US 2014.0007292A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0007292 A1 Cerf et al. (43) Pub. Date: Jan. 2, 2014
(54) NOVEL INSECTICIDAL PROTEINS AND Publication Classification METHODS FOR THEIR USE (51) Int. Cl. (71) Applicant: PIONEER HIBRED C07K I4/2 (2006.01) INTERNATIONAL INC, Johnston, IA GOIN33/68 (2006.01) (US) C07K 6/2 (2006.01) AOIN 43/50 (2006.01) (72) Inventors: David C. Cerf, Palo Alto, CA (US); A6II 45/06 (2006.01) James J. English, San Ramon, CA (US); (52) U.S. Cl. Carol A. Hendrick, Des Moines, IA CPC ...... C07K 14/21 (2013.01); A0IN 43/50 (US); Lu Liu, Palo Alto, CA (US); (2013.01); A61K 45/06 (2013.01); C07K Jarred K. Oral, San Carlos, CA (US); I6/1214 (2013.01); G0IN33/68 (2013.01) Philip A. Patten, Menlo Park, CA (US); USPC ...... 800/279; 536/23.7:435/320.1; 530/350; Barbara A. Rosen, Mountain View, CA 435/252.3; 435/252.34; 435/252.35:435/252.2: (US); Ute Schellenberger, Palo Alto, 435/254.11: 435/254.2:435/254.21; CA (US); Ingrid A. Udranszky, 435/252.33:435/252.31; 514/4.5; 424/94.6; Mountain View, CA (US); Jun-Zhi Wei, 424/94.61; 530/387.9; 800/302:436/501 Palo Alto, CA (US); Genhai Zhu, San (57) ABSTRACT Jose, CA (US) Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, (73) Assignee: Pioneer Hi Bred International Inc, the nucleic acid sequences are useful for preparing plants and Johnston, IA (US) microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and (21) Appl. No.: 13/792,861 seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for Subsequent trans (22) Filed: Mar 11, 2013 formation into organisms of interest, as probes for the isola tion of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling, inhibiting Related U.S. Application Data growth or killing lepidopteran, coleopteran, dipteran, fungal, (60) Provisional application No. 61/667,039, filed on Jul. 2, hemipteran, and nematode pest populations and for produc 2012. ing compositions with insecticidal activity. Patent Application Publication Jan. 2, 2014 Sheet 1 of 7 US 2014/0007292 A1
PIP-1A (1) PSEEN3174 (1) SK PIP-1B (1) S AECFG-592740 (1) NRocoERAVNIIDSKVEor Pput 1063 (1) Pput 1064 (1)
BBBBBBBBBBBBBBBBB BBBBBBBBBBBB 100 PIP-1A (51) PSEEN3174 (51) PIP-1B (51) AECFG-592740 (34) Pput 1063 (21) Pput 1064 (25) DTTYGERCNYD
PIP-1A (99) PSEEN3174 (99) PIP-1B (99) AECFG-592740 (84) TSR TKYEH, Pput 1063 (69) TYKTLSAGDCEIDLSRASG Pput 1064 (72)
BBBBBBBBBBB 151 200 PIP-1A (149) PSEEN3174 (149) PIP-1B (149) AECFG-592740 (133) Pput 1063 (118) Pput 1064 (122)
201 250 PIP-1A (199) :--& PSEEN3174 (199) PIP-1B (199) AECFG-592740 (182) Pput 1063 (167) Pput 1064 (171)
BBBBBBBB BBBBBBBB 251 PIP-1A (243) PSEEN3174 (243) PIP-1B (243) AECFG-592740 (227)
Pput 1063 (211) FNLAYGSGDSGGSFNDQ&ASNRFLQ s Pput 1064 (221) FNSEKSPGRREFSV------Patent Application Publication Jan. 2, 2014 Sheet 2 of 7 US 2014/0007292 A1
Fig. 2
Saturated mutagenesis
3.188 Reverse primer -> -H
--> st Sphi Forwar primer BamHI 3188R
PCR using 3188FI3188R to recover full length and clone to expression vector
Transform to E. Coli host
Pick 96 or more colonies for sequencing to recover variants
Patent Application Publication Jan. 2, 2014 Sheet 4 of 7 US 2014/0007292 A1
Fig. 3B
66 O CGTTAAGTTG GA CGTGAAAT CCCCGGGCTC AACCTGGGAA. CTGCATCCAA AACTGGCGAG CGTTAAGTTG GA CGTGAAAG CCCCGGGCTC AACCTGGGAA CTGCATCCAA AACTGGCGAG
72 O CTAGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG CT AGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG
78O AAGGAACACC AG GGCGAAG GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC AAGGAACACC AG GGCGAAG GCGACCACCT GGACTGATAC GACACTGAG GTGCGAAAGC
8 AO GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC GTGGGGAGCA AACAGGATTA. GATACCCTGG TAGTCCACGC CGTAAA CGAT GTCAACTAGC
9 OO CGTTGGGAGC CT GAGCTCT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG CGTTGGAATC CT GAGATT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG
96.O TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT
1 O2 O GTGGTTTAAT CGAAGCAAC GCGAAGAACC TTACCAGGCC PTGACATCCA ATGAACTTTC GTGGTTTAAT CGAAGCAAC GCGAAGAACC TTACCAGGCC TGACATGCA GAGAACTTTC
108 O CAGAGATGGA TTGGTGCCTT CGGGAACATT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CAGAGATGGA TTGGTGCCTT CGGGAACTCT GACACAGGTG CTGCATGGCT GTCGTCAGCT
114 O CGTGTCGTGA GA GTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA CGTGTCGTGA GAT GTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA
12 OO GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT Patent Application Publication Jan. 2, 2014 Sheet 5 of 7 US 2014/0007292 A1
Fig. 3C
126 O GACGTCAAGT CATCATGGCC CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC GACGTCAAGT CATCATGGCC CTTACCCCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC
1320 AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA. ATCCCATAAA ACCGATCGTA. GTCCGGATCG AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA ATCTCACAAA ACCGATCGTA GTCCGGATCG
138O CAGTCTGCAA CTCGACTGCG TGAAGTCGGA. ATCGCTAGTA ATCGCGAATC AGAATGTCGC CAGTCTGCAA CTCGACTGCG TGAAGTCGGA ATCGCTAGTA ATCGCAAATC AGAATGTTGC
144 O GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC
15 OO CAGAAGTAGC TAGTCTAACC TTCGGGAGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG CAGAAGTAGC TAGTCTAACC TTCGGGGGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG
15 6.O TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GATCAC CTCC TTAATCGACG TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GAT CACCTCC TT
162O ACATCAGCTG CTTCATAAGC TCCCACACGA ATTGCTTGAT TCATTGAAGA AGACGATTGG
1680 GTCTGTAGCT CAGTTGGTTA GAGCGCACCC CTGATAAGGG TGAGGTCGGC AGTTCGAATC
17 OO TGCCCAGACC CACCAATTAC S EO ID NO : 21 6 P. chlororaphis SS 44C4 16S-rDNA SEO ID NO: 217 P. entomophila-L48 16S-rDNA Patent Application Publication Jan. 2, 2014 Sheet 6 of 7 US 2014/0007292 A1
Fig. 4
Plate 1
£3.u.)u.993.
US 2014/0007292 A1 Jan. 2, 2014
NOVEL, INSECTICDAL PROTEINS AND against insect pests, e.g., insecticidal proteins which are METHODS FOR THEIR USE active against a variety of insects in the order Lepidoptera and the order Hemiptera including but not limited to species CROSS REFERENCE belonging to the family Pentatomidae, the family Plataspidae 0001. This utility application claims the benefit U.S. Pro and the family Cydnidae. In addition, there remains a need for visional Application No. 61/667,039, filed Jul. 2, 2012, which biopesticides having activity against a variety of insect pests is incorporated herein by reference in its entirety. that have developed resistance to existing pesticides. REFERENCE TO SEQUENCE LISTING SUMMARY OF THE INVENTION SUBMITTED ELECTRONICALLY 0008 Compositions and methods for conferring pesticidal 0002 The official copy of the sequence listing is submitted activity to bacteria, plants, plant cells, tissues and seeds are electronically via EFS-Web as an ASCII formatted sequence provided. Compositions include nucleic acid molecules listing with a file named "4208 sequence listing..txt created encoding sequences for pesticidal and insecticidal polypep on Mar. 4, 2013, and having a size of 471 kilobytes and is filed tides, vectors comprising those nucleic acid molecules, and concurrently with the specification. The sequence listing con host cells comprising the vectors. Compositions also include tained in this ASCII formatted document is part of the speci the pesticidal polypeptide sequences and antibodies to those fication and is herein incorporated by reference in its entirety. polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and FIELD OF THE INVENTION expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be syn 0003. This disclosure relates to the field of molecular biol thetic sequences that have been designed for expression in an ogy. Provided are novel genes that encode pesticidal proteins. organism including, but not limited to, a microorganism or a These pesticidal proteins and the nucleic acid sequences that plant. Compositions also comprise transformed bacteria, encode them are useful in preparing pesticidal formulations plants, plant cells, tissues and seeds. and in the production of transgenic pest-resistant plants. 0009. In particular, isolated or recombinant nucleic acid molecules are provided encoding Pseudomonas Insecticidal BACKGROUND OF THE INVENTION Protein-1 (PIP-1) polypeptides including amino acid substi 0004 Biological control of insect pests of agricultural sig tutions, amino aciddeletions, amino acid insertions, and frag nificance using a microbial agent, Such as fungi, bacteria or ments thereof, and combinations thereof. Additionally, amino another species of insect affords an environmentally friendly acid sequences corresponding to the PIP-1 polypeptides are and commercially attractive alternative to synthetic chemical encompassed. Provided are an isolated or recombinant pesticides. Generally speaking, the use of biopesticides pre nucleic acid molecule capable of encoding a PIP-1 polypep sents a lower risk of pollution and environmental hazards, and tide of SEQID NO: 2, 101, 102, 103,104,105,106, 107, 108, biopesticides provide greater target specificity than is char 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, acteristic of traditional broad-spectrum chemical insecti 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, cides. In addition, biopesticides often costless to produce and 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, thus improve economic yield for a wide variety of crops. 145, 146, 147, 148, 149, 150, 151, 204, 206, 208, 211, 212, 0005 Certain species of microorganisms of the genus 213, 214, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, Bacillus are known to possess pesticidal activity against a 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, range of insect pests including Lepidoptera, Diptera, 267, 268, 269,298, 299, 300, 301, 302,303, 304, 305, 306, Coleoptera, Hemiptera and others. Bacillus thuringiensis 307, 308,309, 310, 311, 312,313, 314, 315, 316, 317,318, (Bt) and Bacillus popilliae are among the most Successful 319, 320, 321,322,323,324, and 325 as well as amino acid biocontrol agents discovered to date. Insect pathogenicity has Substitutions, amino acid deletions, amino acid insertions, also been attributed to strains of B. larvae, B. lentimorbus, B. and fragments thereof, and combinations thereof. In some sphaericus and B. cereus. Microbial insecticides, particularly embodiments exemplary PIP-1 polypeptides comprise a those obtained from Bacillus strains, have played an impor sequence set forth in of SEQID NO: 2, 101, 102, 103, 104, tant role in agriculture as alternatives to chemical pest control. 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 0006 Crop plants have been developed with enhanced 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, insect resistance by genetically engineering crop plants to 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, produce pesticidal proteins from Bacillus. For example, corn 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 204, and cotton plants have been genetically engineered to pro 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, duce pesticidal proteins isolated from strains of Bt. These 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, genetically engineered crops are now widely used in agricul 263,264, 265, 266, 267, 268, and 269 as well as amino acid ture and have provided the farmer with an environmentally Substitutions, amino acid deletions, amino acid insertions, friendly alternative to traditional insect-control methods. and fragments thereof, and combinations thereof. While they have proven to be very successful commercially, 0010 Also provided are nucleic acid sequences set forth in these genetically engineered, insect-resistant crop plants pro SEQID NO: 1, 152, 153, 154, 155, 156, 157, 158, 159, 160, vide resistance to only a narrow range of the economically 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, important insect pests. In some cases, insects can develop 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, resistance to different insecticidal compounds, which raises 185, 186, 197, 188, 189, 190, 191, 192, 193, 194, 195, 196, the need to identify alternative biological control agents for 197, 198, 199, 200, 201, 202, 203, 205, 207, 220, 221, 222, pest control. 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 0007 Accordingly, there remains a need for new pesti 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 270, 271, cidal proteins with different ranges of insecticidal activity 272,273, 274, 275, 276, 277,278, 279, 280, 281, 282,283, US 2014/0007292 A1 Jan. 2, 2014
284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 0019 5. The recombinant nucleic acid molecule of 296, and 297 as well as variants and fragments thereof encod embodiment 1, 2, 3 or 4, wherein the PIP-1 polypeptide has ing PIP-1 polypeptides. insecticidal activity against an insect pest in the order Lepi 0011. In some embodiments exemplary nucleic acid mol doptera. ecules comprise a sequence set forth in SEQID NO: 152, 153, 0020. 6. The recombinant nucleic acid molecule of 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, embodiment 1, 2, 3, 4 or 5, wherein the nucleic acid molecule 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, is from a Pseudomonas chlororaphis strain. 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 0021 7. The recombinant nucleic acid molecule of 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, embodiment 1, 2, 3, 4, 5 or 6, wherein the Pseudomonas 202, 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, chlororaphis strain comprises a 16S ribosomal DNA having 228, 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, at least about 96.9% identity to SEQID NO: 216. 240,241,242, 243, and 244 as well as variants and fragments 0022 8. The recombinant nucleic acid molecule of thereof encoding PIP-1 polypeptides, as well as variants and embodiment 1, 2, 3, 4, 5, 6 or 7 wherein the Pseudomonas fragments thereof that encode PIP-1 polypeptides. Nucleic chlororaphis strain is SS44C4 deposited under accession it acid sequences that are complementary to a nucleic acid NRRLB-50613. sequence of the embodiments or that hybridize to a sequence 0023 9. The recombinant nucleic acid molecule of of the embodiments are also encompassed. embodiment 1,2,3,4,5,6,7, or 8 wherein the PIP-1 polypep 0012 Methods are provided for producing the polypep tide comprises an amino acid motif as represented by posi tides and for using those polypeptides for controlling, inhib tions 171-183 of SEQID NO: 213. iting growth or killing a Lepidopteran, Coleopteran, nema 0024 10. The recombinant nucleic acid molecule of tode, fungi, Hemipteran and/or Dipteran pests. The embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the PIP-1 transgenic plants of the embodiments express one or more of polypeptide further comprises any one or more amino acid the pesticidal sequences disclosed herein. In various embodi motifs as represented by positions 149-159 of SEQ ID NO: ments, the transgenic plant further comprises one or more 213 and positions 64-79 of SEQID NO: 213. additional genes for insect resistance, for example, one or 0025 11. The recombinant nucleic acid molecule of more additional genes for controlling coleopteran, lepi embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein the PIP-1 dopteran, hemipteran or nematodepests. It will be understood polypeptide comprises a polypeptide having at least 80% by one of skill in the art that the transgenic plant may com identity to the amino acid sequence of SEQID NO: 2. prise any gene imparting an agronomic trait of interest. 0026 12. The recombinant nucleic acid molecule of 0013 Methods for detecting the nucleic acids and embodiment 1,2,3,4,5,6,7,8,9, 10 or 11, wherein the PIP-1 polypeptides of the embodiments in a sample are also polypeptide further comprises any one or more amino acid included. A kit for detecting the presence of a PIP-1 polypep motifs as represented by positions 64-79 of SEQID NO: 213, tide or detecting the presence of a nucleotide sequence encod positions 149-159 of SEQID NO:213, and positions 171-183 ing a PIP-1 polypeptide in a sample is provided. A kit for of SEQ ID NO: 213. detecting the presence of nucleotide sequence encoding a 0027 13. The recombinant nucleic acid molecule of PIP-1 polypeptide may comprise a nucleic acid probe that embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the comprises at least 20 contiguous nucleotides of the nucleotide PIP-1 polypeptide comprises an amino acid sequence of SEQ sequence encoding the PIP-1 polypeptide or a complement ID NO: 211, wherein thereof. A kit for detecting the presence of a PIP-1 polypep Xaa at position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly or tide may comprise an antibody that specifically binds to the Asn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position 20 PIP-1 polypeptide. The kit is provided along with all reagents is Leu or Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at and control samples necessary for carrying out a method for position 22 is Ser, Lys or Arg; Xaaat position 24 is Glnor Ala; detecting the intended agent, as well as instructions for use. Xaa at position 25 is Gly or Ala Xaa at position 26 is Ser or 0014. The compositions and methods of the embodiments Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 30 are useful for the production of organisms with enhanced pest is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position resistance or tolerance. These organisms and compositions 36 is Ala, Seror Val: Xaaat position 38 is Asn, Argor Ser; Xaa comprising the organisms are desirable for agricultural pur at position 42 is Phe or Tyr, Xaa at position 46 is Arg, Lys or poses. The compositions of the embodiments are also useful His;Xaaat position 48 is Gly or Asp;Xaaat position 49 is Phe for generating altered or improved proteins that have pesti or Tyr; Xaa at position 53 is Ser or Gly; Xaa at position 58 is cidal activity or for detecting the presence of PIP-1 polypep Tyror Phe: Xaa at position 60 is Ala or Ser; Xaa at position 63 tides or nucleic acids in products or organisms. is Gln or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position The following embodiments are encompassed by the present 97 is Met or Val: Xaa at position 98 is Asp or Glu, Xaa at disclosure. position 105 is Glin or ASn; Xaa at position 107 is Thr or Ile: 0015 1. A recombinant nucleic acid molecule encoding a Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg PIP-1 polypeptide. or Leu, Xaa at position 120 is Lys, Argor Glin; Xaa at position 0016 2. The recombinant nucleic acid molecule of 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; Xaa at embodiment 1, wherein the PIP-1 polypeptide is orally active. position 125 is ASnor Ser; Xaa at position 127 is Ser, Asn. Thr 0017 3. The recombinant nucleic acid molecule of or Lys; Xaa at position 134 is Gly or Ala; Xaa at position 135 embodiment 1 or 2, wherein the PIP-1 polypeptide has insec is Ser, Asn or Lys; Xaa at position 137 is Asp or Gly; Xaa at ticidal activity against an insect pest in the order Hemiptera. position 141 is Val or Ile: Xaa at position 142 is Gly or Asp; 0.018 4. The recombinant nucleic acid molecule of Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, embodiment 1, 2 or 3, wherein the PIP-1 polypeptide has Thr or Val: Xaa at position 150 is Seror Thr; Xaa at position insecticidal activity against an insect pest in the family Pen 151 is Asn, Arg or Ser; Xaa at position 160 is Thr or Ser; Xaa tatomidae. at position 162 is Ser or Thr; Xaa at position 163 is Asn., Asp US 2014/0007292 A1 Jan. 2, 2014
or Glu; Xaa at position 164 is Seror Thr; Xaa at position 166 Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at is Gln or Glu, Xaa at position 167 is Leu or Met; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaa position 168 is Thr, Lys or Ala; Xaa at position 174 is Ile, Val at position 176 is Tyr, Met, Phe, Leu or Cys; Xaa at position or Met; Xaa at position 175 is Val or Ile: Xaa at position 180 177 is Gln, Ile, Met or Pro; Xaa at position 178 is Val, Cys, is Met or Leu; Xaa at position 191 is Arg or Lys: Xaa at Thr, Pro, Ala, Met, Gln, Phe, Ile, Ser or Lys; Xaa at position position 194 is Gly or Ala; Xaa at position 200 is Asn or Ser; 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, Ala or Gln: Xaa Xaa at position 203 is ASnor Glin; Xaa at position 204 is Thr at position 180 is Met, Leu, Pro, Trp, Asn., Tyr, Gly, Gln, Ala, or Ala; Xaa at position 206 is Gly or Asp; Xaa at position 209 Val, Phe, Ile, Cys or Ser; Xaa at position 181 is Val, Ala, Leu, is Leu or Val; Xaa at position 220 is ASn or Arg; Xaa at Trp, Cys, Thr, Ile or Lys: Xaa at position 182 is Tyr, Phe, Met position 221 is Ser or Lys: Xaa at position 222 is Thr or Arg; or His; Xaa at position 183 is Ala, Met, Val, Thr, Asp, Gly, Xaa at position 226 is Asp, Pro or Glu; Xaa at position 228 is Cys, Ile, Phe, Ser, Gln or Leu; Xaa at position 191 is Arg or Seror Gly: Xaa at position 229 is Lys or ASn; Xaa at position Lys: Xaa at position 194 is Gly or Ala; Xaa at position 195 is 231 is Ile or Val: Xaa at position 232 is Ala, Thr or Glu; and Asin or Tyr; Xaa at position 200 is Asn or Ser; Xaa at position Xaa at position 251 is Gly, Seror Glu; Xaa at position 254 is 203 is Asn or Glin; Xaa at position 204 is Thr or Ala; Xaa at Seror Asn; Xaa at position 258 is Seror Arg; Xaa at position position 206 is Gly or Asp; Xaa at position 209 is Leu or Val; 265 is ASnor Asp; and Xaa at position 266 is Asp or ASn; and Xaa at position 213 is Tyr or Phe: Xaa at position 220 is Asn wherein, 1 to 28 amino acids are optionally deleted from the or Arg; Xaa at position 221 is Seror Lys; Xaa at position 222 N-terminus of the polypeptide. is Thr or Arg; Xaa at position 226 is Asp, Pro or Glu; Xaa at 0028. 14. The recombinant nucleic acid molecule of position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, PIP-1 polypeptide comprises an amino acid sequence of SEQ Thror Glu:Xaaat position 240 is Gln, Arg, Ala, Val, Glu, Met, ID NO: 212, wherein Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Xaa at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys: Xaa at position Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 20 is Leu or Val: Xaa at position 21 is Lys, Ser or ASn; Xaa at is Val, Leu, Ala, Thr, Gly, Cys, Ile, Seror Met; Xaa at position position 22 is Ser, Lys or Arg; Xaa at position 24 is Glin or Ala; 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at Xaa at position 25 is Gly or Ala; Xaa at position 26 is Seror position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, Asn; Xaa at position 27 is Leu, Thror Ala; Xaa at position 28 Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or ASn; Xaa at is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, Ser, His, Cys or Gln; Xaa at position 30 is Ala or Ile: Xaa at ASn, Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys: Xaa at position 35 is Phe or Leu; Xaa at position 36 is Ala, Seror Val; position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, Lys, Xaa at position 38 is Asn, Argor Ser; Xaaat position 42 is Phe Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thror Cys; Xaa at or Tyr; Xaa at position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at Ile, Asp, Gly or Ala; Xaa at position 249 is Asn. Lys, Val, Gly, position 46 is Arg, Lys or His; Xaa at position 48 is Gly or Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, Thr, Pro, Leu, Asp; Xaa at position 49 is Phe, Tyr or Leu; Xaa at position 53 Ala, His or Glin; Xaa at position 251 is Gly, Seror Glu; Xaa at is Seror Gly: Xaa at position 58 is Tyr or Phe: Xaa at position position 254 is Ser or Asn; Xaa at position 258 is Ser or Arg; 60 is Ala or Ser; Xaa at position 63 is Glin or Lys; Xaa at position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or at position 77 is Phe or Tyr; Xaa at position 89 is Pro, Leu, His; Xaa at position 265 is ASn or Asp; and Xaa at position Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys or Lys; Xaa at 266 is Asp or ASn; and wherein, 1 to 28 amino acids are position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, optionally deleted from the N-terminus of the polypeptide. Ala or Thr; Xaa at position 97 is Met or Val: Xaa at position 0029. 15. The recombinant nucleic acid molecule of 98 is Asp or Glu; Xaa at position 105 is Glin or Asn; Xaa at embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the position 107 is Thr or Ile: Xaa at position 108 is Glin or Thr: PIP-1 polypeptide comprises an amino acid sequence of Xaa at position 110 is Arg or Leu, Xaa at position 120 is LyS, (SEQ ID NO: 213), wherein Argor Glin; Xaa at position 121 is Thr or Ser; Xaa at position Xaa at position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, 123 is Thr or Glu; Xaa at position 125 is Asn or Ser; Xaa at Thr, Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp position 127 is Ser, Asn. Thror Lys; Xaaat position 134 is Gly or Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at or Ala; Xaa at position 135 is Ser, ASnor LyS, Xaa at position position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, Val, 137 is Asp or Gly; Xaa at position 141 is Val or Ile: Xaa at Ile or Met; Xaaat position 21 is Lys, Ser, Asn, Arg, Thror Gln; position 142 is Gly or Asp; Xaa at position 144 is Asp or Glu; Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at position 24 Xaaat position 147 is Ile, ThrorVal: Xaaat position 150 is Ser is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly or Ala; Xaa or Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position at position 26 is Ser, Asn. Thr or Glin; Xaa at position 27 is 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at position 28 is Arg, position 163 is Asn., Asp or Glu; Xaa at position 164 is Seror Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, His, Cys Thr; Xaa at position 166 is Glin or Glu; Xaa at position 167 is or Gln; Xaa at position 30 is Ala, Ile, Leu, Val or Met; Xaa at Leu or Met; Xaa at position 168 is Thr, Lys or Ala; Xaa at position 35 is Phe, Leu, Ile, Val or Met; Xaa at position 36 is position 171 is Gly, Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Ala, Ser. Thr, Val, Ile or Leu; Xaa at position 38 is Asn, Arg, Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Ser, Gln, Lys or Thr; Xaa at position 42 is Phe, Tyr, Trp, Leu, Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaa at position 173 is Ile, Val or Met; Xaa at position 43 is Pro, Met, Gly, Gln, Ser, Phe, Gly, His, Leu, Ala, Arg, Asn, Cys, Lys, Trp, Thr, Ser, Tyr Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; or Met; Xaaat position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Xaaat position 46 is Arg, Lys or His;Xaa at position 48 is Gly, US 2014/0007292 A1 Jan. 2, 2014
Asp, Ala or Glu, Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, Ile, Gln, Tyror ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Val or Met; Xaa at position 53 is Ser, Gly, Ala or Thr; Xaa at Arg, Val, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, position 58 is Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Phe, Thr or Lys; Xaa at position 247 is Asn. Leu, Asp, Tyr, Thr, Xaa at position 63 is Gln, Lys, ASn or Arg; Xaa at Ala, Phe, His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa Trp, Thror Cys: Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, at position 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at Ser, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala, Xaa at position position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. 249 is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, Tyr, Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 is Gly, Ser, Thr, Ala, Asp or Glu; Xaa at position 254 is ASn, Ile, Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, Ser, Asn. Thr or Glin; Xaa at position 258 is Ser, Arg, Thr or Val, Leu or Ile: Xaa at position 98 is Asp or Glu, Xaa at Lys: Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, position 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, Ile or His: Xaa at position 265 is Asn., Asp, Gln or Glu; and Leu or Val: Xaa at position 108 is Gln, Thr, Seror Asn; Xaa at Xaa at position 266 is Asp, Asn., Gln or Glu; and wherein, 1 to position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at position 28 amino acids are optionally deleted from the N-terminus of 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thror Ser; the polypeptide. Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at position 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is Ser, ASn, 0030) 16. The recombinant nucleic acid molecule of Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is Gly or Ala; embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or Lys; Xaa at wherein the recombinant nucleic acid molecule comprises a position 137 is Asp, Gly, Glu or Ala; Xaa at position 141 is polynucleotide of SEQID NO: 1, afragment or a complement Val, Ile or Leu; Xaa at position 142 is Gly, Asp, Ala or Glu; thereof. Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, 0031. 17. The recombinant nucleic acid molecule of Thr, Val, Leu, Met or Ser; Xaa at position 150 is Ser or Thr: embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, Xaa at position 151 is ASn, Arg, Ser, Gln, Lys or Thr, Xaa at wherein the PIP-1 polypeptide comprises an amino acid position 160 is Thr or Ser; Xaa at position 162 is Seror Thr: sequence of SEQID NO: 2 or a fragment thereof. Xaa at position 163 is ASn, Asp, Glu or Glin; Xaa at position 0032. 18. The recombinant nucleic acid molecule of 164 is Seror Thr; Xaa at position 166 is Gln, Glu, Asp or ASn; embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, Xaa at position 167 is Leu, Met, Ile, Val: Xaa at position 168 wherein the recombinant nucleic acid molecule hybridizes is Thr, Lys, Ala, Ser, Arg or Gly: Xaa at position 171 is Gly, understringent conditions to a polynucleotide of SEQID NO: Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Xaa at position 172 1 is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, 0033. 19. The recombinant nucleic acid molecule of Cys, Val, Ala or Met; Xaaat position 173 is Phe, Gly. His, Leu, embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr or Met; Xaa at wherein the recombinant nucleic acid molecule comprises a position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Met, Cys, polynucleotide of SEQID NO: 1. Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at position 175 0034 20. A plant or progeny thereof, comprising the is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaaat position 176 recombinant nucleic acid molecule of embodiment 1, 2, 3, 4, is Tyr, Met, Phe, Leu or Cys: Xaa at position 177 is Gln, Ile, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. Metor Pro; Xaa at position 178 is Val, Cys, Thr, Pro, Ala, Met, 0035 21. A plant or progeny thereof stably transformed Gln, Phe, Ile, Seror Lys; Xaa at position 179 is Val, Phe, Thr, with the recombinant nucleic acid molecule of embodiment 1, Ile, Cys, Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. Leu: Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or 0036). 22. The plant of embodiment 20 or 21, wherein the Ser; Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or plant is a monocotyledon. Lys; Xaa at position 182 is Tyr, Phe, Met or His: Xaa at 0037. 23. The plant of embodiment 20 or 21, wherein the position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, plant is a dicotyledon. Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at position 0038 24. The plant of embodiment 20 or 21, wherein the 194 is Gly or Ala; Xaa at position 195 is Asn., Tyr, Gln or Trp; plant is selected from barley, corn, oat, rice, rye, Sorghum, turf Xaa at position 200 is Asn. Ser. Thr or Gln; Xaa at position grass, Sugarcane, wheat, alfalfa, banana, broccoli, bean, cab 203 is ASnor Glin; Xaa at position 204 is Thr, Ala, Seror Gly: bage, canola, carrot, cassava, cauliflower, celery, citrus, cot Xaa at position 206 is Gly, Asp, Ala or Glu; Xaa at position ton, a cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, 209 is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: pea, peanut, pepper, potato, poplar, pine, Sunflower, saf Xaa at position 220 is ASn, Arg, Gln or Lys, Xaa at position flower, soybean, Strawberry, Sugar beet, Sweet potato, 221 is Ser, Lys, Thror Arg; Xaaat position 222 is Thr, Arg, Ser tobacco, tomato ornamental, shrub, nut, chickpea, pigeon or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at pea, millets, hops, and pasture grass plant cells. position 228 is Seror Gly; Xaaat position 229 is Lys, Asn, Arg 0039. 25. The plant of embodiment 20, 21, 22, 23 or 24, or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa at further comprising one or more additional transgenic traits. position 232 is Ala, Thr, Ser, Gly, Asp or Glu, Xaa at position 0040 26. The plant of embodiment 25, wherein the one or 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, Asn. Thr, more additional transgenic trait is selected from insect resis Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 is Arg, Lys, tance, herbicide resistance, fungal resistance, virus resistance Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, Pro, Gly, Leu, or stress tolerance, disease resistance, male sterility, stalk Phe, Thr, Ala or Cys;Xaa at position 242 is ASn, Ala, Arg, LyS, strength, increased yield, modified starches, improved oil His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, profile, balanced amino acids, high lysine or methionine, Leu or Val: Xaa at position 243 is Val, Leu, Ala, Thr, Gly, Cys, increased digestibility, improved fiber quality, and drought Ile, Seror Met; Xaa at position 244 is Leu, Val, Phe, Ile, Met, tolerance. Gln, Cys, Trp or Ala; Xaa at position 245 is Met, Ala, Arg, 0041. 27. An expression cassette, comprising the recom Asp, Glu, Leu, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, binant nucleic acid molecule of embodiment 1,2,3,4,5,6,7, US 2014/0007292 A1 Jan. 2, 2014
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, wherein the increased digestibility, improved fiber quality, flowering, ear nucleic acid is operably linked to one or more regulatory and seed development, enhancement of nitrogen utilization sequences directing expression of the PIP-1 polypeptide. efficiency, altered nitrogen responsiveness, drought resis 0042. 28. A plant, comprising the expression cassette of tance or tolerance, cold resistance or tolerance, salt resistance embodiment 27. or tolerance, and increased yield under stress. 0043. 29. A plant cell, comprising the expression cassette 0064. 44. The plant of embodiment 40, 42 or 43, wherein of embodiment 27. the plant is a monocotyledon. 0044, 30. A recombinant microbial cell, comprising the 0065 45. The plant of embodiment 40, 42 or 43, wherein expression cassette of embodiment 27. the plant is a dicotyledon. 0045 31. Seed or grain of the plant of embodiment 20, 21, 0.066 46. A recombinant PIP-1 polypeptide. 22, 23, 24, 25 or 26 or a progeny thereof, wherein the seed or 0067 47. The recombinant PIP-1 polypeptide of embodi grain comprises the recombinant nucleic acid molecule. ment 46, wherein the PIP-1 polypeptide is orally active. 0046. 32. The seed of embodiment 31, wherein one or more seed treatment has been applied to the seed. 0068 48. The recombinant PIP-1 polypeptide of embodi 0047 33. The seed of embodiment 32, wherein the one or ment 46 or 47, wherein the PIP-1 polypeptide has insecticidal more seed treatment is selected from a herbicide, an insecti activity against an insect pest of the order Hemiptera. cide, a fungicide, a germination inhibitor, a germination 0069 49. The recombinant PIP-1 polypeptide of embodi enhancer, a plant growth regulator, a bactericide, and a nema ment 46, 47 or 48, wherein the PIP-1 polypeptide has insec tocide. ticidal activity against an insect pest of the Pentatomidae 0048, 34. Abiological sample derived from a tissue or seed family. of the plant of embodiment 20, 21, 22, 23, 24, 25 or 26. (0070) 50. The recombinant PIP-1 polypeptide of embodi 0049. 35. A recombinant microorganism, comprising a ment 49, wherein the PIP-1 polypeptide has insecticidal recombinant nucleic acid molecule of embodiment 1, 2, 3, 4, activity against an insect selected from Nezara viridula, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. Halyomorpha haly's, Piezodorus guildini, Euschistus servus, 0050 36. The microorganism of embodiment 35, wherein Acrosternum hilare, Euschistus heros, Euschistus tristigmus, the microorganism is selected from a bacteria, baculovirus, Acrosternum hilare, Dichelops furcatus, Dichelops melacan algae, and fungi. thus, Bagrada hilaris, Megacopta Cribraria, Scaptocoris cas 0051 37. The microorganism of embodiment 36, wherein tanea, Helicoverpa zea Boddie, Pseudoplusia includens the bacteria is selected from a Bacillus, a Pseudomonas, a Walker, and Anticarsia gemmatalis. Clavibacter, a Rhizobium and E. coli. (0071) 51. The recombinant PIP-1 polypeptide of embodi 0052 38. A method for producing a polypeptide with ment 46, 47, 48, 49 or 50, wherein the PIP-1 polypeptide has insecticidal activity, comprising culturing the microorganism insecticidal activity against an insect pest of the order Lepi of embodiment 35, 36 or 37 under conditions in which the doptera. nucleic acid molecule encoding the polypeptide is expressed. (0072 52. The recombinant PIP-1 polypeptide of embodi 0053. 39. A method for expressing in a plant a PIP-1 ment 46, 47, 48, 49, 50 or 51, wherein the PIP-1 polypeptide polypeptide, comprising the steps of: is produced by a Pseudomonas chlororaphis strain. 0054 (a) inserting into the plant cell a nucleic acid (0073 53. The recombinant PIP-1 polypeptide of embodi sequence comprising in the 5' to 3' direction an operably ment 52, wherein the PIP-1 polypeptide is produced by a linked recombinant, double-stranded DNA molecule, Pseudomonas chlororaphis strain having a 16S ribosomal wherein the recombinant double-stranded DNA mol DNA having at least about 96.9% identity to SEQ ID NO: ecule comprises 216. 0055 (i) a promoter that functions in the plant cell; (0074 54. The recombinant PIP-1 polypeptide of embodi 0056 (ii) a nucleic acid molecule encoding a PIP-1 ment 52, wherein the Pseudomonas chlororaphis strain is polypeptide as set forth in embodiment 1, 2, 3, 4, 5, 6, SS44C4 deposited under accession # NRRLB-50613. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; and (0075 55. The recombinant PIP-1 polypeptide of embodi 0057 (iii) a 3' non-translated polynucleotide that ment 46, 47, 48, 49, 50, 51, 52, 53 or 54, wherein the PIP-1 functions in the cells of the plant to cause termination polypeptide comprises an amino acid motif as represented by of transcription; positions 171-183 of SEQID NO: 213. 0.058 (b) obtaining a transformed plant cell comprising (0076 56. The recombinant PIP-1 polypeptide of embodi the nucleic acid sequence of step (a); and ment 55, further comprising any one or more amino acid 0059 (c) generating from the transformed plant cell a motifs as represented by positions 149-159 of SEQ ID NO: plant capable of expressing the PIP-1 polypeptide. 213, and positions 64-79 of SEQID NO: 213. 0060 40. A plant produced by the method of embodiment (0077. 57. The recombinant PIP-1 polypeptide of embodi 39. ment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56, wherein the 0061 41. Seed or grain produced by the plant of embodi PIP-1 polypeptide comprises a polypeptide having at least ment 40. 80% identity to the amino acid sequence of SEQID NO: 2. 0062 42. The plant of embodiment 40, further comprising (0078 58. The recombinant PIP-1 polypeptide of embodi one or more additional transgenic traits. ment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57 wherein 0063 43. The plant of embodiment 42, wherein the one or the PIP-1 polypeptide comprises an amino acid motif as rep more additional transgenic trait is selected from insect resis resented by positions 171-183 of SEQ ID NO: 213 and tance, herbicide resistance, fungal resistance, viral resistance, wherein the PIP-1 polypeptide has at least 80% identity to the stress tolerance, disease resistance, male Sterility, stalk amino acid sequence of SEQID NO: 2. strength, increased yield, modified starches, improved oil (0079 59. The recombinant PIP-1 polypeptide of embodi profile, balanced amino acids, high lysine or methionine, ment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, US 2014/0007292 A1 Jan. 2, 2014 wherein the PIP-1 polypeptide comprises an amino acid Asp; Xaa at position 49 is Phe, Tyr or Leu; Xaa at position 53 sequence of (SEQID NO: 211), wherein is Seror Gly: Xaa at position 58 is Tyr or Phe: Xaa at position Xaa at position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly or 60 is Ala or Ser; Xaa at position 63 is Glin or Lys; Xaa at Asn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position 20 position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa is Leu or Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at at position 77 is Phe or Tyr; Xaa at position 89 is Pro, Leu, position 22 is Ser, Lys or Arg; Xaa at position 24 is Glin or Ala; Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys or Lys; Xaa at Xaa at position 25 is Gly or Ala Xaa at position 26 is Ser or position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 30 Ala or Thr; Xaa at position 97 is Met or Val: Xaa at position is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position 98 is Asp or Glu; Xaa at position 105 is Glin or Asn; Xaa at 36 is Ala, Seror Val: Xaa at position 38 is Asn, Argor Ser; Xaa position 107 is Thr or Ile: Xaa at position 108 is Glin or Thr: at position 42 is Phe or Tyr, Xaa at position 46 is Arg, Lys or Xaa at position 110 is Arg or Leu; Xaa at position 120 is Lys, His;Xaaat position 48 is Gly or Asp;Xaaat position 49 is Phe Argor Glin; Xaa at position 121 is Thr or Ser; Xaa at position or Tyr; Xaa at position 53 is Ser or Gly; Xaa at position 58 is 123 is Thr or Glu; Xaa at position 125 is Asn or Ser; Xaa at Tyror Phe: Xaa at position 60 is Ala or Ser; Xaa at position 63 position 127 is Ser, Asn. Thror Lys;Xaaat position 134 is Gly is Gln or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position or Ala; Xaa at position 135 is Ser, ASnor Lys; Xaa at position 97 is Met or Val: Xaa at position 98 is Asp or Glu, Xaa at 137 is Asp or Gly: Xaa at position 141 is Val or Ile: Xaa at position 105 is Glin or Asn; Xaa at position 107 is Thr or Ile: position 142 is Gly or Asp; Xaa at position 144 is Asp or Glu; Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg Xaaat position 147 is Ile, ThrorVal: Xaaat position 150 is Ser or Leu, Xaa at position 120 is Lys, Arg or Glin; Xaa at position or Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; Xaa at 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at position 125 is ASnor Ser; Xaaat position 127 is Ser, Asn. Thr position 163 is Asn., Asp or Glu; Xaa at position 164 is Seror or Lys; Xaa at position 134 is Gly or Ala; Xaa at position 135 Thr; Xaa at position 166 is Glin or Glu; Xaa at position 167 is is Ser, Asn or Lys; Xaa at position 137 is Asp or Gly; Xaa at Leu or Met; Xaa at position 168 is Thr, Lys or Ala; Xaa at position 141 is Val or Ile: Xaa at position 142 is Gly or Asp; position 171 is Gly, Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Thr or Val: Xaa at position 150 is Seror Thr; Xaa at position Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaaat position 173 is 151 is Asn, Arg or Ser; Xaa at position 160 is Thr or Ser; Xaa Phe, Gly, His, Leu, Ala, Arg, Asn, Cys, Lys, Trp, Thr, Ser, Tyr at position 162 is Ser or Thr; Xaa at position 163 is Asn, Asp or Met; Xaaat position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, or Glu; Xaa at position 164 is Seror Thr; Xaa at position 166 Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at is Gln or Glu, Xaa at position 167 is Leu or Met; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaa position 168 is Thr, Lys or Ala; Xaa at position 174 is Ile, Val at position 176 is Tyr, Met, Phe, Leu or Cys; Xaa at position or Met; Xaa at position 175 is Val or Ile: Xaa at position 180 177 is Gln, Ile, Met or Pro; Xaa at position 178 is Val, Cys, is Met or Leu; Xaa at position 191 is Arg or Lys: Xaa at Thr, Pro, Ala, Met, Gln, Phe, Ile, Ser or Lys; Xaa at position position 194 is Gly or Ala; Xaa at position 200 is Asn or Ser; 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, Ala or Gln: Xaa Xaa at position 203 is ASnor Glin; Xaa at position 204 is Thr at position 180 is Met, Leu, Pro, Trp, Asn., Tyr, Gly, Gln, Ala, or Ala; Xaa at position 206 is Gly or Asp; Xaa at position 209 Val, Phe, Ile, Cys or Ser; Xaa at position 181 is Val, Ala, Leu, is Leu or Val; Xaa at position 220 is ASn or Arg; Xaa at Trp, Cys, Thr, Ile or Lys: Xaa at position 182 is Tyr, Phe, Met position 221 is Ser or Lys: Xaa at position 222 is Thr or Arg; or His; Xaa at position 183 is Ala, Met, Val, Thr, Asp, Gly, Xaa at position 226 is Asp, Pro or Glu; Xaa at position 228 is Cys, Ile, Phe, Ser, Gln or Leu; Xaa at position 191 is Arg or Seror Gly: Xaa at position 229 is Lys or ASn; Xaa at position Lys: Xaa at position 194 is Gly or Ala; Xaa at position 195 is 231 is Ile or Val: Xaa at position 232 is Ala, Thr or Glu; and Asin or Tyr; Xaa at position 200 is Asn or Ser; Xaa at position Xaa at position 251 is Gly, Seror Glu; Xaa at position 254 is 203 is Asn or Glin; Xaa at position 204 is Thr or Ala; Xaa at Seror Asn; Xaa at position 258 is Seror Arg; Xaa at position position 206 is Gly or Asp; Xaa at position 209 is Leu or Val; 265 is ASnor Asp; and Xaa at position 266 is Asp or ASn; and Xaa at position 213 is Tyr or Phe: Xaa at position 220 is Asn wherein, 1 to 28 amino acids are optionally deleted from the or Arg; Xaa at position 221 is Seror Lys; Xaa at position 222 N-terminus of the polypeptide. is Thr or Arg; Xaa at position 226 is Asp, Pro or Glu; Xaa at 0080) 60. The recombinant PIP-1 polypeptide of embodi position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; ment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, wherein the PIP-1 polypeptide comprises an amino acid Thror Glu:Xaaat position 240 is Gln, Arg, Ala, Val, Glu, Met, sequence of SEQID NO: 212, wherein Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at Xaa at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys: Xaa at position or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, 20 is Leu or Val: Xaa at position 21 is Lys, Ser or ASn; Xaa at Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 position 22 is Ser, Lys or Arg; Xaa at position 24 is Glin or Ala; is Val, Leu, Ala, Thr, Gly, Cys, Ile, Seror Met; Xaa at position Xaa at position 25 is Gly or Ala; Xaa at position 26 is Seror 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 28 position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or ASn; Xaa at His, Cys or Gln; Xaa at position 30 is Ala or Ile: Xaa at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, Ser, position 35 is Phe or Leu; Xaa at position 36 is Ala, Seror Val; ASn, Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys: Xaa at Xaa at position 38 is Asn, Argor Ser; Xaaat position 42 is Phe position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, Lys, or Tyr; Xaa at position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thror Cys; Xaa at Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, position 46 is Arg, Lys or His; Xaa at position 48 is Gly or Ile, Asp, Gly or Ala; Xaa at position 249 is Asn. Lys, Val, Gly, US 2014/0007292 A1 Jan. 2, 2014
Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, Thr, Pro, Leu, Gln, Phe, Ile, Seror Lys; Xaa at position 179 is Val, Phe, Thr, Ala, His or Glin; Xaa at position 251 is Gly, Seror Glu; Xaa at Ile, Cys, Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, position 254 is Ser or Asn; Xaa at position 258 is Ser or Arg; Leu: Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or Ser; Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or His; Xaa at position 265 is ASn or Asp; and Xaa at position Lys: Xaa at position 182 is Tyr, Phe, Met or His; Xaa at 266 is Asp or ASn; and wherein, 1 to 28 amino acids are position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, optionally deleted from the N-terminus of the polypeptide. Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at position I0081 61. The recombinant PIP-1 polypeptide of embodi 194 is Gly or Ala; Xaa at position 195 is Asn., Tyr, Gln or Trp; ment 58, wherein the PIP-1 polypeptide comprises an amino Xaa at position 200 is Asn. Ser. Thr or Gln; Xaa at position acid sequence of SEQID NO: 213, wherein 203 is ASnor Glin; Xaa at position 204 is Thr, Ala, Seror Gly: Xaa at position 206 is Gly, Asp, Ala or Glu; Xaa at position Xaa at position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, 209 is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: Thr, Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp Xaa at position 220 is ASn, Arg, Gln or Lys, Xaa at position or Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at 221 is Ser, Lys, Thror Arg; Xaaat position 222 is Thr, Arg, Ser position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, Val, or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at Ile or Met; Xaaat position 21 is Lys, Ser, Asn, Arg, Thror Gln; position 228 is Seror Gly; Xaaat position 229 is Lys, Asn, Arg Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at position 24 or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa at is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly or Ala; Xaa position 232 is Ala, Thr, Ser, Gly, Asp or Glu; Xaa at position at position 26 is Ser, Asn. Thr or Glin; Xaa at position 27 is 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, Asn. Thr, Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at position 28 is Arg, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 is Arg, Lys, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, His, Cys Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, Pro, Gly, Leu, or Gln; Xaa at position 30 is Ala, Ile, Leu, Val or Met; Xaa at Phe, Thr, Ala or Cys;Xaa at position 242 is ASn, Ala, Arg, Lys, position 35 is Phe, Leu, Ile, Val or Met; Xaa at position 36 is His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Ala, Ser. Thr, Val, Ile or Leu; Xaa at position 38 is Asn, Arg, Leu or Val: Xaa at position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ser, Gln, Lys or Thr; Xaa at position 42 is Phe, Tyr, Trp, Leu, Ile, Seror Met; Xaa at position 244 is Leu, Val, Phe, Ile, Met, Ile, Val or Met; Xaa at position 43 is Pro, Met, Gly, Gln, Ser, Gln, Cys, Trp or Ala; Xaa at position 245 is Met, Ala, Arg, Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Asp, Glu, Leu, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Xaaat position 46 is Arg, Lys or His; Xaa at position 48 is Gly, Ile, Gln, Tyror ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Asp, Ala or Glu, Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, Arg, Val, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Val or Met; Xaa at position 53 is Ser, Gly, Ala or Thr; Xaa at Phe, Thr or Lys; Xaa at position 247 is Asn. Leu, Asp, Tyr, position 58 is Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Ala, Phe, His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Thr, Xaa at position 63 is Gln, Lys, ASn or Arg; Xaa at Trp, Thror Cys: Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa Ser, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala, Xaa at position at position 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at 249 is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Tyr, Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, 251 is Gly, Ser, Thr, Ala, Asp or Glu; Xaa at position 254 is ASn, Ile, Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, Ser, Asn. Thr or Glin; Xaa at position 258 is Ser, Arg, Thr or Val, Leu or Ile: Xaa at position 98 is Asp or Glu, Xaa at Lys: Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, position 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, Ile or His: Xaa at position 265 is Asn., Asp, Gln or Glu; and Leu or Val: Xaa at position 108 is Gln, Thr, Seror Asn; Xaa at Xaa at position 266 is Asp, Asn., Gln or Glu; and wherein, 1 to position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at position 28 amino acids are optionally deleted from the N-terminus of 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thror Ser; the polypeptide. Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at position 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is Ser, ASn, I0082 62. The recombinant PIP-1 polypeptide of embodi Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is Gly or Ala; ment 55, comprising an amino acid sequence of SEQID NO: Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or Lys; Xaa at 2 or a fragment thereof. position 137 is Asp, Gly, Glu or Ala; Xaa at position 141 is I0083 63. The recombinant PIP-1 polypeptide of embodi Val, Ile or Leu; Xaa at position 142 is Gly, Asp, Ala or Glu; ment 55, consisting essentially of an amino acid sequence of Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, SEQID NO: 2. Thr, Val, Leu, Met or Ser; Xaa at position 150 is Ser or Thr: I0084) 64. The recombinant PIP-1 polypeptide of embodi Xaa at position 151 is ASn, Arg, Ser, Gln, Lys or Thr, Xaa at ment 55, wherein the PIP-1 polypeptide is encoded by the position 160 is Thr or Ser; Xaa at position 162 is Seror Thr: polynucleotide of SEQID NO: 1. Xaa at position 163 is ASn, Asp, Glu or Glin; Xaa at position I0085 65. The recombinant PIP-1 polypeptide of embodi 164 is Seror Thr; Xaa at position 166 is Gln, Glu, Asp or ASn; ment 46, comprising one or more properties selected from: Xaa at position 167 is Leu, Met, Ile, Val: Xaa at position 168 I0086) a) anamino acid motifas represented by positions is Thr, Lys, Ala, Ser, Arg or Gly: Xaa at position 171 is Gly, 64-79 of SEQID NO: 213; Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Xaa at position 172 0.087 b) an amino acid motif as represented by posi is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, tions 149-159 of SEQID NO: 213; Cys, Val, Ala or Met; Xaaat position 173 is Phe, Gly. His, Leu, 0088 c) anamino acid motifas represented by positions Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr or Met; Xaa at 171-183 of SEQID NO: 213; position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Met, Cys, 0089 e) insecticidal activity againstan insect pest of the Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at position 175 order Hemiptera: is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaaat position 176 0090 f) insecticidal activity againstan insect pest of the is Tyr, Met, Phe, Leu or Cys: Xaa at position 177 is Gln, Ile, order Lepidoptera: Metor Pro; Xaa at position 178 is Val, Cys, Thr, Pro, Ala, Met, 0.091 g) orally active; and US 2014/0007292 A1 Jan. 2, 2014
0092 h) a calculated molecular weight of between of embodiment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, about 15 kD to about 35 kD. 58, 59, 60, 61, 62, 63, 64 or 65. 0093 66. A plant capable of expressing the recombinant 0107 80. A method for controlling an insect pest popula PIP-1 polypeptide of embodiment 46, 47, 48, 49, 50, 51, 52, tion resistant to a pesticidal protein, comprising contacting 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65. the resistant insect pest population with a insecticidally-ef 0094 67. The plant of embodiment 66, wherein the plant is fective amount of the recombinant PIP-1 polypeptide of a monocotyledon. embodiment 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 0095 68. The plant of embodiment 66, wherein the plantis 59, 60, 61, 62, 63, 64 or 65. a dicotyledon. 0108) 81. The method of controlling an insect pest popu 0096 69. The plant of embodiment 66, wherein the plantis lation resistant to an pesticidal protein, comprising contacting selected from barley, corn, oat, rice, rye, Sorghum, turfgrass, the population with a insecticidally-effective amount of the Sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, recombinant PIP-1 polypeptide of embodiment 46, 47, 48. canola, carrot, cassava, cauliflower, celery, citrus, cotton, a 49, 50, 51, 52,53,54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64 or cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, 65, wherein the pesticidal protein is selected from Cry1Ac, peanut, pepper, potato, poplar, pine, Sunflower, safflower, Cry1Ab, Cry1A.105, Cry1Ac, Cry1F, Cry1 Fa2, Cry1F, Soybean, Strawberry, Sugar beet, Sweet potato, tobacco, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, tomato ornamental, shrub, nut, chickpea, pigeon pea, millets, Vip3A, Cry9c, eCry3.1Ab and CBI-Bt. hops, and pasture grasses. 0109) 82. A method for protecting a plant from an insect 0097. 70. The plant of embodiment 66, 67, 68, 69 or 70 pest, comprising expressing in the plant or cell thereof a wherein the plant expresses one or more additional transgenic recombinant PIP-1 polypeptide of embodiment 46, 47, 48. traits. 49, 50, 51, 52,53,54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64 or 0098 71. The plant of embodiment 70, wherein the one or 65. more additional transgenic trait is selected insect resistance, 0110 83. A biologically pure culture of a Pseudomonas herbicide resistance, fungal resistance, viral resistance, stress chlororaphis strain SS44C4 deposited under accession it tolerance, disease resistance, male Sterility, Stalk strength, increased yield, modified starches, improved oil profile, bal NRRLB-50613. anced amino acids, high lysine or methionine, increased 0111 84. A method of isolating a polypeptide having digestibility, improved fiber quality, flowering, ear and seed insecticidal activity from a Pseudomonas chlororaphis strain, development, enhancement of nitrogen utilization efficiency, comprising altered nitrogen responsiveness, drought resistance or toler 0112 a) obtaining a protein cell lysate from a bacterial ance, cold resistance or tolerance, and salt resistance or tol isolate; erance, and increased yield under stress. 0113 b) screening the protein cell lysate for insecticidal 0099 72. A composition, comprising an insecticidally activity; and effective amount of the recombinant PIP-1 polypeptide of 0114 c) isolating an insecticidal protein from the pro embodiment 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, tein cell lysate. 59, 60, 61, 62, 63, 64 or 65. 0115 85. A recombinant receptor to the polypeptide of 0100 73. The composition of embodiment 72, further SEQID NO: 2, SEQID NO:4, SEQID NO:332 or SEQ ID comprising an agriculturally suitable carrier. NO: 6. 0101 74. The composition of embodiment 73, wherein the 0116 86. The recombinant receptor of embodiment 85, carrier is selected from a powder, a dust, pellets, granules, wherein the receptor is isolated from a Hemiptera. spray, emulsion, colloid, and solution. 0117 87. A method of identifying a PIP-1 polypeptide in 0102 75. The composition of embodiment 72, 73 or 74, a biological sample, comprising contacting the biological further comprising one or more herbicides, insecticides or sample with the receptor of embodiment 85 or 86. fungicides. 0118 88. An isolated antibody orantigen-binding portion (0103) 76. The composition of embodiment 75, wherein the thereof, wherein the antibody binds specifically to the PIP-1 one or more insecticides are pesticidal proteins. polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, 0104 77. The composition of embodiment 76, wherein the 55, 56, 57,58, 59, 60, 61, 62, 63, 64 or 65. one or more pesticidal proteins are selected from a Cry1 protein, a Cry2 protein, a Cry3 protein, a Cry4 protein, a Cry5 0119 89. A method of detecting a PIP-1 polypeptide in a protein, a Cry6 protein, a Cry7 protein, a Cry8 protein, a Cry9 biological sample comprising, contacting the protein with the protein, a Cry 15 protein, Cry22 protein, a Cry23 protein, a antibody of embodiment 88. Cry32 protein, a Cry34 protein, a Cry35 protein, a Cry36 I0120 90. A method of isolating a PIP-1 polypeptide in a protein, a Cry37 protein, a Cry43 protein, a Cry46 protein, a biological sample comprising, contacting the protein with the Crys 1 protein, a Crys5 protein, a Cry binary toxin, a Cyt antibody of embodiment 88. protein, a VIP toxin, a SIP protein, an insecticidal lipase, an I0121 91. A method of controlling Lepidoptera and/or insecticidal chitinase, and a Snake Venom protein. Hemiptera insect infestation in a transgenic plant and provid 0105 78. A method for controlling an insect pest popula ing insect resistance management, comprising expressing in tion, comprising contacting the insect pest population with an the plant at least two different insecticidal proteins having insecticidally-effective amount of the recombinant PIP-1 different modes of action. polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, 0.122 92. The method of embodiment 91, wherein one of 55, 56, 57,58, 59, 60, 61, 62, 63, 64 or 65. the at least two insecticidal proteins comprises a PIP-1 0106 79. A method of inhibiting growth or killing an polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, insect pest, comprising contacting the insect pest with a insec 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 insecticidal to ticidally-effective amount of recombinant PIP-1 polypeptide insects in the order Lepidoptera and/or Hemiptera. US 2014/0007292 A1 Jan. 2, 2014
