtP-83CC 9 493-506 413-438 441-490 SO8-SS7 S61-579 S81-630 PtP-83Cd 11 493-506 413-438 441-490 SO8-SS7 S61-579 S81-630 PtP-83Ce 13 493-506 413-438 441-490 SO8-SS7 S61-579 S81-630 US 2016/0347799 A1 Dec. 1, 2016 53
TABLE 2-continued PIP-83Cf 15 493-506 413-438 441-490 SO8-SS7 S61-579 581-630 PtP-83Fa 3 S25-538 445-470 473-522 S40-589 S93-611 613-662 SEQ ID MOTIF MOTIF MOTIF MOTIF MOTIF MOTIF NO: 12 4 16 5 23 3
PtP-83Aa 1 648-683 684-719 723-741 746-795 798-807 809-858 PtP-83 Ca 5 634-669 670-70S 708-726 732-781 784-793 795-844 PIP-83Cb 7 635-670 671-706 709–727 731-780 783-792 794-843 PtP-83 Cc 9 634-669 670-70S 708-726 730-779 782-791 793-842 PtP-83C 11 634-669 670-70S 708-726 730-779 782-791 793-842 PtP-83Ce 13 634-669 670-70S 708-726 730-779 782-791 793-842 PIP-83Cf 15 634-669 670-70S 708-726 731-780 783-792 794-843 PtP-83Fa 3 667-702 703-738 740-758 763-812 815-824 826-875 n.p. = not present
Alignment of Protein Sequences and Secondary Structures MOTIF 11 as represented by an amino acid sequence of the formula KVVKCARPPSPDMMVISAVAEHAL 0170 A customized script was developed to generate WLNDVLLQWQKNESQLMIQGTAEPYNECLAL gapped secondary structure alignment according to the mul LGR (SEQID NO: 656); an amino acid sequence MOTIF 12 tiple protein sequence alignment from step 1 for all proteins. as represented by an amino acid sequence of the formula All aligned protein sequences and structures were concat PTELTIVAWPLGMDTVLAGNLLIAQENAALVL enated into a single FASTA file, and then imported into GLIQLGPSS (SEQ ID NO: 657); an amino acid sequence MEGA for visualization and identification of conserved MOTIF 13 as represented by an amino acid sequence of the Structures. formula RDQIMTHQIMPGSVTVILIVILCRLLQFPIT 0171 In some embodiments a PtIP-83 polypeptide com DGSQAITAT (SEQ ID NO: 658); an amino acid sequence prises an amino acid sequence MOTIF selected from: an MOTIF 14 as represented by an amino acid sequence of the amino acid sequence MOTIF 1 as represented by an amino formula TSIPVEVVTDPSNILLGMQTTVILHIAEL acid sequence of the formula MPIDEMPSEADWSIFVNE (SEQ ID NO: 659); an amino acid sequence MOTIF 15 as IVEAVAEGMPTEVSEVPAVIWKAKCKNMVIAAL represented by an amino acid sequence of the formula GREMSCI (SEQ ID NO: 646); an amino acid sequence EGLREQIFQNRQVARAVLFAVLKAVAMQILAG) MOTIF 2 as represented by an amino acid sequence of the (SEQ ID NO: 660); an amino acid sequence MOTIF 16 as formula PQLQYRMYGNSLIKNIQMAQVAQNYDO represented by an amino acid sequence of the formula EDIFKQFLIKLFIIAQNQILFIG.SYLLQQNKRIA F WITSIRVRIRHLEMIQHIFLAVIQEASG (SEQ ID NO: (SEQ ID NO: 647); an amino acid sequence MOTIF 3 as 661); an amino acid sequence MOTIF 17 as represented by represented by an amino acid sequence of the formula an amino acid sequence of the formula QISELQYED NTFMQMTPFTRWRLRLSASASENAEGLAFPTATA IWVOGLMMLRDIA (SEQ ID NO: 662); an amino acid PLDSTTEQIVIVITFHVTAIR (SEQ ID NO: 648); an sequence MOTIF 18 as represented by an amino acid amino acid sequence MOTIF 4 as represented by an amino sequence of the formula TFTLGSGVTGITSMHGEPSLD acid sequence of the formula DNFTSRHVVKGDIP PWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acid VSLLLDGEDWEFEIPVQAGIGMSSFP (SEQ ID NO: sequence MOTIF 19 as represented by an amino acid 649); an amino acid sequence MOTIF5 as represented by an sequence of the formula MDYSTLYRDLNOIS (SEQ ID amino acid sequence of the formula IIHQPSATROIST NO: 664); an amino acid sequence MOTIF 20 as represented GITIVYILLQGSTIFHDRRRDEEVMTFQAADAPLN by an amino acid sequence of the formula LRLPFMQK FYIQHIYAYRLDTG (SEQ ID NO: 650); an amino acid LHARVIEQNVRKSE (SEQID NO: 665); an amino acid sequence MOTIF 6 as represented by an amino acid sequence MOTIF 21 as represented by an amino acid sequence of the formula SHOADRLAAIQPAVIDLTN sequence of the formula VDSLEQVGQHILIVIGDAP HYLEMATHQMDMRTTRSMILILIGLLNMI (SEQ ID NO: 666); an amino acid sequence MOTIF 22 as LRIONAALMY EY (SEQ ID NO: 651); an amino acid represented by an amino acid sequence of the formula sequence MOTIF 7 as represented by an amino acid IVEQCAVMKIIMIGRFVGSLIW (SEQ ID NO: sequence of the formula VLDRVEFSEVMVIHRMYVRL 667); an amino acid sequence MOTIF 23 as represented by SADLINDIVGELPEIGAEGRKTVKRVLIYVFL an amino acid sequence of the formula TLTNEPSEEQF ADWE (SEQID NO: 652); an amino acid sequence MOTIF (SEQ ID NO: 668); and an amino acid sequence MOTIF 24 8 as represented by an amino acid sequence of the formula as represented by an amino acid sequence of the formula AIDERELQMESFHSAVISQRRQELINDITALIFAKM LPRQSRNISF (SEQ ID NO: 669). DERLMISLQMEEENSRAMEQAQKE M (SEQ ID 0172. In some embodiments a PtIP-83 polypeptide com NO: 653); an amino acid sequence MOTIF 9 as represented prises an amino acid sequence MOTIF selected from: an by an amino acid sequence of the formula FVTAGATAPGA amino acid sequence MOTIF 1 having at least 90% |AVIASAGQAVSIAGQAAQAGLRRWEILEGQ sequence identity to the amino acid sequence as represented LEAVMEWAAVIK (SEQ ID NO: 654); an amino acid by the formula MPIDEMPSEADWSIFVNELIVEAVAE sequence MOTIF 10 as represented by an amino acid GMPTEVSEVPAVIWKAKCKNMVIAALGREMISCII sequence of the formula DGMNWGITIYIYHIGELKETV (SEQ ID NO: 646); an amino acid sequence MOTIF 2 EQRSPLLPSNAILAVWADRCITIITSARHNHIVF having at least 90% sequence identity to the amino acid NAPGRIVII (SEQ ID NO: 655); an amino acid sequence sequence as represented by the formula PQLQYRMYGNS US 2016/0347799 A1 Dec. 1, 2016 54
LIKNIQMAQVAQNYDOEDIFKQFLIKLFILIAQNQI to the amino acid sequence as represented by the formula LFIG.SYLLQQNKRAF (SEQ ID NO: 647); an amino TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF acid sequence MOTIF 3 having at least 90% sequence (SEQ ID NO: 663); an amino acid sequence MOTIF 19 identity to the amino acid sequence as represented by the having at least 90% sequence identity to the amino acid formula NTFMQMTPFTRWRLRLSASASENAEG sequence as represented by the formula MDYSTLYRDLN LAFPTATAPLDSTTEQIVIVITFHVTAIR (SEQ ID QIS (SEQID NO: 664); an amino acid sequence MOTIF 20 NO: 648); an amino acid sequence MOTIF 4 having at least having at least 90% sequence identity to the amino acid 90% sequence identity to the amino acid sequence as sequence as represented by the formula LRLPFMQK represented by the formula DNFTSRHVVKGDIP LHARVIEQNVRKSE (SEQID NO: 665); an amino acid VSLLLDGEDWEFEIPVQAGIGMSSFP (SEQ ID NO: sequence MOTIF 21 having at least 90% sequence identity 649); an amino acid sequence MOTIF5 having at least 90% to the amino acid sequence as represented by the formula sequence identity to the amino acid sequence as represented VDSLEQVGQHILIVIGDAP (SEQ ID NO: 666); an amino acid sequence MOTIF 22 having at least 90% by the formula IIHQPSATRQISTIGITIVYILLQG sequence identity to the amino acid sequence as represented STIFHDRRRDEEVMTFQAADAPLNFYIQHIYAY RLDT. G. (SEQ ID NO: 650); an amino acid sequence by the formula IVIEQCAVMKIIMGRFIVGSLIVV MOTIF6 having at least 90% sequence identity to the amino (SEQ ID NO: 667): an amino acid sequence MOTIF 23 acid sequence as represented by the formula SHOADR having at least 90% sequence identity to the amino acid LAAIQPLAVIDLTNHYLEMATHQMDMRTTRSMI sequence as represented by the formula TLTNEPSEEQF LILIGLLNMILRIQNAALMY EY (SEQID NO: 651); an (SEQ ID NO: 668); and an amino acid sequence MOTIF 24 amino acid sequence MOTIF 7 having at least 90% having at least 90% sequence identity to the amino acid sequence identity to the amino acid sequence as represented sequence as represented by the formula LPROSRNISF by the formula VLDRVEFSEVMVIHRMYVRLISADL (SEQ ID NO: 669). NDTVGELIPEIGAEGIRKIVKRVLIYVFLADWE 0173. In some embodiments a PtIP-83 polypeptide com (SEQ ID NO: 652); an amino acid sequence MOTIF 8 prises an amino acid sequence MOTIF selected from: an having at least 90% sequence identity to the amino acid amino acid sequence MOTIF 1 as represented by an amino sequence as represented by the formula AIDERELQMES acid sequence of the formula MPIDEMPSTEDAD FHSAVISQRRQELNDITAIFAKMIDERLM WSIFVNEIVLEAVAEGMPTEVSEVPAVIWKR) SLQMEEENSRAMEQAQKEM (SEQ ID NO: 653); an AKCKNMVIAALGRE MISCII (SEQ ID NO: 670); an amino acid sequence MOTIF 9 having at least 90% amino acid sequence MOTIF 2 as represented by an amino sequence identity to the amino acid sequence as represented acid sequence of the formula PQLQYRMYGNSLIKRN by the formula FVTAGATAPGAAVIASAGQAV QMAQVAQNYDIQRIEDIFKQRFLKRLFILAVL) SIAGQAAQAGLRRWEILEGQLEAVMEWAAVIK QNQILFIG.SYL LQEQNKRAF (SEQ ID NO: 671); an (SEQ ID NO: 654); an amino acid sequence MOTIF 10 amino acid sequence MOTIF 3 as represented by an amino having at least 90% sequence identity to the amino acid acid sequence of the formula NITKFMQMTPFTRHIWR sequence as represented by the formula DGMNWGITIYI LRLSASASPKAENAKEGLAFPTATAPLDSTT YHIGEIKEIVEQRSPLLPSNAILAVWADRCTIIT EQIVIVAITF HVTAIR (SEQ ID NO: 672); an amino SARHNHIVFINAPGRIVII (SEQ ID NO: 655); an amino acid sequence MOTIF 4 as represented by an amino acid acid sequence MOTIF 11 having at least 90% sequence sequence of the formula DNFTSRHVVKGDIPVISN identity to the amino acid sequence as represented by the LLLDGEGDWEFEIPVQAGIGMSSFP (SEQ ID NO: formula KVVKCARPPSPDMMVISAVAEHAL 673); an amino acid sequence MOTIF5 as represented by an WLNDVLLQWQKNESQLMIQGTAEPYNECLAL amino acid sequence of the formula IIHQPSATROIST LGR (SEQID NO: 656); an amino acid sequence MOTIF 12 GITIVIIYILLQGSTIVFHDRRRDEEQIVIMLITIFP having at least 90% sequence identity to the amino acid QAADAVIPLNFYIQHIYAYRLDTG (SEQ ID NO: sequence as represented by the formula PTELTIVAWPL 674); an amino acid sequence MOTIF 6 as represented by an GMDTVLAGNLLIAOENAALIVLIGLIQLGPSS (SEQID amino acid sequence of the formula SHOADRLAAIQP NO: 657); an amino acid sequence MOTIF 13 having at least |AVIDNLTNIHYFILEMATHQMDMRTTRSIMIL 90% sequence identity to the amino acid sequence as ILGLLNMILMRIQN AALMRYEY (SEQ ID NO: represented by the formula RDQIMTHQMPGSVTVI 675); an amino acid sequence MOTIF 7 as represented by an IVILCRLLQFPITDGSQAITAT (SEQ ID NO: 658); an amino acid sequence of the formula VLDRQIVEF amino acid sequence MOTIF 14 having at least 90% SEVMVIHRMYVINRLISADLINDIVIGAEQLIPE sequence identity to the amino acid sequence as represented GAEGIRKIVKRVLIYVFLADVVE (SEQ ID NO: by the formula TSIPVEWTDPSNILLGMQTTVLH 676); an amino acid sequence MOTIF 8 as represented by an IAEL (SEQ ID NO: 659); an amino acid sequence MOTIF amino acid sequence of the formula AIDERELQMESFH 15 having at least 90% sequence identity to the amino acid SAAVISQRKRQGEELNDTDATIFAKMIDE sequence as represented by the formula EGLREQFQN RLMISLQMEEENS DIRGAMEQAQRIKEM (SEQID RQVARAVLFAVLKAVAMQILAG) (SEQID NO: 660); NO: 677); an amino acid sequence MOTIF 9 as represented an amino acid sequence MOTIF 16 having at least 90% by an amino acid sequence of the formula FVLITAGAT sequence identity to the amino acid sequence as represented APGAAVIASAGQAVSNIAGQAAQAGLRRVVEILE by the formula WITSRVRIRHLEMIQHIFLAVIQEASG GQLEAVMEVVAAVIIK (SEQ ID NO: 678); an amino (SEQ ID NO: 661); an amino acid sequence MOTIF 17 acid sequence MOTIF 10 as represented by an amino acid having at least 90% sequence identity to the amino acid sequence of the formula DIGDIMAINKIWGITIYIV sequence as represented by the formula QISELQYED YHIGAEKEVTEQIRVLISPLLYFIPNISNGINW) IWVOGLMMLRDIA (SEQ ID NO: 662); an amino acid ASPIYLAG VIVIWEADQRICSTIITISAA sequence MOTIF 18 having at least 90% sequence identity |RFMHNIHVTIVFINDIAERPG|RWILIVIIIR (SEQ US 2016/0347799 A1 Dec. 1, 2016
ID NO: 679); an amino acid sequence MOTIF 11 as repre FEIPVQAGIGMSSFP (SEQ ID NO: 673); an amino acid sented by an amino acid sequence of the formula KVVK sequence MOTIF 5 having at least 90% sequence identity to |CARGCPHYPSPIDEMILIMVISAVAGEVHA the amino acid sequence as represented by the formula LI NWLNSDKVLLIQRIWOK NESQHILLMIQGT IIHQPSAITIRQISTIGITIVIIYILLQGSTIVFHDRRR AEPSAYNECLALLGR (SEQ ID NO: 680); an amino DEEQIVIMLITIFPIQAADAVIPLNFYIQHIYAY acid sequence MOTIF 12 as represented by an amino acid RLDTG (SEQ ID NO: 674); an amino acid sequence sequence of the formula (PNITEQLTIVATIWPLGRM MOTIF6 having at least 90% sequence identity to the amino DTVAGINDILLIATIQHEINSAALIVLSIGLITMA acid sequence as represented by the formula SHOADR QLGPQIS PS (SEQ ID NO: 681); an amino acid LAAIQPAVIDNILTNIHYFILEMATHQMDMRTTRS sequence MOTIF 13 as represented by an amino acid MILILIGLLNMILMRIQN AALMRYEY (SEQ ID sequence of the formula RLCIDLWKQNPRIMTP NO: 675); an amino acid sequence MOTIF 7 having at least HQRIMILPGSVTVILIVILCRLLQFPITIDGIGSR) 90% sequence identity to the amino acid sequence as QFRASTADITW (SEQ ID NO: 682); an amino acid represented by the formula VLDRQIVEFSEVM sequence MOTIF 14 as represented by an amino acid VIHRMYVINRLISADLINDIVIGAEQLPEIGAEG sequence of the formula (TAISGVIILPVIEDIWTDPSN) RKIVKRVLIYVFLADVVE (SEQ ID NO: 676); an ILLMIGMOTITSVILHIAEL (SEQ ID NO: 683); an amino acid sequence MOTIF 8 having at least 90% amino acid sequence MOTIF 15 as represented by an amino sequence identity to the amino acid sequence as represented acid sequence of the formula EGLREQIFQNREQVA by the formula AIDERELQMESFHSAAVISQRKRQ RNAIVLFAVLKSASIVAMQILAG) (SEQ ID NO: GEELNDTDATIFAKMDERLMISLQMEEENS 684); an amino acid sequence MOTIF 16 as represented by DIRGAMEQAQRIKEM (SEQ ID NO: 677); an amino an amino acid sequence of the formula WITSRVRIRHLEM acid sequence MOTIF 9 having at least 90% sequence QHIFLAVIQKEASISMIGN (SEQ ID NO: 685); an identity to the amino acid sequence as represented by the amino acid sequence MOTIF 17 as represented by an amino formula FIVLITAGATAPGAAVIASAGQAVSN) acid sequence of the formula QIMSEQLQYED IAGQAAQAGLRRVVEILEGOLEAVMEVVAAVIK IWVOGLMMLRDIMA (SEQ ID NO: 686); an amino (SEQ ID NO: 678); an amino acid sequence MOTIF 10 acid sequence MOTIF 18 as represented by an amino acid having at least 90% sequence identity to the amino acid sequence of the formula TFTLGSGVTGITSMHGEPSLD sequence as represented by the formula DIGDIMAINK PWNGVSLDSASPTAF (SEQ ID NO: 663); an amino acid WGITIYIVIIYHIGAEKEIVEQIRVLISPLLYFIPN) sequence MOTIF 19 as represented by an amino acid SNGINWASPIIIYLAG VIVIWEADQRICSTIIT sequence of the formula MLVDYISKITSKILIYFIRE SAARFMHNIHVTIVFINDIAERPG|RWIVIR DLNOIS (SEQ ID NO: 687); an amino acid sequence (SEQ ID NO: 679); an amino acid sequence MOTIF 11 MOTIF 20 as represented by an amino acid sequence of the having at least 90% sequence identity to the amino acid formula LRHQLPTFMQKLHARITIVQLIRE sequence as represented by the formula KVVKCA QERNFVRKWSSE (SEQ ID NO: 688): an amino RGCPHYPSPIDEMILIMVISAVAGEVHALI acid sequence MOTIF 21 as represented by an amino acid NIWLNSDKVLLIQRIWOK NESQHILMIQGT sequence of the formula VIDNISALEDIQVGSIQH AEPSAYNECLALLGR (SEQ ID NO: 680); an amino ILIVIGDAP (SEQ ID NO: 689); an amino acid sequence acid sequence MOTIF 12 having at least 90% sequence MOTIF 22 as represented by an amino acid sequence of the identity to the amino acid sequence as represented by the formula IVEOHCASIVAMIKIMIGVIRPFI) formula (PNITIEQLTIVATIWPLGRIMDTVLAGINDL VGSLIW (SEQ ID NO: 690); an amino acid sequence LILATIOHENSAALIVLSIGLITMAQLGPQIS PIS MOTIF23 as represented by an amino acid sequence of the (SEQ ID NO: 681); an amino acid sequence MOTIF 13 formula TLTNEQIPSEEQDHF (SEQ ID NO: 691); and having at least 90% sequence identity to the amino acid an amino acid sequence MOTIF 24 as represented by an sequence as represented by the formula RLCDLWK amino acid sequence of the formula LPRSQSRTINIV QNPRIMTPHQRIMILPGSVTVIIVLCRLLQFPIT SF (SEQ ID NO: 692). DGIGISROFRASTADITW (SEQ ID NO: 682); an 0.174. In some embodiments a PtIP-83 polypeptide com amino acid sequence MOTIF 14 having at least 90% prises an amino acid sequence MOTIF selected from: an sequence identity to the amino acid sequence as represented amino acid sequence MOTIF 1 having at least 90% by the formula (TAISGVILIPVEDIVVTDPSNILLMI sequence identity to the amino acid sequence as represented GMQTITSVILHIAEL (SEQ ID NO: 683); an amino acid by the formula MPIDEMPSTEDADWSIFVNELIVL) sequence MOTIF 15 having at least 90% sequence identity EAVAEGMPTEVSEVPAVWKRAKCKNMVIAAL to the amino acid sequence as represented by the formula GRE MISCI (SEQ ID NO: 670); an amino acid sequence EGLREQIFQNREQVARNAIVLFAVLKSASIVA MOTIF2 having at least 90% sequence identity to the amino MQIAG (SEQ ID NO: 684); an amino acid sequence acid sequence as represented by the formula PQLQYRMYG MOTIF 16 having at least 90% sequence identity to the NSLIKRNIQMAQVAQNYDIQREDIFKQRFL amino acid sequence as represented by the formula WITS KRLFILIAVLQNQILFIG.SYL LIQEQNKRAF (SEQ RVRIRHLEMIQHIFLAVIQKEASISMIGN (SEQ ID ID NO: 671); an amino acid sequence MOTIF 3 having at NO: 685); an amino acid sequence MOTIF 17 having at least least 90% sequence identity to the amino acid sequence as 90% sequence identity to the amino acid sequence as represented by the formula NITKFMQMTPFTRHIWRL represented by the formula QIMSEQLQYEDIWVOG RLSASASPKAENAKEGLAFPTATAPLDSTTEQ LMMLRDIMA (SEQ ID NO: 686); an amino acid IVIVAITF HVTAIR (SEQ ID NO: 672); an amino acid sequence MOTIF 18 having at least 90% sequence identity sequence MOTIF 4 having at least 90% sequence identity to to the amino acid sequence as represented by the formula the amino acid sequence as represented by the formula TFTLGSGVTGITSMHGEPSLDPWNGVSLDSASPTAF DNFTSRHVVKGDIPVSNLLLDGEGDWE (SEQ ID NO: 663); an amino acid sequence MOTIF 19 US 2016/0347799 A1 Dec. 1, 2016 56 having at least 90% sequence identity to the amino acid SPLLYFIVPNQS NGTQNWQIASPTIYLVL sequence as represented by the formula MLVDYISK AGVILIVIWEDADQNERICSTITISLVITISATA ITSKILIYFIREDLNQIS (SEQ ID NO: 687); an amino |RFMKHNIHVTILSVFILNDQEAERDKPG|RWK) acid sequence MOTIF 20 having at least 90% sequence IVLIRLVK) (SEQID NO: 702); an amino acid sequence identity to the amino acid sequence as represented by the MOTIF 11 as represented by an amino acid sequence of the formula LRHQLPTFMQKLHARITIVQLIRE formula KVRILIVKRILCARGCKPHYPSPIDE QERNFVRKWSSE (SEQ ID NO: 688): an amino MILVI MVILISAVAGEVDILHALINVQWLNSQ acid sequence MOTIF 21 having at least 90% sequence TDKERVLLQRNKVVQKNRQESQHNLMIV identity to the amino acid sequence as represented by the QGTAEDIPSATYNECLALLGR (SEQ ID NO: 703); an formula VIDNISALEDIQVGSIQHILIVIGDAP amino acid sequence MOTIF 12 as represented by an amino (SEQ ID NO: 689); an amino acid sequence MOTIF 22 acid sequence of the formula PNQITEQDNLTIVATILS having at least 90% sequence identity to the amino acid WPLGRKMDTVAGINDQELLIATSQHNIE sequence as represented by the formula IVEOHCAS NSQTAALIVLSITIGLITMALVSIQLGPQNSPTIS VAIMIKIMIGVIRPFIVGISLIW (SEQ ID NO: (SEQ ID NO: 704); an amino acid sequence MOTIF 13 as 690); an amino acid sequence MOTIF 23 having at least represented by an amino acid sequence of the formula 90% sequence identity to the amino acid sequence as RLCKIVIDLWKEIVRQNPRKMTPHQRIMILV represented by the formula TLTNEQIPSEEQDHF (SEQ PGSVTVIIIVLLCRLLQFPITLVSIDGEIGSRTK) ID NO: 691); and an amino acid sequence MOTIF 24 having QFRNKASTITADESITWS) (SEQ ID NO: 705); an at least 90% sequence identity to the amino acid sequence as amino acid sequence MOTIF 14 as represented by an amino represented by the formula LPRSIQSRTINIVISF (SEQ acid sequence of the formula TASGVTILILVIPVIED ID NO: 692). WTDPISNTQILLMIVIGMQTITSVILHIVIAEL (SEQ 0.175. In some embodiments a PtIP-83 polypeptide com ID NO: 706); an amino acid sequence MOTIF 15 as repre prises an amino acid sequence MOTIF selected from: an sented by an amino acid sequence of the formula EGLR amino acid sequence MOTIF 1 as represented by an amino EQNDIFQNREKDIQVARNKQAVLIFAVLKSRT acid sequence of the formula MPIDEMPSTEDAD ASTIVAMQNILAG) (SEQ ID NO: 707); an amino acid WSIFVNEIVLEAVAEGMPTEVSEVPAVILIWKR) sequence MOTIF 16 as represented by an amino acid AKCKNMVILIAAL GREMISCTI (SEQID NO: 693); an sequence of the formula WITSRVRIRHLEMIQHN.F amino acid sequence MOTIF 2 as represented by an amino |AVILIQKNRELASTISMTIGNQ (SEQ ID NO: 708); acid sequence of the formula PQLQYRMYGNSLIKRN an amino acid sequence MOTIF 17 as represented by an QQMAQVAQNYDIQRNKEDIFKQRNKFLIKRLFI amino acid sequence of the formula QIMLVISEQND (IAVLQNQIL FIVIGSYLLIQENDIQNKRAF (SEQ ID LQYEDIWVOGLMIVIMLIVIRDIMLVA (SEQ ID NO: 694); an amino acid sequence MOTIF 3 as represented NO: 709); an amino acid sequence MOTIF 18 as represented by an amino acid sequence of the formula NITKSRF by an amino acid sequence of the formula TFTLGSGVT MQMTPFT|RHKIWRLRLSASASPKATRENAKR) GITSMHGEPSLDPWNGVSLDSASPTAF (SEQ ID NO: EGLAFPTATAPLIVIDSTTEQ NDIVLIVAILITF 663); an amino acid sequence MOTIF 19 as represented by HVTAIR (SEQ ID NO: 695); an amino acid sequence an amino acid sequence of the formula MLVIDYISKTR MOTIF 4 as represented by an amino acid sequence of the ITSKRLYFIREKDDLNQIS (SEQ ID NO: 710); an formula DNQEFTSRHWKGDEIPVISNTQLLLDG amino acid sequence MOTIF 20 as represented by an amino EGDIDWEFEIPVQAGIGMSSFP (SEQ ID NO: 696); an acid sequence of the formula LRHQKNILPTSFMQKN amino acid sequence MOTIF 5 as represented by an amino RLHARITKLVSIVQLINIIRLVKJEQERNDKINFO acid sequence of the formula IIHQPSATITIRQKNISTIG VRILKKWSRTSETD (SEQ ID NO: 711); an amino ITLVSIVILIYILLQGSTIVLFHDRRRDEEQDNIV acid sequence MOTIF 21 as represented by an amino acid MLIVITIFPIQA ADAVEILPLNFYIQHNIYAY sequence of the formula VDNQEISAT LEDIQVGST RLDTG (SEQ ID NO: 697); an amino acid sequence QHNILVIVIGDEAP (SEQ ID NO: 712); an amino acid MOTIF 6 as represented by an amino acid sequence of the sequence MOTIF 22 as represented by an amino acid formula SHONADRLAAIQPAVILIDNILTNIHYFILE sequence of the formula IVLEQHNDICASTIVAILIM MATHQNIMDMRTTRSKTIMILVILILVIGLLNLM ILVKIMLVIGVILRPKFILVVGILISLTIVW ILVIILMIVIRIQNAALMRILVKYEY (SEQ ID NO: (SEQ ID NO: 713); an amino acid sequence MOTIF 23 as 698); an amino acid sequence MOTIF 7 as represented by an represented by an amino acid sequence of the formula amino acid sequence of the formula VLIDROKNIVEF TLTNEQDNPSEEQDHNIF (SEQ ID NO: 714); and an SEVMVIHRMYVINRLISATDLINDQEIVIGAEQND amino acid sequence MOTIF 24 as represented by an amino LIPEDIGAEGDIRKIVKRIVLIYVFLIVADWE (SEQ acid sequence of the formula LPRSKTIQSRTKSNIVL) ID NO: 699); an amino acid sequence MOTIF 8 as repre SF (SEQ ID NO: 715). sented by an amino acid sequence of the formula ADE 0176). In some embodiments a PtIP-83 polypeptide com RELQMESFHSATAVISQRKRQGENDELNDQE) prises an amino acid sequence MOTIF selected from: an TDSEATSIFLVAKMDERLMI VISLQMEEE amino acid sequence MOTIF 1 having at least 90% NSDQETRGKAMEQAQRNKKEM (SEQ ID NO: sequence identity to the amino acid sequence as represented 700); an amino acid sequence MOTIF 9 as represented by an by the formula MPIDEMPSTEDADWSIFVNEIVL) amino acid sequence of the formula FVLITAGATAPGA EAVAEGMPTEVSEVPAVILWKRAKCKNMVIL) |AVILIASAGQAVISNTQIAGQAAQAGLRRVVEILE AAL GREMISCTI (SEQ ID NO: 693); an amino acid GQNILEAVMEWA AVILIK (SEQ ID NO: 701); an sequence MOTIF 2 having at least 90% sequence identity to amino acid sequence MOTIF 10 as represented by an amino the amino acid sequence as represented by the formula acid sequence of the formula DIGDEMANKOKIWG PQLQYRMYGNSLIKRNQQMAQVAQNYDIQRNK ITLVSIYIVLYHIGAEKERDIVIEQNDIRVLKI EDIFKQRNKFLIKRLFILAVLQNQIL FIVIGSYLL US 2016/0347799 A1 Dec. 1, 2016 57