(0123 93. The method of embodiment 92, wherein one of 0.138 108. A method for expressing in a planta insecti the at least two insecticidal proteins comprises a Cry protein cidal protein, comprising insecticidal to insects in the order Lepidoptera and/or Hemi 0.139 (a) inserting into the plant cell a nucleic acid ptera. sequence comprising in the 5' to 3' direction an operably 0.124 94. A method of reducing likelihood of emergence linked recombinant, double-stranded DNA molecule, of Lepidoptera and/or Hemiptera insect resistance to trans wherein the recombinant, double-stranded DNA mol genic plants expressing in the plants insecticidal proteins to ecule comprises control the insect species, comprising expressing a PIP-1A 0140 (i) a promoter that functions in the plant cell; polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, 0141 (ii) a nucleic acid molecule encoding the pro 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 insecticidal to the tein of SEQID NO: 4; and insect species in combination with an insecticidal protein to 0.142 (iii) a 3' non-translated polynucleotide that the insect species having a different modes of action com functions in the cells of the plant to cause termination pared to the PIP-1A polypeptide. of transcription; 0.125 95. A means for effective Lepidoptera and/or Hemi 0.143 (b) obtaining a transformed plant cell comprising ptera insect resistance management, comprising co-express the nucleic acid sequence of step (a); and ing at high levels in transgenic plants two or more insecticidal 0144 (c) generating from the transformed plant cell a proteins toxic to Lepidoptera and/or Hemiptera insects but plant capable of expressing the protein of SEQID NO: 4. each exhibiting a different mode of effectuating its inhibiting 0145 109. A plant produced by the method of embodi growth or killing activity, wherein the two or more insecti ment 108. cidal proteins comprise a PIP-1 polypeptide of embodiment 0146 110. Seed or grain of the plant of embodiment 109. 46,47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62, 0147 111. The method of embodiment 108, wherein the 63, 64 or 65 and a Cry protein. plant further comprises one or more additional transgenic 0126 96. A method for obtaining regulatory approval for traits. planting or commercialization of plants expressing proteins 0148 112. A plant capable of expressing a recombinant insecticidal to insects in the order Lepidoptera and/or Hemi protein of SEQID NO: 4. ptera, comprising the step of referring to, Submitting or rely 0149 113. A method for controlling an insect pest popu ing on insect assay binding data showing that the PIP-1 lation, comprising contacting the insect pest population with polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, a insecticidally-effective amount of a recombinant protein of 55, 56, 57,58, 59,60, 61, 62, 63, 64 or 65 does not compete SEQID NO: 4. with binding sites for Cry proteins in Such insects. 0150. 114. A method of inhibiting growth or killing an 0127 97. A plant or progeny thereof, comprising the insect pest, comprising contacting the insect pest with a insec recombinant nucleic acid molecule of SEQID NO: 3. ticidally-effective amount of a recombinant protein of SEQ 0128 98. A plant or progeny thereof stably transformed ID NO: 4. with the recombinant nucleic acid molecule of SEQID NO:3. 0151. 115. A method for controlling an insect pest popu 0129 99. The plant or progeny thereof of embodiment 97 lation resistant to a pesticidal protein, comprising contacting or 98, wherein the plant is a monocotyledon. the insect pest population with a insecticidally-effective 0130 100. The plantor progeny thereof of embodiment 97 amount of a recombinant protein of SEQID NO: 4. or 98, wherein the plant is a dicotyledon. 0152 116. A method for protecting a plant from an insect 0131 101. The plant or progeny thereof of embodiment 97 pest, comprising expressing in the plant or cell thereof a or 98, wherein the plant is selected from barley, corn, oat, rice, recombinant insecticidal protein of SEQID NO: 4. rye, Sorghum, turfgrass, Sugarcane, wheat, alfalfa, banana, 0153. 117. A recombinant nucleic acid molecule encoding broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, a insecticidal protein comprising a polypeptide having at least celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, 80% identity to the amino acid sequence of SEQID NO: 6. grape, onion, lettuce, pea, peanut, pepper, potato, poplar, 0154) 118. The recombinant nucleic acid molecule of pine, Sunflower, safflower, soybean, Strawberry, Sugar beet, embodiment 117, wherein the insecticidal protein is orally Sweet potato, tobacco, tomato ornamental, shrub, nut, chick active. pea, pigeon pea, millets, hops, and pasture grasses. 0.155 119. The recombinant nucleic acid molecule of 0132) 102. The plant or progeny thereof of embodiment embodiment 117 or 118, wherein the insecticidal protein has 97, 98.99, 100 or 101, further comprising one or more addi insecticidal activity against an insect pest in the order Hemi tional transgenic traits. ptera. 0.133 103. An expression cassette, comprising the recom 0156 120. The recombinant nucleic acid molecule of binant nucleic acid molecule of SEQ ID NO: 3 or SEQ ID embodiment 119, wherein the insecticidal protein has insec NO:331, wherein the nucleic acid is operably linked to one or ticidal activity against an insect pest in the family Pentatomi more regulatory sequences directing expression of the dae. polypeptide of SEQID NO. 4 or SEQID NO: 332. O157 121. The recombinant nucleic acid molecule of 0134 104. A plant, comprising the expression cassette of embodiment 117, 118, 119 or 120, wherein the insecticidal embodiment 103. protein has insecticidal activity against an insect pest in the 0135 105. A plant cell, comprising the expression cassette order Lepidoptera. of embodiment 103. 0158 122. The recombinant nucleic acid molecule of 0136. 106. A seed or grain of the plant of embodiment 97. embodiment 117, 118, 119, 120 or 121, wherein the nucleic 98.99, 100, 101 or 102, wherein the seed or grain comprises acid molecule is produced by a Pseudomonas entomophila the recombinant nucleic acid molecule of SEQID NO: 3. strain. 0.137 107. The seed of embodiment 106, wherein one or 0159 123. The recombinant nucleic acid molecule of more seed treatment has been applied to the seed. embodiment 117, wherein the insecticidal protein comprises US 2014/0007292 A1 Jan. 2, 2014
an amino acid motif as represented by positions 171-183 of wherein the nucleic acid is operably linked to one or more SEQID NO: 6 or positions 171-183 of SEQID NO: 213. regulatory sequences directing expression of the insecticidal 0160 124. The recombinant nucleic acid molecule of protein. embodiment 123, wherein the insecticidal protein further 0176) 140. A plant, comprising the expression cassette of comprises any one or more amino acid motifs as represented embodiment 139. by positions 149-159 of SEQID NO: 213 and positions 69-79 0177 141. A plant cell, comprising the expression cassette of SEQ ID NO: 213. of embodiment 139. 0161 125. A recombinant insecticidal protein, comprising 0.178 142. Seed or grain of the plant of embodiment 133, a polypeptide having at least 80% identity to the amino acid 134, 135, 136, 137 or 138, wherein the seed or grain com sequence of SEQID NO: 6. prises the recombinant nucleic acid molecule. 0162. 126. The recombinant insecticidal protein of 0179 143. The seed of embodiment 142, wherein one or embodiment 125, wherein the insecticidal protein is orally more seed treatment has been applied to the seed. active. 0180 144. A method for expressing in a planta insecti 0163) 127. The recombinant insecticidal protein of cidal protein, comprising embodiment 125 or 126, wherein the insecticidal protein has 0181 (a) inserting into the plant cell a nucleic acid insecticidal activity against an insect pest in the order Hemi sequence comprising in the 5' to 3' direction an operably ptera. linked recombinant, double-stranded DNA molecule, 0164. 128. The recombinant insecticidal protein of wherein the recombinant, double-stranded DNA mol embodiment 127, wherein the insecticidal protein has insec ecule comprises ticidal activity against an insect pest in the family Pentatomi 0182 (i) a promoter that functions in the plant cell; dae. 0183 (ii) a nucleic acid molecule encoding the insec 0.165 129. The recombinant insecticidal protein of ticidal protein of embodiment 125, 126, 127, 128, embodiment 125, 126, 127 or 128, wherein the insecticidal 129, 130, 131 or 132; and protein has insecticidal activity against an insect pest in the 0.184 (iii) a 3' non-translated polynucleotide that order Lepidoptera. functions in the cells of the plant to cause termination 0166 130. The recombinant insecticidal protein of of transcription; embodiment 125, 126, 127, 128 or 129, wherein the nucleic acid molecule is produced by a Pseudomonas entomophila 0185 (b) obtaining a transformed plant cell comprising strain. the nucleic acid sequence of step (a); and 0167 131. The recombinant insecticidal protein of 0186 (c) generating from the transformed plant cell a embodiment 125, wherein the insecticidal protein comprises plant capable of expressing the insecticidal protein. an amino acid motif as represented by positions 171-183 of 0187 145. A plant produced by the method of embodi SEQID NO: 213. ment 144. 0168 132. The recombinant insecticidal protein of 0188 146. Seed or grain of the plant of embodiment 145. embodiment 131, wherein the insecticidal protein further (0189 147. The method of embodiment 144, wherein the comprises any one or more amino acid motifs as represented plant further comprises one or more additional transgenic by positions 149-159 of SEQ ID NO: 213, and positions traits. 69-79 of SEQID NO: 213. 0.190 148. A plant capable of expressing a recombinant 0169. 133. A plant or progeny thereof, comprising the insecticidal protein of embodiment 125, 126, 127, 128, 129, recombinant nucleic acid molecule of embodiment 117, 118, 130, 131 or 132. 119, 120, 121, 122, 123 or 124. 0191) 149. A method for controlling an insect pest popu 0170 134. A plant or progeny thereof stably transformed lation, comprising contacting the insect pest population with with the recombinant nucleic acid molecule of embodiment an insecticidally-effective amount of a recombinant insecti 117, 118, 119, 120, 121, 122, 123 or 124. cidal protein of embodiment 125, 126, 127, 128, 129, 130, 0171 135. The plant or progeny thereof of embodiment 131 or 132. 133 or 134, wherein the plant is a monocotyledon. 0.192 150. A method of inhibiting growth or killing an 0172 136. The plant or progeny thereof of embodiment insect pest, comprising contacting the insect pest with a insec 133 or 134, wherein the plant is a dicotyledon. ticidally-effective amount of a recombinant insecticidal pro 0173 137. The plant or progeny thereof of embodiment tein of embodiment 125, 126, 127, 128, 129, 130, 131 or 132. 133 or 134, wherein the plant is selected from barley, corn, 0193 151. A method for controlling an insect pest popu oat, rice, rye, Sorghum, turfgrass, Sugarcane, wheat, alfalfa, lation resistant to a pesticidal protein, comprising contacting banana, broccoli, bean, cabbage, canola, carrot, cassava, cau the insect pest population with a pesticidally-effective liflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax, amount of a recombinant protein of embodiment 125, 126, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, pop 127, 128, 129, 130, 131 or 132. lar, pine, Sunflower, safflower, soybean, Strawberry, Sugar 0194 152. A method for protecting a plant from an insect beet, Sweet potato, tobacco, tomato ornamental, shrub, nut, pest, comprising expressing in the plant or cell thereof a chickpea, pigeon pea, millets, hops, and pasture grass plant recombinant pesticidal protein of embodiment 125, 126, 127, cells. 128, 129, 130, 131 or 132. 0.174 138. The plant or progeny thereof of embodiment 0.195 153. A method of controlling Lepidoptera and/or 133, 134, 135, 136 or 137, further comprising one or more Hemiptera insect infestation in a transgenic plant and provid additional transgenic traits. ing insect resistance management, comprising expressing in 0175 139. An expression cassette, comprising the recom the plant at least two different insecticidal proteins having binant nucleic acid molecule encoding the insecticidal pro different modes of action, wherein one of the at least two tein of embodiment 117, 118, 119, 120, 121, 122, 123 or 124, insecticidal proteins comprises a insecticidal protein of US 2014/0007292 A1 Jan. 2, 2014
embodiment 125, 126, 127, 128, 129, 130, 131 or 132, insec insecticidal to insects in the order Lepidoptera and/or Hemi ticidal to insects in the order Lepidoptera and/or Hemiptera. ptera, comprising the step of referring to, Submitting or rely 0.196 154. The method of embodiment 153, wherein one ing on insect assay binding data showing that the insecticidal of the at least two insecticidal proteins comprises a Cry pro protein of SEQID NO: 4 does not compete with binding sites tein insecticidal to insects in the order Lepidoptera and/or for a Cry protein in the insects. Hemiptera. 0.197 155. A method of reducing likelihood of emergence BRIEF DESCRIPTION OF THE FIGURES of Lepidoptera and/or Hemiptera insect species resistance to (0205 FIG. 1 shows the alignment of PIP-1A (SEQID NO: transgenic plants expressing in the plants insecticidal proteins 2); the active insecticidal protein orthologs PSEEN3174 to control the insect species, comprising expressing a first (SEQID NO: 6) and PIP-1B (SEQID NO:4); and the inactive insecticidal protein of embodiment 125, 126, 127, 128, 129, homologs AECFG 592740 (SEQ ID NO: 12); Pput 1063 130, 131 or 132, insecticidal to the insect species in combi (SEQ ID NO: 8); and Pput 1064 (SEQ ID NO: 10). The nation with a second insecticidal protein insecticidal to the motifs amino acids 64-79 of SEQID NO: 2 (motif 1), amino insect species having a different mode of action compared to acids 149-159 of SEQ ID NO: 2 (motif 2), amino acids the first insecticidal protein. 171-183 of SEQID NO: 2 (motif3), and amino acids 240-249 0198 156. A means for effective Lepidoptera and/or of SEQID NO: 2 (motif 4) are indicated in bold and under Hemiptera insect resistance management, comprising co-ex line in the PIP-1A sequence. The predicted secondary struc pressing at high levels in transgenic plants two or more insec tures of selected beta-sheets are indicated with “B” above the ticidal proteins toxic to Lepidoptera and/or Hemiptera insects Sequence. but each exhibiting a different mode of effectuating its inhib 0206 FIG. 2 illustrates a generalized sewing and rescuing iting growth or killing activity, wherein one of the two or more PCR mutagenesis strategy using degenerate oligonucleotides insecticidal proteins comprise a insecticidal protein of to generate partially or fully saturated amino acid substitu embodiment 125, 126, 127, 128, 129, 130, 131 or 132 and one of the two or more insecticidal proteins comprise a Cry pro tions at positions in the PIP-1A protein. tein. 0207 FIG. 3A-3C shows the alignment of Pseudomonas 0199. 157. A method for obtaining regulatory approval for chlororaphis strain SS44C4 16S ribosomal DNA (SEQ ID planting or commercialization of plants expressing proteins NO: 216) and Pseudomonas entomophila L48 16S ribosomal insecticidal to insects in the order Lepidoptera and/or Hemi DNA (SEQID NO:217) having 96.8% identity. Differences ptera, comprising the step of referring to, Submitting or rely between the sequences are indicated in Bold and Underlined. ing on insect assay binding data showing that the insecticidal 0208 FIG. 4 shows the results of the Lygus insecticidal protein of embodiment 125, 126, 127, 128, 129, 130, 131 or activity screening of PIP-1A polypeptide variants having 132 does not compete with binding sites for a Cry protein in multiple amino acid substitutions at residues 240-249 of SEQ the insects. ID NO: 2 (motif 4). The insecticidal activity was scored from 0200 158. A method of controlling Lepidoptera and/or 0 to 8 with 8 being the most active. Hemiptera insect infestation in a transgenic plant and provid (0209 FIG. 5 shows the sequence alignment of PIP-1A ing insect resistance management, comprising expressing in (SEQ ID NO: 2), PIP-1B (SEQ ID NO: 4), PIP-1C (SEQ ID the plant at least two different insecticidal proteins having NO:332) and PSEEN3174 (SEQ ID NO: 6). different modes of action, wherein one of the at least two insecticidal proteins comprises the amino acid sequence of DETAILED DESCRIPTION SEQID NO: 4, insecticidal to insects in the order Lepidoptera 0210. It is to be understood that this invention is not lim and/or Hemiptera. ited to the particular methodology, protocols, cell lines, gen 0201 159. The method of embodiment 158, wherein one era, and reagents described, as such may vary. It is also to be of the at least two insecticidal proteins comprises a Cry pro understood that the terminology used herein is for the purpose tein insecticidal to insects in the order Lepidoptera and/or of describing particular embodiments only, and is not Hemiptera. intended to limit the scope of the present invention. 0202 160. A method of reducing likelihood of emergence 0211. As used herein the singular forms “a”, “and”, and of Lepidoptera and/or Hemiptera insect species resistance to “the include plural referents unless the context clearly dic transgenic plants expressing in the plants insecticidal proteins tates otherwise. Thus, for example, reference to “a cell to control the insect species, comprising expressing the insec includes a plurality of such cells and reference to “the pro ticidal protein of SEQ ID NO. 4 insecticidal to the insect tein’ includes reference to one or more proteins and equiva species in combination with an insecticidal protein insecti lents thereof knownto those skilled in the art, and so forth. All cidal to the insect species having a different modes of action technical and Scientific terms used herein have the same compared to the protein of SEQID NO: 4. meaning as commonly understood to one of ordinary skill in 0203) 161. A means for effective Lepidoptera and/or the art to which this invention belongs unless clearly indi Hemiptera insect resistance management, comprising co-ex cated otherwise. pressing at high levels in transgenic plants two or more insec 0212. The present disclosure is drawn to compositions and ticidal proteins toxic to Lepidoptera and/or Hemiptera insects methods for controlling pests. The methods involve trans but each exhibiting a different mode of effectuating its inhib forming organisms with a nucleic acid sequence encoding a iting growth or killing activity, wherein one of the two or more PIP-1 polypeptide. In particular, the nucleic acid sequences of insecticidal proteins comprise the insecticidal protein of SEQ the embodiments are useful for preparing plants and micro ID NO. 4 and one of the two or more insecticidal proteins organisms that possess pesticidal activity. Thus, transformed comprise a Cry protein. bacteria, plants, plant cells, plant tissues and seeds are pro 0204 162. A method for obtaining regulatory approval for vided. Compositions are pesticidal nucleic acids and proteins planting or commercialization of plants expressing proteins of bacterial species. The nucleic acid sequences find use in the US 2014/0007292 A1 Jan. 2, 2014
construction of expression vectors for Subsequent transfor (Accession if AY197341), Cry1Aa15 (Accession it mation into organisms of interest, as probes for the isolation DQ062690), Cry1Ab1 (Accession # M13898), Cry1Ab2 of other homologous (or partially homologous) genes, and for (Accession # M12661), Cry1Ab3 (Accession # M15271), the generation of altered PIP-1 polypeptides by methods Cry1Ab4 (Accession # D00117), Cry1Ab5 (Accession it known in the art, Such as site directed mutagenesis, domain X04698), Cry1Ab6 (Accession # M37263), Cry1Ab7 (Ac swapping or DNA shuffling. The PIP-1 polypeptides find use cession if X13233), Cry1Ab8 (Accession # M16463), in controlling, inhibiting growth or killing Lepidopteran, Cry1Ab9 (Accession # X54939), Cry1Ab10 (Accession it Coleopteran, Dipteran, fungal, Hemipteran, and nematode A29125), Cry1Ab11 (Accession if I12419), Cry1Ab12 (Ac pest populations and for producing compositions with pesti cession # AF059670), Cry1Ab13 (Accession # AF254640), cidal activity. Insect pests of interest include, but are not Cry1Ab14 (Accession # U94191), Cry1Ab15 (Accession it limited to, the superfamily of stink bugs and other related insects including, but not limited to, species belonging to the AF35.8861), Cry1Ab16 (Accession # AF375608), Cry1Ab17 family Pentatomidae (Nezara viridula, Halyomorpha halys, (Accession if AAT46415), Cry1Ab18 (Accession it Piezodorus guildini, Euschistus servus, Acrosternum hilare, AAQ88259), Cry1Ab19 (Accession # AY847289), Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Cry1Ab20 (Accession it DQ241675), Cry1Ab21 (Accession Dichelops fircatus, Dichelops melacanthus, and Bagrada # EF683163), Cry1Ab22 (Accession # ABW87320), hilaris (Bagrada Bug)), the family Plataspidae (Megacopta Cry1Ab-like (Accession if AF327924), Cry1Ab-like (Acces sion # AF327925), Cry1Ab-like (Accession # AF327926), cribraria—Bean plataspid), and the family Cydnidae (Scap Cry1Ab-like (Accession it DQ781309), Cry1Ac 1 (Accession tocoris Castanea—Root Stink bug) and Lepidoptera species # M11068), Cry1Ac2 (Accession #M35524), Cry1Ac3 (Ac including but not limited to: diamond-back moth, e.g., Heli cession # X54159), Cry1Ac4 (Accession # M73249), coverpa zea Boddie; soybean looper, e.g., Pseudoplusia Cry1Ac5 (Accession if M73248), Cry1AcG (Accession it includens Walker and Velvet bean caterpillar e.g., Anticarsia U43606), Cry1Ac7 (Accession # U87793), Cry1Ac8 (Acces gemmatalis Hübner. sion # U87397), Cry1Ac9 (Accession # U89872), Cry1Ac10 0213. By "pesticidal toxin' or “pesticidal protein' is (Accession if AJO02514), Cry1Ac11 (Accession it intended a toxin that has toxic activity against one or more AJ130970), Cry1Ac12 (Accession if I12418), Cry1Ac13 pests, including, but not limited to, members of the Lepi (Accession it AF 148644), Cry1Ac 14 (Accession it doptera, Diptera, Hemiptera and Coleoptera orders or the AF492767), Cry1Ac15 (Accession # AY122057), Cry1Ac16 Nematoda phylum or a protein that has homology to Such a (Accession if AY730621), Cry1Ac 17 (Accession it protein. Pesticidal proteins have been isolated from organ AY925090), Cry1Ac18 (Accession it DQ023296), Cry1Ac19 isms including, for example, Bacillus sp., Pseudomonas sp., (Accession it DQ195217), Cry1Ac20 (Accession it Photorhabdus sp., Xenorhabdus sp., Clostridium bifermen DQ285666), Cry1Ac21 (Accession # DQ062689), tans and Paenibacillus popilliae. Pesticidal proteins include Cry1Ac22 (Accession # EU282379), Cry1Ac23 (Accession but are not limited to: insecticidal proteins from Pseudomo # AM949588), Cry1Ac24 (Accession # ABL01535), nas sp. such as PSEEN3174 (Monalysin, (2011) PLoS Patho Cry1Ad1 (Accession if M73250), Cry1Ad2 (Accession it gens, 7:1-13), from Pseudomonas protegens strain CHAO and A27531), Cry1Ae1 (Accession#M65252), Cry1Af1 (Acces Pf-5 (previously fluorescens) (Pechy–Tarr, (2008) Environ sion # U82003), Cry1Ag1 (Accession # AF081248), mental Microbiology 10:2368-2386: GenBank Accession Cry1Ahl (Accession if AF281866), Cry1Ah2 (Accession it No. EU400157); from Pseudomonas Taiwanensis (Liu, et al., DQ269474), Cry1Ai1 (Accession # AY174873), Cry1A-like (2010) J. Agric. Food Chem. 58:12343-12349) and from (Accession # AF327927), Cry1 Ba1 (Accession # X06711), Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals Cry 1Ba2 (Accession # X95704), Cry 1Ba3 (Accession it of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell AF368257), Cry1 Ba4 (Accession # AF363025), Cry 1Ba5 Tiss. Organ Cult. 89:159-168); insecticidal proteins from (Accession if AB020894), Cry1 Ba6 (Accession it Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., ABL60921), Cry1 Bb1 (Accession # L32020), Cry1 Bc1 (Ac (2010) The Open Toxinology Journal 3:101-118 and Morgan, cession # Z46442), Cry1Bd1 (Accession it U70726), et al., (2001) Applied and Envir. Micro. 67:2062-2069), U.S. Cry1 Bd2 (Accession # AY138457), Cry 1Be1 (Accession it Pat. No. 6,048,838, and U.S. Pat. No. 6,379,946; and 8-en AF077326), Cry 1Be2 (Accession # AAQ52387), Cry1 Bf1 dotoxins including, but not limited to, the Cry 1, Cry2, Cry3, (Accession i AX189649), Cry 1Bf2 (Accession it Cry4, Crys, Cry6, Cry7, Cry8, Cry9, Cry 10, Cry 11, Cry 12, AAQ52380), Cry1 Bg1 (Accession # AY176063), Cry1Cal Cry13, Cry 14, Cry15, Cry 16, Cry 17, Cry 18, Cry 19, Cry20, (Accession # X07518), Cry1Ca2 (Accession if X13620), Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 1Ca3 (Accession if M73251), Cry 1Ca4 (Accession it Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, A27642), Cry1Ca5(Accession #X96682), Cry1Ca61 (Ac Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, cession # AF215647), Cry1Ca7 (Accession # AYO15492), Cry45, Cry 46, Cry47, Cry49, Cry 51 and Cry55 classes of Cry1 Cab (Accession # AF362020), Cry1Ca9 (Accession it Ö-endotoxin genes and the B. thuringiensis cytolytic Cyt1 and AY078160), Cry1Ca10(Accession #AF540014), Cry1Ca11 Cyt2 genes. Members of these classes of B. thuringiensis (Accession # AY955268), Cry1Cb1 (Accession # M97880), insecticidal proteins include, but are not limited to Cry1Aa1 Cry1Cb2 (Accession # AY007686), Cry1Cb3 (Accession it (Accession # Accession it M11250), Cry1Aa2 (Accession it EU679502), Cry1 Cb-like (Accession # AAX63901), M10917), Cry1Aa3 (Accession # D00348), Cry1Aa4 (Ac Cry 1 Dal (Accession if X54160), Cry 1Da2 (Accession it cession if X13535), Cry1Aa5 (Accession # D17518), I76415), Cry1Db1 (Accession # Z22511), Cry1Db2 (Acces Cry1Aao (Accession # U43605), Cry1Aa7 (Accession it sion # AF358862), Cry1Dc1 (Accession # EF059913), Cry1 AF081790), Cry1Aa8 (Accession # I26149), Cry1Aa9 (Ac Eat (Accession # X53985), Cry1 Ea2 (Accession # X56144), cession # AB026261), Cry1Aa10 (Accession # AF154676), Cry 1 Ea3 (Accession if M73252), Cry1Ea-4 (Accession it Cry1Aa11 (Accession if YO9663), Cry1Aa12 (Accession it U94323), Cry1 Eas (Accession # A15535), Cry1 Eao (Acces AF384211), Cry1Aa13 (Accession # AF510713), Cry1Aa14 sion # AF202531), Cry1 Ea7 (Accession # AAW72936),
US 2014/0007292 A1 Jan. 2, 2014
AF169251), Cry 19Aa1 (Accession # Y07603), Cry 19Bal # AB253419), Crys 1Aa1 (Accession # DQ836184), (Accession #D88381), Cry20Aa1 (Accession # U82518), Cry52Aa1 (Accession#EF613489), Crys3Aa1 (Accession it Cry21 Aa1 (Accession # I32932), Cry21Aa2 (Accession it EF633476), Crys4Aa1 (Accession # EU339367), Crys5Aa1 I66477), Cry21Ba1 (Accession # AB0884.06), Cry22Aal (Accession it EU121521), Crys5Aa2 (Accession it (Accession if I34547), Cry22Aa2 (Accession if AX472772), AAE33526). Cry22Aa3 (Accession # EU715020), Cry22Ab1 (Accession 0214) Examples of 8-endotoxins also include but are not # AAK50456), Cry22Ab2 (Accession # AX472764), limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and Cry22Ba1 (Accession if AX472770), Cry23Aa1 (Accession 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of # AAF76375), Cry24Aa1 (Accession #U88.188), Cry24Bal C.-helix 1 and/or C-helix 2 variants of Cry proteins such as (Accession it BAD32657), Cry24Ca1 (Accession it Cry1A) of U.S. Pat. Nos. 8.304,604 and 8.304,605, Cry 1B of AM158318), Cry25Aa1 (Accession # U88189), Cry26Aal U.S. patent application Ser. No. 10/525,318; Cry 1C of U.S. (Accession it AF 122897), Cry27Aa1 (Accession it Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218, AB023293), Cry28Aa1 (Accession #AF132928), Cry28Aa2 188: Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962, (Accession # AF285775), Cry29Aa1 (Accession it 705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of AJ251977), Cry30Aa1 (Accession # AJ251978), Cry30Bal U.S. Pat. No. 7,064.249); a Cry3A protein including but not (Accession # BAD00052), Cry30Ca1 (Accession it limited to an engineered hybrid insecticidal protein (eHIP) BAD67157), Cry30Da1 (Accession # EF095955), Cry30 created by fusing unique combinations of variable regions Db1 (Accession # BAE80088), Cry30Ea1 (Accession # and conserved blocks of at least two different Cry proteins EU503140), Cry30Fa1 (Accession # EU751609), Cry30Ga1 (US Patent Application Publication Number 2010/0017914); (Accession it EU882064), Cry31 Aa1 (Accession it a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins AB031065), Cry31Aa2 (Accession # AY081052), Cry31Aa3 of US Patent Numbers 7,329,736, 7,449,552, 7,803,943, (Accession if AB250922), Cry31 Aa4 (Accession it 7,476,781, 7,105,332, 7,378.499 and 7.462,760; a Cry9 pro AB274826), Cry31Aa5 (Accession # AB274827), Cry31Ab1 tein such as such as members of the Cry9A, Cry9B, Cry9C, (Accession # AB250923), Cry31Ab2 (Accession it Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Nai AB274825), Cry31 Ac1 (Accession # AB276125), Cry32Aa1 mov, et al., (2008) Applied and Environmental Microbiology (Accession if AY008143), Cry32Ba1 (Accession it 74.7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. BAB78601), Cry32Ca1 (Accession # BAB78602), 6,127,180, 6,624,145 and 6,340,593: a CryET33 and Cry32Dal (Accession it BAB78603), Cry33Aa1 (Accession CryET34 protein of U.S. Pat. Nos. 6,248.535, 6,326,351, # AAL26871), Cry34Aal (Accession # AAG50341), 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 Cry34Aa2 (Accession if AAK64560), Cry34Aa3 (Accession and CryET34 homologs of US Patent Publication Number # AY536899), Cry34Aa-4 (Accession # AY536897), 2006/0191034, 2012/0278954, and PCT Publication Number Cry34Ab1 (Accession if AAG41671), Cry34Ac1 (Accession WO 2012/139004: a Cry35Ab1 protein of U.S. Pat. Nos. # AAG50118), Cry34Ac2 (Accession # AAK64562), 6,083,499, 6,548,291 and 6,340,593: a Cry46 protein, a Cry Cry34Ac3 (Accession # AY536896), Cry34Ba1 (Accession # 51 protein, a Cry binary toxin; a TIC901 or related toxin; AAK64565), Cry34Ba2 (Accession # AY536900), TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, Cry34Ba3 (Accession # AY536898), Cry35Aa1 (Accession # TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI AAG50342), Cry35Aa2 (Accession # AAK64561), 027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757: Cry35Aa3 (Accession # AY536895), Cry35Aa4 (Accession # AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. AY536892), Cry35Ab1 (Accession # AAG41672), No. 7,923,602: AXMI-018, AXMI-020, and AXMI-021 of Cry35Ab2 (Accession #AAK64563), Cry35Ab3 (Accession WO 2006/083891: AXMI-010 of WO 2005/038032: AXMI # AY536891), Cry35Ac1 (Accession # AAG50117), 003 of WO 2005/021585; AXMI-008 of US 2004/0250311; Cry35Ba1 (Accession #AAK64566), Cry35Ba2 (Accession AXMI-006 of US 2004/0216186: AXMI-007 of US 2004/ # AY536894), Cry35Ba3 (Accession # AY536893), 0210965; AXMI-009 of US 2004/0210964: AXMI-014 of US Cry36Aa1 (Accession if AAK64558), Cry37Aa1 (Accession 2004/0197917: AXMI-004 of US 2004/0197916: AXMI-028 # AAF76376), Cry38Aa1 (Accession # AAK64559), and AXMI-029 of WO 2006/119457; AXMI-007, AXMI Cry39Aa1 (Accession it BAB72016), Cry40Aa1 (Accession 008, AXMI-0080r12, AXMI-009, AXMI-014 and AXMI # BAB72018), Cry40Ba1 (Accession # BAC77648), 004 of WO 2004/074462: AXMI-150 of U.S. Pat. No. 8,084, Cry40Ca1 (Accession#EU381045), Cry40Dal (Accession # 416: AXMI-205 of US2011 0023184: AXMI-011, AXMI EU596478), Cry41Aa1 (Accession # AB116649), Cry41Ab1 012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, (Accession it AB 116651), Cry42Aa1 (Accession it AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI AB1 16652), Cry43Aa1 (Accession # AB115422), Cry43Aa2 022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of (Accession if AB176668), Cry43Ba1 (Accession it US 2011/0263488; AXMI-R1 and related proteins of US AB115422), Cry43-like (Accession # AB115422), Cry44Aa 2010/0197592: AXMI221Z, AXMI222z, AXMI223Z, (Accession it BAD08532), Cry45Aa (Accession it AXMI224Z and AXMI225Z of WO 2011/103248: AXMI218, BAD22577), Cry46Aa (Accession it. BAC79010), Cry46Aa2 AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, (Accession if BAG68906), Cry46 Ab (Accession it AXMI229, AXMI230, and AXMI231 of WO1 1/103,247; BAD35170), Cry47Aa (Accession # AY950229), Cry48Aa AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI (Accession if AJ841948), Cry48Aa2 (Accession it 184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AM237205), Cry48Aa3 (Accession # AM237206), Cry48Ab AXMI-030, AXMI-035, and AXMI-045 of US 2010/ (Accession # AM237207), Cry48Ab2 (Accession # 0298211: AXMI-066 and AXMI-076 of US20090144852; AM237208), Cry49Aa (Accession # AJ841948), Cry49Aa2 AXMI 128, AXMI130, AXMI131, AXMI133, AXMI 140, (Accession it AM237201), Cry49Aa3 (Accession it AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AM237203), Cry49Aa4 (Accession # AM237204), AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, Cry49Ab1 (Accession # AM237202), CrysOAa1 (Accession AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, US 2014/0007292 A1 Jan. 2, 2014
AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, a different genus. There are three main types of TC proteins. AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, As referred to herein, Class A proteins (“Protein A') are AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, stand-alone toxins. Class B proteins (“Protein B) and Class AXMI 180, AXMI181, AXMI182, AXMI 185, AXMI 186, C proteins (“Protein C) enhance the toxicity of Class A AXMI 187, AXMI 188, AXMI189 of U.S. Pat. No. 8,318,900; proteins. Examples of Class A proteins are TchA, TcdA, AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, XptA1 and XptA2. Examples of Class B proteins are TcaG, TcdB, XptB1Xb and XptC1 Wi. Examples of Class C proteins AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also AXMI100, AXMI101, AXMI102, AXMI 103, AXMI 104, include spider, Snake and Scorpion Venom proteins. Examples AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, of spider venom peptides include but are not limited to lyc AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, otoxin-1 peptides and mutants thereof (U.S. Pat. No. 8.334, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, 366). AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI 129, 0215. In some embodiments the PIP-1 polypeptides AXMI164, AXMI151, AXMI161, AXMI 183, AXMI132, include amino acid sequences deduced from the full-length AXMI138, AXMI137 of US 2010/0005543; Cry proteins nucleic acid sequences disclosed herein, and amino acid such as Cry1A and Cry3A having modified proteolytic sites sequences that are shorter than the full-length sequences, of U.S. Pat. No. 8.319,019; and a Cry1Ac, Cry2Aa and either due to the use of an alternate downstream start site or Cry1Ca toxin protein from Bacillus thuringiensis strain due to processing that produces a shorter protein having pes VBTS 2528 of US Patent Application Publication Number ticidal activity. Processing may occur in the organism after 2011/0064710. Other Cry proteins are well known to one the protein is expressed in or in the pest after ingestion of the skilled in the art (see, Crickmore, et al., “Bacillus thuringien protein. sis toxin nomenclature' (2011), at lifesci. Sussex.ac.uk/home/ Neil Crickmore/Bt? which can be accessed on the world 0216. Thus, provided herein are novel isolated or recom wide web using the “www’ prefix). The insecticidal activity binant nucleic acid sequences encoding polypeptides that of Cry proteins is well known to one skilled in the art (for confer pesticidal activity. Also provided are the amino acid review, see, van Frannkenhuyzen, (2009).J. Invert. Path. 101: sequences of PIP-1 polypeptides. The protein resulting from 1-16). The use of Cry proteins as transgenic plant traits is well translation of these PIP-1 polypeptide genes allows cells to known to one skilled in the art and Cry-transgenic plants control or kill pests that ingest it. including but not limited to Cry1Ac, Cry1Ac--Cry2Ab. Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, Cry1 F+Cry1Ac, Bacterial Strains Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, 0217. One aspect of the invention pertains to bacterial Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory strains that are capable of expressing a PIP-1 polypeptide. In approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283 Some embodiments the bacterial strain is a Pseudomonas 300 and the CERA (2010) GM Crop Database Center for chlororaphis strain. In some embodiments the bacterial strain Environmental Risk Assessment (CERA), ILSI Research is a biologically pure culture of a Pseudomonas chlororaphis Foundation, Washington D.C. at cera-gmc.org/index. strain SS44C4, deposited on Dec. 1, 2011 under Accession php?action gm crop database which can be accessed on the Number NRRLB-50613 with the Agricultural Research Ser world-wide web using the “www” prefix). More than one vice Culture Collection (NRRL). In some embodiments the pesticidal proteins well known to one skilled in the art can bacterial strain is a biologically pure culture of a Pseudomo also be expressed in plants such as Vip3Ab & Cry1 Fa nas chlororaphis strain having a 16S ribosomal DNA having (US2012/0317682), Cry 1BE & Cry1F (US2012/0311746), at least about 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, (US2012/0317681), Cry1DA & Cry1BE (US2012/ 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 0331590), Cry1DA & Cry1 Fa (US2012/0331589), Cry1AB 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% & Cry1BE (US2012/0324606), and Cry1 Fa & Cry2Aa, or 99.9% sequence identity compared to SEQID NO: 216. Cry1 I or Cry1E (US2012/0324605). Pesticidal proteins also include insecticidal lipases including lipid acylhydrolases of Nucleic Acid Molecules, and Variants and Fragments Thereof U.S. Pat. No. 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys 0218. Another aspect of the invention pertains to isolated Res Commun 15:1406-1413). Pesticidal proteins also include or recombinant nucleic acid molecules comprising nucleic VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos. acid sequences encoding PIP-1 polypeptides and polypep 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and tides or biologically active portions thereof, as well as nucleic 8.237,020, and the like. Other VIP proteins are well known to acid molecules sufficient for use as hybridization probes to one skilled in the art (see, lifesci. Sussex.ac.uk/home/Neil identify nucleic acid molecules encoding proteins with Crickmore/Bt/vip.html which can be accessed on the world regions of sequence homology. As used herein, the term wide web using the “www’ prefix). Pesticidal proteins also “nucleic acid molecule' is intended to include DNA mol include toxin complex (TC) proteins, obtainable from organ ecules (e.g., recombinant DNA, cDNA, genomic DNA, plas isms such as Xenorhabdus, Photorhabdus and Paenibacillus tid DNA, mitochondrial DNA) and RNA molecules (e.g., (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC mRNA) and analogs of the DNA or RNA generated using proteins have “standalone' insecticidal activity and other TC nucleotide analogs. The nucleic acid molecule can be single proteins enhance the activity of the stand-alone toxins pro stranded or double-stranded, but preferably is double duced by the same given organism. The toxicity of a “stand stranded DNA. alone TC protein (from Photorhabdus, Xenorhabdus or 0219. An "isolated' or “recombinant nucleic acid mol Paenibacillus, for example) can be enhanced by one or more ecule (or DNA) is used herein to refer to a nucleic acid TC protein “potentiators’ derived from a source organism of sequence (or DNA) that is no longer in its natural environ US 2014/0007292 A1 Jan. 2, 2014
ment, for example in an in vitro or in a recombinant bacterial protein. A similar method for obtaining enhanced expression or plant host cell. In some embodiments, an "isolated” or of transgenes in monocotyledonous plants is disclosed in U.S. “recombinant nucleic acid is free of sequences (preferably Pat. No. 5,689,052. protein encoding sequences) that naturally flank the nucleic 0223. In some embodiments the nucleic acid molecule acid (i.e., sequences located at the 5' and 3' ends of the nucleic encoding a PIP-1 polypeptide is a polynucleotide having the acid) in the genomic DNA of the organism from which the sequence set forth in SEQ ID NO: 1, 3 or 331 and variants, nucleic acid is derived. For purposes of the disclosure, “iso fragments and complements thereof. By "complement is lated' or “recombinant' when used to refer to nucleic acid intended a nucleic acid sequence that is sufficiently comple molecules excludes isolated chromosomes. For example, in mentary to a given nucleic acid sequence Such that it can various embodiments, the recombinant nucleic acid molecule hybridize to the given nucleic acid sequence to thereby form encoding a PIP-1 polypeptide can contain less than about 5 a stable duplex. In some embodiments the nucleic acid mol kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid ecule encoding a PIP-1 polypeptide is a nucleic acid molecule sequences that naturally flank the nucleic acid molecule in having the sequence set forth in SEQID NO: 1, 3 or 331. The genomic DNA of the cell from which the nucleic acid is corresponding amino acid sequences for the insecticidal pro derived. tein encoded by these nucleic acid sequences are set forth in 0220 A variety of polynucleotides that encode PIP-1 SEQID NO: 2, 4 and 332. polypeptides or related proteins are contemplated. Such poly 0224. In some embodiments the nucleic acid molecule nucleotides are useful for production of PIP-1 polypeptides in encoding a PIP-1 polypeptide is a polynucleotide having a host cells when operably linked to suitable promoter, tran nucleotide sequence encoding a polypeptide comprising an Scription termination and/or polyadenylation sequences. amino acid sequence having at least 80% identity, to the Such polynucleotides are also useful as probes for isolating amino acid sequence of SEQID NO: 2, SEQID NO:4 or SEQ homologous or Substantially homologous polynucleotides ID NO: 332, wherein the polypeptide has pesticidal activity. that encode PIP-1 polypeptides or related proteins. In some embodiments the nucleic acid molecule encoding a 0221) One source of polynucleotides that encode PIP-1 PIP-1 polypeptide is a polynucleotide having a nucleotide polypeptides or related proteins is a Pseudomonas chlorora sequence encoding a polypeptide comprising an amino acid phis strain which contains the PIP-1A polynucleotide of SEQ sequence having at least 80% identity, to the amino acid IDNO: 1 encoding the PIP-1A polypeptide of SEQID NO: 2. sequence of SEQ ID NO: 2, wherein the polypeptide has This polynucleotide sequence was isolated from a Pseudomo pesticidal activity. In some embodiments the nucleic acid nas chlororaphis host and is thus suitable for expression of molecule encoding a PIP-1 polypeptide is a polynucleotide the encoded PIP-1A polypeptide in other bacterial hosts. For having a nucleotide sequence encoding a polypeptide com example, SEQID NO: 1 can be used to express the PIP-1A prising an amino acid sequence having at least 80% identity, protein in bacterial hosts that include but are not limited to an to the amino acid sequence of SEQ ID NO: 4, wherein the Agrobacterium, an Alcaligenes, a Bacillus, an Escherichia, a polypeptide has pesticidal activity. In some embodiments the Salmonella, a Pseudomonas and a Rhizobium bacterial host nucleic acid molecule encoding a PIP-1 polypeptide is a cells. The polynucleotides are also useful as probes for iso polynucleotide having a nucleotide sequence encoding a lating homologous or Substantially homologous polynucle polypeptide comprising an amino acid sequence having at otides that encode PIP-1 polypeptides or related proteins. least 80% identity, to the amino acid sequence of SEQID NO: Such probes can be used to identify homologous or Substan 332, wherein the polypeptide has pesticidal activity. tially homologous polynucleotides derived from Pseudomo 0225. In some embodiments the nucleic acid molecule nas or other bacterial strains. encoding a PIP-1 polypeptide is a polynucleotide having a 0222 Polynucleotides that encode a PIP-1 polypeptide nucleotide sequence encoding a polypeptide comprising an can also be synthesized de novo from a PIP-1 polypeptide amino acid sequence of (SEQ ID NO: 211), wherein Xaa at sequence. The sequence of the polynucleotide gene can be position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly or Asn; deduced from a PIP-1A polypeptide sequence through use of Xaa at position 19 is Asp, Glu or Cys: Xaa at position 20 is the genetic code. Computer programs such as "BackTrans Leu or Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at late” (GCGTM Package. Acclerys, Inc. San Diego, Calif.) can position 22 is Ser, Lys or Arg; Xaaat position 24 is Glnor Ala; be used to convert a peptide sequence to the corresponding Xaa at position 25 is Gly or Ala Xaa at position 26 is Ser or nucleotide sequence encoding the peptide. Examples of Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 30 PIP-1 polypeptide sequences that can be used to obtain cor is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position responding nucleotide encoding sequences include, but are 36 is Ala, Seror Val: Xaaat position 38 is Asn, Argor Ser; Xaa not limited to, the PIP-1 polypeptide sequence of SEQ ID at position 42 is Phe or Tyr, Xaa at position 46 is Arg, Lys or NO: 2. Furthermore, synthetic PIP-1A polynucleotide His;Xaaat position 48 is Gly or Asp;Xaaat position 49 is Phe sequences of the invention can be designed so that they will be or Tyr; Xaa at position 53 is Ser or Gly; Xaa at position 58 is expressed in plants. U.S. Pat. No. 5,500,365 describes a Tyror Phe: Xaa at position 60 is Ala or Ser; Xaa at position 63 method for synthesizing plant genes to improve the expres is Gln or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position sion level of the protein encoded by the synthesized gene. 97 is Met or Val: Xaa at position 98 is Asp or Glu, Xaa at This method relates to the modification of the structural gene position 105 is Glin or ASn; Xaa at position 107 is Thr or Ile: sequences of the exogenous transgene, to cause them to be Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg more efficiently transcribed, processed, translated and or Leu, Xaa at position 120 is Lys, Argor Glin; Xaa at position expressed by the plant. Features of genes that are expressed 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; Xaa at well in plants include elimination of sequences that can cause position 125 is ASnor Ser; Xaa at position 127 is Ser, Asn. Thr undesired intron splicing or polyadenylation in the coding or Lys; Xaa at position 134 is Gly or Ala; Xaa at position 135 region of a gene transcript while retaining Substantially the is Ser, Asn or Lys; Xaa at position 137 is Asp or Gly; Xaa at amino acid sequence of the toxic portion of the insecticidal position 141 is Val or Ile: Xaa at position 142 is Gly or Asp; US 2014/0007292 A1 Jan. 2, 2014
Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Thr or Val: Xaa at position 150 is Seror Thr; Xaa at position Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaaat position 173 is 151 is Asn, Arg or Ser; Xaa at position 160 is Thr or Ser; Xaa Phe, Gly, His, Leu, Ala, Arg, Asn, Cys, Lys, Trp, Thr, Ser, Tyr at position 162 is Ser or Thr; Xaa at position 163 is Asn, Asp or Met; Xaaat position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, or Glu; Xaa at position 164 is Seror Thr; Xaa at position 166 Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at is Gln or Glu, Xaa at position 167 is Leu or Met; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaa position 168 is Thr, Lys or Ala; Xaa at position 174 is Ile, Val at position 176 is Tyr, Met, Phe, Leu or Cys; Xaa at position or Met; Xaa at position 175 is Val or Ile: Xaa at position 180 177 is Gln, Ile, Met or Pro; Xaa at position 178 is Val, Cys, is Met or Leu; Xaa at position 191 is Arg or Lys: Xaa at Thr, Pro, Ala, Met, Gln, Phe, Ile, Ser or Lys; Xaa at position position 194 is Gly or Ala; Xaa at position 200 is Asn or Ser; 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, Ala or Gln: Xaa Xaa at position 203 is ASnor Glin; Xaa at position 204 is Thr at position 180 is Met, Leu, Pro, Trp, Asn., Tyr, Gly, Gln, Ala, or Ala; Xaa at position 206 is Gly or Asp; Xaa at position 209 Val, Phe, Ile, Cys or Ser; Xaa at position 181 is Val, Ala, Leu, is Leu or Val; Xaa at position 220 is ASn or Arg; Xaa at Trp, Cys, Thr, Ile or Lys: Xaa at position 182 is Tyr, Phe, Met position 221 is Ser or Lys: Xaa at position 222 is Thr or Arg; or His; Xaa at position 183 is Ala, Met, Val, Thr, Asp, Gly, Xaa at position 226 is Asp, Pro or Glu; Xaa at position 228 is Cys, Ile, Phe, Ser, Gln or Leu; Xaa at position 191 is Arg or Seror Gly: Xaa at position 229 is Lys or ASn; Xaa at position Lys: Xaa at position 194 is Gly or Ala; Xaa at position 195 is 231 is Ile or Val: Xaa at position 232 is Ala, Thr or Glu; and Asin or Tyr; Xaa at position 200 is Asn or Ser; Xaa at position Xaa at position 251 is Gly, Seror Glu; Xaa at position 254 is 203 is Asn or Glin; Xaa at position 204 is Thr or Ala; Xaa at Seror Asn; Xaa at position 258 is Seror Arg; Xaa at position position 206 is Gly or Asp; Xaa at position 209 is Leu or Val; 265 is ASnor Asp; and Xaa at position 266 is Asp or ASn; and Xaa at position 213 is Tyr or Phe: Xaa at position 220 is Asn wherein, 1 to 28 amino acids are optionally deleted from the or Arg; Xaa at position 221 is Seror Lys; Xaa at position 222 N-terminus of the polypeptide. is Thr or Arg; Xaa at position 226 is Asp, Pro or Glu; Xaa at 0226. In some embodiments the nucleic acid molecule position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; encoding a PIP-1 polypeptide is a polynucleotide having a Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, nucleotide sequence encoding a polypeptide comprising an Thror Glu:Xaaat position 240 is Gln, Arg, Ala, Val, Glu, Met, amino acid sequence of a sequence of SEQ ID NO: 212, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at wherein Xaaat position 2 is Pro or Thr; Xaa at position 3 is Ile position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, or Thr; Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys: Xaa at position Gly or Asn; Xaa at position 19 is Asp, Glu or Cys; Xaa at 242 is ASn, Ala, Arg, Lys. His, Ser, Cys, Glu, Pro, Trp, Gln, position 20 is Leu or Val; Xaaat position 21 is Lys, Seror ASn; Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 Xaa at position 22 is Ser, Lys or Arg; Xaa at position 24 is Gln is Val, Leu, Ala, Thr, Gly, Cys, Ile, Seror Met; Xaa at position or Ala; Xaa at position 25 is Gly or Ala; Xaa at position 26 is 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at Ser or Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or ASn; Xaa at Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala or Ile: Xaa position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, Ser, at position 35 is Phe or Leu; Xaa at position 36 is Ala, Ser or ASn, Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys: Xaa at Val: Xaa at position 38 is Asn, Arg or Ser; Xaa at position 42 position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, Lys, is Phe or Tyr; Xaa at position 43 is Pro, Met, Gly, Gln, Ser, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thror Cys; Xaa at Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, Xaa at position 46 is Arg, Lys or His; Xaa at position 48 is Gly Ile, Asp, Gly or Ala; Xaa at position 249 is Asn. Lys, Val, Gly, or Asp; Xaa at position 49 is Phe, Tyr or Leu; Xaa at position Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, Thr, Pro, Leu, 53 is Ser or Gly: Xaa at position 58 is Tyr or Phe: Xaa at Ala, His or Glin; Xaa at position 251 is Gly, Seror Glu; Xaa at position 60 is Ala or Ser; Xaa at position 63 is Glin or Lys; Xaa position 254 is Ser or Asn; Xaa at position 258 is Ser or Arg; at position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or Xaaat position 77 is Phe or Tyr; Xaa at position 89 is Pro, Leu, His; Xaa at position 265 is ASn or Asp; and Xaa at position Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys or Lys; Xaa at 266 is Asp or Asn. position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, 0227. In some embodiments the nucleic acid molecule Ala or Thr; Xaa at position 97 is Met or Val: Xaa at position encoding a PIP-1 polypeptide is a polynucleotide having a 98 is Asp or Glu; Xaa at position 105 is Glin or Asn; Xaa at nucleotide sequence encoding a polypeptide comprising an position 107 is Thr or Ile: Xaa at position 108 is Glin or Thr: amino acid sequence of a sequence of SEQ ID NO: 213 Xaa at position 110 is Arg or Leu, Xaa at position 120 is LyS, wherein Xaa at position 2 is Pro, Thr or Ser; Xaa at position Argor Glin; Xaa at position 121 is Thr or Ser; Xaa at position 3 is Ile, Thr, Leu, Val, Metor Ser; Xaaat position 6 is Glu, Gly, 123 is Thr or Glu; Xaa at position 125 is Asn or Ser; Xaa at Asp or Ala; Xaa at position 8 is Ser, Gly, Asn. Thror Glin; Xaa position 127 is Ser, Asn. Thror Lys; Xaaat position 134 is Gly at position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, or Ala; Xaa at position 135 is Ser, ASnor LyS, Xaa at position Val, Ile or Met; Xaa at position 21 is Lys, Ser, Asn, Arg, Thr 137 is Asp or Gly; Xaa at position 141 is Val or Ile: Xaa at or Glin; Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at position 142 is Gly or Asp; Xaa at position 144 is Asp or Glu; position 24 is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly Xaaat position 147 is Ile, ThrorVal: Xaaat position 150 is Ser or Ala; Xaa at position 26 is Ser, Asn. Thr or Glin; Xaa at or Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position position 27 is Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, position 163 is Asn., Asp or Glu; Xaa at position 164 is Seror Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala, Ile, Leu, Thr; Xaa at position 166 is Glin or Glu; Xaa at position 167 is Val or Met; Xaa at position 35 is Phe, Leu, Ile, Valor Met; Xaa Leu or Met; Xaa at position 168 is Thr, Lys or Ala; Xaa at at position 36 is Ala, Ser, Thr, Val, Ile or Leu; Xaa at position position 171 is Gly, Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; 38 is Asn, Arg, Ser, Gln, Lys or Thr; Xaa at position 42 is Phe, US 2014/0007292 A1 Jan. 2, 2014
Tyr, Trp, Leu, Ile, Val or Met; Xaa at position 43 is Pro, Met, Ile, Seror Met; Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gly, Gln, Ser, Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Gln, Cys, Trp or Ala; Xaa at position 245 is Met, Ala, Arg, Glu or Cys; Xaa at position 46 is Arg, Lys or His; Xaa at Asp, Glu, Leu, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, position 48 is Gly, Asp, Ala or Glu; Xaa at position 49 is Phe, Ile, Gln, Tyror ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Tyr, Trp, Leu, Ile, Val or Met; Xaa at position 53 is Ser, Gly, Arg, Val, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Ala or Thr; Xaa at position 58 is Tyr or Phe, Xaa at position 60 Phe, Thr or Lys; Xaa at position 247 is Asn. Leu, Asp, Tyr, is Ala, Ser, Gly or Thr; Xaa at position 63 is Gln, Lys, Asn or Ala, Phe, His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Arg; Xaa at position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val Trp, Thror Cys: Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala, Xaa at position or Ser; Xaa at position 77 is Phe, Tyr, Trp, Leu, Ile, Valor Met; 249 is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Tyr, Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, Trp, 251 is Gly, Ser, Thr, Ala, Asp or Glu; Xaa at position 254 is Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa at position Ser, Asn. Thr or Glin; Xaa at position 258 is Ser, Arg, Thr or 97 is Met, Val, Leu or Ile: Xaa at position 98 is Asp or Glu; Lys: Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Xaa at position 105 is Glin or ASn; Xaa at position 107 is Thr, Ile or His: Xaa at position 265 is Asn., Asp, Gln or Glu; and Ile, Ser, Leu or Val: Xaaat position 108 is Gln, Thr, Seror ASn; Xaa at position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at Xaa at position 266 is Asp, ASn, Gln or Glu. position 120 is Lys, Arg, Gln or ASn; Xaa at position 121 is 0228. In some embodiments the nucleic acid molecules Thror Ser; Xaa at position 123 is Thr, Glu, Seror Asp; Xaa at encode a PIP-1 polypeptide having a nucleotide sequence position 125 is Asn. Ser, Gln or Thr; Xaaat position 127 is Ser, encoding a polypeptide comprising one or more amino acid Asn. Thr, Gln, Lys, Seror Arg; Xaa at position 134 is Gly or motifs selected from i) amino acids 64-79 of SEQID NO: 2, Ala; Xaa at position 135 is Ser, Asn. Thr, Gln, Argor Lys; Xaa amino acids 64-79 of SEQID NO: 211, amino acids 64-79 of at position 137 is Asp, Gly, Glu or Ala; Xaa at position 141 is SEQ ID NO: 212 or amino acids 64-79 of SEQID NO: 213, Val, Ile or Leu; Xaa at position 142 is Gly, Asp, Ala or Glu; ii) amino acids 149-159 of SEQ ID NO: 2, amino acids Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, 149-159 of SEQID NO: 211, amino acids 149-159 of SEQID Thr, Val, Leu, Met or Ser; Xaa at position 150 is Ser or Thr: NO: 212 or amino acids 149-159 of SEQ ID NO: 213, iii) Xaa at position 151 is ASn, Arg, Ser, Gln, Lys or Thr, Xaa at amino acids 171-183 of SEQID NO: 2, amino acids 171-183 position 160 is Thr or Ser; Xaa at position 162 is Seror Thr: of SEQID NO: 211, amino acids 171-183 of SEQID NO: 212 Xaa at position 163 is ASn, Asp, Glu or Glin; Xaa at position or amino acids 171-183 of SEQID NO: 213, and iv) amino 164 is Seror Thr; Xaa at position 166 is Gln, Glu, Asp or ASn; acids 240-249 of SEQID NO: 2, amino acids 240-249 of SEQ Xaa at position 167 is Leu, Met, Ile, Val: Xaa at position 168 ID NO: 211, amino acids 240-249 of SEQ ID NO: 212 or is Thr, Lys, Ala, Ser, Arg or Gly: Xaa at position 171 is Gly, amino acids 240-249 of SEQID NO: 213. In some embodi Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Xaa at position 172 ments the nucleic acid molecules encode a PIP-1 polypeptide is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, having a nucleotide sequence encoding a polypeptide com Cys, Val, Ala or Met; Xaaat position 173 is Phe, Gly. His, Leu, prising an amino acid as represented by positions 171-183 of Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr or Met; Xaa at SEQID NO: 213 wherein at least one amino acid at positions position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Met, Cys, 171-183 of SEQID NO: 213 are not identical to amino acids Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at position 175 at positions 171-183 of SEQID NO: 6. is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaaat position 176 0229. In some embodiments the nucleic acid molecules is Tyr, Met, Phe, Leu or Cys: Xaa at position 177 is Gln, Ile, encode a PIP-1 polypeptide having a nucleotide sequence Metor Pro; Xaa at position 178 is Val, Cys, Thr, Pro, Ala, Met, encoding a polypeptide comprising an amino acid sequence Gln, Phe, Ile, Seror Lys; Xaa at position 179 is Val, Phe, Thr, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Ile, Cys, Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Leu: Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or 98%, 99% or greater identity to the amino acid sequence set Ser; Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or forth in SEQID NO: 2, SEQID NO: 6 or SEQID NO.4 and Lys; Xaa at position 182 is Tyr, Phe, Met or His: Xaa at wherein the polypeptide comprises one or more amino acid position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, motifs selected from i) amino acids 64-79 of SEQID NO: 2, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at position amino acids 64-79 of SEQID NO: 211, amino acids 64-79 of 194 is Gly or Ala; Xaa at position 195 is Asn., Tyr, Gln or Trp; SEQ ID NO: 212 or amino acids 64-79 of SEQID NO: 213, Xaa at position 200 is Asn. Ser. Thr or Gln; Xaa at position ii) amino acids 149-159 of SEQ ID NO: 2, amino acids 203 is ASnor Glin; Xaa at position 204 is Thr, Ala, Seror Gly: 149-159 of SEQID NO: 211, amino acids 149-159 of SEQID Xaa at position 206 is Gly, Asp, Ala or Glu; Xaa at position NO: 212 or amino acids 149-159 of SEQ ID NO: 213, iii) 209 is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: amino acids 171-183 of SEQID NO: 2, amino acids 171-183 Xaa at position 220 is ASn, Arg, Gln or Lys, Xaa at position of SEQID NO: 211, amino acids 171-183 of SEQID NO: 212 221 is Ser, Lys, Thror Arg; Xaaat position 222 is Thr, Arg, Ser or amino acids 171-183 of SEQID NO: 213, and iv) amino or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at acids 240-249 of SEQID NO: 2, amino acids 240-249 of SEQ position 228 is Seror Gly; Xaaat position 229 is Lys, Asn, Arg ID NO: 211, amino acids 240-249 of SEQ ID NO: 212 or or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa at amino acids 240-249 of SEQID NO: 213. position 232 is Ala, Thr, Ser, Gly, Asp or Glu, Xaa at position 0230. In some embodiments the nucleic acid molecules 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, Asn. Thr, encode a PIP-1 polypeptide having a nucleotide sequence Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 is Arg, Lys, encoding a polypeptide comprising an amino acid sequence Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, Pro, Gly, Leu, having at least 80% identity to the amino acid sequence set Phe, Thr, Ala or Cys;Xaa at position 242 is ASn, Ala, Arg, LyS, forth in SEQID NO: 2, SEQID NO: 6 or SEQID NO.4 and His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, wherein the polypeptide comprises one or more amino acid Leu or Val: Xaa at position 243 is Val, Leu, Ala, Thr, Gly, Cys, motifs selected from i) amino acids 64-79 of SEQID NO: 2, US 2014/0007292 A1 Jan. 2, 2014
amino acids 64-79 of SEQID NO: 211, amino acids 64-79 of 0234. In some embodiments the nucleic acid molecules SEQ ID NO: 212 or amino acids 64-79 of SEQID NO: 213, encode a PIP-1 polypeptide having a nucleotide sequence ii) amino acids 149-159 of SEQ ID NO: 2, amino acids encoding a polypeptide comprising an amino acid sequence 149-159 of SEQID NO: 211, amino acids 149-159 of SEQID having at least 80% identity to the amino acid sequence set NO: 212 or amino acids 149-159 of SEQ ID NO: 213, iii) forth in SEQ ID NO: 2, and wherein the polypeptide com amino acids 171-183 of SEQID NO: 2, amino acids 171-183 prises one or more amino acid motifs selected from i) amino of SEQID NO: 211, amino acids 171-183 of SEQID NO: 212 acids 64-79 of SEQID NO:2 or amino acids 64-79 of SEQID or amino acids 171-183 of SEQ ID NO: 213, and iv) amino NO: 213, ii) amino acids 149-159 of SEQID NO: 2 or amino acids 240-249 of SEQID NO: 2, amino acids 240-249 of SEQ acids 149-159 of SEQID NO: 213, iii) amino acids 171-183 ID NO: 211, amino acids 240-249 of SEQ ID NO: 212 or of SEQID NO: 2 oramino acids 171-183 of SEQID NO: 213 amino acids 240-249 of SEQID NO: 213. and iv) amino acids 240-249 of SEQID NO: 2 or amino acids 0231. In some embodiments the nucleic acid molecules 240-249 of SEQID NO: 213. encode a PIP-1 polypeptide having a nucleotide sequence 0235. In some embodiments exemplary nucleic acid mol encoding a polypeptide comprising an amino acid sequence ecules encode a PIP-1 polypeptide of SEQID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 98%, 99% or greater identity to the amino acid sequence set 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, forth in SEQ ID NO: 2 and wherein the polypeptide com 151, 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, prises one or more amino acid motifs selected from i) amino 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, acids 64-79 of SEQID NO: 2, amino acids 64-79 of SEQ ID 261, 262, 263,264, 265, 266, 267, 268, 269,298, 299, 300, NO: 211, amino acids 64-79 of SEQ ID NO: 212 or amino 301,302,303, 304, 305,306, 307, 308,309, 310, 311, 312, acids 64-79 of SEQID NO: 213, ii) amino acids 149-159 of 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323,324, SEQ ID NO: 2, amino acids 149-159 of SEQ ID NO: 211, and 325 as well as amino acid substitutions, amino acid amino acids 149-159 of SEQ ID NO: 212 or amino acids deletions, amino acid insertions and fragments thereof and 149-159 of SEQ ID NO: 213, iii) amino acids 171-183 of combinations thereof. SEQ ID NO: 2, amino acids 171-183 of SEQ ID NO: 211, 0236. In some embodiments exemplary nucleic acid mol amino acids 171-183 of SEQ ID NO: 212 or amino acids ecules encode a PIP-1 polypeptide of SEQID NO: 101, 102, 171-183 of SEQID NO: 213, and iv) amino acids 240-249 of 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, SEQ ID NO: 2, amino acids 240-249 of SEQ ID NO: 211, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, amino acids 240-249 of SEQ ID NO: 212 or amino acids 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 240-249 of SEQID NO: 213. 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 0232. In some embodiments the nucleic acid molecules 151, 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, encode a PIP-1 polypeptide having a nucleotide sequence 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, encoding a polypeptide comprising an amino acid sequence 261, 262, 263,264, 265, 266, 267, 268, and 269 as well as having at least 80% identity to the amino acid sequence set amino acid Substitutions, deletions, insertions and fragments forth in SEQ ID NO: 2 and wherein the polypeptide com thereof and combinations thereof. prises one or more amino acid motifs selected from i) amino 0237. In some embodiments exemplary nucleic acid mol acids 64-79 of SEQID NO: 2, amino acids 64-79 of SEQ ID ecules comprise a sequence set forth in SEQID NO: 152, 153, NO: 211, amino acids 64-79 of SEQ ID NO: 212 or amino 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, acids 64-79 of SEQID NO: 213, ii) amino acids 149-159 of 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, SEQ ID NO: 2, amino acids 149-159 of SEQ ID NO: 211, 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, amino acids 149-159 of SEQ ID NO: 212 or amino acids 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 149-159 of SEQ ID NO: 213, iii) amino acids 171-183 of 202, 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, SEQ ID NO: 2, amino acids 171-183 of SEQ ID NO: 211, 228, 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, amino acids 171-183 of SEQ ID NO: 212 or amino acids 240, 241, 242, 243, 244, 270, 271, 272,273, 274, 275, 276, 171-183 of SEQID NO: 213, and iv) amino acids 240-249 of 277,278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, SEQ ID NO: 2, amino acids 240-249 of SEQ ID NO: 211, 289, 290, 291, 292, 293, 294, 295, 296, and 297 as well as amino acids 240-249 of SEQ ID NO: 212 or amino acids variants and fragments thereof encoding PIP-1 polypeptides. 240-249 of SEQID NO: 213. 0238. In some embodiments exemplary nucleic acid mol 0233. In some embodiments the nucleic acid molecules ecules comprise a sequence set forth in SEQID NO: 152, 153, encode a PIP-1 polypeptide having a nucleotide sequence 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, encoding a polypeptide comprising an amino acid sequence 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 98%, 99% or greater identity to the amino acid sequence set 202, 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, forth in SEQ ID NO: 2, and wherein the polypeptide com 228, 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, prises one or more amino acid motifs selected from i) amino 240,241,242, 243, and 244 as well as variants and fragments acids 64-79 of SEQID NO:2 or amino acids 64-79 of SEQID thereof encoding PIP-1 polypeptides. NO: 213, ii) amino acids 149-159 of SEQID NO: 2 or amino 0239. In some embodiments the nucleic acid molecules acids 149-159 of SEQID NO: 213, iii) amino acids 171-183 encode a PIP-1 polypeptide of Table 4, Table 6, Table 9, Table of SEQID NO: 2 oramino acids 171-183 of SEQID NO: 213 12, Table 13, Table 14 and/or Table 16, combinations of the and iv) amino acids 240-249 of SEQID NO: 2 or amino acids amino acid Substitutions thereof and deletions and/or inser 240-249 of SEQID NO: 213. tions thereof. US 2014/0007292 A1 Jan. 2, 2014 20