QENDIQNKRAF (SEQ ID NO: 694); an amino acid least 90% sequence identity to the amino acid sequence as sequence MOTIF3 having at least 90% sequence identity to represented by the formula EGLREQNDFQNREKDIQ the amino acid sequence as represented by the formula VARNKQAVLIFAVLKSRTASTIVAMQNILAG) NITKSRFMQMTPFTIRHKIWRLRLSASASPKATREN (SEQ ID NO: 707); an amino acid sequence MOTIF 16 AKREGILAFPTATAPLIVIDSTTEQ NDIVLIVAIL having at least 90% sequence identity to the amino acid ITFHVTAIR (SEQ ID NO: 695); an amino acid sequence sequence as represented by the formula WITS MOTIF4 having at least 90% sequence identity to the amino RVRIRHLEMIQHNIFLAVILIOKNRELASTISMT acid sequence as represented by the formula DNQEFTS IGNQ (SEQ ID NO: 708); an amino acid sequence MOTIF RHWKGDEIPVISNTQLLLDGEGDIDWEFEIPVQ 17 having at least 90% sequence identity to the amino acid AGIGMSSFP (SEQ ID NO: 696); an amino acid sequence sequence as represented by the formula QIMLVISEQND MOTIF5 having at least 90% sequence identity to the amino LQYEDIWVOGLMIVIMLIVIRDIMLVA (SEQ ID acid sequence as represented by the formula IIHQPSATT NO: 709); an amino acid sequence MOTIF 18 having at least RQKNISTIGITLVSIVILIYILLQGSTIVLFHDRRR 90% sequence identity to the amino acid sequence as DEEQDNIVIMLIVITIFPIQA ADAVEILPLNFY represented by the formula TFTLGSGVTGITSMH QHNIYAYRLDTG (SEQ ID NO: 697); an amino acid GEPSLDPWNGVSLDSASPTAF (SEQ ID NO: 663); an sequence MOTIF 6 having at least 90% sequence identity to amino acid sequence MOTIF 19 having at least 90% the amino acid sequence as represented by the formula sequence identity to the amino acid sequence as represented SHQNADRLAAIQPAVILIDNILTNHYFILEMAT by the formula MLVIDYSKTRITSKRLYFIREKD HQNMDMRTTRSKTIMILVILILVIGLLNLM ILV DLNOIS (SEQ ID NO: 710); an amino acid sequence LMIVIRIQNAALMRILVKYEY (SEQ ID NO: 698); an MOTIF 20 having at least 90% sequence identity to the amino acid sequence MOTIF 7 having at least 90% amino acid sequence as represented by the formula sequence identity to the amino acid sequence as represented LRHQKNILPTSFMQKNRLHARITKLVSIVQLIN IRLVKEQERNDKINFOIVRILKKWSR TISETD by the formula VLIDRQKNIVEFSEVMVIHRMYVIN (SEQ ID NO: 711); an amino acid sequence MOTIF 21 RLSATDLINDQEIVIGADEQNDLIPEDIGAEGD having at least 90% sequence identity to the amino acid RKTVKRVLIIYVFLIVADWE (SEQ ID NO: 699); an sequence as represented by the formula VDNQESATL amino acid sequence MOTIF 8 having at least 90% EDIQVGSTIQHNILVIVIGDEAP (SEQID NO: 712); sequence identity to the amino acid sequence as represented an amino acid sequence MOTIF 22 having at least 90% by the formula AIDERELQMESFHSATAVISQRKR sequence identity to the amino acid sequence as represented QGENDELINDQEIITDSEATSIFLVAKMIDER LMI VISLQMEEENSDQETRGKAMEQAQRNK by the formula IVLEQHNDICASTIVAILIMILVK KEM (SEQ ID NO: 700); an amino acid sequence MOTIF IMLVIGVILIRPKFILVVGILISLTIVIW (SEQ ID 9 having at least 90% sequence identity to the amino acid NO: 713); an amino acid sequence MOTIF23 having at least sequence as represented by the formula FIVLITAGATAP 90% sequence identity to the amino acid sequence as GAAVILIASAGQAVISNTQIAGQAAQAGLRRV represented by the formula TLTNEQDNPSEEQDHNIF VEILEGQNILEAVMEVVA AIVILIK (SEQ ID NO: 701): (SEQ ID NO: 714); and an amino acid sequence MOTIF 24 an amino acid sequence MOTIF 10 having at least 90% having at least 90% sequence identity to the amino acid sequence identity to the amino acid sequence as represented sequence as represented by the formula LPRSKTIQSRTK by the formula DIGDEMANKOKIWGITLVSIYIVL) SNIVLISF (SEQ ID NO: 715). YHIGAEKERDIVIEQNDIRVLKIISPLLYFIV 0177. In some embodiment a PtIP-83 polypeptide com PNQS NGTQNWQIASPTIIIYLVILLAGVILIV prises, sequentially from the N-terminus to the C-terminus, WEDADQNERICSTITISLVITISATARFMKHN an amino acid sequence MOTIF selected from: MOTIF 19 HV TILSVFILNDQEAERDKPG|RWKLIVL) (SEQ ID NO: 664, SEQ ID NO: 687 or SEQID NO: 710), IRLVK) (SEQ ID NO: 702); an amino acid sequence MOTIF 7 (SEQ ID NO: 652, SEQ ID NO: 676 or SEQ ID MOTIF 11 having at least 90% sequence identity to the NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQID NO: 682 amino acid sequence as represented by the formula KVRIL or SEQID NO: 705), MOTIF 20 (SEQID NO: 665, SEQID VKRILICAIRGCKPHYPSPIDEMILVI MVILISAV NO: 688 or SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: AGILEVDILHALLINVQWLNSQ TDKERVLL 655, SEQ ID NO: 679 or SEQ ID NO: 702), MOTIF 18 QRNKJVVQKNRQESQHNLMIVIQGTAED (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 669, SEQID PSATYNECLALLGR (SEQ ID NO: 703); an amino acid NO: 692 or SEQ ID NO: 715), MOTIF 14 (SEQ ID NO: sequence MOTIF 12 having at least 90% sequence identity 659, SEQ ID NO: 683 or SEQ ID NO: 706), MOTIF 11 to the amino acid sequence as represented by the formula (SEQ ID NO: 656, SEQ ID NO: 680 or SEQID NO: 703), PNQITEQDNILTIVATILSWPLGRKIMDTVLAG) MOTIF 22 (SEQID NO: 667, SEQID NO: 690 or SEQ ID NDQELLIATSQHNIENSQTAALIVLSITIGLIT NO: 713), MOTIF 2 (SEQID NO: 647, SEQID NO: 671 or MALVSIQLGPQNSPTIS (SEQ ID NO: 704); an amino SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ ID acid sequence MOTIF 13 having at least 90% sequence NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: identity to the amino acid sequence as represented by the 660, SEQID NO: 684 or SEQID NO: 707), MOTIF 9 (SEQ formula RLCKIVIDLWKEIVRQNPRKMTPHQR ID NO: 654, SEQID NO: 678 or SEQID NO: 701), MOTIF MILVIPGSVTVIIIVLLCRLLQFPITLVSIDGEIG 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: SRTKQFRNKLASTITADESITWS) (SEQ ID NO: 712), MOTIF 1 (SEQID NO: 646, SEQID NO: 670 or SEQ 705); an amino acid sequence MOTIF 14 having at least ID NO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: 90% sequence identity to the amino acid sequence as 686 or SEQID NO: 709), MOTIF 6 (SEQID NO: 651, SEQ represented by the formula TAISGVTILILVIPVIED ID NO: 675 or SEQID NO: 698), MOTIF 12 (SEQ ID NO: VVTDPISNTQILLMIVIGMQTITSVILHIVIAEL (SEQ 657, SEQID NO: 681 or SEQID NO: 704), MOTIF4 (SEQ ID NO: 706); an amino acid sequence MOTIF 15 having at ID NO: 649, SEQID NO: 673 or SEQID NO: 696), MOTIF US 2016/0347799 A1 Dec. 1, 2016
16 (SEQ ID NO: 661, SEQ ID NO: 685 or SEQ ID NO: the amino acid sequence MOTIFs: MOTIF 19 (SEQ ID NO: 708), MOTIF5 (SEQ ID NO: 650, SEQID NO: 674 or SEQ 664, SEQID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQ ID NO: ID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF 691 or SEQID NO: 714), and MOTIF 3 (SEQID NO: 648, 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQ ID NO: SEQ ID NO: 672 or SEQ ID NO: 695). 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or 0178. In some embodiments a PtIP-83 polypeptide com SEQ ID NO: 711), MOTIF 14 (SEQ ID NO: 659, SEQ ID prises, sequentially from the N-terminus to the C-terminus, NO: 683 or SEQID NO: 706), MOTIF 2 (SEQID NO: 647, an amino acid sequence MOTIF selected from: MOTIF 19 SEQ ID NO: 671 or SEQ ID NO: 694), MOTIF 8 (SEQ ID (SEQ ID NO: 664, SEQ ID NO: 687 or SEQ ID NO: 710), NO: 653, SEQID NO: 677 or SEQID NO: 700), MOTIF 15 MOTIF 7 (SEQ ID NO: 652, SEQ ID NO: 676 or SEQ ID (SEQ ID NO: 660, SEQ ID NO: 684 or SEQID NO: 707), NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 682 MOTIF 9 (SEQ ID NO: 654, SEQ ID NO: 678 or SEQ ID or SEQID NO: 705), MOTIF 20 (SEQID NO: 665, SEQID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQID NO: 689 NO: 688 or SEQ ID NO: 711), MOTIF 14 (SEQ ID NO: or SEQID NO: 712), MOTIF 1 (SEQID NO: 646, SEQID 659, SEQID NO: 683 or SEQID NO: 706), MOTIF 2 (SEQ NO: 670 or SEQ ID NO: 693), MOTIF 17 (SEQ ID NO: ID NO: 647, SEQID NO: 671 or SEQID NO: 694), MOTIF 662, SEQID NO: 686 or SEQID NO: 709), MOTIF 6 (SEQ 8 (SEQID NO: 653, SEQID NO: 677 or SEQID NO: 700), ID NO: 651, SEQID NO: 675 or SEQID NO: 698), MOTIF MOTIF 15 (SEQID NO: 660, SEQID NO: 684 or SEQ ID 12 (SEQ ID NO: 657, SEQ ID NO: 681 or SEQ ID NO: NO: 707), MOTIF 9 (SEQID NO: 654, SEQID NO: 678 or 704), MOTIF4 (SEQID NO: 649, SEQID NO: 673 or SEQ SEQ ID NO: 701), MOTIF 21 (SEQ ID NO: 666, SEQ ID ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID NO: NO: 689 or SEQID NO: 712), MOTIF 1 (SEQID NO: 646, 685 or SEQID NO: 708), MOTIF5 (SEQID NO: 650, SEQ SEQID NO: 670 or SEQID NO: 693), MOTIF 17 (SEQ ID ID NO: 674 or SEQID NO: 697), MOTIF 23 (SEQ ID NO: NO: 662, SEQID NO: 686 or SEQID NO: 709), MOTIF 6 668, SEQ ID NO: 691 or SEQID NO: 714), and MOTIF 3 (SEQ ID NO: 651, SEQ ID NO: 675 or SEQ ID NO: 698), (SEQ ID NO: 648, SEQ ID NO: 672 or SEQID NO: 695). MOTIF 12 (SEQID NO: 657, SEQID NO: 681 or SEQ ID 0181. In some embodiments a PtIP-83 polypeptide com NO: 704), MOTIF4 (SEQID NO: 649, SEQID NO: 673 or prises, sequentially from the N-terminus to the C-terminus: SEQ ID NO: 696), MOTIF 16 (SEQ ID NO: 661, SEQ ID a Region A of between about 200 to about 300 amino acids NO: 685 or SEQID NO: 708), MOTIF5 (SEQID NO: 650, in length comprising an amino acid sequence MOTIF of SEQID NO: 674 or SEQID NO: 697), MOTIF 23 (SEQ ID MOTIF 19 (SEQID NO: 664, SEQID NO: 687 or SEQ ID NO: 668, SEQ ID NO: 691 or SEQ ID NO: 714), and NO: 710), MOTIF 7 (SEQID NO: 652, SEQID NO: 676 or MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: 672 or SEQ ID SEQ ID NO: 699), MOTIF 13 (SEQ ID NO: 658, SEQ ID NO: 695). NO: 682 or SEQ ID NO: 705), MOTIF 20 (SEQ ID NO: 0179. In some embodiments a PtIP-83 polypeptide com 665, SEQ ID NO: 688 or SEQ ID NO: 711), MOTIF 10 prises, sequentially from the N-terminus to the C-terminus, (SEQ ID NO: 655, SEQ ID NO: 679 or SEQID NO: 702), the amino acid sequence MOTIFs: MOTIF 19 (SEQ ID NO: MOTIF 18 (SEQ ID NO: 663), MOTIF 24 (SEQ ID NO: 664, SEQID NO: 687 or SEQID NO: 710), MOTIF 7 (SEQ 669, SEQID NO: 692 or SEQID NO: 715), and/or MOTIF ID NO: 652, SEQID NO: 676 or SEQID NO: 699), MOTIF 14 having a predominantly nonconserved secondary struc 13 (SEQ ID NO: 658, SEQ ID NO: 682 or SEQ ID NO: ture; a Region B of between about 380 to about 465 amino 705), MOTIF 20 (SEQ ID NO: 665, SEQ ID NO: 688 or acids in length comprising an amino acid sequence MOTIF SEQ ID NO: 711), MOTIF 10 (SEQ ID NO: 655, SEQ ID of MOTIF 22 (SEQ ID NO: 667, SEQID NO: 690 or SEQ NO: 679 or SEQ ID NO: 702), MOTIF 18 (SEQ ID NO: ID NO: 713), MOTIF 2 (SEQID NO: 647, SEQID NO: 671 663), MOTIF 24 (SEQ ID NO: 669, SEQ ID NO: 692 or or SEQ ID NO: 694), MOTIF 8 (SEQ ID NO: 653, SEQ ID SEQ ID NO: 715), MOTIF 14 (SEQ ID NO: 659, SEQ ID NO: 677 or SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: NO: 683 or SEQ ID NO: 706), MOTIF 11 (SEQ ID NO: 660, SEQID NO: 684 or SEQID NO: 707), MOTIF 9 (SEQ 656, SEQ ID NO: 680 or SEQ ID NO: 703), MOTIF 22 ID NO: 654, SEQID NO: 678 or SEQID NO: 701), MOTIF (SEQ ID NO: 667, SEQ ID NO: 690 or SEQ ID NO: 713), 21 (SEQ ID NO: 666, SEQ ID NO: 689 or SEQ ID NO: MOTIF 2 (SEQ ID NO: 647, SEQ ID NO: 671 or SEQ ID 712), MOTIF 1 (SEQID NO: 646, SEQID NO: 670 or SEQ NO: 694), MOTIF8 (SEQID NO: 653, SEQID NO: 677 or ID NO: 693), MOTIF 17 (SEQ ID NO: 662, SEQ ID NO: SEQ ID NO: 700), MOTIF 15 (SEQ ID NO: 660, SEQ ID 686 or SEQID NO: 709), MOTIF 6 (SEQID NO: 651, SEQ NO: 684 or SEQID NO: 707), MOTIF9 (SEQID NO: 654, ID NO: 675 or SEQ ID NO: 698), and/or MOTIF 12 and SEQID NO: 678 or SEQID NO: 701), MOTIF 21 (SEQ ID having a predominately alpha helical structure; and a Region NO: 666, SEQID NO: 689 or SEQID NO: 712), MOTIF 1 C of between about 150 to about 180 amino acids in length (SEQ ID NO: 646, SEQID NO: 670 or SEQ ID NO: 693), comprising an amino acid sequence MOTIF of MOTIF 16 MOTIF 17 (SEQID NO: 662, SEQID NO: 686 or SEQ ID (SEQ ID NO: 661, SEQ ID NO: 685 or SEQID NO: 708), NO: 709), MOTIF 6 (SEQID NO: 651, SEQID NO: 675 or MOTIF 5 (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID SEQ ID NO: 698), MOTIF 12 (SEQ ID NO: 657, SEQ ID NO: 697), MOTIF 23 (SEQ ID NO: 668, SEQID NO: 691 NO: 681 or SEQID NO: 704), MOTIF4 (SEQID NO: 649, or SEQ ID NO: 714), and/or MOTIF 3 having a consensus SEQID NO: 673 or SEQID NO: 696), MOTIF 16 (SEQ ID secondary structure comprising predominately beta Strand NO: 661, SEQID NO: 685 or SEQID NO: 708), MOTIF5 Structure. (SEQ ID NO: 650, SEQ ID NO: 674 or SEQ ID NO: 697), 0182. In some embodiments a PtIP-83 polypeptide com MOTIF 23 (SEQID NO: 668, SEQID NO: 691 or SEQ ID prises sequentially from the N-terminus to the C-terminus: a NO: 714), and MOTIF 3 (SEQ ID NO: 648, SEQ ID NO: Region A of between about 200 to about 300 amino acids in 672 or SEQ ID NO: 695). length having predominantly a nonconserved secondary 0180. In some embodiments a PtIP-83 polypeptide com structure; a Region B of between about 380 to about 465 prises, sequentially from the N-terminus to the C-terminus, amino acids in length having a consensus secondary struc US 2016/0347799 A1 Dec. 1, 2016 59 ture comprising 8 to 10 segments of predominately alpha predominately alpha helical structure; and a Region C of helical structure; and a Region C of between about 150 to between about 150 to about 180 amino acids in length about 180 amino acids in length having a consensus sec having a consensus secondary structure comprising seven ondary structure comprising 6 to 8 segments of predomi segments of predominately beta strand structure. nately beta strand structure. As used herein “predominantly 0186. In some embodiments a PtIP-83 polypeptide com a nonconserved secondary structure” means that the regions prises sequentially from the N-terminus to the C-terminus: a of secondary structure don't consistently align within the Region A of between about 200 to about 300 amino acids in family of PtIP polypeptides. As used herein “predominately length having a predominantly nonconserved secondary alpha helical structure” means that secondary structure pre structure; a Region B of between about 380 to about 465 diction may have one or more gap of between 1 to 4 amino amino acids in length having a consensus secondary struc acids of coil and/or beta Strand structure intervening in the ture comprising sequentially: i) an alpha helix-1 of between alpha helix structure. As used herein “predominately beta about 10 and about 26 amino acids in length; ii) a coil-1 of Strand structure” means that secondary structure prediction between about 2 and about 8 amino acids in length flanked may have a gap of between 1 to 4 amino acids of coil and/or by alpha helix-1 and alpha helix-2; iii) an alpha helix-2 of alpha helix structure intervening in the beta Strand structure. between about 15 and about 24 amino acids in length; iv) a In some embodiments the secondary structure is generated coil-2 of between about 4 and about 14 amino acids in length by the PSIPRED, top ranked secondary structure prediction flanked by alpha helix-2 and alpha helix-3; v) an alpha helix method (Jones D T. (1999).J. Mol. Biol. 292: 195-202). 3 of between about 15 and about 27 amino acids in length; 0183 In some embodiments a PtIP-83 polypeptide com vi) a coil-3 of between about 11 and about 13 amino acids prises sequentially from the N-terminus to the C-terminus: a in length flanked by alpha helix-3 and alpha helix-4; vii) an Region A of between about 200 to about 300 amino acids in alpha helix-4 of about 180 amino acids in length; viii) a length having a predominantly nonconserved secondary coil-4 of between about 4 and about 5 amino acids in length structure; a Region B of between about 380 to about 465 flanked by alpha helix-4 and alpha helix-5; ix) an alpha amino acids in length having a consensus secondary struc helix-5 of between about 50 and about 54 amino acids in ture comprising nine segments of predominately alpha heli length; x) a coil-5 of between about 11 and about 17 amino cal structure; and a Region C of between about 150 to about acids in length flanked by alpha helix-5 and alpha helix-6: 180 amino acids in length having a consensus secondary xi) an alpha helix-6 of between about 15 and about 16 amino structure comprising seven segments of predominately beta acids in length; xii) a coil-6 of between about 6 and about 9 Strand structure. amino acids in length flanked by alpha helix-6 and alpha 0184. In some embodiments a PtIP-83 polypeptide com helix-7: xiii) an alpha helix-7 of between about 49 and about prises sequentially from the N-terminus to the C-terminus: a 55 amino acids in length; xiv) a coil-7 of between about 3 Region A of between about 200 to about 300 amino acids in and about 8 amino acids in length flanked by alpha helix-7 length having a flexible consensus secondary structure, and alpha helix-8: xv) an alpha helix-8 of between about 33 wherein the Region. A comprises a conserved beta Strand 1 and about 36 amino acids in length; xvi) a coil-8 of between (B1a) of between about 4 and about 12 amino acids in length about 14 and about 16 amino acids in length flanked by within about amino acid residue 30 to about amino acid alpha helix-8 and alpha helix-9: xvii) an alpha helix-9 of residue 130 from the N-terminus of the PtIP-83 polypeptide: between about 16 and about 23 amino acids in length; xviii) a Region B of between about 380 to about 465 amino acids a coil-9 of between about 21 and about 28 amino acids in in length having a consensus secondary structure comprising length flanked by alpha helix-9 and Region C; and a Region nine segments of predominately alpha helical structure; and C of between about 150 to about 180 amino acids in length a Region C of between about 150 to about 180 amino acids having a consensus secondary structure comprising seven in length having a consensus secondary structure comprising segments of predominately beta strand structure. seven segments of predominately beta Strand structure. As 0187. In some embodiments a PtIP-83 polypeptide com used herein, the term “about when used in the context of the prises sequentially from the N-terminus to the C-terminus: a lowerfupper limit of the length of a secondary structural Region A of between about 200 to about 300 amino acids in element means the greater of -/+an integer of up to -/-20% length having a predominantly nonconserved secondary of the length of the secondary structural element or -/+1 structure; a Region B of between about 380 to about 465 amino acid. By means of example, a secondary structure amino acids in length having a consensus secondary struc element of between about 3 amino acids and about 23 amino ture comprising nine segments of predominately alpha heli acids in length means a secondary structure element of cal structure; and a Region C of between about 150 to about between 2 and 27 amino acids in length. 180 amino acids in length having a consensus secondary 0185. In some embodiments a PtIP-83 polypeptide com structure comprising sequentially: i) a beta Strand-1 (B1) of prises sequentially from the N-terminus to the C-terminus: a between about 3 amino acids and about 5 amino acids in Region A of between about 200 to about 300 amino acids in length; ii) a coil of between about 13 amino acids and about length having a flexible consensus secondary structure, 17 amino acids in length; iii) a beta strand-2 (B2) of between wherein the Region. A comprises a conserved beta Strand 1 about 7 amino acids and about 11 amino acids in length; iv) (B1a) of between about 4 and about 12 amino acids in length, a coil of between about 17 amino acids and about 23 amino a coil of between about 10 and about 20 amino acids in acids in length; v) a beta strand-3 (B3) of between about 5 length and a beta strand 2 (B1b) of between about 4 and amino acids and about 7 amino acids in length; vi) a coil of about 12 amino acids in length, within about amino acid between about 12 amino acids and about 14 amino acids in residue 30 to about amino acid residue 165 from the N-ter length; vii) a beta strand-4 (B4) of between about 5 amino minus of the PtIP-83 polypeptide; a Region B of between acids and about 6 amino acids in length; viii) a coil of about 380 to about 465 amino acids in length having a between about 2 amino acids and about 7 amino acids in consensus secondary structure comprising nine segments of length; ix) a beta strand-5 (B5) of between about 5 amino US 2016/0347799 A1 Dec. 1, 2016 60 acids and about 7 amino acids in length; X) a coil of between 0189 In some embodiments a PtIP-83 polypeptide com about 26 amino acids and about 28 amino acids in length; xi) prises sequentially from the N-terminus to the C-terminus: a a beta strand-6 (B6) of between about 5 amino acids and Region A of between about 200 to about 300 amino acids in about 7 amino acids in length; xii) a coil of between about length having a flexible consensus secondary structure, 16 amino acids and about 20 amino acids in length; and xiii) wherein the Region. A comprises a conserved beta Strand 1 a beta strand-1 (37) of between about 13 amino acids and (B1a) of between about 4 and about 12 amino acids in length about 17 amino acids in length. within about amino acid residue 30 to about amino acid 0188 In some embodiments a PtIP-83 polypeptide com residue 130 from the N-terminus of the PtIP-83 polypeptide: prises sequentially from the N-terminus to the C-terminus: a a Region B of between about 380 to about 465 amino acids Region A of between about 200 to about 300 amino acids in in length having a consensus secondary structure comprising length having a predominantly nonconserved secondary sequentially: i) an alpha helix-1 of between about 10 and structure; a Region B of between about 380 to about 465 about 26 amino acids in length; ii) a coil-1 of between about amino acids in length having a consensus secondary struc 2 and about 8 amino acids in length flanked by alpha helix-1 ture comprising sequentially: i) an alpha helix-1 of between and alpha helix-2; iii) an alpha helix-2 of between about 15 about 10 and about 26 amino acids in length; ii) a coil-1 of and about 24 amino acids in length; iv) a coil-2 of between between about 2 and about 8 amino acids in length flanked about 4 and about 14 amino acids in length flanked by alpha by alpha helix-1 and alpha helix-2; iii) an alpha helix-2 of helix-2 and alpha helix-3; v) an alpha helix 3 of between between about 15 and about 24 amino acids in length; iv) a about 15 and about 27 amino acids in length; vi) a coil-3 of coil-2 of between about 4 and about 14 amino acids in length between about 11 and about 13 amino acids in length flanked flanked by alpha helix-2 and alpha helix-3; v) an alpha helix by alpha helix-3 and alpha helix-4; vii) an alpha helix-4 of 3 of between about 15 and about 27 amino acids in length; about 24 180 amino acids in length; viii) a coil-4 of between vi) a coil-3 of between about 11 and about 13 amino acids about 4 and about 5 amino acids in length flanked by alpha in length flanked by alpha helix-3 and alpha helix-4; vii) an helix-4 and alpha helix-5; ix) an alpha helix-5 of between alpha helix-4 of about 24 180 amino acids in length; viii) a about 50 and about 54 amino acids in length; x) a coil-5 of coil-4 of between about 4 and about 5 amino acids in length between about 11 and about 17 amino acids in length flanked flanked by alpha helix-4 and alpha helix-5; ix) an alpha by alpha helix-5 and alpha helix-6; xi) an alpha helix-6 of helix-5 of between about 50 and about 54 amino acids in between about 15 and about 16 amino acids in length; xii) length: X) a coil-5 of between about 11 and about 17 amino a coil-6 of between about 6 and about 9 amino acids in acids in length flanked by alpha helix-5 and alpha helix-6: length flanked by alpha helix-6 and alpha helix-7: xiii) an xi) an alpha helix-6 of between about 15 and about 16 amino alpha helix-7 of between about 49 and about 55 amino acids acids in length; xii) a coil-6 of between about 6 and about 9 in length; xiv) a coil-7 of between about 3 and about 8 amino amino acids in length flanked by alpha helix-6 and alpha acids in length flanked by alpha helix-7 and alpha helix-8; helix-7: xiii) an alpha helix-7 of between about 49 and about XV) an alpha helix-8 of between about 33 and about 36 55 amino acids in length; xiv) a coil-7 of between about 3 amino acids in length; xvi) a coil-8 of between about 14 and and about 8 amino acids in length flanked by alpha helix-7 about 16 amino acids in length flanked by alpha helix-8 and and alpha helix-8: xv) an alpha helix-8 of between about 33 alpha helix-9; xvii) an alpha helix-9 of between about 16 and and about 36 amino acids in length; xvi) a coil-8 of between about 23 amino acids in length; xviii) a coil-9 of between about 14 and about 16 amino acids in length flanked by about 21 and about 28 amino acids in length flanked by alpha helix-8 and alpha helix-9: xvii) an alpha helix-9 of alpha helix-9 and Region C; and a Region C of between between about 16 and about 23 amino acids in length; xviii) a coil-9 of between about 21 and about 28 amino acids in about 150 to about 180 amino acids in length having a length flanked by alpha helix-9 and Region C; and a Region consensus secondary structure comprising sequentially: i) a C of between about 150 to about 180 amino acids in length beta strand-1 (B1) of between about 3 amino acids and about having a consensus secondary structure comprising sequen 5 amino acids in length; ii) a coil of between about 13 amino tially: i) a beta strand-1 (B1) of between about 3 amino acids acids and about 17 amino acids in length; iii) a beta Strand-2 and about 5 amino acids in length; ii) a coil of between about (B2) of between about 7 amino acids and about 11 amino 13 amino acids and about 17 amino acids in length; iii) a beta acids in length; iv) a coil of between about 17 amino acids strand-2 (12) of between about 7 amino acids and about 11 and about 23 amino acids in length; v) a beta strand-3 (B3) amino acids in length; iv) a coil of between about 17 amino of between about 5 amino acids and about 7 amino acids in acids and about 23 amino acids in length; v) a beta Strand-3 length; vi) a coil of between about 12 amino acids and about (B3) of between about 5 amino acids and about 7 amino 14 amino acids in length; vii) a beta Strand-4 (B4) of between acids in length; vi) a coil of between about 12 amino acids about 5 amino acids and about 6 amino acids in length; viii) and about 14 amino acids in length; vii) a beta Strand-4 (34) a coil of between about 2 amino acids and about 7 amino of between about 5 amino acids and about 6 amino acids in acids in length; ix) a beta strand-5 (B5) of between about 5 length; viii) a coil of between about 2 amino acids and about amino acids and about 7 amino acids in length; X) a coil of 7 amino acids in length; ix) a beta strand-5 (B5) of between between about 26 amino acids and about 28 amino acids in about 5 amino acids and about 7 amino acids in length; X) length; xi) a beta strand-6 (B6) of between about 5 amino a coil of between about 26 amino acids and about 28 amino acids and about 7 amino acids in length; xii) a coil of acids in length; xi) a beta strand-6 (B6) of between about 5 between about 16 amino acids and about 20 amino acids in amino acids and about 7 amino acids in length; xii) a coil of length; and xiii) a beta strand-1 (37) of between about 13 between about 16 amino acids and about 20 amino acids in amino acids and about 17 amino acids in length. length; and xiii) a beta strand-1 (37) of between about 13 0190. In some embodiments a PtIP-83 polypeptide has a amino acids and about 17 amino acids in length. calculated molecular weight of between about 70 kD and US 2016/0347799 A1 Dec. 1, 2016
about 120 kD, between about 75 kD and about 110 kD, and referred to as inteins, which catalyze their own excision between about 80 kD and about 105 kD, and between about through three coordinated reactions at the N-terminal and 85 kD and about 105 kD. C-terminal splice junctions: an acyl rearrangement of the 0191 In some embodiments the PtIP-83 polypeptide has N-terminal cysteine or serine; a transesterfication reaction a modified physical property. As used herein, the term between the two termini to form a branched ester or thioester “physical property” refers to any parameter suitable for intermediate and peptide bond cleavage coupled to cycliza describing the physical-chemical characteristics of a protein. tion of the intein C-terminal asparagine to free the intein As used herein, "physical property of interest' and “property (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094. The of interest are used interchangeably to refer to physical elucidation of the mechanism of protein splicing has led to properties of proteins that are being investigated and/or a number of intein-based applications (Comb, et al., U.S. modified. Examples of physical properties include, but are Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247; not limited to net Surface charge and charge distribution on Camarero and Muir, (1999).J. Amer: Chem. Soc. 121:5597 the protein surface, net hydrophobicity and hydrophobic 5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al., residue distribution on the protein Surface, Surface charge (1998) Nucleic Acids Res. 26:5109-5115; Chong, et al., density, Surface hydrophobicity density, total count of Sur (1998) J. Biol. Chem. 273: 10567-10577; Cotton, et al., face ionizable groups, Surface tension, protein size and its (1999) J. Am. Chem. Soc. 121:1100-1101: Evans, et al., distribution in Solution, melting temperature, heat capacity, (1999) J. Biol. Chem. 274: 18359-18363; Evans, et al., and second virial coefficient. Examples of physical proper (1999).J. Biol. Chem. 274:3923-3926; Evans, et al., (1998) ties also include, but are not limited to solubility, folding, Protein Sci. 7:2256-2264: Evans, et al., (2000) J. Biol. stability, and digestibility. In some embodiments the PtIP-83 Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS polypeptide has increased digestibility of proteolytic frag Lett. 459:166-172: Mathys, et al., (1999) Gene 231:1-13; ments in an insect gut. Models for digestion by simulated Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543 gastric fluids are known to one skilled in the art (Fuchs, R. 3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA L. and J. D. Astwood. Food Technology 50: 83-88, 1996: 95:6705-6710; Otomo, et al., (1999) Biochemistry Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 38:16040-16044: Otomo, et al., (1999) J. Biolmol. NMR 1996; Fu T J et al J. Agric Food Chem. 50: 7154-7160, 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 2002). 96:13638-13643; Severinov and Muir, (1998).J. Biol. Chem. 0.192 In some embodiments variants include polypep 273:16205-16209; Shingledecker, et al., (1998) Gene 207: tides that differ in amino acid sequence due to mutagenesis. 187-195; Southworth, et al., (1998) EMBO.J. 17:918-926; Variant proteins encompassed by the disclosure are biologi Southworth, et al., (1999) Biotechniques 27:110-120: Wood, cally active, that is they continue to possess the desired et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., biological activity (i.e. pesticidal activity) of the native (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231; Wu, et protein. In some embodiment the variant will have at least al., (1998b) Biochim Biophy's Acta 1387:422-432; Xu, et al., about 10%, at least about 30%, at least about 50%, at least (1999) Proc. Natl. Acad. Sci. USA 96:388-393: Yamazaki, et about 70%, at least about 80% or more of the insecticidal al., (1998) J. Am. Chem. Soc., 120:5591-5592). For the activity of the native protein. In some embodiments, the application of inteins in plant transgenes, see, Yang, et al., variants may have improved activity over the native protein. (Transgene Res 15:583-593 (2006)) and Evans, et al., (Annu. 0193 Bacterial genes quite often possess multiple Rev. Plant Biol. 56:375-392 (2005)). methionine initiation codons in proximity to the start of the (0195 In another aspect the PtIP-83 polypeptide may be open reading frame. Often, translation initiation at one or encoded by two separate genes where the intein of the more of these start codons will lead to generation of a precursor protein comes from the two genes, referred to as functional protein. These start codons can include ATG a split-intein, and the two portions of the precursor are codons. However, bacteria Such as Bacillus sp. also recog joined by a peptide bond formation. This peptide bond nize the codon GTG as a start codon, and proteins that formation is accomplished by intein-mediated trans-splic initiate translation at GTG codons contain a methionine at ing. For this purpose, a first and a second expression cassette the first amino acid. On rare occasions, translation in bac comprising the two separate genes further code for inteins terial systems can initiate at a TTG codon, though in this capable of mediating protein trans-splicing. By trans-splic event the TTG encodes a methionine. Furthermore, it is not ing, the proteins and polypeptides encoded by the first and often determined a priori which of these codons are used second fragments may be linked by peptide bond formation. naturally in the bacterium. Thus, it is understood that use of Trans-splicing inteins may be selected from the nucleolar one of the alternate methionine codons may also lead to and organellar genomes of different organisms including generation of pesticidal proteins. These pesticidal proteins eukaryotes, archaebacteria and eubacteria. Inteins that may are encompassed in the present disclosure and may be used be used for are listed at neb.com/neb/inteins.html, which can in the methods of the present disclosure. It will be under be accessed on the world-wide web using the “www.” stood that, when expressed in plants, it will be necessary to prefix). The nucleotide sequence coding for an intein may be alter the alternate start codon to ATG for proper translation. split into a 5' and a 3' part that code for the 5' and the 3' part 0194 In another aspect the PtIP-83 polypeptide may be of the intein, respectively. Sequence portions not necessary expressed as a precursor protein with an intervening for intein splicing (e.g. homing endonuclease domain) may sequence that catalyzes multi-step, post translational protein be deleted. The intein coding sequence is split Such that the splicing. Protein splicing involves the excision of an inter 5' and the 3' parts are capable of trans-splicing. For selecting vening sequence from a polypeptide with the concomitant a suitable splitting site of the intein coding sequence, the joining of the flanking sequences to yield a new polypeptide considerations published by Southworth, et al., (1998) (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168). EMBO.J. 17:918-926 may be followed. In constructing the This intervening sequence or protein splicing element, first and the second expression cassette, the 5' intein coding US 2016/0347799 A1 Dec. 1, 2016 62 sequence is linked to the 3' end of the first fragment coding SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID for the N-terminal part of the PtIP-83 polypeptide and the 3' NO: 13, SEQID NO: 15, SEQID NO: 17, SEQID NO: 19, intein coding sequence is linked to the 5' end of the second SEQ ID NO: 21, SEQID NO. 23, SEQ ID NO: 716, SEQ fragment coding for the C-terminal part of the PtIP-83 ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ ID polypeptide. NO: 757, SEQID NO: 758, SEQ ID NO: 759, SEQID NO: 0196. In general, the trans-splicing partners can be 760, SEQID NO: 761, SEQID NO: 762, SEQID NO: 763, designed using any split intein, including any naturally SEQID NO: 764, SEQID NO: 765, SEQID NO: 766, SEQ occurring or artificially-split split intein. Several naturally ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769. occurring split inteins are known, for example: the split (0199 The development of recombinant DNA methods intein of the DnaB gene of Synechocystis sp. PCC6803 (see, has made it possible to study the effects of sequence trans Wu, et al., (1998) Proc Natl AcadSci USA. 95(16): 9226-31 position on protein folding, structure and function. The and Evans, et al., (2000) J Biol Chem. 275(13):9091-4 and approach used in creating new sequences resembles that of of the DnaB gene from Nostoc punctiforme (see, Iwai, et al., naturally occurring pairs of proteins that are related by linear (2006) FEBS Lett. 580(7): 1853-8). Non-split inteins have reorganization of their amino acid sequences (Cunningham, been artificially split in the laboratory to create new split et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222: inteins, for example: the artificially split Ssp DnaB intein Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841; (see, Wu, et al., (1998) Biochim Biophy's Acta. 1387:422-32) Schimming, et al., (1992) Eur: J. Biochem. 204:13-19; and split Sce VMA intein (see, Brenzel, et al., (2006) Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127 Biochemistry. 45(6): 1571-8) and an artificially split fungal 130; MacGregor, et al., (1996) FEBS Lett. 378:263-266). mini-intein (see, Elleuche, et al., (2007) Biochem Biophys The first in vitro application of this type of rearrangement to Res Commun. 355(3):830-4). There are also intein databases proteins was described by Goldenberg and Creighton (J. available that catalogue known inteins (see for example the Mol. Biol. 165:407-413, 1983). In creating a circular per online-database available at: bioinformatics. Weizmann.ac.il/ muted variant a new N-terminus is selected at an internal site pietro/inteins/Inteinstable.html, which can be accessed on (breakpoint) of the original sequence, the new sequence the world-wide web using the “www’ prefix). having the same order of amino acids as the original from 0.197 Naturally-occurring non-split inteins may have the breakpoint until it reaches an amino acid that is at or near endonuclease or other enzymatic activities that can typically the original C-terminus. At this point the new sequence is be removed when designing an artificially-split split intein. joined, either directly or through an additional portion of Such mini-inteins or minimized split inteins are well known sequence (linker), to an amino acid that is at or near the in the art and are typically less than 200 amino acid residues original N-terminus and the new sequence continues with long (see, Wu, et al., (1998) Biochim Biophy's Acta. 1387: the same sequence as the original until it reaches a point that 422-32). Suitable split inteins may have other purification is at or near the amino acid that was N-terminal to the enabling polypeptide elements added to their structure, breakpoint site of the original sequence, this residue forming provided that such elements do not inhibit the splicing of the the new C-terminus of the chain. The length of the amino split intein or are added in a manner that allows them to be acid sequence of the linker can be selected empirically or removed prior to splicing. Protein splicing has been reported with guidance from structural information or by using a using proteins that comprise bacterial intein-like (BIL) combination of the two approaches. When no structural domains (see, Amitai, et al., (2003) Mol Microbiol. 47:61 information is available, a small series of linkers can be 73) and hedgehog (Hog) auto-processing domains (the latter prepared for testing using a design whose length is varied in is combined with inteins when referred to as the Hog/intein order to span a range from 0 to 50 A and whose sequence is superfamily or HINT family (see, Dassa, et al., (2004) J Biol chosen in order to be consistent with Surface exposure Chem. 279:32001-7) and domains such as these may also be (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. used to prepare artificially-split inteins. In particular, non 20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157: splicing members of Such families may be modified by 105-132; solvent exposed surface area, Lee and Richards, molecular biology methodologies to introduce or restore (1971).J. Mol. Biol. 55:379-400) and the ability to adopt the splicing activity in Such related species. Recent studies necessary conformation without deranging the configuration demonstrate that splicing can be observed when a N-termi of the pesticidal polypeptide (conformationally flexible: nal split intein component is allowed to react with a C-ter Karplus and Schulz, (1985) Naturwissenschaften 72:212 minal split intein component not found in nature to be its 213). Assuming an average of translation of 2.0 to 3.8 A per "partner'; for example, splicing has been observed utilizing residue, this would mean the length to test would be between partners that have as little as 30 to 50% homology with the 0 to 30 residues, with 0 to 15 residues being the preferred “natural splicing partner (see, Dassa, et al., (2007) Bio range. Exemplary of Such an empirical series would be to chemistry. 46(1):322-30). Other such mixtures of disparate construct linkers using a cassette sequence such as Gly-Gly split intein partners have been shown to be unreactive one Gly-Ser repeated n times, where n is 1, 2, 3 or 4. Those with another (see, Brenzel, et al., (2006) Biochemistry. skilled in the art will recognize that there are many such 45(6): 1571-8). However, it is within the ability of a person sequences that vary in length or composition that can serve skilled in the relevant art to determine whether a particular as linkers with the primary consideration being that they be pair of polypeptides is able to associate with each other to neither excessively long nor short (cf., Sandhu, (1992) provide a functional intein, using routine methods and Critical Rev. Biotech. 12:437-462); if they are too long, without the exercise of inventive skill. entropy effects will likely destabilize the three-dimensional (0198 In another aspect the PtIP-83 polypeptide is a fold, and may also make folding kinetically impractical, and circular permuted variant. In certain embodiments the PtIP if they are too short, they will likely destabilize the molecule 83 polypeptide is a circular permuted variant of the poly because of torsional or steric strain. Those skilled in the peptide of SEQ ID NO: 1, SEQ ID NO:3, SEQID NO: 5, analysis of protein structural information will recognize that US 2016/0347799 A1 Dec. 1, 2016