0240 Also provided are nucleic acid molecules that Substance (Such as, for example, a protein) that can be mea encode transcription and/or translation products that are Sub Sured by, but is not limited to, insect mortality, insect weight sequently spliced to ultimately produce functional PIP-1 loss, insect repellency, and other behavioral and physical polypeptide. Splicing can be accomplished in vitro or in Vivo, changes of an insect after feeding and exposure for an appro and can involve cis- or trans-splicing. The Substrate for splic priate length of time. Thus, an organism or substance having ing can be polynucleotides (e.g., RNA transcripts) or insecticidal activity adversely impacts at least one measur polypeptides. An example of cis-splicing of a polynucleotide able parameter of insect fitness. For example, “insecticidal is where an intron inserted into a coding sequence is removed proteins’ are proteins that display insecticidal activity by and the two flanking exon regions are spliced to generate a themselves or in combination with other proteins. PIP-1 polypeptide encoding sequence. An example of trans 0242. As used herein, the term "pesticidally effective splicing would be where a polynucleotide is encrypted by amount connotes a quantity of a Substance or organism that separating the coding sequence into two or more fragments has pesticidal activity when present in the environment of a that can be separately transcribed and then spliced to form the pest. For each Substance or organism, the pesticidally effec full-length pesticidal encoding sequence. The use of a splic tive amount is determined empirically for each pest affected ing enhancer sequence, which can be introduced into a con in a specific environment. Similarly, an “insecticidally effec struct, can facilitate splicing either in cis or trans-splicing of tive amount may be used to refer to a "pesticidally effective polypeptides (U.S. Pat. Nos. 6,365.377 and 6,531,316). Thus, amount when the pest is an insect pest. in some embodiments the polynucleotides do not directly 0243. By “retains activity” is intended that the PIP-1A encode a full-length PIP-1 polypeptide, but rather encode a polypeptide has at least about 10%, at least about 30%, at least fragment or fragments of a PIP-1 polypeptide. These poly about 50%, at least about 70%, 80%, 90%, 95% or higher of nucleotides can be used to express a functional PIP-1 the insecticidal activity compared to the full-length PIP-1A polypeptide through a mechanism involving splicing, where polypeptide (SEQID NO:2). In one embodiment, the insec splicing can occur at the level of polynucleotide (e.g., intron/ ticidal activity is against a Lepidoptera species. In another exon) and/or polypeptide (e.g., intein/extein). This can be embodiment, the insecticidal activity is against a Hemiptera useful, for example, in controlling expression of pesticidal species. activity, since functional pesticidal polypeptide will only be 0244. In some embodiments a fragment of a nucleic acid expressed if all required fragments are expressed in an envi sequence encoding a PIP-1 polypeptide encoding a biologi ronment that permits splicing processes to generate func cally active portion of a protein will encode at least about 15, tional product. In another example, introduction of one or 25, 30, 50, 75, 100, 125, 150, 175, 200 or 250, contiguous more insertion sequences into a polynucleotide can facilitate amino acids or up to the total number of amino acids present recombination with a low homology polynucleotide; use of in a full-length PIP-1 polypeptide of the embodiments. In an intron or intein for the insertion sequence facilitates the Some embodiments, the fragment is an N-terminal or a C-ter removal of the intervening sequence, thereby restoring func minal truncation of at least about 1,2,3,4,5,6,7,8,9, 10, 11, tion of the encoded variant. 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acids 0241) Nucleic acid molecules that are fragments of these relative to SEQID NO: 2, 3 or 4 or variants thereof, e.g., by nucleic acid sequences encoding PIP-1 polypeptides are also proteolysis, insertion of a start codon, deletion of the codons encompassed by the embodiments. By “fragment” is encoding the deleted amino acids with the concomitant inser intended a portion of the nucleic acid sequence encoding a tion of a stop codon or by insertion of a stop codon in the PIP-1 polypeptide. A fragment of a nucleic acid sequence coding sequence. In some embodiments, the fragments may encode a biologically active portion of a PIP-1 polypep encompassed herein result from the removal of the N-termi tide or it may be a fragment that can be used as a hybridization nal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, probe or PCR primer using methods disclosed below. Nucleic 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33,34 or more acid molecules that are fragments of a nucleic acid sequence amino acids relative to SEQ ID NO: 2, 3 or 4 or variants encoding a PIP-1 polypeptide comprise at least about 50, 100, thereof, e.g., by proteolysis or by insertion of a start codon in 200, 300, 400, 500, 600 or 700, contiguous nucleotides or up the coding sequence. to the number of nucleotides present in a full-length nucleic 0245. In some embodiments the PIP-1 polypeptides are acid sequence encoding a PIP-1 polypeptide disclosed herein, encoded by a nucleic acid sequence Sufficiently identical to depending upon the intended use. By "contiguous nucle the nucleic acid sequence of SEQ ID NO: 1, 3 or 5. By otides is intended nucleotide residues that are immediately “sufficiently identical' is intended an amino acid or nucleic adjacent to one another. Fragments of the nucleic acid acid sequence that has at least about 50%, 55%, 60%. 65%, sequences of the embodiments will encode protein fragments 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, that retain the biological activity of the PIP-1 polypeptide 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and, hence, retain insecticidal activity. As used herein, the 98%, 99% or greater sequence identity compared to a refer term "pesticidal activity” refers to activity of an organism or ence sequence using one of the alignment programs described a Substance (such as, for example, a protein) that can be herein using standard parameters. In some embodiments the measured by, but is not limited to, pest mortality, pest weight sequence homology identity is against the full length loss, pest repellency, and other behavioral and physical sequence of the polynucleotide encoding a PIP-1 polypeptide changes of a pest after feeding and exposure for an appropri or against the full length sequence of a PIP-1 polypeptide. In ate length of time. Thus, an organism or Substance having some embodiments the PIP-1 polypeptide has at least about pesticidal activity adversely impacts at least one measurable 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, parameter of pest fitness. For example, “pesticidal proteins’ 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, are proteins that display pesticidal activity by themselves or 94%.95%,96%.97%.98%,99% or greater sequence identity in combination with other proteins. As used herein, the term compared to SEQID NO: 2, SEQID NO: 4, SEQID NO:332 “insecticidal activity” refers to activity of an organism or a or SEQID NO: 6. One of skill in the art will recognize that US 2014/0007292 A1 Jan. 2, 2014
these values can be appropriately adjusted to determine cor of the GCG Wisconsin Genetics Software Package, Version responding identity of proteins encoded by two nucleic acid 10 (available from Accelrys, Inc., 9685 Scranton Rd., San sequences by taking into account codon degeneracy, amino Diego, Calif., USA). When utilizing the ALIGN program for acid similarity, reading frame positioning, and the like. comparing amino acid sequences, a PAM 120 weight residue 0246 To determine the percent identity of two amino acid table, a gap length penalty of 12 and a gap penalty of 4 can be sequences or of two nucleic acids, the sequences are aligned used. for optimal comparison purposes. The percent identity 0249. Another non-limiting example of a mathematical between the two sequences is a function of the number of algorithm utilized for the comparison of sequences is the identical positions shared by the sequences (i.e., percent algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. identity=number of identical positions/total number of posi 48(3):443-453, used GAP Version 10 software to determine tions (e.g., overlapping positions)x100). In one embodiment, sequence identity or similarity using the following default the two sequences are the same length. In another embodi parameters: % identity and % similarity for a nucleic acid ment, the comparison is across the entirety of the reference sequence using GAP Weight of 50 and Length Weight of 3 sequence (e.g., across the entirety of SEQID NO: 1,331 or 3 and the nwsgapdna.cmpii scoring matrix; % identity or % or across the entirety of one of SEQID NO: 2,332 or 4). The similarity for an amino acid sequence using GAP weight of 8 percent identity between two sequences can be determined and length weight of 2, and the BLOSUM62 scoring pro using techniques similar to those described below, with or gram. Equivalent programs may also be used. By "equivalent without allowing gaps. In calculating percent identity, typi program' is intended any sequence comparison program that, cally exact matches are counted. for any two sequences in question, generates an alignment 0247 The determination of percent identity between two having identical nucleotide residue matches and an identical sequences can be accomplished using a mathematical algo percent sequence identity when compared to the correspond rithm. A non-limiting example of a mathematical algorithm ing alignment generated by GAPVersion 10. utilized for the comparison of two sequences is the algorithm 0250. The embodiments also encompass nucleic acid mol of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA ecules encoding variants of PIP-1 polypeptide. “Variants of 87:2264, modified as in Karlin and Altschul, (1993) Proc. the PIP-1 polypeptide encoding nucleic acid sequences Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is include those sequences that encode the PIP-1 polypeptides incorporated into the BLASTN and BLASTX programs of disclosed herein but that differ conservatively because of the Altschul, et al., (1990).J. Mol. Biol. 215:403. BLAST nucle degeneracy of the genetic code as well as those that are otide searches can be performed with the BLASTN program, sufficiently identical as discussed above. Naturally occurring score=100, wordlength=12, to obtain nucleic acid sequences allelic variants can be identified with the use of well-known homologous to pesticidal-like nucleic acid molecules. molecular biology techniques, such as polymerase chain BLAST protein searches can be performed with the BLASTX reaction (PCR) and hybridization techniques as outlined program, score 50, wordlength 3, to obtain amino acid below. Variant nucleic acid sequences also include syntheti sequences homologous to pesticidal protein molecules. To cally derived nucleic acid sequences that have been gener obtain gapped alignments for comparison purposes, Gapped ated, for example, by using site-directed mutagenesis but BLAST (in BLAST 2.0) can be utilized as described in Alts which still encode the PIP-1 polypeptides disclosed as dis chul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, cussed below. PSI-Blast can be used to perform an iterated search that 0251. The skilled artisan will further appreciate that detects distant relationships between molecules. See. Alts changes can be introduced by mutation of the nucleic acid chul, et al., (1997) supra. When utilizing BLAST, Gapped sequences thereby leading to changes in the amino acid BLAST, and PSI-Blast programs, the default parameters of sequence of the encoded PIP-1 polypeptides, without altering the respective programs (e.g., BLASTX and BLASTN) can the biological activity of the proteins. Thus, variant nucleic be used. Alignment may also be performed manually by acid molecules can be created by introducing one or more inspection. nucleotide substitutions, additions or deletions into the cor 0248. Another non-limiting example of a mathematical responding nucleic acid sequence disclosed herein, such that algorithm utilized for the comparison of sequences is the one or more amino acid Substitutions, additions or deletions ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids are introduced into the encoded protein. Mutations can be Res. 22:4673-4680). ClustalW compares sequences and introduced by Standard techniques, such as site-directed aligns the entirety of the amino acid or DNA sequence and mutagenesis and PCR-mediated mutagenesis. Such variant thus can provide data about the sequence conservation of the nucleic acid sequences are also encompassed by the present entire amino acid sequence. The ClustalW algorithm is used invention. in several commercially available DNA/amino acid analysis 0252 Alternatively, variant nucleic acid sequences can be software packages, such as the ALIGNX module of the Vector made by introducing mutations randomly along all or part of NTI Program Suite (Invitrogen Corporation, Carlsbad, the coding sequence, such as by Saturation mutagenesis and Calif.). After alignment of amino acid sequences with Clust the resultant mutants can be screened for ability to confer alW, the percent amino acid identity can be assessed. A non pesticidal activity to identify mutants that retain activity. Fol limiting example of a Software program useful for analysis of lowing mutagenesis, the encoded protein can be expressed ClustalW alignments is GENEDOCTM. GENEDOCTM (Karl recombinantly, and the activity of the protein can be deter Nicholas) allows assessment of amino acid (or DNA) simi mined using standard assay techniques. larity and identity between multiple proteins. Another non 0253) The polynucleotides of the disclosure and frag limiting example of a mathematical algorithm utilized for the ments thereofare optionally used as substrates for a variety of comparison of sequences is the algorithm of Myers and recombination and recursive recombination reactions, in Miller, (1988) CABIOS 4:11-17. Such an algorithm is incor addition to standard cloning methods as set forth in, e.g., porated into the ALIGN program (version 2.0), which is part Ausubel, Berger and Sambrook, i.e., to produce additional US 2014/0007292 A1 Jan. 2, 2014 22 pesticidal polypeptide homologues and fragments thereof and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, with desired properties. A variety of such reactions are et al., (1995) Gene, 164:49-53: Stemmer, (1995) Science known, including those developed by the inventors and their 270:1510; Stemmer, (1995) Bio/Technology 13:549-553; co-workers. Methods for producing a variant of any nucleic Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) acid listed herein comprising recursively recombining Such PNAS USA 91:10747-10751. polynucleotide with a second (or more) polynucleotide, thus forming a library of variant polynucleotides are also embodi 0258 Mutational methods of generating diversity include, ments of the disclosure, as are the libraries produced, the cells for example, site-directed mutagenesis (Ling, et al., (1997) comprising the libraries, and any recombinant polynucleotide Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods produces by Such methods. Additionally, Such methods Mol Biol 57:369-374; Smith, (1985) Ann Rev. Genet 19:423 optionally comprise selecting a variant polynucleotide from 462. Botstein and Shortle, (1985) Science 229:1193–1201; Such libraries based on pesticidal activity, as is wherein Such Carter, (1986) Biochem J 237: 1-7 and Kunkel, (1987) “The recursive recombination is done in vitro or in vivo. efficiency of oligonucleotide directed mutagenesis' in Nucleic Acids & Molecular Biology (Eckstein and Lilley, 0254. A variety of diversity generating protocols, includ eds. Springer Verlag, Berlin)); mutagenesis using uracil con ing nucleic acid recursive recombination protocols are avail taining templates (Kunkel, (1985) PNAS USA 82:488-492; able and fully described in the art. The procedures can be used Kunkel, et al., (1987) Methods Enzymol 154:367-382 and separately, and/or in combination to produce one or more Bass, et al., (1988) Science 242:240-245); oligonucleotide variants of a nucleic acid or set of nucleic acids, as well as directed mutagenesis (Zoller and Smith, (1983) Methods variants of encoded proteins. Individually and collectively, Enzymol 100:468-500; Zoller and Smith, (1987) Methods these procedures provide robust, widely applicable ways of Enzymol 154:329-350; Zoller and Smith, (1982) Nucleic generating diversified nucleic acids and sets of nucleic acids Acids Res 10:6487-6500), phosphorothioate-modified DNA (including, e.g., nucleic acid libraries) useful, e.g., for the mutagenesis (Taylor, et al., (1985) NuclAcids Res 13:8749 engineering or rapid evolution of nucleic acids, proteins, 8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 pathways, cells and/or organisms with new and/or improved (1985); Nakamaye and Eckstein (1986) Nucl Acids Res characteristics. 14:9679–9698; Sayers, et al., (1988) Nucl Acids Res 16:791 0255 While distinctions and classifications are made in 802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); the course of the ensuing discussion for clarity, it will be mutagenesis using gapped duplex DNA (Kramer, et al., appreciated that the techniques are often not mutually exclu (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, sive. Indeed, the various methods can be used singly or in (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) combination, in parallel or in series, to access diverse Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl Acids sequence variants. Res 16:6987-6999). 0256 The result of any of the diversity generating proce dures described herein can be the generation of one or more 0259. Additional suitable methods include point mis nucleic acids, which can be selected or screened for nucleic match repair (Kramer, et al., (1984) Cell 38:879-887), acids with or which confer desirable properties or that encode mutagenesis using repair-deficient host strains (Carter, et al., proteins with or which confer desirable properties. Following (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) diversification by one or more of the methods herein or oth Methods in Enzymol 154:382-403), deletion mutagenesis erwise available to one of skill, any nucleic acids that are (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res produced can be selected for a desired activity or property, 14:5115), restriction-selection and restriction-purification e.g. pesticidal activity or, such activity at a desired pH, etc. (Wells, et al., (1986) Phil Trans RSoc Lond A317:415-423), This can include identifying any activity that can be detected, mutagenesis by total gene synthesis (Nambiar, et al., (1984) for example, in an automated or automatable format, by any Science 223:1299-1301: Sakamar and Khorana, (1988) Nucl of the assays in the art, see, e.g., discussion of Screening of Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315 insecticidal activity, infra. A variety of related (or even unre 323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305 lated) properties can be evaluated, in serial or in parallel, at 3316), double-strand break repair (Mandecki, (1986) PNAS the discretion of the practitioner. USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 0257 Descriptions of a variety of diversity generating pro 4:450-455). Additional details on many of the above methods cedures for generating modified nucleic acid sequences, e.g., can be found in Methods Enzymol Volume 154, which also those coding for polypeptides having pesticidal activity or describes useful controls for trouble-shooting problems with fragments thereof, are found in the following publications various mutagenesis methods. and the references cited therein: Soong, et al., (2000) Nat 0260 Additional details regarding various diversity gen Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Target erating methods can be found in the following US Patents, ing 4:1-4; Ness et al. (1999) Nat Biotechnol 17:893-896: PCT Publications and Applications and EPO Publications: Changet al. (1999) Nat Biotechnol 17:793-797; Minshulland U.S. Pat. No. 5,723,323, U.S. Pat. No. 5,763,192, U.S. Pat. Stemmer, (1999) Curr Opin Chem Biol 3:284-290; Chris No. 5,814,476, U.S. Pat. No. 5,817,483, U.S. Pat. No. 5,824, tians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et 514, U.S. Pat. No. 5,976,862, U.S. Pat. No. 5,605,793, U.S. al., (1998) Nature 391:288-291; Crameri, et al., (1997) Nat Pat. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. No. Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA 5,834,252, U.S. Pat. No. 5,837,458, WO 1995/22625, WO 94:4504-4509: Patten, et al., (1997) Curr Opin Biotechnol 1996/33207, WO 1997/20078, WO 1997/35966, WO 1999/ 8:724-733; Crameri, et al., (1996) Nat Med 2:100-103; 41402, WO 1999/41383, WO 1999/41369, WO 1999/41368, Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, al., (1996).J Mol Biol 255:373-386; Stemmer, (1996) “Sexual WO 1998/31837, WO 1998/27230, WO 1998/27230, WO PCR and Assembly PCRIn: The Encyclopedia of Molecular 2000/00632, WO 2000/09679, WO 1998/42832, WO 1999/ Biology. VCH Publishers, New York. pp. 447-457; Crameri 29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, US 2014/0007292 A1 Jan. 2, 2014
WO 2000/18906, WO 2000/04190, WO 2000/42561, WO 0265. In hybridization methods, all or part of the pesticidal 2000/42559, WO 2000/42560, WO 2001/23401, and PCT/ nucleic acid sequence can be used to screen cDNA or USO1/06775. genomic libraries. Methods for construction of such cDNA 0261 The nucleotide sequences of the embodiments can and genomic libraries are generally known in the art and are also be used to isolate corresponding sequences from other disclosed in Sambrook and Russell. (2001), supra. The so organisms, particularly other bacteria, particularly a called hybridization probes may be genomic DNA fragments, Pseudomonas species and more particularly a Pseudomonas cDNA fragments, RNA fragments or other oligonucleotides, chlororaphis strain. In this manner, methods such as PCR, and may be labeled with a detectable group such as 32P or any hybridization and the like can be used to identify such other detectable marker, such as other radioisotopes, a fluo sequences based on their sequence homology to the rescent compound, an enzyme oran enzyme co-factor. Probes sequences set forth herein. Sequences that are selected based for hybridization can be made by labeling synthetic oligo on their sequence identity to the entire sequences set forth nucleotides based on the known PIP-1 polypeptide-encoding herein or to fragments thereof are encompassed by the nucleic acid sequence disclosed herein. Degenerate primers embodiments. Such sequences include sequences that are designed on the basis of conserved nucleotides or amino acid orthologs of the disclosed sequences. The term “orthologs' residues in the nucleic acid sequence or encoded amino acid refers to genes derived from a common ancestral gene and sequence can additionally be used. The probe typically com which are found in different species as a result of speciation. prises a region of nucleic acid sequence that hybridizes under Genes found in different species are considered orthologs stringent conditions to at least about 12, at least about 25, at when their nucleotide sequences and/or their encoded protein least about 50, 75, 100, 125, 150, 175 or 200 consecutive sequences share substantial identity as defined elsewhere nucleotides of nucleic acid sequence encoding a PIP-1 herein. Functions of orthologs are often highly conserved polypeptide of the disclosure or a fragment or variant thereof. among species. Methods for the preparation of probes for hybridization are 0262. In a PCR approach, oligonucleotide primers can be generally known in the art and are disclosed in Sambrook and designed for use in PCR reactions to amplify corresponding Russell, (2001), supra, herein incorporated by reference. DNA sequences from cDNA or genomic DNA extracted from 0266 For example, an entire nucleic acid sequence, any organism of interest. Methods for designing PCR primers encoding a PIP-1 polypeptide, disclosed herein or one or and PCR cloning are generally known in the art and are more portions thereof, may be used as a probe capable of disclosed in Sambrook, et al., (1989) Molecular Cloning. A specifically hybridizing to corresponding nucleic acid Laboratory Manual (2d ed., Cold Spring Harbor Laboratory sequences encoding PIP-1 polypeptide-like sequences and Press, Plainview, N.Y.), hereinafter “Sambrook”. See also, messenger RNAs. To achieve specific hybridization under a Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods variety of conditions, such probes include sequences that are and Applications (Academic Press, New York); Innis and unique and are preferably at least about 10 nucleotides in Gelfand, eds. (1995) PCR Strategies (Academic Press, New length or at least about 20 nucleotides in length. Such probes York); and Innis and Gelfand, eds. (1999) PCR Methods may be used to amplify corresponding pesticidal sequences Manual (Academic Press, New York). Known methods of from a chosen organism by PCR. This technique may be used PCR include, but are not limited to, methods using paired to isolate additional coding sequences from a desired organ primers, nested primers, single specific primers, degenerate ism or as a diagnostic assay to determine the presence of primers, gene-specific primers, Vector-specific primers, par coding sequences in an organism. Hybridization techniques tially-mismatched primers, and the like. include hybridization screening of plated DNA libraries (ei 0263. To identify potential PIP-1 polypeptides from bac ther plaques or colonies; see, for example, Sambrook, et al., terial collections, the bacterial cell lysates can be screened (1989) Molecular Cloning: A Laboratory Manual (2d ed., with antibodies generated against PIP-1A (SEQ ID NO: 2), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, PSEEN3174 (SEQID NO: 6), PIP-1B (SEQID NO: 4) and N.Y.). PIP-1C (SEQ ID NO: 332) proteins using Western blotting 0267. Hybridization of such sequences may be carried out and/or ELISA methods. This type of assays can be performed under stringent conditions. By “stringent conditions' or in a high throughput fashion. Positive samples can be further “stringent hybridization conditions' is intended conditions analyzed by various techniques such as antibody based pro under which a probe will hybridize to its target sequence to a tein purification and identification. Methods of generating detectably greater degree than to other sequences (e.g., at antibodies are well known in the art as discussed infra. least 2-fold over background). Stringent conditions are 0264. Alternatively, mass spectrometry based protein sequence-dependent and will be different in different circum identification method can be used to identify homologs of stances. By controlling the stringency of the hybridization PIP-1A (SEQ ID NO: 2) using protocols in the literatures and/or washing conditions, target sequences that are 100% (Patterson, (1998), 10(22):1-24, Current Protocol in Molecu complementary to the probe can be identified (homologous lar Biology published by John Wiley & Son Inc). Specifically, probing). Alternatively, stringency conditions can be adjusted LC-MS/MS based protein identification method is used to to allow some mismatching in sequences so that lower associate the MS data of given cell lysate or desired molecular degrees of similarity are detected (heterologous probing). weight enriched samples (excised from SDS-PAGE gel of Generally, a probe is less than about 1000 nucleotides in relevant molecular weight bands to PIP-1A protein) with length, preferably less than 500 nucleotides in length. sequence information of PIP-1A (SEQ ID NO: 2) and its 0268 Typically, stringent conditions will be those in homologs. Any match in peptide sequences indicates the which the salt concentration is less than about 1.5 MNaion, potential of having the homologous proteins in the samples. typically about 0.01 to 1.0 M Na ion concentration (or other Additional techniques (protein purification and molecular salts) at pH 7.0 to 8.3 and the temperature is at least about 30° biology) can be used to isolate the protein and identify the C. for short probes (e.g., 10 to 50 nucleotides) and at least sequences of the homologs. about 60° C. for long probes (e.g., greater than 50 nucle US 2014/0007292 A1 Jan. 2, 2014 24 otides). Stringent conditions may also be achieved with the insecticidal activity against one or more insect pests of the addition of destabilizing agents such as formamide. Exem Lepidoptera and/or Hemiptera orders compared to, and plary low stringency conditions include hybridization with a including, the protein of SEQID NO: 2, and is sufficiently buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS homologous to, and includes, the protein of SEQID NO: 2. A (sodium dodecyl sulphate) at 37° C., and a wash in 1x to variety of PIP-1 polypeptides are contemplated. One source 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 of polypeptides that encode a PIP-1 polypeptide or related to 55° C. Exemplary moderate stringency conditions include proteins is a Pseudomonas chlororaphis strain which com hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS prises the polynucleotide of SEQ ID NO: 1 encoding the at 37° C., and a wash in 0.5x to 1XSSC at 55 to 60° C. PIP-1 polypeptide of SEQID NO: 2. Exemplary high Stringency conditions include hybridization 0271 As used herein, the terms “protein.” “peptide mol in 50% formamide, 1 MNaCl, 1% SDS at 37°C., and a wash ecule' or “polypeptide' includes any molecule that com in 0.1xSSC at 60 to 65° C. Optionally, wash buffers may prises five or more amino acids. It is well known in the art that comprise about 0.1% to about 1% SDS. Duration of hybrid protein, peptide or polypeptide molecules may undergo ization is generally less than about 24 hours, usually about 4 modification, including post-translational modifications, to about 12 hours. Such as, but not limited to, disulfide bond formation, glyco 0269 Specificity is typically the function of post-hybrid Sylation, phosphorylation or oligomerization. Thus, as used ization washes, the critical factors being the ionic strength herein, the terms “protein.” “peptide molecule' or “polypep and temperature of the final wash solution. For DNA-DNA tide' includes any protein that is modified by any biological hybrids, the Tm can be approximated from the equation of or non-biological process. The terms "amino acid' and Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: “amino acids’ refer to all naturally occurring L-amino acids. Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)- 0272. A “recombinant protein’ is used to refer to a protein 500/L: where M is the molarity of monovalent cations, 96 GC that is no longer in its natural environment, for example in is the percentage of guanosine and cytosine nucleotides in the vitro or in a recombinant bacterial or plant host cell. A PIP-1 DNA,% form is the percentage of formamide in the hybrid polypeptide that is substantially free of cellular material ization solution, and L is the length of the hybrid in base pairs. includes preparations of protein having less than about 30%, The Tm is the temperature (under defined ionic strength and 20%, 10% or 5% or less (by dry weight) of non-pesticidal pH) at which 50% of a complementary target sequence protein (also referred to hereinas a “contaminating protein'). hybridizes to a perfectly matched probe. Tm is reduced by 0273 "Fragments’ or “biologically active portions' about 1° C. for each 1% of mismatching; thus, Tm, hybrid include polypeptide fragments comprising amino acid ization, and/or wash conditions can be adjusted to hybridize sequences sufficiently identical to a PIP-1 polypeptide and to sequences of the desired identity. For example, if that exhibit insecticidal activity. "Fragments' or “biologi sequences with 90% identity are sought, the Tm can be cally active portions' include polypeptide fragments com decreased 10° C. Generally, stringent conditions are selected prising amino acid sequences Sufficiently identical to the to be about 5° C. lower than the thermal melting point (Tm) amino acid sequence set forth in SEQID NO: 2, 4,332 and 6 for the specific sequence and its complement at a defined including but not limited to SEQ ID NO. 204, 206 and 208 ionic strength and pH. However, severely stringent conditions and that exhibit insecticidal activity. A biologically active can utilize a hybridization and/or wash at 1, 2, 3 or 4°C. lower portion of a PIP-1 polypeptide can be a polypeptide that is, for than the thermal melting point (Tm); moderately stringent example, 10, 25, 50, 100, 150, 200, 250 or more amino acids conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 in length. Such biologically active portions can be prepared or 10° C. lower than the thermal melting point (Tm): low by recombinant techniques and evaluated for insecticidal stringency conditions can utilize a hybridization and/or wash activity. As used here, a fragment comprises at least 8 con at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting tiguous amino acids of a PIP-1 polypeptide. In some embodi point (Tm). Using the equation, hybridization and wash com ments afragment comprises at least 8 contiguous amino acids positions, and desired Tm, those of ordinary skill will under of SEQ ID NO: 2 or 4. In some embodiments a fragment stand that variations in the stringency of hybridization and/or comprises at least 8 contiguous amino acids of SEQID NO: wash solutions are inherently described. If the desired degree 2. In some embodiments a fragment comprises at least 8 of mismatching results in a Tm of less than 45° C. (aqueous contiguous amino acids of SEQID NO: 4. The embodiments solution) or 32° C. (formamide solution), it is preferred to encompass other fragments, however, Such as any fragment in increase the SSC concentration so that a higher temperature the protein greater than about 10, 20, 30, 50, 100, 150, 200, can be used. An extensive guide to the hybridization of 250 or more amino acids. nucleic acids is found in Tijssen, (1993) Laboratory Tech niques in Biochemistry and Molecular Biology Hybridiza 0274. In some embodiments, the fragment is an N-termi nal and/or a C-terminal truncation of at least about 1, 2, 3, 4, tion with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or N.Y.); and Ausubel, et al., eds. (1995) Current Protocols in more amino acids relative to SEQID NO: 2 or 4 or variants Molecular Biology, Chapter 2 (Greene Publishing and Wiley thereof e.g., by proteolysis, by insertion of a start codon, by Interscience, New York). See, Sambrook, et al., (1989) deletion of the codons encoding the deleted amino acids and Molecular Cloning: A Laboratory Manual (2d ed., Cold concomitant insertion of a start codon and/or insertion of a Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). stop codon. In some embodiments, the fragments encom passed herein result from the removal of the N-terminal 1, 2, Proteins and Variants and Fragments. Thereof 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, (0270. Pseudomonas Insecticidal Protein-1 (PIP-1) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33,34 or more amino polypeptides are also encompassed by the disclosure. By acids relative to SEQID NO: 2 or 4, and variants thereof (e.g., “Pseudomonas Insecticidal Protein-1”. “PIP-1 polypeptide' SEQID NO. 204, 206,208 and 330), e.g., by proteolysis or by or “PIP-1 protein’ is intended a polypeptide that retains insertion of a start codon, by deletion of the codons encoding US 2014/0007292 A1 Jan. 2, 2014
the deleted amino acids and concomitant insertion of a start Tyror Phe: Xaa at position 60 is Ala or Ser; Xaa at position 63 codon. In particular embodiments the proteolytic cleavage is Gln or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position site is between Ser34 and Asn35 of SEQID NO: 2 or variants 97 is Met or Val: Xaa at position 98 is Asp or Glu, Xaa at thereof. In some embodiments the truncation is of the first 34 position 105 is Glin or ASn; Xaa at position 107 is Thr or Ile: amino acids of SEQID NO: 2 resulting in a PIP-1 polypeptide Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg from amino acids 35-271 of SEQID NO: 2. It is well known or Leu, Xaa at position 120 is Lys, Argor Glin; Xaa at position in the art that polynucleotides encoding the truncated PIP-1 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; Xaa at polypeptides can be engineered to add a start codon at the position 125 is ASnor Ser; Xaa at position 127 is Ser, Asn. Thr N-terminus Such as ATG encoding methionine or methionine or Lys; Xaa at position 134 is Gly or Ala; Xaa at position 135 followed by an alanine. It is also well known in the art that is Ser, Asn or Lys; Xaa at position 137 is Asp or Gly; Xaa at depending on what host the PIP-1 polypeptide is expressed in position 141 is Val or Ile: Xaa at position 142 is Gly or Asp; the methionine may be partially of completed processed off. Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, 0275. In some embodiments fragments, biologically Thr or Val: Xaa at position 150 is Seror Thr; Xaa at position active portions of SEQ ID NO: 2 or 4, including but not 151 is Asn, Arg or Ser; Xaa at position 160 is Thr or Ser; Xaa limited to SEQID NO: 101, 102, 103, 104, 105, 106, 107, at position 162 is Ser or Thr; Xaa at position 163 is Asn., Asp 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or Glu; Xaa at position 164 is Seror Thr; Xaa at position 166 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, is Gln or Glu, Xaa at position 167 is Leu or Met; Xaa at 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, position 168 is Thr, Lys or Ala; Xaa at position 174 is Ile, Val 144, 145, 146, 147, 148, 149, 150, 151, 204, 206, 208, 211, or Met; Xaa at position 175 is Val or Ile: Xaa at position 180 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, 252, 253, is Met or Leu; Xaa at position 191 is Arg or Lys: Xaa at 254, 255, 256, 257, 258, 259,260, 261, 262, 263,264, 265, position 194 is Gly or Ala; Xaa at position 200 is Asn or Ser; 266, 267, 268, and 269, as well as amino acid substitutions, Xaa at position 203 is ASnor Gln; Xaa at position 204 is Thr deletions and/or insertions thereofare also provided, and may or Ala; Xaa at position 206 is Gly or Asp; Xaa at position 209 be used to practice the methods of the disclosure. is Leu or Val; Xaa at position 220 is ASn or Arg; Xaa at 0276 By variants is intended proteins or polypeptides position 221 is Ser or Lys: Xaa at position 222 is Thr or Arg; having an amino acid sequence that is at least about 50%, Xaa at position 226 is Asp, Pro or Glu; Xaa at position 228 is 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, Seror Gly: Xaa at position 229 is Lys or ASn; Xaa at position 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 231 is Ile or Val: Xaa at position 232 is Ala, Thr or Glu; and 95%, 96%, 97%, 98% or 99% identical to the parental amino Xaa at position 251 is Gly, Seror Glu; Xaa at position 254 is acid sequence. In some embodiments a PIP-1 polypeptide has Seror Asn; Xaa at position 258 is Seror Arg; Xaa at position at least about 60%, 65%, about 70%, 75%, at least about 80%, 265 is ASnor Asp; and Xaa at position 266 is Asp or ASn; and 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, amino acid deletions, amino acid insertions, and fragments 91%, 92%.93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereof, and combinations thereof. across the entire length of the amino acid sequence of SEQID 0279. In some embodiments a PIP-1 polypeptide com NO: 2, SEQID NO:332 or SEQID NO: 4. In some embodi prises an amino acid sequence of SEQID NO: 211 having 1, ments a PIP-1 polypeptide has at least about 80%, 81%, 82%, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the 38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, entire length of the amino acid sequence of SEQID NO: 2. 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, in any 0277. In some embodiments a PIP-1 polypeptide com combination, at residues designated by Xaa in SEQID NO: prises an amino acid sequence having at least 80% identity, to 211 compared to the native amino acid at the corresponding the amino acid sequence of SEQID NO: 2, SEQID NO:332 position of SEQID NO: 2. or SEQ ID NO:4, wherein the polypeptide has insecticidal 0280. In some embodiments a PIP-1 polypeptide com activity. In some embodiments a PIP-1 polypeptide com prises an amino acid sequence of SEQID NO: 211 having 1, prises an amino acid sequence having at least 80% identity to 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, the amino acid sequence of SEQID NO: 2, SEQID NO:332 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, or SEQ ID NO: 4, wherein the polypeptide has insecticidal 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53 or activity. In some embodiments a PIP-1 polypeptide com 54 amino acid Substitutions, in any combination, at residues prises an amino acid sequence having at least 80% identity to designated by Xaa in SEQID NO: 211 compared to the native the amino acid sequence of SEQ ID NO: 2, wherein the amino acid at the corresponding position of SEQID NO: 2. polypeptide has insecticidal activity. 0281. In some embodiments a PIP-1 polypeptide com 0278. In some embodiments a PIP-1 polypeptide com prises an amino acid sequence of SEQID NO: 212, wherein prises an amino acid sequence of SEQID NO: 211, wherein Xaa at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: Xaa at position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly or Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly Asn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position 20 or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position is Leu or Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at 20 is Leu or Val: Xaa at position 21 is Lys, Ser or ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position 24 is Glin or Ala; position 22 is Ser, Lys or Arg; Xaaat position 24 is Glnor Ala; Xaa at position 25 is Gly or Ala Xaa at position 26 is Ser or Xaa at position 25 is Gly or Ala; Xaa at position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 30 Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 28 is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, 36 is Ala, Seror Val: Xaa at position 38 is Asn, Argor Ser; Xaa His, Cys or Gln; Xaa at position 30 is Ala or Ile: Xaa at at position 42 is Phe or Tyr, Xaa at position 46 is Arg, Lys or position 35 is Phe or Leu; Xaa at position36 is Ala, Seror Val; His;Xaaat position 48 is Gly or Asp;Xaaat position 49 is Phe Xaaat position 38 is Asn, Argor Ser; Xaaat position 42 is Phe or Tyr; Xaa at position 53 is Ser or Gly; Xaa at position 58 is or Tyr; Xaa at position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, US 2014/0007292 A1 Jan. 2, 2014 26
Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, position 46 is Arg, Lys or His; Xaa at position 48 is Gly or Ile, Asp, Gly or Ala; Xaa at position 249 is Asn. Lys, Val, Gly, Asp; Xaa at position 49 is Phe, Tyr or Leu; Xaa at position 53 Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, Thr, Pro, Leu, is Seror Gly: Xaa at position 58 is Tyr or Phe: Xaa at position Ala, His or Glin; Xaa at position 251 is Gly, Seror Glu; Xaa at 60 is Ala or Ser; Xaa at position 63 is Glin or Lys; Xaa at position 254 is Ser or Asn; Xaa at position 258 is Ser or Arg; position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or at position 77 is Phe or Tyr; Xaa at position 89 is Pro, Leu, His; Xaa at position 265 is ASn or Asp; and Xaa at position Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys or Lys; Xaa at 266 is Asp or ASn; and amino acid deletions, amino acid position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, insertions, and fragments thereof, and combinations thereof. Ala or Thr; Xaa at position 97 is Met or Val: Xaa at position 0282. In some embodiments a PIP-1 polypeptide com 98 is Asp or Glu; Xaa at position 105 is Glin or Asn; Xaa at prises an amino acid sequence of SEQID NO: 212 having 1, position 107 is Thr or Ile: Xaa at position 108 is Glin or Thr: 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Xaa at position 110 is Arg or Leu, Xaa at position 120 is LyS, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, Argor Glin; Xaa at position 121 is Thr or Ser; Xaa at position 38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 123 is Thr or Glu; Xaa at position 125 is Asn or Ser; Xaa at 55, 56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71, position 127 is Ser, Asn. Thror Lys; Xaaat position 134 is Gly 72, 73,74, 75,76, 77,78, 79,80, 81, 82, 83, 84,85, 86, 87, 88 or Ala; Xaa at position 135 is Ser, ASnor LyS, Xaa at position or 89 amino acid substitutions, in any combination, at resi 137 is Asp or Gly; Xaa at position 141 is Val or Ile: Xaa at dues designated by Xaa in SEQID NO: 212 compared to the position 142 is Gly or Asp; Xaa at position 144 is Asp or Glu; native amino acid at the corresponding position of SEQ ID Xaaat position 147 is Ile, ThrorVal: Xaaat position 150 is Ser NO: 2. or Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position 0283. In some embodiments a PIP-1 polypeptide com 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at prises an amino acid sequence of SEQID NO: 212 having 1, position 163 is Asn., Asp or Glu; Xaa at position 164 is Seror 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Thr; Xaa at position 166 is Glin or Glu; Xaa at position 167 is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, Leu or Met; Xaa at position 168 is Thr, Lys or Ala; Xaa at 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53 or position 171 is Gly, Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; 54 amino acid Substitutions, in any combination, at residues Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. designated by Xaa in SEQID NO: 212 compared to the native Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaa at position 173 is amino acid at the corresponding position of SEQID NO: 2. Phe, Gly. His, Leu, Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr 0284. In some embodiments a PIP-1 polypeptide com or Met; Xaaat position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, prises an amino acid sequence of SEQID NO: 213 wherein Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at Xaa at position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, position 175 is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaa Thr, Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp at position 176 is Tyr, Met, Phe, Leu or Cys; Xaa at position or Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at 177 is Gln, Ile, Met or Pro; Xaa at position 178 is Val, Cys, position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, Val, Thr, Pro, Ala, Met, Gln, Phe, Ile, Ser or Lys; Xaa at position Ile or Met; Xaaat position 21 is Lys, Ser, Asn, Arg, Thror Gln; 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, Ala or Gln: Xaa Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at position 24 at position 180 is Met, Leu, Pro, Trp, Asn., Tyr, Gly, Gln, Ala, is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly or Ala; Xaa Val, Phe, Ile, Cys or Ser; Xaa at position 181 is Val, Ala, Leu, at position 26 is Ser, Asn. Thr or Glin; Xaa at position 27 is Trp, Cys, Thr, Ile or Lys: Xaa at position 182 is Tyr, Phe, Met Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at position 28 is Arg, or His: Xaa at position 183 is Ala, Met, Val, Thr, Asp, Gly, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, His, Cys Cys, Ile, Phe, Ser, Gln or Leu; Xaa at position 191 is Arg or or Gln; Xaa at position 30 is Ala, Ile, Leu, Val or Met; Xaa at Lys; Xaa at position 194 is Gly or Ala; Xaa at position 195 is position 35 is Phe, Leu, Ile, Val or Met; Xaa at position 36 is Asin or Tyr; Xaa at position 200 is Asn or Ser; Xaa at position Ala, Ser. Thr, Val, Ile or Leu; Xaa at position 38 is Asn, Arg, 203 is Asn or Glin; Xaa at position 204 is Thr or Ala; Xaa at Ser, Gln, Lys or Thr; Xaa at position 42 is Phe, Tyr, Trp, Leu, position 206 is Gly or Asp; Xaa at position 209 is Leu or Val; Ile, Val or Met; Xaa at position 43 is Pro, Met, Gly, Gln, Ser, Xaa at position 213 is Tyr or Phe: Xaa at position 220 is Asn Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Cys; or Arg; Xaa at position 221 is Seror Lys; Xaa at position 222 Xaaat position 46 is Arg, Lys or His;Xaa at position 48 is Gly, is Thr or Arg; Xaa at position 226 is Asp, Pro or Glu; Xaa at Asp, Ala or Glu, Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, position 228 is Seror Gly: Xaa at position 229 is Lys or ASn; Val or Met; Xaa at position 53 is Ser, Gly, Ala or Thr; Xaa at Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, position 58 is Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Thror Glu:Xaa at position 240 is Gln, Arg, Ala, Val, Glu, Met, Thr, Xaa at position 63 is Gln, Lys, ASn or Arg; Xaa at Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Val or Ser; Xaa position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, at position 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys: Xaa at position position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 ASn, Ile, Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, is Val, Leu, Ala, Thr, Gly, Cys, Ile, Seror Met; Xaa at position Val, Leu or Ile: Xaa at position 98 is Asp or Glu, Xaa at 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at position 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, Leu or Val: Xaa at position 108 is Gln, Thr, Seror Asn; Xaa at Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or ASn; Xaa at position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at position position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, Ser, 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thror Ser; ASn, Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys: Xaa at Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at position position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, LyS, 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is Ser, Asn. Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thror Cys; Xaa at Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is Gly or Ala; US 2014/0007292 A1 Jan. 2, 2014 27
Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or Lys; Xaa at 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, position 137 is Asp, Gly, Glu or Ala; Xaa at position 141 is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, Val, Ile or Leu; Xaa at position 142 is Gly, Asp, Ala or Glu; 38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, 55, 56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71, Thr, Val, Leu, Met or Ser; Xaa at position 150 is Ser or Thr: 72, 73,74, 75,76, 77,78, 79,80, 81, 82, 83, 84,85, 86, 87, 88 Xaa at position 151 is ASn, Arg, Ser, Gln, Lys or Thr, Xaa at or 89 amino acid substitutions, in any combination, at resi position 160 is Thr or Ser; Xaa at position 162 is Seror Thr: dues designated by Xaa in SEQID NO: 213 compared to the Xaa at position 163 is ASn, Asp, Glu or Glin; Xaa at position native amino acid at the corresponding position of SEQ ID 164 is Seror Thr; Xaa at position 166 is Gln, Glu, Asp or ASn; NO: 2. Xaa at position 167 is Leu, Met, Ile, Val: Xaa at position 168 0286. In some embodiments a PIP-1 polypeptide com is Thr, Lys, Ala, Ser, Arg or Gly: Xaa at position 171 is Gly, prises an amino acid sequence of SEQID NO: 213 having 1, Leu, Gln, Met, Cys, Asn., Asp, Seror Ala; Xaa at position 172 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, is Thr, Gly. His, Phe, Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, Cys, Val, Ala or Met; Xaaat position 173 is Phe, Gly. His, Leu, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53 or Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr or Met; Xaa at 54 amino acid Substitutions, in any combination, at residues position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Met, Cys, designated by Xaa in SEQID NO: 213 compared to the native Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at position 175 amino acid at the corresponding position of SEQID NO: 2. is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaaat position 176 0287. In some embodiments a PIP-1 polypeptide com is Tyr, Met, Phe, Leu or Cys: Xaa at position 177 is Gln, Ile, prises one or more amino acid motifs selected from i) an Metor Pro; Xaa at position 178 is Val, Cys, Thr, Pro, Ala, Met, amino acid motif represented by amino acids at positions Gln, Phe, Ile, Seror Lys; Xaa at position 179 is Val, Phe, Thr, 64-79 of SEQID NO: 2, amino acids 64-79 of SEQID NO: Ile, Cys, Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, 211, amino acids 64-79 of SEQID NO: 212 or amino acids Leu: Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or 64-79 of SEQID NO: 213, ii) an amino acid motifrepresented Ser; Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or by amino acids at positions 149-159 of SEQID NO: 2, amino Lys; Xaa at position 182 is Tyr, Phe, Met or His: Xaa at acids 149-159 of SEQID NO: 211, amino acids 149-159 of position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, SEQID NO: 212 oramino acids 149-159 of SEQIDNO: 213, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at position iii) an amino acid motif represented by amino acids at posi 194 is Gly or Ala; Xaa at position 195 is Asn., Tyr, Gln or Trp; tions 171-183 of SEQID NO:2, amino acids 171-183 of SEQ Xaa at position 200 is ASn, Ser. Thr or Gln; Xaa at position ID NO: 211, amino acids 171-183 of SEQ ID NO: 212 or 203 is ASnor Glin; Xaa at position 204 is Thr, Ala, Seror Gly: amino acids 171-183 of SEQID NO: 213, and iv) an amino Xaa at position 206 is Gly, Asp, Ala or Glu; Xaa at position acid motif represented by amino acids at positions 240-249 of 209 is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: SEQ ID NO: 2, amino acids 240-249 of SEQ ID NO: 211, Xaa at position 220 is ASn, Arg, Gln or Lys, Xaa at position amino acids 240-249 of SEQ ID NO: 212 or amino acids 221 is Ser, Lys, Thror Arg; Xaaat position 222 is Thr, Arg, Ser 240-249 of SEQIDNO: 213. In some embodiments the PIP-1 or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at polypeptide comprises an amino acid as represented by posi position 228 is Seror Gly; Xaaat position 229 is Lys, Asn, Arg tions 171-183 of SEQID NO: 213 wherein at least one amino or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa at acid at positions 171-183 of SEQID NO: 213 are not identical position 232 is Ala, Thr, Ser, Gly, Asp or Glu, Xaa at position to amino acids at positions 171-183 of SEQID NO: 6. 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, Asn. Thr, 0288. In some embodiments a PIP-1 polypeptide com Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 is Arg, Lys, prises an amino acid sequence having at least 80% identity to Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, Pro, Gly, Leu, the amino acid sequence set forth in SEQID NO: 2, SEQID Phe, Thr, Ala or Cys;Xaa at position 242 is ASn, Ala, Arg, LyS, NO: 332 or SEQID NO. 4 and comprises one or more amino His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, acid motifs selected from i) an amino acid motif represented Leu or Val: Xaa at position 243 is Val, Leu, Ala, Thr, Gly, Cys, by amino acids at positions 64-79 of SEQ ID NO: 2, amino Ile, Seror Met; Xaa at position 244 is Leu, Val, Phe, Ile, Met, acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ Gln, Cys, Trp or Ala; Xaa at position 245 is Met, Ala, Arg, ID NO: 212 or amino acids 64-79 of SEQID NO: 213, ii) an Asp, Glu, Leu, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, amino acid motif represented by amino acids at positions Ile, Gln, Tyr or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, 149-159 of SEQID NO: 2, amino acids 149-159 of SEQID Arg, Val, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, NO: 211, amino acids 149-159 of SEQID NO: 212 or amino Phe, Thr or Lys; Xaa at position 247 is Asn. Leu, Asp, Tyr, acids 149-159 of SEQID NO: 213, iii) an amino acid motif Ala, Phe, His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, represented by amino acids at positions 171-183 of SEQID Trp, Thror Cys: Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, NO: 2, amino acids 171-183 of SEQID NO: 211, amino acids Ser. His, Cys, Leu, Trp, Ile, Asp, Gly or Ala, Xaa at position 171-183 of SEQID NO: 212 or amino acids 171-183 of SEQ 249 is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, ID NO: 213, and iv) an amino acid motif represented by Tyr, Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position amino acids at positions 240-249 of SEQ ID NO: 2, amino 251 is Gly, Ser, Thr, Ala, Asp or Glu; Xaa at position 254 is acids 240-249 of SEQID NO: 211, amino acids 240-249 of Ser, Asn. Thr or Glin; Xaa at position 258 is Ser, Arg, Thr or SEQID NO: 212 oramino acids 240-249 of SEQIDNO: 213. Lys; Xaa at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, 0289. In some embodiments a PIP-1 polypeptide com Ile or His; Xaa at position 265 is Asn., Asp, Gln or Glu; and prises an amino acid sequence having at least 80% identity to Xaa at position 266 is Asp, ASn, Gln or Glu, and amino acid the amino acid sequence set forth in SEQID NO: 2, SEQID deletions, amino acid insertions and fragments thereof, and NO: 332 or SEQID NO. 4 and comprises one or more amino combinations thereof. acid motifs selected from i) an amino acid motif represented 0285. In some embodiments a PIP-1 polypeptide com by amino acids at positions 64-79 of SEQ ID NO: 2, amino prises an amino acid sequence of SEQID NO: 213 having 1, acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ US 2014/0007292 A1 Jan. 2, 2014 28
ID NO: 212 or amino acids 64-79 of SEQID NO: 213, ii) an 0295. In some embodiments a PIP-1 polypeptide does not amino acid motif represented by amino acids at positions have the amino acid sequence of SEQ ID NO: 4. In some 149-159 of SEQID NO: 2, amino acids 149-159 of SEQID embodiments a PIP-1 polypeptide does not have the amino NO: 211, amino acids 149-159 of SEQID NO: 212 or amino acid sequence of SEQID NO: 6. acids 149-159 of SEQID NO: 213, iii) an amino acid motif 0296. In some embodiments a PIP-1 polypeptide has a represented by amino acids at positions 171-183 of SEQID calculated molecular weight of between about 15 kD and NO: 2, amino acids 171-183 of SEQID NO: 211, amino acids about 35 kD, between about 19 kD and about 35kD, between 171-183 of SEQID NO: 212 or amino acids 171-183 of SEQ about 21 kD and about 35kD, between about 23 kD and about ID NO: 213, and iv) amino acids 240-249 of SEQID NO: 2, 35kD, between about 25 kD and about 32 kD, between about amino acids 240-249 of SEQID NO: 211, amino acids 240 27 kD and about 32 kD, between about 28 kD and about 32 249 of SEQID NO: 212 or amino acids 240-249 of SEQID kD, between about 29 kD and about 32 kD, between about 30 NO: 213. kD and about 31 kD or about 30.5 kD. 0290. In some embodiments the amino acid motifs repre 0297. In some embodiments a PIP-1 polypeptide is sented by i) amino acids 64-79 of SEQID NO: 2, amino acids encoded by a nucleic acid molecule that hybridizes under 64-79 of SEQID NO: 211, amino acids 64-79 of SEQID NO: stringent conditions to the nucleic acid molecule of SEQID 212 or amino acids 64-79 of SEQID NO: 213, ii) amino acids NO: 1 or 3. Variants include polypeptides that differinamino 149-159 of SEQID NO: 2, amino acids 149-159 of SEQID acid sequence due to mutagenesis. Variant proteins encom NO: 211, amino acids 149-159 of SEQID NO: 212 or amino passed by the disclosure are biologically active, that is they acids 149-159 of SEQID NO: 213, iii) amino acids 171-183 continue to possess the desired biological activity (i.e. pesti of SEQID NO: 2, amino acids 171-183 of SEQID NO: 211, cidal activity) of the native protein. By “retains activity” is amino acids 171-183 of SEQ ID NO: 212 or amino acids intended that the variant will have at least about 30%, at least 171-183 of SEQID NO: 213, and iv) amino acids 240-249 of about 50%, at least about 70% or at least about 80% of the SEQ ID NO: 2, amino acids 240-249 of SEQ ID NO: 211, insecticidal activity of the native protein. In some embodi amino acids 240-249 of SEQ ID NO: 212 or amino acids ments, the variants may have improved activity over the 240-249 of SEQ ID NO: 213, the amino acid motif may native protein. optional have a deletion of one or more amino acids within the 0298 Bacterial genes quite often possess multiple motif, a insertion of one or more amino acids within the motif methionine initiation codons in proximity to the start of the or combinations thereof. open reading frame. Often, translation initiation at one or 0291. In some embodiments exemplary PIP-1 polypep more of these start codons will lead to generation of a func tides are encoded by the polynucleotide sequence set forth in tional protein. These start codons can include ATG codons. SEQID NO: 152, 153,154, 155, 156,157,158, 159, 160,161, For example, SEQ ID NO: 215 represent alternate start site 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, protein encoded by SEQID NO: 1. However, bacteria such as 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, Bacillus sp. also recognize the codon GTG as a start codon, 186, 197, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, and proteins that initiate translation at GTG codons contain a 198, 199, 200, 201, 202, 203, 205, 207, 220, 221, 222, 223, methionine at the first amino acid. On rare occasions, trans 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, lation in bacterial systems can initiate at a TTG codon, though 236, 237,238,239, 240, 241, 242, 243, 244, 270, 271, 272, in this event the TTG encodes a methionine. Furthermore, it is 273, 274, 275,276, 277,278, 279, 280, 281, 282,283, 284, not often determined a priori which of these codons are used 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, naturally in the bacterium. Thus, it is understood that use of and 297 as well as variants and fragments thereof encoding one of the alternate methionine codons may also lead to PIP-1 polypeptides. generation of pesticidal proteins. These pesticidal proteins 0292. In some embodiments exemplary nucleic acid mol are encompassed in the present disclosure and may be used in ecules comprise a sequence set forth in SEQID NO: 152, 153, the methods of the present disclosure. It will be understood 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, that, when expressed in plants, it will be necessary to alter the 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, alternate start codon to ATG for proper translation. 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 0299. In another aspect the PIP-1 polypeptide may be 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, expressed as a precursor protein with an intervening sequence 202, 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, that catalyzes multi-step, post translational protein splicing. 228, 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, Protein splicing involves the excision of an intervening 240,241,242, 243, and 244 as well as variants and fragments sequence from a polypeptide with the concomitant joining of thereof encoding PIP-1 polypeptides. the flanking sequences to yield a new polypeptide (Chong, et al., (1996).J. Biol. Chem. 271:22159-22168). This interven 0293. In some embodiments a PIP-1 polypeptide includes ing sequence or protein splicing element, referred to as variants where an amino acid that is part of a proteolytic inteins, which catalyze their own excision through three coor cleavage site is changed to another amino acid to eliminate or dinated reactions at the N-terminal and C-terminal splice alter the proteolytic cleavage at that site. In some embodi junctions: an acyl rearrangement of the N-terminal cysteine ments the proteolytic cleavage is by a protease in the insect or Serine; a transesterification reaction between the two ter gut. In other embodiments the proteolytic cleavage is by a mini to form a branched ester or thioester intermediate and plant protease in the transgenic plant. peptide bond cleavage coupled to cyclization of the intein 0294. In some embodiments exemplary PIP-1 polypep C-terminal asparagine to free the intein (Evans, et al., (2000) tides are the polypeptides shown in Table 4, Table 6, Table 9, J. Biol. Chen. 275:9091-9094. The elucidation of the mecha Table 12, Table 13, Table 14 and/or Table 16 and combina nism of protein splicing has led to a number of intein-based tions of the amino substitutions thereofas well as deletions, applications (Comb, et al., U.S. Pat. No. 5,496.714; Comb, et and or insertions and fragments thereof. al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999).J. US 2014/0007292 A1 Jan. 2, 2014 29