using the distance between the chain ends, defined as the protein. When experimental structural information is not distance between the c-alpha carbons, can be used to define available or is not feasible to obtain, methods are also the length of the sequence to be used or at least to limit the available to analyze the primary amino acid sequence in number of possibilities that must be tested in an empirical order to make predictions of protein tertiary and secondary selection of linkers. They will also recognize that it is structure, Solvent accessibility and the occurrence of turns sometimes the case that the positions of the ends of the and loops. Biochemical methods are also sometimes appli polypeptide chain are ill-defined in structural models cable for empirically determining Surface exposure when derived from X-ray diffraction or nuclear magnetic reso direct structural methods are not feasible; for example, using nance spectroscopy data, and that when true, this situation the identification of sites of chain scission following limited will therefore need to be taken into account in order to proteolysis in order to infer surface exposure (Gentile and Salvatore, (1993) Eur: J. Biochem. 218:603-621). Thus properly estimate the length of the linker required. From using either the experimentally derived structural informa those residues whose positions are well defined are selected tion or predictive methods (e.g., Srinivisan and Rose, (1995) two residues that are close in sequence to the chain ends, and Proteins. Struct., Funct. & Genetics 22:81-99) the parental the distance between their c-alpha carbons is used to calcu amino acid sequence is inspected to classify regions accord late an approximate length for a linker between them. Using ing to whether or not they are integral to the maintenance of the calculated length as a guide, linkers with a range of secondary and tertiary structure. The occurrence of number of residues (calculated using 2 to 3.8 A per residue) sequences within regions that are known to be involved in are then selected. These linkers may be composed of the periodic secondary structure (alpha and 3-10 helices, paral original sequence, shortened or lengthened as necessary, and lel and anti-parallel beta sheets) are regions that should be when lengthened the additional residues may be chosen to avoided. Similarly, regions of amino acid sequence that are be flexible and hydrophilic as described above; or optionally observed or Pt|P- to have a low degree of solvent exposure the original sequence may be substituted for using a series are more likely to be part of the so-called hydrophobic core of linkers, one example being the Gly-Gly-Gly-Ser cassette of the protein and should also be avoided for selection of approach mentioned above; or optionally a combination of amino and carboxyl termini. In contrast, those regions that the original sequence and new sequence having the appro are known or PtIP- to be in surface turns or loops, and priate total length may be used. Sequences of pesticidal especially those regions that are known not to be required for polypeptides capable of folding to biologically active states biological activity, are the preferred sites for location of the can be prepared by appropriate selection of the beginning extremes of the polypeptide chain. Continuous stretches of (amino terminus) and ending (carboxyl terminus) positions amino acid sequence that are preferred based on the above from within the original polypeptide chain while using the criteria are referred to as a breakpoint region. Polynucle linker sequence as described above. Amino and carboxyl otides encoding circular permuted PtIP-83 polypeptides termini are selected from within a common stretch of with new N-terminus/C-terminus which contain a linker sequence, referred to as a breakpoint region, using the region separating the original C-terminus and N-terminus guidelines described below. A novel amino acid sequence is can be made essentially following the method described in thus generated by selecting amino and carboxyl termini from Mullins, et al., (1994) J. Am. Chem. Soc. 116:5529-5533. within the same breakpoint region. In many cases the Multiple steps of polymerase chain reaction (PCR) ampli selection of the new termini will be such that the original fications are used to rearrange the DNA sequence encoding position of the carboxyl terminus immediately preceded that the primary amino acid sequence of the protein. Polynucle of the amino terminus. However, those skilled in the art will otides encoding circular permuted PtIP-83 polypeptides recognize that selections of termini anywhere within the with new N-terminus/C-terminus which contain a linker region may function, and that these will effectively lead to region separating the original C-terminus and N-terminus either deletions or additions to the amino or carboxyl can be made based on the tandem-duplication method portions of the new sequence. It is a central tenet of described in Horlick, et al., (1992) Protein Eng. 5:427-431. molecular biology that the primary amino acid sequence of Polymerase chain reaction (PCR) amplification of the new a protein dictates folding to the three-dimensional structure N-terminus/C-terminus genes is performed using a tandemly necessary for expression of its biological function. Methods duplicated template DNA. are known to those skilled in the art to obtain and interpret three-dimensional structural information using X-ray diffrac 0200. In another aspect fusion proteins are provided that tion of single protein Crystals or nuclear magnetic resonance include within its amino acid sequence an amino acid spectroscopy of protein solutions. Examples of structural sequence comprising a Pt(P-83 polypeptide including but information that are relevant to the identification of break not limited to the polypeptide of SEQ ID NO: 1, SEQ ID point regions include the location and type of protein NO:3, SEQID NO:5, SEQID NO:7, SEQID NO:9, SEQ secondary structure (alpha and 3-10 helices, parallel and ID NO: 11, SEQID NO: 13, SEQID NO: 15, SEQID NO: anti-parallel beta sheets, chain reversals and turns, and 17, SEQID NO: 19, SEQID NO:21, SEQID NO. 23, SEQ loops; Kabsch and Sander, (1983) Biopolymers 22:2577 ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID 2637; the degree of solvent exposure of amino acid residues, NO: 756, SEQID NO: 757, SEQ ID NO: 758, SEQID NO: the extent and type of interactions of residues with one 759, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, another (Chothia, (1984) Ann. Rev. Biochem, 53:537-572) SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQ and the static and dynamic distribution of conformations ID NO: 766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID along the polypeptide chain (Alber and Mathews, (1987) NO: 769, and active fragments thereof. Methods Enzymol. 154:51 1-533). In some cases additional 0201 Methods for design and construction of fusion information is known about solvent exposure of residues; proteins (and polynucleotides encoding same) are known to one example is a site of post-translational attachment of those of skill in the art. Polynucleotides encoding a PtIP-83 carbohydrate which is necessarily on the surface of the polypeptide may be fused to signal sequences which will US 2016/0347799 A1 Dec. 1, 2016 64 direct the localization of the Pt|P-83 polypeptide to particu sites on both parent molecules and their Subsequent joining lar compartments of a prokaryotic or eukaryotic cell and/or through recombinant DNA techniques may result in the direct the secretion of the PtIP-83 polypeptide of the addition of one or more extra amino acids between the embodiments from a prokaryotic or eukaryotic cell. For transit peptide and the second protein. This generally does example, in E. coli, one may wish to direct the expression of not affect targeting activity as long as the transit peptide the protein to the periplasmic space. Examples of signal cleavage site remains accessible and the function of the sequences or proteins (or fragments thereof) to which the second protein is not altered by the addition of these extra PtIP-83 polypeptide may be fused in order to direct the amino acids at its N-terminus. Alternatively, one skilled in expression of the polypeptide to the periplasmic space of the art can create a precise cleavage site between the transit bacteria include, but are not limited to, the pelB signal peptide and the second protein (with or without its initiator sequence, the maltose binding protein (MBP) signal methionine) using gene synthesis (Stemmer, et al., (1995) Gene 164:49-53) or similar methods. In addition, the transit sequence, MBP, the ompA signal sequence, the signal peptide fusion can intentionally include amino acids down sequence of the periplasmic E. coli heat-labile enterotoxin stream of the cleavage site. The amino acids at the N-ter B-Subunit and the signal sequence of alkaline phosphatase. minus of the mature protein can affect the ability of the Several vectors are commercially available for the construc transit peptide to target proteins to plastids and/or the tion of fusion proteins which will direct the localization of efficiency of cleavage following protein import. This may be a protein, Such as the pMAL series of vectors (particularly dependent on the protein to be targeted. See, e.g., Comai, et the pMAL-p series) available from New England Biolabs. In a specific embodiment, the PtIP-83 polypeptide may be al., (1988) J. Biol. Chem. 263(29): 15104-9. fused to the pelB pectate lyase signal sequence to increase 0202 In some embodiments fusion proteins are provide the efficiency of expression and purification of Such poly comprising a PtIP-83 polypeptide and an insecticidal poly peptides in Gram-negative bacteria (see, U.S. Pat. Nos. peptide joined by an amino acid linker. In some embodi 5,576,195 and 5,846,818). Plant plastid transit peptide/ ments fusion proteins are provided represented by a formula polypeptide fusions are well known in the art (see, U.S. Pat. selected from the group consisting of: No. 7,193,133). Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also well known in the art. The plastid transit peptide is generally fused N-ter wherein R' is a PtIP-83 polypeptide, R is a protein of minal to the polypeptide to be targeted (e.g., the fusion interest. The R' polypeptide is fused either directly or partner). In one embodiment, the fusion protein consists through a linker (L) segment to the Ripolypeptide. The term essentially of the plastid transit peptide and the PtIP-83 “directly defines fusions in which the polypeptides are polypeptide to be targeted. In another embodiment, the joined without a peptide linker. Thus “L” represents a fusion protein comprises the plastid transit peptide and the chemical bound or polypeptide segment to which both R' polypeptide to be targeted. In Such embodiments, the plastid and R are fused in frame, most commonly L is a linear transit peptide is preferably at the N-terminus of the fusion peptide to which R' and R are bound by amide bonds protein. However, additional amino acid residues may be linking the carboxy terminus of R' to the amino terminus of N-terminal to the plastid transit peptide providing that the Land carboxy terminus of L to the amino terminus of R. By fusion protein is at least partially targeted to a plastid. In a “fused in frame' is meant that there is no translation specific embodiment, the plastid transit peptide is in the termination or disruption between the reading frames of R' N-terminal half, N-terminal third or N-terminal quarter of and R. The linking group (L) is generally a polypeptide of the fusion protein. Most or all of the plastid transit peptide between 1 and 500 amino acids in length. The linkers joining is generally cleaved from the fusion protein upon insertion the two molecules are preferably designed to (1) allow the into the plastid. The position of cleavage may vary slightly two molecules to fold and act independently of each other, between plant species, at different plant developmental (2) not have a propensity for developing an ordered second stages, as a result of specific intercellular conditions or the ary structure which could interfere with the functional particular combination of transit peptide/fusion partner used. domains of the two proteins, (3) have minimal hydrophobic In one embodiment, the plastid transit peptide cleavage is or charged characteristic which could interact with the homogenous such that the cleavage site is identical in a functional protein domains and (4) provide steric separation population of fusion proteins. In another embodiment, the of R' and R such that R' and R could interact simultane plastid transit peptide is not homogenous, such that the ously with their corresponding receptors on a single cell. cleavage site varies by 1-10 amino acids in a population of Typically surface amino acids in flexible protein regions fusion proteins. The plastid transit peptide can be recombi include Gly, Asn and Ser. Virtually any permutation of nantly fused to a second protein in one of several ways. For amino acid sequences containing Gly, ASn and Ser would be example, a restriction endonuclease recognition site can be expected to satisfy the above criteria for a linker sequence. introduced into the nucleotide sequence of the transit peptide Other neutral amino acids, such as Thr and Ala, may also be at a position corresponding to its C-terminal end and the used in the linker sequence. Additional amino acids may also same or a compatible site can be engineered into the be included in the linkers due to the addition of unique nucleotide sequence of the protein to be targeted at its restriction sites in the linker sequence to facilitate construc N-terminal end. Care must be taken in designing these sites tion of the fusions. to ensure that the coding sequences of the transit peptide and 0203. In some embodiments the linkers comprise the second protein are kept “in frame' to allow the synthesis sequences selected from the group of formulas: (Gly-Ser), of the desired fusion protein. In some cases, it may be (Gly Ser), (GlysSer), (Gly, Ser), or (AlaGlySer), where n preferable to remove the initiator methionine codon of the is an integer. One example of a highly-flexible linker is the second protein when the new restriction site is introduced. (GlySer)-rich spacer region present within the pill protein of The introduction of restriction endonuclease recognition the filamentous bacteriophages, e.g. bacteriophages M13 or US 2016/0347799 A1 Dec. 1, 2016
fa (Schaller, et al., 1975). This region provides a long, ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID flexible spacer region between two domains of the pill NO: 765, SEQID NO: 766, SEQ ID NO: 767, SEQID NO: Surface protein. Also included are linkers in which an 768 or SEQ ID NO: 769. endopeptidase recognition sequence is included. Such a 0206. In some embodiments a PtIP-83 polypeptide vari cleavage site may be valuable to separate the individual ant comprises one or more amino acid Substitution, of Table components of the fusion to determine if they are properly 13, Table 14, Table 15, Table 16, Table 17, Table 18, Table folded and active in vitro. Examples of various endopepti 20, Table 21, Table 23, Table 24 or combinations thereof, dases include, but are not limited to, Plasmin, Enterokinase, compared to the native amino acid of PtIP-83Aa (SEQ ID Kallikerin, Urokinase, Tissue Plasminogen activator, clostri NO: 1) at the corresponding residue. pain, Chymosin, Collagenase, Russell's Viper Venom Pro 0207. In some embodiments a PtIP-83 polypeptide vari tease, Postproline cleavage enzyme, V8 protease. Thrombin ant is selected from but not limited to any one of SEQ ID and factor Xa. In some embodiments the linker comprises NO:236-299, SEQ ID NO:334-367, SEQID NO:398-427, the amino acids EEKKN (SEQ ID NO: 37) from the SEQ ID NO. 518-607, and SEQ ID NO: 728-737. multi-gene expression vehicle (MGEV), which is cleaved by 0208. In some embodiments a PtIP-83 polypeptide com vacuolar proteases as disclosed in US Patent Application prises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. Publication Number US 2007/0277263. In other embodi 15 or more amino acids from the N-terminus of the Pt|P-83 ments, peptide linker segments from the hinge region of polypeptide relative to the amino acid position of any one of heavy chain immunoglobulins IgG, IgA, IgM, Ig|D or IgE SEQID NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: provide an angular relationship between the attached poly 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ peptides. Especially useful are those hinge regions where the ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQID NO: cysteines are replaced with serines. Linkers of the present 21, SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, disclosure include sequences derived from murine IgG SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, SEQ gamma 2b hinge region in which the cysteines have been ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID changed to serines. The fusion proteins are not limited by the NO: 761, SEQID NO: 762, SEQ ID NO: 763, SEQID NO: form, size or number of linker sequences employed and the 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, only requirement of the linker is that functionally it does not SEQ ID NO: 768 or SEQ ID NO: 769. interfere adversely with the folding and function of the 0209. In some embodiments a PtIP-83 polypeptide com individual molecules of the fusion. prises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 0204. In another aspect chimeric PtIP-83 polypeptides 15 or more amino acids from the C-terminus of the PtP-83 are provided that are created through joining two or more polypeptide relative to the amino acid position of any one of portions of PtIP-83 genes, which originally encoded sepa SEQID NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: rate PtIP-83 proteins to create a chimeric gene. The trans 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ lation of the chimeric gene results in a single chimeric ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQID NO: PtIP-83 polypeptide with regions, motifs or domains derived 21, SEQ ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, from each of the original polypeptides. In certain embodi SEQID NO: 755, SEQID NO: 756, SEQID NO: 757, SEQ ments the chimeric protein comprises portions, motifs or ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID domains of PtIP-83 polypeptides of SEQID NO: 1, SEQ ID NO: 761, SEQID NO: 762, SEQ ID NO: 763, SEQID NO: NO:3, SEQID NO. 5, SEQID NO:7, SEQID NO:9, SEQ 764, SEQID NO: 765, SEQID NO: 766, SEQID NO: 767, ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQID NO: SEQ ID NO: 768 or SEQ ID NO: 769. 17, SEQID NO: 19, SEQID NO: 21, SEQID NO. 23, SEQ 0210 Methods for such manipulations are generally ID NO: 716, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID known in the art. For example, amino acid sequence variants NO: 756, SEQID NO: 757, SEQID NO: 758, SEQID NO: of a PtIP-83 polypeptide can be prepared by mutations in the 759, SEQID NO: 760, SEQID NO: 761, SEQID NO: 762, DNA. This may also be accomplished by one of several SEQID NO: 763, SEQID NO: 764, SEQID NO: 765, SEQ forms of mutagenesis and/or in directed evolution. In some ID NO: 766, SEQID NO: 767, SEQID NO: 768 or SEQID aspects, the changes encoded in the amino acid sequence NO: 769, in any combination. will not substantially affect the function of the protein. Such 0205. It is recognized that DNA sequences may be altered variants will possess the desired pesticidal activity. How by various methods, and that these alterations may result in ever, it is understood that the ability of a PtIP-83 polypeptide DNA sequences encoding proteins with amino acid to confer pesticidal activity may be improved by the use of sequences different than that encoded by the wild-type (or Such techniques upon the compositions of this disclosure. native) pesticidal protein. In some embodiments a PtIP-83 0211 For example, conservative amino acid substitutions polypeptide may be altered in various ways including amino may be made at one or more, Pt|P-, nonessential amino acid acid substitutions, deletions, truncations and insertions of residues. A “nonessential amino acid residue is a residue one or more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, that can be altered from the wild-type sequence of a PtIP-83 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, without altering the biological activity. A “conservative 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or amino acid substitution' is one in which the amino acid more amino acid Substitutions, deletions and/or insertions or residue is replaced with an amino acid residue having a combinations thereof compared to any one of SEQ ID NO: similar side chain. Families of amino acid residues having 1, SEQ ID NO:3, SEQ ID NO. 5, SEQID NO: 7, SEQ ID similar side chains have been defined in the art. These NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, families include: amino acids with basic side chains (e.g., SEQID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID lysine, arginine, histidine); acidic side chains (e.g., aspartic NO: 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID NO: acid, glutamic acid); polar, negatively charged residues and 755, SEQID NO: 756, SEQID NO: 757, SEQID NO: 758, their amides (e.g., aspartic acid, asparagine, glutamic, acid, SEQID NO: 759, SEQID NO: 760, SEQID NO: 761, SEQ glutamine; uncharged polar side chains (e.g., glycine, US 2016/0347799 A1 Dec. 1, 2016 66 asparagine, glutamine, serine, threonine, tyrosine, cysteine); 0214. It is also understood in the art that the substitution Small aliphatic, nonpolar or slightly polar residues (e.g., of like amino acids can be made effectively on the basis of Alanine, serine, threonine, proline, glycine); nonpolar side hydrophilicity. U.S. Pat. No. 4,554,101, states that the chains (e.g., alanine, Valine, leucine, isoleucine, proline, greatest local average hydrophilicity of a protein, as gov phenylalanine, methionine, tryptophan); large aliphatic, erned by the hydrophilicity of its adjacent amino acids, nonpolar residues (e.g., methionine, leucine, isoleucine, correlates with a biological property of the protein. valine, cystine); beta-branched side chains (e.g., threonine, 0215. As detailed in U.S. Pat. No. 4,554,101, the follow valine, isoleucine); aromatic side chains (e.g., tyrosine, ing hydrophilicity values have been assigned to amino acid phenylalanine, tryptophan, histidine); large aromatic side residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0. chains (e.g., tyrosine, phenylalanine, tryptophan). 1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2): glutamine (+0.2); glycine (O); threonine (-0.4); proline 0212 Amino acid substitutions may be made in noncon (-0.5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); served regions that retain function. In general. Such Substi methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine tutions would not be made for conserved amino acid resi (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan dues or for amino acid residues residing within a conserved (-3.4). motif, where such residues are essential for protein activity. 0216 Alternatively, alterations may be made to the pro Examples of residues that are conserved and that may be tein sequence of many proteins at the amino or carboxy essential for protein activity include, for example, residues terminus without Substantially affecting activity. This can that are identical between all proteins contained in an include insertions, deletions or alterations introduced by alignment of similar or related toxins to the sequences of the modern molecular methods, such as PCR, including PCR embodiments (e.g., residues that are identical in an align amplifications that alter or extend the protein coding ment of homologous proteins). Examples of residues that are sequence by virtue of inclusion of amino acid encoding conserved but that may allow conservative amino acid sequences in the oligonucleotides utilized in the PCR ampli Substitutions and still retain activity include, for example, fication. Alternatively, the protein sequences added can residues that have only conservative substitutions between include entire protein-coding sequences, such as those used all proteins contained in an alignment of similar or related commonly in the art to generate protein fusions. Such fusion toxins to the sequences of the embodiments (e.g., residues proteins are often used to (1) increase expression of a protein that have only conservative substitutions between all pro of interest (2) introduce a binding domain, enzymatic activ teins contained in the alignment homologous proteins). ity or epitope to facilitate either protein purification, protein However, one of skill in the art would understand that detection or other experimental uses known in the art (3) functional variants may have minor conserved or noncon target secretion or translation of a protein to a Subcellular served alterations in the conserved residues. Guidance as to organelle. Such as the periplasmic space of Gram-negative appropriate amino acid substitutions that do not affect bio bacteria, mitochondria or chloroplasts of plants or the endo logical activity of the protein of interest may be found in the plasmic reticulum of eukaryotic cells, the latter of which model of Dayhoff, et al., (1978) Atlas of Protein Sequence often results in glycosylation of the protein. and Structure (Natl. Biomed. Res. Found. Washington, 0217 Variant nucleotide and amino acid sequences of the D.C.), herein incorporated by reference. In making Such disclosure also encompass sequences derived from muta changes, the hydropathic index of amino acids may be genic and recombinogenic procedures Such as DNA shuf considered. The importance of the hydropathic amino acid fling. With such a procedure, one or more different PtIP-83 index in conferring interactive biologic function on a protein polypeptide coding regions can be used to create a new is generally understood in the art (Kyte and Doolittle, (1982) PtIP-83 polypeptide possessing the desired properties. In J Mol Biol. 157(1):105-32). It is accepted that the relative this manner, libraries of recombinant polynucleotides are hydropathic character of the amino acid contributes to the generated from a population of related sequence polynucle secondary structure of the resultant protein, which in turn otides comprising sequence regions that have substantial defines the interaction of the protein with other molecules, sequence identity and can be homologously recombined in for example, enzymes, Substrates, receptors, DNA, antibod vitro or in vivo. For example, using this approach, sequence ies, antigens, and the like. motifs encoding a domain of interest may be shuffled 0213. It is known in the art that certain amino acids may between a pesticidal gene and other known pesticidal genes be substituted by other amino acids having a similar hydro to obtain a new gene coding for a protein with an improved pathic index or score and still result in a protein with similar property of interest, Such as an increased insecticidal activ biological activity, i.e., still obtain a biological functionally ity. Strategies for such DNA shuffling are known in the art. equivalent protein. Each amino acid has been assigned a See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. hydropathic index on the basis of its hydrophobicity and USA 91:10747-10751; Stemmer, (1994) Nature 370:389 charge characteristics (Kyte and Doolittle, ibid). These are: 391; Crameri, et al., (1997) Nature Biotech. 15:436-438: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509: Cra nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); meri, et al., (1998) Nature 391:288-291; and U.S. Pat. Nos. tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine 5,605,793 and 5,837,458. (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); 0218 Domain swapping or shuffling is another mecha asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In nism for generating altered PtIP-83 polypeptides. Domains making such changes, the Substitution of amino acids whose may be swapped between PtIP-83 polypeptides resulting in hydropathic indices are within +2 is preferred, those which hybrid or chimeric toxins with improved insecticidal activity are within +1 are particularly preferred, and those within or target spectrum. Methods for generating recombinant +0.5 are even more particularly preferred. proteins and testing them for pesticidal activity are well US 2016/0347799 A1 Dec. 1, 2016 67 known in the art (see, for example, Naimov, et al., (2001) tration, centrifugation, sedimentation, or concentration of a Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., culture of cells comprising the polypeptide. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., 0227 Such compositions disclosed above may be (1990).J. Biol. Chem. 265:20923-20930; Rang, et al.,91999) obtained by the addition of a Surface-active agent, an inert Appl. Environ. Microbiol. 65:2918-2925). carrier, a preservative, a humectant, a feeding stimulant, an 0219. Alignment of PtIP-83 homologs (FIG. 2) allows for attractant, an encapsulating agent, a binder, an emulsifier, a identification of residues that are highly conserved among dye, a UV protectant, a buffer, a flow agent or fertilizers, natural homologs in this family. micronutrient donors, or other preparations that influence 0220. In some embodiments PtP-83 polypeptides are plant growth. One or more agrochemicals including, but not provided comprising an amino acid sequence having at least limited to, herbicides, insecticides, fungicides, bactericides, 80%, at least 85%, at least 90%, at least 95%, at least 96%, nematocides, molluscicides, acaricides, plant growth regu at least 97%, at least 98% or at least 99% sequence identity lators, harvest aids, and fertilizers, can be combined with to any one of SEQ ID NO: 786-888. carriers, Surfactants or adjuvants customarily employed in 0221. In some embodiments PtP-83 polypeptides are the art of formulation or other components to facilitate provide comprising the amino acid sequence of any one of product handling and application for particular target pests. SEQ ID NO: 786-888. Suitable carriers and adjuvants can be solid or liquid and 0222. In some embodiments the PtIP-83 polypeptide is correspond to the Substances ordinarily employed in formu not the amino acid sequence of any one of SEQ ID NO: lation technology, e.g., natural or regenerated mineral Sub 786-888. stances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. The active ingredients of the embodi Compositions ments are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. 0223 Compositions comprising a PtIP-83 polypeptide of For example, the compositions of the embodiments may be the disclosure are also embraced. In some embodiments the applied to grain in preparation for or during storage in a composition comprises a PtIP-83 polypeptide of SEQ ID grain bin or silo, etc. The compositions of the embodiments NO: 1, SEQID NO:3, SEQID NO:5, SEQID NO: 7, SEQ may be applied simultaneously or in Succession with other ID NO:9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: compounds. Methods of applying an active ingredient of the 15, SEQID NO: 17, SEQID NO: 19, SEQ ID NO: 21, SEQ embodiments or an agrochemical composition of the ID NO. 23, SEQ ID NO: 716, SEQ ID NO: 754, SEQ ID embodiments that contains at least one of the Cyt1 A variant NO: 755, SEQID NO: 756, SEQID NO: 757, SEQID NO: polypeptides produced by the bacterial strains of the 758, SEQID NO: 759, SEQID NO: 760, SEQID NO: 761, embodiments include, but are not limited to, foliar applica SEQID NO: 762, SEQID NO: 763, SEQID NO: 764, SEQ tion, seed coating, and soil application. The number of ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID applications and the rate of application depend on the NO: 768 or SEQ ID NO: 769 or a variant thereof. In some intensity of infestation by the corresponding pest. embodiments the composition comprises a PtIP-83 fusion 0228 Suitable surface-active agents include, but are not protein. limited to, anionic compounds such as a carboxylate of for 0224. In some embodiments compositions are provided example, a metal; a carboxylate of a long chain fatty acid; comprising a Pt(P-83 polypeptide comprising an amino acid an N-acylsarcosinate; mono or di-esters of phosphoric acid sequence of any one of SEQID NO: 236-299, SEQID NO: with fatty alcohol ethoxylates or salts of such esters; fatty 334-367, SEQID NO:398-427, SEQID NO:518-607, SEQ alcohol Sulfates such as sodium dodecyl Sulfate, Sodium ID NO: 640-645, and SEQ ID NO: 728-737 or a variant octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty thereof. alcohol Sulfates; ethoxylated alkylphenol Sulfates; lignin 0225. In some embodiments compositions are provide Sulfonates; petroleum Sulfonates; alkyl aryl Sulfonates Such comprising a Pt(P-83 polypeptide comprising the amino acid as alkyl-benzene Sulfonates or lower alkylnaphtalene Sul sequence of any one of SEQ ID NO: 786-888 or a variant fonates, e.g., butyl-naphthalene Sulfonate; salts of sulfonated thereof. naphthalene-formaldehyde condensates; salts of Sulfonated 0226. In some embodiments agricultural compositions of phenol-formaldehyde condensates; more complex Sul PtIP-83 polypeptides are disclosed. In the embodiments, a fonates Such as the amide Sulfonates, e.g., the Sulfonated transformed microorganism (which includes whole organ condensation product of oleic acid and N-methyl taurine; or isms, cells, spore(s), PtIP-83 polypeptide(s), pesticidal com the dialkyl SulfoSuccinates, e.g., the Sodium sulfonate of ponent(s), pest-impacting component(s), variant(s), living or dioctyl succinate. Non-ionic agents include condensation dead cells and cell components, including mixtures of living products offatty acid esters, fatty alcohols, fatty acid amides and dead cells and cell components, and including broken or fatty-alkyl- or alkenyl-substituted phenols with ethylene cells and cell components) or an isolated PtIP-83 polypep oxide, fatty esters of polyhydric alcohol ethers, e.g., Sorbitan tide(s) can be formulated with an acceptable carrier into a fatty acid esters, condensation products of Such esters with pesticidal composition(s) that is, for example, a Suspension, ethylene oxide, e.g., polyoxyethylene Sorbitar fatty acid a solution, an emulsion, a dusting powder, a dispersible esters, block copolymers of ethylene oxide and propylene granule or pellet, a wettable powder, and an emulsifiable oxide, acetylenic glycols such as 2.4.7.9-tetraethyl-5-decyn concentrate, an aerosol or spray, an impregnated granule, an 4,7-diol, or ethoxylated acetylenic glycols. Examples of a adjuvant, a coatable paste, a colloid, and also encapsulations cationic Surface-active agent include, for instance, an ali in, for example, polymer Substances. Such formulated com phatic mono-, di-, or polyamine Such as an acetate, naph positions may be prepared by Such conventional means as thenate or oleate; or oxygen-containing amine Such as an desiccation, lyophilization, homogenization, extraction, fil amine oxide of polyoxyethylene alkylamine; an amide US 2016/0347799 A1 Dec. 1, 2016 linked amine prepared by the condensation of a carboxylic odology, for example the standard somatic cell hybridization acid with a di- or polyamine; or a quaternary ammonium technique of Kohler and Milstein, (1975) Nature 256:495. salt. Other techniques for producing monoclonal antibody can 0229. Examples of inert materials include but are not also be employed Such as viral or oncogenic transformation limited to inorganic minerals such as kaolin, phyllosilicates, of B lymphocytes. An animal system for preparing hybrido carbonates, sulfates, phosphates, Mica, Amorphous Silica mas is a murine system. Immunization protocols and tech Gel, talc, clay, Volcanic ash or botanical materials such as niques for isolation of immunized splenocytes for fusion are cork, powdered corncobs, peanut hulls, rice hulls, and known in the art. Fusion partners (e.g., murine myeloma walnut shells. Kaolins such as kaolinite, dickite, nacrite, cells) and fusion procedures are also known. The antibody anauxite, halloysite and endellite are useful as carrier mate and monoclonal antibodies of the disclosure can be prepared rials. Montmorillonites, such as beidellite, nontronite, mont by utilizing a PtIP-83 polypeptide as antigens. morillonite, hectorite, Saponite, Sauconite and bentonite are 0232 A kit for detecting the presence of a PtIP-83 useful as carrier materials. Vermiculites such as biotite are polypeptide or detecting the presence of a nucleotide useful as carrier materials. sequence encoding a Pt(P-83 polypeptide in a sample is 0230. The compositions of the embodiments can be in a provided. In one embodiment, the kit provides antibody Suitable form for direct application or as a concentrate of based reagents for detecting the presence of a PtIP-83 primary composition that requires dilution with a suitable polypeptide in a tissue sample. In another embodiment, the quantity of water or other diluent before application. The kit provides labeled nucleic acid probes useful for detecting pesticidal concentration will vary depending upon the nature the presence of one or more polynucleotides encoding of the particular formulation, specifically, whether it is a PtIP-83 polypeptide. The kit is provided along with appro concentrate or to be used directly. The composition contains priate reagents and controls for carrying out a detection 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or method, as well as instructions for use of the kit. 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, Receptor Identification and Isolation for example, about 0.01 lb-5.0 lb. per acre when in dry form and at about 0.01 pts.