Amer: Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 580(7):1853-8). Non-split inteins have been artificially split 192:271-281, Chong, et al., (1998) Nucleic Acids Res. in the laboratory to create new split inteins, for example: the 26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273: artificially split Ssp DnaB intein (see, Wu, et al., (1998) 10567-10577; Cotton, et al., (1999).J. Am. Chem. Soc. 121: Biochim Biophy's Acta 1387:422-32) and split Sce VMA 1100-1101: Evans, et al., (1999).J. Biol. Chem. 274:18359 intein (see, Brenzel, et al., (2006) Biochemistry 45(6): 1571 18363; Evans, et al., (1999).J. Biol. Chem. 274:3923-3926: 8) and an artificially split fungal mini-intein (see, Elleuche, et Evans, et al., (1998) Protein Sci. 7:2256-2264: Evans, et al., al., (2007) Biochem Biophy's Res Commun 355(3):830-4). (2000).J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun, There are also intein databases available that catalogue (1999) FEBS Lett. 459:166-172: Mathys, et al., (1999) Gene known inteins (see, for example the online-database available 231:1-13; Mills, et al., (1998) Proc. Natl. Acad. Sci. USA at: bioinformatics. Weizmann.ac.il/pietro/inteins/Intein 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA stable.html, which can be accessed on the world-wide web 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040 using the “www’ prefix). 16044: Otomo, et al., (1999).J. Biolmol. NMR 14:105-114; 0302 Naturally-occurring non-split inteins may have Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638 endonuclease or other enzymatic activities that can typically 13643: Severinov and Muir, (1998) J. Biol. Chem. 273: be removed when designing an artificially-split split intein. 16205-16209; Shingledecker, et al., (1998) Gene 207:187 Such mini-inteins or minimized split inteins are well known 195; Southworth, et al., (1998) EMBO J. 17:918-926; in the art and are typically less than 200 amino acid residues Southworth, et al., (1999) Biotechniques 27:110-120: Wood, long (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422 et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) 32). Suitable split inteins may have other purification Proc. Natl. Acad. Sci. USA95:9226-9231; Wu, et al., (1998b) enabling polypeptide elements added to their structure, pro Biochim Biophy's Acta 1387:422-432; Xu, et al., (1999) Proc. vided that such elements do not inhibit the splicing of the split Natl. Acad. Sci. USA 96:388-393:Yamazaki, et al., (1998).J. intein or are added in a manner that allows them to be Am. Chem. Soc. 120:5591-5592). For the application of removed prior to splicing. Protein splicing has been reported inteins in plant transgenes see Yang, J, et al., (Transgene Res using proteins that comprise bacterial intein-like (BIL) 15:583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. domains (see, Amitai, et al., (2003) Mol Microbiol 47:61-73) 56:375-392, (2005)). and hedgehog (Hog) auto-processing domains (the latter is 0300. In another aspect the PIP-1 polypeptide may be combined with inteins when referred to as the Hog/intein encoded by two separate genes where the intein of the pre superfamily or HINT family (see, Dassa, et al., (2004) J Biol cursor protein comes from the two genes, referred to as a Chem, 27932001-7) and domains such as these may also be split-intein and the two portions of the precursor are joined by used to prepare artificially-split inteins. In particular, non a peptide bond formation. This peptide bond formation is splicing members of Such families may be modified by accomplished by intein-mediated trans-splicing. For this pur molecular biology methodologies to introduce or restore pose, a first and a second expression cassette comprising the splicing activity in Such related species. Recent studies dem two separate genes further code for inteins capable of medi onstrate that splicing can be observed when a N-terminal split ating protein trans-splicing. By trans-splicing, the proteins intein component is allowed to react with a C-terminal split and polypeptides encoded by the first and second fragments intein component not found in nature to be its “partner'; for may be linked by peptide bond formation. Trans-splicing example, splicing has been observed utilizing partners that inteins may be selected from the nucleolar and organellar have as little as 30 to 50% homology with the “natural genomes of different organisms including eukaryotes, splicing partner (see, Dassa, et al., (2007) Biochemistry archaebacteria and eubacteria. Inteins that may be used for 46(1):322-30). Other such mixtures of disparate split intein are listed at neb.com/neb/inteins.html, which can be accessed partners have been shown to be unreactive one with another on the world-wide web using the “www’ prefix). The nucle (see, Brenzel, et al., 2006 Biochemistry 45(6):1571-8). How otide sequence coding for an intein may be split into a 5' and ever, it is within the ability of a person skilled in the relevant a 3' part that code for the 5' and the 3' part of the intein, art to determine whether a particular pair of polypeptides is respectively. Sequence portions not necessary for intein splic able to associate with each other to provide a functional ing (e.g., homing endonuclease domain) may be deleted. The intein, using routine methods and without the exercise of intein coding sequence is split Such that the 5' and the 3' parts inventive skill. are capable of trans-splicing. For selecting a Suitable splitting 0303. In another aspect the PIP-1 polypeptide is a circular site of the intein coding sequence, the considerations pub permuted variant. In certain embodiments the PIP-1 polypep lished by Southworth, et al., (1998) EMBO.J. 17:918-926 tide is a circular permuted variant of the polypeptide of SEQ may be followed. In constructing the first and the second ID NO: 2, 4, 101, 102, 103,104,105,106, 107, 108, 109, 110, expression cassette, the 5' intein coding sequence is linked to 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, the 3' end of the first fragment coding for the N-terminal part 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, of the PIP-1 polypeptide and the 3' intein coding sequence is 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, linked to the 5' end of the second fragment coding for the 147, 148, 149, 150, 151, 204, 206, 208, 211, 212, 213, 214, C-terminal part of the PIP-1 polypeptide. 245, 246, 247, 248, 249, 250, 251, 252,253,254, 255, 256, 0301 In general, the trans-splicing partners can be 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, designed using any split intein, including any naturally-oc 269,298, 299, 300, 301,302,303, 304,305,306, 307, 308, curring or artificially-split split intein. Several naturally-oc 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, curring split inteins are known, for example: the split intein of 321,322,323,324,325, and 332. In certain embodiments the the DnaB gene of Synechocystis sp. PCC6803 (see, Wu, et al., PIP-1 polypeptide is a circular permuted variant of the (1998) Proc Natl AcadSci USA 95(16): 9226-31 and Evans, et polypeptide of SEQ ID NO: 2, 4, 101, 102, 103, 104, 105, al., (2000).J Biol Chem 275(13):9091-4 and of the DnaB gene 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, US 2014/0007292 A1 Jan. 2, 2014 30
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, selection of linkers. They will also recognize that it is some 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 204, 206, times the case that the positions of the ends of the polypeptide 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, chain are ill-defined in structural models derived from X-ray 252,253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, diffraction or nuclear magnetic resonance spectroscopy data, 264, 265, 266, 267,268,269, and 332. and that when true, this situation will therefore need to be 0304. The development of recombinant DNA methods has taken into account in order to properly estimate the length of made it possible to study the effects of sequence transposition the linker required. From those residues whose positions are on protein folding, structure and function. The approach used well defined are selected two residues that are close in in creating new sequences resembles that of naturally occur sequence to the chain ends, and the distance between their ring pairs of proteins that are related by linear reorganization c-alpha carbons is used to calculate an approximate length for of their amino acid sequences (Cunningham, et al., (1979) a linker between them. Using the calculated length as a guide, Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and linkers with a range of number of residues (calculated using 2 Erfle, (1990).J. Bacteriol. 172:3837-3841; Schimming, et al., to 3.8 A per residue) are then selected. These linkers may be (1992) Eur: J. Biochem. 204:13-19: Yamiuchi and Minami composed of the original sequence, shortened or lengthened kawa, (1991) FEBS Lett. 260:127-130; MacGregor, et al., as necessary, and when lengthened the additional residues (1996) FEBS Lett. 378:263-266). The first in vitro application may be chosen to be flexible and hydrophilic as described of this type of rearrangement to proteins was described by above; or optionally the original sequence may be substituted Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). for using a series of linkers, one example being the Gly-Gly In creating a circular permuted variant a new N-terminus is Gly-Ser cassette approach mentioned above; or optionally a selected at an internal site (breakpoint) of the original combination of the original sequence and new sequence hav sequence, the new sequence having the same order of amino ing the appropriate total length may be used. Sequences of acids as the original from the breakpoint until it reaches an pesticidal polypeptides capable of folding to biologically amino acid that is at or near the original C-terminus. At this active states can be prepared by appropriate selection of the point the new sequence is joined, either directly or through an beginning (amino terminus) and ending (carboxyl terminus) additional portion of sequence (linker), to an amino acid that positions from within the original polypeptide chain while is at or near the original N-terminus and the new sequence using the linker sequence as described above. Amino and continues with the same sequence as the original until it carboxyl termini are selected from within a common stretch reaches a point that is at or near the amino acid that was of sequence, referred to as a breakpoint region, using the N-terminal to the breakpoint site of the original sequence, this guidelines described below. A novel amino acid sequence is residue forming the new C-terminus of the chain. The length thus generated by selecting amino and carboxyl termini from of the amino acid sequence of the linker can be selected within the same breakpoint region. In many cases the selec empirically or with guidance from structural information or tion of the new termini will be such that the original position by using a combination of the two approaches. When no of the carboxyl terminus immediately preceded that of the structural information is available, a small series of linkers amino terminus. However, those skilled in the art will recog can be prepared for testing using a design whose length is nize that selections of termini anywhere within the region varied in order to span a range from 0 to 50 A and whose may function, and that these will effectively lead to either sequence is chosen in order to be consistent with Surface deletions or additions to the amino or carboxyl portions of the exposure (hydrophilicity, Hopp and Woods, (1983) Mol. new sequence. It is a central tenet of molecular biology that Immunol. 20:483-489: Kyte and Doolittle, (1982) J. Mol. the primary amino acid sequence of a protein dictates folding Biol. 157:105-132; solvent exposed surface area, Lee and to the three-dimensional structure necessary for expression of Richards, (1971).J. Mol. Biol. 55:379-400) and the ability to its biological function. Methods are known to those skilled in adopt the necessary conformation without deranging the con the art to obtain and interpret three-dimensional structural figuration of the pesticidal polypeptide (conformationally information using X-ray diffraction of single protein Crystals flexible; Karplus and Schulz, (1985) Naturwissenschaften or nuclear magnetic resonance spectroscopy of protein solu 72:212-213. Assuming an average of translation of 2.0 to 3.8 tions. Examples of structural information that are relevant to A per residue, this would mean the length to test would be the identification of breakpoint regions include the location between 0 to 30 residues, with 0 to 15 residues being the and type of protein secondary structure (alpha and 3-10 heli preferred range. Exemplary of Such an empirical series would ces, parallel and anti-parallel beta sheets, chain reversals and be to construct linkers using a cassette sequence Such as turns, and loops; Kabsch and Sander, (1983) Biopolymers Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3 or 4. 22:2577-2637; the degree of solvent exposure of amino acid Those skilled in the art will recognize that there are many residues, the extent and type of interactions of residues with Such sequences that vary in length or composition that can one another (Chothia, (1984) Ann. Rev. Biochem. 53:537 serve as linkers with the primary consideration being that they 572) and the static and dynamic distribution of conformations be neither excessively long nor short (cf., Sandhu, (1992) along the polypeptide chain (Alber and Mathews, (1987) Critical Rev. Biotech. 12:437-462); if they are too long, Methods Enzymol. 154:51 1-533). In some cases additional entropy effects will likely destabilize the three-dimensional information is known about solvent exposure of residues; one fold, and may also make folding kinetically impractical, and example is a site of post-translational attachment of carbohy if they are too short, they will likely destabilize the molecule drate which is necessarily on the surface of the protein. When because of torsional or steric strain. Those skilled in the experimental structural information is not available or is not analysis of protein structural information will recognize that feasible to obtain, methods are also available to analyze the using the distance between the chain ends, defined as the primary amino acid sequence in order to make predictions of distance between the c-alpha carbons, can be used to define protein tertiary and secondary structure, solvent accessibility the length of the sequence to be used or at least to limit the and the occurrence of turns and loops. Biochemical methods number of possibilities that must be tested in an empirical are also sometimes applicable for empirically determining US 2014/0007292 A1 Jan. 2, 2014
Surface exposure when direct structural methods are not fea 0307. In another aspect fusion proteins are provided com sible; for example, using the identification of sites of chain prising a PIP-1 polypeptide and a second pesticidal polypep Scission following limited proteolysis in order to infer Surface tide Such a Cry protein. Methods for design and construction exposure (Gentile and Salvatore, (1993) Eur: J. Biochem. of fusion proteins (and polynucleotides encoding same) are 218:603-621). Thus using either the experimentally derived known to those of skill in the art. Polynucleotides encoding a structural information or predictive methods (e.g., Srinivisan PIP-1 polypeptide may be fused to signal sequences which and Rose, (1995) Proteins. Struct., Funct. & Genetics 22:81– will direct the localization of the PIP-1 polypeptide to par 99) the parental amino acid sequence is inspected to classify ticular compartments of a prokaryotic or eukaryotic cell and/ regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occur or direct the secretion of the PIP-1 polypeptide of the embodi rence of sequences within regions that are known to be ments from a prokaryotic or eukaryotic cell. For example, in involved in periodic secondary structure (alpha and 3-10 heli E. coli, one may wish to direct the expression of the protein to ces, parallel and anti-parallel beta sheets) are regions that the periplasmic space. Examples of signal sequences or pro should be avoided. Similarly, regions of amino acid sequence teins (or fragments thereof) to which the PIP-1 polypeptide that are observed or predicted to have a low degree of solvent may be fused in order to direct the expression of the polypep exposure are more likely to be part of the so-called hydro tide to the periplasmic space of bacteria include, but are not phobic core of the protein and should also be avoided for limited to, the pelB signal sequence, the maltose binding selection of amino and carboxyl termini. In contrast, those protein (MBP) signal sequence, MBP the omp A signal regions that are known or predicted to be in Surface turns or sequence, the signal sequence of the periplasmic E. coli heat loops, and especially those regions that are known not to be labile enterotoxin B-subunit, and the signal sequence of alka required for biological activity, are the preferred sites for line phosphatase. Several vectors are commercially available location of the extremes of the polypeptide chain. Continuous for the construction of fusion proteins which will direct the stretches of amino acid sequence that are preferred based on localization of a protein, such as the pMAL series of vectors the above criteria are referred to as a breakpoint region. Poly (particularly the pMAL-p series) available from New nucleotides encoding circular permuted PIP-1 polypeptides England Biolabs. In a specific embodiment, the PIP-1 with new N-terminus/C-terminus which contain a linker polypeptide may be fused to the pelB pectate lyase signal region separating the original C-terminus and N-terminus can sequence to increase the efficiency of expression and purifi be made essentially following the method described in Mul cation of Such polypeptides in Gram-negative bacteria (see, lins, et al., (1994).J. Am. Chem. Soc. 116:5529-5533. Multiple U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant plastid transit steps of polymerase chain reaction (PCR) amplifications are peptide/polypeptide fusions are well known in the art (see, used to rearrange the DNA sequence encoding the primary U.S. Pat. No. 7, 193,133). Apoplast transit peptides such as amino acid sequence of the protein. Polynucleotides encod rice or barley alpha-amylase secretion signal are also well ing circular permuted PIP-1 polypeptides with new N-termi known in the art. The plastid transit peptide is generally fused nuS/C-terminus which contain a linker region separating the N-terminal to the polypeptide to be targeted (e.g., the fusion original C-terminus and N-terminus can be made based on the partner). In one embodiment, the fusion protein consists tandem-duplication method described in Horlick, et al., essentially of the peptide transit plastid and the PIP-1 (1992) Protein Eng. 5:427-431. Polymerase chain reaction polypeptide to be targeted. In another embodiment, the fusion (PCR) amplification of the new N-terminus/C-terminus protein comprises the peptide transit plastid and the polypep genes is performed using a tandemly duplicated template tide to be targeted. In Such embodiments, the plastid transit DNA. peptide is preferably at the N-terminus of the fusion protein. However, additional amino acid residues may be N-terminal 0305. In another aspect fusion proteins are provided that to the plastid transit peptide providing that the fusion protein include within its amino acid sequence an amino acid is at least partially targeted to a plastid. In a specific embodi sequence comprising a PIP-1 polypeptide including but not ment, the plastid transit peptide is in the N-terminal half, limited to the polypeptide of SEQID NO: 2, 4, 101, 102, 103, N-terminal third or N-terminal quarter of the fusion protein. 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, Most or all of the plastid transit peptide is generally cleaved 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, from the fusion protein upon insertion into the plastid. The 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, position of cleavage may vary slightly between plant species, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, at different plant developmental stages, as a result of specific 204, 206, 208,211, 212, 213, 214, 245, 246, 247, 248, 249, intercellular conditions or the particular combination of tran 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, sit peptide/fusion partner used. In one embodiment, the plas 262, 263,264, 265, 266, 267,268, 269,298, 299, 300, 301, tid transit peptide cleavage is homogenous such that the 302,303, 304,305, 306, 307, 308,309, 310, 311, 312, 313, cleavage site is identical in a population of fusion proteins. In 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, another embodiment, the plastid transit peptide is not homog and 332. enous, such that the cleavage site varies by 1-10 amino acids 0306 In some embodiments fusion proteins comprises a in a population of fusion proteins. The plastid transit peptide PIP-1 polypeptide of SEQ ID NO: 2, 4, 101, 102, 103, 104, can be recombinantly fused to a second protein in one of 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, several ways. For example, a restriction endonuclease recog 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, nition site can be introduced into the nucleotide sequence of 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, the transit peptide at a position corresponding to its C-termi 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 204, nal end and the same or a compatible site can be engineered 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, into the nucleotide sequence of the protein to be targeted at its 251, 252,253,254, 255, 256, 257, 258, 259, 260, 261, 262, N-terminal end. Care must be taken in designing these sites to 263,264, 265,266, 267,268,269,332, and active fragments ensure that the coding sequences of the transit peptide and the thereof. second protein are kept “in frame' to allow the synthesis of US 2014/0007292 A1 Jan. 2, 2014 32 the desired fusion protein. In some cases, it may be preferable be included in the linkers due to the addition of unique restric to remove the initiator methionine codon of the second pro tion sites in the linker sequence to facilitate construction of tein when the new restriction site is introduced. The introduc the fusions. tion of restriction endonuclease recognition sites on both 0311. In some embodiments the linkers comprise parent molecules and their Subsequent joining through sequences selected from the group of formulas: (Gly-Ser), recombinant DNA techniques may result in the addition of (Gly Ser), (GlysSer), (Gly, Ser), or (AlaGlySer), where n one or more extra amino acids between the transit peptide and is an integer. One example of a highly-flexible linker is the the second protein. This generally does not affect targeting (GlySer)-rich spacer region present within the pill protein of activity as long as the transit peptide cleavage site remains the filamentous bacteriophages, e.g., bacteriophages M13 or accessible and the function of the second protein is not altered fl (Schaller, et al., 1975). This region provides along, flexible by the addition of these extra amino acids at its N-terminus. spacer region between two domains of the pill Surface protein. Alternatively, one skilled in the art can create a precise cleav Also included are linkers in which an endopeptidase recog nition sequence is included. Such a cleavage site may be age site between the transit peptide and the second protein valuable to separate the individual components of the fusion (with or without its initiator methionine) using gene synthesis to determine if they are properly folded and active in vitro. (Stemmer, et al., (1995) Gene 164:49-53) or similar methods. Examples of various endopeptidases include, but are not lim In addition, the transit peptide fusion can intentionally ited to, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue include amino acids downstream of the cleavage site. The Plasminogen activator, clostripain, Chymosin, Collagenase, amino acids at the N-terminus of the mature protein can affect Russell's Viper Venom Protease, Postproline cleavage the ability of the transit peptide to target proteins to plastids enzyme, V8 protease, Thrombin and factor Xa. In some and/or the efficiency of cleavage following protein import. embodiments the linker comprises the amino acids EEKKN This may be dependent on the protein to be targeted. See, e.g., from the multi-gene expression vehicle (MGEV), which is Comai, et al., (1988).J. Biol. Chem. 263(29): 15104-9. cleaved by vacuolar proteases as disclosed in US 2007/ 0277263. In other embodiments, peptide linker segments 0308. In some embodiments fusion proteins are provide from the hinge region of heavy chain immunoglobulins IgG, comprising a PIP-1 polypeptide, a pesticidal protein Such as a IgA, IgM, Ig|D or IgE provide an angular relationship between Cry protein, and an amino acid linker. the attached polypeptides. Especially useful are those hinge 0309. In some embodiments fusion proteins are provided regions where the cysteines are replaced with serines. Pre represented by a formula selected from the group consisting ferred linkers of the present invention include sequences of derived from murine IgG gamma2bhinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker 0310 where R' is a PIP-1 polypeptide, R is a pesticidal sequences employed and the only requirement of the linker is protein with a different but complementary activity to the that functionally it does not interfere adversely with the fold PIP-1 polypeptide, including but not limited to Cry proteins: ing and function of the individual molecules of the fusion. a polypeptide that increases the solubility and/or stability of 0312. In another aspect chimeric PIP-1 polypeptide are the PIP-1 polypeptide; or a transit peptide or leader sequence. provided that are created through joining two or more por The R' polypeptide is fused either directly or through a linker tions of genes, which originally encoded separate insecticidal segment to the R polypeptide. The term “directly defines proteins from different species, to create achimeric gene. The fusions in which the polypeptides are joined without a peptide translation of the chimeric gene results in a single chimeric linker. Thus L represents a chemical bound or polypeptide pesticidal polypeptide with regions, motifs or domains segment to which both R" and R are fused in frame, most derived from each of the original polypeptides. In certain commonly L is a linear peptide to which RandR are bound embodiments the chimeric protein comprises portions, motifs by amide bonds linking the carboxy terminus of R' to the or domains of PIP-1A (SEQ ID NO: 2) and orthologs amino terminus of L and carboxy terminus of L to the amino PSEEN3174 (SEQ ID NO: 6), PIP-1C (SEQ ID NO:332), terminus of R. By “fused in frame' is meant that there is no and PIP-1B (SEQ ID NO: 4) in any combination. In certain translation termination or disruption between the reading embodiments the chimeric insecticidal polypeptide includes frames of R' and R. The linking group (L) is generally a but not limited to the polypeptides of SEQID NO: 101, 102, polypeptide of between 1 and 500 amino acids in length. The 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, linkers joining the two molecules are preferably designed to 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, (1) allow the two molecules to fold and act independently of 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, each other, (2) not have a propensity for developing an 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, ordered secondary structure which could interfere with the 151, 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, functional domains of the two proteins, (3) have minimal 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, hydrophobic or charged characteristic which could interact 261, 262, 263,264, 265, 266, 267,268, 269, and 332. with the functional protein domains and (4) provide steric 0313. It is recognized that DNA sequences may be altered separation of R' and R such that R' and R could interact by various methods, and that these alterations may result in simultaneously with their corresponding receptors on a single DNA sequences encoding proteins with amino acid cell. Typically surface amino acids in flexible protein regions sequences different than that encoded by the wild-type (or include Gly, Asn and Ser. Virtually any permutation of amino native) pesticidal protein. These proteins may be altered in acid sequences containing Gly, ASn and Ser would be various ways including amino acid substitutions, deletions, expected to satisfy the above criteria for a linker sequence. truncations, and insertions of one or more amino acids, Other neutral amino acids, such as Thr and Ala, may also be including up to 2, 3, 4, 5, 6,7,8,9, 10, 15, 20, 25, 30, 35, 40 used in the linker sequence. Additional amino acids may also 45, 50, about 55, 60, 65,70, 75,80, 85,90, 100,105,110, 115, US 2014/0007292 A1 Jan. 2, 2014
120, 125, 130, 135, 140, 145, 150, 155 or more amino acid contained in an alignment of similar or related toxins to the Substitutions, deletions and/or insertions or combinations sequences of the embodiments (e.g., residues that have only thereof compared to SEQ ID NO: 2 or 4 including but not conservative Substitutions between all proteins contained in limited to SEQID NO: 101, 102, 103, 104, 105, 106, 107, the alignment homologous proteins). However, one of skill in 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, the art would understand that functional variants may have 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, minor conserved or nonconserved alterations in the con 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, served residues. Guidance as to appropriate amino acid Sub 144, 145, 146, 147, 148, 149, 150, 151, 204, 206, 208, 211, stitutions that do not affect biological activity of the protein of 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, 252, 253, interest may be found in the model of Dayhoff, et al., (1978) 254, 255, 256, 257, 258, 259,260, 261, 262, 263,264, 265, Atlas of Protein Sequence and Structure (Natl. Biomed. Res. 266, 267, 268,269, and 332. In some embodiments a PIP-1 Found. Washington, D.C.), herein incorporated by reference. polypeptide comprises the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9. 0316. In making such changes, the hydropathic index of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, amino acids may be considered. The importance of the hydro 27, 28 or more amino acids from the N-terminus of the PIP-1 pathic amino acid index in conferring interactive biologic polypeptide relative to the amino acid position of SEQ ID function on a protein is generally understood in the art (Kyte NO: 2. Methods for such manipulations are generally known and Doolittle, (1982) J Mol Biol. 157(1):105-32). It is in the art. For example, amino acid sequence variants of a accepted that the relative hydropathic character of the amino PIP-1 polypeptide can be prepared by mutations in the DNA. acid contributes to the secondary structure of the resultant This may also be accomplished by one of several forms of protein, which in turn defines the interaction of the protein mutagenesis and/or in directed evolution. In some aspects, the with other molecules, for example, enzymes, Substrates, changes encoded in the amino acid sequence will not Substan receptors, DNA, antibodies, antigens and the like. tially affect the function of the protein. Such variants will 0317. It is known in the art that certain amino acids may be possess the desired pesticidal activity. However, it is under Substituted by otheramino acids having a similar hydropathic stood that the ability of a PIP-1 polypeptide to confer pesti index or score and still result in a protein with similar bio cidal activity may be improved by the use of Such techniques logical activity, i.e., still obtain a biological functionally upon the compositions of this disclosure. equivalent protein. Each amino acid has been assigned a 0314 For example, conservative amino acid substitutions hydropathic index on the basis of its hydrophobicity and may be made at one or more, predicted, nonessential amino charge characteristics (Kyte and Doolittle, ibid). These are: acid residues. A “nonessential amino acid residue is a resi isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala due that can be altered from the wild-type sequence of a PIP-1 nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala polypeptide without altering the biological activity. A "con nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); servative amino acid substitution' is one in which the amino tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine acid residue is replaced with an amino acid residue having a (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); similar side chain. Families of amino acid residues having asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In mak similar side chains have been defined in the art. These fami ing Such changes, the Substitution of amino acids whose lies include: amino acids with basic side chains (e.g., lysine, hydropathic indices are within +2 is preferred, those which arginine, histidine); acidic side chains (e.g., aspartic acid, are within +1 are particularly preferred and those within +0.5 glutamic acid); polar, negatively charged residues and their are even more particularly preferred. amides (e.g., aspartic acid, asparagine, glutamic, acid, 0318. It is also understood in the art that the substitution of glutamine; uncharged polar side chains (e.g., glycine, aspar like amino acids can be made effectively on the basis of agine, glutamine, serine, threonine, tyrosine, cysteine); Small hydrophilicity. U.S. Pat. No. 4,554,101, states that the great aliphatic, nonpolar or slightly polar residues (e.g., Alanine, est local average hydrophilicity of a protein, as governed by serine, threonine, proline, glycine); nonpolar side chains the hydrophilicity of its adjacentamino acids, correlates with (e.g., alanine, Valine, leucine, isoleucine, proline, phenylala a biological property of the protein. nine, methionine, tryptophan); large aliphatic, nonpolar resi 0319. As detailed in U.S. Pat. No. 4,554,101, the follow dues (e.g., methionine, leucine, isoleucine, Valine, cystine); ing hydrophilicity values have been assigned to amino acid beta-branched side chains (e.g., threonine, Valine, isoleu residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); cine); aromatic side chains (e.g., tyrosine, phenylalanine, glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2): tryptophan, histidine); large aromatic side chains (e.g., glutamine (+0.2); glycine (O); threonine (-0.4); proline (-0. tyrosine, phenylalanine, tryptophan). 5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); 0315 Amino acid substitutions may be made in noncon methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine served regions that retain function. In general. Such substitu (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3. tions would not be made for conserved amino acid residues or 4). for amino acid residues residing within a conserved motif. 0320 Alternatively, alterations may be made to the protein where such residues are essential for protein activity. sequence of many proteins at the amino or carboxy terminus Examples of residues that are conserved and that may be without substantially affecting activity. This can include essential for protein activity include, for example, residues insertions, deletions or alterations introduced by modern that are identical between all proteins contained in an align molecular methods, such as PCR, including PCR amplifica ment of similar or related toxins to the sequences of the tions that alter or extend the protein coding sequence by virtue embodiments (e.g., residues that are identical in an alignment of inclusion of amino acid encoding sequences in the oligo of homologous proteins). Examples of residues that are con nucleotides utilized in the PCR amplification. Alternatively, served but that may allow conservative amino acid substitu the protein sequences added can include entire protein-cod tions and still retain activity include, for example, residues ing sequences, such as those used commonly in the art to that have only conservative substitutions between all proteins generate protein fusions. Such fusion proteins are often used US 2014/0007292 A1 Jan. 2, 2014 34 to (1) increase expression of a protein of interest (2) introduce (SEQ ID NO: 6). The person skilled in the art will be able to a binding domain, enzymatic activity or epitope to facilitate use comparisons to other proteins or functional assays to either protein purification, protein detection or other experi further define motifs. High throughput Screening can be used mental uses known in the art (3) target secretion or translation to test variations of those motifs to determine the role of of a protein to a Subcellular organelle. Such as the periplasmic specific residues. Given that knowledge for several motifs, space of Gram-negative bacteria, mitochondria or chloro one can then define the requirements for a functional protein. plasts of plants or the endoplasmic reticulum of eukaryotic Knowledge of the motifs allows the skilled artisan to design cells, the latter of which often results in glycosylation of the sequence variations that would not impact function. protein. 0325 This line of investigation was pursued in Examples 0321. In some embodiments, the PIP-1 polypeptide com 9-11. Alignment of homologues of SEQID NO: 2, 4 and 6 prises an amino acid sequence of SEQ ID NO: 2 having an allowed identification of residues that are highly conserved amino acid Substitutions compared to the native amino acid of among natural homologues in this family (FIG. 1). In SEQID NO: 2 at one or more residues selected from positions example 9 saturation mutagenesis was used to make and test 2, 3, 6, 8, 19, 20, 21, 22, 24, 25, 26, 27, 28, 30, 35, 36,38, 42, most or all possible substitutions at each of 6 conserved 43,46,48,49, 53,60, 63, 66,77, 89,93, 97,98, 105,108, 110, residues. These mutants were tested for activity and a number 120, 121, 123, 125, 127, 134, 135, 137, 141, 142, 144, 147, of active Substitutions not present among the homologues 150, 151, 160, 162, 163, 164, 166, 167, 168, 171, 172, 173, were identified providing an understanding of the functional 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 194, 195, constraints at these residues. In Example 10 four motifs were 200, 203, 204, 209, 213, 220, 221, 222, 226, 228, 229, 231, identified among the most conserved regions in the alignment 232, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, of SEQID NO: 2, 4 and 6. To further characterize the func 254, 258, 259, 265 and 266 of SEQ ID NO: 2. In specific tional constraints on these sequence motifs, they were com embodiments, the substitution is an alanine for the native pared to a set of three distant homologues (AECFG 592740 amino acid at the recited position(s). Also encompassed are (SEQ ID NO: 12), Pput 1063 (SEQID NO: 8), and Pput the nucleic acid sequence(s) encoding the variant protein or 1064 (SEQ ID NO: 10) that have no detectable insecticidal polypeptide. activity (FIG. 1). These homologues are deemed to fall within 0322 Variant nucleotide and amino acid sequences of the the same PFAM as SEQID NO: 2, 4 and 6 and thus are likely disclosure also encompass sequences derived from to share the same overall fold. The sequences corresponding mutagenic and recombinogenic procedures such as DNA to these four motifs from these distant homologues were shuffling. With such a procedure, one or more different PIP-1 swapped into the PIP-1A backbone. The data presented in polypeptide coding regions can be used to create a new PIP-1 Example 10 demonstrates that these motifs are under rela polypeptide possessing the desired properties. In this manner, tively stringent functional constraints, as most of the motif libraries of recombinant polynucleotides are generated from a Swaps from the distant homologues resulted in loss of func population of related sequence polynucleotides comprising tion. In Example 11 the functional constraints on two of these sequence regions that have Substantial sequence identity and motifs were further examined by performing Saturation can be homologously recombined in vitro or in vivo. For mutagenesis on all residues in motifs 3 and 4. example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene Antibodies and other known pesticidal genes to obtain a new gene coding 0326 Antibodies to a PIP-1 polypeptide of the embodi for a protein with an improved property of interest, such as an ments or to variants or fragments thereof, are also encom increased insecticidal activity. Strategies for such DNA shuf passed. Methods for producing antibodies are well known in fling are known in the art. See, for example, Stemmer, (1994) the art (see, for example, Harlow and Lane, (1988) Antibod Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, ies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997).J. Mol. Biol. 272: Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265). 336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 0327. A kit for detecting the presence of a PIP-1 polypep 94:4504-4509: Crameri, et al., (1998) Nature 391:288-291 tide, or detecting the presence of a nucleotide sequence and U.S. Pat. Nos. 5,605,793 and 5,837,458. encoding a PIP-1 polypeptide, in a sample is provided. In one 0323 Domain swapping or shuffling is another mecha embodiment, the kit provides antibody-based reagents for nism for generating altered PIP-1 polypeptides. Domains detecting the presence of a PIP-1 polypeptide in a tissue may be swapped between PIP-1 polypeptides, resulting in sample. In another embodiment, the kit provides labeled hybrid or chimeric toxins with improved pesticidal activity or nucleic acid probes useful for detecting the presence of one or target spectrum. Methods for generating recombinant pro more polynucleotides encoding PIP-1 polypeptide(s). The kit teins and testing them for pesticidal activity are well known in is provided along with appropriate reagents and controls for the art (see, for example, Naimov, et al., (2001) Appl. Environ. carrying out a detection method, as well as instructions for Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. use of the kit Environ. Microbiol. 62:1537-1543; Ge, et al., (1991).J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Receptor Identification and Isolation Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. 0328. Receptors to the PIP-1 polypeptide of the embodi Microbiol. 65:2918-2925). ments or to variants or fragments thereof, are also encom 0324 Both DNA shuffling and site directed mutagenesis passed. Methods for identifying receptors are well known in were used to define polypeptide sequences that possess pes the art (see, Hofmann, et. al., (1988) Eur: J. Biochem. 173: ticidal activity. In Example 8 DNA shuffling was used to 85-91; Gill, et al., (1995).J. Biol. Chem. 27277-27282) can be generate a library of active variants by recombination of the employed to identify and isolate the receptor that recognizes diversity present in PIP-1A (SEQID NO:2) and PSEEN3174 the PIP-1 polypeptides using the brush-border membrane US 2014/0007292 A1 Jan. 2, 2014
vesicles from Susceptible insects. In addition to the radioac erally, operably linked means that the nucleic acid sequences tive labeling method listed in the cited literatures, PIP-1 being linked are contiguous and where necessary to join two polypeptide can be labeled with fluorescent dye and other protein coding regions in the same reading frame. The con common labels such as streptavidin. Brush-border membrane struct may additionally contain at least one additional gene to vesicles (BBMV) of susceptible insects such as soybean be cotransformed into the organism. Alternatively, the addi looper and Stink bugs can be prepared according to the pro tional gene(s) can be provided on multiple DNA constructs. tocols listed in the references and separated on SDS-PAGE 0332 Such a DNA construct is provided with a plurality of gel and blotted on suitable membrane. Labeled PIP-1 restriction sites for insertion of the PIP-1 polypeptide gene polypeptides can be incubated with blotted membrane of sequence to be under the transcriptional regulation of the BBMV and labeled the PIP-1 polypeptides can be identified regulatory regions. The DNA construct may additionally con with the labeled reporters. Identification of protein band(s) tain selectable marker genes. that interact with the PIP-1 polypeptides can be detected by 0333. The DNA construct will generally include in the 5' N-terminal amino acid gas phase sequencing or mass spec to 3' direction of transcription: a transcriptional and transla trometry based protein identification method (Patterson, tional initiation region (i.e., a promoter), a DNA sequence of (1998) 10(22):1-24, Current Protocol in Molecular Biology the embodiments and a transcriptional and translational ter published by John Wiley & Son Inc). Once the protein is mination region (i.e., termination region) functional in the identified, the corresponding gene can be cloned from organism serving as a host. The transcriptional initiation genomic DNA or cDNA library of the susceptible insects and region (i.e., the promoter) may be native, analogous, foreign binding affinity can be measured directly with the PIP-1 or heterologous to the host organism and/or to the sequence of polypeptides. Receptor function for insecticidal activity by the embodiments. Additionally, the promoter may be the the PIP-1 polypeptides can be verified by accomplished by natural sequence or alternatively a synthetic sequence. The RNAi type of gene knock out method (Rajagopal, et al., term “foreign' as used herein indicates that the promoter is (2002).J. Biol. Chem. 277:46849-46851). not found in the native organism into which the promoter is introduced. Where the promoter is “foreign” or "heterolo Nucleotide Constructs, Expression Cassettes and Vectors gous' to the sequence of the embodiments, it is intended that 0329. The use of the term “nucleotide constructs' herein is the promoter is not the native or naturally occurring promoter not intended to limit the embodiments to nucleotide con for the operably linked sequence of the embodiments. As used structs comprising DNA. Those of ordinary skill in the art will herein, a chimeric gene comprises a coding sequence oper recognize that nucleotide constructs particularly polynucle ably linked to a transcription initiation region that is heter otides and oligonucleotides composed of ribonucleotides and ologous to the coding sequence. Where the promoter is a combinations of ribonucleotides and deoxyribonucleotides native or natural sequence, the expression of the operably may also be employed in the methods disclosed herein. The linked sequence is altered from the wild-type expression, nucleotide constructs, nucleic acids, and nucleotide which results in an alteration in phenotype. sequences of the embodiments additionally encompass all 0334. In some embodiments the DNA construct may also complementary forms of Such constructs, molecules and include a transcriptional enhancer sequence. As used herein, sequences. Further, the nucleotide constructs, nucleotide the term an "enhancer refers to a DNA sequence which can molecules and nucleotide sequences of the embodiments stimulate promoter activity and may be an innate element of encompass all nucleotide constructs, molecules and the promoter or a heterologous element inserted to enhance sequences which can be employed in the methods of the the level or tissue-specificity of a promoter. Various enhanc embodiments for transforming plants including, but not lim ers are known in the art including for example, introns with ited to, those comprised of deoxyribonucleotides, ribonucle gene expression enhancing properties in plants (US Patent otides and combinations thereof. Such deoxyribonucleotides Application Publication Number 2009/0144863, the ubiq and ribonucleotides include both naturally occurring mol uitin intron (i.e., the maize ubiquitin intron 1 (see, for ecules and synthetic analogues. The nucleotide constructs, example, NCBI sequence S94464)), the omega enhancer or nucleic acids, and nucleotide sequences of the embodiments the omega prime enhancer (Gallie, et al., (1989) Molecular also encompass all forms of nucleotide constructs including, Biology of RNA ed. Cech (Liss, New York) 237-256 and but not limited to, single-stranded forms, double-stranded Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S forms, hairpins, stem-and-loop structures and the like. enhancer (see, e.g., Benfey, et al., (1990) EMBO.J. 9:1685 0330. A further embodiment relates to a transformed 96) and the enhancers of U.S. Pat. No. 7,803,992 may also be organism such as an organism selected from plant and insect used, each of which is incorporated by reference. The above cells, bacteria, yeast, baculovirus, protozoa, nematodes and list of transcriptional enhancers is not meant to be limiting. algae. The transformed organism comprises a DNA molecule Any appropriate transcriptional enhancer can be used in the of the embodiments, an expression cassette comprising the embodiments. DNA molecule or a vector comprising the expression cas 0335 The termination region may be native with the tran sette, which may be stably incorporated into the genome of Scriptional initiation region, may be native with the operably the transformed organism. linked DNA sequence of interest, may be native with the plant 0331. The sequences of the embodiments are provided in host or may be derived from another source (i.e., foreign or DNA constructs for expression in the organism of interest. heterologous to the promoter, the sequence of interest, the The construct will include 5' and 3' regulatory sequences plant host or any combination thereof). operably linked to a sequence of the embodiments. The term 0336 Convenient termination regions are available from “operably linked as used herein refers to a functional linkage the Ti-plasmid of A. tumefaciens, such as the octopine Syn between a promoter and a second sequence, wherein the thase and nopaline synthase termination regions. See also, promoter sequence initiates and mediates transcription of the Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144: DNA sequence corresponding to the second sequence. Gen Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) US 2014/0007292 A1 Jan. 2, 2014 36
Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 0340. By “signal sequence' is intended a sequence that is 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Bal known or Suspected to result in cotranslational or post-trans las, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, lational peptide transport across the cell membrane. In et al., (1987) Nucleic Acid Res. 15:9627-9639. eukaryotes, this typically involves Secretion into the Golgi 0337. Where appropriate, a nucleic acid may be optimized apparatus, with some resulting glycosylation. Insecticidal for increased expression in the host organism. Thus, where toxins of bacteria are often synthesized as protoxins, which the host organism is a plant, the synthetic nucleic acids can be are protolytically activated in the gut of the target pest synthesized using plant-preferred codons for improved (Chang, (1987) Methods Enzymol. 153:507-516). In some expression. See, for example, Campbell and Gowri, (1990) embodiments, the signal sequence is located in the native Plant Physiol. 92: 1-11 for a discussion of host-preferred sequence or may be derived from a sequence of the embodi codon usage. For example, although nucleic acid sequences ments. By “leader sequence' is intended any sequence that of the embodiments may be expressed in both monocotyle when translated, results in an amino acid sequence Sufficient donous and dicotyledonous plant species, sequences can be to trigger co-translational transport of the peptide chain to a modified to account for the specific codon preferences and Subcellular organelle. Thus, this includes leader sequences GC content preferences of monocotyledons or dicotyledons targeting transport and/or glycosylation by passage into the as these preferences have been shown to differ (Murray et al. endoplasmic reticulum, passage to vacuoles, plastids includ (1989) Nucleic Acids Res. 17:477-498). Thus, the maize ing chloroplasts, mitochondria and the like. Nuclear-encoded preferred codon for a particular amino acid may be derived proteins targeted to the chloroplast thylakoid lumen compart from known gene sequences from maize. Maize codon usage ment have a characteristic bipartite transit peptide, composed for 28 genes from maize plants is listed in Table 4 of Murray, of a stromal targeting signal peptide and a lumen targeting et al., Supra. Methods are available in the art for synthesizing signal peptide. The stromal targeting information is in the plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380, amino-proximal portion of the transit peptide. The lumen 831, and 5,436,391 and Murray, et al., (1989) Nucleic Acids targeting signal peptide is in the carboxyl-proximal portion of Res. 17:477-498, herein incorporated by reference. the transit peptide, and contains all the information for tar 0338. Additional sequence modifications are known to geting to the lumen. Recent research in proteomics of the enhance gene expression in a cellular host. These include higher plant chloroplast has achieved in the identification of elimination of sequences encoding spurious polyadenylation numerous nuclear-encoded lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell signals, exon-intron splice site signals, transposon-like 12:319-341, 2000; Brickeretal. Biochim. Biophy's Acta 1503: repeats, and other well-characterized sequences that may be 350-356, 2001), the lumen targeting signal peptide of which deleterious to gene expression. The GC content of the can potentially be used in accordance with the present inven sequence may be adjusted to levels average for a given cellu tion. About 80 proteins from Arabidopsis, as well as homolo lar host, as calculated by reference to known genes expressed gous proteins from spinach and garden pea, are reported by in the host cell. The term "host cell as used herein refers to a Kieselbach et al., Photosynthesis Research, 78:249-264, cell which contains a vector and Supports the replication 2003. In particular, table 2 of this publication, which is incor and/or expression of the expression vector is intended. Host porated into the description herewith by reference, discloses cells may be prokaryotic cells such as E. coli or eukaryotic 85 proteins from the chloroplast lumen, identified by their cells such as yeast, insect, amphibian or mammalian cells or accession number (see also US Patent Application Publica monocotyledonous or dicotyledonous plant cells. An tion 2009/09044298). In addition, the recently published example of a monocotyledonous host cell is a maize host cell. draft version of the rice genome (Goffetal, Science 296:92 When possible, the sequence is modified to avoid predicted 100, 2002) is a suitable source for lumen targeting signal hairpin secondary mRNA structures. peptide which may be used in accordance with the present 0339. The expression cassettes may additionally contain 5' invention. leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and 0341 Suitable chloroplast transit peptides (CTP) are well include: picornavirus leaders, for example, EMCV leader known to one skilled in the art including chimeric CTPs (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvi sativa 1-deoxy-D xylulose-5-Phosphate Synthase oryza rus leaders, for example, TEV leader (Tobacco Etch Virus) sativa-Superoxide dismutase Oryza sativa-Soluble starch Syn (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader thase Oryza sativa-NADP-dependent Malic acid enzyme (Maize Dwarf Mosaic Virus), human immunoglobulin heavy oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldo chain binding protein (BiP) (Maceak, et al., (1991) Nature lase 2 oryza sativa-L-AScorbate peroxidase 5 oryza sativa 353:90-94); untranslated leader from the coat protein mRNA Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea ofalfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Mays-beta-glucosidase, Zea Mays-Malate dehydrogenase, Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech Zea Mays Thioredoxin M-type US Patent Application Publi (Liss, New York), pp. 237-256) and maize chlorotic mottle cation 2012/0304336). virus leader (MCMV) (Lommel, et al., (1991) Virology 0342. The PIP-1 polypeptide gene to be targeted to the 81:382-385). See also, Della-Cioppa, et al., (1987) Plant chloroplast may be optimized for expression in the chloro Physiol. 84:965-968. Such constructs may also contain a plast to account for differences in codon usage between the “signal sequence' or "leader sequence' to facilitate co-trans plant nucleus and this organelle. In this manner, the nucleic lational or post-translational transport of the peptide to cer acids of interest may be synthesized using chloroplast-pre tain intracellular structures such as the chloroplast (or other ferred codons. See, for example, U.S. Pat. No. 5,380,831, plastid), endoplasmic reticulum or Golgi apparatus. herein incorporated by reference. US 2014/0007292 A1 Jan. 2, 2014 37
0343. In preparing the expression cassette, the various bertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750, DNA fragments may be manipulated so as to provide for the 386 (nematode-inducible) and the references cited therein. Of DNA sequences in the proper orientation and, as appropriate, particular interest is the inducible promoter for the maize in the proper reading frame. Toward this end, adapters or PRms gene, whose expression is induced by the pathogen linkers may be employed to join the DNA fragments or other Fusarium moniliforme (see, for example, Cordero, et al., manipulations may be involved to provide for convenient (1992) Physiol. Mol. Plant Path. 41: 189-200). restriction sites, removal of superfluous DNA, removal of 0348 Chemical-regulated promoters can be used to restriction sites or the like. For this purpose, in vitro mutagen modulate the expression of a gene in a plant through the esis, primer repair, restriction, annealing, resubstitutions, application of an exogenous chemical regulator. Depending e.g., transitions and transversions, may be involved. upon the objective, the promoter may be a chemical-inducible 0344) A number of promoters can be used in the practice of promoter, where application of the chemical induces gene the embodiments. The promoters can be selected based on the expression or a chemical-repressible promoter, where appli desired outcome. The nucleic acids can be combined with cation of the chemical represses gene expression. Chemical constitutive, tissue-preferred, inducible or other promoters inducible promoters are known in the art and include, but are for expression in the host organism. Suitable constitutive not limited to, the maize ln 2-2 promoter, which is activated promoters for use in a plant host cell include, for example, the by benzenesulfonamide herbicide safeners, the maize GST core promoter of the Rsyn? promoter and other constitutive promoter, which is activated by hydrophobic electrophilic promoters disclosed in WO 1999/43838 and U.S. Pat. No. compounds that are used as pre-emergent herbicides, and the 6,072,050; the core CaMV35S promoter (Odell, et al., (1985) tobacco PR-1a promoter, which is activated by salicylic acid. Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Other chemical-regulated promoters of interest include ste Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant roid-responsive promoters (see, for example, the glucocorti Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant coid-inducible promoter in Schena, et al., (1991) Proc. Natl. Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor: Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Appl. Genet. 81:581–588); MAS (Velten, et al., (1984) EMBO Plant J. 14(2):247-257) and tetracycline-inducible and tetra J.3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and cycline-repressible promoters (see, for example, Gatz, et al., the like. Other constitutive promoters include, for example, (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. those discussed in U.S. Pat. Nos. 5,608, 149; 5,608,144: 5,814,618 and 5,789,156), herein incorporated by reference. 5,604,121 : 5,569,597; 5,466,785; 5,399,680: 5,268,463: 0349 Tissue-preferred promoters can be utilized to target 5,608,142 and 6,177,611. enhanced PIP-1 polypeptide expression within a particular 0345 Depending on the desired outcome, it may be ben plant tissue. Tissue-preferred promoters include those dis eficial to express the gene from an inducible promoter. Of cussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; particular interest for regulating the expression of the nucle Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; otide sequences of the embodiments in plants are wound Hansen, et al., (1997) Mol. Gen. Genet. 254(3):337-343; Rus inducible promoters. Such wound-inducible promoters, may sell, et al., (1997) Transgenic Res. 6(2): 157-168; Rinehart, et respond to damage caused by insect feeding, and include al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., Rev. Phytopath. 28:425-449: Duan, et al., (1996) Nature Bio (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., technology 14:494-498); wun1 and wun2, U.S. Pat. No. (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) 5,428, 148; win1 and win2 (Stanford, et al., (1989) Mol. Gen. Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Genet. 215:200-208); systemin (McGurl, et al., (1992) Sci Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) ence 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara Mol. Biol. 22:783-792: Eckelkamp, et al., (1993) FEBS Let Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters ters 323:73-76); MPI gene (Corderok, et al., (1994) Plant J. can be modified, if necessary, for weak expression. 6(2): 141-150) and the like, herein incorporated by reference. 0350 Leaf-preferred promoters are known in the art. See, 0346 Additionally, pathogen-inducible promoters may be for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; employed in the methods and nucleotide constructs of the Kwon, et al., (1994) Plant Physiol. 105:357-67: Yamamoto, embodiments. Such pathogen-inducible promoters include et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., those from pathogenesis-related proteins (PR proteins), (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. which are induced following infection by a pathogen; e.g., PR Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) Proc. proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. Natl. Acad. Sci. USA 90(20):9586-9590. See, for example, Redolfi, et al., (1983) Neth, J. Plant Pathol. 0351 Root-preferred or root-specific promoters are 89:245-254; Uknes, et al., (1992) Plant Cell 4:645-656 and known and can be selected from the many available from the Van Loon, (1985) Plant Mol. Virol. 4:111-116. See also, WO literature or isolated de novo from various compatible spe 1999/43819, herein incorporated by reference. cies. See, for example, Hire, et al., (1992) Plant Mol. Biol. 0347 Of interest are promoters that are expressed locally 20(2):207-218 (soybean root-specific glutamine synthetase at or near the site of pathogen infection. See, for example, gene); Keller and Baumgartner, (1991) Plant Cell 3(10): Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, 1051-1061 (root-specific control element in the GRP 1.8 gene et al., (1989) Molecular Plant-Microbe Interactions 2:325 of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3): 331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 433-443 (root-specific promoter of the mannopine synthase 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA (1991) Plant Cell3(1):11-22 (full-length cDNA clone encod 93:14972-14977. See also, Chen, et al., (1996) Plant J. ing cytosolic glutamine synthetase (GS), which is expressed 10:955-966: Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA in roots and root nodules of soybean). See also, Bogusz, et al., 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201: Sie (1990) Plant Cell 207):633-641, where two root-specific pro US 2014/0007292 A1 Jan. 2, 2014 moters isolated from hemoglobin genes from the nitrogen Where a promoter drives expression at unacceptably high fixing nonlegume Parasponia andersonii and the related non levels, portions of the promoter sequence can be deleted or nitrogen-fixing nonlegume Trema tomentosa are described. modified to decrease expression levels. The promoters of these genes were linked to a 6-glucu 0354. Such weak constitutive promoters include, for ronidase reporter gene and introduced into both the nonle example the core promoter of the Rsyn7 promoter (WO 1999/ gume Nicotiana tabacum and the legume Lotus corniculatus, 43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV and in both instances root-specific promoteractivity was pre promoter, and the like. Other constitutive promoters include, served. Leach and Aoyagi, (1991) describe their analysis of for example, those disclosed in U.S. Pat. Nos. 5,608, 149: the promoters of the highly expressed roC and roll D root 5,608,144; 5,604,121 : 5,569,597; 5,466,785; 5,399,680; inducing genes of Agrobacterium rhizogenes (see, Plant Sci 5,268,463; 5,608,142 and 6,177,611, herein incorporated by ence (Limerick) 79(1):69-76). They concluded that enhancer reference. and tissue-preferred DNA determinants are dissociated in those promoters. Teen, et al., (1989) used gene fusion to lacz 0355 The above list of promoters is not meant to be lim to show that the Agrobacterium T-DNA gene encoding iting. Any appropriate promoter can be used in the embodi octopine synthase is especially active in the epidermis of the mentS. root tip and that the TR2 gene is root specific in the intact 0356. Generally, the expression cassette will comprise a plant and stimulated by wounding in leaf tissue, an especially selectable marker gene for the selection of transformed cells. desirable combination of characteristics foruse with an insec Selectable marker genes are utilized for the selection of trans ticidal or larvicidal gene (see, EMBO.J. 8(2):343-350). The formed cells or tissues. Marker genes include genes encoding TR1 gene fused to mptII (neomycin phosphotransferase II) antibiotic resistance, such as those encoding neomycin phos showed similar characteristics. Additional root-preferred photransferase II (NEO) and hygromycin phosphotransferase promoters include the VfBNOD-GRP3 gene promoter (HPT), as well as genes conferring resistance to herbicidal (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and compounds, such as glufosinate ammonium, bromoxynil. rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4): imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). 681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; Additional examples of Suitable selectable marker genes 5,633,363; 5,459,252: 5,401,836; 5,110,732 and 5,023,179. include, but are not limited to, genes encoding resistance to 0352 “Seed-preferred” promoters include both “seed chloramphenicol (Herrera Estrella, et al., (1983) EMBO.J. specific' promoters (those promoters active during seed 2:987-992); methotrexate (Herrera Estrella, et al., (1983) development such as promoters of seed storage proteins) as Nature 303:209-213 and Meijer, et al., (1991) Plant Mol. well as “seed-germinating promoters (those promoters Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. active during seed germination). See, Thompson, et al., Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, (1989) BioEssays 10:108, herein incorporated by reference. et al., (1996) Transgenic Res. 5:131-137); bleomycin (Hille, Such seed-preferred promoters include, but are not limited to, et al., (1990) Plant Mol. Biol. 7:171-176); sulfonamide Cim 1 (cytokinin-induced message); cz 19B1 (maize 19 kDa (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bro Zein); and milps (myo-inositol-1-phosphate synthase) (see, moxynil (Stalker, et al., (1988) Science 242:419–423); gly U.S. Pat. No. 6,225,529, herein incorporated by reference). phosate (Shaw, et al., (1986) Science 233:478-481 and U.S. Gamma-Zein and Glb-1 are endosperm-specific promoters. patent application Ser. Nos. 10/004.357 and 10/427,692); For dicots, seed-specific promoters include, but are not lim phosphinothricin (DeBlock, et al., (1987) EMBO.J. 6:2513 ited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku, K. D. and 2518). Seegenerally, Yarranton, (1992) Curr. Opin. Biotech. Goldberg, R. B. Plant Cell 1:1079-1093, 1989), bean 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. B-phaseolin, napin, B-conglycinin, glycinin 1, soybean lectin, Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; cruciferin, and the like. For monocots, seed-specific promot Reznikoff, (1992) Mol. Microbiol. 6:2419-2422: Barkley, et ers include, but are not limited to, maize 15 kDa Zein, 22 kDa al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell Zein, 27 kDa Zein, g-Zein, waxy, shrunken 1, shrunken 2, 48:555-566; Brown, et al., (1987) Cell 49:603-612: Figge, et globulin 1, etc. See also, WO 2000/12733, where seed-pre al., (1988) Cell 52:713-722: Deuschle, et al., (1989) Proc. ferred promoters from endl and end2 genes are disclosed; Natl. Acad. Sci. USA 86:5400-5404: Fuerst, et al., (1989) herein incorporated by reference. In dicots, seed specific pro Proc. Natl. Acad. Sci. USA 86:2549-2553: Deuschle, et al., moters include but are not limited to seed coat promoter from (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, Arabidopsis, pFBAN; and the early seed promoters from Ara University of Heidelberg; Reines, et al., (1993) Proc. Natl. bidopsis, p26, p63, and p63tr (U.S. Pat. Nos. 7.294,760 and Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. 7,847,153). A promoter that has “preferred expression in a Cell. Biol. 10:3343-3356: Zambretti, et al., (1992) Proc. Natl. particular tissue is expressed in that tissue to a greater degree Acad. Sci. USA 89:3952-3956: Bairn, et al., (1991) Proc. than in at least one other plant tissue. Some tissue-preferred Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) promoters show expression almost exclusively in the particu Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, lar tissue. (1989) Topics Mol. Struc. Biol. 10:143-162: Degenkolb, et 0353 Where low level expression is desired, weak pro al., (1991) Antimicrob. Agents Chemother: 35:1591-1595: moters will be used. Generally, the term “weak promoter as Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; used herein refers to a promoter that drives expression of a Bonin, (1993) Ph.D. Thesis, University of Heidelberg: Gos coding sequence at a low level. By low level expression at sen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; levels of about 1/1000 transcripts to about 1/100,000 tran Oliva, et al., (1992) Antimicrob. Agents Chemother: 36:913 scripts to about 1/500,000 transcripts is intended. Alterna 919; Hlavka, et al., (1985) Handbook of Experimental Phar tively, it is recognized that the term “weak promoters' also macology, Vol. 78 (Springer-Verlag, Berlin) and encompasses promoters that drive expression in only a few 0357 Gill, et al., (1988) Nature 334:721-724. Such dis cells and not in others to give a total low level of expression. closures are herein incorporated by reference. US 2014/0007292 A1 Jan. 2, 2014 39
0358. The above list of selectable marker genes is not Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. meant to be limiting. Any selectable marker gene can be used Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et in the embodiments. al., (1985) in The Experimental Manipulation of Ovule Tis sues, ed. Chapman, et al., (Longman, New York), pp. 197-209 Plant Transformation (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415 0359 The methods of the embodiments involve introduc 418 and Kaeppler, et al., (1992) Theor: Appl. Genet. 84:560 ing a polypeptide or polynucleotide into a plant. “Introduc 566 (whisker-mediated transformation); D Halluin, et al., ing is intended to mean presenting to the plant the polynucle (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., otide or polypeptide in Such a manner that the sequence gains (1993) Plant Cell Reports 12:250-255 and Christou and Ford, access to the interior of a cell of the plant. The methods of the (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., embodiments do not depend on a particular method for intro (1996) Nature Biotechnology 14:745-750 (maize via Agro ducing a polynucleotide or polypeptide into a plant, only that bacterium tumefaciens); all of which are herein incorporated the polynucleotide or polypeptides gains access to the interior by reference. of at least one cell of the plant. Methods for introducing 0362. In specific embodiments, the sequences of the polynucleotide or polypeptides into plants are known in the embodiments can be provided to a plant using a variety of art including, but not limited to, stable transformation meth transient transformation methods. Such transient transforma ods, transient transformation methods and virus-mediated tion methods include, but are not limited to, the introduction methods. of the PIP-1 polypeptide or variants and fragments thereof 0360 "Stable transformation' is intended to mean that the directly into the plant or the introduction of the PIP-1 nucleotide construct introduced into a plant integrates into the polypeptide transcript into the plant. Such methods include, genome of the plant and is capable of being inherited by the for example, microinjection or particle bombardment. See, progeny thereof. “Transient transformation' is intended to for example, Crossway, et al., (1986) Mol Gen. Genet. 202: mean that a polynucleotide is introduced into the plant and 179-185: Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, does not integrate into the genome of the plant or a polypep et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, tide is introduced into a plant. By “plant' is intended whole et al., (1994) The Journal of Cell Science 107: 775-784, all of plants, plant organs (e.g., leaves, Sterns, roots, etc.), seeds, which are herein incorporated by reference. Alternatively, the plant cells, propagules, embryos and progeny of the same. PIP-1 polypeptide polynucleotide can be transiently trans Plant cells can be differentiated or undifferentiated (e.g. cal formed into the plant using techniques known in the art. Such lus, suspension culture cells, protoplasts, leaf cells, root cells, techniques include viral vector system and the precipitation phloem cells, and pollen). of the polynucleotide in a manner that precludes Subsequent 0361 Transformation protocols as well as protocols for release of the DNA. Thus, transcription from the particle introducing nucleotide sequences into plants may vary bound DNA can occur, but the frequency with which it is depending on the type of plant or plant cell, i.e., monocot or released to become integrated into the genome is greatly dicot, targeted for transformation. Suitable methods of intro reduced. Such methods include the use of particles coated ducing nucleotide sequences into plant cells and Subsequent with polyethylimine (PEI: Sigma #P3143). insertion into the plant genome include microinjection 0363 Methods are known in the art for the targeted inser (Crossway, et al., (1986) Biotechniques 4:320-334), elec tion of a polynucleotide at a specific location in the plant troporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA genome. In one embodiment, the insertion of the polynucle 83:5602-5606), Agrobacterium-mediated transformation otide at a desired genomic location is achieved using a site (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer specific recombination system. See, for example, WO 1999/ (Paszkowski, et al., (1984) EMBO.J. 3:2717-2722) and bal 25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 listic particle acceleration (see, for example, U.S. Pat. Nos. and WO 1999/25853, all of which are herein incorporated by 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et reference. Briefly, the polynucleotide of the embodiments can al., (1995) in Plant Cell, Tissue, and Organ Culture. Funda be contained in transfer cassette flanked by two non-identical mental Methods, ed. Gamborg and Phillips, (Springer-Verlag, recombination sites. The transfer cassette is introduced into a Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926) plant have stably incorporated into its genome a target site and Lecl transformation (WO 2000/28058). For potato trans which is flanked by two non-identical recombination sites formation see, Tu, et al., (1998) Plant Molecular Biology that correspond to the sites of the transfer cassette. An appro 37:829-838 and Chong, et al., (2000) Transgenic Research priate recombinase is provided and the transfer cassette is 9:71-78. Additional transformation procedures can be found integrated at the target site. The polynucleotide of interest is in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; thereby integrated at a specific chromosomal position in the Sanford, et al., (1987) Particulate Science and Technology plant genome. 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 0364 Plant transformation vectors may be comprised of 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technol one or more DNA vectors needed for achieving plant trans ogy 6:923-926 (soybean): Finer and McMullen, (1991) In formation. For example, it is a common practice in the art to Vitro Cell Dev. Biol. 27P:175-182 (soybean): Singh, et al., utilize plant transformation vectors that are comprised of (1998) Theor: Appl. Genet. 96:319-324 (soybean); Datta, et more than one contiguous DNA segment. These vectors are al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., often referred to in the art as “binary vectors'. Binary vectors (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize): as well as vectors with helperplasmids are most often used for Klein, et al., (1988) Biotechnology 6:559-563 (maize): U.S. Agrobacterium-mediated transformation, where the size and Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, et al., complexity of DNA segments needed to achieve efficient (1988) Plant Physiol. 91:440-444 (maize): Fromm, et al., transformation is quite large, and it is advantageous to sepa (1990) Biotechnology 8:833-839 (maize): Hooykaas-Van rate functions onto separate DNA molecules. Binary vectors Slogteren, et al., (1984) Nature (London) 311:763-764; U.S. typically contain a plasmid vector that contains the cis-acting US 2014/0007292 A1 Jan. 2, 2014 40 sequences required for T-DNA transfer (such as left border 0367 The cells that have been transformed may be grown and right border), a selectable marker that is engineered to be into plants in accordance with conventional ways. See, for capable of expression in a plant cell, and a "gene of interest' example, McCormick, et al., (1986) Plant Cell Reports 5:81– (a gene engineered to be capable of expression in a plant cell 84. These plants may then be grown, and either pollinated for which generation of transgenic plants is desired). Also with the same transformed strain or different strains and the present on this plasmid vector are sequences required for resulting hybrid having constitutive or inducible expression bacterial replication. The cis-acting sequences are arranged in of the desired phenotypic characteristic identified. Two or a fashion to allow efficient transfer into plant cells and expres more generations may be grown to ensure that expression of sion therein. For example, the selectable marker gene and the the desired phenotypic characteristic is stably maintained and pesticidal gene are located between the left and right borders. inherited and then seeds harvested to ensure that expression Often a second plasmid vector contains the trans-acting fac of the desired phenotypic characteristic has been achieved. tors that mediate T-DNA transfer from Agrobacterium to 0368. The nucleotide sequences of the embodiments may plant cells. This plasmid often contains the virulence func be provided to the plant by contacting the plant with a virus or tions (Vir genes) that allow infection of plant cells by Agro viral nucleic acids. Generally, such methods involve incorpo bacterium, and transfer of DNA by cleavage at border rating the nucleotide construct of interest within a viral DNA sequences and Vir-mediated DNA transfer, as is understood in or RNA molecule. It is recognized that the recombinant pro the art (Hellens and Mullineaux, (2000) Trends in Plant Sci teins of the embodiments may be initially synthesized as part ence 5:446-451). Several types of Agrobacterium strains (e.g. of a viral polyprotein, which later may be processed by pro LBA4404, GV3101, EHA101, EHA105, etc.) can be used for teolysis in vivo or in vitro to produce the desired PIP-1 plant transformation. The second plasmid vector is not nec polypeptide. It is also recognized that Such a viral polypro essary for transforming the plants by other methods such as tein, comprising at least a portion of the amino acid sequence microprojection, microinjection, electroporation, polyethyl of a PIP-1 polypeptide of the embodiments, may have the ene glycol, etc. desired pesticidal activity. Such viral polyproteins and the 0365. In general, plant transformation methods involve nucleotide sequences that encode for them are encompassed transferring heterologous DNA into target plant cells (e.g., by the embodiments. Methods for providing plants with immature or mature embryos, Suspension cultures, undiffer nucleotide constructs and producing the encoded proteins in entiated callus, protoplasts, etc.), followed by applying a the plants, which involve viral DNA or RNA molecules are maximum threshold level of appropriate selection (depend known in the art. See, for example, U.S. Pat. Nos. 5,889, 191; ing on the selectable marker gene) to recover the transformed 5,889, 190; 5,866,785: 5,589,367 and 5,316,931, herein plant cells from a group of untransformed cell mass. Follow incorporated by reference. ing integration of heterologous foreign DNA into plant cells, 0369 Methods for transformation of chloroplasts are one then applies a maximum threshold level of appropriate known in the art. See, for example, Svab, et al., (1990) Proc. selection in the medium to kill the untransformed cells and Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) separate and proliferate the putatively transformed cells that Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, Survive from this selection treatment by transferring regularly (1993) EMBO.J. 12:601-606. The method relies on particle to a fresh medium. By continuous passage and challenge with gun delivery of DNA containing a selectable marker and appropriate selection, one identifies and proliferates the cells targeting of the DNA to the plastid genome through homolo that are transformed with the plasmid vector. Molecular and gous recombination. Additionally, plastid transformation can biochemical methods can then be used to confirm the pres be accomplished by transactivation of a silent plastid-borne ence of the integrated heterologous gene of interest into the transgene by tissue-preferred expression of a nuclear-en genome of the transgenic plant. coded and plastid-directed RNA polymerase. Such a system 0366 Explants are typically transferred to a fresh supply has been reported in McBride, et al., (1994) Proc. Natl. Acad. of the same medium and cultured routinely. Subsequently, the Sci., USA 91:7301-73.05. transformed cells are differentiated into shoots after placing 0370. The embodiments further relate to plant-propagat on regeneration medium Supplemented with a maximum ing material of a transformed plant of the embodiments threshold level of selecting agent. The shoots are then trans including, but not limited to, seeds, tubers, corms, bulbs, ferred to a selective rooting medium for recovering rooted leaves, and cuttings of roots and shoots. shoot or plantlet. The transgenic plantlet then grows into a 0371. The embodiments may be used for transformation mature plant and produces fertile seeds (e.g., Hiei, et al., of any plant species, including, but not limited to, monocots (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) and dicots. Examples of plants of interest include, but are not Nature Biotechnology 14:745-750). Explants are typically limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. transferred to a fresh supply of the same medium and cultured rapa, B. juncea), particularly those Brassica species useful as routinely. A general description of the techniques and meth Sources of seed oil, alfalfa (Medicago sativa), rice (Oryza ods for generating transgenic plants are found in Ayres and sativa), rye (Secale cereale), Sorghum (Sorghum bicolor, Sor Park, (1994) Critical Reviews in Plant Science 13:219-239 ghum vulgare), millet (e.g., pearl millet (Pennisetum glau and Bommineni and Jauhar, (1997) Maydica 42:107-120. cum), proso millet (Panicum miliaceum), foxtail millet (Se Since the transformed material contains many cells; both taria italica), finger millet (Eleusine Coracana)), Sunflower transformed and non-transformed cells are present in any (Helianthus annuus), Safflower (Carthamus tinctorius), piece of Subjected target callus or tissue or group of cells. The wheat (Triticum aestivum), soybean (Glycine max), tobacco ability to kill non-transformed cells and allow transformed (Nicotiana tabacum), potato (Solanum tuberosum), peanuts cells to proliferate results in transformed plant cultures. (Arachis hypogaea), cotton (Gossypium barbadense, Gos Often, the ability to remove non-transformed cells is a limi sypium hirsutum), Sweet potato (Ipomoea batatus), cassava tation to rapid recovery of transformed plant cells and Suc (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos cessful generation of transgenic plants. nucifera), pineapple (Ananas comosus), citrus trees (Citrus US 2014/0007292 A1 Jan. 2, 2014
spp.), cocoa (Theobroma Cacao), tea (Camellia sinensis), Evaluation of Plant Transformation banana (Musa spp.), avocado (Persea americana), fig (Ficus 0375 Following introduction of heterologous foreign Casica), guava (Psidium guajava), mango (Mangifera DNA into plant cells, the transformation or integration of indica), olive (Olea europaea), papaya (Carica papaya), heterologous gene in the plant genome is confirmed by vari cashew (Anacardium Occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), Sugar beets (Beta ous methods such as analysis of nucleic acids, proteins and vulgaris), Sugarcane (Saccharum spp.), oats, barley, Veg metabolites associated with the integrated gene. etables ornamentals and conifers. 0376 PCR analysis is a rapid method to screen trans formed cells, tissue or shoots for the presence of incorporated 0372 Vegetables include tomatoes (Lycopersicon escu gene at the earlier stage before transplanting into the soil lentum), lettuce (e.g., Lactuca sativa), green beans (Phaseo (Sambrook and Russell, (2001) Molecular Cloning: A Labo lus vulgaris), lima beans (Phaseolus limensis), peas ratory Manual. Cold Spring Harbor Laboratory Press, Cold (Lathyrus spp.), and members of the genus Cucumis such as Spring Harbor, N.Y.). PCR is carried out using oligonucle cucumber (C. sativus), cantaloupe (C. cantalupensis), and otide primers specific to the gene of interest or Agrobacterium muskmelon (C. melo). Ornamentals include azalea (Rhodo vector background, etc. dendron spp.), hydrangea (Macrophylla hydrangea), hibiscus 0377 Plant transformation may be confirmed by Southern (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa blot analysis of genomic DNA (Sambrook and Russell, spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), (2001) supra). In general, total DNA is extracted from the carnation (Dianthus caryophyllus), poinsettia (Euphorbia transformant, digested with appropriate restriction enzymes, pulcherrima), and chrysanthemum. Conifers that may be fractionated in an agarose gel and transferred to a nitrocellu employed in practicing the embodiments include, for lose or nylon membrane. The membrane or “blot' is then example, pines such as loblolly pine (Pinus taeda), slash pine probed with, for example, radiolabeled 32P target DNA frag (Pinus elliotil), ponderosa pine (Pinus ponderosa), lodgepole ment to confirm the integration of introduced gene into the pine (Pinus contorta) and Monterey pine (Pinus radiata); plant genome according to standard techniques (Sambrook Douglas-fir (Pseudotsuga menziesii); Western hemlock and Russell. (2001) supra). (Tsuga Canadensis); Sitka spruce (Picea glauca); redwood 0378. In Northern blot analysis, RNA is isolated from (Sequoia sempervirens); true firs such as silver fir (Abies specific tissues of transformant, fractionated in a formalde amabilis) and balsam fir (Abies balsamea); and cedars such as hyde agarose gel, and blotted onto a nylon filter according to Western red cedar (Thuja plicata) and Alaska yellow-cedar standard procedures that are routinely used in the art (Sam (Chamaecyparis nootkatensis). Plants of the embodiments brook and Russell. (2001) supra). Expression of RNA include crop plants (for example, corn, alfalfa, Sunflower, encoded by the pesticidal gene is then tested by hybridizing Brassica, Soybean, cotton, safflower, peanut, Sorghum, the filter to a radioactive probe derived from a pesticidal gene, wheat, millet, tobacco, etc.). Such as corn and Soybean plants. by methods known in the art (Sambrook and Russell. (2001) 0373 Turfgrasses include, but are not limited to: annual Supra). bluegrass (Poa annua); annual ryegrass (Lolium multiflo 0379 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 presence (Festuca rubra); colonial bentgrass (Agrostis tenuis); creep of protein encoded by the pesticidal gene by standard proce ing bentgrass (Agrostis palustris); crested wheatgrass (Agro dures (Sambrook and Russell, 2001, supra) using antibodies pyron desertorum); fairway wheatgrass (Agropyron Cris that bind to one or more epitopes present on the PIP-1 tatum); hard fescue (Festuca longifolia); Kentucky bluegrass polypeptide. (Poa pratensis); orchardgrass (Dactyls glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); red Stacking of Traits in Transgenic Plant top (Agrostis alba); rough bluegrass (Poa trivialis); sheep 0380 Transgenic plants may comprise a stack of one or fescue (Festuca ovina); Smooth bromegrass (Bromus iner more insecticidal polynucleotides disclosed herein with one mis); tall fescue (Festuca arundinacea); timothy (Phleum or more additional polynucleotides resulting in the produc pratense); Velvet bentgrass (Agrostis canina); weeping alka tion or Suppression of multiple polypeptide sequences. Trans ligrass (Puccinelia distans); western wheatgrass (Agropyron genic plants comprising stacks of polynucleotide sequences Smithii); Bermuda grass (Cynodon spp.); St. Augustine grass can be obtained by either or both of traditional breeding (Stenotaphrum secundatum); Zoysia grass (Zoysia spp.); methods or through genetic engineering methods. These Bahia grass (Paspalum notatum); carpet grass (Axonopus afi methods include, but are not limited to, breeding individual nis); centipedegrass (Eremochloa Ophiuroides): kikuyu grass lines each comprising a polynucleotide of interest, transform (Pennisetum clandesinum); seashore paspalum (Paspalum ing a transgenic plant comprising a gene disclosed herein vaginatum); bluegramma (Bouteloua gracilis); buffalo grass with a Subsequent gene, and co-transformation of genes into (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipen a single plant cell. As used herein, the term "stacked' includes dula). having two or more traits present in the same plant (e.g., both 0374 Plants of interest include grain plants that provide traits are incorporated into the nuclear genome, one trait is seeds of interest, oil-seed plants, and leguminous plants. incorporated into the nuclear genome and one trait is incor Seeds of interest include grain seeds, such as corn, wheat, porated into the genome of a plastid or both traits are incor barley, rice, Sorghum, rye, millet, etc. Oil-seed plants include porated into the genome of a plastid). In one non-limiting cotton, soybean, Safflower, Sunflower, Brassica, maize, example, 'stacked traits’ comprise a molecular stack where alfalfa, palm, coconut, flax, castor, olive etc. Leguminous the sequences are physically adjacent to each other. A trait, as plants include beans and peas. Beans include guar, locust used herein, refers to the phenotype derived from a particular bean, fenugreek, soybean, garden beans, cowpea, mungbean, sequence or groups of sequences. Co-transformation of genes lima bean, fava bean, lentils, chickpea, etc. can be carried out using single transformation vectors com US 2014/0007292 A1 Jan. 2, 2014 42 prising multiple genes or genes carried separately on multiple ATCCTM Accession Numbers 40098, 67136, 31995 and vectors. If the sequences are stacked by genetically trans 31998. Other non-limiting examples of Bacillus thuringien forming the plants, the polynucleotide sequences of interest sis transgenes being genetically engineered are given in the can be combined at any time and in any order. The traits can following patents and patent applications and hereby are be introduced simultaneously in a co-transformation protocol incorporated by reference for this purpose: U.S. Pat. Nos. with the polynucleotides of interest provided by any combi 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, nation of transformation cassettes. For example, if two 6,060,594, 6,063,597, 6,077,824, 6,620,988, 6,642,030, sequences will be introduced, the two sequences can be con 6,713,259, 6,893,826, 7,105,332; 7,179,965, 7,208.474; tained in separate transformation cassettes (trans) or con 7,227,056, 7,288,643, 7,323,556, 7,329,736, 7,449,552, tained on the same transformation cassette (cis). Expression 7,468,278, 7,510,878, 7,521,235, 7,544,862, 7,605,304, of the sequences can be driven by the same promoter or by 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, different promoters. In certain cases, it may be desirable to 7,858,849, and WO 1991/14778; WO 1999/31248: WO introduce a transformation cassette that will Suppress the 2001/12731: WO 1999/24581 and WO 1997/40162. expression of the polynucleotide of interest. This may be combined with any combination of other suppression cas 0385 Genes encoding pesticidal proteins may also be settes or overexpression cassettes to generate the desired stacked including but are not limited to: insecticidal proteins combination of traits in the plant. It is further recognized that from Pseudomonas sp. such as PSEEN3174 (Monalysin, polynucleotide sequences can be stacked at a desired (2011) PLOS Pathogens, 7:1-13), from Pseudomonas prote genomic location using a site-specific recombination system. gens strain CHAO and Pf-5 (previously fluorescens) (Pechy Tarr, (2008) Environmental Microbiology 10:2368-2386: See, for example, WO 1999/25821, WO 1999/25854, WO GenBank Accession No. EU400157); from Pseudomonas 1999/25840, WO 1999/25855 and WO 1999/25853, all of Taiwanensis (Liu, et al., (2010) J. Agric. Food Chem. which are herein incorporated by reference. 58:12343-12349) and from Pseudomonas pseudoalcligenes 0381. In some embodiments the polynucleotides encoding (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and the PIP-1 polypeptides disclosed herein, alone or stacked Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89:159-168); with one or more additional insect resistance traits can be insecticidal proteins from Photorhabdus sp. and Xenorhab stacked with one or more additional input traits (e.g., herbi dus sp. (Hinchliffe, et al., (2010) The Open Toxinology Jour cide resistance, fungal resistance, virus resistance or stress nal 3:101-118 and Morgan, et al., (2001) Applied and Envir. tolerance, disease resistance, male Sterility, Stalk strength, Micro. 67:2062-2069), U.S. Pat. No. 6,048,838, and U.S. Pat. and the like) or output traits (e.g., increased yield, modified No. 6,379,946; and 8-endotoxins including, but not limited starches, improved oil profile, balanced amino acids, high to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, lysine or methionine, increased digestibility, improved fiber Cry 10, Cry 11, Cry12, Cry 13, Cry 14, Cry 15, Cry 16, Cry 17, quality, drought resistance, and the like). Thus, the polynucle Cry 18, Cry 19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, otide embodiments can be used to provide a complete agro Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, nomic package of improved crop quality with the ability to Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, flexibly and cost effectively control any number of agronomic Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 pests. and Crys5 classes of 5-endotoxin genes and the B. thuring 0382 Transgenes useful for stacking include but are not iensis cytolytic Cyt1 and Cyt2 genes. Members of these limited to: classes of B. thuringiensis insecticidal proteins include, but 1. Transgenes that Confer Resistance to Insects or Disease are not limited to Cry1Aa1 (Accession # Accession it and that Encode: M11250), Cry1Aa2 (Accession # M10917), Cry1Aa3 (Ac 0383 (A) Plant disease resistance genes. Plant defenses cession # D00348), Cry1Aa4 (Accession if X13535), are often activated by specific interaction between the product Cry1Aa5 (Accession # D17518), Cry1Aao (Accession it of a disease resistance gene (R) in the plant and the product of U43605), Cry1Aa7 (Accession # AF081790), Cry1Aa8 (Ac a corresponding avirulence (AVr) gene in the pathogen. A cession #126149), Cry1Aa9 (Accession if AB026261), plant variety can be transformed with cloned resistance gene Cry1Aa10(Accession it. AF154676), Cry1Aa11 (Accession it to engineer plants that are resistant to specific pathogen Y09663), Cry1Aa12 (Accession # AF384211), Cry1Aa13 strains. See, for example, Jones, et al., (1994) Science 266: (Accession it AF510713), Cry1Aa14 (Accession it 789 (cloning of the tomato Cf-9 gene for resistance to Cla AY197341), Cry1Aa15 (Accession it DQ062690), Cry1Ab1 dosporium fillvum); Martin, et al., (1993) Science 262:1432 (Accession # M13898), Cry1Ab2 (Accession # M12661), (tomato Pto gene for resistance to Pseudomonas Syringae pv. Cry1Ab3 (Accession if M15271), Cry1Ab4 (Accession it tomato encodes a protein kinase); Mindrinos, et al., (1994) D00117), Cry1Ab5 (Accession # X04698), Cry1Ab6 (Ac Cell 78: 1089 (Arabidopsis RSP2 gene for resistance to cession # M37263), Cry1Ab7 (Accession #X13233), Pseudomonas syringae), McDowell and Woffenden, (2003) Cry1Ab8 (Accession ii M16463), Cry1Ab9 (Accession it Trends Biotechnol. 21 (4):178-83 and Toyoda, et al., (2002) X54939), Cry1Ab10 (Accession # A29125), Cry1Ab11 (Ac Transgenic Res. 11 (6):567-82. A plant resistant to a disease is cession if I12419), Cry1Ab12 (Accession # AF059670), one that is more resistant to a pathogen as compared to the Cry1Ab13 (Accession if AF254640), Cry1Ab14 (Accession wild type plant. # U94191), Cry1Ab15 (Accession # AF358861), Cry1Ab16 0384 (B) Genes encoding a Bacillus thuringiensis pro (Accession # AF375608), Cry1Ab17 (Accession # tein, a derivative thereof or a synthetic polypeptide modeled AAT46415), Cry1Ab18 (Accession # AAQ88259), thereon. See, for example, Geiser, et al., (1986) Gene 48: 109, Cry1Ab19 (Accession # AY847289), Cry1Ab20 (Accession who disclose the cloning and nucleotide sequence of a Bt # DQ241675), Cry1Ab21 (Accession # EF683163), delta-endotoxin gene. Moreover, DNA molecules encoding Cry1Ab22 (Accession # ABW87320), Cry1Ab-like (Acces delta-endotoxin genes can be purchased from AmericanType sion # AF327924), Cry1Ab-like (Accession # AF327925), Culture Collection (Rockville, Md.), for example, under Cry1Ab-like (Accession if AF327926), Cry1Ab-like (Acces
US 2014/0007292 A1 Jan. 2, 2014 44
EF219060), Crys Ba1 (Accession # U19725), CrysBa2 (Ac Db1 (Accession # BAE80088), Cry30Ea1 (Accession it cession # EU121522), Cry6Aa1 (Accession # L07022), EU503140), Cry30Fa1 (Accession # EU751609), Cry30Ga1 Cry6Aa2 (Accession it AF499736), Cry6Aa3 (Accession it (Accession it EU882064), Cry31Aa1 (Accession it DQ835612), Cry6Ba1 (Accession # L07024), Cry7Aa1 (Ac AB031065), Cry31Aa2 (Accession # AY081052), Cry31Aa3 cession # M64478), Cry7Ab1 (Accession # U04367), (Accession if AB250922), Cry31Aa4 (Accession it Cry7Ab2 (Accession # U04368), Cry7Ab3 (Accession # BI AB274826), Cry31Aa5 (Accession # AB274827), Cry31Ab1 1015188), Cry7Ab4 (Accession # EU380678), Cry7Ab5 (Accession # AB250923), Cry31Ab2 (Accession # (Accession # ABX79555), Cry7Ab6 (Accession it AB274825), Cry31 Ac1 (Accession # AB276125), Cry32Aa1 FJ194973), Cry7Ba1 (Accession if ABB70817), Cry7Ca1 (Accession if AY008143), Cry32Ba1 (Accession it (Accession # EF486.523), Cry8Aa1 (Accession # U04364), BAB78601), Cry32Ca1 (Accession # BAB78602), Cry8Ab1 (Accession # EU044830), Cry8Bal (Accession # Cry32Dal (Accession it BAB78603), Cry33Aa1 (Accession U04365), Cry8Bb1 (Accession # AX543924), Cry8Bc1 (Ac # AAL26871), Cry34Aa1 (Accession # AAG50341), cession # AX543926), Cry8Ca1 (Accession # U04366), Cry34Aa2 (Accession if AAK64560), Cry34Aa3 (Accession Cry8Ca2 (Accession # AAR98783), Cry8Ca3 (Accession # # AY536899), Cry34Aa-4 (Accession # AY536897), EU625349), Cry8Dal (Accession # AB089299), Cry8Da2 Cry34Ab1 (Accession if AAG41671), Cry34Ac1 (Accession (Accession # BD133574), Cry8Da3 (Accession it # AAG50118), Cry34Ac2 (Accession # AAK64562), BD133575), Cry8 Db1 (Accession # AB303980), Cry8Ea1 Cry34Ac3 (Accession # AY536896), Cry34Ba1 (Accession it (Accession if AY329081), Cry8Ea2 (Accession it AAK64565), Cry34Ba2 (Accession # AY536900), EU047597), Cry8Fa1 (Accession # AY551093), Cry8Ga1 Cry34Ba3 (Accession # AY536898), Cry35Aa1 (Accession it (Accession # AY590188), Cry8Ga2 (Accession it AAG50342), Cry35Aa2 (Accession #AAK64561), DQ318860), Cry8Ga3 (Accession # FJ 198072), Cry8Hal Cry35Aa3 (Accession # AY536895), Cry35Aa4 (Accession it (Accession # EF465532), Cry8Ial (Accession # EU381044), AY536892), Cry35Ab1 (Accession # AAG41672), Cry8Ja1 (Accession it EU625348), Cry8 like (Accession it Cry35Ab2 (Accession #AAK64563), Cry35Ab3 (Accession ABS53003), Cry9Aa1 (Accession #X58120), Cry9Aa2 (Ac # AY536891), Cry35Ac1 (Accession # AAG50117), cession # X58534), Cry9Aa like (Accession # AAQ52376), Cry35Ba1 (Accession #AAK64566), Cry35Ba2 (Accession Cry9Ba1 (Accession # X75019), Cry9Bb1 (Accession # # AY536894), Cry35Ba3 (Accession # AY536893), AY758316), Cry9Ca1 (Accession # Z37527), Cry9Ca2 (Ac Cry36Aa1 (Accession if AAK64558), Cry37Aa1 (Accession cession # AAQ52375), Cry9Dal (Accession # D85560), # AAF76376), Cry38Aa1 (Accession # AAK64559), Cry9Da2 (Accession # AF042733), Cry9 Db1 (Accession # Cry39Aa1 (Accession it BAB72016), Cry40Aa1 (Accession AY971349), Cry9Ea1 (Accession # AB01 1496), Cry9Ea2 # BAB72018), Cry40Ba1 (Accession # BAC77648), (Accession #AF358863), Cry9Ea3 (Accession # EF157307), Cry40Ca1 (Accession#EU381045), Cry40Dal (Accession it Cry9Ea4 (Accession # EU760456), Cry9Eas (Accession # EU596478), Cry41Aa1 (Accession # AB116649), Cry41Ab1 EU789519), Cry9Eaé (Accession # EU887516), Cry9Eb1 (Accession it AB 116651), Cry42Aa1 (Accession it (Accession i AX189653), Cry9Ec1 (Accession it AB1 16652), Cry43Aa1 (Accession # AB115422), Cry43Aa2 AF093107), Cry9Ed1 (Accession # AY973867), Cry9 like (Accession if AB176668), Cry43Ba1 (Accession it (Accession # AF093107), Cry 10Aa1 (Accession # M12662), AB115422), Cry43-like (Accession # AB115422), Cry44Aa Cry 10Aa2 (Accession if E00614), Cry10Aa3 (Accession it (Accession it BAD08532), Cry45Aa (Accession it AL731825), Cry 10A like (Accession it DQ167578), BAD22577), Cry46Aa (Accession it. BAC79010), Cry46Aa2 Cry 11 Aa1 (Accession if M31737), Cry 11 Aa2 (Accession it (Accession if BAG68906), Cry46Ab (Accession it M22860), Cry11 Aa3 (Accession # AL731825), Cry11 Aa BAD35170), Cry47Aa (Accession # AY950229), Cry48Aa like (Accession it DQ166531), Cry 11 Ba1 (Accession it (Accession if AJ841948), Cry48Aa2 (Accession it X86902), Cry11 Bb1 (Accession # AF017416), Cry12Aa1 AM237205), Cry48Aa3 (Accession # AM237206), Cry48Ab (Accession # L07027), Cry13Aa1 (Accession # L07023), (Accession # AM237207), Cry48Ab2 (Accession # Cry 14Aa1 (Accession # U13955), Cry 15Aa1 (Accession it AM237208), Cry49Aa (Accession # AJ841948), Cry49Aa2 M76442), Cry16Aa1 (Accession #X94146), Cry 17Aa1 (Ac (Accession it AM237201), Cry49Aa3 (Accession it cession # X99478), Cry18Aa1 (Accession # X99049), AM237203), Cry49Aa4 (Accession # AM237204), Cry18Bal (Accession #AF169250), Cry18Ca1 (Accession # Cry49Ab1 (Accession # AM237202), CrysOAa1 (Accession AF169251), Cry 19Aa1 (Accession # Y07603), Cry 19Bal # AB253419), Crys 1Aa1 (Accession # DQ836184), (Accession # D88381), Cry20Aa1 (Accession # U82518), Cry52Aa1 (Accession#EF613489), Crys3Aa1 (Accession it Cry21 Aa1 (Accession # I32932), Cry21Aa2 (Accession it EF633476), Crys4Aa1 (Accession # EU339367), Crys5Aa1 I66477), Cry21Ba1 (Accession # AB0884.06), Cry22Aal (Accession it EU121521), Crys5Aa2 (Accession it (Accession if I34547), Cry22Aa2 (Accession if AX472772), AAE33526). Cry22Aa3 (Accession # EU715020), Cry22Ab1 (Accession 0386 Examples of 8-endotoxins also include but are not # AAK50456), Cry22Ab2 (Accession # AX472764), limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and Cry22Ba1 (Accession if AX472770), Cry23Aa1 (Accession 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of # AAF76375), Cry24Aa1 (Accession # U88.188), Cry24Bal C.-helix 1 and/or C-helix 2 variants of Cry proteins such as (Accession it BAD32657), Cry24Ca1 (Accession it Cry1A) of U.S. Pat. Nos. 8.304,604 and 8,304,605, Cry 1B of AM158318), Cry25Aa1 (Accession # U88189), Cry26Aal U.S. patent application Ser. No. 10/525,318; Cry 1C of U.S. (Accession it AF 122897), Cry27Aa1 (Accession it Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218, AB023293), Cry28Aa1 (Accession #AF132928), Cry28Aa2 188: Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962, (Accession # AF285775), Cry29Aa1 (Accession it 705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of AJ251977), Cry30Aa1 (Accession # AJ251978), Cry30Bal U.S. Pat. No. 7,064.249); a Cry3A protein including but not (Accession # BAD00052), Cry30Ca1 (Accession it limited to an engineered hybrid insecticidal protein (eHIP) BAD67157), Cry30Da1 (Accession # EF095955), Cry30 created by fusing unique combinations of variable regions US 2014/0007292 A1 Jan. 2, 2014
and conserved blocks of at least two different Cry proteins VBTS 2528 of US Patent Application Publication Number (US Patent Application Publication Number 2010/0017914); 2011/0064710. Other Cry proteins are well known to one a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins skilled in the art (see, Crickmore, et al., “Bacillus thuringien of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476, sis toxin nomenclature' (2011), at lifesci. Sussex.ac.uk/home/ 781, 7,105,332, 7,378.499 and 7.462,760; a Cry9 protein Neil Crickmore/Bt? which can be accessed on the world such as such as members of the Cry9A, Cry9B, Cry9C, wide web using the “www’ prefix). The insecticidal activity Cry9D, Cry9E, and Cry9F families; a Cry 15 protein of Nai of Cry proteins is well known to one skilled in the art (for mov, et al., (2008) Applied and Environmental Microbiology review, see, van Frannkenhuyzen, (2009).J. Invert. Path. 101: 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 1-16). The use of Cry proteins as transgenic plant traits is well 6,127,180, 6,624,145 and 6,340,593: a CryET33 and known to one skilled in the art and Cry-transgenic plants CryET34 protein of U.S. Pat. Nos. 6,248.535, 6,326,351, including but not limited to Cry1Ac, Cry1Ac--Cry2Ab. 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, Cry1F+Cry1Ac, and CryET34 homologs of US Patent Publication Number Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, 2006/0191034, 2012/0278954, and PCT Publication Number Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory WO 2012/139004: a Cry35Ab1 protein of U.S. Pat. Nos. approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283 6,083,499, 6,548,291 and 6,340,593: a Cry46 protein, a Cry 300 and the CERA (2010) GM Crop Database Center for 51 protein, a Cry binary toxin; a TIC901 or related toxin; Environmental Risk Assessment (CERA), ILSI Research TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, Foundation, Washington D.C. at cera-gmc.org/index. TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI php?action gm crop database which can be accessed on the 027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757: world-wide web using the “www” prefix). More than one AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. pesticidal proteins well known to one skilled in the art can No. 7,923,602: AXMI-018, AXMI-020, and AXMI-021 of also be expressed in plants such as Vip3Ab & Cry1 Fa WO 2006/083891: AXMI-010 of WO 2005/038032: AXMI 003 of WO 2005/021585; AXMI-008 of US 2004/0250311; (US2012/0317682), Cry 1BE & Cry1F (US2012/0311746), AXMI-006 of US 2004/0216186: AXMI-007 of US 2004/ Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa 0210965; AXMI-009 of US 2004/0210964: AXMI-014 of US (US2012/0317681), Cry 1DA & Cry1BE (US2012/ 2004/0197917: AXMI-004 of US 2004/0197916: AXMI-028 0331590), Cry1DA & Cry1 Fa (US2012/0331589), Cry1AB and AXMI-029 of WO 2006/119457: AXMI-007, AXMI & Cry 1BE (US2012/0324606), and Cry1Fa & Cry2Aa, 008, AXMI-0080r12, AXMI-009, AXMI-014 and AXMI Cry 1I or Cry1E (US2012/0324605). Pesticidal proteins also 004 of WO 2004/074462: AXMI-150 of U.S. Pat. No. 8,084, include insecticidal lipases including lipid acylhydrolases of 416: AXMI-205 of US20110023184: AXMI-011, AXMI U.S. Pat. No. 7,491,869, and cholesterol oxidases such as 012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, from Streptomyces (Purcell et al. (1993) Biochem Biophys AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI Res Commun 15:1406-1413). Pesticidal proteins also include 022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos. US 2011/0263488; AXMI-R1 and related proteins of US 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 2010/0197592: AXMI221Z, AXMI222z, AXMI223Z, 8.237,020, and the like. Other VIP proteins are well known to AXMI224Z and AXMI225Z of WO 2011/103248: AXMI218, one skilled in the art (see, lifesci. Sussex.ac.uk/home/Neil AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, Crickmore/Bt/vip.html which can be accessed on the world AXMI229, AXMI230, and AXMI231 of WO1 1/103,247; wide web using the “www’ prefix). Pesticidal proteins also AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI include toxin complex (TC) proteins, obtainable from organ 184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, isms such as Xenorhabdus, Photorhabdus and Paenibacillus AXMI-030, AXMI-035, and AXMI-045 of US 2010/ (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC 0298211: AXMI-066 and AXMI-076 of US20090144852; proteins have “standalone' insecticidal activity and other TC AXMI 128, AXMI130, AXMI131, AXMI133, AXMI 140, proteins enhance the activity of the stand-alone toxins pro AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, duced by the same given organism. The toxicity of a “stand AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, alone' TC protein (from Photorhabdus, Xenorhabdus or AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, Paenibacillus, for example) can be enhanced by one or more AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, TC protein “potentiators’ derived from a source organism of AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, a different genus. There are three main types of TC proteins. AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, As referred to herein, Class A proteins (“Protein A') are AXMI 180, AXMI181, AXMI182, AXMI 185, AXMI 186, stand-alone toxins. Class B proteins (“Protein B) and Class AXMI 187, AXMI 188, AXMI189 of U.S. Pat. No. 8,318,900; C proteins (“Protein C) enhance the toxicity of Class A AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, proteins. Examples of Class A proteins are TchA, TcdA, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, XptA1 and XptA2. Examples of Class B proteins are TcaG, AXMI100, AXMI101, AXMI102, AXMI 103, AXMI 104, TcdB, XptB1Xb and XptC1 Wi. Examples of Class C proteins AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, include spider, Snake and Scorpion Venom proteins. Examples AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, of spider venom peptides include but are not limited to lyc AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI 129, otoxin-1 peptides and mutants thereof (U.S. Pat. No. 8.334, AXMI164, AXMI151, AXMI161, AXMI 183, AXMI132, 366). AXMI138, AXMI137 of US 2010/0005543; Cry proteins 0387 (C) A polynucleotide encoding an insect-specific such as Cry1A and Cry3A having modified proteolytic sites hormone or pheromone such as an ecdysteroid and juvenile of U.S. Pat. No. 8.319,019; and a Cry1Ac, Cry2Aa and hormone, a variant thereof, a mimetic based thereon or an Cry1Ca toxin protein from Bacillus thuringiensis strain antagonistoragonist thereof. See, for example, the disclosure US 2014/0007292 A1 Jan. 2, 2014 46 by Hammock, et al., (1990) Nature 344:458, of baculovirus effected by the virus from which the coat protein gene is expression of cloned juvenile hormone esterase, an inactiva derived, as well as by related viruses. See, Beachy, et al., tor of juvenile hormone. (1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated 0388 (D) A polynucleotide encoding an insect-specific resistance has been conferred upon transformed plants peptide which, upon expression, disrupts the physiology of against alfalfa mosaic virus, cucumber mosaic virus, tobacco the affected pest. For example, see the disclosures of Regan, streak virus, potato virus X, potato virus Y, tobacco etch virus, (1994).J. Biol. Chem. 269:9 (expression cloning yields DNA tobacco rattle virus and tobacco mosaic virus. Id. coding for insect diuretic hormone receptor); Pratt, et al., 0395 (K) A gene encoding an insect-specific antibody or (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allosta an immunotoxin derived therefrom. Thus, an antibody tar tin is identified in Diploptera puntata); Chattopadhyay, et al., geted to a critical metabolic function in the insect gut would (2004) Critical Reviews in Microbiology 30(1):33-54, Zjaw inactivate an affected enzyme, killing the insect. Cf. Taylor, et iony, (2004).J Nat Prod 67(2):300-310; Carlini and Grossi al., Abstract #497, SEVENTH INTL SYMPOSIUM ON de-Sa (2002) Toxicon 40(11):1515-1539;Ussufetal. (2001) MOLECULAR PLANT-MICROBE INTERACTIONS (Ed Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira (2004) inburgh, Scotland, 1994) (enzymatic inactivation in trans Toxicon 44(4):385-403. See also, U.S. Pat. No. 5.266,317 to genic tobacco via production of single-chain antibody frag Tomalski, et al., who disclose genes encoding insect-specific ments). toxins. 0396 (L) A gene encoding a virus-specific antibody. See, 0389 (E) A polynucleotide encoding an enzyme respon for example, Tavladoraki, et al., (1993) Nature 366:469, who sible for a hyperaccumulation of a monterpene, a sesquiter show that transgenic plants expressing recombinant antibody pene, a steroid, hydroxamic acid, a phenylpropanoid deriva genes are protected from virus attack. tive or another non-protein molecule with insecticidal 0397) (M) A polynucleotide encoding a developmental activity. arrestive protein produced in nature by a pathogen or a para 0390 (F) A polynucleotide encoding an enzyme involved site. Thus, fungal endo alpha-1,4-D-polygalacturonases in the modification, including the post-translational modifi facilitate fungal colonization and plant nutrient release by cation, of a biologically active molecule; for example, a gly solubilizing plant cell wall homo-alpha-1,4-D-galacturonase. colytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a See, Lamb, et al., (1992) Bio/Technology 10:1436. The clon nuclease, a cyclase, a transaminase, an esterase, a hydrolase, ing and characterization of a gene which encodes a bean a phosphatase, a kinase, a phosphorylase, a polymerase, an endopolygalacturonase-inhibiting protein is described by elastase, a chitinase and a glucanase, whether natural or syn Toubart, et al., (1992) Plant J. 2:367. thetic. See, PCT Application WO 1993/02197 in the name of 0398 (N) A polynucleotide encoding a developmental Scott, et al., which discloses the nucleotide sequence of a arrestive protein produced in nature by a plant. For example, callase gene. DNA molecules which contain chitinase-encod Logemann, et al., (1992) Bio/Technology 10:305, have shown ing sequences can be obtained, for example, from the ATCC that transgenic plants expressing the barley ribosome-inacti under Accession Numbers 39637 and 67152. See also, Vating gene have an increased resistance to fungal disease. Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691, who teach the nucleotide sequence of a cDNA encoding 0399 (O) Genes involved in the Systemic Acquired Resis tobacco hookworm chitinase and Kawalleck, et al., (1993) tance (SAR) Response and/or the pathogenesis related genes. Plant Molec. Biol. 21:673, who provide the nucleotide Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, sequence of the parsley ubi4-2 polyubiquitin gene and U.S. (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich, Pat. Nos. 6,563,020; 7,145,060 and 7,087,810. (2003) Cell 113(7):815-6. 0391 (G) A polynucleotide encoding a molecule that 04.00 (P) Antifungal genes (Cornelissen and Melchers, stimulates signal transduction. For example, see the disclo (1993) Pl. Physiol. 101:709-712 and Parijs, et al., (1991) sure by Botella, et al., (1994) Plant Molec. Biol. 24:757, of Planta 183:258-264 and Bushnell, et al., (1998) Can. J. of nucleotide sequences for mung bean calmodulin cDNA Plant Path. 20(2):137-149. Also see, U.S. application Ser. clones and Griess, et al., (1994) Plant Physiol. 104: 1467, who Nos. 09/950,933; 11/619,645; 11/657,710; 1 1/748,994; provide the nucleotide sequence of a maize calmodulin 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. cDNA clone. LysM Receptor-like kinases for the perception of chitin frag 0392 (H) A polynucleotide encoding a hydrophobic ments as a first step in plant defense response against fungal moment peptide. See, PCT Application WO 1995/16776 and pathogens (US 2012/0110696). U.S. Pat. No. 5,580,852 disclosure of peptide derivatives of 04.01 (Q) Detoxification genes, such as for fumonisin, Tachyplesin which inhibit fungal plant pathogens) and PCT beauvericin, moniliformin and Zearalenone and their struc Application WO 1995/18855 and U.S. Pat. No. 5,607.914 turally related derivatives. For example, see, U.S. Pat. Nos. (teaches synthetic antimicrobial peptides that confer disease 5,716,820; 5,792,931; 5,798.255; 5,846,812: 6,083,736; resistance). 6,538,177; 6,388,171 and 6,812,380. 0393 (I) A polynucleotide encoding a membrane per 0402 (R) A polynucleotide encoding a Cystatin and cys mease, a channel former or a channel blocker. For example, teine proteinase inhibitors. See, U.S. Pat. No. 7,205.453. see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of (0403 (S) Defensingenes. See, WO 2003/0008.63 and U.S. heterologous expression of a cecropin-beta lytic peptide ana Pat. Nos. 6,911,577; 6,855,865; 6,777,592 and 7,238,781. log to render transgenic tobacco plants resistant to 0404 (T) Genes conferring resistance to nematodes. See, Pseudomonas Solanacearum. e.g., PCT Application Number WO 1996/30517: PCT Appli 0394 (J) A gene encoding a viral-invasive protein or a cation Number WO 1993/19181, WO 2003/033651 and complex toxin derived therefrom. For example, the accumu Urwin, et al., (1998) Planta204:472-479, Williamson, (1999) lation of viral coat proteins in transformed plant cells imparts Curr Opin Plant Bio. 2(4):327-31; U.S. Pat. Nos. 6,284.948 resistance to viral infection and/or disease development and 7.301,069 and miR164 genes (WO 2012/058266). US 2014/0007292 A1 Jan. 2, 2014 47
04.05 (U) Genes that confer resistance to Phytophthora Patent No. 0 242 246 and 0 242 236 to Leemans, et al. De Root Rot, such as the Rps 1. Rps 1-a, Rps 1-b, Rps 1-c. Rps Greef, et al., (1989) Bio/Technology 7:61, describe the pro 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c. Rps duction of transgenic plants that express chimeric bar genes 4. Rps 5, Rps 6. Rps 7 and other Rps genes. See, for example, coding for phosphinothricin acetyl transferase activity. See Shoemaker, et al., Phytophthora Root Rot Resistance Gene also, U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874, Mapping in Soybean, Plant Genome IV Conference, San 265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 Diego, Calif. (1995). B1 and 5,879,903, which are incorporated herein by reference 0406 (V) Genes that conferresistance to Brown Stem Rot, for this purpose. Exemplary genes conferring resistance to such as described in U.S. Pat. No. 5.689,035 and incorporated phenoxy proprionic acids and cyclohexones, such as Sethoxy by reference for this purpose. dim and haloxyfop, are the Acc1-S1, Acc1-52 and Acc1-53 0407 (W) Genes that confer resistance to Colletotrichum, genes described by Marshall, et al., (1992) Theor: Appl. such as described in US Patent Application Publication US Genet. 83:435. 2009/0035765 and incorporated by reference for this pur 0410 (C) A polynucleotide encoding a protein for resis pose. This includes the Rcg locus that may be utilized as a tance to herbicide that inhibits photosynthesis, such as a single locus conversion. triazine (psbA and gS+genes) and a benzonitrile (nitrilase 2. Transgenes that Confer Resistance to a Herbicide, for gene). Przibilla, et al., (1991) Plant Cell 3:169 describe the Example: transformation of Chlamydomonas with plasmids encoding 0408 (A) A polynucleotide encoding resistance to a her mutant psbA genes. Nucleotide sequences for nitrilase genes bicide that inhibits the growing point or meristem, Such as an are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA imidazolinone or a Sulfonylurea. Exemplary genes in this molecules containing these genes are available under ATCC category code for mutant ALS and AHAS enzyme as Accession Numbers 53435, 67441 and 67442. Cloning and described, for example, by Lee, et al., (1988) EMBO.J. 7:1241 expression of DNA coding for a glutathione S-transferase is and Miki, et al., (1990) Theor: Appl. Genet. 80:449, respec described by Hayes, et al., (1992) Biochem. J. 285: 173. tively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141, 0411 (D) A polynucleotide encoding a protein for resis 870; 5,767,361; 5,731, 180; 5,304.732; 4,761,373; 5,331,107; tance to Acetohydroxy acid synthase, which has been found 5,928,937 and 5,378,824; U.S. patent application Ser. No. to make plants that express this enzyme resistant to multiple 1 1/683,737 and International Publication WO 1996/33270. types of herbicides, has been introduced into a variety of 04.09 (B) A polynucleotide encoding a protein for resis plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246: tance to Glyphosate (resistance imparted by mutant 419). Other genes that confer resistance to herbicides include: 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA a gene encoding a chimeric protein of rat cytochrome genes, respectively) and other phosphono compounds such as P4507A1 and yeast NADPH-cytochrome P450 oxidoreduc glufosinate (phosphinothricin acetyl transferase (PAT) and tase (Shiota, et al., (1994) Plant Physiol 106:17), genes for Streptomyces hygroscopicus phosphinothricin acetyl trans glutathione reductase and Superoxide dismutase (Aono, et al., ferase (bar) genes) and pyridinoxy or phenoxy proprionic (1995) Plant Cell Physiol 36:1687, and genes for various acids and cyclohexones (ACCase inhibitor-encoding genes). phosphotransferases (Datta, et al., (1992) Plant Mol Biol See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., 20:619). which discloses the nucleotide sequence of a form of EPSPS 0412 (E) A polynucleotide encoding resistance to a her which can confer glyphosate resistance. U.S. Pat. No. 5,627, bicide targeting Protoporphyrinogen oxidase (protox) which 061 to Barry, et al., also describes genes encoding EPSPS is necessary for the production of chlorophyll. The protox enzymes. See also, U.S. Pat. Nos. 6,566,587; 6,338,961: enzyme serves as the target for a variety of herbicidal com 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783: pounds. These herbicides also inhibit growth of all the differ 4,971,908: 5,312,910; 5,188,642; 5,094,945, 4,940,835; ent species of plants present, causing their total destruction. 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; The development of plants containing altered protox activity 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E which are resistant to these herbicides are described in U.S. and 5,491,288 and International Publications EP 1173580; Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373 and WO 2001/66704; EP 1173581 and EP 1173582, which are International Publication WO 2001/12825. incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express agene encod 0413 (F) The aad-1 gene (originally from Sphingobium ing a glyphosate oxido-reductase enzyme as described more herbicidovorans) encodes the aryloxyalkanoate dioxygenase fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are (AAD-1) protein. The trait confers tolerance to 2,4-dichlo incorporated herein by reference for this purpose. In addition rophenoxyacetic acid and aryloxyphenoxypropionate (com glyphosate resistance can be imparted to plants by the over monly referred to as “fop herbicides such as quizalofop) expression of genes encoding glyphosate N-acetyltrans herbicides. The aad-1 gene, itself, for herbicide tolerance in ferase. See, for example, U.S. Pat. Nos. 7.462.481; 7,405,074 plants was first disclosed in WO 2005/107437 (see also, US and US Patent Publication Number US 2008/0234.130). A 2009/0093366). The aad-12 gene, derived from Delftia aci DNA molecule encoding a mutant aroA gene can be obtained dovorans, which encodes the aryloxyalkanoate dioxygenase under ATCC Accession Number 39256 and the nucleotide (AAD-12) protein that confers tolerance to 2,4-dichlorophe sequence of the mutant gene is disclosed in U.S. Pat. No. noxyacetic acid and pyridyloxyacetate herbicides by deacti 4.769,061 to Comai. European Patent Application Number 0 Vating several herbicides with an aryloxyalkanoate moiety, 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to including phenoxy auxin (e.g., 2,4-D, MCPA), as well as Goodman, et al. disclose nucleotide sequences of glutamine pyridyloxy auxins (e.g., fluoroxypyr, triclopyr). synthetase genes which confer resistance to herbicides Such 0414 (G) A polynucleotide encoding a herbicide resistant as L-phosphinothricin. The nucleotide sequence of a phos dicamba monooxygenase disclosed in US Patent Application phinothricin-acetyl-transferase gene is provided in European Publication 2003/0135879 for imparting dicamba tolerance; US 2014/0007292 A1 Jan. 2, 2014 48
0415 (H) A polynucleotide molecule encoding bromoxy 0430 (2) Modulating a gene that reduces phytate content. nil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for In maize, this, for example, could be accomplished, by clon imparting bromoxynil tolerance; ing and then re-introducing DNA associated with one or more 0416) (I) A polynucleotide molecule encoding phytoene of the alleles, such as the LPA alleles, identified in maize (crt1) described in Misawa, et al., (1993) Plant J. 4:833-840 mutants characterized by low levels of phytic acid, Such as in and in Misawa, et al., (1994) Plant J. 6:481-489 for norflu WO 2005/113778 and/or by altering inositol kinase activity razon tolerance. as in WO 2002/059324, US 2003/0009011, WO 2003/ 0417 3. Transgenes that Confer or Contribute to an 027243, US 2003/0079247, WO 1999/05298, U.S. Pat. No. Altered Grain Characteristic, 6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, WO 2002/059324, US 2003/0079247, WO 1998/45448, WO Such as: 1999/55882, WO 2001/04147. 0431 (C) Altered carbohydrates effected, for example, by 0418 (A) Altered fatty acids, for example, by altering a gene for an enzyme that affects the branching 0419 (1) Down-regulation of stearoyl-ACP to increase pattern of starch or, a gene altering thioredoxin Such as NTR stearic acid content of the plant. See, KnultZon, et al., (1992) and/or TRX (see, (see, U.S. Pat. No. 6,531,648 which is Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 incorporated by reference for this purpose) and/or a gamma (Genes to Alter Lipid Profiles in Corn), Zein knock out or mutant such as cs27 or TUSC27 or en27 0420 (2) Elevating oleic acid via FAD-2 gene modifica (see, U.S. Pat. No. 6,858,778 and US 2005/0160488, US tion and/or decreasing linolenic acid via FAD-3 gene modi 2005/0204418; which are incorporated by reference for this fication (see, U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 purpose). See, Shiroza, et al., (1988) J. Bacteriol. 170:810 and WO 1993/11245), (nucleotide sequence of Streptococcus mutant fructosyltrans 0421 (3) Altering conjugated linolenic or linoleic acid ferase gene), Steinmetz, et al., (1985) Mol. Gen. Genet. 200: content, such as in WO 2001/12800, 220 (nucleotide sequence of Bacillus subtilis levansucrase 0422 (4) Altering LEC1, AGP Dek1, Superall, mill ps, gene), Pen, et al., (1992) Bio/Technology 10:292 (production and various Ipa genes Such as Ipal, Ipa3, hpt or hggt. For of transgenic plants that express Bacillus licheniformis alpha example, see, WO 2002/42424, WO 1998/22604, WO 2003/ amylase), Elliot, et al., (1993) Plant Molec. Biol. 21:515 011015, WO 2002/057439, WO 2003/011015, U.S. Pat. Nos. (nucleotide sequences of tomato invertase genes), Søgaard, et 6,423,886, 6,197.561, 6,825,397 and US Patent Application al., (1993).J. Biol. Chem. 268:22480 (site-directed mutagen Publication Numbers US 2003/0079247, US 2003/0204870 esis of barley alpha-amylase gene) and Fisher, et al., (1993) and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci. Plant Physiol. 102: 1045 (maize endosperm starch branching 92:562O-5624. enzyme II), WO 1999/10498 (improved digestibility and/or 0423 (5) Genes encoding delta-8 desaturase for making starch extraction through modification of UDP-D-xylose long-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058, 4-epimerase, Fragile 1 and 2. Refl, HCHL., C4H), U.S. Pat. 571 and 8.338,152), delta-9 desaturase for lowering saturated No. 6,232,529 (method of producing high oil seed by modi fats (U.S. Pat. No. 8,063,269), Primula A6-desaturase for fication of starch levels (AGP)). The fatty acid modification improving omega-3 fatty acid profiles. genes mentioned herein may also be used to affect starch 0424 (6) Isolated nucleic acids and proteins associated content and/or composition through the interrelationship of with lipid and Sugar metabolism regulation, in particular, the starch and oil pathways. lipid metabolism protein (LMP) used in methods of produc 0432 (D) Altered antioxidant content or composition, ing transgenic plants and modulating levels of seed storage Such as alteration of tocopherol or tocotrienols. For example, compounds including lipids, fatty acids, starches or seed Stor see, U.S. Pat. No. 6,787,683, US 2004/0034886 and WO age proteins and use in methods of modulating the seed size, 2000/68393 involving the manipulation of antioxidant levels seed number, seed weights, root length and leaf size of plants and WO 2003/082899 through alteration of a homogentisate (EP2404499). geranylgeranyl transferase (hggt). 0425 (7) Altering expression of a High-Level Expression 0433 (E) Altered essential seed amino acids. For example, of Sugar-Inducible 2 (HSI2) protein in the plant to increase or see, U.S. Pat. No. 6,127.600 (method of increasing accumu decrease expression of HSI2 in the plant. Increasing expres lation of essential amino acids in seeds), U.S. Pat. No. 6,080, sion of HSI2 increases oil content while decreasing expres 913 (binary methods of increasing accumulation of essential sion of HSI2 decreases abscisic acid sensitivity and/or amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), increases drought resistance (US 2012/0066794). WO 1999/40209 (alteration of amino acid compositions in 0426 (8) Expression of cytochrome b5 (Cb5) alone or seeds), WO 1999/29882 (methods for altering amino acid with FAD2 to modulate oil content in plant seed, particularly content of proteins), U.S. Pat. No. 5,850,016 (alteration of to increase the levels of omega-3 fatty acids and improve the amino acid compositions in seeds), WO 1998/20133 (pro ratio of omega-6 to omega-3 fatty acids (US Patent Applica teins with enhanced levels of essential amino acids), U.S. Pat. tion Publication Number 2011/019 1904). No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 0427 (9) Nucleic acid molecules encoding wrinkled 1-like (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid polypeptides for modulating sugar metabolism (U.S. Pat. No. biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increased 8,217,223). lysine and threonine), U.S. Pat. No. 6,441.274 (plant tryp 0428 B) Altered phosphorus content, for example, by the tophan synthase beta subunit), U.S. Pat. No. 6,346,403 (me 0429 (1) Introduction of a phytase-encoding gene would thionine metabolic enzymes), U.S. Pat. No. 5,939,599 (high enhance breakdown of phytate, adding more free phosphate sulfur), U.S. Pat. No. 5,912,414 (increased methionine), WO to the transformed plant. For example, see Van Hartingsveldt, 1998/56935 (plant amino acid biosynthetic enzymes), WO et al., (1993) Gene 127:87, for a disclosure of the nucleotide 1998/45458 (engineered seed protein having higher percent sequence of an Aspergillus niger phytase gene. age of essential amino acids), WO 1998/42831 (increased US 2014/0007292 A1 Jan. 2, 2014 49 lysine), U.S. Pat. No. 5,633,436 (increasing sulfur amino acid 013227, WO 2003/013228, WO 2003/014327, WO 2004/ content), U.S. Pat. No. 5,559,223 (synthetic storage proteins 031349, WO 2004/076638, WO 1998.09521; with defined structure containing programmable levels of 0442 (B) WO 199938977 describing genes, including essential amino acids for improvement of the nutritional CBF genes and transcription factors effective in mitigating value of plants), WO 1996/01905 (increased threonine), WO the negative effects of freezing, high salinity and drought on 1995/15392 (increased lysine), US 2003/0163838, US 2003/ plants, as well as conferring other positive effects on plant O150014, US 2004/0068767, U.S. Pat. No. 6,803,498, WO phenotype; 2001 (79.516. 0443) (C) US 2004/0148654 and WO 2001/36596 where 4. Genes that Control Male-Sterility: abscisic acid is altered in plants resulting in improved plant 0434. There are several methods of conferring genetic phenotype such as increased yield and/or increased tolerance male sterility available. Such as multiple mutant genes at to abiotic stress; separate locations within the genome that confer male steril 0444 (D) WO 2000/006341, WO 2004/090143, U.S. Pat. ity, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727.219 to Nos. 7,531,723 and 6,992.237 where cytokinin expression is Brar, et al., and chromosomal translocations as described by modified resulting in plants with increased stress tolerance, Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. In addi Such as drought tolerance, and/or increased yield. Also see, tion to these methods, Albertsen, et al., U.S. Pat. No. 5,432, WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S. 068, describe a system of nuclear male sterility which Pat. No. 6,084,153, WO 200164898, U.S. Pat. No. 6,177,275 includes: identifying a gene which is critical to male fertility; and U.S. Pat. No. 6,107,547 (enhancement of nitrogen utili silencing this native gene which is critical to male fertility; Zation and altered nitrogen responsiveness); removing the native promoter from the essential male fertility 0445 (E) For ethylene alteration, see, US 2004/0128719, gene and replacing it with an inducible promoter, inserting US 2003/0166197 and WO 2000/32761; this genetically engineered gene back into the plant; and thus 0446 (F) For plant transcription factors or transcriptional creating a plant that is male sterile because the inducible regulators of abiotic stress, see e.g., US 2004/0098764 or US promoter is not “on” resulting in the male fertility gene not 2004/0078852; being transcribed. Fertility is restored by inducing or turning 0447 (G) Genes that increase expression of vacuolar “on”, the promoter, which in turn allows the gene that confers pyrophosphatase such as AVP1 (U.S. Pat. No. 8,058,515) for male fertility to be transcribed. increased yield; nucleic acid encoding a HSFA4 or a HSFA5 0435 (A) Introduction of a deacetylase gene under the (Heat Shock Factor of the class A4 or A5) polypeptides, an control of a tapetum-specific promoter and with the applica oligopeptide transporter protein (OPT4-like) polypeptide; a tion of the chemical N-Ac-PPT (WO 01/29237). plastochron2-like (PLA2-like) polypeptide or a Wuschel 0436 (B) Introduction of various stamen-specific promot related homeobox 1-like (WOX1-like) polypeptide (US Patent Application Publication Number US 2011/0283420): ers (WO 1992/13956, WO 1992/13957). 0448 (H) Down regulation of polynucleotides encoding 0437 (C) Introduction of the barnase and the barstar gene poly (ADP-ribose) polymerase (PARP) proteins to modulate (Paul, et al., (1992) Plant Mol. Biol. 19:611-622). programmed cell death (U.S. Pat. No. 8,058,510) for 0438. For additional examples of nuclear male and female increased vigor, sterility systems and genes, see also, U.S. Pat. Nos. 5,859, 0449 (I) Polynucleotide encoding DTP21 polypeptides 341; 6,297,426; 5,478,369; 5,824,524; 5,850,014; and 6,265, for conferring drought resistance (US Patent Publication 640; all of which are hereby incorporated by reference. Number US 2011/0277181); 5. Genes that Create a Site for Site Specific DNA Integration. 0450 (J) Nucleotide sequences encoding ACC Synthase 3 0439. This includes the introduction of FRT sites that may (ACS3) proteins for modulating development, modulating be used in the FLP/FRT system and/or LOX sites that may be response to stress and modulating stress tolerance (US Patent used in the Cre/LOXp system. For example, see LyZnik, et al., Pub. No. US20100287669). (2003) Plant Cell Rep. 21:925-932 and WO 1999/25821, 0451 (K) Polynucleotides that encode proteins that confer which are hereby incorporated by reference. Other systems a drought tolerance phenotype (DTP) for conferring drought that may be used include the Gin recombinase of phage Mu resistance (WO 2012/058528). (Maeser, et al., (1991) Vicki Chandler, The Maize Handbook 0452 (L) Tocopherol cyclase (TC) genes for conferring ch. 118 (Springer-Verlag 1994), the Pin recombinase of E. drought and salt tolerance (US Patent Application Publication coli (Enomoto, et al., 1983) and the R/RS system of the pSRi Number 2012/0272352). plasmid (Araki, et al., 1992). 0453 (M) CAAX amino terminal family proteins for 6. Genes that Affect Abiotic Stress Resistance stress tolerance (U.S. Pat. No. 8.338,661). 0440 Including but not limited to flowering, ear and seed 0454 (N) Mutations in the SAL1 encoding gene have development, enhancement of nitrogen utilization efficiency, increased stress tolerance, including increased drought resis altered nitrogen responsiveness, drought resistance or toler tant (US Patent Application Publication Number 2010/ ance, cold resistance or tolerance, and salt resistance or tol 0257633). erance, and increased yield under stress. 0455 (O) Expression of a nucleic acid sequence encoding 0441 (A) For example, see, WO 2000/73475 where water a polypeptide selected from the group consisting of GRF use efficiency is altered through alteration of malate: U.S. Pat. polypeptide, RAA1-like polypeptide, SYR polypeptide, Nos. 5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417,428, ARKL polypeptide, and YTP polypeptide increasing yield 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO 2000/ related traits (US Patent Application Publication Number 060089, WO 2001/026459, WO 2001/035725, WO 2001/ 2011/0061133). 034726, WO 2001/035727, WO 2001/036444, WO 2001/ 0456 (P) Modulating expression in a plant of a nucleic 036597, WO 2001/036598, WO 2002/015675, WO 2002/ acid encoding a Class III Trehalose Phosphate Phosphatase 0.17430, WO 2002/077185, WO 2002/079403, WO 2003/ (TPP) polypeptide for enhancing yield-related traits in plants, US 2014/0007292 A1 Jan. 2, 2014 50 particularly increasing seed yield (US Patent Application fying the plant's root architecture (US Patent Application Publication Number 2010/0024067). Publication Number 2009/0064373). 0457. Other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, 8. Genes that Confer Plant Digestibility. plant growth and/or plant structure, can be introduced or 0464) (A) Altering the level of xylan present in the cell introgressed into plants, see e.g., WO 1997/49811 (LHY), wall of a plant by modulating expression of Xylan synthase WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No. (U.S. Pat. No. 8,173.866). 6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/ 14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1), 0465. In some embodiment the stacked trait may be a trait WO 2000/44918 (VRN2), WO 1999/49064 (GI), WO 2000/ or event that has received regulatory approval including but 46358 (FR1), WO 1997/29123, U.S. Pat. No. 6,794,560, U.S. not limited to the events in Table 1A-1F. Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and Rht), and WO 2004/076638 and WO 2004/031349 (transcription fac TABLE 1A tors). Triticum aestivum Wheat 7. Genes that Confer Increased Yield 0458 (A) A transgenic crop plant transformed by a Event Company Description 1-AminoCyclopropane-1-Carboxylate Deaminase-like AP2OSCL BASF Inc. Selection for a mutagenized version of the Polypeptide (ACCDP) coding nucleic acid, wherein expres enzyme acetohydroxyacid synthase (AHAS), sion of the nucleic acid sequence in the crop plant results in also known as acetolactate synthase (ALS) or the plants increased root growth, and/or increased yield, acetolactate pyruvate-lyase. and/or increased tolerance to environmental stress as com AP602CL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), pared to a wild type variety of the plant (U.S. Pat. No. 8,097. also known as acetolactate synthase (ALS) or 769). acetolactate pyruvate-lyase. 0459 (B) Over-expression of maize zinc finger protein BW255-2, BASF Inc. Selection for a mutagenized version of the gene (Zm-ZFP1) using a seed preferred promoter has been BW238-3 enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or shown to enhance plant growth, increase kernel number and acetolactate pyruvate-lyase. total kernel weight per plant (US 2012/0079623). BWT BASF Inc. Tolerance to imidazolinone herbicides induced 0460 (C) Constitutive over-expression of maize lateral by chemical mutagenesis of the organ boundaries (LOB) domain protein (Zm-LOBDP1) has acetohydroxyacid synthase (AHAS) gene using been shown to increase kernel number and total kernel weight Sodium azide. per plant (2012/0079622). MON71800 Monsanto Glyphosate tolerant wheat variety produced by Company inserting a modified 5-enolpyruvylshikimate-3- 0461 (D) Enhancing yield-related traits in plants by phosphate synthase (EPSPS) encoding gene modulating expression in a plant of a nucleic acid encoding a from the soil bacterium Agrobacterium VIM1 (Variant in Methylation 1)-like polypeptide oraVTC2 tumefaciens, strain CP4. like (GDP-L-galactose phosphorylase) polypeptide or a SWP965001 Cyanamid Selection for a mutagenized version of the DUF1685 polypeptide or an ARF6-like (Auxin Responsive Crop enzyme acetohydroxyacid synthase (AHAS), Factor) polypeptide (WO 2012/038893). Protection also known as acetolactate synthase (ALS) or 0462 (E) Modulating expression in a plant of a nucleic acetolactate pyruvate-lyase. Teal 11A BASF Inc. Selection for a mutagenized version of the acid encoding a Step 20-like polypeptide or a homologue enzyme acetohydroxyacid synthase (AHAS), thereof gives plants having increased yield relative to control also known as acetolactate synthase (ALS) or plants (EP2431472). acetolactate pyruvate-lyase. 0463 (F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptides and homologs thereof for modi TABLE 1B Glycine max L. Soybean Event Company Description A2704-12, A2704-21, Bayer CropScience Glufosinate ammonium herbicide tolerant ASS47-35 (Aventis CropScience soybean produced by inserting a modified (AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. ASS47-127 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis CropScience soybean produced by inserting a modified (AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introduced csir1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacid synthase protein that confers tolerance to imidazolinone herbicides due to a point mutation that results in a single amino acid Substitution in which the US 2014/0007292 A1 Jan. 2, 2014 51
TABLE 1 B-continued
Glycine max L. Soybean
Event Company Description
serine residue at position 653 is replaced by asparagine (S653N). DP-3 OS423 Pioneer Hi-Bred High oleic acid soybean produced by inserting International Inc. additional copies of a portion of the omega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing of the endogenous omega-6 desaturase gene (FAD2-1). Pioneer Hi-Bred Soybean event with two herbicide tolerance International Inc. genes: glyphosate N-acetlytransferase, which detoxifies glyphosate, and a modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleic acid soybean produced by inserting a Agricultural Products second copy of the fatty acid desaturase (GmFad2-1) encoding gene from soybean, which resulted in silencing of the endogenous host gene. GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean variety produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens. GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis soybean produced by inserting a modified CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. MON877O1 Monsanto Company Resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmataiis) and soybean looper (Pseudoplitsia includens). MON877O1 x Monsanto Company Glyphosate herbicide tolerance through MON89788 expression of the EPSPS encoding gene from A. tumefaciens strain CP4, and resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatais) and soybean looper (Pseudoplitsia includens) via expression of the Cry1Ac encoding gene from B. thiringiensis. MON89788 Monsanto Company Glyphosate-tolerant soybean produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA (epsps) gene from Agrobacterium timefaciens CP4. OT96-15 Agriculture & Agri-Food Low linolenic acid soybean produced through Canada traditional cross-breeding to incorporate the novel trait from a naturally occurring fan1 gene mutant that was selected for low linolenic acid. Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis soybean produced by inserting a modified CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicals. US 2014/0007292 A1 Jan. 2, 2014 52
TABLE 1C TABLE 1D Medicago Saiva Alfalfa Heianthus annuus Sunflower Event Company Description Event Company Description J101, J163 Monsanto Glyphosate herbicide tolerant alfalfa (lucerne) Company produced by inserting a gene encoding the X81359 BASF Inc. Tolerance to imidazolinone herbicides by and Forage enzyme 5-enolypyruvylshikimate-3-phosphate Genetics synthase (EPSPS) from the CP4 strain of selection of a naturally occurring mutant. International Agrobacterium timefaciens.
TABLE 1E Oryza sativa Rice Event Company Description CL121, CL141, CFX51 BASF Inc. Tolerance to the imidazolinone herbicide, imazethalpyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using sodium azide. LLRICE06, LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). LLRICE6O1 Bayer CropScience Glufosinate ammonium herbicide tolerant rice (Aventis produced by inserting a modified CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to the imidazolinone herbicide, imazethalpyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).
TABLE1F Zea mass L. Maize Event Company Description 176 Syngenta Seeds, Inc. Insect-resistant maize produced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp. kunstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). 3751IR Pioneer Hi-Bred Selection of somaclonal variants by culture of International Inc. embryos on imidazolinone containing media. 676,678,680 Pioneer Hi-Bred Male-sterile and glufosinate ammonium herbicide International Inc. tolerant maize produced by inserting genes encoding DNA adenine methylase and phosphinothricin acetyltransferase (PAT) from Escherichia coli and Streptomyces viridochromogenes, respectively. B16 (DLL25) Dekalb Genetics Glufosinate ammonium herbicide tolerant maize Corporation produced by inserting the gene encoding phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicnis. Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maize produced by inserting the Cry1Ab gene from Bacilius thiringiensis Subsp. ikirstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11 x GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD unique identifier: MON-OOO21-9). US 2014/0007292 A1 Jan. 2, 2014 53
TABLE 1 F-continued Zea maps L. Maize Event Company Description BT11 XMIR162 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier: SYN-IR162-4). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacilius thairingiensis Subsp. ikirstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Resistance to other lepidopteran pests, including H. zea, S.frugiperda, A. ipsilon, and S. albicosta, is derived from MIR162, which contains the vip3Aa gene from Bacilius thuringiensis strain AB88. BT11 x MIR162 x Syngenta Seeds, Inc. Bacilius thuringiensis Cry1Ab delta-endotoxin MIR604 protein and the genetic material necessary for its production (via elements of vector pZO1502) in Event Bt11 corn (OECD Unique Identifier: SYN BTO11-1) x Bacilius thuringiensis Vip3Aa20 insecticidal protein and the genetic material necessary for its production (via elements of vector pNOV1300) in Event MIR162 maize (OECD Unique Identifier: SYN-IR162-4) x modified Cry3A protein and the genetic material necessary for its production (via elements of vector pZM26) in Event MIR604 corn (OECD Unique Identifier: SYN-IR6O4-5). BT11 x Syngenta Seeds, Resistance to coleopteran pests, particularly corn rootworm pests MIR162 x Inc. (Diabroica spp.) and several lepidopteran pests of corn, including MIR604 x European corn borer (ECB, Ostrinia nubialis), corn earworm GA21 (CEW, Helicoverpa zea), fall army worm (FAW. Spodoptera fugiperda), and black cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate and glufosinate-ammonium containing herbicides. BT11 x Syngenta Seeds, Stacked insect resistant and herbicide tolerant maize produced MIR604 Inc. by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD unique identifier: SYN-IR6O5-5). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacilius thiringiensis Subsp. ikairs taki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mGry3A gene from Bacilius thiringiensis. BT11 x Syngenta Seeds, Stacked insect resistant and herbicide tolerant maize produced MIR604 x Inc. by conventional cross breeding of parental lines BT11 (OECD GA21 unique identifier: SYN-BTO11-1), MIR604 (OECD unique identifier: SYN-IR6O5-5) and GA21 (OECD unique identifier: MON-OOO21-9). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacilius thiringiensis Subsp. ikairs taki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mGry3A gene from Bacilius thuringiensis. Tolerance to glyphosate herbicide is derived from GA21 which contains a modified EPSPS gene from maize. CBH-351 Aventis Insect-resistant and glufosinate ammonium herbicide tolerant CropScience maize developed by inserting genes encoding Cry9C protein from Bacilius thuringiensis subsp. tolworthi and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. DAS DOW AgroSciences Lepidopteran insect resistant and glufosinate ammonium O6275-8 LLC herbicide-tolerant maize variety produced by inserting the Cry1F gene from Bacilius thairingiensis varaizawai and the phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicals. DAS DOW AgroSciences Corn rootworm-resistant maize produced by inserting the 591.22-7 LLC and Pioneer Hi Cry34Ab1 and Cry35Ab1 genes from Bacilius thuringiensis strain Bred International PS149B1. The PAT encoding gene from Streptomyces Inc. viridochromogenes was introduced as a selectable marker. US 2014/0007292 A1 Jan. 2, 2014 54
TABLE 1 F-continued Zea maps L. Maize Event Company Description DAS- DOW AgroSciences Stacked insect resistant and herbicide tolerant maize produced S9122-7 x LLC and Pioneer Hi- by conventional cross breeding of parental lines DAS-59122-7 NK603 Bred International (OECD unique identifier: DAS-59122-7) with NK603 (OECD Inc. unique identifier: MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacilius thuringiensis strain PS149B1. Tolerance to glyphosate herbicide is derived from NK603. DAS-591.22-7 x DOW AgroSciences Stacked insect resistant and herbicide tolerant maize produced TC1507 x LLC and Pioneer by conventional cross breeding of parental lines DAS-59122-7 NK603 Hi-Bred (OECD unique identifier: DAS-59122-7) and TC1507 (OECD International Inc. unique identifier: DAS-O15O7-1) with NK603 (OECD unique identifier: MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacilius thuringiensis strain PS149B1. Lepidopteran resistance and tolerance to glufosinate ammonium herbicide is derived from TC1507. Tolerance to glyphosate herbicide is derived from NK603. DBT418 Dekalb Genetics Insect-resistant and glufosinate ammonium herbicide tolerant Corporation maize developed by inserting genes encoding Cry1AC protein from Bacilius thiringiensis subsp kistaki and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus DK4O4SR BASF Inc. Somaclonal variants with a modified acetyl-CoA-carboxylase (ACCase) were selected by culture of embryos on sethoxydim enriched medium. Event 3272 Syngenta Seeds, Maize line expressing a heat stable alpha-amylase gene Inc. amy797E for use in the dry-grind ethanol process. The phosphomannose isomerase gene from E. coli was used as a selectable marker. Event 98.140 Pioneer Hi-Bred Maize event expressing tolerance to glyphosate herbicide, via International Inc. expression of a modified bacterial glyphosate N acetlytransferase, and ALS-inhibiting herbicides, vial expression of a modified form of the maize acetolactate synthase enzyme. EXP191OIT Syngenta Seeds, Tolerance to the imidazolinone herbicide, imazethalpyr, Inc. (formerly induced by chemical mutagenesis of the acetolactate synthase Zeneca Seeds) (ALS) enzyme using ethyl methanesulfonate (EMS). GA21 Syngenta Seeds, Introduction, by particle bombardment, of a modified 5 Inc. (formerly enolpyruvylshikimate-3-phosphate synthase (EPSPS), an Zeneca Seeds) enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. GA21 x Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid MON810 derived from conventional cross-breeding of the parental lines GA21 (OECD identifier: MON-OOO21-9) and MON810 (OECD identifier: MON-OO81O-6). IT Pioneer Hi-Bred Tolerance to the imidazolinone herbicide, imazethalpyr, was International Inc. obtained by in vitro selection of Somaclonal variants. LYO38 Monsanto Company Altered amino acid composition, specifically elevated levels of lysine, through the introduction of the cordap A gene, derived from Corynebacterium glutamictim, encoding the enzyme dihydrodipicolinate synthase (cDHDPS). MIR162 Syngenta Seeds, Insect-resistant maize event expressing a Vip3A protein from Inc. Bacilius thuringiensis and the Escherichia coli PMI selectable marker MIR604 Syngenta Seeds, Corn rootworm resistant maize produced by transformation Inc. with a modified Cry3A gene. The phosphomannose isomerase gene from E. coli was used as a selectable marker. MIR604 x Syngenta Stacked insect resistant and herbicide tolerant maize produced by GA21 Seeds, Inc. conventional cross breeding of parental lines MIR604 (OECD unique identifier: SYN-IR6O5-5) and GA21 (OECD unique identifier: MON OOO21-9). Corn rootworm-resistance is derived from MIR604 which contains the mGry3A gene from Bacilius thiringiensis. Tolerance to glyphosate herbicide is derived from GA21. MON801 OO Monsanto Insect-resistant maize produced by inserting the Cry1Ab gene from Company Bacilius thiringiensis subsp. ikirstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). MON802 Monsanto Insect-resistant and glyphosate herbicide tolerant maize produced by Company inserting the genes encoding the Cry1Ab protein from Bacilius thiringiensis and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi- Resistance to European corn borer (Ostrinia nubialis) by introduction Bred of a synthetic Cry1Ab gene. Glyphosate resistance via introduction of International he bacterial version of a plant enzyme, 5-enolpyruvylshikimate-3- Inc. phosphate synthase (EPSPS). US 2014/0007292 A1 Jan. 2, 2014 55
TABLE 1 F-continued Zea maps L. Maize Event Company Description MON810 Monsanto Insect-resistant maize produced by inserting a truncated form of the Company Cry1Ab gene from Bacilius thuringiensis subsp. kunstaki HD-1. The genetic modification affords resistance to attack by the European corn borer (ECB). MON810 x Monsanto Stacked insect resistant and enhanced lysine content maize derived LYO38 Company from conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) and LYO38 (OECD identifier: REN-OOO38-3). MON810 x Monsanto Stacked insect resistant and glyphosate tolerant maize derived from MON88O17 Company conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) and MON88017 (OECD identifier: MON 88O17-3). European corn borer (ECB) resistance is derived from a truncated form of the Cry1Ab gene from Bacilius thuringiensis subsp. kurstaki HD-1 present in MON810. Corn rootworm resistance is derived from the Cry3Bb1 gene from Bacilius thuringiensis Subspecies kumamotoensis strain EG4691 present in MON88017. Glyphosate tolerance is derived from a 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4 present in MON88017. MON832 Monsanto Introduction, by particle bombardment, of glyphosate oxidase (GOX) Company and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. MON863 Monsanto Corn root worm resistant maize produced by inserting the Cry3Bb1 Company gene from Bacilius thairingiensis Subsp. iki imamotoensis. MON863 x Monsanto Stacked insect resistant corn hybrid derived from conventional cross MON810 Company breeding of the parental lines MON863 (OECD identifier: MON OO863-5) and MON810 (OECD identifier: MON-OO81O-6) MON863 x Monsanto Company Stacked insect resistant and herbicide tolerant MON810 x corn hybrid derived from conventional cross NK603 breeding of the stacked hybrid MON-OO863-5 x MON-OO81O-6 and NK603 (OECD identifier: MON-OO6O3-6). MON863 x Monsanto Company Stacked insect resistant and herbicide tolerant NK603 corn hybrid derived from conventional cross breeding of the parental lines MON863 (OECD identifier: MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460 Monsanto Company MON 87460 was developed to provide reduced yield loss under water-limited conditions compared to conventional maize. Efficacy in MON 87460 is derived by expression of the inserted Bacilius subtilis cold shock protein B (CspB). MON88O17 Monsanto Company Corn rootworm-resistant maize produced by inserting the Cry3Bb1 gene from Bacilius thiringiensis Subspecies kinamotoensis strain EG4691. Glyphosate tolerance derived by inserting a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maize event expressing two different insecticidal proteins from Bacilius thiringiensis providing resistance to number of lepidopteran pests. MON89034 x Monsanto Company Stacked insect resistant and glyphosate tolerant MON88O17 maize derived from conventional cross-breeding of the parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017 (OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects is derived from two Cry genes present in MON89043. Corn rootworm resistance is derived from a single Cry genes and glyphosate tolerance is derived from the 5 enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens present in MON88017. MON89034 x Monsanto Company Stacked insect resistant and herbicide tolerant NK603 maize produced by conventional crossbreeding of parental lines MON89034 (OECD identifier: MON-89O34-3) with NK603 (OECD unique identifier: MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two Cry 9. enes present in MON89043. Tolerance to 9. yphosate herbicide is derived from NK603. US 2014/0007292 A1 Jan. 2, 2014 56
TABLE 1 F-continued Zea maps L. Maize Event Company Description MON89034 x Monsanto Company and Mycogen Stacked insect resistant and herbicide tolerant TC1507 x Seeds co Dow AgroSciences LLC maize produced by conventional crossbreeding MON88O17 x of parental lines: MON89034, TC1507, DAS- MON88017, and DAS-59122. Resistance to the 591.22-7 above-ground and below-ground insect pests and tolerance to glyphosate and glufosinate ammonium containing herbicides. MS3 Bayer CropScience (Aventis Malesterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilitis amyloiquefaciens; PPT resistance was via PPT acetyltransferase (PAT). MS6 Bayer CropScience (Aventis Malesterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilitis amyloiquefaciens; PPT resistance was via PPT acetyltransferase (PAT). NK603 Monsanto Company Introduction, by particle bombardment, of a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. NK6O3 x Monsanto Company Stacked insect resistant and herbicide tolerant MON810 corn hybrid derived from conventional cross breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and MON810 (OECD identifier: MON-OO81O-6). NK6O3 x Monsanto Company Stacked glufosinate ammonium and glyphosate T25 herbicide tolerant maize hybrid derived from conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and T25 (OECD identifier: ACS-ZM003-2). T14, T25 Bayer CropScience (Aventis Glufosinate herbicide tolerant maize produced by CropScience(AgrEvo)) inserting the phosphinothricin N-acetyltransferase (PAT) encoding gene from the aerobic actinomycete Streptomyces viridochromogenes. T2S X Bayer CropScience (Aventis Stacked insect resistant and herbicide tolerant MON810 CropScience(AgrEvo)) corn hybrid derived from conventional cross breeding of the parental lines T25 (OECD identifier: ACS-ZMOO3-2) and MON810 (OECD identifier: MON-OO81O-6). TC1507 Mycogen (co Dow AgroSciences); Insect-resistant and glufosinate ammonium Pioneer (cio DuPont) herbicide tolerant maize produced by inserting the Cry1F gene from Bacilius thuringiensis var. aizawai and the phosphinothricin N acetyltransferase encoding gene from Streptomyces viridochromogenes. TC1507 x DOW AgroSciences LLC and Stacked insect resistant and herbicide tolerant DAS- Pioneer Hi-Bred International Inc. maize produced by conventional cross breeding of 591.22-7 parental lines TC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECD unique identifier: DAS-59122-7). Resistance to lepidopteran insects is derived from TC1507 due the presence of the Cry1F gene from Bacilius thiringiensis var. aizawai. Corn rootworm resistance is derived from DAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacilius thuringiensis strain PS149B1. Tolerance to glufosinate ammonium herbicide is derived from TC1507 from the phosphinothricin N acetyltransferase encoding gene from Streptomyces viridochromogenes. TC1507 x DOW AgroSciences LLC Stacked insect resistant and herbicide tolerant NK603 corn hybrid derived from conventional cross breeding of the parental lines 1507 (OECD identifier: DAS-O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).
0466 Other events with regulatory approval are well Gene Silencing known to one skilled in the art and can be found at the Center for Environmental Risk Assessment (cera-gmc.org/ 0467. In some embodiments the stacked trait may be in the '?action gm crop database, which can be accessed using the form of silencing of one or more polynucleotides of interest www prefix). resulting in Suppression of one or more target pest polypep US 2014/0007292 A1 Jan. 2, 2014 57 tides. In some embodiments the silencing is achieved through 0475 Another variation describes the use of plant viral the use of a suppression DNA construct. sequences to direct the Suppression of proximal mRNA 0468. In some embodiments one or more of the PIP-1, encoding sequences (PCT Publication WO 1998/36083). PIP-1A (SEQ ID NO: 2), PSEEN3174 (SEQ ID NO: 6), 0476 Recent work has described the use of “hairpin' PIP-1C (SEQ ID NO: 332), and PIP-1B (SEQ ID NO: 4) structures that incorporate all or part, of an mRNA encoding polypeptides or fragments or variants thereof may be stacked sequence in a complementary orientation that results in a with one or more polynucleotides encoding one or more potential “stem-loop' structure for the expressed RNA (PCT polypeptides having insecticidal activity or agronomic traits Publication Number WO 1999/53050). In this case the stem is as set forth Supra and optionally may further include one or formed by polynucleotides corresponding to the gene of more polynucleotides providing for gene silencing of one or interest inserted in either sense or anti-sense orientation with more target polynucleotides as discussed infra. respect to the promoter and the loop is formed by some 0469 “Suppression DNA construct” is a recombinant polynucleotides of the gene of interest, which do not have a DNA construct which when transformed or stably integrated complement in the construct. This increases the frequency of into the genome of the plant, results in “silencing of a target coSuppression or silencing in the recovered transgenic plants. gene in the plant. The target gene may be endogenous or For review of hairpin suppression see, Wesley, et al., (2003) transgenic to the plant. “Silencing, as used herein with Methods in Molecular Biology, Plant Functional Genomics: respect to the target gene, refers generally to the Suppression Methods and Protocols 236:273-286. of levels of mRNA or protein/enzyme expressed by the target 0477. A construct where the stem is formed by at least 30 gene, and/or the level of the enzyme activity or protein func nucleotides from a gene to be suppressed and the loop is tionality. The term "suppression' includes lower, reduce, formed by a random nucleotide sequence has also effectively decline, decrease, inhibit, eliminate and prevent. “Silencing been used for suppression (WO 1999/61632). or “gene silencing does not specify mechanism and is inclu 0478. The use of poly-Tand poly-A sequences to generate sive, and not limited to, anti-sense, coSuppression, viral-Sup the stem in the stem-loop structure has also been described pression, hairpin Suppression, stem-loop Suppression, RNAi (WO 2002/00894). based approaches and Small RNA-based approaches. 0479. Yet another variation includes using synthetic 0470 A suppression DNA construct may comprise a repeats to promote formation of a stem in the stem-loop region derived from a target gene of interest and may com structure. Transgenic organisms prepared with Such recom prise all or part of the nucleic acid sequence of the sense binant DNA fragments have been shown to have reduced Strand (or antisense strand) of the target gene of interest. levels of the protein encoded by the nucleotide fragment Depending upon the approach to be utilized, the region may forming the loop as described in PCT Publication Number be 100% identical or less than 100% identical (e.g., at least WO 2002/00904. 50% or any integer between 51% and 100% identical) to all or 0480 RNA interference refers to the process of sequence part of the sense Strand (or antisense Strand) of the gene of specific post-transcriptional gene silencing in animals medi interest. ated by short interfering RNAs (siRNAs) (Fire, et al., (1998) 0471 Suppression DNA constructs are well-known in the Nature 391:806). The corresponding process in plants is com art, are readily constructed once the target gene of interest is monly referred to as post-transcriptional gene silencing selected, and include, without limitation, coSuppression con (PTGS) or RNA silencing and is also referred to as quelling in structs, antisense constructs, viral-suppression constructs, fungi. The process of post-transcriptional gene silencing is hairpin Suppression constructs, stem-loop Suppression con thought to be an evolutionarily-conserved cellular defense structs, double-stranded RNA-producing constructs, and mechanism used to prevent the expression of foreign genes more generally, RNAi (RNA interference) constructs and and is commonly shared by diverse flora and phyla (Fire, et small RNA constructs such as siRNA (short interfering RNA) al., (1999) Trends Genet. 15:358). Such protection from for constructs and miRNA (microRNA) constructs. eign gene expression may have evolved in response to the 0472 “Antisense inhibition” refers to the production of production of double-stranded RNAs (dsRNAs) derived from antisense RNA transcripts capable of suppressing the expres viral infection or from the random integration of transposon sion of the target protein. elements into a host genome via a cellular response that 0473) “Antisense RNA” refers to an RNA transcript that is specifically destroys homologous single-stranded RNA of complementary to all or part of a target primary transcript or viral genomic RNA. The presence of dsRNA in cells triggers mRNA and that blocks the expression of a target isolated the RNAi response through a mechanism that has yet to be nucleic acid fragment (U.S. Pat. No. 5,107,065). The comple fully characterized. mentarity of an antisense RNA may be with any part of the 0481. The presence of long dsRNAs in cells stimulates the specific gene transcript, i.e., at the 5' non-coding sequence, 3' activity of a ribonuclease III enzyme referred to as dicer. non-coding sequence, introns or the coding sequence. Dicer is involved in the processing of the dsRNA into short 0474 “Cosuppression” refers to the production of sense pieces of dsRNA known as short interfering RNAs (siRNAs) RNA transcripts capable of suppressing the expression of the (Berstein, et al., (2001) Nature 409:363). Short interfering target protein. “Sense' RNA refers to RNA transcript that RNAs derived from dicer activity are typically about 21 to includes the mRNA and can be translated into protein within about 23 nucleotides in length and comprise about 19 base a cellor in vitro. CoSuppression constructs in plants have been pair duplexes (Elbashir, et al., (2001) Genes Dev. 15:188). previously designed by focusing on overexpression of a Dicer has also been implicated in the excision of 21- and nucleic acid sequence having homology to a native mRNA, in 22-nucleotide small temporal RNAs (stRNAs) from precur the sense orientation, which results in the reduction of all sor RNA of conserved structure that are implicated in trans RNA having homology to the overexpressed sequence (see, lational control (Hutvagner, et al., (2001) Science 293:834). Vaucheret, et al. (1998) Plant J. 16:651-659 and Gura, (2000) The RNAi response also features an endonuclease complex, Nature 404:804-808). commonly referred to as an RNA-induced silencing complex US 2014/0007292 A1 Jan. 2, 2014
(RISC), which mediates cleavage of single-stranded RNA target sequences or from different regions of the same target having sequence complementarity to the antisense strand of sequence can be employed. For example, the Suppressor the siRNA duplex. Cleavage of the target RNA takes place in enhancer elements employed can comprise fragments of the the middle of the region complementary to the antisense target sequence derived from different region of the target strand of the siRNA duplex (Elbashir, et al., (2001) Genes sequence (i.e., from the 3'UTR, coding sequence, intron, and/ Dev. 15:188). In addition, RNA interference can also involve or 5' UTR). Further, the suppressor enhancer element can be Small RNA (e.g., miRNA) mediated gene silencing, presum contained in an expression cassette, as described elsewhere ably through cellular mechanisms that regulate chromatin herein, and in specific embodiments, the Suppressor enhancer structure and thereby prevent transcription of target gene element is on the same or on a different DNA vector or sequences (see, e.g., Allshire, (2002) Science 297: 1818 construct as the silencing element. The Suppressor enhancer 1819; Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, element can be operably linked to a promoter as disclosed (2002) Science 297:2215-2218; and Hall, et al., (2002) Sci herein. It is recognized that the Suppressor enhancer element ence 297:2232–2237). As such, miRNA molecules of the can be expressed constitutively or alternatively, it may be invention can be used to mediate gene silencing via interac produced in a stage-specific manner employing the various tion with RNA transcripts or alternately by interaction with inducible or tissue-preferred or developmentally regulated particular gene sequences, wherein Such interaction results in promoters that are discussed elsewhere herein. gene silencing either at the transcriptional or post-transcrip 0485. In specific embodiments, employing both a silenc tional level. ing element and the Suppressor enhancer element the sys 0482 Methods and compositions are further provided temic production of RNAi occurs throughout the entire plant. which allow for an increase in RNAi produced from the In further embodiments, the plant or plant parts of the inven silencing element. In such embodiments, the methods and tion have an improved loading of RNAi into the phloem of the compositions employ a first polynucleotide comprising a plant than would be observed with the expression of the silencing element for a target pest sequence operably linked silencing element construct alone and, thus provide better to a promoteractive in the plant cell; and, a second polynucle control of phloem feeding insects by an RNAi approach. In otide comprising a Suppressor enhancer element comprising specific embodiments, the plants, plant parts, and plant cells the target pest sequence or an active variant or fragment of the invention can further be characterized as allowing for thereof operably linked to a promoter active in the plant cell. the production of a diversity of RNAi species that can The combined expression of the silencing element with Sup enhance the effectiveness of disrupting target gene expres pressor enhancer element leads to an increased amplification S1O. of the inhibitory RNA produced from the silencing element 0486 In specific embodiments, the combined expression over that achievable with only the expression of the silencing of the silencing element and the Suppressor enhancer element element alone. In addition to the increased amplification of increases the concentration of the inhibitory RNA in the plant the specific RNAi species itself, the methods and composi cell, plant, plant part, plant tissue orphloem over the level that tions further allow for the production of a diverse population is achieved when the silencing element is expressed alone. of RNAi species that can enhance the effectiveness of dis 0487. As used herein, an “increased level of inhibitory rupting target gene expression. As such, when the Suppressor RNA' comprises any statistically significant increase in the enhancer element is expressed in a plant cell in combination level of RNAi produced in a plant having the combined with the silencing element, the methods and composition can expression when compared to an appropriate control plant. allow for the systemic production of RNAi throughout the For example, an increase in the level of RNAi in the plant, plant; the production of greater amounts of RNAi than would plant part or the plant cell can comprise at least about a 1%, be observed with just the silencing element construct alone; about a 1%-5%, about a 5%-10%, about a 10%-20%, about a and, the improved loading of RNAi into the phloem of the 20%-30%, about a 30%-40%, about a 40%-50%, about a plant, thus providing better control of phloemfeeding insects 50%-60%, about 60-70%, about 70%–80%, about a 80%- by an RNAi approach. Thus, the various methods and com 90%, about a 90%-100% or greater increase in the level of positions provide improved methods for the delivery of RNAi in the plant, plant part, plant cell or phloem when inhibitory RNA to the target organism. See, for example, US compared to an appropriate control. In other embodiments, 2009/O1880O8. the increase in the level of RNAi in the plant, plant part, plant 0483. As used herein, a “suppressor enhancer element' cell or phloem can comprise at least about a 1 fold, about a 1 comprises a polynucleotide comprising the target sequence to fold-5 fold, about a 5 fold.-10 fold, about a 10 fold-20 fold, be suppressed or an active fragment or variant thereof. It is about a 20 fold-30 fold, about a 30 fold-40 fold, about a 40 recognize that the Suppressor enhancer element need not be fold-50 fold, about a 50 fold-60 fold, about 60 fold-70 fold, identical to the target sequence, but rather, the Suppressor about 70 fold-80 fold, about a 80 fold-90 fold, about a 90 enhancer element can comprise a variant of the target fold-100 fold or greater increase in the level of RNAi in the sequence, so long as the Suppressor enhancer element has plant, plant part, plant cell or phloem when compared to an Sufficient sequence identity to the target sequence to allow for appropriate control. Examples of combined expression of the an increased level of the RNAi produced by the silencing silencing element with Suppressor enhancer element for the element over that achievable with only the expression of the control of Stinkbugs and Lygus can be found in US 2011/ silencing element. Similarly, the Suppressor enhancer ele O3O1223 and US 2009/O1921.17. ment can comprise afragment of the target sequence, wherein 0488. Some embodiments relate to down-regulation of the fragment is of Sufficient length to allow for an increased expression of target genes in insect pest species by interfering level of the RNAi produced by the silencing element over that ribonucleic acid (RNA) molecules. WO 2007/074405 achievable with only the expression of the silencing element. describes methods of inhibiting expression of target genes in 0484. It is recognized that multiple suppressor enhancer invertebrate pests including Colorado potato beetle. WO elements from the same target sequence or from different 2005/110068 describes methods of inhibiting expression of US 2014/0007292 A1 Jan. 2, 2014 59 target genes in invertebrate pests including in particular West 0490 Microorganism hosts that are known to occupy the ern corn rootworm as a means to control insect infestation. “phytosphere' (phylloplane, phyllosphere, rhizosphere, and/ Furthermore, WO 2009/091864 describes compositions and or rhizoplana) of one or more crops of interest may be methods for the Suppression of target genes from insect pest selected. These microorganisms are selected so as to be species including pests from the Lygus genus. Nucleic acid capable of Successfully competing in the particular environ molecules including RNAi for targeting the vacuolar ATPase ment with the wild-type microorganisms, provide for stable H subunit, useful for controlling a coleopteran pest popula maintenance and expression of the gene expressing the PIP-1 tion and infestation as described in US Patent Application polypeptide, and desirably, provide for improved protection Publication 2012/O198586. WO 2012/055982 describes ribo of the pesticide from environmental degradation and inacti nucleic acid (RNA or double stranded RNA) that inhibits or Vation. down regulates the expression of a target gene that encodes: 0491 Such microorganisms include bacteria, algae, and an insect ribosomal protein Such as the ribosomal protein fungi. Of particular interest are microorganisms such as bac L19, the ribosomal protein L40 or the ribosomal protein teria, e.g., Alcaligenes, Pseudomonas, Erwinia, Serratia, 527A, an insect proteasome subunit Such as the Rpnó protein, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit Rhodopseudomonas, Methylius, Agrobacterium, Aceto protein or the Pros beta 2 protein; an insect B-coatomer of the bacter; Lactobacillus, Arthrobacter, Azotobacter, Leuconos COPI vesicle, the y-coatomer of the COPI vesicle, the toc, and Alcaligenes, fungi, particularly yeast, e.g., Saccha B'-coatomer protein orthe-coatomer of the COPI vesicle: an romyces, Cryptococcus, Kluyveromyces, Sporobolomyces, insect Tetraspanine 2A protein which is a putative transmem Rhodotorula, and Aureobasidium. Of particular interest are brane domain protein; an insect protein belonging to the actin Such phytosphere bacterial species as Alcaligenes faecalis, family Such as Actin 5C, an insect ubiquitin-5E protein; an Pseudomonas Syringae, Pseudomonas fluorescens, Serratia insect Sec23 protein which is a GTPase activator involved in narceScens, Acetobacter xylinum, Agrobacteria, intracellular protein transport; an insect crinkled protein Rhodopseudomonas spheroides, Xanthomonas campestris, which is an unconventional myosin which is involved in Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli motor activity; an insect crooked neck protein which is and Azotobacter vinelandii and phytosphere yeast species involved in the regulation of nuclear alternative mRNA splic Such as Rhodotorula rubra, R. glutinis, R. marina, R. auran ing; an insect vacuolar H+-ATPase G-subunit protein; and an tiaca, Cryptococcus albidus, C. difluens, C. laurentii, Sac insect Tbp-1 such as Tat-binding protein. US Patent Applica charomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolo tion Publications 2012/029750 and 2012/0322660 describe myces roseus, S. odorus, Kluyveromyces veronae, and an interfering ribonucleic acid (RNA or double stranded Aureobasidium pollulans. Of particular interest are the pig RNA) that functions upon uptake by an insect pest species to mented microorganisms. Host organisms of particular inter down-regulate expression of a target gene in said insect pest, est include yeast, Such as Rhodotorula spp., Aureobasidium wherein the RNA comprises at least one silencing element spp., Saccharomyces spp. (Such as S. cerevisiae), Sporobolo wherein the silencing element is a region of double-stranded myces spp., phylloplane organisms such as Pseudomonas RNA comprising annealed complementary strands, one spp. (Such as P. aeruginosa, Pfluorescens, P. chlororaphis), Strand of which comprises or consists of a sequence of nucle Erwinia spp., and Flavobacterium spp., and other Such organ otides which is at least partially complementary to a target isms, including Agrobacterium tumefaciens, E. coli, Bacillus nucleotide sequence within the target gene. US Patent Appli subtilis, and the like. cation Publication 2012/0164205 describe potential targets 0492 Genes encoding the PIP-1 polypeptides of the for interfering double stranded ribonucleic acids for inhibit embodiments can be introduced into microorganisms that ing invertebrate pests including: a Chd3 Homologous multiply on plants (epiphytes) to deliver PIP-1 polypeptides Sequence, a Beta-Tubulin Homologous Sequence, a 40 kDa to potential target pests. Epiphytes, for example, can be gram V-ATPase Homologous Sequence, a EF 1a Homologous positive or gram-negative bacteria. Sequence, a 26S Proteosome Subunit p28 Homologous 0493 Root-colonizing bacteria, for example, can be iso Sequence, a Juvenile Hormone Epoxide Hydrolase Homolo lated from the plant of interest by methods known in the art. gous Sequence, a Swelling Dependent Chloride Channel Pro Specifically, a Bacillus cereus Strain that colonizes roots can tein Homologous Sequence, a Glucose-6-Phosphate 1-Dehy be isolated from roots of a plant (see, for example, Handels drogenase Protein Homologous Sequence, an Act 42A man, et al., (1991) Appl. Environ. Microbiol. 56:713-718). Protein Homologous Sequence, a ADP-Ribosylation Factor 1 Genes encoding the PIP-1 polypeptides of the embodiments Homologous Sequence, a Transcription Factor IIB Protein can be introduced into a root-colonizing Bacillus cereus by Homologous Sequence, a Chitinase Homologous Sequences, standard methods known in the art. a Ubiquitin Conjugating Enzyme Homologous Sequence, a 0494 Genes encoding PIP-1 polypeptides can be intro Glyceraldehyde-3-Phosphate Dehydrogenase Homologous duced, for example, into the root-colonizing Bacillus by Sequence, an Ubiquitin B Homologous Sequence, a Juvenile means of electro transformation. Specifically, genes encoding Hormone Esterase Homolog, and an Alpha Tubuliln Homolo the PIP-1 polypeptides can be cloned into a shuttle vector, for gous Sequence. example, pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:211-218. The shuttle vector pHT3101 containing Use in Pesticidal Control the coding sequence for the particular PIP-1 polypeptide gene can, for example, be transformed into the root-colonizing 0489 General methods for employing strains comprising Bacillus by means of electroporation (Lerecius, et al., (1989) a nucleic acid sequence of the embodiments or a variant FEMS Microbiol. Letts. 60:211-218). thereof, in pesticide control or in engineering other organisms 0495 Expression systems can be designed so that PIP-1 as pesticidal agents are known in the art. See, for example polypeptides are secreted outside the cytoplasm of gram U.S. Pat. No. 5,039,523 and EPO480762A2. negative bacteria, Such as E. coli, for example. Advantages of US 2014/0007292 A1 Jan. 2, 2014 60 having PIP-1 polypeptides secreted are: (1) avoidance of adjuvants can be solid or liquid and correspond to the Sub potential cytotoxic effects of the PIP-1 polypeptide stances ordinarily employed in formulation technology, e.g. expressed; and (2) improvement in the efficiency of purifica natural or regenerated mineral Substances, solvents, dispers tion of the PIP-1 polypeptide, including, but not limited to, ants, wetting agents, tackifiers, binders or fertilizers. Like increased efficiency in the recovery and purification of the wise the formulations may be prepared into edible “baits” or protein per Volume cell broth and decreased time and/or costs fashioned into pest “traps' to permit feeding or ingestion by of recovery and purification per unit protein. a target pest of the pesticidal formulation. 0496 PIP-1 polypeptides can be made to be secreted in E. 0500 Methods of applying an active ingredient oran agro coli, for example, by fusing an appropriate E. coli signal chemical composition that contains at least one of the PIP-1 peptide to the amino-terminal end of the PIP-1 polypeptide. polypeptides produced by the bacterial strains include leaf Signal peptides recognized by E. coli can be found in proteins application, seed coating and Soil application. The number of already known to be secreted in E. coli, for example the applications and the rate of application depend on the inten OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437 sity of infestation by the corresponding pest. 2442). Omp A is a major protein of the E. coli outer mem 0501. The composition may be formulated as a powder, brane, and thus its signal peptide is thought to be efficient in dust, pellet, granule, spray, emulsion, colloid, Solution or the translocation process. Also, the OmpA signal peptide Such like, and may be prepared by Such conventional means does not need to be modified before processing as may be the as desiccation, lyophilization, homogenation, extraction, fil case for other signal peptides, for example lipoprotein signal tration, centrifugation, sedimentation or concentration of a peptide (Duffaud, et al., (1987) Meth. Enzymol. 153:492). culture of cells comprising the polypeptide. In all Such com 0497 PIP-1 polypeptides of the embodiments can be fer positions that contain at least one Such pesticidal polypeptide, mented in a bacterial host and the resulting bacteria processed the polypeptide may be present in a concentration of from and used as a microbial spray in the same manner that Bt about 1% to about 99% by weight. strains have been used as insecticidal sprays. In the case of a 0502 Lepidopteran, dipteran, heteropteran, nematode, PIP-1 polypeptide(s) that is secreted from Bacillus, the secre hemiptera or coleopteran pests may be killed or reduced in tion signal is removed or mutated using procedures known in numbers in a given area by the methods of the disclosure or the art. Such mutations and/or deletions prevent secretion of may be prophylactically applied to an environmental area to the PIP-1 polypeptide(s) into the growth medium during the prevent infestation by a susceptible pest. Preferably the pest fermentation process. The PIP-1 polypeptides are retained ingests or is contacted with, apesticidally-effective amount of within the cell, and the cells are then processed to yield the the polypeptide. By "pesticidally-effective amount” is encapsulated PIP-1 polypeptides. Any suitable microorgan intended an amount of the pesticide that is able to bring about ism can be used for this purpose. Pseudomonas has been used death to at least one pest or to noticeably reduce pest growth, to express Bt toxins as encapsulated proteins and the resulting feeding or normal physiological development. This amount cells processed and sprayed as an insecticide (Gaertner, et al., will vary depending on Such factors as, for example, the (1993), in: Advanced Engineered Pesticides, ed. Kim). specific target pests to be controlled, the specific environ 0498 Alternatively, the PIP-1 polypeptides are produced ment, location, plant, crop or agricultural site to be treated, the by introducing a heterologous gene into a cellular host. environmental conditions, and the method, rate, concentra Expression of the heterologous gene results, directly or indi tion, stability, and quantity of application of the pesticidally rectly, in the intracellular production and maintenance of the effective polypeptide composition. The formulations may pesticide. These cells are then treated under conditions that also vary with respect to climatic conditions, environmental prolong the activity of the toxin produced in the cell when the considerations, and/or frequency of application and/or sever cell is applied to the environment of target pest(s). The result ity of pest infestation. ing product retains the toxicity of the toxin. These naturally 0503. The pesticide compositions described may be made encapsulated PIP-1 polypeptides may then be formulated in by formulating either the bacterial cell, Crystal and/or spore accordance with conventional techniques for application to Suspension or isolated protein component with the desired the environment hosting a target pest, e.g., soil, water, and agriculturally-acceptable carrier. The compositions may be foliage of plants. See, for example EPA 0192319, and the formulated prior to administration in an appropriate means references cited therein. Such as lyophilized, freeze-dried, desiccated or in an aqueous carrier, medium or Suitable diluent, such as Saline or other Pesticidal Compositions buffer. The formulated compositions may be in the form of a 0499. In some embodiments the active ingredients can be dust or granular material or a suspension in oil (vegetable or applied in the form of compositions and can be applied to the mineral) or water or oil/water emulsions or as a wettable crop area or plant to be treated, simultaneously or in Succes powder or in combination with any other carrier material Sion, with other compounds. These compounds can be fertil Suitable for agricultural application. Suitable agricultural car izers, weed killers, Cryoprotectants, Surfactants, detergents, riers can be solid or liquid and are well known in the art. The pesticidal soaps, dormant oils, polymers, and/or time-release term 'agriculturally-acceptable carrier covers all adjuvants, or biodegradable carrier formulations that permit long-term inert components, dispersants, Surfactants, tackifiers, bind dosing of a target area following a single application of the ers, etc. that are ordinarily used in pesticide formulation tech formulation. They can also be selective herbicides, chemical nology; these are well known to those skilled in pesticide insecticides, Virucides, microbicides, amoebicides, pesti formulation. The formulations may be mixed with one or cides, fungicides, bacteriocides, nematocides, molluscicides more Solidor liquid adjuvants and prepared by various means, or mixtures of several of these preparations, if desired, e.g., by homogeneously mixing, blending and/or grinding the together with further agriculturally acceptable carriers, Sur pesticidal composition with Suitable adjuvants using conven factants or application-promoting adjuvants customarily tional formulation techniques. Suitable formulations and employed in the art of formulation. Suitable carriers and application methods are described in U.S. Pat. No. 6,468.523, US 2014/0007292 A1 Jan. 2, 2014
herein incorporated by reference. The plants can also be traZamide, Imazosulfuron, Mefenacet, Oxaziclomefone, treated with one or more chemical compositions, including Pyrazosulfuron, Pyributicarb, Quinclorac. Thiobencarb, one or more herbicide, insecticides or fungicides. Exemplary Indanofan, Flufenacet, FentraZamide, Halosulfuron, Oxazi chemical compositions include: Fruits/Vegetables Herbi clomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyri cides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, bac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Tefuryltrione, OxadiaZone, Fenoxaprop, Pyrimisulfan; Rice Halo sulfuron Gowan, Paraquat, PropyZamide, Sethoxydim, Insecticides: Diazinon, Fenitrothion, Fenobucarb, Monocro Butafenacil, Halosulfuron, Indaziflam: Fruits/Vegetables tophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imi Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, dacloprid, Isoprocarb. Thiacloprid, Chromafenozide. Thia Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, cloprid, Dinotefuran, Clothianidin, Ethiprole, Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl. Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamec Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, tin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Metha Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, midophos, Etofenprox. Triazophos, 4-(6-Chlorpyridin-3- Clothianidin, Spirodiclofen, Gamma-cyhalothrin, yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on, Spiromesifen, Spinosad, Rynaxypyr, CyaZypyr, Spinoteram, Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-me Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, thyl, AZOxystrobin, Carpropamid, Edifenphos, FerimZone, Thiodicarb. Metaflumizone, Sulfoxaflor, Cyflumetofen, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyro Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, quilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxaniil, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamec Simeconazole, Tiadinil; Cotton Herbicides: Diuron, Flu tin-benzoate, lindoxacarb. Forthiazate, Fenamiphos, Cadusa ometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, phos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox, Methomyl, CarfentraZone, Clethodim, Fluazifop-butyl, Glyphosate, 4-(6-Chlorpyridin-3-yl)methyl(2,2-difluorethyl)amino Norflurazon, Pendimethalin, Pyrithiobac-sodium, Triflox furan-2(5H)-on; Fruits/Vegetables Fungicides: Carbenda ysulfuron, Tepraloxydim, Glufosinate, Flumioxazin. Thidi Zim, Chlorothalonil, EBDCs, Sulphur. Thiophanate-methyl, aZuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyri AZOxystrobin, Cymoxanil, FluaZinam, Fosetyl, Iprodione, fos, Cypermethrin, Deltamethrin, Malathion, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Monocrotophos, Abamectin, Acetamiprid, Emamectin Ben Ethaboxam, 1 provalicarb, Trifloxystrobin, Fenhexamid, Zoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spi Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxam nosad. Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, ide, Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid: Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Ryn Cereals Herbicides: Isoproturon, Bromoxynil, loxynil, Phe axypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thia noxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, methoxam, Thiacloprid, Dinetofuran, Flubendiamide, Fenoxaprop, Florasulam, Fluoroxypyr. Metsulfuron, Triasul CyaZypyr, Spinosad, Spinotoram, gamma Cyhalothrin, furon, Flucarbazone, Iodosulfuron, Propoxycarbazone, 4-(6-Chlorpyridin-3-yl)methyl(2,2-difluorethyl)amino Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Ami furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, dosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfu Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazo ron, Sulfosulfuron, Pyrasulfotole, PyroxSulam, Flufenacet, phos, Endosulfan; Cotton Fungicides: Etridiazole, Metal Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbenda axyl, Quintozene: Soybean Herbicides: Alachlor, Bentazone, Zim, Chlorothalonil, AZOxystrobin, Cyproconazole, Cyprodi Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, nil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Qui Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, noxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Imazaquin, Imazethapyr. (S-)Metolachlor, Metribuzin, Pen Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothiocona dimethalin, Tepraloxydim, Glufosinate: Soybean Insecti Zole, Fluoxastrobin: Cereals Insecticides: Dimethoate, cides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, Imidacloprid, Clothianidin. Thiamethoxam, Thiacloprid, B-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thia Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, methoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphy CyaZypyr, Spinosad, Spinotoram, Emamectin-Benzoate, riphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Fipronil, Ethiprole, Deltamethrin, B-Cyfluthrin, gamma and Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bro lambda Cyhalothrin, 4-(6-Chlorpyridin-3-yl)methyl(2.2- moxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethena difluorethyl)aminofuran-2(5H)-on, Spirotetramat, Spinodi mid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, clofen, Triflumuron, Flonicamid. Thiodicarb, beta-Cy Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sul fluthrin, Soybean Fungicides: AZOxystrobin, Cyproconazole, cotrione, Foramsulfuron, ToprameZone, Tembotrione, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Saflufenacil. Thiencarbazone, Flufenacet, Pyroxasulfon; Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Herbicides: Chloridazon, Desmedipham, Ethiofumesate, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Ter Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, bufos. Thiamethoxam, Clothianidin, Spiromesifen, Fluben Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, diamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imida B-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflu cloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetami moron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr. prid, Dinetofuran, Deltamethrin, B-Cyfluthrin, gamma/ Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methio lambda Cyhalothrin, 4-(6-Chlorpyridin-3-yl)methyl(2.2- carb, Spirodiclofen, Spirotetramat; Maize Fungicides: Feni difluorethyl)aminofuran-2(5H)-on, Tefluthrin, Rynaxypyr. tropan, Thiram, Prothioconazole, Tebuconazole, Triflox Cyaxypyr. Fipronil, Carbofuran; Canola Herbicides: Clopy ystrobin; Rice Herbicides: Butachlor, Propanil, ralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metaza AZimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fen chlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, US 2014/0007292 A1 Jan. 2, 2014 62
Clethodim, Tepraloxydim; Canola Fungicides: AZOX medinalis Guenee (rice leafroller); Desmia funeralis Hübner ystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, (grape leaffolder); Diaphania hyalinata Linnaeus (melon VincloZolin; Canola Insecticides: Carbofuran organophos worm); D. nitidalis Stoll (pickleworm); Diatraea grandi phates, Pyrethroids. Thiacloprid, Deltamethrin, Imidaclo Osella Dyar (southwestern corn borer), D. Saccharalis Fabri prid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofu cius (Surgarcane borer); Eoreuma loftini Dyar (Mexican rice ran, B-Cyfluthrin, gamma and lambda Cyhalothrin, tau borer); Ephestia elutella Hübner (tobacco (cacao) moth); Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Galleria mellonella Linnaeus (greater wax moth); Herpeto Flubendiamide, Rynaxypyr, Cyazypyr, 4-(6-Chlorpyridin gramma licarsisalis Walker (Sod webworm); Homoeosoma 3-yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on. electellum Hulst (Sunflower moth); Elasmopalpus lignosellus 0504. In some embodiments the herbicide is Atrazine, Zeller (lesser cornstalk borer); Achroia grisella Fabricius Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfu (lesser wax moth); Loxostege Sticticalis Linnaeus (beet web ron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, worm); Orthaga thyrisalis Walker (tea tree web moth); Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumiox Maruca testulalis Geyer (bean pod borer); Plodia interpunc azin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-meto tella Hübner (Indian meal moth); Scirpophaga incertulas lachlor, Hexazinne or combinations thereof. Walker (yellow stem borer); Udea rubigalis Guenee (celery 0505. In some embodiments the insecticide is Esfenvaler leaftier); and leafrollers, budworms, seed worms, and fruit ate, Chlorantraniliprole, Methomyl, lindoxacarb, Oxamyl or worms in the family Tortricidae Acleris gloverana Walsing combinations thereof. ham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm): Archips argyrospila Walker Pesticidal and Insecticidal Activity (fruit tree leaf roller); A. rosana Linnaeus (European leaf 0506 “Pest includes but is not limited to, insects, fungi, roller); and other Archips species, Adoxophyes Orana Fischer bacteria, nematodes, mites, ticks, and the like. Insect pests von Rosslerstamm (summer fruit tortrix moth); Cochylis hos include insects selected from the orders Coleoptera, Diptera, pes Walsingham (banded sunflower moth): Cydia latifer Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemi reana Walsingham (filbertworm): C. pomonella Linnaeus ptera orthroptera, Thysanoptera, Dermaptera, Isoptera, Ano (coding moth); Platynota flavedana Clemens (variegated lea plura, Siphonaptera, Trichoptera, etc., particularly Lepi froller); P Stultana Walsingham (omnivorous leafroller); doptera, and Hemiptera. Lobesia botrana Denis & Schiffermuller (European grape 0507 Those skilled in the art will recognize that not all vine moth); Spilonota ocellana Denis & Schiffermuller (eye compounds are equally effective against all pests. Com spotted bud moth); Endopiza viteana Clemens (grape berry pounds of the embodiments display activity against insect moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota pests, which may include economically important agro salubricola Meyrick (Brazilian apple leafroller); Grapholita nomic, forest, greenhouse, nursery ornamentals, food and molesta Busck (oriental fruit moth); Suleima helianthana fiber, public and animal health, domestic and commercial Riley (Sunflower bud moth); Argyrotaenia spp., Choristo structure, household and stored product pests. neura spp. 0508 Larvae of the order Lepidoptera include, but are not 0509 Selected other agronomic pests in the order Lepi limited to, armyworms, cutworms, loopers, and heliothines in doptera include, but are not limited to. Alsophila pometaria the family Noctuidae Spodoptera frugiperda JE Smith (fall Harris (fall cankerworm); Anarsia lineatella Zeller (peach armyworm): S. exigua Hübner (beet armyworm): S. litura twig borer); Anisota Senatoria J. E. Smith (orange striped Fabricius (tobacco cutworm, cluster caterpillar); Mamestra oakworm); Antheraea pernyi Guérin-Meneville (Chinese configurata Walker (bertha armyworm); M. brassicae Lin Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Buc naeus (cabbage moth); Agrotis ipsilon Hufnagel (black cut culatrix thurberiella Busck (cotton leaf perforator); Collas worm); A. Orthogonia Morrison (western cutworm); A. sub eurytheme Boisduval (alfalfa caterpillar); Datana integer terranea Fabricius (granulate cutworm); Alabama argillacea rima Grote & Robinson (walnut caterpillar); Dendrolimus Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage Sibiricus Tschetwerikov (Siberian silk moth), Ennomos sub looper); Pseudoplusia includens Walker (soybean looper); signaria Hübner (elm spanworm): Erannis tiliaria Harris Anticarsia gemmatalis Hübner (velvetbean caterpillar); (linden looper); Euproctis chrysorrhoea Linnaeus (browntail Hypena scabra Fabricius (green cloverworm); Heliothis vire moth); Harrisina americana Guérin-Meneville (grapeleaf scens Fabricius (tobacco budworm); Pseudaletia unipuncta skeletonizer); Hemileuca Oliviae Cockrell (range caterpillar); Haworth (armyworm); Athetis mindara Barnes and Mcdun Hyphantria cunea Drury (fall webworm); Keiferia lycoper nough (rough skinned cutworm); Euxoa messoria Harris Sicella Walsingham (tomato pinworm), Lambdina fiscellaria (darksided cutworm); Earias insulana Boisduval (spiny boll fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugu worm); E. Vittella Fabricius (spotted bollworm); Helicoverpa brosa Hulst (Western hemlock looper); Leucoma salicis Lin armigera Hübner (American bollworm); H. zea Boddie (corn naeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); earworm or cotton bollworm); Melanchra picta Harris (Zebra Manduca quinquemaculata Haworth (five spotted hawk caterpillar); Egira (Xylomyges) curialis Grote (citrus cut moth, tomato hornworm); M. sexta Haworth (tomato horn worm); borers, casebearers, webworms, coneworms, and worm, tobacco hornworm); Operophtera brumata Linnaeus skeletonizers from the family Pyralidae Ostrinia nubilalis (winter moth); Paleacrita vernata Peck (spring canker Hübner (European corn borer); Amyelois transitella Walker worm); Papilio Cresphontes Cramer (giant Swallowtail (naval orangeworm); Anagasta kuehniella Zeller (Mediterra orange dog); Phryganidia Californica Packard (California nean flour moth); Cadra cautella Walker (almond moth); oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Chilo suppressalis Walker (rice stem borer); C. partellus, Phyllonorycter blancardella Fabricius (spotted tentiform (Sorghum borer); Corcyra cephalonica Stainton (rice moth); leafminer); Pieris brassicae Linnaeus (large white butterfly): Crambus caliginosellus Clemens (corn root webworm): C. Prapae Linnaeus (small white butterfly); P napi Linnaeus teterrellus Zincken (bluegrass webworm): Cnaphalocrocis (green veined white butterfly): Platyptilia carduidactyla US 2014/0007292 A1 Jan. 2, 2014
Riley (artichoke plume moth); Plutella xylostella Linnaeus ovinus Linnaeus (keds); and other Brachycera, mosquitoes (diamondback moth); Pectinophora gossypiella Saunders Aedes spp.; Anopheles spp., Culex spp., black flies Prosimu (pink bollworm); Pontia protodice Boisduval & Leconte lium spp., Simulium spp.; biting midges, sand flies, Sciarids, (Southern cabbageworm); Sabulodes aegrotata Guenee (om and other Nematocera. nivorous looper); Schizura concinna J. E. Smith (red humped 0512 Included as insects of interest are adults and nymphs caterpillar); Sitotroga cerealella Olivier (Angoumois grain of the orders Hemiptera and Homoptera such as, but not moth); Thaumetopoea pityocampa Schiffermuller (pine pro limited to, adelgids from the family Adelgidae, plant bugs cessionary caterpillar); Tineola bisselliella Hummel (web from the family Miridae, cicadas from the family Cicadidae, bing clothesmoth); Tuta absoluta Meyrick (tomato leaf leafhoppers, Empoasca spp.; from the family Cicadellidae, miner); Yponomeuta padella Linnaeus (ermine moth); planthoppers from the families Cixiidae, Flatidae, Fulgoroi Heliothis subflexia Guenee; Malacosoma spp. and Orgvia spp. dea, 1ssidae and Delphacidae, treehoppers from the family 0510. Of interest are larvae and adults of the order Membracidae, psyllids from the family Psyllidae, whiteflies Coleoptera including weevils from the families Anthribidae, from the family Aleyrodidae, aphids from the family Aphid Bruchidae, and Curculionidae (including, but not limited to: idae, phylloxera from the family Phylloxeridae, mealybugs Anthonomus grandis Boheman (boll weevil); Lissorhoptrus from the family Pseudococcidae, scales from the families Oryzophilus Kuschel (rice water weevil); Sitophilus grana Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, rius Linnaeus (granary weevil); S. Oryzae Linnaeus (rice wee Eriococcidae ortheziidae, Phoenicococcidae and Margar vil); Hypera punctata Fabricius (clover leaf weevil); Cylin odidae, lace bugs from the family Tingidae, Stink bugs from drocopturus adspersus LeConte (Sunflower stem weevil); the family Pentatomidae, cinch bugs, Blissus spp.; and other Smicronyx filvus LeConte (red sunflower seed weevil); S. seed bugs from the family Lygaeidae, Spittlebugs from the SOrdidus LeConte (gray Sunflower seed weevil); Sphenopho family Cercopidae squash bugs from the family Coreidae, rus maidis Chittenden (maize billbug)); flea beetles, cucum and red bugs and cotton stainers from the family Pyrrhoc ber beetles, rootworms, leaf beetles, potato beetles, and leaf oridae. miners in the family Chrysomelidae (including, but not 0513 Agronomically important members from the order limited to: Leptinotarsa decemlineata Say (Colorado potato Homoptera further include, but are not limited to: Acyrthisi beetle); Diabrotica virgifera virgifera LeConte (western corn phon pisum Harris (pea aphid); Aphis Craccivora Koch (cow rootworm): D. barberi Smith & Lawrence (northern corn pea aphid); A. fabae Scopoli (black bean aphid); A. gossypii rootworm); D. undecimpunctata howardi Barber (Southern Glover (cotton aphid, melon aphid); A. maidiradicis Forbes corn rootworm); Chaetocnema pulicaria Melsheimer (corn (corn root aphid); A. pomi De Geer (apple aphid); A. spirae flea beetle); Phyllotreta cruciferae Goeze (corn flea beetle); cola Patch (spirea aphid); Aulacorthum Solani Kaltenbach Colaspis brunnea Fabricius (grape colaspis); Oulema mel (foxglove aphid); Chaetosiphon fragaefolii Cockerell (Straw anopus Linnaeus (cereal leaf beetle); Zygogramma exclama berry aphid); Diuraphis noxia Kurdumov/Mordvilko (Rus tionis Fabricius (sunflower beetle)); beetles from the family sian wheat aphid); Dysaphis plantaginea Paaserini (rosy Coccinellidae (including, but not limited to: Epilachna apple aphid); Eriosoma lanigerum Hausmann (woolly apple varivestis Mulsant (Mexican bean beetle)); chafers and other aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); beetles from the family Scarabaeidae (including, but not lim Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis ited to: Popillia japonica Newman (Japanese beetle); Cyclo erysimi Kaltenbach (turnip aphid); Metopolophium dirrho cephala borealis Arrow (northern masked chafer, white dum Walker (cereal aphid); Macrosiphum euphorbiae Tho grub); C. immaculate Olivier (southern masked chafer, white mas (potato aphid); Myzus persicae Sulzer (peach-potato grub); Rhizotrogus maialis Razoumowsky (European cha aphid, greenpeach aphid); Nasonovia ribisnigri Mosley (let fer); Phyllophaga crimita Burmeister (white grub): Ligyrus tuce aphid); Pemphigus spp. (root aphids and gall aphids); gibbosus De Geer (carrot beetle)); carpet beetles from the Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Lin family Dermestidae; wireworms from the family Elateridae, naeus (bird cherry-oat aphid); Schizaphis graminum Rondani Eleodes spp., Melanotus spp.; Conoderus spp., Limonius (greenbug); Sipha flava Forbes (yellow Sugarcane aphid); spp.; Agriotes spp., Ctenicera spp., Aeolus spp., bark beetles Sitobion avenae Fabricius (English grain aphid); Therioaphis from the family Scolytidae and beetles from the family Tene maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii brionidae. Boyer de Fonscolombe (black citrus aphid); and T. citricida 0511 Adults and immatures of the order Diptera are of Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phyl interest, including leafminers Agromyza parvicornis Loew loxera devastatrix Pergande (pecan phylloxera); Bemisia (cornblotch leafminer); midges (including, but not limited to: tabaci Gennadius (tobacco whitefly, sweetpotato whitefly): Contarinia sorghicola Coquillett (Sorghum midge); May B. argentifolii Bellows & Perring (silverleaf whitefly): etiola destructor Say (Hessian fly); Sitodiplosis mosellana Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes Géhin (wheat midge); Neolasioptera murtfeldtiana Felt, abutiloneus (bandedwinged whitefly) and T vaporariorum (sunflower seed midge)); fruit flies (Tephritidae), Oscinella Westwood (greenhouse whitefly); Empoasca fabae Harris frit Linnaeus (fruit flies); maggots (including, but not limited (potato leafhopper); Laodelphax striatellus Fallen (Smaller to: Delia platura Meigen (seedcorn maggot); D. coarctata brown planthopper); Macrolestes quadrilineatus Forbes (as Fallen (wheat bulb fly); and other Delia spp., Meromyza ter leafhopper); Nephotettix cinticeps Uhler (green leafhop americana Fitch (wheat stem maggot); Musca domestica per); N. nigropictus Stal (rice leafhopper); Nilaparvata Linnaeus (house flies); Fannia canicularis Linnaeus, Ffemo lugens Stal (brown planthopper); Peregrinus maidis Ash ralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus mead (corn planthopper); Sogatella furcifera Horvath (white (stable flies)); face flies, horn flies, blow flies, Chrysomya backed planthopper); Sogatodes Orizicola Muir (rice delpha spp., Phormia spp.; and other muscoid fly pests, horse flies cid), Tiphlocyba pomaria McAtee (white apple leafhopper); Tabanus spp., botflies Gastrophilus spp., Oestrus spp., cattle Erythroneoura spp. (grape leafhoppers); Magicicada Septen grubs Hypoderma spp.; deerflies Chrysops spp.; Mellophagus decim Linnaeus (periodical cicada); Icerya purchasi Maskell US 2014/0007292 A1 Jan. 2, 2014 64
(cottony cushion scale); Ouadraspidiotus perniciosus Com Fabricius (black widow spider); and centipedes in the order stock (San Jose scale); Planococcus citri Risso (citrus mea Scutigeromorpha Such as Scutigera Coleoptrata Linnaeus lybug); Pseudococcus spp. (other mealybug complex); (house centipede). Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri 0519 Insect pest of interest include the Superfamily of Ashmead (persimmon psylla). Stink bugs and other related insects including but not limited 0514 Agronomically important species of interest from to species belonging to the family Pentatomidae (Nezara the order Hemiptera include, but are not limited to: Acroster viridula, Halyomorpha haly's, Piezodorus guildini, Euschis nun hilare Say (green Stink bug); Anasa tristis De Geer tus servus, Acrosternum hilare, Euschistus heros, Euschistus (squash bug); Blissus leucopterus leucopterus Say (chinch tristigmus, Acrosternum hilare, Dichelops fircatus, Dich bug); Corythuca gossypii Fabricius (cotton lace bug); Cyr elops melacanthus, and Bagrada hilaris (Bagrada Bug)), the topeltis modesta Distant (tomato bug); Dysdercus suturellus family Plataspidae (Megacopta cribraria—Bean plataspid), Herrich-Schäffer (cotton stainer); Euschistus servus Say and the family Cydnidae (Scaptocoris Castanea—Root Stink (brown stink bug); E. variolarius Palisot de Beauvois (one bug); and Lepidoptera species including but not limited to: spotted Stink bug); Graptostethus spp. (complex of seed diamond-back moth, e.g., Helicoverpa zea Boddie; soybean bugs); Leptoglossus Corculus Say (leaf-footed pine seedbug); looper, e.g., Pseudoplusia includens Walker, and Velvet bean Lygus lineolaris Palisot de Beauvois (tarnished plant bug), L. caterpillar e.g., Anticarsia gemmatalis Hübner. Hesperus Knight (Western tarnished plant bug); L. pratensis 0520 Methods for measuring pesticidal activity are well Linnaeus (common meadow bug); L. rugulipennis Poppius known in the art. See, for example, Czapla and Lang, (1990) (European tarnished plant bug); Lygocoris pabulinus Lin J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) naeus (common green capsid); Nezara viridula Linnaeus Biochem. J. 252: 199-206; Marrone, et al., (1985).J. of Eco (Southern green Stink bug); Oebalus pugnax Fabricius (rice nomic Entomology 78:290–293 and U.S. Pat. No. 5,743,477, Stink bug). Oncopeltus fasciatus Dallas (large milkweed all of which are herein incorporated by reference in their bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper). entirety. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985).J. of Eco 0515. Furthermore, embodiments may be effective against nomic Entomology 78:290–293. Such assays can include con Hemiptera such, Calocoris norvegicus Gmelin (strawberry tacting plants with one or more pests and determining the bug); Orthops campestris Linnaeus; Plesiocoris rugicollis plant’s ability to survive and/or cause the death of the pests. Fallen (apple capsid), Cyrtopeltis modestus Distant (tomato 0521. Nematodes include parasitic nematodes such as bug); Cyrtopeltis notatus Distant (Suckfly); Spanagonicus root-knot, cyst, and lesion nematodes, including Heterodera albofasciatus Reuter (whitemarked fleahopper); Diaphno spp., Meloidogyne spp., and Globodera spp.; particularly coris chlorionis Say (honeylocust plant bug); Labopidicola members of the cyst nematodes, including, but not limited to, alli Knight (onion plant bug); Pseudatomoscelis seriatus Heterodera glycines (soybean cyst nematode); Heterodera Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid Schachtii (beet cyst nematode); Heterodera avenae (cereal plant bug); Poecilocapsus lineatus Fabricius (four-lined plant cyst nematode); and Globodera rostochiensis and Globodera bug); Nysius ericae Schilling (false chinch bug); Nysius pailida (potato cyst nematodes). Lesion nematodes include raphanus Howard (false chinch bug); Nezara viridula Lin Pratylenchus spp. naeus (Southern green Stink bug); Eurygaster spp.; Coreidae spp., Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Seed Treatment Reduviidae spp.; and Cimicidae spp. 0516. Also included are adults and larvae of the order 0522 To protect and to enhance yield production and trait Acari (mites) such as Aceria to Sichella Keifer (wheat curl technologies, seed treatment options can provide additional mite); Petrobia latens Müller (brown wheat mite); spider crop plan flexibility and cost effective control against insects, mites and red mites in the family Tetranychidae, Panonychus weeds and diseases. Seed material can be treated, typically ulmi Koch (European red mite); Tetranychus urticae Koch Surface treated, with a composition comprising combinations (two spotted spider mite); (T. mcdanieli McGregor of chemical or biological herbicides, herbicide safeners, (McDaniel mite); T cinnabarinus Boisduval (carmine spider insecticides, fungicides, germination inhibitors and enhanc mite); T turkestani Ugarov & Nikolski (strawberry spider ers, nutrients, plant growth regulators and activators, bacteri mite); flat mites in the family Tenuipalpidae, Brevipalpus cides, nematocides, avicides and/or molluscicides. These lewisi McGregor (citrus flat mite); rust and bud mites in the compounds are typically formulated together with further family Eriophyidae and other foliar feeding mites and mites carriers, Surfactants or application-promoting adjuvants cus important in human and animal health, i.e. dust mites in the tomarily employed in the art of formulation. The coatings family Epidermoptidae, follicle mites in the family Demodi may be applied by impregnating propagation material with a cidae, grain mites in the family Glycyphagidae, ticks in the liquid formulation or by coating with a combined wet or dry order Ixodidae. Ixodes Scapularis Say (deer tick); I. holocy formulation. Examples of the various types of compounds clus Neumann (Australian paralysis tick); Dermacentor vari that may be used as seed treatments are provided in The abilis Say (American dog tick); Amblyomma americanum Pesticide Manual: A World Compendium, C. D. S. Tomlin Linnaeus (lone star tick); and scab and itch mites in the Ed., Published by the British Crop Production Council, which families Psoroptidae, Pyemotidae, and Sarcoptidae. is hereby incorporated by reference. 0523 Some seed treatments that may be used on crop seed 0517. Insect pests of the order Thysanura are of interest, include, but are not limited to, one or more of abscisic acid, Such as Lepisma saccharina Linnaeus (silverfish); Thermo acilbenzolar-5-methyl, avermectin, amitrol, azaconazole, bia domestica Packard (firebrat). aZospirillum, azadirachtin, azoxystrobin, bacillus spp. (in 0518. Additional arthropod pests covered include: spiders cluding one or more of cereus, firmus, megaterium, pumilis, in the order Araneae Such as Loxosceles reclusa Gertsch & sphaericus, Subtilis and/or thuringiensis), bradyrhizobium Mulaik (brown recluse spider); and the Latrodectus mactans spp. (including one or more of betae, canariense, elkanii, US 2014/0007292 A1 Jan. 2, 2014 iriomotense, japonicum, liaonigense, pachyrhizi and/or fit pests, producing pests more Susceptible to predator attack yuanmingense), captan, carboxin, chitosan, clothianidin, or deterring the pests from eating the plant. copper, cyaZypyr, difenoconazole, etidiazole, fipronil, flu 0527. In some embodiments methods are provided for dioxonil, fluoxastrobin, fluguinconazole, flurazole, flux controlling an insect pest population resistant to a pesticidal ofenim, harpin protein, imazalil, imidacloprid, ipconazole, protein, comprising contacting the insect pest population isoflavenoids, lipo-chitooligosaccharide, mancoZeb, manga with an insecticidally-effective amount of a recombinant nese, maneb, mefenoxam, metalaxyl, metconazole, myclobu PIP-1 polypeptide. In some embodiments methods are pro tanil, PCNB, penflufen, penicillium, penthiopyrad, per vided for controlling an insect pest population resistant to a methrine, picoxystrobin, prothioconazole, pyraclostrobin, pesticidal protein, comprising contacting the insect pest rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB, tebu population with an insecticidally-effective amount of a conazole, thiabendazole, thiamethoxam, thiocarb, thiram, recombinant pesticidal protein of SEQID NO: 6 or a variant tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, thereof. triticonazole and/or zinc. PCNB seed coat refers to EPA reg 0528. In some embodiments methods are provided for istration number 002935.00419, containing quintozen and ter protecting a plant from an insect pest, comprising expressing razole. TCMTB refers to 2-(thiocyanomethylthio) benzothia in the plant or cell thereof a recombinant PIP-1 polypeptide. Zole. In some embodiments methods are provided for protecting a 0524 Seed varieties and seeds with specific transgenic plant from an insect pest, comprising expressing in the plant traits may be tested to determine which seed treatment or cell thereof a recombinant pesticidal protein of SEQ ID options and application rates may complement such varieties NO: 6 or variants thereof. and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head Smut Susceptibility Insect Resistance Management (IRM) Strategies may benefit from the use of a seed treatment that provides 0529 Expression of B. thuringiensis 8-endotoxins in protection against head Smut, a variety with good yield poten transgenic corn plants has proven to be an effective means of tial but cyst nematode susceptibility may benefit from the use controlling agriculturally important insect pests (Perlak, et of a seed treatment that provides protection against cyst al., 1990; 1993). However, insects have evolved that are resis nematode, and so on. Likewise, a variety encompassing a tant to B. thuringiensis Ö-endotoxins expressed in transgenic transgenic trait conferring insect resistance may benefit from plants. Such resistance, should it become widespread, would the second mode of action conferred by the seed treatment, a clearly limit the commercial value of germplasm containing Variety encompassing a transgenic trait conferring herbicide genes encoding such B. thuringiensis Ö-endotoxins. resistance may benefit from a seed treatment with a safener 0530 One way to increasing the effectiveness of the trans that enhances the plants resistance to that herbicide, etc. Fur genic insecticides against target pests and contemporane ther, the good root establishment and early emergence that ously reducing the development of insecticide-resistant pests results from the proper use of a seed treatment may result in is to use provide non-transgenic (i.e., non-insecticidal pro more efficient nitrogen use, a better ability to withstand tein) refuges (a section of non-insecticidal crops/corn) for use drought and an overall increase in yield potential of a variety with transgenic crops producing a single insecticidal protein or varieties containing a certain trait when combined with a active against target pests. The United States Environmental seed treatment. Protection Agency (epa.gov/oppbppdl/biopesticides/pipS/bt corn refuge 2006.htm, which can be accessed using the Methods for Inhibiting Growth or Killing an Insect Pest and www prefix) publishes the requirements for use with trans Controlling an Insect Population genic crops producing a single Bt protein active against target pests. In addition, the National Corn Growers Association, on 0525. In some embodiments methods are provided for their website: (incga.com/insect-resistance-management inhibiting growth or killing an insect pest, comprising con fact-sheet-bt-corn, which can be accessed using the www tacting the insect pest with an insecticidally-effective amount prefix) also provides similar guidance regarding refuge of a recombinant PIP-1 polypeptide. In some embodiments requirements. Due to losses to insects within the refuge area, methods are provided for inhibiting growth or killing an larger refuges may reduce overall yield. insect pest, comprising contacting the insect pest with an 0531. Another way of increasing the effectiveness of the insecticidally-effective amount of a recombinant pesticidal transgenic insecticides against target pests and contempora protein of SEQID NO: 6 or a variant thereof. neously reducing the development of insecticide-resistant 0526 In some embodiments methods are provided for pests would be to have a repository of insecticidal genes that controlling an insect pest population, comprising contacting are effective against groups of insect pests and which mani the insect pest population with an insecticidally-effective fest their effects through different modes of action. amount of a recombinant PIP-1 polypeptide. In some embodi 0532 Expression in a plant of two or more insecticidal ments methods are provided for controlling an insect pest compositions toxic to the same insect species, each insecti population, comprising contacting the insect pest population cide being expressed at efficacious levels would be another with an insecticidally-effective amount of a recombinant pes way to achieve control of the development of resistance. This ticidal protein of SEQID NO: 6 or a variant thereof. As used is based on the principle that evolution of resistance against herein, by “controlling a pest population' or “controls a pest' two separate modes of action is far more unlikely than only is intended any effect on a pest that results in limiting the one. Roush for example, outlines two-toxin strategies, also damage that the pest causes. Controlling a pest includes, but called "pyramiding or 'stacking for management of insec is not limited to, killing the pest, inhibiting development of ticidal transgenic crops. (The Royal Society. Phil. Trans. R. the pest, altering fertility or growth of the pest in Such a Soc. Lond. B. (1998)353:777-1786). Stacking or pyramiding manner that the pest provides less damage to the plant, of two different proteins each effective against the target pests decreasing the number of offspring produced, producing less and with little or no cross-resistance can allow for use of a US 2014/0007292 A1 Jan. 2, 2014 66 smaller refuge. The U.S. Environmental Protection Agency the two or more insecticidal proteins comprise a PIP-1 requires significantly less (generally 5%) structured refuge of polypeptide and a Cry protein. Also provided are means for non-Bt corn be planted than for single trait products (gener effective Lepidoptera and/or Hemiptera insect resistance ally 20%). There are various ways of providing the IRM management of transgenic plants, comprising co-expressing effects of a refuge, including various geometric planting pat at high levels in the plants two or more insecticidal proteins terns in the fields and in-bag seed mixtures, as discussed toxic to Lepidoptera and/or Hemiptera insects but each exhib further by Roush. iting a different mode of effectuating its inhibiting growth or 0533. In some embodiments the PIP-1 polypeptides of the activity, wherein the two or more insecticidal proteins com disclosure are useful as an insect resistance management prise a protein of SEQID NO: 6 or variants thereofanda Cry strategy in combination (i.e., pyramided) with other pesti protein. cidal proteins include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, 0542. In addition, methods are provided for obtaining and the like. regulatory approval for planting or commercialization of 0534 Provided are methods of controlling Lepidoptera plants expressing proteins insecticidal to insects in the order and/or Hemiptera insect infestation(s) in a transgenic plant Lepidoptera and/or Hemiptera, comprising the step of refer that promote insect resistance management, comprising ring to, Submitting or relying on insect assay binding data expressing in the plant at least two different insecticidal pro showing that the PIP-1 polypeptide does not compete with teins having different modes of action. binding sites for Cry proteins in Such insects. In addition, 0535 In some embodiments the methods of controlling methods are provided for obtaining regulatory approval for Lepidoptera and/or Hemiptera insect infestation in a trans planting or commercialization of plants expressing proteins genic plant and promoting insect resistance management the insecticidal to insects in the order Lepidoptera and/or Hemi at least one of the insecticidal proteins comprise a PIP-1 ptera, comprising the step of referring to, Submitting or rely polypeptide insecticidal to insects in the order Lepidoptera and/or Hemiptera. ing on insect assay binding data showing that the protein of 0536. In some embodiments the methods of controlling SEQ ID NO: 6 or variant thereof does not compete with Lepidoptera and/or Hemiptera insect infestation in a trans binding sites for Cry proteins in Such insects. genic plant and promoting insect resistance management the at least one of the insecticidal proteins comprises a protein of Methods for Increasing Plant Yield SEQIDNO: 6 or variants thereof, insecticidal to insects in the order Lepidoptera and/or Hemiptera. 0543 Methods for increasing plant yield are provided. 0537. In some embodiments the methods of controlling The methods comprise providing a plant or plant cell express Lepidoptera and/or Hemiptera insect infestation in a trans ing a polynucleotide encoding the pesticidal polypeptide genic plant and promoting insect resistance management sequence disclosed herein and growing the plant or a seed comprise expressing in the transgenic planta PIP-1 polypep thereof in a field infested with a pest against which the tide and a Cry protein insecticidal to insects in the order polypeptide has pesticidal activity. In some embodiments, the Lepidoptera and/or Hemiptera having different modes of polypeptide has pesticidal activity against a lepidopteran, action. coleopteran, dipteran, hemipteran or nematode pest, and the 0538. In some embodiments the methods of controlling field is infested with a lepidopteran, hemipteran, coleopteran, Lepidoptera and/or Hemiptera insect infestation in a trans dipteran or nematode pest. genic plant and promoting insect resistance management comprise in the transgenic planta protein of SEQID NO: 6 or (0544. As defined herein, the "yield” of the plant refers to variants thereofanda Cry protein insecticidal to insects in the the quality and/or quantity of biomass produced by the plant. order Lepidoptera and/or Hemiptera having different modes By “biomass” is intended any measured plant product. An of action. increase in biomass production is any improvement in the 0539 Also provided are methods of reducing likelihood of yield of the measured plant product. Increasing plant yield emergence of Lepidoptera and/or Hemiptera insect resistance has several commercial applications. For example, increasing to transgenic plants expressing in the plants insecticidal pro plant leafbiomass may increase the yield of leafy vegetables teins to control the insect species, comprising expression of a for human or animal consumption. Additionally, increasing PIP-1 polypeptide insecticidal to the insect species in com leaf biomass can be used to increase production of plant bination with a second insecticidal protein to the insect spe derived pharmaceutical or industrial products. An increase in cies having different modes of action. yield can comprise any statistically significant increase 0540 Also provided are methods of reducing likelihood of including, but not limited to, at least a 1% increase, at least a emergence of Lepidoptera and/or Hemiptera insect resistance 3% increase, at least a 5% increase, at least a 10% increase, at to transgenic plants expressing in the plants insecticidal pro least a 20% increase, at least a 30%, at least a 50%, at least a teins to control the insect species, comprising expression of a 70%, at least a 100% or a greater increase in yield compared protein of SEQID NO: 6 or variants thereof, insecticidal to to a plant not expressing the pesticidal sequence. the insect species in combination with a second insecticidal protein to the insect species having different modes of action. 0545. In specific methods, plant yield is increased as a 0541. Also provided are means for effective Lepidoptera result of improved pest resistance of a plant expressing a and/or Hemiptera insect resistance management of transgenic PIP-1 polypeptide disclosed herein. Expression of the PIP-1 plants, comprising co-expressing at high levels in the plants polypeptide results in a reduced ability of a pest to infest or two or more insecticidal proteins toxic to Lepidoptera and/or feed on the plant, thus improving plant yield. Hemiptera insects but each exhibiting a different mode of 0546. The following examples are offered by way of illus effectuating its inhibiting growth or killing activity, wherein tration and not by way of limitation. US 2014/0007292 A1 Jan. 2, 2014 67
EXPERIMENTALS sion with PBS buffer. The lysis product was centrifuged and the soluble fraction retained and stored at 4°C. overnight to Example 1 allow insoluble chlororaphin products to precipitate. The remaining Supernatant was filtered sequentially through 25 Identification of an Insecticidal Protein Active um, 8 um, 5um, 1.2 um and 0.45 um filters to remove the Against Lygus from Strain SS44C4 majority of the Crystalline material. The soluble cell lysate (0547. The Lygus active protein PIP-1A was identified by was adjusted to 1.2 Mammonium Sulfate and loaded onto an protein purification, N-terminal amino acid sequencing, PCR Ether column (ToyopearlTM Ether-650S, Tosoh Bioscience cloning from Pseudomonas chlororaphis strain SS44C4 as LLC, 3604 Horizon Drive, Suite 100, King of Prussia, Pa. follows: 19406) of appropriate size. A linear gradient was run from 1.2 0548. Insecticidal activity against Lygus (Lygus hesperus) Mammonium sulfate to 0.6 Mammonium sulfate over 15 was observed from a cell lysate of SS44C4 grown in Trypti column Volumes. The elution peak fractions containing pro case Soy medium (Tryptone 17 g/L, enzymatic digest of Soy tein of interest were then concentrated via a spin concentrator. meal 3 g/L, Dextrose 2.5 g/L, Sodium Chloride 5 g/L, The concentrate was then buffer exchanged into 25 mM Tris K2HPO4 2.5 g/L) and cultured overnight at 30° C. This pH 8 to remove ammonium sulfate using a 7000 MWCO insecticidal activity exhibited heat and proteinase sensitivity indicating proteinaceous nature. ZebaTM desalting column (Thermo Fisher Scientific Inc., 747 0549 Lygus (Lygus hesperus) bioassays were conducted Meridian Rd, Rockford, Ill. 61101). The concentrated and using the cell lysate samples mixed with insect diet (Bio-Serv desalted protein was then loaded onto a Mono OTM column F964.4B) in each well of a 96 well bioassay plate (BD Fal (cat #17-5166-01, GE Healthcare). Optimum elution and conTM 353910). A variable number of Lygus hesperus second purity was achieved by application of a linear gradient from 0 instar nymphs (2 to 7) were placed into each well of a 96 well to 400 mM NaCl. plate. The assay was run four days at 25° C. and then was 0552. The active fraction pool from the MonoQTM purifi scored for insect mortality and stunting of insect growth. A cation was subjected to N-terminal sequencing. The protein series of concentrations of the purified protein sample was pool was run on SDS-PAGE, transferred to a PVDF mem assayed against those insects and concentrations for 50% brane, and stained with CoomassieTM Blue dye. Four bands mortality (LC50) or inhibition of 50% of the individuals were present on the membrane. All were successfully identi (1050) were calculated. The Lygus bioassay results for PIP fied by N-terminal sequencing with a single sequence per 1A is shown in Table 2. band. The N-terminal amino acid sequence of two protein 0550 Genomic DNA was extracted with a Sigma Bacte bands were BLAST searched against the NCBI database and rial Genomic DNA Extraction Kit (Cat # NA2110-KT, a hypothetical protein (PSEEN3174) from a genome Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178) according to the manufactures instructions. The DNA con sequence of Pseudomonas entomophila (Vodovar, N et al. centration was determined using a NanoDrop Spectropho (2006) Nat. Biotechnol. 24 (6), 673-679) was identified as a tometer (Thermo Scientific, 3411 Silverside Road, Bancroft homology match (FIG.1). The PSEEN3174 gene, was cloned Building, Suite 100, Wilmington, Del. 19810) and the by PCR using primers ATACATATGACGATCAAGGAA genomic DNA was diluted to 40 ng/ul with sterile water. A 25 GAGCTG (SEQ ID NO: 13) and TTGGATCCT ul PCR reaction was set up by combining 80 ng genomic CAATAACGGCGATGAGGATCGTTGTAG (SEQ ID NO: DNA, 2 ul (5uM) 16S ribosomal DNA primers TACCTTGT 14). PCR with the cloning primers (SEQ ID NO: 13 and 14) TACGACTT (SEQ ID NO: 209) and AGAGTTTGATC was performed against the SS44C4 genomic DNA prepara MTGGCTCAG (SEQ ID NO: 210), 1 u1 10 cmM dNTP, 1 x tion, and a band of the expected molecular weight was iso PhusionTM HF buffer, and 1 unit of PhusionTM High-Fidelity lated. DNA Polymerase (New England Biolabs, Cat #M0530L,240 0553. The resulting PCR product was DNA sequenced and County Road, Ipswich, Mass. 01938-2723). The PCR reac coupled with MS/MS spectra from in-gel digests showed this tion was run in MJ Research PTC-200 Thermo Cycler (Bio gene product having the DNA sequence of SEQ ID NO: 1 Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, encoding a protein designated herein as “PIP-1A, having the Calif., 94547, USA) with the following program: 96° C. 1 amino acid sequence of SEQ ID NO: 2. The PSEEN3174 min:30 cycles of 96° C. 15 seconds, 52° C.2 minutes and 72° gene has the DNA sequence set forth in SEQID NO: 5 and C. 2 minutes; 72° C. 10 minutes; and hold on 4°C. The PCR encodes an amino acid sequence having the amino acid products were purified with QiaGuickR DNA purification Kit sequence set forth in SEQID NO: 6. Using the PIP-1A (SEQ (Cat #28104, QIAGENR, Inc., 27220 Turnberry Lane, Valen ID NO: 2) and PSEEN3174 (SEQID NO: 6) sequence infor cia, Calif. 91355). The purified PCR sample was DNA mation another homologous gene, SPBB 340380 (anno sequenced and the resulting 16S ribosomal DNA sequence tated as a hypothetical protein from Dendroctonus frontalis was BLAST searched against the NCBI database which indi Bacterial community), was identified by BLAST search from cated that SS44C4 is a Pseudomonas chlororaphis strain. The the Department of Energy Joint Genomic Institute website Pseudomonas chlororaphis strain SS44C4 was deposited on (igi.doe.gov/, which can be accessed on the worldwide web Dec. 1, 2011 under accession#NRRLB-50613 with the Agri using the “www” prefix). The SPBB 340380 coding cultural Research Service Culture Collection (NRRL), 1815 sequence was generated by back translation of protein North University Street, Peoria, Ill. 61604, (nrrl.ncaurusda. sequence using PSEEN3174 (SEQ ID NO: 5) codon usage gov, which can be accessed on the world-wide web using the and the gene was synthesized. The SPBB 340380 coding “www” prefix). sequence has the DNA sequence set forth in SEQID NO: 3 0551. The cell pellet of an overnight culture from a single and encodes an amino acid sequence, designated herein as colony of SS44C4 grown in LB Broth at 30°C. was lyzed in “PIP-1B, having the amino acid sequence set forth in SEQ a French Press at ~20,000 psi in a single pass after resuspen ID NO: 4. US 2014/0007292 A1 Jan. 2, 2014 68
Example 2 ticidal proteins on a variety of Lepidoptera species (European E. coli Expression of PIP-1A, PSEEN3174 and corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa PIP-1B zea), black cutworm (Agrotis ipsilon), fall armyworm 0554. The three coding sequences, PIP-1A (SEQID NO: (Spodoptera frugiperda), Soybean looper (Pseudoplusia 1); PSEEN3174 (SEQID NO:5);& PIP-1B (SEQID NO:3), includens) and Velvet bean caterpillar (Anticarsia gemmata were subcloned into an E. coli expression vector pMALTM lis)), a Coleoptera specie (Western corn rootworm (Di (New England Biolabs, 240 County Road, Ipswich, Mass. abrotica virgifera) 01938-2723) having a 6xHis tag added to the Maltose Bind ing Protein and transformed into E. coli for recombinant 0556 Lepidoptera feeding assays were conducted on an protein expression. E. coli cells transformed with the expres artificial diet containing the cell lysates of bacterial Strains in sion constructs were grown overnight at 37°C. with carbeni a 96 well plate set up. The cell lysate was incorporated with cillin selection and then inoculated to a fresh 2XYT medium the Lepidopteran-specific artificial diet in a ratio of 1:2 cell (1:250) and further grown to ODoo -0.8. IPTG was then lysate to diet mixture. Neonate larvae were placed in each added and the cells were grown further at 37° C. for another well to feed ad libitum for 5 days. Results were expressed as 6 hours or transferred to 16°C. for overnightgrowth to induce positive for larvae reactions such as stunting and or mortality. protein expression. The E. coli expressed proteins were puri Results were expressed as negative if the larvae were similar fied either by Amylose resin (New England Biolabs, 240 to the negative control that is feeding diet to which the above County Road, Ipswich, Mass. 01938-2723) or Ni-NTA aga buffer only has been applied. Cell lysates was assayed on rose (Cat. No. K950-01, Invitrogen, 3175 Staley Road, Grand European corn borer (Ostrinia nubilalis), corn earworm Island, N.Y. 14072), according to the manufacturer's proto (Helicoverpa zea), black cutworm (Agrotis ipsilon), fall cols. armyworm (Spodoptera frugiperda), Soybean looper Example 3 (Pseudoplusia includens) and Velvet bean caterpillar (Antic arsia gemmatalis). A series of concentrations of the purified Lepidoptera and Coleoptera Assays with Purified protein sample was assayed against those insects and concen Proteins trations for 50% mortality (LC50) or inhibition of 50% of the 0555 Insecticidal activity bioassay screens were con individuals (IC50) were calculated. The insecticidal activity ducted on the cell lysate to evaluate the effects of the insec for PIP-1A and PSEEN3174 are shown in Table 2. TABLE 2
PIP-1A (SEQID NO: 2) PSEEN3174 (SEQID NO: 6)
Insect dose effect dose effect
Lygus 40 ppm LC-SO 40 ppm LC-SO Brown marmorated Stink bug 150 ppm LCSO 100 ppm LCSO Southern green Stink bug, 700 ppm single dose, 85% 620 ppm single dose, 75% adult mortality at 6 days mortality at 6 days Southern green Stink bug, 250 ppm single dose, 99% nottested nymphs mortality at 4 days Southern green Stink bug, 100 ppm LCSO 100 ppm LCSO nymphs Colorado potato beetle 875 ppm inactive 875 ppm inactive Diamond back moth 122 ng/cm LC-50 20.5 ng/cm LC-50 Diamond back moth 66.7 ng/cm2 IC-50 12.8 ng/cm2 IC50 Diamond back moth-Cry1A 205 ng/cm LC-50 15.9 ng/cm LC-50 resistant Diamond back moth-Cry1A 59.9 mg/cm2 IC-50 8.7 ng/cm2 IC50 resistant Western Corn Root Worm 200 ug/cm mild stunting 90 ug'cm mild stunting Soybean looper 21.3 ppm LC-SO 44.8 ppm LC-SO Soybean looper 10.0 ppm C-SO 18.8 ppm C-SO Velvet bean caterpillar 14.0 ppm LC-SO 45.8 ppm LC-SO Velvet bean caterpillar 3.9 ppm C-SO 11.8 ppm C-SO Cornear worm ~200 ppm C-SO ~200 ppm C-SO Fall army worm ~200 ppm C-SO ~200 ppm C-SO European corn borer 700 ppm inactive >400 ppm C-SO Black cut worm ~300 ppm C-SO ~200 ppm C-SO Black bean aphid inactive highest dose 200 ppm inactive highest dose 200 ppm Pea aphid (oral dose) 260 ug/ml LC-50 Day1, 90% 161 ug?ml LC-50 Day 2, 90% mortality on day 2 mortality on day 3 US 2014/0007292 A1 Jan. 2, 2014 69
0557 Coleoptera feeding assays were conducted on an insects were dead was the LC50. The results for PIP-1A (SEQ artificial diet containing the cell lysates of bacterial strains. ID NO: 2) and PSEEN3174 (SEQ ID NO: 6) are shown in The cell lysate was incorporated with the coleopteran-spe Table 2. cific artificial diet in a ratio of 1:5 cell lysate to diet mixture. Western corn rootworm (Diabrotica virgifera) neonate larvae Example 6 were placed in each well to feed ad libitum for 5 days. Results Colorado Potato Beetle (Leptinotarsa decemlineata) were expressed as positive for larvae reactions such as stunt Bioassay with Purified Proteins ing and or mortality. Results were expressed as negative if the larvae were similar to the negative control that is feeding diet 0560 20ul of cell lysate samples were mixed with 75ul of to which the above buffer only has been applied. A series of modified Coleopteran diet (Bio-Serv F980OB) in each well of concentrations of the purified protein sample was assayed a 96 well bioassay plate (BD FalconTM 353910) and allowed to Solidify. A single neonate larva was placed in each well and against those insects and concentrations for 50% mortality the plate sealed with a Mylar covering. Holes were punched in (LC50) or inhibition of 50% of the individuals (1050) were calculated. The results for PIP-1A and PSEEN3174 are the Mylar sheet and the plate incubated at 25°C. for four days. shown in Table 2. The bioassay was scored for insect mortality and stunting of growth. The results for PIP-1A (SEQ ID NO: 2) and Example 4 PSEEN3174 (SEQID NO: 6) are shown in Table 2. Example 7 Aphid Oral Feeding Assays with Purified Proteins Cross-Resistance Test in Diamondback Moth 0558 Membrane feeding assays as described (Li, et al., (Plutella xylostella) with Purified Proteins (2011) Journal of Invertebrate Pathology 107:69-78) were 0561. A diet overlay assay similar to Wang, et al., ((2007) used to assess the toxicity of PIP-1A and PSEEN3174, for Appl. Environ. Microbiol. 73:1199-1207) was used for testing mulated in PBS pH 7.4. Briefly, the individual proteins were the LC50 and IC50 of the sample on susceptible and Cry1A mixed with filter-sterilized complete artificial diet as resistant diamondback moth (DBM, Plutella xylostella). For described in Febvay, et al., (1988), Can. J. Zool. 66:2449 neonate bioassays, an aliquot of PIP-1A (SEQ ID NO: 2) 2453) to a final concentration of up to 1250 micrograms/ml. sample solution was applied to the surface (-7 cm) of 5 ml This diet (100 ul) was placed on stretched parafilm pulled artificial diet (Southland Products Inc.) in a 30-ml insect tightly across a 3 cm cell culture plate with a 1 cm hole on one rearing cup. Each bioassay included seven 2x consecutive side of the plate. A second layer of stretched parafilm was dilutions from 500 ng/cm of the PIP-1A (SEQ ID NO: 2) applied to form a thin film of diet exposed to aphids through sample and the negative control, with three replications for the 1 cm hole. Around 30 second instar pea or green peach each concentration. The PIP-1A (SEQ ID NO: 2) protein aphids were transferred to each plate, with three replicates for dilutions were prepared by mixing PIP-1A protein (SEQ ID each toxin. The same number of aphids were fed on diet only, NO: 2) with appropriate amount of PBS buffer solution as a control treatment. All plates were incubated at 24°C. with (Fisher Scientific Inc). Neonate larvae (<24 h after hatch) an 18:6 light:dark photoperiod. Mortality was scored every were placed in each assaying cup. Mortality and larval growth 24 hours and dead aphids were removed. The artificial diet inhibition (defined as inhibition if larvae did not enter second was replaced every 3 days. Data were analyzed by one-way instar within 4 days) by each sample were scored after 4 days ANOVA. The results for PIP-1A (SEQ ID NO: 2) and of feeding on the treated diet at 27°C. Concentrations for PSEEN3174 (SEQID NO: 6) are shown in Table 2. 50% mortality (LC50) or inhibition of 50% of the individuals (IC50) were calculated based on probit analysis. The results Example 5 (Table 3) showed no cross-resistance (resistance ratio<2) for PIP-1A (SEQID NO: 2) to Cry1A in diamondback moth. Southern Green Stinkbug (Nezara viridula) and Brown Marmorated Stinkbug (Halvomorpha haly) TABLE 3 Bioassay with Purified Proteins DBMStrain LCIC ng/cm 95% FL Resistance Ratio 0559 40 ul of the cell lysate samples were mixed with 360 Susceptible LC 122.5 80.8-172.3 1.O ul of the diet (Bio-Serv F964.4B). 10 to 15 newly molted instar IC 66.71 42.2O-98.21 1.O Cry1A-Res LC 205.3 145.7-285.1 1.7 nymphs were placed in polystyrene Petridishes (100 mmx20 IC 59.94 36.90-88.64 O.90 mm) lined with moist Whatman(R) filter paper (100 mm diam eter). The bioassay was incubated at 25°C. for four days. The bioassay was scored for insect mortality and stunting of Example 8 growth. To generate IC50 or LC50 data, a series of concen trations of purified proteins were assayed against insects and Creation and Identification of PIP-1A Variants the concentration at which 50% of insects experienced severe 0562 Libraries of modified PIP-1A polynucleotides were damage was the IC50 and the concentration at which 50% of generated using recursive sequence recombination methods US 2014/0007292 A1 Jan. 2, 2014 70
(Crameri, et al., (1998) Nature. 391:288-291; Stemmer, assay and approximately 1000 clones expressed a polypep (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Ness, et. tide at significant levels and were active as clear cell lysates in al., (2002) Nature Biotechnology 20:1251-1255), also known the Lygus bioassay. Lygus bioassays were conducted using as gene shuffling methods. To increase the crossover points the cell lysates at 100 ppm concentration of the PIP-1 between the two genes, codons of PIP-1A (SEQ ID NO: 1) polypeptide. The concentrations of PIP-1 polypeptides were were modified using the codon usage of PSEEN3174 (SEQ estimated using densitometry method of SDS-PAGE with ID NO: 5) as the template while the protein sequences are not BSA as standard using program Phoretix ID (Totall ab Ltd changed. The modified PIP-1A coding sequence was named Keel House, Garth Heads, Newcastle upon Tyne NE1 2JE). as PIP-1A Synth (SEQID NO: 15) and was synthesized. The Of the active clones, 50 were DNA sequenced (SEQID NOS: DNA sequence identity between those two genes was 152-202) and the amino acid sequence (SEQID NOS: 101 increased from 78% to 87% after the modification. To per 151) of the encoded PIP-1 polypeptide was determined. Table form the classic gene family shuffling, random DNA frag 4 shows the percent homology of the PIP-1 polypeptides ments of both PIP-1A Synth (SEQ ID NO: 15) and (SEQID NOs: 101-151) to PIP-1A (SEQID NO: 2). For each PSEEN3174 (SEQ ID NO: 5) were generated by limited of the sequences in Table 4 only those positions and the nuclease digestion. DNA fragments with molecular weights corresponding amino acids where PIP-1A (SEQID NO: 2), of 50 to 200 base pairs of both genes were recovered from PSEEN3174 (SEQ ID NO: 6) and the PIP-1 polypeptide agarose gel. The isolated DNA fragments were assembled on differ are shown. Amino acid substitutions were also identi a thermo cycler with polymerase and rescued by cloning fied at positions 3, 6, 49, 213, 249 (shaded) of PIP-1A (SEQ primers franking both termini. The libraries were cloned as ID NO: 2) which arent the corresponding amino acid of Maltose-Binding-Protein fusions into pMAL(R)-c2x (NEB) PSEEN3174 (SEQ ID NO: 6). These results demonstrate a and transformed into E. coli cells. Approximately 5000 diverse set of PIP-1A polypeptide variants that have insecti clones from the shuffled libraries were screened in the Lygus cidal activity.
TABLE 4
Identity
PIP-1A (SEQID NO: 2) D0274242 SEQID NO: DO274246 S D NO:
DO274234 S D D0274322 SEQI 48 D D0274327 SEQ 51 D D0274236 SEQIDE NO: 04 US 2014/0007292 A1 Jan. 2, 2014 71
TABLE 4-continued
4 D D O 2 7 2 5 8
DO274 DO274248 S EQ 14 9 D D0274251 SEQID NO: 11 6 8 9 D DO274262 SEEQID NO: 2 2 89 PSEEN3174 (SEQID NO: 6) 79
% Identity Sequence 98 105 108110 151 Name to PIP
DO274
DO274 D DO274322
N US 2014/0007292 A1 Jan. 2, 2014 72
TABLE 4-continued
DO274 DO274 262 89 PSEEN3
% Sequence Identity 167 168174 175 18O 2002O3 204 209 220 221 222 Name to PIP
PIP-1A 100 Q L T I V M G N N T N S T D D0274242 97 E M K A 246 97 so A D 267 97 D 273 97 M K S D 276 97 D D0274308 96 S Q A N V T
DO274234 95 M K D D0274322 95 D DO274327 95 E M K D D027429 94 E M K D 238 93 D 270 92 D D0274311 92 E M K DO274247 91 DO274258 91 E D DO274264 91 V I L A S V
US 2014/0007292 A1 Jan. 2, 2014 74
TABLE 4-continued
US 2014/0007292 A1 Jan. 2, 2014
TABLE 4-continued
D DO27428O 88 D D0274306 88 E D D0274316 8 8 D D0274323 8 8 E D 239 87 E D 260 87 D 2 D D02743 D D0274282 86 E D DO274 2O 86 E
D02743O2 85 E D0274240 84 E D0274252 84 E
255 83 E 243 82 281 82 DO274313 E M K PSEEN3174 E M K
Example 9 NO: 6) and PIP-1B (SEQ ID NO: 4), and three inactive orthologs, AECFG 592740 (SEQ ID NO: 12), Pput 1063 Identification of Amino Acid Positions Affecting the (SEQ ID NO: 8) and Pput 1064 (SEQID NO: 10) is shown Protein Stability and Function in FIG. 1. Secondary structure features of PIP-1A (SEQ ID 0563 BLAST searching the Department of Energy Joint NO: 2) were obtained using program Garnier (EMBOSS Genomic Institute website (www.jgi.doe.gov/) and NCBI Explorer) (Garnier, et al., (1978).J. Mol. Biol. 120:97-120) database using the PIP-1A (SEQID NO: 2) and PSEEN3174 (SEQ ID NO: 6) sequence revealed information regarding and selected structure features shown above the alignment of three additional genes having lower homology: AECFG FIG. 1. Six positions (P43, W66, P89, Y93, Y176, F259 of 592740 (2035954615—annotated as a hypothetical protein SEQ ID NO: 2) were selected for saturated mutagenesis Acromyrmex echination fungus garden), Pput 1063 (Ac analysis. Mutants were generated using degenerate oligos cession # ABQ77224: Gene ID:5191350 annotated as a (Table 5) for each site using sewing and rescuing PCR strat hypothetical protein Pseudomonas putida F1) and Pput egy of two overlapping fragments of N-terminus (no muta 1064 (Accession # ABQ77225; Gene ID:5191351–anno tion) and C-terminus (with mutations) gene) for each site tated as a hypothetical protein Pseudomonas putida F1). using sewing and rescuing PCR strategy of two overlapping The AECFG 592740 coding sequence has the DNA fragments of N-terminus (no mutation) and C-terminus (with sequence set forth in SEQID NO: 11 and encodes a polypep mutations) gene) as illustrated in FIG. 2. The rescued mutant tide, having the amino acid sequence set forth in SEQID NO: libraries were cloned into the Maltose-Binding-Protein 12. The Pput 1063 coding sequence has the DNA sequence fusions of pMAL(R)-c2x (NEB). Individual mutations were set forth in SEQID NO: 7 and encodes the polypeptide set identified by sequencing 96 clones of each library. The forth in SEQID NO:8. The Pput 1064 coding sequence has respective variant proteins were expressed in E. coli, and cell the DNA sequence set forth in SEQID NO:9 and encodes the lysates tested in the Lygus assay as described previously for polypeptide set forth in SEQ ID NO: 10. The AECFG PIP-1A (SEQ ID NO: 2). Table 6 shows for each mutated 592740 (SEQID NO: 11), Pput 1063 (SEQID NO: 7), and position the amino acid substitutions identified those substi Pput 1064 (SEQ ID NO: 9) genes were synthesized, the tutions that expressed soluble protein, and those Substitutions respective proteins were expressed as Maltose binding pro that were active in the Lygus assay and/or the Soybean looper teinfusions in E. coli, and cell lysates were tested in the Lygus assay with a minimal score of 4 or greater out of total maximal assay as described previously for PIP-1A (SEQ ID NO: 2). score of 8. The expression and activity of the substitutions in The AECFG 592740 (SEQ ID NO: 12), Pput 1063 (SEQ parenthesis were not determined. Substitutions indicated ID NO: 8), and Pput 1064 (SEQ ID NO: 8) proteins were with an “*” had significantly reduced soluble expression. inactive in the Lygus assay. This data demonstrate that the amino substitutions indicated 0564. The protein sequence alignment of three active in Table 6 as “Active mutants’ can be made while retaining homologs, PIP-1A (SEQID NO: 2), PSEEN3174 (SEQ ID activity.
US 2014/0007292 A1 Jan. 2, 2014 77
TABLE 7 PIP-1A PIP-1B PSEEN3174 Pput 1063 AECFG 592740 Pput 1064 PIP-1A 93 79 23 37 36 PIP-1B 79 26 38 35 PSEEN3174 24 36 34 Pput 1063 22 23 AECFG 592740 36 Pput 1064
0566. The chimeras were generated using a sewing PCR expressed and functionally tested in Lygus insect bioassays. strategy with fragments of N-terminus and C-terminus of the Table 9 shows the amino acid sequence for each of the four wild type PIP-1A with overlapping oligonucleotides (Table motifs (underlined in FIG. 1) from PIP-1A and the corre 8) coding for the replaced sequence of inactive proteins. sponding amino acid sequence based on the alignment (FIG. 1) with AECFG 592740 (SEQ ID NO:12), Pput 1063 0567 The rescued PCR products containing the replace (SEQID NO: 8), and Pput 1064 (SEQID NO: 10) that were ments were cloned into the pMAL expression vector as substituted. In Table 9 the differences between the respective described above for PIP-1A. The resulting chimeras were sequences are in indicated in “bold' and “underlining”. TABLE 8 Replaced Oligo Motif from ale Sequence 1. AECFG 59 55Mot1R CATGTCACCGTAGATGGTACCGCCACGTACCCAGCAGCCTTGCTTGGG 274 O SEO ID NO: 33 55Mot1F GGTACCATCTACGGTGACATGTGGATCGGAAGCAGAATTGGGGCACCTAC AC SEQ ID NO: 34 Pput 1O 63Mot1R GAAGCCACCGTAGACGGTTTCGCCTTCTATCCAGCAGCCTTGCTTGGGC 1063 SEO ID NO : 35 1O 63Mot GAAACCGTCTACGGTGGCTCGGTTTCCCCAAGCAGAATTGGGGCACCTAC SEO ID NO: 36 Pput 1064Mot1R ACGGACGTCACCGTAGGTGGTATCGGCATCTACCCAGCAGCCTTGCTTGG 1064 SEO ID NO : 37 1064ydt.1F ACCACCTACGGTGACGTCCGTGCGGCAAGCAGAATTGGGGCACCTAC SEO ID NO: 38 2 AECFG 59 55Mot2R ACCATGCACGCCTTCAGTGTAACTGGATCCAATTGAGATATCCGAACC 274 O SEO ID NO : 39 55Mot2F TACACTGAAGGCGGCATGGTTCGAACACGTTCAGCAATAGCACTCAATTG SEO ID NO: 40 Pput 1O 63Mot2R CAGAGGACGCCATTCTTCAGCACTGCATCCAATTGAGATATCCGAACC 1063 SEO ID NO: 41 1063Mot2F GCTGAAGAATGGCGTCCTCGCGACGTTCAGCAATAGCACTCAATTG SEO ID NO: 42 Pput 1064Mot2R CGAACCAGACACGGTTTCACTGACACTGAATCCAATTGAGATATCCG 1064 SEO ID NO : 43 1064Mot2F AGTGAAACCGTGTCTGGTTCGGAGACGTTCAGCAATAGCACTCAATTG SEO ID NO: 44
3 AECFG 59 55Mot3R ATGCATCTGATAGAAGTTGTAGATGCCAGGACCAGTCAATTGAGTG 274 O SEO ID NO: 45 55Mot3F TACAACTTCTATCAGATGCATATGGTTTTTGCGCACAACGCCACTTCTG SEO ID NO: 46 Pput 1063Mot3R CTGATACGCGACGTAGCATTCAGGACCAGTCAATTGAGTGCTATT 1063 SEO ID NO : 47 1063Mot3F GAAIGCTACGTCGCGTATCAGCTTAAACTGGTTTATGCGCACAACGCCACT TC SEO ID NO : 48 Pput 1064Mot3R ATGAACCTGATACACCATGATGGTGCCAGGACCAGTCAATTGAG 1064 SEO ID NO: 49 1064Mot3F ATCATGGTGTATCAGGTTCATATGGTTTATGCGCACAACGCCAC SEO ID NO : 50
4. AECFG 59 55Mot4R GTTGTCCATCAACACAGCGCGCTGAACAGTATCCCAATCCAG 274 O SEO ID NO: 51 55Mot4F CGCGCTGTGTTGATGGACAACTACAAGCCAGGCAGCAATAGTGGGCAC SEO ID NO : 52 Pput 1O 63ylot 4R. GGCCAGGTTGAAGATAAGGTGGTAAACAGTATCCCAATCCAGCGGC 1063 SEO ID NO : 53 1063Mot4F CACCTTATCTTCAACCTGGCCTACGGCCCAGGCAGCAATAGTGGGCACTTC SEO ID NO: 54 US 2014/0007292 A1 Jan. 2, 2014 78
TABLE 8- continued Replaced Oligo Motif from ale Sequence
Pput 1064Mot 4R. CTGGTTGAACAACACAGCCTGGTTAACAGTATCCCAATCCAGCGGC 1064 SEO ID NO. 55 1064Mot4F CAGGCTGTGTTGTTCAACCAGGAGGAGCCAGGCAGCAATAGTGGGCACTTC SEO ID NO: 56
TABLE 9
Soluble Replaced PIP-1A WT amino Amino acids protein Motif from Oligos acid sequence replaced expressed Activity 1. AECFG 59 55Mot1R GCWVDGITVYGDIFIG GCWVRGGTIYGDMWIW No No 274 O 55Mot1F a.a. 64 - 79 of a.a. 49- 64 of SEQ ID NO: 2 SEO ID NO: 12 Pput1063 1063Mot1R GCWIEGETVYGGFGFP No No 1063Mot1F a.a. 34 - 49 of SEO ID NO: 8 Pput1064 1064Mot1R GCWVDADTTYGDVRCG No No 1064Mot1F a.a. 37-52 of SEO ID NO: 10
2 AECFG 59 55Mot2R FSNSESWSTTO SSYTEGWHGSN No No 274 O 55Mot2F a.a. 149-159 of a.a. 133-143 of SEQ ID NO: 2 SEO ID NO: 12 Pput1063 1063Mot2R CS-AEEWRPLS No No 1063Mot2F a.a. 118-127 of SEO ID NO: 8 Pput1064 1064Mot2R FSVSETVSGSE No No 1064Mot2F a.a. 122-132 of SEO ID NO: 10
3 AECFG 59 55Mot3R GTFIVYOVVMVYA GIYNFYQMHMVFA No No 274 O 55Mot3F a.a. 171-183 of a.a. 155-167 of SEQ ID NO: 2 SEO ID NO: 12 Pput1063 1063Mot3R ECYWAYQLKLWYA No No 1063Mot3F a.a. 139-151 of SEO ID NO: 8 Pput1064 1064Mot3R GTIMVYQVHMVYA No No 1064Mot3F a.a. 144-156 of SEO ID NO: 10
4. AECFG 59 55Mot4R ORNVLMENYN QRAVLMDNYK Yes Yes 274 O 55Mot 4F a.a. 240-249 of a.a. 224-233 of SEQ ID NO: 2 SEO ID NO: 12 Pput1063 1063Mot4R YELFNLAY No No 1063Mot4F a.a. 2 O8 - 217 of SEO ID NO: 8 Pput1064 1064Mot4R NQAVLFNQFE No No 1064Mot4F a.a. 217-227 of SEO ID NO: 10
0568 Table 9 also indicates if the resulting proteins were were selected to further define the roles of the regions in soluble when expressed as a MAL fusion in E. coli. and were insecticidal functions. To further define the permitted active in the Lygus assay. sequence variation within those two selected motifs, satu 0569. As indicated in Table 9, all but one of these chimeras rated mutagenesis was designed for each position of the had reduced expression of soluble protein and was inactive in motifs using the mutagenesis oligonucleotides as shown in the bioassay indicating that these four motifs have functional Tables 10 and 11 for motifs 3 and 4 respectively. The variants constraints. were generated using a similar strategy as described in Example 9. Tables 12 and 13 show for each mutated position Example 11 the amino acid substitutions identified, those substitutions that expressed soluble protein, and those substitutions that Saturated Mutagenesis of Motifs to Define Sequence were active in the Lygus assay and/or the Soybean looper Variations that Retain Insecticidal Activity assay with a minimal score of 4 or greater out of total maximal score of 8. This data demonstrate that the amino substitutions 0570. Two motifs, amino acids 171 to 183 (motif3) and indicated in Tables 12 and 13 as “Active mutants' can be amino acids 240 to 249 (motif4) of PIP-1A (SEQID NO: 2) made while retaining activity.
US 2014/0007292 A1 Jan. 2, 2014 81
TABLE 13 dentified Soluble expressed Position mutations mutants Active mutants Q240 R, A, V. E., M. G., R, A, V, E, M, G, D*, R. A. V. E., M. G. D. D, W, N, T, I, S, W, N, T, I, S, F, H, W, N, T, I, S, F, H, F, H, C, L. Y. P. K C, L,Y, K C, L,Y, K R241 K, E, Q, S., I, V, K, E, Q, S., I, V, D, K, E, Q, S., I, V, D, D,Y, M, N, H., P. Y, M, N, H., P. G., L, Y, M, N, H., P. G. L., G, L, F, T, A, C, W F, T, A, C, W F, T, A, C, W N242 R, K, H, S, C, A, R, K, H, S, C, A, E, R, K, H, S, C, A, E, E. P. W. Q, T, F, P*, W, Q, T, F.Y. M., PW, Q, T, F.Y. M., Y. M., D, V, G, L, I D. V. G. L., I D, G, L, I W243 P, L, Q, E, A, F, L, A, T, G, C, I, S. L, A, T, G, C, I, S, N, Y, T, W, G, C, M, M, , R, S, H, K, M, D L244 V, F, I, S, M.Y. V, F, I, M, W, Q, A, V, F, I, M, Q, C, W W, P, Q, H, T, K, C. E, A, N, C, R, G, D M245 A, R, D, E, L., P. A, R, D, E, L., P, S, A, R, D, E, L., P, S, S.W. G. V. K., F, W. G. V. K., F, C, T, W. G. V. K., F, C, T, C, T, H, I, Q, Y, N H, I, Q, Y, N H, I, Q, Y, N E246 G, S, I, A., L, V, Y, D, G, R, V. A. W. Y, D, G, R, V, A, W, H. W. R.Y. C, D, Q, S, N, I, L., M. C., Q, S, N, I, L., M. C., N, Q, P, M, F, T, K P, H, F, T, K P, H, F, T, K N247 L., D.Y.A., F. H., L., D.Y.A., F. H. R. L., D.Y.A., F. H., R, R, K, Q, G. V., I, K, Q, G. V., I, S, E, K, Q, G. V., I, S, E, S, E, P. M, W, T, C P. M, W, T, C P. M, W, T, C Y248 V, T, E, F, S, H, V, T, E, F, S, H, C, V, T, E, F, S, H, C, C, N, L., G, K, A, N, L, G, A, W, I, D, P L. W., I, D, G, A W. R., I, D, P, Q, (M) N249 V. G. M. D. K. C. V. G. M., D, K, C, F, V. G. M., D, K, C, F, F, R, E, W, Y, S, R, E, W, Y, S, I, T, R, E, W, Y, S, I, T, I, T, P, L, A, H, Q P, L, A, H, Q P, L, A,
Example 12 Example 13 Defined Protein Sequences of Fragments Retaining Transient Expression and Insect Bioassay on Activity Transient Leaf Tissues (0572. A series of truncated variants of PIP-1A (SEQ ID NO: 2) are generated in 5 amino acid increments from both 0571. Both PIP-1A (SEQ ID NO: 2) and PSEEN3174 ends by PCR cloning for the first and/or last 30 amino acids. (SEQ ID NO: 6) as MBP fusions and alone were cloned into The truncated genes are cloned to the same expression system a transient expression vector under control of a viral promoter as listed above. Recombinant proteins of those truncated ver pDMMV (Day, et. al., (1999) Plant Mol. Biol. 40.771-782). sions of PIP-1A are assayed with insects and minimal length The agro-infiltration method of introducing an Agrobacte of the protein is defined with the variant still retains detectible rium cell Suspension to plant cells of intact tissues so that insecticidal activity. reproducible infection and Subsequent plant derived trans gene expression may be measured or studied is well known in Example 14 the art (Kapila, et. al., (1997) Plant Science 122:101-108). N-Terminal Truncation Variants Briefly, young plantlets of Phaseolus vulgaris or Glycine (0573. The PIP-1A (SEQID NO: 2), PSEEN3174 (SEQID max, were agro-infiltrated with normalized bacterial cell cul NO: 6) and PIP-1B (SEQ ID NO: 4) proteins were digested tures oftestand control strains. Leaf discs were generated and with a limited Trypsin digestion (1 part of Trypsin vs. 100 infested with 3 neonates of both Soy Bean Looper (SBL) parts of purified protein). The resulting N-terminal trypsin (Pseudoplusia includes) or Velvet bean caterpillar (VBC) truncated variants, PIP-1AT1 (SEQ ID NO. 204), (Velvet Anticarsia gemmatalis) with two control leaf discs PSEEN3174T1 (SEQID NO: 206), PIP-1BT1 (SEQID NO: generated with Agrobacterium only. The consumption of 208), have amino acids 1-28 deleted compared to the respec green leaf tissues was scored after two days infestation. The tive full length proteins by N-terminal Amino Acid sequenc transiently expressed PIP-1A (SEQ ID NO: 2) and ing. The PIP-1AT1 (SEQID NO. 204), PSEEN3174T1 (SEQ PSEEN3174 (SEQID NO: 6) protected leaf discs from con ID NO: 206), PIP-1BT1 (SEQID NO. 208) were assayed in sumption by the infested SBL and VBC insects while the total the Lygus assay and found to have substantially the same green tissue consumption was observed for the two negative activity as the respective full length proteins. controls. Transient protein expressions of both PIP-1A (SEQ ID NO: 2) and PSEEN3174 (SEQID NO: 6) were confirmed Example 15 by Mass spectrometry based protein identification method using extracted protein lysates from infiltrate leaf tissues Proteolytic Cleavage Site Variants (Patterson, (1998) 10(22): 1-24, Current Protocol in Molecu (0574. The arginine (R) at position 28 of PIP-1A was lar Biology published by John Wiley & Son Inc). mutated to alter the trypsin cleavage site. The variants were US 2014/0007292 A1 Jan. 2, 2014 82 generated using a similar strategy as described in Example 9 Example 16 using the saturation mutagenesis primers R28R (SEQID NO: Multiple Residue Motif 4 PIP-1A Variants 218), and R28F (SEQIDNO: 219). Table 14 shows the amino (0575. To further explore the role of motif 4 (amino acids acid substitutions identified, those substitutions that 240 to 249 of PIP-1A (SEQ ID NO: 2), a series of variants expressed soluble protein, and those substitutions that were were generated with multiple amino acid Substitutions in active in the Lygus assay with a minimal score of 4 or greater motif4. The variants were generated using a similar mutagen out of total maximal score of 8. This data demonstrate that the esis strategy as described in Example 9 using the mutagenesis amino substitutions indicated in Table 14 as 'Active mutants' primer Motif 4-Comb-F CCGCTGGATTGGGATACTGT can be made to eliminate a proteolytic cleavage site while TVWWNGCHAYDTTWTKDTKGRKNAYTWT retaining activity. NAYCCAGGCAGC AATAGTGGGCACTTC (SEQID NO: 326) paired with primer 3188R GGATGTGCTGCAAGGC TABLE 1.4 GATTAAG (SEQID NO:327) and Comb-RAACAGTATC CCAATCCAGCGG (SEQ ID NO:328) paired with 3188F Identified Soluble expressed Lygus Active CAGACTGTCGATGAAGCCCTGAAAG (SEQ ID NO: Position mutations Mutants mutants 329). The mutagenesis primer Motif 4-Comb-F was designed to be partially degenerate at residues 240-249 of PIP-1A R28 S, K, T, V, G, A, S, K, T, V, G, A, S, K, T, V, G, (SEQID NO: 2) resulting in selected amino acid substitutions M, D, W, P, L, H, M, D, W, P, L, H, A, M., D, W, P. at each residues. Table 15 shows the degenerate codon encod C, Q, C, Q, L, H, C, Q, ing each of residues 240-249 and the possible resulting amino acids. In Table 15 the native amino acid is indicated in bold and underlining.
TABLE 1.5
Degenerate Residue codon Degeneracy Resulting amino acids*
24 O WWW W = A G OR C Glin, Lys, Glu, Asp, Ile, W = A OR T Wall, Asn His and Lieu.
241 NGC N = G, A, T OR C Arg, Ser, Gly, and Cys
242 HAY H = A, C OR T Asn His, and Tyr Y = C OR T
243 DTT D = A G OR T Wall, Ile and Phe
244 WTK W = A OR T Leu, Met, Ile and Phe K = G OR
245 DTK D = A G OR T Met, Ile, Wall, Leu and K = G OR T Phe
246 GRK R = A OR G Glu, Gly and Asp K = G OR
247 NAY N = G, A, T OR C Asn., Asp, Tyr and His Y = C, OR
248 TWT W = A OR T Tyr and Phe
249 NAY N = G, A, T OR C Asn., Asp, Tyr and His Y = C, OR US 2014/0007292 A1 Jan. 2, 2014
0576. The resulting polynucleotides encoding the PIP-1A TABLE 16 - continued variant polypeptides were expressed as MBP fusions in E. coli and screened as cleared lysates in a 96 well format (3 of Soluble plates) for Lygus insecticidal activity as described in Example Variant Amino acids seq. mutations expression 1 and scored for activity on a scale of 0 to 8 (see FIG. 4). The clones encoding the variant PIP-polypeptides having Lygus 2E1 NRHVLVDNFY 5 Yes SEQ ID (a.a. 240-249 insecticidal activity ranging from 4 to 8 were DNA sequenced NO: 254 of SEQ ID NO: 254) (SEQID NO: 220, SEQID NO: 221, SEQID NO: 222, SEQ IDNO:223, SEQIDNO: 224, SEQID NO: 225, SEQID NO: 2E12 WSNVLIDDFD 7 Yes 226, SEQID NO: 227, SEQID NO: 228, SEQID NO: 229, SEQ ID (a.a. 240-249 SEQID NO. 230, SEQID NO. 231, SEQID NO. 232, SEQ NO: 255 of SEO ID NO: 255) IDNO. 233, SEQIDNO. 234, SEQID NO. 235, SEQID NO: 2F4. vsHVMMEDYD 6 Yes 236, SEQID NO. 237, SEQID NO. 238, SEQID NO. 239, SEQ ID (a.a. 240-249 SEQID NO: 240, SEQID NO: 241, SEQID NO: 242, SEQ NO: 256 of SEQ ID NO: 256) ID NO: 243, and SEQID NO: 244) to determine the identity 2F8 NSHILVGNYD 7 Yes of the amino acid substitutions at residues 240-249 of the SEQ ID (a.a. 240-249 PIP1A polypeptides of SEQID NO: 245, SEQID NO: 246, NO: 257 of SEO ID NO: 257) SEQID NO: 247, SEQID NO. 248, SEQID NO. 249, SEQ 2G5 NSYVMIENFY 7 Yes IDNO: 250, SEQIDNO: 251, SEQID NO:252, SEQID NO: SEQ ID (a.a. 240-249 253, SEQID NO: 254, SEQID NO: 255, SEQID NO: 256, NO: 258 of SEO ID NO: 258) SEQID NO: 257, SEQID NO: 258, SEQID NO: 259, SEQ 2G6 NCNIIMENYD 5 Yes IDNO: 260, SEQIDNO:261, SEQID NO. 262, SEQID NO: SEQ ID (a.a. 240-249 263, SEQID NO: 264, SEQID NO: 265, SEQID NO: 266, NO: 259 of SEO ID NO: 259) SEQID NO: 267, SEQID NO: 268, and SEQID NO: 269, which are shown in Table 16. The motif 4 amino acid substi 3A2 IRYIFIDNFD 8 Yes tutions compared to PIP-1A (SEQID NO: 2) are indicated in SEQ ID (a.a. 240-249 bold and underlining. NO: 26 O of SEQ ID NO: 260) 3A1O VRNVLVENYH 3 Yes TABLE 16 SEQ ID (a.a. 240-249 NO: 261 of SEQ ID NO: 261) of Soluble Variant Amino acids seq. mutations expression 3C7 QRYVLIDNFY 5 Yes SEQ ID (a.a. 240-249 PIP-1A ORNVLMENYN O Yes NO: 262 of SEQ ID NO: 262) (a.a. 240-249 of SEQ ID NO: 2) 3E3 LSHFMLGNFN 8 Yes SEQ ID (a.a. 240-249 A3 NSYVLLDYYY 7 Yes NO: 263 of SEQ ID NO: 263) SEO ID (a.a. 240-249 NO: 245 of SEQ ID NO: 245) 3F1 RCNVLMGDFD 6 Yes SEQ ID (a.a. 240-249 E3 NCYIFMEYYD 7 Yes NO: 264 of SEQ ID NO: 264) SEO ID (a.a. 240-249 NO: 246 of SEQ ID NO: 246) 3F2 IGNVMWGDFD 8 Yes SEQ ID (a.a. 240-249 F5 NCYIMMENFD 7 Yes NO: 265 of SEQ ID NO: 265) SEO ID (a.a. 240-249 NO: 247 of SEO ID NO: 247) 3F6 QCYVLIENFH 5 Yes SEQ ID (a.a. 240-249 B9 QCNVLFDNFH 5 Yes NO: 266 of SEQ ID NO: 266) SEO ID (a.a. 240-249 NO: 248 of SEQ ID NO: 248) 3F12 VCNVLMEHFY 5 Yes SEQ ID (a.a. 240-249 C10 QGYVLVDNFN 5 Yes NO: 267 of SEO ID NO: 267) SEO ID (a.a. 240-249 NO: 249 of SEQ ID NO: 249) 3G7 VRNVFFDYFD 7 Yes SEQ ID (a.a. 240-249 A11 NRYVFFGNYD 6 Yes NO: 268 of SEQ ID NO: 268) SEO ID (a.a. 240-249 NO: 25 O of SEO ID NO: 25O) 3F4 WSYLFDNFH 8 Yes SEQ ID (a.a. 240-249 2A2 QCNIMIGYFD 8 Yes NO: 269 of SEQ ID NO: 269) SEO ID (a.a. 240-249 NO: 251 of SEQ ID NO: 251)
2G1. QGNVLMENYN 1. Yes 0577. The clones encoding the variant PIP-1A polypep SEO ID (a.a. 240-249 tides having Lygus insecticidal activity ranging from 0 to 4 NO: 252 of SEQ ID NO: 252) were DNA sequenced (SEQID NO: 270, SEQID NO: 271, 2C7 VSNILVGNFN 6 Yes SEQID NO. 272, SEQID NO: 273, SEQID NO: 274, SEQ SEO ID (a.a. 240-249 IDNO: 275, SEQIDNO:276, SEQID NO: 277, SEQID NO: NO: 253 of SEO ID NO: 253) 278, SEQID NO: 279, SEQID NO: 280, SEQID NO. 281, SEQID NO: 282, SEQID NO: 283, SEQID NO: 284, SEQ US 2014/0007292 A1 Jan. 2, 2014
IDNO: 285, SEQIDNO:286, SEQID NO: 287, SEQID NO: SEQID NO:319, SEQID NO:320, SEQID NO:321, SEQ 288, SEQID NO: 289, SEQID NO: 290, SEQID NO: 291, ID NO:322, SEQID NO:323, SEQID NO:324, and SEQID SEQID NO. 292, SEQID NO: 293, SEQID NO: 294, SEQ NO: 325, which are shown in Table 17. Protein expression ID NO: 295, SEQ ID NO: 296, and SEQ ID NO: 297), to analysis by SDS-PAGE (data not shown) revealed that the determine the identity of the amino acid substitutions at resi variant proteins with Lygus insecticidal activity from 0 to 4 dues 240-249 of the PIP-1A polypeptides SEQID NO: 298, affect soluble expression (protein folding and solubility) in E. SEQID NO: 299, SEQID NO:300, SEQID NO:301, SEQ coli with the proteins accumulating as insoluble fraction of IDNO:302, SEQIDNO:303, SEQID NO:304, SEQID NO: the cleared lysate. The loss of activity from the multiple 305, SEQID NO:306, SEQID NO:307, SEQID NO:308, substitutions in motif 4 appears to be from the lack of soluble SEQID NO:309, SEQID NO:310, SEQID NO:311, SEQ expressed proteins in the E. coli expression system. Motif 4 IDNO:312, SEQIDNO:313, SEQID NO:314, SEQID NO: appears to be tolerant to multiple amino acid Substitution 315, SEQID NO:316, SEQID NO:317, SEQID NO:318, while remaining active. TABLE 1.7
of Soluble Variants Amino acids seq. mutations expression PIP-1A QRNVLMENYN (a.a. 240-249 O yes of SEQ ID NO: 2)
Bf HSYWFIDNYN (a.a. 240-249 6 No SEO ID NO: 298 of SEQ ID NO : 298)
C 7 WCNFFFGDFD (a.a. 240-249 9 No SEO ID NO : 299 of SEO ID NO : 299)
D7 KRYFMMIGYFH (a.a. 240-249 8 No SEO ID NO: 3 OO of SEQ ID NO: 3 OO)
E7 CHVFIGYFY (a.a. 240-249 9 No SEO ID NO: 3 O1 of SEQ ID NO: 301)
E7 EGNFFWGNFD (a.a. 240-249 8 No SEO ID NO: 3 O2 of SEQ ID NO: 3O2)
A8 IRYFILEDYN (a.a. 240-249 6 No SEO ID NO: 303 of SEQ ID NO: 303)
B8 LGYFMWEDFD (a.a. 240-249 9 No SEO ID NO: 304 of SEQ ID NO: 3O4)
C8 KGNVLVEYYN (a.a. 240-249 4. No SEO ID NO : 3 O5 of SEO ID NO : 305)
D8 LSNVIMGHFY (a.a. 240-249 7 No SEO ID NO: 306 of SEQ ID NO: 306)
E8 WSYWFFGHFD (a.a. 24 O-249 9 No SEO ID NO : 3 O7 of SEO ID NO : 307)
G8 DGYLWGNFD (a.a. 240-249 8 No SEO ID NO: 3O8 of SEQ ID NO: 3O8)
A9 NGNIFLDHFD (a.a. 240-249 9 No SEO ID NO : 3 O9 of SEO ID NO : 3 O9)
D9 ICYFDDYH (a.a. 240-249 9 No SEO ID NO: 310 of SEQ ID NO : 31 O)
F9 NSNFLFENFH (a.a. 240-249 6 No SEO ID NO: 311 of SEQ ID NO : 311)
D10 CHILIGDYN (a.a. 240-249 7 No SEO ID NO: 312 of SEQ ID NO : 312)
E10 HCNVIVDYYN (a.a. 240-249 6 No SEO ID NO: 313 of SEQ ID NO : 313)
F10 EGYVMFGYFN (a.a. 240-249 8 No SEO ID NO: 314 of SEQ ID NO : 314)
B11 WCYLVEYYH (a.a. 240-249 7 No SEO ID NO: 315 of SEQ ID NO : 315) US 2014/0007292 A1 Jan. 2, 2014
TABLE 1.7-continued
of Soluble Variants Amino acids seq. mutations expression
C11 LRHVMFGNYY (a.a. 24 O-249 6 No SEO ID NO: 316 of SEQ ID NO : 316)
D11 NRNIFFDDYY (a.a. 240-249 7 No SEO ID NO : 317 of SEO ID NO : 317)
E11 KGYVMWGDFN (a.a. 24 O-249 8 No SEO ID NO: 318 of SEQ ID NO : 318)
F11 LGNFFLGYYN (a.a. 240-249 7 No SEO ID NO: 319 of SEQ ID NO : 31.9)
H11 LSNVLIDNFY (a.a. 240-249 6 No SEO ID NO: 32O of SEQ ID NO: 32O)
Al2 NCYFIWDDYN (a.a. 240-249 8 No SEO ID NO: 321 of SEQ ID NO: 321)
B12 ISYVFVEDFH (a.a. 240-249 8 No SEO ID NO: 322 of SEQ ID NO: 322)
D12 NIHIMIEYYH (a.a. 24 O-249 8 No SEO ID NO: 323 of SEQ ID NO: 323)
E12 IGHFMLDYYH (a.a. 24 O-249 9 No SEO ID NO: 324 of SEQ ID NO: 324) G12 IcyVMVGNYH (a.a. 24 O-249 7 No SEO ID NO: 325 of SEO ID NO : 325)
Example 17 Example 18 Transformation of Maize by Particle Bombardment Identification of an Insecticidal Protein Active and Regeneration of Transgenic Plants Against Lygus from Strain JH19887-2 0579. Immature maize embryos from greenhouse donor plants are bombarded with a DNA molecule containing the 0578 A Blast search of a proprietary genomic contig toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably library of a Pseudomonas Protegens strain JH19887-2 against linked to an ubiquitin promoter and the selectable marker the PIP-1 polynucleotide sequence of SEQID NO: 1 identi gene PAT (Wohleben, et al., (1988) Gene 70: 25-37), which fied a polynucleotide of SEQ ID NO: 331, encoding a confers resistance to the herbicide Bialaphos. Alternatively, polypeptide of SEQ ID NO: 332 (herein referred to as PIP the selectable marker gene is provided on a separate DNA 1C) having 82% sequence identity to PIP-1A (SEQ ID NO: molecule. Transformation is performed as follows. Media 2). Table 18 shows the % sequence identity between PIP-1C recipes follow below. (SEQ ID NO: 332) and PIP-1A (SEQ ID NO: 2), PIP-1B (SEQ ID NO: 4), and PSEEN3174 (SEQ ID NO: 6). FIG.5 Preparation of Target Tissue shows the sequence alignment of PIP-1A (SEQ ID NO: 2), 0580. The ears are husked and surface sterilized in 30% PIP-1B (SEQ ID NO: 4), PIP-1C (SEQ ID NO: 332) and CLOROXTM bleach plus 0.5% Micro detergent for 20 min PSEEN3174 (SEQ ID NO: 6). PIP-1C was expressed in E. utes, and rinsed two times with sterile water. The immature coli in the same way as PIP-1A. The purified PIP-1C was embryos are excised and placed embryo axis side down assayed against Soybean looper (SBL) and lygus in diet based (scutellum side up), 25 embryos per plate, on 560Y medium assays. PIP-1C (SEQ ID NO: 332) demonstrated killing for 4 hours and then aligned within the 2.5 cm target Zone in activity against both SBL and lygus demonstrating insecti preparation for bombardment. cidal spectrum similar to PIP-1A (SEQ ID NO: 2). Preparation of DNA TABLE 18 0581. A plasmid vector comprising a nucleotide sequence (e.g., SEQ ID NO: 1) operably linked to an ubiquitin pro PSEEN3174 PIP-1C PIP-1A PIP-1B SEQID SEQID moter is made. For example, a suitable transformation vector SEQ ID NO: 2 SEQID NO: 4 NO: 6 NO:332 comprises a UBI1 promoter from Zea mays, a 5' UTR from UBI1 and a UBI1 intron, in combination with a Pintermi PIP-1A 93% 79% 82% PIP-1B 79% 84% nator. The vector additionally contains a PAT selectable PSEEN3174 80% marker gene driven by a CAMV35S promoter and includes a PIP-1C CAMV35S terminator. Optionally, the selectable marker can reside on a separate plasmid. A DNA molecule comprising a toxin nucleotide sequence as well as a PATselectable marker US 2014/0007292 A1 Jan. 2, 2014 is precipitated onto 1.1 um (average diameter) tungsten pel 0590 Plant regeneration medium (288J) comprises 4.3 lets using a CaCl2 precipitation procedure as follows: g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins 0582 100 uL prepared tungsten particles in water stock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0583. 10 uL (1 lug) DNA in Tris EDTA buffer (1 lug total 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to DNA) volume with polished D-1HO) (Murashige and Skoog. 0584 100 uL 2.5 M CaCl, (1962) Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 0585) 10 uL 0.1 M spermidine mg/L Zeatin, 60 g/L Sucrose, and 1.0 mL/L of 0.1 mM abscisic 0586 Each reagent is added sequentially to a tungsten acid (brought to volume with polisheddl HO after adjusting particle Suspension, while maintained on the multitube Vor to pH 5.6); 3.0 g/L GelriteTM (added after bringing to volume texer. The final mixture is sonicated briefly and allowed to with dl HO); and 1.0 mg/L indoleacetic acid and 3.0 mg/L incubate under constant vortexing for 10 minutes. After the Bialaphos (added after Sterilizing the medium and cooling to precipitation period, the tubes are centrifuged briefly, liquid 60 C). removed, washed with 500 mL 100% ethanol, and centri 0591 Hormone-free medium (272V) comprises 4.3 g/L fuged for 30 seconds. Again the liquidis removed, and 105ul MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock 100% ethanol is added to the final tungsten particle pellet. For solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, particle gun bombardment, the tungsten/DNA particles are 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to briefly sonicated and 10 uL spotted onto the center of each volume with polisheddl H2O), 0.1 g/L myo-inositol, and 40.0 macrocarrier and allowed to dry about 2 minutes before bom g/L sucrose (brought to volume with polished d1 HO after bardment. adjusting pH to 5.6); and 6 g/L Bacto-agar (added after bring ing to volume with polisheddl H2O), sterilized and cooled to Particle Gun Treatment 60° C. 0587. The sample plates are bombarded at level #4 in Example 19 particle gun iHE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from Agrobacterium-Mediated Transformation of Maize each tube of prepared particles/DNA. and Regeneration of Transgenic Plants Subsequent Treatment 0592 For Agrobacterium-mediated transformation of maize with a toxin nucleotide sequence (e.g., SEQID NO: 1), 0588. Following bombardment, the embryos are kept on the method of Zhao can be used (U.S. Pat. No. 5,981,840 and 560Y medium for 2 days, then transferred to 560R selection PCT Patent Publication Number WO 1998/32326; the con medium containing 3 mg/liter Bialaphos, and Subcultured tents of which are hereby incorporated by reference). Briefly, every 2 weeks. After approximately 10 weeks of selection, immature embryos are isolated from maize and the embryos selection-resistant callus clones are transferred to 288J contacted with a Suspension of Agrobacterium under condi medium to initiate plant regeneration. Following Somatic tions whereby the bacteria are capable of transferring the embryo maturation (2-4 weeks), well-developed somatic nucleotide sequence (e.g. SEQID NO: 1) to at least one cell embryos are transferred to medium for germination and trans of at least one of the immature embryos (step 1: the infection ferred to the lighted culture room. Approximately 7-10 days step). In this step the immature embryos can be immersed in later, developing plantlets are transferred to 272V hormone an Agrobacterium Suspension for the initiation of inoculation. free medium in tubes for 7-10 days until plantlets are well The embryos are co-cultured for a time with the Agrobacte established. Plants are then transferred to inserts in flats rium (step 2: the co-cultivation step). The immature embryos (equivalent to 2.5" pot) containing potting soil and grown for can be cultured on Solid medium following the infection step. 1 week in a growth chamber, Subsequently grown an addi Following this co-cultivation period an optional “resting tional 1-2 weeks in the greenhouse, then transferred to classic step is contemplated. In this resting step, the embryos are 600 pots (1.6 gallon) and grown to maturity. Plants are moni incubated in the presence of at least one antibiotic known to tored and scored for expression of the toxin by assays known inhibit the growth of Agrobacterium without the addition of a in the art or as described above. selective agent for plant transformants (step 3: resting step). The immature embryos can be cultured on solid medium with Bombardment and Culture Media antibiotic, but without a selecting agent, for elimination of 0589 Bombardment medium (560Y) comprises 4.0 g/L Agrobacterium and for a resting phase for the infected cells. N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vita Next, inoculated embryos are cultured on medium containing min Mix (1000xSIGMA-1511), 0.5 mg/L thiamine HCl, a selective agent and growing transformed callus is recovered 120.0 g/L sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline (step 4: the selection step). The immature embryos are cul (brought to volume with deionized HO following adjustment tured on Solid medium with a selective agent resulting in the to pH 5.8 with KOH); 2.0 g/L GelriteTM (added after bringing selective growth of transformed cells. The callus is then to volume with dl HO); and 8.5 mg/L silver nitrate (added regenerated into plants (step 5: the regeneration step), and after sterilizing the medium and cooling to room tempera calli grown on selective medium can be cultured on Solid ture). Selection medium (560R) comprises 4.0 g/L N6 basal medium to regenerate the plants. salts (SIGMA C-1416), 1.0 mL/L Eriksson’s Vitamin Mix (1000xSIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L Example 19 sucrose, and 2.0 mg/L 2,4-D (brought to volume withdl HO following adjustment to pH 5.8 with KOH);3.0 g/L GelriteTM Transformation of Soybean Embryos (added after bringing to volume withdl HO); and 0.85 mg/L 0593. Soybean embryos are bombarded with a plasmid silver nitrate and 3.0 mg/L Bialaphos (both added after ster containing a nucleotide sequence (e.g., SEQID NO: 1) oper ilizing the medium and cooling to room temperature). ably linked to a pinll promoter as follows. To induce somatic US 2014/0007292 A1 Jan. 2, 2014 embryos, cotyledons, 3-5 mm in length dissected from Sur formed, necrotic embryogenic clusters. Isolated green tissue face-sterilized, immature seeds of an appropriate Soybean is removed and inoculated into individual flasks to generate cultivar are cultured in the light or dark at 26° C. on an new, clonally propagated, transformed embryogenic Suspen appropriate agar medium for six to ten weeks. Somatic sion cultures. Each new line may be treated as an independent embryos producing secondary embryos are then excised and transformation event. These Suspensions can then be subcul placed into a Suitable liquid medium. After repeated selection tured and maintained as clusters of immature embryos or for clusters of Somatic embryos that multiplied as early, regenerated into whole plants by maturation and germination globular-staged embryos, the Suspensions are maintained as described below. of individual somatic embryos. 0594 Soybean embryogenic suspension cultures can be 0600 All publications and patent applications mentioned maintained in 35 mL liquid media on a rotary shaker, 150 in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publi rpm, at 26°C. with florescent lights on a 16:8 hour day/night cations and patent applications are herein incorporated by schedule. Cultures are subcultured every two weeks by inocu reference to the same extent as if each individual publication lating approximately 35 mg of tissue into 35 mL of liquid or patent application was specifically and individually indi medium. cated to be incorporated by reference. 0595 Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombard 0601 Although the foregoing disclosure has been described in some detail by way of illustration and example ment (Klein, et al., (1987) Nature (London) 327:70-73, U.S. for purposes of clarity of understanding, it will be obvious Pat. No. 4,945.050). A DuPont Biolistic PDS1000/HE instru that certain changes and modifications may be practiced ment (helium retrofit) can be used for these transformations. within the scope of the appended claims. 0596. A selectable marker gene that can be used to facili tate Soybean transformation is a transgene composed of the 0602. The above description of various illustrated 35S promoter from Cauliflower Mosaic Virus (Odell, et al., embodiments of the invention is not intended to be exhaustive (1985) Nature 313:810-812), the hygromycin phosphotrans or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are ferase gene from plasmid plR225 (from E. coli; Gritz, et al., described herein for illustrative purposes, various equivalent (1983) Gene 25:179-188), and the 3' region of the nopaline modifications are possible within the scope of the invention, synthase gene from the T-DNA of the Tiplasmid of Agrobac as those skilled in the relevant art will recognize. The teach terium tumefaciens. The expression cassette comprising a ings provided herein of the invention can be applied to other toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably purposes, other than the examples described above. The linked to the pinll promoter can be isolated as a restriction invention may be practiced in ways other than those particu fragment. This fragment can then be inserted into a unique larly described in the foregoing description and examples. restriction site of the vector carrying the marker gene. Numerous modifications and variations of the invention are 0597 To 50 uL of a 60 mg/mL 1 lum gold particle suspen possible in light of the above teachings and, therefore, are sion is added (in order): 5 LL DNA (1 lug/LL), 20LL spermi within the scope of the appended claims. dine (0.1M), and 50 uL CaCl (2.5M). The particle prepara 0603 These and other changes may be made to the inven tion is then agitated for three minutes, spun in a microfuge for tion in light of the above detailed description. In general, in 10 seconds and the supernatant removed. The DNA-coated the following claims, the terms used should not be construed particles are then washed once in 400 uL 70% ethanol and to limit the invention to the specific embodiments disclosed in resuspended in 40 uL of anhydrous ethanol. The DNA/par the specification and the claims. ticle Suspension can be Sonicated three times for one second 0604 Certain teachings related to PIP polynucleotides each. Five microliters of the DNA-coated gold particles are and polypeptides were disclosed in U.S. Provisional patent then loaded on each macro carrier disk. application No. 61/667,039, filed Jul. 2, 2012, the disclosure 0598. Approximately 300-400 mg of a two-week-old sus of which is herein incorporated by reference in its entirety. pension culture is placed in an empty 60x15 mm petri dish 0605. The entire disclosure of each document cited (in and the residual liquid removed from the tissue with a pipette. cluding patents, patent applications, journal articles, For each transformation experiment, approximately 5-10 abstracts, manuals, books, or other disclosures) in the Back plates of tissue are normally bombarded. Membrane rupture ground of the Invention, Detailed Description, and Examples pressure is set at 1100 psi, and the chamber is evacuated to a is herein incorporated by reference in their entireties. vacuum of 28 inches mercury. The tissue is placed approxi 0606. The above examples are put forth so as to provide mately 3.5 inches away from the retaining screen and bom those of ordinary skill in the art with a complete disclosure barded three times. Following bombardment, the tissue can and description of how to make and use the Subject invention, be divided in half and placed back into liquid and cultured as and are not intended to limit the scope of what is regarded as described above. the invention. Efforts have been made to ensure accuracy with 0599 Five to seven days post bombardment the liquid respect to the numbers used (e.g. amounts, temperature, con media may be exchanged with fresh media, and eleven to centrations, etc.) but some experimental errors and deviations twelve days post-bombardment with fresh media containing should be allowed for. Unless otherwise indicated, parts are 50 mg/mL hygromycin. This selective media can be refreshed parts by weight, molecular weight is average molecular weekly. Seven to eight weeks post-bombardment, green, weight; temperature is in degrees centigrade; and pressure is transformed tissue may be observed growing from untrans at or near atmospheric. US 2014/0007292 A1 Jan. 2, 2014 88
SEQUENCE LISTING
<16O is NUMBER OF SEO ID NOS: 332
<210s, SEQ ID NO 1 &211s LENGTH: 816 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas chlororaphis
<4 OOs, SEQUENCE: 1 atgc.cgat.ca aggaagagct gagcc agcct caaagt catt catcgaact tacgacctg 6 O aaaagtgagc aaggaagttct ccdc.gc.cgct ttgacat coa actittgctgg caact tcgat 12 O Cagttcc.caa ctaagcgtgg toggctittgcg atcgacagot acctgctgga ttacagcgc.g 18O
Cccaa.gcaag gctgctgggt agatggcatt accgt.ctacg gtgacat citt tat cigcaa.g 24 O cagaattggg gcacct acac togc.ccggtc tittgcct acc togcagtacat gigacaccatt 3OO tccatt Cogc agcaggtgac acagacticgc agctat cagt tact aaggg acacaccaaa 360 acgttcacga C caatgtcag cqC caaatac agcgttggag gtag tattga catcgtcaac 42O gtcggttcgg at atct caat tatt cagt alacagtgaat cctggtctac tacgcagacg 48O ttcagdaata gcact caatt gactggtcct ggcacct tca togtgitatica ggttgttatg 54 O gtttatgcgc acaacgc.cac ttctg.cgggc aggcagaatg gtaatgcctt cqcct acaac 6OO aagaccaata citgtcggctic goggctggac ttgtactatt tdtctgc cat cacticagaac 660 agtacggt cattgtcgattic cagcaaggcc atcgc.gc.cgc tiggattggga tactgttcag 72 O cgcaatgtgt tatggagaa ctaca accca ggcagcaata gtgggcactt Cagtttcgac 78O tggagcgcct acaacgatcc ticatcgc.cgt tattga 816
<210s, SEQ ID NO 2 &211s LENGTH: 271 212. TYPE: PRT <213> ORGANISM: Pseudomonas chlororaphis <4 OOs, SEQUENCE: 2 Met Pro Ile Lys Glu Glu Lieu Ser Glin Pro Glin Ser His Ser Ile Glu 1. 5 1O 15 Lieu. Asp Asp Lieu Lys Ser Glu Glin Gly Ser Lieu. Arg Ala Ala Lieu. Thir 2O 25 3O Ser Asn Phe Ala Gly Asn Phe Asp Glin Phe Pro Thr Lys Arg Gly Gly 35 4 O 45 Phe Ala Ile Asp Ser Tyr Lieu. Lieu. Asp Tyr Ser Ala Pro Lys Glin Gly SO 55 6 O Cys Trp Val Asp Gly Ile Thr Val Tyr Gly Asp Ile Phe Ile Gly Lys 65 70 7s 8O Gln Asn Trp Gly Thr Tyr Thr Arg Pro Val Phe Ala Tyr Lieu. Glin Tyr 85 90 95 Met Asp Thir Ile Ser Ile Pro Glin Glin Val Thr Glin Thr Arg Ser Tyr 1OO 105 11 O Gln Lieu. Thir Lys Gly His Thr Lys Thr Phe Thir Thr Asn Val Ser Ala 115 12 O 125 Llys Tyr Ser Val Gly Gly Ser Ile Asp Ile Val Asn Val Gly Ser Asp 13 O 135 14 O
Ile Ser Ile Gly Phe Ser Asn Ser Glu Ser Trp Ser Thr Thr Glin Thr 145 150 155 160
US 2014/0007292 A1 Jan. 2, 2014 90
- Continued
Cys Trp Val Asp Gly Ile Thr Val Tyr Gly Asp Ile Phe Ile Gly Lys 65 70 7s 8O Gln Asn Trp Gly Thr Tyr Thr Arg Pro Val Phe Ala Tyr Lieu. Glin Tyr 85 90 95 Met Asp Thir Ile Ser Ile Pro Glin Glin Val Thr Glin Thr Arg Ser Tyr 1OO 105 11 O Gln Lieu. Thir Lys Gly His Thr Lys Thr Phe Thir Thr Ser Val Thr Ala 115 12 O 125 Llys Tyr Ser Val Gly Gly Ser Ile Gly Ile Val Asn Val Gly Ser Asp 13 O 135 14 O Ile Ser Val Gly Phe Ser Ser Ser Glu Ser Trp Ser Thr Thr Glin Thr 145 150 155 160 Phe Ser Glu Ser Thr Gln Leu Ala Gly Pro Gly Thr Phe Ile Val Tyr 1.65 17O 17s Glin Val Val Lieu Val Tyr Ala His Asn Ala Thir Ser Ala Gly Arg Glin 18O 185 19 O Asn Gly Asn Ala Phe Ala Tyr Asn Llys Thr Glin Thr Val Gly Ser Arg 195 2OO 2O5 Lieu. Asp Leu Tyr Tyr Lieu Ser Ala Ile Thr Glin Asn Ser Thr Val Ile 21 O 215 22O Val Glu Ser Ser Lys Ala Ile Ala Pro Lieu. Asp Trp Asp Thr Val Glin 225 23 O 235 24 O Arg Asn Val Lieu Met Glu Asn Tyr Asn Pro Ser Ser Asn. Ser Gly His 245 250 255 Phe Ser Phe Asp Trp Ser Ala Tyr Asn Asp Pro His Arg Arg Tyr 26 O 265 27 O
<210s, SEQ ID NO 5 &211s LENGTH: 816 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas entomophila <4 OOs, SEQUENCE: 5 atgacgat.ca aggaagagct gggc.ca.gc.ct caaagccatt catcgaact ggacgaggtg 6 O agcaaggagg cc.gcaagtac gcgggcc.gcg ttgactitcca acctgtctgg cc.gct tcgac 12 O
Cagtaccc.ga C caagaaggg cactittgcg atcgatggitt atttgctgga ctacagctica 18O
Cccaa.gcaag gttgctgggt ggacggitatic actgtctatg gcgatat ct a catcggcaa.g 24 O
Cagaactggg gCacttatac cc.gc.ccggtg tttgcct acc tacagtatgt ggaalaccatc 3OO tccatt coac agaatgtgac gaccaccctic agctato agc tigaccaaggg gcatacccgt. 360 t cct tcgaga C cagtgtcaa cqC caagtac agcgttggcg cca acataga tat citcaac 42O gtgggttcgg agattt CCaC cq99tttacc cqcagcgagt cctggtc. cac cacgcagtcg 48O ttcaccgata ccaccgagat gaaggggcca gggacgttcg tcatttacca ggtcgtgctg 54 O gtgtatgcgc acaacgc.cac Ctcggcaggg C9gcagaatg C caatgcctt cqcct acagc 6OO aaaacc cagg cagtgggctic gcgggtggac ttgtact act titcggc.cat tacccagcgc 660 aagcgggt catcgttc.cgt.c gag caatgcc gtcacgc.cgc tiggactggga tacggtgcaa. 72 O cgcaacgtgc tigatggaaaa ctaca accca ggcagtalaca gcgga cactt Cagct tcgac 78O tggagtgcct acaacgatcc ticatcgc.cgt tattga 816 US 2014/0007292 A1 Jan. 2, 2014 91
- Continued <210s, SEQ ID NO 6 &211s LENGTH: 271 212. TYPE: PRT <213> ORGANISM: Pseudomonas entomophila
<4 OOs, SEQUENCE: 6 Met Thr Ile Lys Glu Glu Lieu. Gly Glin Pro Glin Ser His Ser Ile Glu 1. 5 1O 15 Lieu. Asp Glu Val Ser Lys Glu Ala Ala Ser Thr Arg Ala Ala Lieu. Thir 2O 25 3O Ser Asn Lieu. Ser Gly Arg Phe Asp Glin Tyr Pro Thr Llys Lys Gly Asp 35 4 O 45 Phe Ala Ile Asp Gly Tyr Lieu. Lieu. Asp Tyr Ser Ser Pro Lys Glin Gly SO 55 6 O Cys Trp Val Asp Gly Ile Thr Val Tyr Gly Asp Ile Tyr Ile Gly Lys 65 70 7s 8O Gln Asn Trp Gly Thr Tyr Thr Arg Pro Val Phe Ala Tyr Lieu. Glin Tyr 85 90 95 Val Glu Thir Ile Ser Ile Pro Glin Asn Val Thir Thr Thr Lieu. Ser Tyr 1OO 105 11 O Gln Lieu. Thir Lys Gly His Thr Arg Ser Phe Glu Thir Ser Val Asn Ala 115 12 O 125 Llys Tyr Ser Val Gly Ala Asn. Ile Asp Ile Val Asn Val Gly Ser Glu 13 O 135 14 O Ile Ser Thr Gly Phe Thr Arg Ser Glu Ser Trp Ser Thr Thr Glin Ser 145 150 155 160 Phe Thr Asp Thir Thr Glu Met Lys Gly Pro Gly Thr Phe Val Ile Tyr 1.65 17O 17s Glin Val Val Lieu Val Tyr Ala His Asn Ala Thir Ser Ala Gly Arg Glin 18O 185 19 O Asn Ala Asn Ala Phe Ala Tyr Ser Lys Thr Glin Ala Val Gly Ser Arg 195 2OO 2O5 Val Asp Lieu. Tyr Tyr Lieu. Ser Ala Ile Thr Glin Arg Lys Arg Val Ile 21 O 215 22O Val Pro Ser Ser Asn Ala Val Thr Pro Leu Asp Trp Asp Thr Val Glin 225 23 O 235 24 O Arg Asn Val Lieu Met Glu Asn Tyr Asn Pro Gly Ser Asn. Ser Gly His 245 250 255 Phe Ser Phe Asp Trp Ser Ala Tyr Asn Asp Pro His Arg Arg Tyr 26 O 265 27 O
<210s, SEQ ID NO 7 &211s LENGTH: 720 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas putida <4 OO > SEQUENCE: 7 atgacaggct tcgagcgttt gttcacccgat gcgttc.cccg ttittaaacgg tt catacctg 6 O attgaaaggt acctgcticag cacggacgag titt catcctg gatgttggat agaaggtgaa 12 O accgt.ctacg gtgggtttgg gttt CCttica ggaaaaaaga agg tattgac cc.gc.ccggitt 18O titcgcc tact tcgactacgt giggcaccitat aaaac attaa gtgctggaga citgtgaaatt 24 O gatctgtc.cc gtgc.ca.gtgg gCatgagg to ttittgcac atgatgc.cga aggcttittct 3OO gcgc.cgagtg gaattgggct ggtaagcgta aagttcagat C tectcitc.cgg Ctgctctgcc 360 US 2014/0007292 A1 Jan. 2, 2014 92
- Continued gaagagtggc ggc.cgittatc atcggttggg cat accgtgc gcgtagcggg agctgaatgc 42O tatgtggcct accagttgaa actggtct at gcgcattggg taaaa.caggg catgcc.ca.g 48O tgct Ctgagc tigttcaaggt acago'ccgtg cgtgtgcaag gcgacaacaa aggcgttitt C 54 O titcCtttctt CC9tggccac agacctgatg tdgg taggac atggttctgga taacaccaaa 6OO gcqc caatat cacgacaggc gttatat cac ctdatatt ca atc.ttgctta toggcgcagog 660 ggtgacgc.cg gctggagttt taatgat cag gcggc.ca.gca accoct tcct gcaat attga 72 O
<210s, SEQ ID NO 8 &211s LENGTH: 239 212. TYPE: PRT <213> ORGANISM: Pseudomonas putida <4 OOs, SEQUENCE: 8 Met Thr Gly Phe Glu Arg Lieu Ser Pro Asp Ala Phe Pro Val Lieu. Asn 1. 5 1O 15 Gly Ser Tyr Lieu. Ile Glu Arg Tyr Lieu. Leu Ser Thr Asp Glu Phe His 2O 25 3O Pro Gly Cys Trp Ile Glu Gly Glu Thr Val Tyr Gly Gly Phe Gly Phe 35 4 O 45 Pro Ser Gly Lys Lys Llys Val Lieu. Thr Arg Pro Val Phe Ala Tyr Phe SO 55 6 O Asp Tyr Val Gly Thr Tyr Llys Thr Lieu. Ser Ala Gly Asp Cys Glu. Ile 65 70 7s 8O Asp Lieu. Ser Arg Ala Ser Gly His Glu Val Trp Phe Ala His Asp Ala 85 90 95 Glu Gly Phe Ser Ala Pro Ser Gly Ile Gly Lieu Val Ser Val Lys Ser 1OO 105 11 O Asp Lieu. Lieu. Ser Gly Cys Ser Ala Glu Glu Trp Arg Pro Lieu. Ser Ser 115 12 O 125 Val Gly His Thr Val Arg Val Ala Gly Ala Glu. Cys Tyr Val Ala Tyr 13 O 135 14 O Glin Lieu Lys Lieu Val Tyr Ala His Trp Wall Lys Glin Gly Asp Ala Glin 145 150 155 160 Cys Ser Glu Lieu. Phe Llys Val Glin Pro Val Arg Val Glin Gly Asp Asn 1.65 17O 17s Lys Gly Val Phe Phe Leu Ser Ser Val Ala Thr Asp Leu Met Trp Val 18O 185 19 O Gly His Gly Ser Asp Asn. Thir Lys Ala Pro Ile Ser Arg Glin Ala Lieu. 195 2OO 2O5 Tyr His Lieu. Ile Phe Asn Lieu Ala Tyr Gly Ala Ala Gly Asp Ala Gly 21 O 215 22O Trp Ser Phe Asin Asp Glin Ala Ala Ser Asn Arg Phe Lieu. Glin Tyr 225 23 O 235
<210s, SEQ ID NO 9 &211s LENGTH: 729 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas putida <4 OOs, SEQUENCE: 9 atgaggltd at attcaatgag catttgatgaatgaaatca gcc.gg taccC cctgaaaaga 6 O gggit ctitt.cg aaatcgagca gtacctgata ggtgat cagt to atgc.cgg ttgctgggtg 12 O