-10 pts. per acre when in liquid form. 0233 Receptors to the PtIP-83 polypeptide of the embodiments or to variants or fragments thereof, are also Antibodies encompassed. Methods for identifying receptors are well known in the art (see, Hofmann, et. al., (1988) Eur: J. 0231. Antibodies to a PtIP-83 polypeptide of the embodi Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. ments or to variants or fragments thereof are also encom 27277-27282) can be employed to identify and isolate the passed. The antibodies of the disclosure include polyclonal receptor that recognizes the Pt|P-83 polypeptide using the and monoclonal antibodies as well as fragments thereof brush-border membrane vesicles from susceptible insects. In which retain their ability to bind to PtIP-83 polypeptide addition to the radioactive labeling method listed in the cited found in the insect gut. An antibody, monoclonal antibody or literatures, PtIP-83 polypeptide can be labeled with fluores fragment thereof is said to be capable of binding a molecule cent dye and other common labels such as streptavidin. if it is capable of specifically reacting with the molecule to Brush-border membrane vesicles (BBMV) of susceptible thereby bind the molecule to the antibody, monoclonal insects such as soybean looper and Stink bugs can be antibody or fragment thereof. The term “antibody’ (Ab) or prepared according to the protocols listed in the references “monoclonal antibody’ (Mab) is meant to include intact and separated on SDS-PAGE gel and blotted on suitable molecules as well as fragments or binding regions or membrane. Labeled PtIP-83 polypeptide can be incubated domains thereof (such as, for example, Fab and F(ab).sub.2 with blotted membrane of BBMV and labeled the PtF-83 fragments) which are capable of binding hapten. Such polypeptide can be identified with the labeled reporters. fragments are typically produced by proteolytic cleavage, Identification of protein band(s) that interact with the PtIP Such as papain or pepsin. Alternatively, hapten-binding 83 polypeptide can be detected by N-terminal amino acid fragments can be produced through the application of gas phase sequencing or mass spectrometry based protein recombinant DNA technology or through synthetic chemis identification method (Patterson, (1998) 10.22, 1-24, Cur try. Methods for the preparation of the antibodies of the rent Protocol in Molecular Biology published by John Wiley present disclosure are generally known in the art. For & Son Inc). Once the protein is identified, the corresponding example, see, Antibodies, A Laboratory Manual, Ed Harlow gene can be cloned from genomic DNA or cDNA library of and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. the Susceptible insects and binding affinity can be measured (1988), as well as the references cited therein. Standard directly with the PtIP-83 polypeptide. Receptor function for reference works setting forth the general principles of immu insecticidal activity by the PtIP-83 polypeptide can be nology include: Klein, J. Immunology: The Science of verified by accomplished by RNAi type of gene knock out Cell-Noncell Discrimination, John Wiley & Sons, N.Y. method (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849 (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: 46851). A New Dimension in Biological Analyses, Plenum Press, N.Y. (1980) and Campbell, “Monoclonal Antibody Technol ogy.” In Laboratory Techniques in Biochemistry and Nucleotide Constructs, Expression Cassettes and Vectors Molecular Biology, Vol. 13, Burdon, et al., (eds.), Elsevier, 0234. The use of the term “nucleotide constructs' herein Amsterdam (1984). See also, U.S. Pat. Nos. 4,196,265; is not intended to limit the embodiments to nucleotide 4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and constructs comprising DNA. Those of ordinary skill in the 4,720,459. PtIP-83 polypeptide antibodies or antigen-bind art will recognize that nucleotide constructs particularly ing portions thereof can be produced by a variety of tech polynucleotides and oligonucleotides composed of ribo niques, including conventional monoclonal antibody meth nucleotides and combinations of ribonucleotides and deoxy US 2016/0347799 A1 Dec. 1, 2016 69 ribonucleotides may also be employed in the methods dis sequence of the embodiments, it is intended that the pro closed herein. The nucleotide constructs, nucleic acids, and moter is not the native or naturally occurring promoter for nucleotide sequences of the embodiments additionally the operably linked sequence of the embodiments. As used encompass all complementary forms of Such constructs, herein, a chimeric gene comprises a coding sequence oper molecules, and sequences. Further, the nucleotide con ably linked to a transcription initiation region that is heter structs, nucleotide molecules, and nucleotide sequences of ologous to the coding sequence. Where the promoter is a the embodiments encompass all nucleotide constructs, mol native or natural sequence, the expression of the operably ecules, and sequences which can be employed in the meth linked sequence is altered from the wild-type expression, ods of the embodiments for transforming plants including, which results in an alteration in phenotype. but not limited to, those comprised of deoxyribonucleotides, 0239. In some embodiments the DNA construct may also ribonucleotides, and combinations thereof. Such deoxyribo include a transcriptional enhancer sequence. As used herein, nucleotides and ribonucleotides include both naturally the term an "enhancer refers to a DNA sequence which can occurring molecules and synthetic analogues. The nucleo stimulate promoter activity, and may be an innate element of tide constructs, nucleic acids, and nucleotide sequences of the promoter or a heterologous element inserted to enhance the embodiments also encompass all forms of nucleotide the level or tissue-specificity of a promoter. Various enhanc constructs including, but not limited to, single-stranded ers are known in the art including for example, introns with forms, double-stranded forms, hairpins, stem-and-loop gene expression enhancing properties in plants (US Patent structures and the like. Application Publication Number 2009/0144863, the ubiq 0235 A further embodiment relates to a transformed uitin intron (i.e., the maize ubiquitin intron 1 (see, for organism such as an organism selected from plant and insect example, NCBI sequence S94464)), the omega enhancer or cells, bacteria, yeast, baculovirus, protozoa, nematodes and the omega prime enhancer (Gallie, et al., (1989) Molecular algae. The transformed organism comprises a DNA mol Biology of RNA ed. Cech (Liss, New York) 237-256 and ecule of the embodiments, an expression cassette compris Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S ing the DNA molecule or a vector comprising the expression enhancer (see, e.g., Benfey, et al., (1990) EMBO.J. 9:1685 cassette, which may be stably incorporated into the genome 96) and the enhancers of U.S. Pat. No. 7,803,992 may also of the transformed organism. be used, each of which is incorporated by reference. The 0236. In some embodiments transgenic host cells are above list of transcriptional enhancers is not meant to be provide transformed with a polynucleotide encoding a PtIP limiting. Any appropriate transcriptional enhancer can be 83 polypeptide of the disclosure. In some embodiments the used in the embodiments. host cell is a plant cell. In some embodiments the host cell 0240. The termination region may be native with the is a bacteria. transcriptional initiation region, may be native with the 0237. The sequences of the embodiments are provided in operably linked DNA sequence of interest, may be native DNA constructs for expression in the organism of interest. with the plant host or may be derived from another source The construct will include 5' and 3' regulatory sequences (i.e., foreign or heterologous to the promoter, the sequence operably linked to a sequence of the embodiments. The term of interest, the plant host or any combination thereof). “operably linked as used herein refers to a functional 0241 Convenient termination regions are available from linkage between a promoter and a second sequence, wherein the Ti-plasmid of A. tumefaciens, such as the octopine the promoter sequence initiates and mediates transcription of synthase and nopaline synthase termination regions. See the DNA sequence corresponding to the second sequence. also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141 Generally, operably linked means that the nucleic acid 144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., sequences being linked are contiguous and where necessary (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant to join two protein coding regions in the same reading frame. Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158: The construct may additionally contain at least one addi Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and tional gene to be cotransformed into the organism. Alterna Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639. tively, the additional gene(s) can be provided on multiple 0242. Where appropriate, a nucleic acid may be opti DNA constructs. mized for increased expression in the host organism. Thus, 0238. Such a DNA construct is provided with a plurality where the host organism is a plant, the synthetic nucleic of restriction sites for insertion of the PtIP-83 polypeptide acids can be synthesized using plant-preferred codons for gene sequence to be under the transcriptional regulation of improved expression. See, for example, Campbell and the regulatory regions. The DNA construct may additionally Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of contain selectable marker genes. The DNA construct will host-preferred codon usage. For example, although nucleic generally include in the 5' to 3’ direction of transcription: a acid sequences of the embodiments may be expressed in transcriptional and translational initiation region (i.e., a both monocotyledonous and dicotyledonous plant species, promoter), a DNA sequence of the embodiments, and a sequences can be modified to account for the specific codon transcriptional and translational termination region (i.e., preferences and GC content preferences of monocotyledons termination region) functional in the organism serving as a or dicotyledons as these preferences have been shown to host. The transcriptional initiation region (i.e., the promoter) differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). may be native, analogous, foreign or heterologous to the Thus, the maize-preferred codon for a particular amino acid host organism and/or to the sequence of the embodiments. may be derived from known gene sequences from maize. Additionally, the promoter may be the natural sequence or Maize codon usage for 28 genes from maize plants is listed alternatively a synthetic sequence. The term “foreign' as in Table 4 of Murray, et al., supra. Methods are available in used herein indicates that the promoter is not found in the the art for synthesizing plant-preferred genes. See, for native organism into which the promoter is introduced. example, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Where the promoter is “foreign' or "heterologous to the Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and US 2016/0347799 A1 Dec. 1, 2016 70
Liu H et al. Mol Bio Rep. 37:677-684, 2010, herein incor TABLE 4 porated by reference. A Zea maize codon usage table can be TTT F 21.2 (10493) also found at kazusa.or.jp/codon/cgi-bin/show.codon. TTC F 21.2 (10487) cgi?species-4577, which can be accessed using the www TTA L 9.2 (4545) prefix. Table 3 shows a maize optimal codon analysis TTG L 22.9 (11340) CTT L 23.9 (11829) (adapted from Liu Het al. Mol Bio Rep. 37:677-684, 2010). CTC L 7.1 (8479) CTA L 8.5 (4216) TABLE 3 CTG L 2.7 (6304) ATT I 25.1 (12411) Amino High Low ATC I 6.3 (8071) Acid Codon Count RSCU Count RSCU ATA I 2.9 (6386) ATG M 22.7 (11218) Phe UUU 115 O.04 2,301 22 GTT V 26.1 (12911) UUC: 5,269 1.96 1,485 O.78 GTC V 1.9 (5894) Ser UCU 176 O.13 2,498 48 GTA V 7.7 (3803) UCC: 3,489 2.48 1,074 O.63 GTG V 21.4 (10610) UCA 104 O.O7 2,610 54 TAT Y 5.7 (7779) UCG: 1975 1.4 670 0.4 TAC Y 4.9 (7367) AGU 77 O.OS 1,788 O6 TAA : O.9 (463) AGC: 2,617 1.86 1,514 O.89 TAG : O.S (263) Leu UUA 10 O.O1 1,326 0.79 CAT H 4.0 (6930) UUG. 174 O.09 2,306 37 CAC H 1.6 (5759) CUU 223 O.11 2,396 43 CAA Q 2O.S (10162) CUC: 5,979 3.08 1,109 O.66 CAG Q 6.2 (8038) CUA 106 O.OS 1,280 O.76 AAT N 22.4 (11088) CUG: 5,161 2.66 1,646 O.98 AAC N 22.8 (11284) Pro CCU 427 O.22 1,900 47 AAA K 26.9 (13334) CCC: 3,035 1.59 6O1 O.47 AAG K 35.9 (17797) CCA 311 O16 2,140 66 GAT D 32.4 (16040) CCG: 3,846 2.02 513 0.4 GAC D 20.4 (10097) Ile AUU 138 O.09 2,388 3 GAA E 33.2 (16438) AUC: 4,380 2.85 1,353 O.74 GAG E 33.2 (16426) TCT S 18.4 (9107) AUA 88 O.O6 1,756 O.96 TCC S 12.9 (6409) Thr ACU 136 O.09 1990 43 TCA S 15.6 (7712) ACC: 3,398 2.25 991 O.71 TCG S 4.8 (2397) ACA 133 O.09 2,075 .5 CCT P 18.9 (9358) ACG* 2,378 1.57 495 O.36 CCC P 10.1 (5010) Wall GUU 182 O.O7 2,595 S1 CCA P 19.1 (9461) GUC: 4,584 1.82 1,096 O.64 CCG P 4.7 (2312) GUA 74 O.O3 1,325 O.77 ACT T 17.1 (8490) GUG* 5,257 2.08 1,842 O7 ACC T 14.3 (7100) Ala GCU 629 O.17 3,063 59 ACA T 14.9 (7391) GCC: 8,057 2.16 1,136 O.S9 ACG T 4.3 (2147) GCA 369 O.1 2,872 49 GCT A. 26.7 (13201) GCG: 5,835 57 630 O.33 GCC A. 16.2 (8026) Tyr UAU 71 O.04 1,632 22 GCA A. 21.4 (10577) UAC: 3,841 .96 1,041 O.78 GCG A. 6.3 (3123) His CAU 131 O.09 1,902 36 TGT C 8.1 (3995) TGC C 8.O (3980) CAC: 2,800 91 897 O.64 TGA : 1.O (480) Cys UGU 52 O.04 1,233 .12 TGG W 13.0 (6412) UGC: 2,291 .96 963 O.88 CGT R 6.6 (3291) Gln CAA 99 O.OS 2,312 .04 CGC R 6.2 (3093) CAG: 3,557 95 2,130 O.96 CGA R 4.1 (2018) Arg CGU 153 O.12 751 O.74 CGG R 3.1 (1510) CGC: 4,278 3.25 466 O46 AGT S 12.6 (6237) CGA 92 O.O7 659 O.65 AGC S 11.3 (5594) CGG* 1,793 36 631 O.62 AGA R 14.8 (7337) AGA 83 O.O6 1,948 1.91 AGG R 13.3 (6574) AGG* 1,493 .14 1,652 1.62 GGT G 20.9 (10353) ASn AAU 131 O.O7 3,074 1.26 GGC G 13.4 (6650) AAC 3,814 .93 1,807 O.74 GGA G 22.3 (11022) Lys AAA 130 O.OS 3,215 O.98 GGG G 13.0 (6431) AAG* 5,047 95 3,340 1.02 Asp GAU 312 O.09 4,217 1.38 GAC: 6,729 91 1,891 O.62 0243 A Glycine max codon usage table is shown in Table Gly GGU 363 O.13 2,301 1.35 4 and can also be found at kazusa.or.jp/codon/cgi-bin/ GGC: 7,842 2.91 1,282 0.75 showcodon.cgi?species=3847&aa=1&style=N, which can GGA 397 O.15 2,044 1.19 be accessed using the www prefix. GGG* 2, 186 O.81 1,215 O.71 0244. In some embodiments the recombinant nucleic acid Glu GAA 193 O.O6 4,080 1.1 molecule encoding a PtIP-83 polypeptide has maize opti GAG* 6,010 1.94 3,307 O.9 mized codons. Codon usage was compared using Chi squared contingency test to identify optimal codons, 0245 Additional sequence modifications are known to Codons that occur significantly more often (P\0.01) are indicated with an asterisk. enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation US 2016/0347799 A1 Dec. 1, 2016
signals, exon-intron splice site signals, transposon-like lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, repeats, and other well-characterized sequences that may be 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et deleterious to gene expression. The GC content of the al. Biochim. Biophy's Acta 1503:350-356, 2001), the lumen sequence may be adjusted to levels average for a given targeting signal peptide of which can potentially be used in cellular host, as calculated by reference to known genes accordance with the present disclosure. About 80 proteins expressed in the host cell. The term “host cell as used from Arabidopsis, as well as homologous proteins from herein refers to a cell which contains a vector and Supports spinach and garden pea, are reported by Kieselbach et al., the replication and/or expression of the expression vector is Photosynthesis Research, 78:249-264, 2003. In particular, intended. Host cells may be prokaryotic cells such as E. coli Table 2 of this publication, which is incorporated into the or eukaryotic cells such as yeast, insect, amphibian or description herewith by reference, discloses 85 proteins mammalian cells or monocotyledonous or dicotyledonous from the chloroplast lumen, identified by their accession plant cells. An example of a monocotyledonous host cell is number (see also US Patent Application Publication 2009/ a maize host cell. When possible, the sequence is modified 09044298). In addition, the recently published draft version to avoid PtIP-hairpin secondary mRNA structures. of the rice genome (Goffetal, Science 296:92-100, 2002) is 0246 The expression cassettes may additionally contain a Suitable source for lumen targeting signal peptide which 5' leader sequences. Such leader sequences can act to may be used in accordance with the present disclosure. enhance translation. Translation leaders are known in the art 0248 Suitable chloroplast transit peptides (CTP) are well and include: picornavirus leaders, for example, EMCV known to one skilled in the art also include chimeric CTPs leader (Encephalomyocarditis 5' noncoding region) (Elroy comprising but not limited to, an N-terminal domain, a Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126 central domain or a C-terminal domain from a CTP from 6.130); potyvirus leaders, for example, TEV leader (Tobacco Oryza sativa 1-deoxy-D xylulose-5-Phosphate Synthase Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), Oryza sativa-Superoxide dismutase Oryza sativa-Soluble MDMV leader (Maize Dwarf Mosaic Virus), human immu starch synthase oryza sativa-NADP-dependent Malic acid noglobulin heavy-chain binding protein (BiP) (Macejak, et enzyme Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate al., (1991) Nature 353:90-94); untranslated leader from the Aldolase 2 oryza sativa-L-AScorbate peroxidase 5 oryza coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) sativa-Phosphoglucan water dikinase, Zea Mays (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate virus leader (TMV) (Gallie, et al., (1989) in Molecular dehydrogenase, Zea Mays Thioredoxin M-type US Patent Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) Application Publication 2012/0304336). and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, 0249. The PtIP-83 polypeptide gene to be targeted to the et al., (1987) Plant Physiol. 84:965-968. Such constructs chloroplast may be optimized for expression in the chloro may also contain a 'signal sequence' or "leader sequence' plast to account for differences in codon usage between the to facilitate co-translational or post-translational transport of plant nucleus and this organelle. In this manner, the nucleic the peptide to certain intracellular structures such as the acids of interest may be synthesized using chloroplast chloroplast (or other plastid), endoplasmic reticulum or preferred codons. See, for example, U.S. Pat. No. 5,380,831, Golgi apparatus. herein incorporated by reference. 0247 “Signal sequence' as used herein refers to a 0250 In preparing the expression cassette, the various sequence that is known or Suspected to result in cotransla DNA fragments may be manipulated so as to provide for the tional or post-translational peptide transport across the cell DNA sequences in the proper orientation and, as appropri membrane. In eukaryotes, this typically involves secretion ate, in the proper reading frame. Toward this end, adapters into the Golgi apparatus, with Some resulting glycosylation. or linkers may be employed to join the DNA fragments or Insecticidal toxins of bacteria are often synthesized as other manipulations may be involved to provide for conve protoxins, which are protolytically activated in the gut of the nient restriction sites, removal of superfluous DNA, removal target pest (Chang, (1987) Methods Enzymol. 153:507-516). of restriction sites or the like. For this purpose, in vitro In some embodiments, the signal sequence is located in the mutagenesis, primer repair, restriction, annealing, resubsti native sequence or may be derived from a sequence of the tutions, e.g., transitions and transversions, may be involved. embodiments. “Leader sequence' as used herein refers to 0251 A number of promoters can be used in the practice any sequence that when translated, results in an amino acid of the embodiments. The promoters can be selected based on sequence Sufficient to trigger co-translational transport of the the desired outcome. The nucleic acids can be combined peptide chain to a Subcellular organelle. Thus, this includes with constitutive, tissue-preferred, inducible or other pro leader sequences targeting transport and/or glycosylation by moters for expression in the host organism. Suitable consti passage into the endoplasmic reticulum, passage to vacu tutive promoters for use in a plant host cell include, for oles, plastids including chloroplasts, mitochondria, and the example, the core promoter of the Rsyn? promoter and other like. Nuclear-encoded proteins targeted to the chloroplast constitutive promoters disclosed in WO 1999/43838 and thylakoid lumen compartment have a characteristic bipartite U.S. Pat. No. 6,072,050; the core CaMV 35S promoter transit peptide, composed of a stromal targeting signal (Odell, et al., (1985) Nature 313:810-812); rice actin (McEl peptide and a lumen targeting signal peptide. The stromal roy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Chris targeting information is in the amino-proximal portion of the tensen, et al., (1989) Plant Mol. Biol. 12:619-632 and transit peptide. The lumen targeting signal peptide is in the Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); carboxyl-proximal portion of the transit peptide, and con pEMU (Last, et al., (1991) Theor: Appl. Genet. 81:581–588): tains all the information for targeting to the lumen. Recent MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS research in proteomics of the higher plant chloroplast has promoter (U.S. Pat. No. 5,659,026) and the like. Other achieved in the identification of numerous nuclear-encoded constitutive promoters include, for example, those discussed US 2016/0347799 A1 Dec. 1, 2016 72 in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121 : 5,569, moters (see, for example, Gatz, et al., (1991) Mol. Gen. 597; 5,466,785; 5,399,680: 5,268,463; 5,608,142 and 6,177, Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789, 611. 156), herein incorporated by reference. 0252) Depending on the desired outcome, it may be 0256 Tissue-preferred promoters can be utilized to target beneficial to express the gene from an inducible promoter. enhanced PtIP-83 polypeptide expression within a particular Of particular interest for regulating the expression of the plant tissue. Tissue-preferred promoters include those dis nucleotide sequences of the embodiments in plants are cussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; wound-inducible promoters. Such wound-inducible promot Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; ers, may respond to damage caused by insect feeding, and Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343: include potato proteinase inhibitor (pin II) gene (Ryan, Russell, et al., (1997) Transgenic Res. 6(2): 157-168; Rine (1990) Ann. Rev. Phytopath. 28:425-449: Duan, et al., hart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van (1996) Nature Biotechnology 14:494-498); wun1 and wun2, Camp, et al., (1996) Plant Physiol. 112(2):525-535: U.S. Pat. No. 5,428, 148; win1 and win2 (Stanford, et al., Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778: et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, al., (1993) Plant Mol. Biol. 22:783-792: Eckelkamp, et al., et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et (1993) FEBS Letters 323:73-76); MPI gene (Corderok, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and al., (1994) Plant J. 6(2):141-150) and the like, herein Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such incorporated by reference. promoters can be modified, if necessary, for weak expres 0253) Additionally, pathogen-inducible promoters may S1O. be employed in the methods and nucleotide constructs of the 0257 Leaf-preferred promoters are known in the art. See, embodiments. Such pathogen-inducible promoters include for example, Yamamoto, et al., (1997) Plant J. 12(2):255 those from pathogenesis-related proteins (PR proteins), 265; Kwon, et al., (1994) Plant Physiol. 105:357-67: Yama which are induced following infection by a pathogen; e.g., moto, et al., (1994) Plant Cell Physiol. 35(5):773-778: PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) etc. See, for example, Redolfi, et al., (1983) Neth, J. Plant Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. 645-656 and Van Loon, (1985) Plant Mol. Virol. 4:111-116. 0258 Root-preferred or root-specific promoters are See also, WO 1999/43819, herein incorporated by reference. known and can be selected from the many available from the 0254 Of interest are promoters that are expressed locally literature or isolated de novo from various compatible at or near the site of pathogen infection. See, for example, species. See, for example, Hire, et al., (1992) Plant Mol. Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, Biol. 2002):207-218 (soybean root-specific glutamine syn et al., (1989) Molecular Plant-Microbe Interactions 2:325 thetase gene); Keller and Baumgartner, (1991) Plant Cell 331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 3(10): 1051-1061 (root-specific control element in the GRP 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA Biol. 14(3):433-443 (root-specific promoter of the man 93:14972-14977. See also, Chen, et al., (1996) Plant J. nopine synthase (MAS) gene of Agrobacterium tumefa 10:955-966: Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA ciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201; length cDNA clone encoding cytosolic glutamine synthetase Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. (GS), which is expressed in roots and root nodules of 5,750,386 (nematode-inducible) and the references cited soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7): therein. Of particular interest is the inducible promoter for 633-641, where two root-specific promoters isolated from the maize PRms gene, whose expression is induced by the hemoglobin genes from the nitrogen-fixing nonlegume pathogen Fusarium moniliforme (see, for example, Cordero, Parasponia andersonii and the related non-nitrogen-fixing et al., (1992) Physiol. Mol. Plant Path. 41.189-200). nonlegume Trema tomentosa are described. The promoters 0255 Chemical-regulated promoters can be used to of these genes were linked to a B-glucuronidase reporter modulate the expression of a gene in a plant through the gene and introduced into both the nonlegume Nicotiana application of an exogenous chemical regulator. Depending tabacum and the legume Lotus corniculatus, and in both upon the objective, the promoter may be a chemical-induc instances root-specific promoter activity was preserved. ible promoter, where application of the chemical induces Leach and Aoyagi, (1991) describe their analysis of the gene expression or a chemical-repressible promoter, where promoters of the highly expressed rolC and roll) root application of the chemical represses gene expression. inducing genes of Agrobacterium rhizogenes (see, Plant Chemical-inducible promoters are known in the art and Science (Limerick) 79(1):69-76). They concluded that include, but are not limited to, the maize In2-2 promoter, enhancer and tissue-preferred DNA determinants are disso which is activated by benzenesulfonamide herbicide safen ciated in those promoters. Teeri, et al., (1989) used gene ers, the maize GST promoter, which is activated by hydro fusion to lacZ to show that the Agrobacterium T-DNA gene phobic electrophilic compounds that are used as pre-emer encoding octopine synthase is especially active in the epi gent herbicides, and the tobacco PR-la promoter, which is dermis of the root tip and that the TR2 gene is root specific activated by Salicylic acid. Other chemical-regulated pro in the intact plant and stimulated by wounding in leaf tissue, moters of interest include Steroid-responsive promoters (see, an especially desirable combination of characteristics for use for example, the glucocorticoid-inducible promoter in with an insecticidal or larvicidal gene (see, EMBO.J. 8(2): Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88: 10421 343-350). The TR1 gene fused to nptII (neomycin phos 10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) photransferase II) showed similar characteristics. Additional and tetracycline-inducible and tetracycline-repressible pro root-preferred promoters include the VfBNOD-GRP3 gene US 2016/0347799 A1 Dec. 1, 2016
promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759 to herbicidal compounds, such as glufosinate ammonium, 772) and rolB promoter (Capana, et al., (1994) Plant Mol. bromoxynil, imidazolinones and 2,4-dichlorophenoxyac Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876: etate (2,4-D). Additional examples of suitable selectable 5,750,386; 5,633,363; 5,459,252: 5,401,836; 5,110,732 and marker genes include, but are not limited to, genes encoding 5,023,179. Arabidopsis thaliana root-preferred regulatory resistance to chloramphenicol (Herrera Estrella, et al., sequences are disclosed in US201301 17883. (1983) EMBO.J. 2:987-992); methotrexate (Herrera Estrella, 0259) “Seed-preferred” promoters include both “seed et al., (1983) Nature 303:209-213 and Meijer, et al., (1991) specific' promoters (those promoters active during seed Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., development Such as promoters of seed storage proteins) as (1987) Mol. Gen. Genet. 210:86-91); spectinomycin well as “seed-germinating promoters (those promoters (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131 active during seed germination). See, Thompson, et al., 137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. (1989) BioEssays 10:108, herein incorporated by reference. 7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Such seed-preferred promoters include, but are not limited Mol. Biol. 15:127-136); bromoxynil (Stalker, et al., (1988) to, Cim 1 (cytokinin-induced message); cz19B1 (maize 19 Science 242:419–423); glyphosate (Shaw, et al., (1986) kDa Zein); and milps (myo-inositol-1-phosphate synthase) Science 233:478-481 and U.S. patent application Ser. Nos. (see, U.S. Pat. No. 6,225.529, herein incorporated by refer 10/004.357 and 10/427,692); phosphinothricin (DeBlock, et ence). Gamma-Zein and Glb-1 are endosperm-specific pro al., (1987) EMBO.J. 6:2513-2518). Seegenerally, Yarranton, moters. For dicots, seed-specific promoters include, but are (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et Goldberg, (1989) Plant Cell 1:1079-1093), bean B-phaseo al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. lin, napin, B-conglycinin, glycinin 1, Soybean lectin, cruci 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. ferin, and the like. For monocots, seed-specific promoters 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., include, but are not limited to, maize 15 kDa Zein, 22 kDa (1987) Cell 49:603-612: Figge, et al., (1988) Cell 52:713 Zein, 27 kDa Zein, g-Zein, waxy, shrunken 1, shrunken 2, 722: Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA globulin 1, etc. See also, WO 2000/12733, where seed 86:5400-5404: Fuerst, et al., (1989) Proc. Natl. Acad. Sci. preferred promoters from endl and end2 genes are dis USA 86:2549-2553: Deuschle, et al., (1990) Science 248: closed; herein incorporated by reference. In dicots, seed 480-483; Gossen, (1993) Ph.D. Thesis, University of specific promoters include but are not limited to seed coat Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA promoter from Arabidopsis, pBAN; and the early seed 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. promoters from Arabidopsis, p26, p.63, and p63tr (U.S. Pat. 10:3343-3356: Zambretti, et al., (1992) Proc. Natl. Acad. Nos. 7.294,760 and 7,847,153). A promoter that has “pre Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. ferred expression in a particular tissue is expressed in that Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) tissue to a greater degree than in at least one other plant Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, tissue. Some tissue-preferred promoters show expression (1989) Topics Mol. Struc. Biol. 10:143-162: Degenkolb, et almost exclusively in the particular tissue. al., (1991) Antimicrob. Agents Chemother: 35:1591-1595: 0260. Where low level expression is desired, weak pro Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; moters will be used. Generally, the term “weak promoter as Bonin, (1993) Ph.D. Thesis, University of Heidelberg: Gos used herein refers to a promoter that drives expression of a sen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; coding sequence at a low level. By low level expression at Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913 levels of between about 1/1000 transcripts to about /100,000 919; Hlavka, et al., (1985) Handbook of Experimental transcripts to about /500,000 transcripts is intended. Alterna Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et tively, it is recognized that the term “weak promoters' also al., (1988) Nature 334:721-724. Such disclosures are herein encompasses promoters that drive expression in only a few incorporated by reference. The above list of selectable cells and not in others to give a total low level of expression. marker genes is not meant to be limiting. Any selectable Where a promoter drives expression at unacceptably high marker gene can be used in the embodiments. levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. Plant Transformation 0261 Such weak constitutive promoters include, for 0263. The methods of the embodiments involve introduc example the core promoter of the Rsyn? promoter (WO ing a polypeptide or polynucleotide into a plant. “Introduc 1999/43838 and U.S. Pat. No. 6,072,050), the core 35S ing is as used herein means presenting to the plant the CaMV promoter, and the like. Other constitutive promoters polynucleotide or polypeptide in Such a manner that the include, for example, those disclosed in U.S. Pat. Nos. sequence gains access to the interior of a cell of the plant. 5,608, 149; 5,608,144; 5,604,121 : 5,569,597; 5,466,785; The methods of the embodiments do not depend on a 5,399,680: 5,268,463; 5,608,142 and 6,177,611, herein particular method for introducing a polynucleotide or poly incorporated by reference. The above list of promoters is not peptide into a plant, only that the polynucleotide or poly meant to be limiting. Any appropriate promoter can be used peptides gains access to the interior of at least one cell of the in the embodiments. plant. Methods for introducing polynucleotide or polypep 0262 Generally, the expression cassette will comprise a tides into plants are known in the art including, but not selectable marker gene for the selection of transformed cells. limited to, stable transformation methods, transient trans Selectable marker genes are utilized for the selection of formation methods, and virus-mediated methods. transformed cells or tissues. Marker genes include genes 0264 “Stable transformation' is as used herein means encoding antibiotic resistance, such as those encoding neo that the nucleotide construct introduced into a plant inte mycin phosphotransferase II (NEO) and hygromycin phos grates into the genome of the plant and is capable of being photransferase (HPT), as well as genes conferring resistance inherited by the progeny thereof. “Transient transformation' US 2016/0347799 A1 Dec. 1, 2016 74 as used herein means that a polynucleotide is introduced into Gen. Genet. 202:179-185: Nomura, et al., (1986) Plant Sci. the plant and does not integrate into the genome of the plant 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. or a polypeptide is introduced into a plant. “Plant’ as used 91:2176-2180 and Hush, et al., (1994) The Journal of Cell herein refers to whole plants, plant organs (e.g., leaves, Science 107:775-784, all of which are herein incorporated stems, roots, etc.), seeds, plant cells, propagules, embryos by reference. Alternatively, the PtIP-83 polypeptide poly and progeny of the same. Plant cells can be differentiated or nucleotide can be transiently transformed into the plant undifferentiated (e.g. callus, Suspension culture cells, proto using techniques known in the art. Such techniques include plasts, leaf cells, root cells, phloem cells and pollen). viral vector system and the precipitation of the polynucle 0265 Transformation protocols as well as protocols for otide in a manner that precludes Subsequent release of the introducing nucleotide sequences into plants may vary DNA. Thus, transcription from the particle-bound DNA can depending on the type of plant or plant cell, i.e., monocot or occur, but the frequency with which it is released to become dicot, targeted for transformation. Suitable methods of intro integrated into the genome is greatly reduced. Such methods ducing nucleotide sequences into plant cells and Subsequent include the use of particles coated with polyethylimine (PEI; insertion into the plant genome include microinjection Sigma #P3143). (Crossway, et al., (1986) Biotechniques 4:320-334), elec 0267 Methods are known in the art for the targeted troporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA insertion of a polynucleotide at a specific location in the 83:5602-5606), Agrobacterium-mediated transformation plant genome. In one embodiment, the insertion of the (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene trans polynucleotide at a desired genomic location is achieved fer (Paszkowski, et al., (1984) EMBO.J. 3:2717-2722) and using a site-specific recombination system. See, for ballistic particle acceleration (see, for example, U.S. Pat. example, WO 1999/25821, WO 1999/25854, WO 1999/ Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782: 25840, WO 1999/25855 and WO 1999/25853, all of which Tomes, et al., (1995) in Plant Cell, Tissue, and Organ are herein incorporated by reference. Briefly, the polynucle Culture. Fundamental Methods, ed. Gamborg and Phillips, otide of the embodiments can be contained in transfer (Springer-Verlag, Berlin) and McCabe, et al., (1988) Bio cassette flanked by two non-identical recombination sites. technology 6:923-926) and Led transformation (WO The transfer cassette is introduced into a plant have stably 00/28058). For potato transformation see, Tu, et al., (1998) incorporated into its genome a target site which is flanked by Plant Molecular Biology 37:829-838 and Chong, et al., two non-identical recombination sites that correspond to the (2000) Transgenic Research 9:71-78. Additional transfor sites of the transfer cassette. An appropriate recombinase is mation procedures can be found in Weissinger, et al., (1988) provided and the transfer cassette is integrated at the target Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Par site. The polynucleotide of interest is thereby integrated at a ticulate Science and Technology 5:27-37 (onion); Christou, specific chromosomal position in the plant genome. et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, 0268 Plant transformation vectors may be comprised of et al., (1988) Bio/Technology 6:923-926 (soybean): Finer one or more DNA vectors needed for achieving plant and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 transformation. For example, it is a common practice in the (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319 art to utilize plant transformation vectors that are comprised 324 (soybean); Datta, et al., (1990) Biotechnology 8:736 of more than one contiguous DNA segment. These vectors 740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA are often referred to in the art as “binary vectors'. Binary 85:4305-4309 (maize): Klein, et al., (1988) Biotechnology vectors as well as vectors with helper plasmids are most 6:559-563 (maize): U.S. Pat. Nos. 5,240,855; 5,322,783 and often used for Agrobacterium-mediated transformation, 5,324,646; Klein, et al., (1988) Plant Physiol. 91:440-444 where the size and complexity of DNA segments needed to (maize): Fromm, et al., (1990) Biotechnology 8:833-839 achieve efficient transformation is quite large, and it is (maize): Hooykaas-Van Slogteren, et al., (1984) Nature advantageous to separate functions onto separate DNA (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); molecules. Binary vectors typically contain a plasmid vector Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:53.45 that contains the cis-acting sequences required for T-DNA 5349 (Liliaceae); De Wet, et al., (1985) in The Experimental transfer (such as left border and right border), a selectable Manipulation of Ovule Tissues, ed. Chapman, et al., (Long marker that is engineered to be capable of expression in a man, New York), pp. 197-209 (pollen); Kaeppler, et al., plant cell, and a "gene of interest' (a gene engineered to be (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., capable of expression in a plant cell for which generation of (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transgenic plants is desired). Also present on this plasmid transformation); DHalluin, et al., (1992) Plant Cell 4:1495 vector are sequences required for bacterial replication. The 1505 (electroporation); Li, et al., (1993) Plant Cell Reports cis-acting sequences are arranged in a fashion to allow 12:250-255 and Christou and Ford, (1995) Annals of Botany efficient transfer into plant cells and expression therein. For 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnol example, the selectable marker gene and the pesticidal gene ogy 14:745-750 (maize via Agrobacterium tumefaciens); all are located between the left and right borders. Often a of which are herein incorporated by reference. second plasmid vector contains the trans-acting factors that 0266. In specific embodiments, the sequences of the mediate T-DNA transfer from Agrobacterium to plant cells. embodiments can be provided to a plant using a variety of This plasmid often contains the virulence functions (Vir transient transformation methods. Such transient transfor genes) that allow infection of plant cells by Agrobacterium, mation methods include, but are not limited to, the intro and transfer of DNA by cleavage at border sequences and duction of the PtIP-83 polynucleotide or variants and frag Vir-mediated DNA transfer, as is understood in the art ments thereof directly into the plant or the introduction of (Hellens and Mullineaux, (2000) Trends in Plant Science the PtIP-83 polypeptide transcript into the plant. Such meth 5:446-451). Several types of Agrobacterium strains (e.g. ods include, for example, microinjection or particle bom LBA4404, GV3101, EHA101, EHA105, etc.) can be used bardment. See, for example, Crossway, et al., (1986) Mol for plant transformation. The second plasmid vector is not US 2016/0347799 A1 Dec. 1, 2016 necessary for transforming the plants by other methods such synthesized as part of a viral polyprotein, which later may be as microprojection, microinjection, electroporation, polyeth processed by proteolysis in vivo or in vitro to produce the ylene glycol, etc. desired PtIP-83 polypeptide. It is also recognized that such 0269. In general, plant transformation methods involve a viral polyprotein, comprising at least a portion of the transferring heterologous DNA into target plant cells (e.g., amino acid sequence of a Pt(P-83 of the embodiments, may immature or mature embryos, Suspension cultures, undiffer have the desired pesticidal activity. Such viral polyproteins entiated callus, protoplasts, etc.), followed by applying a and the nucleotide sequences that encode for them are maximum threshold level of appropriate selection (depend encompassed by the embodiments. Methods for providing ing on the selectable marker gene) to recover the trans plants with nucleotide constructs and producing the encoded formed plant cells from a group of untransformed cell mass. proteins in the plants, which involve viral DNA or RNA Following integration of heterologous foreign DNA into molecules are known in the art. See, for example, U.S. Pat. plant cells, one then applies a maximum threshold level of Nos. 5,889, 191; 5,889, 190; 5,866,785: 5,589,367 and 5,316, appropriate selection in the medium to kill the untrans 931; herein incorporated by reference. formed cells and separate and proliferate the putatively 0273 Methods for transformation of chloroplasts are transformed cells that survive from this selection treatment known in the art. See, for example, Svab, et al., (1990) Proc. by transferring regularly to a fresh medium. By continuous Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, passage and challenge with appropriate selection, one iden (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and tifies and proliferates the cells that are transformed with the Maliga, (1993) EMBO.J. 12:601-606. The method relies on plasmid vector. Molecular and biochemical methods can particle gun delivery of DNA containing a selectable marker then be used to confirm the presence of the integrated and targeting of the DNA to the plastid genome through heterologous gene of interest into the genome of the trans homologous recombination. Additionally, plastid transfor genic plant. mation can be accomplished by transactivation of a silent 0270 Explants are typically transferred to a fresh supply plastid-borne transgene by tissue-preferred expression of a of the same medium and cultured routinely. Subsequently, nuclear-encoded and plastid-directed RNA polymerase. the transformed cells are differentiated into shoots after Such a system has been reported in McBride, et al., (1994) placing on regeneration medium Supplemented with a maxi Proc. Natl. Acad. Sci. USA 91:7301-73.05. mum threshold level of selecting agent. The shoots are then 0274 The embodiments further relate to plant-propagat transferred to a selective rooting medium for recovering ing material of a transformed plant of the embodiments rooted shoot or plantlet. The transgenic plantlet then grows including, but not limited to, seeds, tubers, corms, bulbs, into a mature plant and produces fertile seeds (e.g., Hiei, et leaves and cuttings of roots and shoots. al., (1994) The Plant Journal 6:271-282; Ishida, et al., 0275. The embodiments may be used for transformation (1996) Nature Biotechnology 14:745-750). Explants are of any plant species, including, but not limited to, monocots typically transferred to a fresh Supply of the same medium and dicots. Examples of plants of interest include, but are not and cultured routinely. A general description of the tech limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. niques and methods for generating transgenic plants are rapa, B. juncea), particularly those Brassica species useful found in Ayres and Park, (1994) Critical Reviews in Plant as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza Science 13:219-239 and Bommineni and Jauhar, (1997) sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Maydica 42:107-120. Since the transformed material con Sorghum vulgare), millet (e.g., pearl millet (Pennisetum tains many cells; both transformed and non-transformed glaucum), proso millet (Panicum miliaceum), foxtail millet cells are present in any piece of Subjected target callus or (Setaria itaica), finger millet (Eleusine coracana)), Sun tissue or group of cells. The ability to kill non-transformed flower (Helianthus annuus), safflower (Carthamus tincto cells and allow transformed cells to proliferate results in rius), wheat (Triticum aestivum), soybean (Glycine max), transformed plant cultures. Often, the ability to remove tobacco (Nicotiana tabacum), potato (Solanum tuberosum), non-transformed cells is a limitation to rapid recovery of peanuts (Arachis hypogaea), cotton (Gossypium bar transformed plant cells and Successful generation of trans badense, Gossypium hirsutum), Sweet potato (Ipomoea genic plants. batatus), cassava (Manihot esculenta), coffee (Coffea spp.), 0271 The cells that have been transformed may be grown coconut (Cocos nucifera), pineapple (Ananas comosus), into plants in accordance with conventional ways. See, for citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea example, McCormick, et al., (1986) Plant Cell Reports (Camelia sinensis), banana (Musa spp.), avocado (Persea 5:81-84. These plants may then be grown, and either polli americana), fig (Ficus casica), guava (Psidium guajava), nated with the same transformed strain or different strains, mango (Mangifera indica), olive (Olea europaea), papaya and the resulting hybrid having constitutive or inducible (Carica papaya), cashew (Anacardium occidentale), maca expression of the desired phenotypic characteristic identi damia (Macadamia integrifolia), almond (Prunus amygd fied. Two or more generations may be grown to ensure that alus), Sugar beets (Beta vulgaris), Sugarcane (Saccharum expression of the desired phenotypic characteristic is stably spp.), oats, barley, vegetables ornamentals, and conifers. maintained and inherited and then seeds harvested to ensure 0276 Vegetables include tomatoes (Lycopersicon escu that expression of the desired phenotypic characteristic has lentum), lettuce (e.g., Lactuca sativa), green beans (Phaseo been achieved. lus vulgaris), lima beans (Phaseolus limensis), peas (Lathy 0272. The nucleotide sequences of the embodiments may rus spp.), and members of the genus Cucumis such as be provided to the plant by contacting the plant with a virus cucumber (C. sativus), cantaloupe (C. cantalupensis), and or viral nucleic acids. Generally, such methods involve musk melon (C. melo). Ornamentals include azalea (Rho incorporating the nucleotide construct of interest within a dodendron spp.), hydrangea (Macrophylla hydrangea), viral DNA or RNA molecule. It is recognized that the hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips recombinant proteins of the embodiments may be initially (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia US 2016/0347799 A1 Dec. 1, 2016 76 hybrida), carnation (Dianthus caryophyllus), poinsettia (Eu (2001) supra). In general, total DNA is extracted from the phorbia pulcherrima), and chrysanthemum. Conifers that transformant, digested with appropriate restriction enzymes, may be employed in practicing the embodiments include, for fractionated in an agarose gel and transferred to a nitrocel example, pines such as loblolly pine (Pinus taeda), slash lulose or nylon membrane. The membrane or “blot' is then pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), probed with, for example, radiolabeled 32P target DNA lodgepole pine (Pinus contorta), and Monterey pine (Pinus fragment to confirm the integration of introduced gene into radiata); Douglas-fir (Pseudotsuga menziesii); Western the plant genome according to standard techniques (Sam hemlock (Tsuga Canadensis); Sitka spruce (Picea glauca); brook and Russell. (2001) supra). redwood (Sequoia sempervirens); true firs such as silver fir 0282. In Northern blot analysis, RNA is isolated from (Abies amabilis) and balsam fir (Abies balsamea); and specific tissues of transformant, fractionated in a formalde cedars such as Western red cedar (Thuja plicata) and Alaska hyde agarose gel, and blotted onto a nylon filter according yellow-cedar (Chamaecyparis mootkatensis). Plants of the to standard procedures that are routinely used in the art embodiments include crop plants (for example, corn, alfalfa, (Sambrook and Russell. (2001) supra). Expression of RNA Sunflower, Brassica, Soybean, cotton, safflower, peanut, sor encoded by the pesticidal gene is then tested by hybridizing ghum, wheat, millet, tobacco, etc.). Such as corn and Soy the filter to a radioactive probe derived from a pesticidal bean plants. gene, by methods known in the art (Sambrook and Russell, 0277 Turfgrasses include, but are not limited to: annual (2001) supra). bluegrass (Poa annua); annual ryegrass (Lolium multiflo 0283 Western blot, biochemical assays and the like may rum); Canada bluegrass (Poa compressa); Chewings fescue be carried out on the transgenic plants to confirm the (Festuca rubra); colonial bentgrass (Agrostis tenuis); creep presence of protein encoded by the pesticidal gene by ing bentgrass (Agrostis palustris); crested wheatgrass (Agro standard procedures (Sambrook and Russell, 2001, supra) pyron desertorum); fairway wheatgrass (Agropyron Crista using antibodies that bind to one or more epitopes present on tum); hard fescue (Festuca longifolia); Kentucky bluegrass the PtIP-83 polypeptide. (Poa pratensis); orchardgrass (Dactylis glomerata); peren nial ryegrass (Lolium perenne); red fescue (Festuca rubra); Stacking of Traits in Transgenic Plant redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); Smooth bromegrass (Bromus 0284 Transgenic plants may comprise a stack of one or inermis); tall fescue (Festuca arundinacea); timothy more insecticidal polynucleotides disclosed herein with one (Phleum pratense); Velvet bentgrass (Agrostis canina); or more additional polynucleotides resulting in the produc weeping alkaligrass (Puccinelia distans); western wheat tion or suppression of multiple polypeptide sequences. grass (Agropyron Smithii); Bermuda grass (Cynodon spp.); Transgenic plants comprising stacks of polynucleotide St. Augustine grass (Stenotaphrum secundatum); Zoysia sequences can be obtained by either or both of traditional grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet breeding methods or through genetic engineering methods. grass (Axonopus affinis); centipede grass (Eremochloa Ophi These methods include, but are not limited to, breeding uroides): kikuyu grass (Pennisetum clandesinum); seashore individual lines each comprising a polynucleotide of inter paspalum (Paspalum vaginatum); blue gramma (Bouteloua est, transforming a transgenic plant comprising a gene gracilis); buffalo grass (Buchloe dactyloids); Sideoats disclosed herein with a Subsequent gene and co-transforma gramma (Bouteloua curtipendula). tion of genes into a single plant cell. As used herein, the term 0278 Plants of interest include grain plants that provide “stacked' includes having the multiple traits present in the seeds of interest, oil-seed plants, and leguminous plants. same plant (i.e., both traits are incorporated into the nuclear Seeds of interest include grain seeds, such as corn, wheat, genome, one trait is incorporated into the nuclear genome barley, rice, sorghum, rye, millet, etc. Oil-seed plants include and one trait is incorporated into the genome of a plastid or cotton, soybean, Safflower, Sunflower, Brassica, maize, both traits are incorporated into the genome of a plastid). In alfalfa, palm, coconut, flax, castor, olive, etc. Leguminous one non-limiting example, "stacked traits' comprise a plants include beans and peas. Beans include guar, locust molecular stack where the sequences are physically adjacent bean, fenugreek, soybean, garden beans, cowpea, mung to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. bean, lima bean, fava bean, lentils, chickpea, etc. Co-transformation of genes can be carried out using single Evaluation of Plant Transformation transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are 0279. Following introduction of heterologous foreign stacked by genetically transforming the plants, the poly DNA into plant cells, the transformation or integration of nucleotide sequences of interest can be combined at any heterologous gene in the plant genome is confirmed by time and in any order. The traits can be introduced simul various methods such as analysis of nucleic acids, proteins taneously in a co-transformation protocol with the poly and metabolites associated with the integrated gene. nucleotides of interest provided by any combination of 0280 PCR analysis is a rapid method to screen trans transformation cassettes. For example, if two sequences will formed cells, tissue or shoots for the presence of incorpo be introduced, the two sequences can be contained in rated gene at the earlier stage before transplanting into the separate transformation cassettes (trans) or contained on the soil (Sambrook and Russell, (2001) Molecular Cloning: A same transformation cassette (cis). Expression of the Laboratory Manual. Cold Spring Harbor Laboratory Press, sequences can be driven by the same promoter or by Cold Spring Harbor, N.Y.). PCR is carried out using oligo different promoters. In certain cases, it may be desirable to nucleotide primers specific to the gene of interest or Agro introduce a transformation cassette that will Suppress the bacterium vector background, etc. expression of the polynucleotide of interest. This may be 0281 Plant transformation may be confirmed by South combined with any combination of other suppression cas ern blot analysis of genomic DNA (Sambrook and Russell, settes or overexpression cassettes to generate the desired US 2016/0347799 A1 Dec. 1, 2016 77 combination of traits in the plant. It is further recognized that (2011) PLOS Pathogens, 7:1-13), from Pseudomonas pro polynucleotide sequences can be stacked at a desired tegens strain CHAO and Pf-5 (previously fluorescens) genomic location using a site-specific recombination sys (Pechy–Tarr, (2008) Environmental Microbiology 10:2368 tem. See, for example, WO 1999/25821, WO 1999/25854, 2386: GenBank Accession No. EU400157); from WO 1999/25840, WO 1999/25855 and WO 1999/25853, all Pseudomonas Taiwanensis (Liu, et al., (2010).J. Agric. Food of which are herein incorporated by reference. Chem. 58:12343-12349) and from Pseudomonas pseudoal 0285. In some embodiments the polynucleotides encod cligenes (Zhang, et al., (2009) Annals of Microbiology ing the Pt|P-83 polypeptide disclosed herein, alone or 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. stacked with one or more additional insect resistance traits 89:159-168); insecticidal proteins from Photorhabdus sp. can be stacked with one or more additional input traits (e.g., and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open herbicide resistance, fungal resistance, virus resistance, Toxinology Journal 3:101-118 and Morgan, et al., (2001) stress tolerance, disease resistance, male Sterility, stalk Applied and Envir. Micro. 67:2062-2069), U.S. Pat. No. strength, and the like) or output traits (e.g., increased yield, 6,048,838, and U.S. Pat. No. 6,379,946; a PIP-1 polypeptide modified Starches, improved oil profile, balanced amino of US Patent Publication US20140007292; an AfIP-1A acids, high lysine or methionine, increased digestibility, and/or AflP-1B polypeptide of US Patent Publication improved fiber quality, drought resistance, and the like). US20140033361; a PHI-4 polypeptide of US patent Publi Thus, the polynucleotide embodiments can be used to pro cation US20140274885 or PCT Patent Publication vide a complete agronomic package of improved crop WO2014/150914; a PIP-47 polypeptide of PCT Serial Num quality with the ability to flexibly and cost effectively ber PCT/US14/51063, a PIP-72 polypeptide of PCT Serial control any number of agronomic pests. Number PCT/US14/55128, and 8-endotoxins including, but Transgenes useful for stacking include but are not limited to: not limited to, the Cry 1, Cry2, Cry3, Cry4, Crys, Cry6, 1. Transgenes that Confer Resistance to Insects or Disease Cry7, Cry8, Cry9, Cry 10, Cry 11, Cry 12, Cry13, Cry 14, and that Encode: Cry 15, Cry 16, Cry 17, Cry 18, Cry 19, Cry20, Cry21, Cry22, (A) Plant disease resistance genes. Plant defenses are often Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, activated by specific interaction between the product of a Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, disease resistance gene (R) in the plant and the product of a Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, corresponding avirulence (AVr) gene in the pathogen. A Cry47, Cry49, Crys0, Crys1, Crys2, Cry53, Cry 54, Crys5, plant variety can be transformed with cloned resistance gene Crys6, Crys7, Crys8, Crys9, Cry60, Cry61, Cry62, Cry63, to engineer plants that are resistant to specific pathogen Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, strains. See, for example, Jones, et al., (1994) Science and Cry 72 classes of Ö-endotoxin genes and the B. thuringi 266:789 (cloning of the tomato Cf-9 gene for resistance to ensis cytolytic Cyt1 and Cyt2 genes. Members of these Cladosporium fulvum); Martin, et al., (1993) Science 262: classes of B. thuringiensis insecticidal proteins include, but 1432 (tomato Pto gene for resistance to Pseudomonas Syrin are not limited to Cry1Aa1 (Accession if AAA22353); gae pv. tomato encodes a protein kinase); Mindrinos, et al., Cry1Aa2 (Accession # Accession if AAA22552); Cry1Aa3 (1994) Cell 78: 1089 (Arabidopsis RSP2 gene for resistance (Accession it BAA00257); Cry1Aa4 (Accession it to Pseudomonas Syringae), McDowell and Woffenden, CAA31886); Cry1Aa5 (Accession # BAAO4468); Cry1Aaé (2003) Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (Accession if AAA86265); Cry1Aa7 (Accession it (2002) Transgenic Res. 11(6):567-82. A plant resistant to a AAD46139); Cry1Aa8 (Accession # I26149); Cry1Aa9 disease is one that is more resistant to a pathogen as (Accession it BAA77213); Cry1Aa10 (Accession it compared to the wild type plant. AAD55382); Cry1Aa11 (Accession #CAA70856); (B) Genes encoding a Bacillus thuringiensis protein, a Cry1Aa12 (Accession if AAP80146); Cry1Aa13 (Accession derivative thereof or a synthetic polypeptide modeled # AAM44305); Cry1Aa14 (Accession if AAP40639): thereon. See, for example, Geiser, et al., (1986) Gene Cry1Aa15 (Accession if AAY66993); Cry1Aa16 (Accession 48: 109, who disclose the cloning and nucleotide sequence of # HQ439776): Cry1Aa17 (Accession # HQ439788): a Bt delta-endotoxin gene. Moreover, DNA molecules Cry1Aa18 (Accession # HQ439790): Cry1Aa19 (Accession encoding delta-endotoxin genes can be purchased from # HQ685121); Cry1Aa20 (Accession # JF340156); American Type Culture Collection (Rockville, Md.), for Cry1Aa21 (Accession if JN651496); Cry1Aa22 (Accession example, under ATCCR) Accession Numbers 40098, 67136, # KC158223); Cry1Ab1 (Accession # AAA22330): 31995 and 31998. Other non-limiting examples of Bacillus Cry1Ab2 (Accession if AAA22613); Cry1Ab3 (Accession it thuringiensis transgenes being genetically engineered are AAA22561); Cry1Ab4 (Accession # BAAO0071); Cry1Ab5 given in the following patents and patent applications and (Accession # CAA28405); Cry1Ab6 (Accession it hereby are incorporated by reference for this purpose: U.S. AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 Pat. Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; (Accession if AAA22551); Cry1Ab9 (Accession it 6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, CAA38701); Cry1Ab10 (Accession # A29125); Cry1Ab11 6,642,030, 6,713,259, 6,893,826, 7,105,332; 7,179,965, (Accession if I12419); Cry1Ab12 (Accession if AAC64003); 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Acces 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862, sion # AAG16877); Cry1Ab15 (Accession # AAO13302); 7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Acces 7,790,846, 7,858,849 and WO 1991/14778; WO 1999/ sion if AAT46415); Cry1Ab18 (Accession # AAQ88259); 3.1248: WO 2001/12731: WO 1999/24581 and WO 1997/ Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Acces 4O162. sion # ABB72460); Cry1Ab21 (Accession # ABS18384); 0286 Genes encoding pesticidal proteins may also be Cry1Ab22 (Accession # ABW87320): Cry1Ab23 (Acces stacked including but are not limited to: insecticidal proteins sion # HQ439777); Cry1Ab24 (Accession # HQ439778): from Pseudomonas sp. such as PSEEN3174 (Monalysin, Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession