US0096.8873OB2

(12) United States Patent (10) Patent No.: US 9,688,730 B2 Cerf et al. (45) Date of Patent: Jun. 27, 2017

(54) INSECTICIDAL PROTEINS AND METHODS FOREIGN PATENT DOCUMENTS FOR THEIR USE WO WO 2011084622 A1 * T 2011 (71) Applicant: PIONEER HI BRED INTERNATIONAL INC, Johnston, IA OTHER PUBLICATIONS (US) Moura (2008) Novel Insights Into the Digestive Vacuole Biology of (72) Inventors: David C. Cerf, Palo Alto, CA (US); the Malarial Parasite Plasmodium Falciparum, Dissertation, Albert James J. English, San Ramon, CA Einstein College of Medicine.* (US); Carol A. Hendrick, Des Moines, Guo et al (2004), Proc. Natl. Acad. Sci. USA vol. 101 pp. 9205 921 O.* IA (US); Lu Liu, Palo Alto, CA (US); Ngo et al. In the Protein Folding Problem and Tertiary Structure Jarred K. Oral, San Carlos, CA (US); Prediction, 1994, Merz et al. (ed.), Birkhauser, Boston, MA, pp. Philip A. Patten, Menlo Park, CA 491-495. (US); Barbara A. Rosen, Mountain Szczesny et al (2011) PLoS ONE 6(6): e20349.* View, CA (US); Ute Schellenberger, Feris et al (2010) GenBank accession NZ ATLPO 1000030.* Palo Alto, CA (US); Ingrid A. Ness et al. Nature Biotechnology (2002) 20:1251-1255.* Udranszky, Mountain View, CA (US); Opota et al. PLoS Pathogens (2011) 7(9): 1-13.* Jun-Zhi Wei, Palo Alto, CA (US); Rose et al Nucleic Acids Research (1998) 26:1628-1635.* Genhai Zhu, San Jose, CA (US) Opota et al. PLoS Pathogens (2011) 7(9): 1-13, figure S1.* Selvapandiyan et al. Applied and Environmental Microbiology, (73) Assignee: PIONEER HI-BRED (2001) 67: 5855-5858.* INTERNATIONAL, INC., Johnston, Goral, et al., “Gaupsin, an Insecticidal and Fungicidal Preparation IA (US) from Strains of Pseudomonas aureofaciens”. Applied Biochemistry and Microbiology, vol. 35, No. 5, pp. 530-532 (1999). (*) Notice: Subject to any disclaimer, the term of this Li, et al., “Agrobacterium-mediated genetic transformation of patent is extended or adjusted under 35 Elymus breviaristatus with Pseudomonas pseudoalcaligenes insec U.S.C. 154(b) by 840 days. ticidal protein gene'. Plant Cell Tiss Organ Cult, vol. 89, pp. 159-168 (2007). (21) Appl. No.: 13/792,861 Pechy-Tarr, et al. ; “Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated Stains of Pseudomonas (22) Filed: Mar. 11, 2013 flueorescens”. Environmental Microbiology, vol. 10, pp. 2368-2386 (2008). (65) Prior Publication Data Peix, et al "Reclassification of Pseudomonas aurantiaca as a synonym of Pseudomonas chlororaphis and proposal of three US 2014/OOO7292 A1 Jan. 2, 2014 subspecies, P. chlororaphis subsp. chlororaphis subsp. nov., P. chlororaphis subsp. aureofaciens Subsp. nov., comb. nov. and P chlororaphis Subsp. aurantiaca Subsp. nov... comb. nov.” Interna Related U.S. Application Data tional Journal of Systematic and Evolutionary Microbiology, vol. (60) Provisional application No. 61/667,039, filed on Jul. 57, pp. 1286-1290 (2007). 2, 2012. (Continued) (51) Int. Cl. Primary Examiner — David H Kruse C07K I4/2 (2006.01) (74) Attorney, Agent, or Firm — Pioneer Hi Bred Int'l, AOIN 43/50 (2006.01) Inc. A6 IK 45/06 (2006.01) (57) ABSTRACT C07K 6/12 (2006.01) GOIN 33/68 (2006.01) Compositions and methods for controlling pests are pro (52) U.S. Cl. vided. The methods involve transforming organisms with a CPC ...... C07K 14/21 (2013.01); A0IN 43/50 nucleic acid sequence encoding an insecticidal protein. In (2013.01); A61K 45/06 (2013.01); C07K particular, the nucleic acid sequences are useful for prepar I6/1214 (2013.01); G0IN 33/68 (2013.01); ing plants and microorganisms that possess insecticidal C07K 2319/24 (2013.01) activity. Thus, transformed bacteria, plants, plant cells, plant (58) Field of Classification Search tissues and seeds are provided. Compositions are insecti None cidal nucleic acids and proteins of bacterial species. The See application file for complete search history. sequences find use in the construction of expression vectors for Subsequent transformation into organisms of interest, as (56) References Cited probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in U.S. PATENT DOCUMENTS controlling, inhibiting growth or killing lepidopteran, cole opteran, dipteran, fungal, hemipteran, and nematode pest 5,965,428 A * 10/1999 Gilmer et al...... 435/252.3 6,172,184 B1 1/2001 Collmer et al. populations and for producing compositions with insecti 2006, O168683 A1 7/2006 Hey et al. cidal activity. 2009 OO68159 A1 3/2009 Baum et al. 2012/0311745 A1 12/2012 Meade et al. 14 Claims, 7 Drawing Sheets US 9,688,730 B2 Page 2

(56) References Cited

OTHER PUBLICATIONS Sezen, et al. “Study of the bacterial flora as a biological control agent of Agelastica alni L. (Coleoptera: Chrysomelidae).” Biologia Bratislava, vol. 59, pp. 327-331 (2004). Vodovar, et al., “Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudominas entomophila', Nature Biotechnology, vol. 24. No. 6, pp. 673-679 (2006). Opota, et al., “Monalysin, a Novel B-Pore-Forming Toxin from the Drosophila Pathogen Pseudomonas entomphila, Contributes to Host Intestinal Damage and Lethality”. PLoS Pathogens, vol. 7 (9), pp. 1-13 (2011). GenBank Accession No. ABQ77224.1. GenBank Accession No. ABQ77225.1. International Search Report for International Application No. PCT/ US2013/047760. Written Opinion for International Application No. PCT/US2013/ O47760. * cited by examiner U.S. Patent Jun. 27, 2017 Sheet 1 of 7 US 9,688,730 B2

PIP-1A (1) PSEEN3174 (1) PIP-1B (1) AECFG-592740 (1) Pput 1063 (1) ---EGFERLSPEAEPVLNGSYL Pput 1064 (1) YSMSDLMNEISRPLK

BBBBBBBBBBBBBBBBB BBBBBBBBBBBB

PIP-1A (51) PSEEN3174 (51) PIP-1B (51) AECFG-592740 (34) Pput 1063 (21) ISRYLESTDEFH--PGCW ESEYGGFGFPSGs: Swat. Pput 1064 (25) IsoyIsGDQLH---AGC TTYGERCNYD

BBB 10 PIP-1A (99) PSEEN3174 (99) PIP-1B (99) T: AECEG-592740 (84) TREATKYHVE Pput 1063 (69) TYKTLSAGDCEIDLSRAS GEEVWSAH Pput 1064 (72) TERSNVTEHEREVVSCEGFS: BBBBBBBBBBB 151 200 PP-1A (149) PSEEN3174 (149) PIP-1B (149) AECFG-592740 (133) Pput 1063 (118) AHWvKobACSELK Pput 1064 (122) -VLSGYVP BBB 250 PIP-1A (199) PSEEN3174 (199) PIP-1B (199) AECFG-592740 (182) SS Pput 1063 (167) Pput 1064 (171) xks

BBBBBBBB 251

PIP-1A (243) PSEEN3174 (243) PIP-1B (243) AECFG-592740 (227) Pput 1063 (211) : Pput 1064 (221) Si U.S. Patent Jun. 27, 2017 Sheet 2 of 7 US 9,688,730 B2

Fig. 2

Saturated mutagenesis

3188F Reverse primer -> 4

--> -H Sphi Forwar primer BamHt 3188R

- I Sewing PCR

PCR using 3188FF3188R to recover full length and clone to expression vector

Transform to E. coli host

Pick 96 or more colonies for sequencing to recover variants U.S. Patent Jun. 27, 2017 Sheet 3 of 7 US 9,688,730 B2

Fig. 3A

6 O AAACCCAAAG ATGTTTGAAC TGAAGAGTTT GATCATGGCT CAGATTGAAC GCTGGCGGCA GCT CAGATTGAAC GCTGGCGGCA

12 O GGCCTAACAC ATGCAAGTCG AGCGGTAGAG AGAAGCTTGC TCTCTTGAG AGCGGCGGAC GGCCTAACAC ATGCAAGTCG AGCGGATGAC GGGAGCTTGC CCTTGATTC AGCGGCGGAC

18O GGGTGAGTAA TGCCTAGGAA TCTGCCTGGT AGTGGGGGAT AACGTCCGGA AACGGACGCT GGGTGAGTAA TGCCTAGGAA TCTCCCTGGT AGTGGGGGAC AACGTTTCGA AAGGAACGCT

24 O AATACCGCAT ACGTCCTACG GGAGAAAGCA GGGGACCTTC GGGCCTTGCG CTATCAGATG AATACCGCAT ACGTCCTACG GGAGAAAGCA GGGGACCTTC GGGCCTTGCG CTATCARATG

3OO AGCCTAGGTC GGATTAGCTA GTTGGTGAGG TAATGGCTCA CCAAGGCGAC GATCCGTAAC AGCCTAGGTC GGATTAGCTA GTTGGKGGGG TAATGGCTCA CCAAGGCGAC GATCCGTAAC

360 TGGTCTGAGA GGATGATCAG TCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG TGGTYTGAGA GGATGATCAG TCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG

420 GCAGCAGTGG GGAATATTGG ACAATGGGCG AAAGCCTGAT CCAGCCATGC CGCGTGTGTG GCAGCAGTGG GGAATATTGG ACAATGGGCG AAAGCCTGAT CCAGCCATGC CGCGTGTGTG

48 O AAGAAGGTCT TCGGATTGTA AAGCACTTTA AGTTGGGAGG AAGGGTACTT ACCTAATACG AARAAGGTCT TCGGATTGTA AAGCACTTTA AGTTGGGAGG AAGGGCAGTA AGTTAATACC

54 O TGAGTATTTT GACGTTACCG ACAGAATAAG CACCGGCTAA CTCTCTCCCA GCAGCCGCGG TTGCTGTTTT GACGTTACCG ACAGAATAAG CACCGGCTAA CTCTGTGCCA GCAGCCGCGG

6OO TAATACAGAG GG GCAAGCG TTAATCGGAA TTACTGGGCG TAAAGCGCGC GTAGGTGGTT TAATACAGAG GG GCAAGCG TTAATCGGAA TTACTGGGCG TAAAGCGCGC GTAGGTGGTT U.S. Patent Jun. 27, 2017 Sheet 4 of 7 US 9,688,730 B2

Fig. 3B

66O CGTTAAGTTG GATGTGAAAT CCCCGGGCTC AACCTGGGAA CTGCATCCAA AACTGGCGAG CGTTAAGTTG GATGTGAAAG CCCCGGGCTC AACCTGGGAA CTG CATCCAA AACTGGCGAG

720 CTAGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG CTAGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG

78O AAGGAACACC AGTGGCGAAG GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC AAGGAACACC AGTGGCGAAG GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC

84 O GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC

9 OO CGTTGGGAGC CTTGAGCTCT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG CGTTGGAATC CTTGAGATTT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG

96.O TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT

1 O2O GTGGTTTAAT TCGAAGCAAC GCGAAGAACC TTACCAGGCC TTGACATCCA ATGAACTTTC GTGGTTTAAT TCGAAGCAAC GCGAAGAACC TTACCAGGCC TTGACATGCA GAGAACTTTC

1 O8 O CAGAGATGGA TTGGTGCCTT CGGGAACATT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CAGAGATGGA TTGGTGCCTT CGGGAACTCT GACACAGGTG CTGCATGGCT GTCGTCAGCT

1140 CGTGTCGTGA GATGTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA CGTGTCGTGA GATGTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA

12 OO GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT U.S. Patent Jun. 27, 2017 Sheet S of 7 US 9,688,730 B2

Fig. 3C

1260 GACGTCAAGT CATCATGGCC CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC GACGTCAAGT CATCATGGCC CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC

132O AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA ATCCCATAAA ACCGATCGTA GTCCGGATCG AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA ATCTCACAAA ACCGATCGTA GTCCGGATCG

1380 CAGTCTGCAA CTCGACTGCG TGAAGTCGGA ATCGCTAGTA ATCGCGAATC AGAATGTCGC CAGTCTGCAA CTCGACTGCG TGAAGTCGGA ATCGCTAGTA ATCGCAAATC AGAATGTTGC

1440 GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC

1500 CAGAAGTAGC TAGTCTAACC TTCGGGAGGA CGGTT ACCAC GGTGTGATTC ATGACTGGGG CAGAAGTAGC TAGTCTAACC TTCGGGGGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG

15 6.O TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GATCACCTCC TTAATCGACG TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GAT CACCTCC

162O ACATCAGCTG CTTCATAAGC TCCCACACGA ATTGCTTGAT TCATTGAAGA AGA CGATTGG

168O GTCTGTAGCT CAGTTGGTTA GAGCGCACCC CTGATAAGGG TGAGGTCGGC AGTTCGAATC

17 () () TGCCCAGACC CACCAATTAC SEO ID NO : 21 6 P. chlororaphis SS44C4 16S-rDNA SEO ID NO: 21 P. entomophila-L48 16S-rDNA U.S. Patent Jun. 27, 2017 Sheet 6 of 7 US 9,688,730 B2

Fig. 4

Plate 1

**•

U.S. Patent Jun. 27, 2017 Sheet 7 Of 7 US 9,688,730 B2

Fig. 5

1 5 O PIP-1A (1) MPIKEELSQPQSHSIELD LRAITSNFGNFDos PTKRGSA PIP-1B (1) M3:IKEELNQPQSHSIELD : PIP-1C (1) KEELGOPOSHSIELD ...... : PSEEN3174 (1) SIKEELSQPQSHSIELDESKEASTRASITSNSGRFDQSPTKGDEA

PIP-1A (51) IDSYLED KGCWVDGITVYGDISIGKQNWGTYTRPVFAYLOY PIP-1B (51) KGCWVDGITVYGDISIGKQNWGTYTRPVFAYLOY PIP-1C (51) LLDSSPKKGCWVDGITVYGDISIGKQNWGTYTRPVFAYIQY. PSEEN3174 (51) IDGYLLDssPKGCWVDGITVYGDISIGKQNWGTYTRPVFAYLOYSTI

PIP-1A (101) PIP-1B (101) PIP-1C (101) PSEEN3174 (101)

PIP-1A (151) PIP-1B (151) Tr.... PIP-1c (151) is S S. VYAHNATSAGONNAFAYS PSEEN3174 (151) YQSVVYAHNATSAGSONNAFAYS

250 PIP-1A (201) SSRDIYYLSAITQ rvissKAirpLDWDrvoRNVIMSNYNipIV.SSKAEPIDWDTVQRNVLMSNYNP PIP-1B (201) E. i s PIP-1c (201) KTVDSRDIYYLSAITQ PSEEN3174 (201) KTAVSRDIYYLSAITORKRVIVPSSSASTPIDWDTVQRNVLMENYNP 251 271 PIP-1A (251) SNSGHFSFDWSA PIP-1B (251) SSNSGHFSFDWSA PIP-1C (251) ESNNGHEREDWSAY PSEEN3174 (251) SNSGHESEDWSAYSPHRRY US 9,688,730 B2 1. 2 INSECTICIDAL PROTEINS AND METHODS which raises the need to identify alternative biological FOR THEIR USE control agents for pest control. Accordingly, there remains a need for new pesticidal CROSS REFERENCE proteins with different ranges of insecticidal activity against insect pests, e.g., insecticidal proteins which are active This utility application claims the benefit U.S. Provisional against a variety of insects in the order Lepidoptera and the Application No. 61/667,039, filed Jul. 2, 2012, which is order Hemiptera including but not limited to species belong incorporated herein by reference in its entirety. ing to the family Pentatomidae, the family Plataspidae and the family Cydnidae. In addition, there remains a need for REFERENCE TO SEQUENCE LISTING 10 biopesticides having activity against a variety of insect pests SUBMITTED ELECTRONICALLY that have developed resistance to existing pesticides. The official copy of the sequence listing is Submitted SUMMARY OF THE INVENTION electronically via EFS-Web as an ASCII formatted sequence 15 Compositions and methods for conferring pesticidal listing with a file named "4208 sequence listing..txt cre activity to bacteria, plants, plant cells, tissues and seeds are ated on Mar. 4, 2013, and having a size of 471 kilobytes and provided. Compositions include nucleic acid molecules is filed concurrently with the specification. The sequence encoding sequences for pesticidal and insecticidal polypep listing contained in this ASCII formatted document is part of tides, vectors comprising those nucleic acid molecules, and the specification and is herein incorporated by reference in host cells comprising the vectors. Compositions also include its entirety. the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in FIELD OF THE INVENTION DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and This disclosure relates to the field of molecular biology. 25 plants. The nucleotide or amino acid sequences may be Provided are novel genes that encode pesticidal proteins. synthetic sequences that have been designed for expression These pesticidal proteins and the nucleic acid sequences that in an organism including, but not limited to, a microorgan encode them are useful in preparing pesticidal formulations ism or a plant. Compositions also comprise transformed and in the production of transgenic pest-resistant plants. bacteria, plants, plant cells, tissues and seeds. 30 In particular, isolated or recombinant nucleic acid mol BACKGROUND OF THE INVENTION ecules are provided encoding Pseudomonas Insecticidal Protein-1 (PIP-1) polypeptides including amino acid substi Biological control of insect pests of agricultural signifi tutions, amino acid deletions, amino acid insertions, and cance using a microbial agent, Such as fungi, bacteria or fragments thereof, and combinations thereof. Additionally, another species of insect affords an environmentally friendly 35 amino acid sequences corresponding to the PIP-1 polypep and commercially attractive alternative to synthetic chemi tides are encompassed. Provided are an isolated or recom cal pesticides. Generally speaking, the use of biopesticides binant nucleic acid molecule capable of encoding a PIP-1 presents a lower risk of pollution and environmental haZ polypeptide of SEQID NO: 2, 101, 102, 103, 104,105,106, ards, and biopesticides provide greater target specificity than 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, is characteristic of traditional broad-spectrum chemical 40 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, insecticides. In addition, biopesticides often cost less to 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, produce and thus improve economic yield for a wide variety 143, 144, 145, 146, 147,148, 149, 150, 151, 204, 206, 208, of crops. 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, 252, Certain species of microorganisms of the genus Bacillus 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, are known to possess pesticidal activity against a range of 45 265, 266, 267,268, 269,298, 299, 300, 301,302,303, 304, insect pests including Lepidoptera, Diptera, Coleoptera, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacil 317, 318, 319, 320, 321, 322, 323,324, and 325 as well as lus popilliae are among the most Successful biocontrol amino acid Substitutions, amino acid deletions, amino acid agents discovered to date. Insect pathogenicity has also been insertions, and fragments thereof, and combinations thereof. attributed to strains of B. larvae, B. lentimorbus, B. spha 50 In some embodiments exemplary PIP-1 polypeptides com ericus and B. cereus. Microbial insecticides, particularly prise a sequence set forth in of SEQID NO: 2, 101, 102, 103, those obtained from Bacillus strains, have played an impor 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, tant role in agriculture as alternatives to chemical pest 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, control. 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, Crop plants have been developed with enhanced insect 55 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, resistance by genetically engineering crop plants to produce 204, 206, 208,211, 212, 213, 214, 245, 246, 247, 248, 249, pesticidal proteins from Bacillus. For example, corn and 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260,261, cotton plants have been genetically engineered to produce 262, 263,264, 265, 266, 267,268, and 269 as well as amino pesticidal proteins isolated from strains of Bt. These geneti acid substitutions, amino acid deletions, amino acid inser cally engineered crops are now widely used in agriculture 60 tions, and fragments thereof, and combinations thereof. and have provided the farmer with an environmentally Also provided are nucleic acid sequences set forth in SEQ friendly alternative to traditional insect-control methods. ID NO: 1, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, While they have proven to be very successful commercially, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, these genetically engineered, insect-resistant crop plants 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, provide resistance to only a narrow range of the economi 65 186, 197, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, cally important insect pests. In some cases, insects can 198, 199, 200, 201, 202, 203, 205, 207, 220, 221, 222, 223, develop resistance to different insecticidal compounds, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, US 9,688,730 B2 3 4 236, 237,238,239, 240, 241, 242, 243, 244, 270, 271, 272, 5. The recombinant nucleic acid molecule of embodiment 273, 274, 275,276, 277,278, 279, 280, 281, 282,283, 284, 1, 2, 3 or 4, wherein the PIP-1 polypeptide has insecticidal 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, activity against an insect pest in the order Lepidoptera. and 297 as well as variants and fragments thereof encoding 6. The recombinant nucleic acid molecule of embodiment PIP-1 polypeptides. 5 1, 2, 3, 4 or 5, wherein the nucleic acid molecule is from a In some embodiments exemplary nucleic acid molecules Pseudomonas chlororaphis strain. comprise a sequence set forth in SEQID NO: 152, 153, 154, 7. The recombinant nucleic acid molecule of embodiment 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 1, 2, 3, 4, 5 or 6, wherein the Pseudomonas chlororaphis 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, strain comprises a 16S ribosomal DNA having at least about 10 96.9% identity to SEQ ID NO: 216. 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 190, 8. The recombinant nucleic acid molecule of embodiment 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 1, 2, 3, 4, 5, 6 or 7 wherein the Pseudomonas chlororaphis 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, 228, strain is SS44C4 deposited under accession it NRRLB 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, 240, 50613. 241, 242, 243, and 244 as well as variants and fragments 15 9. The recombinant nucleic acid molecule of embodiment thereof encoding PIP-1 polypeptides, as well as variants and 1, 2, 3, 4, 5, 6, 7, or 8 wherein the PIP-1 polypeptide fragments thereof that encode PIP-1 polypeptides. Nucleic comprises an amino acid motif as represented by positions acid sequences that are complementary to a nucleic acid 171-183 of SEQ ID NO: 213. sequence of the embodiments or that hybridize to a sequence 10. The recombinant nucleic acid molecule of embodi of the embodiments are also encompassed. ment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the PIP-1 polypeptide Methods are provided for producing the polypeptides and further comprises any one or more amino acid motifs as for using those polypeptides for controlling, inhibiting represented by positions 149-159 of SEQ ID NO: 213 and growth or killing a Lepidopteran, Coleopteran, nematode, positions 64-79 of SEQ ID NO: 213. fungi, Hemipteran and/or Dipteran pests. The transgenic 11. The recombinant nucleic acid molecule of embodi plants of the embodiments express one or more of the 25 ment 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein the PIP-1 pesticidal sequences disclosed herein. In various embodi polypeptide comprises a polypeptide having at least 80% ments, the transgenic plant further comprises one or more identity to the amino acid sequence of SEQ ID NO: 2. additional genes for insect resistance, for example, one or 12. The recombinant nucleic acid molecule of embodi more additional genes for controlling coleopteran, lepi ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, wherein the PIP-1 dopteran, hemipteran or nematode pests. It will be under 30 polypeptide further comprises any one or more amino acid stood by one of skill in the art that the transgenic plant may motifs as represented by positions 64-79 of SEQ ID NO: comprise any gene imparting an agronomic trait of interest. 213, positions 149-159 of SEQID NO: 213, and positions 171-183 of SEQ ID NO: 213. Methods for detecting the nucleic acids and polypeptides 13. The recombinant nucleic acid molecule of embodi of the embodiments in a sample are also included. A kit for 35 ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the PIP-1 detecting the presence of a PIP-1 polypeptide or detecting polypeptide comprises an amino acid sequence of SEQ ID the presence of a nucleotide sequence encoding a PIP-1 NO: 211, wherein polypeptide in a sample is provided. A kit for detecting the Xaa at position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly presence of nucleotide sequence encoding a PIP-1 polypep or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at tide may comprise a nucleic acid probe that comprises at 40 position 20 is Leu or Val: Xaa at position 21 is Lys, Ser or least 20 contiguous nucleotides of the nucleotide sequence ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position encoding the PIP-1 polypeptide or a complement thereof. A 24 is Glin or Ala; Xaa at position 25 is Gly or Ala Xaa at kit for detecting the presence of a PIP-1 polypeptide may position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or comprise an antibody that specifically binds to the PIP-1 Ala; Xaa at position 30 is Ala or Ile: Xaa at position 35 is polypeptide. The kit is provided along with all reagents and 45 Phe or Leu; Xaa at position 36 is Ala, Ser or Val: Xaa at control samples necessary for carrying out a method for position 38 is Asn, Arg or Ser; Xaa at position 42 is Phe or detecting the intended agent, as well as instructions for use. Tyr, Xaa at position 46 is Arg, Lys or His; Xaa at position The compositions and methods of the embodiments are 48 is Gly or Asp; Xaa at position 49 is Phe or Tyr; Xaa at useful for the production of organisms with enhanced pest position 53 is Ser or Gly; Xaa at position 58 is Tyr or Phe: resistance or tolerance. These organisms and compositions 50 Xaa at position 60 is Ala or Ser; Xaa at position 63 is Gln comprising the organisms are desirable for agricultural or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position 97 purposes. The compositions of the embodiments are also is Met or Val: Xaa at position 98 is Asp or Glu; Xaa at useful for generating altered or improved proteins that have position 105 is Glin or ASn; Xaa at position 107 is Thr or Ile: pesticidal activity or for detecting the presence of PIP-1 Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg polypeptides or nucleic acids in products or organisms. 55 or Leu; Xaa at position 120 is Lys, Arg or Glin; Xaa at The following embodiments are encompassed by the present position 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; disclosure. Xaa at position 125 is Asn or Ser; Xaa at position 127 is Ser, 1. A recombinant nucleic acid molecule encoding a PIP-1 Asn. Thr or Lys: Xaa at position 134 is Gly or Ala; Xaa at polypeptide. position 135 is Ser, Asn or Lys: Xaa at position 137 is Asp 2. The recombinant nucleic acid molecule of embodiment 60 or Gly: Xaa at position 141 is Val or Ile: Xaa at position 142 1, wherein the PIP-1 polypeptide is orally active. is Gly or Asp; Xaa at position 144 is Asp or Glu, Xaa at 3. The recombinant nucleic acid molecule of embodiment position 147 is Ile, Thr or Val: Xaa at position 150 is Ser or 1 or 2, wherein the PIP-1 polypeptide has insecticidal Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position activity against an insect pest in the order Hemiptera. 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at 4. The recombinant nucleic acid molecule of embodiment 65 position 163 is Asn, Asp or Glu; Xaa at position 164 is Ser 1, 2 or 3, wherein the PIP-1 polypeptide has insecticidal or Thr; Xaa at position 166 is Glin or Glu, Xaa at position activity against an insect pest in the family Pentatomidae. 167 is Leu or Met; Xaa at position 168 is Thr, Lys or Ala; US 9,688,730 B2 5 6 Xaa at position 174 is Ile, Val or Met; Xaa at position 175 position 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, is Val or Ile: Xaa at position 180 is Met or Leu; Xaa at Ser or Lys; Xaa at position 179 is Val, Phe, Thr, Ile, Cys, position 191 is Arg or Lys; Xaa at position 194 is Gly or Ala; Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, Leu, Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 5 Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at Lys: Xaa at position 182 is Tyr, Phe, Met or His: Xaa at position 220 is ASnor Arg; Xaa at position 221 is Seror Lys; position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Xaa at position 222 is Thr or Arg; Xaa at position 226 is Asp, Ser, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at Pro or Glu; Xaa at position 228 is Seror Gly: Xaa at position position 194 is Gly or Ala; Xaa at position 195 is Asn or Tyr; 229 is Lys or Asn; Xaa at position 231 is Ile or Val: Xaa at 10 Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn position 232 is Ala, Thr or Glu; and Xaa at position 251 is or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 Gly, Ser or Glu, Xaa at position 254 is Ser or Asn; Xaa at is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at position 258 is Seror Arg; Xaa at position 265 is Asn or Asp; position 213 is Tyr or Phe: Xaa at position 220 is Asn or Arg; and Xaa at position 266 is Asp or Asn; and wherein, 1 to 28 Xaa at position 221 is Ser or Lys; Xaa at position 222 is Thr amino acids are optionally deleted from the N-terminus of 15 or Arg; Xaa at position 226 is Asp, Pro or Glu, Xaa at the polypeptide. position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; 14. The recombinant nucleic acid molecule of embodi Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the PIP-1 Thr or Glu, Xaa at position 240 is Gln, Arg, Ala, Val, Glu, polypeptide comprises an amino acid sequence of SEQ ID Met, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu: NO: 212, wherein Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Xaa at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: Tyr, Met, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly at position 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 20 is Leu or Val: Xaa at position 21 is Lys, Ser or position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position 25 Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp 24 is Gln or Ala; Xaa at position 25 is Gly or Ala; Xaa at or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Glu, Leu, position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Ile, Gln, Tyr Ala; Xaa at position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Met, Asp, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position 30 or Lys, Xaa at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, 36 is Ala, Ser or Val: Xaa at position 38 is Asn, Arg or Ser; His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr Xaa at position 42 is Phe or Tyr; Xaa at position 43 is Pro, or Cys: Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser, Met, Gly, Gln, Ser, Thr, Arg, Val, Leu, LyS, Asp, Ala, ASn, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala; Xaa at position 249 Phe, Trp, Glu or Cys; Xaa at position 46 is Arg, Lys or His: is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Xaa at position 48 is Gly or Asp; Xaa at position 49 is Phe, 35 Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 Tyr or Leu; Xaa at position 53 is Ser or Gly; Xaa at position is Gly, Ser or Glu; Xaa at position 254 is Ser or ASn; Xaa at 58 is Tyr or Phe: Xaa at position 60 is Ala or Ser; Xaa at position 258 is Ser or Arg; Xaa at position 259 is Phe, Trp, position 63 is Glin or Lys: Xaa at position 66 is Trp, Tyr, Phe, Tyr, Cys, Met, Leu, Val, Ile or His: Xaa at position 265 is Arg, Lys, His, Ile, Val or Ser; Xaa at position 77 is Phe or ASn or Asp, and Xaa at position 266 is Asp or ASn; and Tyr; Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, 40 wherein, 1 to 28 amino acids are optionally deleted from the Ala, Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, N-terminus of the polypeptide. Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa at 15. The recombinant nucleic acid molecule of embodi position 97 is Met or Val; Xaa at position 98 is Asp or Glu; ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the PIP-1 Xaa at position 105 is Gln or Asn; Xaa at position 107 is Thr polypeptide comprises an amino acid sequence of (SEQ ID or Ile: Xaa at position 108 is Glin or Thr; Xaa at position 110 45 NO: 213), wherein is Arg or Leu, Xaa at position 120 is Lys, Arg or Glin; Xaa Xaa at position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, at position 121 is Thr or Ser; Xaa at position 123 is Thr or Thr, Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp Glu; Xaa at position 125 is Asn or Ser; Xaa at position 127 or Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa is Ser, Asn. Thr or Lys: Xaa at position 134 is Gly or Ala; at position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, Xaa at position 135 is Ser, Asn or Lys; Xaa at position 137 50 Val, Ile or Met; Xaa at position 21 is Lys, Ser, Asn, Arg, Thr 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 position 24 is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly Glu; Xaa at position 147 is Ile, Thr or Val: Xaa at position or Ala; Xaa at position 26 is Ser, Asn. Thr or Glin; Xaa at 150 is Seror Thr; Xaa at position 151 is Asn, Arg or Ser; Xaa position 27 is Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at at position 160 is Thr or Ser; Xaa at position 162 is Ser or 55 position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, Thr; Xaa at position 163 is Asn, Asp or Glu, Xaa at position Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala, Ile, 164 is Ser or Thr; Xaa at position 166 is Glin or Glu; Xaa at Leu, Val or Met; Xaa at position 35 is Phe, Leu, Ile, Val or position 167 is Leu or Met; Xaa at position 168 is Thr, Lys Met; Xaa at position 36 is Ala, Ser, Thr, Val, Ile or Leu; Xaa or Ala; Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn. at position 38 is Asn, Arg, Ser, Gln, Lys or Thr; Xaa at Asp, Ser or Ala; Xaa at position 172 is Thr, Gly. His, Phe, 60 position 42 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, Val, Leu, Xaa at position 173 is Phe, Gly. His, Leu, Ala, Arg, Asn. Cys, LyS, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at position 46 Lys, Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, is Arg, Lys or His; Xaa at position 48 is Gly, Asp, Ala or Glu; Gly, Arg, ASn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa Ser. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, 65 at position 53 is Ser, Gly, Ala or Thr; Xaa at position 58 is Lys, Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Thr; Xaa or Cys; Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at at position 63 is Gln, Lys, ASn or Arg; Xaa at position 66 is US 9,688,730 B2 7 8 Trp, Tyr, Phe, Arg, Lys. His, Ile, Val or Ser; Xaa at position Trp, Ile, Asp, Gly or Ala; Xaa at position 249 is ASn, LyS, 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at position 89 Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 is Gly, or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Ser. Thr, Ala, Asp or Glu; Xaa at position 254 is Ser, Asn. Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, Val, Thr or Gln; Xaa at position 258 is Ser, Arg, Thr or Lys: Xaa Leu or Ile: Xaa at position 98 is Asp or Glu; Xaa at position at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, Leu His; Xaa at position 265 is Asn, Asp, Gln or Glu; and Xaa or Val: Xaa at position 108 is Gln, Thr, Ser or Asn; Xaa at 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 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thr or 10 of the polypeptide. Ser; Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at 16. The recombinant nucleic acid molecule of embodi position 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 wherein Ser, Asn. Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is the recombinant nucleic acid molecule comprises a poly Gly or Ala; Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or nucleotide of SEQ ID NO: 1, a fragment or a complement Lys; Xaa at position 137 is Asp, Gly, Glu or Ala; Xaa at 15 thereof. position 141 is Val, Ile or Leu; Xaa at position 142 is Gly, 17. The recombinant nucleic acid molecule of embodi Asp, Ala or Glu, Xaa at position 144 is Asp or Glu, Xaa at ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, wherein position 147 is Ile, Thr, Val, Leu, Met or Ser; Xaa at position the PIP-1 polypeptide comprises an amino acid sequence of 150 is Ser or Thr; Xaa at position 151 is Asn, Arg, Ser, Gln, SEQ ID NO: 2 or a fragment thereof. Lys or Thr; Xaa at position 160 is Thr or Ser; Xaa at position 18. The recombinant nucleic acid molecule of embodi 162 is Ser or Thr; Xaa at position 163 is Asn., Asp, Glu or ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, wherein Gln; Xaa at position 164 is Ser or Thr; Xaa at position 166 the recombinant nucleic acid molecule hybridizes under is Gln, Glu, Asp or Asn; Xaa at position 167 is Leu, Met, Ile, stringent conditions to a polynucleotide of SEQ ID NO: 1. Val: Xaa at position 168 is Thr, Lys, Ala, Ser, Arg or Gly; 19. The recombinant nucleic acid molecule of embodi Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn, Asp, Ser 25 ment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, wherein or Ala; Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, the recombinant nucleic acid molecule comprises a poly Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaa at nucleotide of SEQ ID NO: 1. position 173 is Phe, Gly. His, Leu, Ala, Arg, ASn, Cys, LyS, 20. A plant or progeny thereof, comprising the recombi Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, Gly, nant nucleic acid molecule of embodiment 1, 2, 3, 4, 5, 6, 7, Arg, Asn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser, 30 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, 21. A plant or progeny thereof stably transformed with the Leu or Met; Xaaat position 176 is Tyr, Met, Phe, Leu or Cys: recombinant nucleic acid molecule of embodiment 1, 2, 3, 4, Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, Seror Lys: 22. The plant of embodiment 20 or 21, wherein the plant Xaa at position 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, 35 is a monocotyledon. Ala or Glin; Xaa at position 180 is Met, Leu: Pro, Trp, Asn. 23. The plant of embodiment 20 or 21, wherein the plant Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; Xaa at position is a dicotyledon. 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or Lys; Xaa at 24. The plant of embodiment 20 or 21, wherein the plant position 182 is Tyr, Phe, Met or His; Xaa at position 183 is is selected from barley, corn, oat, rice, rye, Sorghum, turf Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, Gln or Leu: 40 grass, Sugarcane, wheat, alfalfa, banana, broccoli, bean, Xaa at position 191 is Arg or Lys; Xaa at position 194 is Gly cabbage, canola, carrot, cassava, cauliflower, celery, citrus, or Ala; Xaa at position 195 is Asn. Tyr, Gln or Trp; Xaa at cotton, a cucurbit, eucalyptus, flax, garlic, grape, onion, position 200 is Asn. Ser. Thr or Glin; Xaa at position 203 is lettuce, pea, peanut, pepper, potato, poplar, pine, Sunflower, Asin or Glin; Xaa at position 204 is Thr, Ala, Ser or Gly; Xaa safflower, soybean, Strawberry, Sugar beet, Sweet potato, at position 206 is Gly, Asp, Ala or Glu; Xaa at position 209 45 tobacco, tomato ornamental, shrub, nut, chickpea, pigeon is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: Xaa pea, millets, hops, and pasture grass plant cells. at position 220 is ASn, Arg, Gln or Lys, Xaa at position 221 25. The plant of embodiment 20, 21, 22, 23 or 24, further is Ser, Lys, Thr or Arg; Xaa at position 222 is Thr, Arg, Ser comprising one or more additional transgenic traits. or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at 26. The plant of embodiment 25, wherein the one or more position 228 is Ser or Gly; Xaa at position 229 is Lys, Asn. 50 additional transgenic trait is selected from insect resistance, Arg or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa herbicide resistance, fungal resistance, virus resistance or at position 232 is Ala, Thr, Ser, Gly, Asp or Glu; Xaa at stress tolerance, disease resistance, male Sterility, stalk position 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, strength, increased yield, modified starches, improved oil Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 profile, balanced amino acids, high lysine or methionine, is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, 55 increased digestibility, improved fiber quality, and drought Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa at position 242 is tolerance. Asn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, 27. An expression cassette, comprising the recombinant Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 is nucleic acid molecule of embodiment 1, 2, 3, 4, 5, 6, 7, 8, Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; Xaa at position 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, wherein the nucleic 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at 60 acid is operably linked to one or more regulatory sequences position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, directing expression of the PIP-1 polypeptide. Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or Asn; Xaa 28. A plant, comprising the expression cassette of embodi at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, ment 27. Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys; Xaa 29. A plant cell, comprising the expression cassette of at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, LyS, 65 embodiment 27. Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr or Cys; Xaa 30. A recombinant microbial cell, comprising the expres at position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, sion cassette of embodiment 27. US 9,688,730 B2 10 31. Seed or grain of the plant of embodiment 20, 21, 22. 48. The recombinant PIP-1 polypeptide of embodiment 23, 24, 25 or 26 or a progeny thereof, wherein the seed or 46 or 47, wherein the PIP-1 polypeptide has insecticidal grain comprises the recombinant nucleic acid molecule. activity against an insect pest of the order Hemiptera. 32. The seed of embodiment 31, wherein one or more seed 49. The recombinant PIP-1 polypeptide of embodiment treatment has been applied to the seed. 5 46, 47 or 48, wherein the PIP-1 polypeptide has insecticidal 33. The seed of embodiment 32, wherein the one or more activity against an insect pest of the Pentatomidae family. seed treatment is selected from a herbicide, an insecticide, a 50. The recombinant PIP-1 polypeptide of embodiment fungicide, a germination inhibitor, a germination enhancer, 49, wherein the PIP-1 polypeptide has insecticidal activity a plant growth regulator, a bactericide, and a nematocide. against an insect selected from Nezara viridula, Halyomor 10 pha haly's, Piezodorus guildini, Euschistus servus, Acroster 34. A biological sample derived from a tissue or seed of nun hilare, Euschistus heros, Euschistus tristigmus, Acros the plant of embodiment 20, 21, 22, 23, 24, 25 or 26. ternum hilare, Dichelops furcatus, Dichelops melacanthus, 35. A recombinant microorganism, comprising a recom Bagrada hilaris, Megacopta cribraria, Scaptocoris cas binant nucleic acid molecule of embodiment 1, 2, 3, 4, 5, 6, tanea, Helicoverpa zea Boddie, Pseudoplusia includens 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. 15 Walker, and Anticarsia gemmatalis. 36. The microorganism of embodiment 35, wherein the 51. The recombinant PIP-1 polypeptide of embodiment microorganism is selected from a bacteria, baculovirus, 46, 47, 48, 49 or 50, wherein the PIP-1 polypeptide has algae, and fungi. insecticidal activity against an insect pest of the order 37. The microorganism of embodiment 36, wherein the Lepidoptera. bacteria is selected from a Bacillus, a Pseudomonas, a 52. The recombinant PIP-1 polypeptide of embodiment Clavibacter, a Rhizobium and E. coli. 46, 47, 48, 49, 50 or 51, wherein the PIP-1 polypeptide is 38. A method for producing a polypeptide with insecti produced by a Pseudomonas chlororaphis strain. cidal activity, comprising culturing the microorganism of 53. The recombinant PIP-1 polypeptide of embodiment embodiment 35, 36 or 37 under conditions in which the 52, wherein the PIP-1 polypeptide is produced by a nucleic acid molecule encoding the polypeptide is 25 Pseudomonas chlororaphis strain having a 16S ribosomal expressed. DNA having at least about 96.9% identity to SEQ ID NO: 39. A method for expressing in a plant a PIP-1 polypep 216. tide, comprising the steps of: 54. The recombinant PIP-1 polypeptide of embodiment (a) inserting into the plant cell a nucleic acid sequence 52, wherein the Pseudomonas chlororaphis strain is SS44C4 comprising in the 5' to 3’ direction an operably linked 30 deposited under accession # NRRLB-50613. recombinant, double-stranded DNA molecule, wherein 55. The recombinant PIP-1 polypeptide of embodiment the recombinant double-stranded DNA molecule com 46, 47, 48, 49, 50, 51, 52, 53 or 54, wherein the PIP-1 prises polypeptide comprises an amino acid motif as represented (i) a promoter that functions in the plant cell; by positions 171-183 of SEQ ID NO: 213. (ii) a nucleic acid molecule encoding a PIP-1 polypep 35 56. The recombinant PIP-1 polypeptide of embodiment tide as set forth in embodiment 1, 2, 3, 4, 5, 6, 7, 8, 55, further comprising any one or more amino acid motifs as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; and represented by positions 149-159 of SEQ ID NO: 213, and (iii) a 3' non-translated polynucleotide that functions in positions 64-79 of SEQ ID NO: 213. the cells of the plant to cause termination of tran 57. The recombinant PIP-1 polypeptide of embodiment Scription; 40 46,47, 48, 49, 50, 51, 52,53,54, 55 or 56, wherein the PIP-1 (b) obtaining a transformed plant cell comprising the polypeptide comprises a polypeptide having at least 80% nucleic acid sequence of step (a); and identity to the amino acid sequence of SEQ ID NO: 2. (c) generating from the transformed plant cell a plant 58. The recombinant PIP-1 polypeptide of embodiment capable of expressing the PIP-1 polypeptide. 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57 wherein the 40. A plant produced by the method of embodiment 39. 45 PIP-1 polypeptide comprises an amino acid motif as repre 41. Seed or grain produced by the plant of embodiment sented by positions 171-183 of SEQ ID NO: 213 and 40. wherein the PIP-1 polypeptide has at least 80% identity to 42. The plant of embodiment 40, further comprising one the amino acid sequence of SEQ ID NO: 2. or more additional transgenic traits. 59. The recombinant PIP-1 polypeptide of embodiment 43. The plant of embodiment 42, wherein the one or more 50 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, wherein additional transgenic trait is selected from insect resistance, the PIP-1 polypeptide comprises an amino acid sequence of herbicide resistance, fungal resistance, viral resistance, (SEQ ID NO: 211), wherein stress tolerance, disease resistance, male Sterility, stalk Xaa at position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly strength, increased yield, modified starches, improved oil or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at profile, balanced amino acids, high lysine or methionine, 55 position 20 is Leu or Val: Xaa at position 21 is Lys, Ser or increased digestibility, improved fiber quality, flowering, ear ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position and seed development, enhancement of nitrogen utilization 24 is Glin or Ala; Xaa at position 25 is Gly or Ala Xaa at efficiency, altered nitrogen responsiveness, drought resis position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or tance or tolerance, cold resistance or tolerance, Salt resis Ala; Xaa at position 30 is Ala or Ile: Xaa at position 35 is tance or tolerance, and increased yield under stress. 60 Phe or Leu; Xaa at position 36 is Ala, Ser or Val: Xaa at 44. The plant of embodiment 40, 42 or 43, wherein the position 38 is Asn, Arg or Ser; Xaa at position 42 is Phe or plant is a monocotyledon. Tyr, Xaa at position 46 is Arg, Lys or His; Xaa at position 45. The plant of embodiment 40, 42 or 43, wherein the 48 is Gly or Asp; Xaa at position 49 is Phe or Tyr; Xaa at plant is a dicotyledon. position 53 is Ser or Gly; Xaa at position 58 is Tyr or Phe: 46. A recombinant PIP-1 polypeptide. 65 Xaa at position 60 is Ala or Ser; Xaa at position 63 is Gln 47. The recombinant PIP-1 polypeptide of embodiment or Lys: Xaa at position 77 is Phe or Tyr; Xaa at position 97 46, wherein the PIP-1 polypeptide is orally active. is Met or Val: Xaa at position 98 is Asp or Glu; Xaa at US 9,688,730 B2 11 12 position 105 is Glin or Asn; Xaa at position 107 is Thr or Ile: Glu; Xaa at position 147 is Ile, Thr or Val; Xaa at position Xaa at position 108 is Glin or Thr; Xaa at position 110 is Arg 150 is Seror Thr; Xaa at position 151 is Asn, Arg or Ser; Xaa or Leu; Xaa at position 120 is Lys, Arg or Glin; Xaa at at position 160 is Thr or Ser; Xaa at position 162 is Ser or position 121 is Thr or Ser; Xaa at position 123 is Thr or Glu; Thr; Xaa at position 163 is Asn, Asp or Glu; Xaa at position Xaa at position 125 is Asn or Ser; Xaa at position 127 is Ser, 5 164 is Ser or Thr; Xaa at position 166 is Glin or Glu; Xaa at Asn. Thr or Lys: Xaa at position 134 is Gly or Ala; Xaa at position 167 is Leu or Met; Xaa at position 168 is Thr, Lys position 135 is Ser, Asn or Lys: Xaa at position 137 is Asp or Ala; Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn. or Gly: Xaa at position 141 is Val or Ile: Xaa at position 142 Asp, Ser or Ala; Xaa at position 172 is Thr, Gly. His, Phe, is Gly or Asp; Xaa at position 144 is Asp or Glu, Xaa at Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; position 147 is Ile, Thr or Val: Xaa at position 150 is Ser or 10 Xaa at position 173 is Phe, Gly. His, Leu, Ala, Arg, Asn. Cys, Thr, Xaa at position 151 is ASn, Arg or Ser; Xaa at position Lys, Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, 160 is Thr or Ser; Xaa at position 162 is Ser or Thr; Xaa at Gly, Arg, ASn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, position 163 is Asn., Asp or Glu; Xaa at position 164 is Ser Ser. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, or Thr; Xaa at position 166 is Glin or Glu, Xaa at position Lys, Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu 167 is Leu or Met; Xaa at position 168 is Thr, Lys or Ala; 15 or Cys; Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at Xaa at position 174 is Ile, Val or Met; Xaa at position 175 position 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, is Val or Ile: Xaa at position 180 is Met or Leu; Xaa at Ser or Lys; Xaa at position 179 is Val, Phe, Thr, Ile, Cys, position 191 is Arg or Lys; Xaa at position 194 is Gly or Ala; Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, Leu, Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at Lys: Xaa at position 182 is Tyr, Phe, Met or His: Xaa at position 220 is ASnor Arg; Xaa at position 221 is Seror Lys; position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Xaa at position 222 is Thr or Arg; Xaa at position 226 is Asp, Ser, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at Pro or Glu; Xaa at position 228 is Seror Gly: Xaa at position position 194 is Gly or Ala; Xaa at position 195 is Asn or Tyr; 229 is Lys or Asn; Xaa at position 231 is Ile or Val: Xaa at 25 Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn position 232 is Ala, Thr or Glu; and Xaa at position 251 is or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 Gly, Ser or Glu, Xaa at position 254 is Ser or Asn; Xaa at is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at position 258 is Seror Arg; Xaa at position 265 is Asn or Asp; position 213 is Tyr or Phe: Xaa at position 220 is Asn or Arg; and Xaa at position 266 is Asp or Asn; and wherein, 1 to 28 Xaa at position 221 is Ser or Lys; Xaa at position 222 is Thr amino acids are optionally deleted from the N-terminus of 30 or Arg; Xaa at position 226 is Asp, Pro or Glu, Xaa at the polypeptide. position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; 60. The recombinant PIP-1 polypeptide of embodiment Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, wherein Thr or Glu, Xaa at position 240 is Gln, Arg, Ala, Val, Glu, the PIP-1 polypeptide comprises an amino acid sequence of Met, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu: SEQ ID NO: 212, wherein 35 Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Xaa at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: Tyr, Met, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly at position 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 20 is Leu or Val: Xaa at position 21 is Lys, Ser or position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position 40 Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp 24 is Gln or Ala; Xaa at position 25 is Gly or Ala; Xaa at or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Glu, Leu, position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Ile, Gln, Tyr Ala; Xaa at position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Met, Asp, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position 45 or Lys, Xaa at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, 36 is Ala, Ser or Val: Xaa at position 38 is Asn, Arg or Ser; His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr Xaa at position 42 is Phe or Tyr; Xaa at position 43 is Pro, or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser, Met, Gly, Gln, Ser, Thr, Arg, Val, Leu, LyS, Asp, Ala, ASn, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala; Xaa at position 249 Phe, Trp, Glu or Cys; Xaa at position 46 is Arg, Lys or His: is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Xaa at position 48 is Gly or Asp; Xaa at position 49 is Phe, 50 Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 Tyr or Leu; Xaa at position 53 is Ser or Gly; Xaa at position is Gly, Ser or Glu; Xaa at position 254 is Ser or ASn; Xaa at 58 is Tyr or Phe: Xaa at position 60 is Ala or Ser; Xaa at position 258 is Ser or Arg; Xaa at position 259 is Phe, Trp, position 63 is Glin or Lys: Xaa at position 66 is Trp, Tyr, Phe, Tyr, Cys, Met, Leu, Val, Ile or His: Xaa at position 265 is Arg, Lys, His, Ile, Val or Ser; Xaa at position 77 is Phe or ASn or Asp, and Xaa at position 266 is Asp or ASn; and Tyr; Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, 55 wherein, 1 to 28 amino acids are optionally deleted from the Ala, Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, N-terminus of the polypeptide. Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa at 61. The recombinant PIP-1 polypeptide of embodiment position 97 is Met or Val; Xaa at position 98 is Asp or Glu; 58, wherein the PIP-1 polypeptide comprises an amino acid Xaa at position 105 is Gln or Asn; Xaa at position 107 is Thr sequence of SEQ ID NO: 213, wherein Xaa at position 2 is or Ile: Xaa at position 108 is Glin or Thr; Xaa at position 110 60 Pro, Thr or Ser; Xaa at position 3 is Ile, Thr, Leu, Val, Met is Arg or Leu, Xaa at position 120 is Lys, Arg or Glin; Xaa or Ser; Xaa at position 6 is Glu, Gly, Asp or Ala; Xaa at at position 121 is Thr or Ser; Xaa at position 123 is Thr or position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at position 19 Glu; Xaa at position 125 is Asn or Ser; Xaa at position 127 is Asp, Glu or Cys: Xaa at position 20 is Leu, Val, Ile or Met; is Ser, Asn. Thr or Lys: Xaa at position 134 is Gly or Ala; Xaa at position 21 is Lys, Ser, ASn, Arg, Thr or Glin; Xaa at Xaa at position 135 is Ser, Asn or Lys; Xaa at position 137 65 position 22 is Ser, Lys, Arg or Thr, Xaa at position 24 is Gln, is Asp or Gly; Xaa at position 141 is Val or Ile: Xaa at Gly, Asn or Ala; Xaa at position 25 is Gly or Ala; Xaa at position 142 is Gly or Asp; Xaa at position 144 is Asp or position 26 is Ser, Asn. Thror Glin; Xaa at position 27 is Leu, US 9,688,730 B2 13 14 Thr, Ala, Ser, Ile, Val or Met; Xaa at position 28 is Arg, Ser, or Leu; Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Lys, Thr, Val, Gly, Ala, Met, Asp, Trp, Pro, Leu, His, Cys or Val, Asp, Tyr, Met, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Gln; Xaa at position 30 is Ala, Ile, Leu, Val or Met; Xaa at Cys; Xaa at position 242 is ASn, Ala, Arg, Lys. His, Ser, Cys, position 35 is Phe, Leu, Ile, Val or Met; Xaa at position 36 Glu, Pro, Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val; is Ala, Ser. Thr, Val, Ile or Leu; Xaa at position 38 is Asn. Xaa at position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser Arg, Ser, Gln, Lys or Thr; Xaa at position 42 is Phe, Tyr, Trp, or Met; Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gln, Leu, Ile, Val or Met; Xaa at position 43 is Pro, Met, Gly, Gln, Cys, Trp or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Ser, Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, Trp, Glu or Glu, Leu, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Ile, Cys; Xaa at position 46 is Arg, Lys or His; Xaa at position Gln, Tyr or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, 48 is Gly, Asp, Ala or Glu; Xaa at position 49 is Phe, Tyr, 10 Arg, Val, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Trp, Leu, Ile, Val or Met; Xaa at position 53 is Ser, Gly, Ala Phe, Thr or Lys; Xaa at position 247 is Asn. Leu, Asp, Tyr, or Thr; Xaa at position 58 is Tyr or Phe: Xaa at position 60 Ala, Phe, His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, is Ala, Ser, Gly or Thr; Xaa at position 63 is Gln, Lys, Asn Trp, Thr or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, or Arg; Xaa at position 66 is Trp, Tyr, Phe, Arg, Lys, His, Ile, Phe, Ser. His, Cys, Leu, Trp, Ile, Asp, Gly or Ala; Xaa at Val or Ser; Xaa at position 77 is Phe, Tyr, Trp, Leu, Ile, Val 15 position 249 is Asn., Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, or Met; Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Glu, Trp, Tyr, Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa Met, Ala, Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, at position 251 is Gly, Ser, Thr, Ala, Asp or Glu; Xaa at Cys, Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa position 254 is Ser, Asn. Thr or Glin; Xaa at position 258 is at position 97 is Met, Val, Leu or Ile: Xaa at position 98 is Ser, Arg, Thr or Lys; Xaa at position 259 is Phe, Trp, Tyr, Asp or Glu; Xaa at position 105 is Glin or ASn; Xaa at Cys, Met, Leu, Val, Ile or His; Xaa at position 265 is Asn. position 107 is Thr, Ile, Ser, Leu or Val; Xaa at position 108 Asp, Gln or Glu, and Xaa at position 266 is Asp, ASn, Gln is Gln, Thr, Ser or ASn; Xaa at position 110 is Arg, Leu, LyS, or Glu; and wherein, 1 to 28 amino acids are optionally Ile, Val or Met; Xaa at position 120 is Lys, Arg, Gln or ASn; deleted from the N-terminus of the polypeptide. Xaa at position 121 is Thr or Ser; Xaa at position 123 is Thr, 62. The recombinant PIP-1 polypeptide of embodiment Glu, Ser or Asp; Xaa at position 125 is Asn. Ser, Gln or Thr: 25 55, comprising an amino acid sequence of SEQID NO: 2 or Xaa at position 127 is Ser, Asn. Thr, Gln, Lys, Ser or Arg; a fragment thereof. Xaa at position 134 is Gly or Ala; Xaa at position 135 is Ser, 63. The recombinant PIP-1 polypeptide of embodiment ASn, Thr, Gln, Arg or Lys, Xaa at position 137 is Asp, Gly, 55, consisting essentially of an amino acid sequence of SEQ Glu or Ala; Xaa at position 141 is Val, Ile or Leu; Xaa at ID NO: 2. position 142 is Gly, Asp, Ala or Glu, Xaa at position 144 is 30 64. The recombinant PIP-1 polypeptide of embodiment Asp or Glu; Xaa at position 147 is Ile, Thr, Val, Leu, Met or 55, wherein the PIP-1 polypeptide is encoded by the poly Ser; Xaa at position 150 is Seror Thr; Xaa at position 151 nucleotide of SEQ ID NO: 1. is Asn, Arg, Ser, Gln, Lys or Thr; Xaa at position 160 is Thr 65. The recombinant PIP-1 polypeptide of embodiment or Ser; Xaa at position 162 is Seror Thr; Xaa at position 163 46, comprising one or more properties selected from: is Asn., Asp, Glu or Glin; Xaa at position 164 is Ser or Thr: 35 a) an amino acid motif as represented by positions 64-79 Xaa at position 166 is Gln, Glu, Asp or ASn; Xaa at position of SEQ ID NO: 213; 167 is Leu, Met, Ile, Val: Xaa at position 168 is Thr, Lys, b) an amino acid motif as represented by positions 149 Ala, Ser, Arg or Gly; Xaa at position 171 is Gly, Leu, Gln, 159 of SEQ ID NO: 213; Met, Cys, Asn., Asp, Ser or Ala; Xaa at position 172 is Thr, c) an amino acid motif as represented by positions 171 Gly. His, Phe, Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, Cys, 40 183 of SEQ ID NO: 213; Val, Ala or Met; Xaa at position 173 is Phe, Gly. His, Leu, e) insecticidal activity against an insect pest of the order Ala, Arg, Asn. Cys, Lys, Trp, Thr, Ser, Tyr or Met; Xaa at Hemiptera: position 174 is Ile, Val, Gly, Arg, Asn, Ala, Gln, Met, Cys, f) insecticidal activity against an insect pest of the order Leu, Phe, Tyr, Lys, Glu, Ser. His or Thr; Xaa at position 175 Lepidoptera; is Val, Ile, Ala, Cys, Glu, Lys, Leu or Met; Xaa at position 45 g) orally active; and 176 is Tyr, Met, Phe, Leu or Cys; Xaa at position 177 is Gln, h) a calculated molecular weight of between about 15 kD Ile, Met or Pro; Xaa at position 178 is Val, Cys, Thr, Pro, to about 35 kD. Ala, Met, Gln, Phe, Ile, Ser or Lys; Xaa at position 179 is 66. A plant capable of expressing the recombinant PIP-1 Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, Ala or Glin; Xaa at polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, position 180 is Met, Leu: Pro, Trp, Asn., Tyr, Gly, Gln, Ala, 50 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65. Val, Phe, Ile, Cys or Ser; Xaa at position 181 is Val, Ala, Leu, 67. The plant of embodiment 66, wherein the plant is a Trp, Cys, Thr, Ile or Lys: Xaa at position 182 is Tyr, Phe, Met monocotyledon. or His; Xaa at position 183 is Ala, Met, Val, Thr, Asp, Gly, 68. The plant of embodiment 66, wherein the plant is a Cys, Ile, Phe, Ser, Gln or Leu; Xaa at position 191 is Arg or dicotyledon. Lys; Xaa at position 194 is Gly or Ala; Xaa at position 195 55 69. The plant of embodiment 66, wherein the plant is is Asn., Tyr, Gln or Trp; Xaa at position 200 is Asn. Ser. Thr selected from barley, corn, oat, rice, rye, Sorghum, turfgrass, or Glin; Xaa at position 203 is Asn or Glin; Xaa at position Sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, 204 is Thr, Ala, Ser or Gly; Xaa at position 206 is Gly, Asp, canola, carrot, cassava, cauliflower, celery, citrus, cotton, a Ala or Glu; Xaa at position 209 is Leu, Val, Ile or Met; Xaa cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, at position 213 is Tyr or Phe: Xaa at position 220 is Asn, Arg, 60 peanut, pepper, potato, poplar, pine, Sunflower, safflower, Gln or Lys; Xaa at position 221 is Ser, Lys, Thr or Arg; Xaa Soybean, Strawberry, Sugar beet, Sweet potato, tobacco, at position 222 is Thr, Arg, Ser or Lys; Xaa at position 226 tomato ornamental, shrub, nut, chickpea, pigeon pea, mil is Asp, Pro, Glu or Gln; Xaa at position 228 is Ser or Gly: lets, hops, and pasture grasses. Xaa at position 229 is LyS, ASn, Arg or Glin; Xaa at position 70. The plant of embodiment 66, 67, 68, 69 or 70 wherein 231 is Ile, Val, Leu or Met; Xaa at position 232 is Ala, Thr, 65 the plant expresses one or more additional transgenic traits. Ser, Gly, Asp or Glu; Xaa at position 240 is Gln, Arg, Ala, 71. The plant of embodiment 70, wherein the one or more Val, Glu, Met, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys additional transgenic trait is selected insect resistance, her US 9,688,730 B2 15 16 bicide resistance, fungal resistance, viral resistance, stress 84. A method of isolating a polypeptide having insecti tolerance, disease resistance, male Sterility, Stalk strength, cidal activity from a Pseudomonas chlororaphis strain, increased yield, modified starches, improved oil profile, comprising balanced amino acids, high lysine or methionine, increased a) obtaining a protein cell lysate from a bacterial isolate; digestibility, improved fiber quality, flowering, ear and seed b) screening the protein cell lysate for insecticidal activ development, enhancement of nitrogen utilization efficiency, ity; and altered nitrogen responsiveness, drought resistance or toler c) isolating an insecticidal protein from the protein cell ance, cold resistance or tolerance, and salt resistance or lysate. tolerance, and increased yield under stress. 85. A recombinant receptor to the polypeptide of SEQID 72. A composition, comprising an insecticidally-effective 10 NO: 2, SEQID NO. 4, SEQID NO:332 or SEQ ID NO: 6. 86. The recombinant receptor of embodiment 85, wherein amount of the recombinant PIP-1 polypeptide of embodi the receptor is isolated from a Hemiptera. ment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 87. A method of identifying a PIP-1 polypeptide in a 60, 61, 62, 63, 64 or 65. biological sample, comprising contacting the biological 73. The composition of embodiment 72, further compris 15 sample with the receptor of embodiment 85 or 86. ing an agriculturally suitable carrier. 88. An isolated antibody or antigen-binding portion 74. The composition of embodiment 73, wherein the thereof, wherein the antibody binds specifically to the PIP-1 carrier is selected from a powder, a dust, pellets, granules, polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, spray, emulsion, colloid, and Solution. 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65. 75. The composition of embodiment 72, 73 or 74, further 89. A method of detecting a PIP-1 polypeptide in a comprising one or more herbicides, insecticides or fungi biological sample comprising, contacting the protein with cides. the antibody of embodiment 88. 76. The composition of embodiment 75, wherein the one 90. A method of isolating a PIP-1 polypeptide in a or more insecticides are pesticidal proteins. biological sample comprising, contacting the protein with 77. The composition of embodiment 76, wherein the one 25 the antibody of embodiment 88. or more pesticidal proteins are selected from a Cry1 protein, 91. A method of controlling Lepidoptera and/or Hemip a Cry2 protein, a Cry3 protein, a Cry4 protein, a Crys tera insect infestation in a transgenic plant and providing protein, a Cry6 protein, a Cry7 protein, a Cry8 protein, a insect resistance management, comprising expressing in the Cry9 protein, a Cry 15 protein, Cry22 protein, a Cry23 plant at least two different insecticidal proteins having protein, a Cry32 protein, a Cry34 protein, a Cry35 protein, 30 different modes of action. a Cry36 protein, a Cry37 protein, a Cry43 protein, a Cry46 92. The method of embodiment 91, wherein one of the at protein, a Crys1 protein, a Crys5 protein, a Cry binary toxin, least two insecticidal proteins comprises a PIP-1 polypeptide of embodiment 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, a Cyt protein, a VIP toxin, a SIP protein, an insecticidal 58, 59, 60, 61, 62, 63, 64 or 65 insecticidal to insects in the lipase, an insecticidal chitinase, and a Snake venom protein. 35 order Lepidoptera and/or Hemiptera. 78. A method for controlling an insect pest population, 93. The method of embodiment 92, wherein one of the at comprising contacting the insect pest population with an least two insecticidal proteins comprises a Cry protein insecticidally-effective amount of the recombinant PIP-1 insecticidal to insects in the order Lepidoptera and/or polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, Hemiptera. 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65. 40 94. A method of reducing likelihood of emergence of 79. A method of inhibiting growth or killing an insect pest, Lepidoptera and/or Hemiptera insect resistance to transgenic comprising contacting the insect pest with a insecticidally plants expressing in the plants insecticidal proteins to con effective amount of recombinant PIP-1 polypeptide of trol the insect species, comprising expressing a PIP-1A embodiment 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, 59, 60, 61, 62, 63, 64 or 65. 45 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 insecticidal to the 80. A method for controlling an insect pest population insect species in combination with an insecticidal protein to resistant to a pesticidal protein, comprising contacting the the insect species having a different modes of action com resistant insect pest population with a insecticidally-effec pared to the PIP-1A polypeptide. tive amount of the recombinant PIP-1 polypeptide of 95. A means for effective Lepidoptera and/or Hemiptera embodiment 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 50 insect resistance management, comprising co-expressing at 59, 60, 61, 62, 63, 64 or 65. high levels in transgenic plants two or more insecticidal 81. The method of controlling an insect pest population proteins toxic to Lepidoptera and/or Hemiptera insects but resistant to an pesticidal protein, comprising contacting the each exhibiting a different mode of effectuating its inhibiting population with a insecticidally-effective amount of the growth or killing activity, wherein the two or more insecti recombinant PIP-1 polypeptide of embodiment 46, 47, 48. 55 cidal proteins comprise a PIP-1 polypeptide of embodiment 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 65, wherein the pesticidal protein is selected from 62, 63, 64 or 65 and a Cry protein. Cry1Ac, Cry1Ab, Cry1A.105, Cry1Ac, Cry1F, Cry1 Fa2, 96. A method for obtaining regulatory approval for plant Cry1F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, ing or commercialization of plants expressing proteins Cry35Ab1, Vip3A, Cry9c, eCry3.1Ab and CBI-Bt. 60 insecticidal to insects in the order Lepidoptera and/or 82. A method for protecting a plant from an insect pest, Hemiptera, comprising the step of referring to, Submitting or comprising expressing in the plant or cell thereof a recom relying on insect assay binding data showing that the PIP-1 binant PIP-1 polypeptide of embodiment 46, 47, 48, 49, 50. polypeptide of embodiment 46,47, 48,49, 50, 51, 52,53,54, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65. 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 does not compete 83. A biologically pure culture of a Pseudomonas chlo 65 with binding sites for Cry proteins in Such insects. roraphis strain SS44C4 deposited under accession it 97. A plant or progeny thereof, comprising the recombi NRRLB-50613. nant nucleic acid molecule of SEQ ID NO: 3. US 9,688,730 B2 17 18 98. A plant or progeny thereof stably transformed with the 116. A method for protecting a plant from an insect pest, recombinant nucleic acid molecule of SEQ ID NO: 3. comprising expressing in the plant or cell thereof a recom 99. The plant or progeny thereof of embodiment 97 or 98, binant insecticidal protein of SEQ ID NO: 4. wherein the plant is a monocotyledon. 117. A recombinant nucleic acid molecule encoding a 100. The plant or progeny thereof of embodiment 97 or 5 insecticidal protein comprising a polypeptide having at least 98, wherein the plant is a dicotyledon. 80% identity to the amino acid sequence of SEQID NO: 6. 101. The plant or progeny thereof of embodiment 97 or 118. The recombinant nucleic acid molecule of embodi 98, wherein the plant is selected from barley, corn, oat, rice, ment 117, wherein the insecticidal protein is orally active. rye, Sorghum, turfgrass, Sugarcane, wheat, alfalfa, banana, 119. The recombinant nucleic acid molecule of embodi broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, 10 ment 117 or 118, wherein the insecticidal protein has insec celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, ticidal activity against an insect pest in the order Hemiptera. grape, onion, lettuce, pea, peanut, pepper, potato, poplar, 120. The recombinant nucleic acid molecule of embodi pine, Sunflower, saflower, soybean, Strawberry, Sugar beet, ment 119, wherein the insecticidal protein has insecticidal Sweet potato, tobacco, tomato ornamental, shrub, nut, chick activity against an insect pest in the family Pentatomidae. pea, pigeon pea, millets, hops, and pasture grasses. 15 121. The recombinant nucleic acid molecule of embodi 102. The plant or progeny thereof of embodiment 97,98, ment 117, 118, 119 or 120, wherein the insecticidal protein 99, 100 or 101, further comprising one or more additional has insecticidal activity against an insect pest in the order transgenic traits. Lepidoptera. 103. An expression cassette, comprising the recombinant 122. The recombinant nucleic acid molecule of embodi nucleic acid molecule of SEQID NO:3 or SEQID NO:331, 2O ment 117, 118, 119, 120 or 121, wherein the nucleic acid wherein the nucleic acid is operably linked to one or more molecule is produced by a Pseudomonas entomophila strain. regulatory sequences directing expression of the polypeptide 123. The recombinant nucleic acid molecule of embodi of SEQ ID NO. 4 or SEQ ID NO: 332. ment 117, wherein the insecticidal protein comprises an 104. A plant, comprising the expression cassette of amino acid motif as represented by positions 171-183 of embodiment 103. 25 SEQ ID NO: 6 or positions 171-183 of SEQ ID NO: 213. 105. A plant cell, comprising the expression cassette of 124. The recombinant nucleic acid molecule of embodi embodiment 103. ment 123, wherein the insecticidal protein further comprises 106. A seed or grain of the plant of embodiment 97, 98, any one or more amino acid motifs as represented by 99, 100, 101 or 102, wherein the seed or grain comprises the positions 149-159 of SEQ ID NO: 213 and positions 69-79 recombinant nucleic acid molecule of SEQ ID NO: 3. 30 of SEQ ID NO: 213. 107. The seed of embodiment 106, wherein one or more 125. A recombinant insecticidal protein, comprising a seed treatment has been applied to the seed. polypeptide having at least 80% identity to the amino acid 108. A method for expressing in a plant a insecticidal sequence of SEQ ID NO: 6. protein, comprising 126. The recombinant insecticidal protein of embodiment (a) inserting into the plant cell a nucleic acid sequence 35 125, wherein the insecticidal protein is orally active. comprising in the 5' to 3’ direction an operably linked 127. The recombinant insecticidal protein of embodiment recombinant, double-stranded DNA molecule, wherein 125 or 126, wherein the insecticidal protein has insecticidal the recombinant, double-stranded DNA molecule com activity against an insect pest in the order Hemiptera. prises 128. The recombinant insecticidal protein of embodiment (i) a promoter that functions in the plant cell; 40 127, wherein the insecticidal protein has insecticidal activity (ii) a nucleic acid molecule encoding the protein of against an insect pest in the family Pentatomidae. SEQ ID NO: 4; and 129. The recombinant insecticidal protein of embodiment (iii) a 3' non-translated polynucleotide that functions in 125, 126, 127 or 128, wherein the insecticidal protein has the cells of the plant to cause termination of tran insecticidal activity against an insect pest in the order Scription; 45 Lepidoptera. (b) obtaining a transformed plant cell comprising the 130. The recombinant insecticidal protein of embodiment nucleic acid sequence of step (a); and 125, 126, 127, 128 or 129, wherein the nucleic acid molecule (c) generating from the transformed plant cell a plant is produced by a Pseudomonas entomophila strain. capable of expressing the protein of SEQ ID NO: 4. 131. The recombinant insecticidal protein of embodiment 109. A plant produced by the method of embodiment 108. 50 125, wherein the insecticidal protein comprises an amino 110. Seed or grain of the plant of embodiment 109. acid motif as represented by positions 171-183 of SEQ ID 111. The method of embodiment 108, wherein the plant NO: 213. further comprises one or more additional transgenic traits. 132. The recombinant insecticidal protein of embodiment 112. A plant capable of expressing a recombinant protein 131, wherein the insecticidal protein further comprises any of SEQ ID NO: 4. 55 one or more amino acid motifs as represented by positions 113. A method for controlling an insect pest population, 149-159 of SEQ ID NO: 213, and positions 69-79 of SEQ comprising contacting the insect pest population with a ID NO 213. insecticidally-effective amount of a recombinant protein of 133. A plant or progeny thereof, comprising the recom SEQ ID NO: 4. binant nucleic acid molecule of embodiment 117, 118, 119, 114. A method of inhibiting growth or killing an insect 60 120, 121, 122, 123 or 124. pest, comprising contacting the insect pest with a insecti 134. A plant or progeny thereof stably transformed with cidally-effective amount of a recombinant protein of SEQID the recombinant nucleic acid molecule of embodiment 117, NO: 4. 118, 119, 120, 121, 122, 123 or 124. 115. A method for controlling an insect pest population 135. The plant or progeny thereof of embodiment 133 or resistant to a pesticidal protein, comprising contacting the 65 134, wherein the plant is a monocotyledon. insect pest population with a insecticidally-effective amount 136. The plant or progeny thereof of embodiment 133 or of a recombinant protein of SEQ ID NO: 4. 134, wherein the plant is a dicotyledon. US 9,688,730 B2 19 20 137. The plant or progeny thereof of embodiment 133 or 152. A method for protecting a plant from an insect pest, 134, wherein the plant is selected from barley, corn, oat, rice, comprising expressing in the plant or cell thereof a recom rye, Sorghum, turfgrass, Sugarcane, wheat, alfalfa, banana, binant pesticidal protein of embodiment 125, 126, 127, 128, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, 129, 130, 131 or 132. celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, 153. A method of controlling Lepidoptera and/or Hemip grape, onion, lettuce, pea, peanut, pepper, potato, poplar, tera insect infestation in a transgenic plant and providing pine, Sunflower, saflower, soybean, Strawberry, Sugar beet, insect resistance management, comprising expressing in the Sweet potato, tobacco, tomato ornamental, shrub, nut, chick plant at least two different insecticidal proteins having pea, pigeon pea, millets, hops, and pasture grass plant cells. different modes of action, wherein one of the at least two 138. The plant or progeny thereof of embodiment 133, 10 insecticidal proteins comprises a insecticidal protein of 134, 135, 136 or 137, further comprising one or more embodiment 125, 126, 127, 128, 129, 130, 131 or 132, additional transgenic traits. insecticidal to insects in the order Lepidoptera and/or 139. An expression cassette, comprising the recombinant Hemiptera. nucleic acid molecule encoding the insecticidal protein of 154. The method of embodiment 153, wherein one of the embodiment 117, 118, 119, 120, 121, 122, 123 or 124, 15 at least two insecticidal proteins comprises a Cry protein wherein the nucleic acid is operably linked to one or more insecticidal to insects in the order Lepidoptera and/or regulatory sequences directing expression of the insecticidal Hemiptera. protein. 155. A method of reducing likelihood of emergence of 140. A plant, comprising the expression cassette of Lepidoptera and/or Hemiptera insect species resistance to embodiment 139. transgenic plants expressing in the plants insecticidal pro 141. A plant cell, comprising the expression cassette of teins to control the insect species, comprising expressing a embodiment 139. first insecticidal protein of embodiment 125, 126, 127, 128, 142. Seed or grain of the plant of embodiment 133, 134, 129, 130, 131 or 132, insecticidal to the insect species in 135, 136, 137 or 138, wherein the seed or grain comprises combination with a second insecticidal protein insecticidal the recombinant nucleic acid molecule. 25 to the insect species having a different mode of action 143. The seed of embodiment 142, wherein one or more compared to the first insecticidal protein. seed treatment has been applied to the seed. 156. A means for effective Lepidoptera and/or Hemiptera 144. A method for expressing in a plant a insecticidal insect resistance management, comprising co-expressing at protein, comprising high levels in transgenic plants two or more insecticidal (a) inserting into the plant cell a nucleic acid sequence 30 proteins toxic to Lepidoptera and/or Hemiptera insects but comprising in the 5' to 3’ direction an operably linked each exhibiting a different mode of effectuating its inhibiting recombinant, double-stranded DNA molecule, wherein growth or killing activity, wherein one of the two or more the recombinant, double-stranded DNA molecule com insecticidal proteins comprise a insecticidal protein of prises embodiment 125, 126, 127, 128, 129, 130, 131 or 132 and (i) a promoter that functions in the plant cell; 35 one of the two or more insecticidal proteins comprise a Cry (ii) a nucleic acid molecule encoding the insecticidal protein. protein of embodiment 125, 126, 127, 128, 129, 130, 157. A method for obtaining regulatory approval for 131 or 132; and planting or commercialization of plants expressing proteins (iii) a 3' non-translated polynucleotide that functions in insecticidal to insects in the order Lepidoptera and/or the cells of the plant to cause termination of tran 40 Hemiptera, comprising the step of referring to, Submitting or Scription; relying on insect assay binding data showing that the insec (b) obtaining a transformed plant cell comprising the ticidal protein of embodiment 125, 126, 127, 128, 129, 130, nucleic acid sequence of step (a); and 131 or 132 does not compete with binding sites for a Cry (c) generating from the transformed plant cell a plant protein in the insects. capable of expressing the insecticidal protein. 45 158. A method of controlling Lepidoptera and/or Hemip 145. A plant produced by the method of embodiment 144. tera insect infestation in a transgenic plant and providing 146. Seed or grain of the plant of embodiment 145. insect resistance management, comprising expressing in the 147. The method of embodiment 144, wherein the plant plant at least two different insecticidal proteins having further comprises one or more additional transgenic traits. different modes of action, wherein one of the at least two 148. A plant capable of expressing a recombinant insec 50 insecticidal proteins comprises the amino acid sequence of ticidal protein of embodiment 125, 126, 127, 128, 129, 130, SEQID NO: 4, insecticidal to insects in the order Lepidop 131 or 132. tera and/or Hemiptera. 149. A method for controlling an insect pest population, 159. The method of embodiment 158, wherein one of the comprising contacting the insect pest population with an at least two insecticidal proteins comprises a Cry protein insecticidally-effective amount of a recombinant insecticidal 55 insecticidal to insects in the order Lepidoptera and/or protein of embodiment 125, 126, 127, 128, 129, 130, 131 or Hemiptera. 132. 160. A method of reducing likelihood of emergence of 150. A method of inhibiting growth or killing an insect Lepidoptera and/or Hemiptera insect species resistance to pest, comprising contacting the insect pest with a insecti transgenic plants expressing in the plants insecticidal pro cidally-effective amount of a recombinant insecticidal pro 60 teins to control the insect species, comprising expressing the tein of embodiment 125, 126, 127, 128, 129, 130, 131 or insecticidal protein of SEQ ID NO. 4 insecticidal to the 132. insect species in combination with an insecticidal protein 151. A method for controlling an insect pest population insecticidal to the insect species having a different modes of resistant to a pesticidal protein, comprising contacting the action compared to the protein of SEQ ID NO: 4. insect pest population with a pesticidally-effective amount 65 161. A means for effective Lepidoptera and/or Hemiptera of a recombinant protein of embodiment 125, 126, 127, 128, insect resistance management, comprising co-expressing at 129, 130, 131 or 132. high levels in transgenic plants two or more insecticidal US 9,688,730 B2 21 22 proteins toxic to Lepidoptera and/or Hemiptera insects but forming organisms with a nucleic acid sequence encoding a each exhibiting a different mode of effectuating its inhibiting PIP-1 polypeptide. In particular, the nucleic acid sequences growth or killing activity, wherein one of the two or more of the embodiments are useful for preparing plants and insecticidal proteins comprise the insecticidal protein of microorganisms that possess pesticidal activity. Thus, trans SEQ ID NO. 4 and one of the two or more insecticidal formed bacteria, plants, plant cells, plant tissues and seeds proteins comprise a Cry protein. are provided. Compositions are pesticidal nucleic acids and 162. A method for obtaining regulatory approval for proteins of bacterial species. The nucleic acid sequences find planting or commercialization of plants expressing proteins use in the construction of expression vectors for Subsequent insecticidal to insects in the order Lepidoptera and/or transformation into organisms of interest, as probes for the Hemiptera, comprising the step of referring to, Submitting or 10 isolation of other homologous (or partially homologous) relying on insect assay binding data showing that the insec genes, and for the generation of altered PIP-1 polypeptides ticidal protein of SEQ ID NO. 4 does not compete with by methods known in the art, such as site directed muta binding sites for a Cry protein in the insects. genesis, domain swapping or DNA shuffling. The PIP-1 polypeptides find use in controlling, inhibiting growth or BRIEF DESCRIPTION OF THE FIGURES 15 killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran, and nematode pest populations and for produc FIG. 1 shows the alignment of PIP-1A (SEQ ID NO: 2): ing compositions with pesticidal activity. Insect pests of the active insecticidal protein orthologs PSEEN3174 (SEQ interest include, but are not limited to, the superfamily of ID NO: 6) and PIP-1B (SEQ ID NO: 4); and the inactive Stink bugs and other related insects including, but not limited homologs AECFG 592740 (SEQ ID NO: 12); Pput 1063 to, species belonging to the family Pentatomidae (Nezara (SEQ ID NO: 8); and Pput 1064 (SEQ ID NO: 10). The viridula, Halyomorpha haly's, Piezodorus guildini, Euschis motifs amino acids 64-79 of SEQ ID NO: 2 (motif 1), tus servus, Acrosternum hilare, Euschistus heros, Euschistus amino acids 149-159 of SEQ ID NO: 2 (motif 2), amino tristigmus, Acrosternum hilare, Dichelops fircatus, Dich acids 171-183 of SEQID NO: 2 (motif3), and amino acids elops melacanthus, and Bagrada hilaris (Bagrada Bug)), 240-249 of SEQ ID NO: 2 (motif 4) are indicated in bold 25 the family Plataspidae (Megacopta cribraria Bean and underline in the PIP-1A sequence. The predicted sec plataspid), and the family Cydnidae (Scaptocoris cas ondary structures of selected beta-sheets are indicated with tanea—Root Stink bug) and Lepidoptera species including “B” above the sequence. but not limited to: diamond-back moth, e.g., Helicoverpa FIG. 2 illustrates a generalized sewing and rescuing PCR zea Boddie; soybean looper, e.g., Pseudoplusia includens mutagenesis strategy using degenerate oligonucleotides to 30 Walker and Velvet bean caterpillar e.g., Anticarsia gemmata generate partially or fully saturated amino acid Substitutions lis Hübner. at positions in the PIP-1A protein. By "pesticidal toxin” or "pesticidal protein' is intended a FIG. 3A-3C shows the alignment of Pseudomonas chlo toxin that has toxic activity against one or more pests, roraphis strain SS44C4 16S ribosomal DNA (SEQ ID NO: including, but not limited to, members of the Lepidoptera, 216) and Pseudomonas entomophila L48 16S ribosomal 35 Diptera, Hemiptera and Coleoptera orders or the Nematoda DNA (SEQID NO:217) having 96.8% identity. Differences phylum or a protein that has homology to such a protein. between the sequences are indicated in Bold and Under Pesticidal proteins have been isolated from organisms lined. including, for example, Bacillus sp., Pseudomonas sp., FIG. 4 shows the results of the Lygus insecticidal activity Photorhabdus sp., Xenorhabdus sp., Clostridium bifermen screening of PIP-1A polypeptide variants having multiple 40 tans and Paenibacillus popilliae. Pesticidal proteins include amino acid substitutions at residues 240-249 of SEQID NO: but are not limited to: insecticidal proteins from Pseudomo 2 (motif 4). The insecticidal activity was scored from 0 to 8 nas sp. such as PSEEN3174 (Monalysin, (2011) PLOS with 8 being the most active. Pathogens, 7:1-13), from Pseudomonas protegens Strain FIG. 5 shows the sequence alignment of PIP-1A (SEQID CHAO and Pf-5 (previously fluorescens) (Pechy–Tarr, (2008) NO: 2), PIP-1B (SEQID NO:4), PIP-1C (SEQID NO:332) 45 Environmental Microbiology 10:2368-2386: GenBank and PSEEN3174 (SEQ ID NO: 6). Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010).J. Agric. Food Chem. 58:12343-12349) DETAILED DESCRIPTION and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., It is to be understood that this invention is not limited to 50 (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecti the particular methodology, protocols, cell lines, genera, and cidal proteins from Photorhabdus sp. and Xenorhabdus sp. reagents described, as Such may vary. It is also to be (Hinchliffe, et al., (2010) The Open Toxinology Journal understood that the terminology used herein is for the 3:101-118 and Morgan, et al., (2001) Applied and Envir. purpose of describing particular embodiments only, and is Micro. 67:2062-2069), U.S. Pat. No. 6,048,838, and U.S. not intended to limit the scope of the present invention. 55 Pat. No. 6,379,946; and Ö-endotoxins including, but not As used herein the singular forms “a”, “and”, and “the limited to, the Cry1, Cry2, Cry3, Cry4, Crys, Cry6, Cry7. include plural referents unless the context clearly dictates Cry8, Cry9, Cry 10, Cry 11, Cry 12, Cry 13, Cry 14, Cry 15, otherwise. Thus, for example, reference to “a cell includes Cry 16, Cry 17, Cry 18, Cry 19, Cry20, Cry21, Cry22, Cry23, a plurality of such cells and reference to “the protein’ Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, includes reference to one or more proteins and equivalents 60 Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, thereof known to those skilled in the art, and so forth. All Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, technical and Scientific terms used herein have the same Cry49, Cry 51 and Crys5 classes of 8-endotoxin genes and meaning as commonly understood to one of ordinary skill in the B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Mem the art to which this invention belongs unless clearly indi bers of these classes of B. thuringiensis insecticidal proteins cated otherwise. 65 include, but are not limited to Cry1Aa1 (Accession # Acces The present disclosure is drawn to compositions and sion # M11250), Cry1Aa2 (Accession # M10917), Cry1Aa3 methods for controlling pests. The methods involve trans (Accession # D00348), Cry1Aa4 (Accession if X13535),

US 9,688,730 B2 27 28 # EF633476), Crys4Aa1 (Accession # EU339367), AXMI 182, AXMI 185, AXMI186, AXMI187, AXMI 188, Crys5Aa1 (Accession # EU121521), Cry55Aa2 (Accession AXIMI189 of U.S. Pat. No. 8,318,900; AXMI079, # AAE33526). AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, Examples of 8-endotoxins also include but are not limited AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858, 5 AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, 849; a DIG-3 or DIG-11 toxin (N-terminal deletion of AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, C.-helix 1 and/or C-helix 2 variants of Cry proteins such as AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, Cry1A) of U.S. Pat. Nos. 8.304,604 and 8.304,605, Cry 1B AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, of U.S. patent application Ser. No. 10/525,318; Cry1C of AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 10 AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, 6.218, 188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982: AXMI137 of US 2010/0005543; Cry proteins such as 6,962,705 and 6,713,063): a Cry2 protein such as Cry2Ab Cry1A and Cry3A having modified proteolytic sites of U.S. protein of U.S. Pat. No. 7,064.249); a Cry3A protein includ Pat. No. 8.319,019; and a Cry1Ac, Cry2Aa and Cry1Ca ing but not limited to an engineered hybrid insecticidal toxin protein from Bacillus thuringiensis strain VBTS 2528 protein (eHIP) created by fusing unique combinations of 15 of US Patent Application Publication Number 2011/ variable regions and conserved blocks of at least two dif 0064710. Other Cry proteins are well known to one skilled ferent Cry proteins (US Patent Application Publication in the art (see, Crickmore, et al., “Bacillus thuringiensis Number 2010/0017914); a Cry4 protein; a Crys protein; a toxin nomenclature' (2011), at lifesci.sussex.ac.uk/home/ Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, Neil Crickmore/Bt? which can be accessed on the world 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378.499 and wide web using the “www’ prefix). The insecticidal activity 7.462.760; a Cry9 protein such as such as members of the of Cry proteins is well known to one skilled in the art (for Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families: review, see, van Frannkenhuyzen, (2009) J. Invert. Path. a Cry 15 protein of Naimov, et al., (2008) Applied and 101:1-16). The use of Cry proteins as transgenic plant traits Environmental Microbiology 74.71.45-7151; a Cry22, a is well known to one skilled in the art and Cry-transgenic Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 25 plants including but not limited to Cry1Ac, Cry1Ac-- and 6,340,593: a CryET33 and CryET34 protein of U.S. Pat. Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, Cry1 F+ Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, and 7,504,229; a CryET33 and CryET34 homologs of US Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have Patent Publication Number 2006/0191034, 2012/0278954, received regulatory approval (see, Sanahuja, (2011) Plant and PCT Publication Number WO 2012/139004: a 30 Biotech Journal 9:283-300 and the CERA (2010) GM Crop Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 Database Center for Environmental Risk Assessment and 6,340,593: a Cry46 protein, a Cry 51 protein, a Cry (CERA), ILSI Research Foundation, Washington D.C. at binary toxin; a TIC901 or related toxin: TIC807 of US cera-gmc.org/index.php?action gm crop database which 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, can be accessed on the world-wide web using the “www.” TIC127, TIC128 of PCT US 2006/033867; AXMI-027, 35 prefix). More than one pesticidal proteins well known to one AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757: skilled in the art can also be expressed in plants such as AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. Vip3Ab & Cry1 Fa (US2012/0317682), Cry 1BE & Cry1F No. 7,923,602: AXMI-018, AXMI-020, and AXMI-021 of (US2012/0311746), Cry1CA & Cry1AB (US2012/ WO 2006/083891: AXMI-010 of WO 2005/038032; AXMI 0311745), Cry1F & CryCa (US2012/0317681), Cry1DA & 003 of WO 2005/021585; AXMI-008 of US 2004/0250311; 40 Cry 1BE (US2012/0331590), Cry1DA & Cry1 Fa (US2012/ AXMI-006 of US 2004/0216186: AXMI-007 of US 2004/ 0331589), Cry1AB & Cry1BE (US2012/0324606), and 0210965; AXMI-009 of US 2004/0210964: AXMI-014 of Cry1 Fa & Cry2Aa, Cry1 I or Cry1E (US2012/0324605). US 2004/0197917; AXMI-004 of US 2004/0197916: Pesticidal proteins also include insecticidal lipases including AXMI-028 and AXMI-029 of WO 2006/119457; AXMI lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and cho 007, AXMI-008, AXMI-0080r12, AXMI-009, AXMI-014 45 lesterol oxidases such as from Streptomyces (Purcell et al. and AXMI-004 of WO 2004/074462: AXMI-150 of U.S. (1993) Biochem Biophy's Res Commun 15:1406-1413). Pes Pat. No. 8,084,416: AXMI-205 of US20110023184: AXMI ticidal proteins also include VIP (vegetative insecticidal 011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,279, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the 034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and 50 like. Other VIP proteins are well known to one skilled in the AXMI-064 of US 2011/0263488; AXMI-R1 and related art (see, lifesci. Sussex.ac.uk/home/Neil Crickmore/Bt/ proteins of US 2010/0197592: AXMI221Z, AXMI222z, Vip.html which can be accessed on the world-wide web AXMI223Z, AXMI224Z and AXMI225Z of WO 2011/ using the “www’ prefix). Pesticidal proteins also include 103248: AXMI218, AXMI219, AXMI220, AXMI226, toxin complex (TC) proteins, obtainable from organisms AXMI227, AXMI228, AXMI229, AXMI230, and 55 such as Xenorhabdus, Photorhabdus and Paenibacillus (see, AXMI231 of WO11/103,247; AXMI-115, AXMI-113, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. have “stand alone' insecticidal activity and other TC pro 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, teins enhance the activity of the stand-alone toxins produced and AXMI-045 of US 2010/0298211: AXMI-066 and by the same given organism. The toxicity of a “stand-alone” AXMI-076 of US20090144852: AXMI128, AXMI130, 60 TC protein (from Photorhabdus, Xenorhabdus or Paeniba AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, cillus, for example) can be enhanced by one or more TC AXMI 143, AXMI144, AXMI146, AXMI148, AXMI149, protein “potentiators' derived from a source organism of a AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, different genus. There are three main types of TC proteins. AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, As referred to herein, Class A proteins (“Protein A') are AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, 65 stand-alone toxins. Class B proteins (“Protein B) and Class AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, C proteins (“Protein C) enhance the toxicity of Class A AXMI177, AXMI178, AXMI179, AXMI 180, AXMI181, proteins. Examples of Class A proteins are TcbA, TcdA, US 9,688,730 B2 29 30 XptA1 and XptA2. Examples of Class B proteins are TcaG, acid molecule encoding a PIP-1 polypeptide can contain less TcdB, XptB1Xb and XptC1 Wi. Examples of Class C pro than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of teins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins nucleic acid sequences that naturally flank the nucleic acid also include spider, Snake and Scorpion venom proteins. molecule in genomic DNA of the cell from which the nucleic Examples of spider venom peptides include but are not acid is derived. limited to lycotoxin-1 peptides and mutants thereof (U.S. A variety of polynucleotides that encode PIP-1 polypep Pat. No. 8,334,366). tides or related proteins are contemplated. Such polynucle In some embodiments the PIP-1 polypeptides include otides are useful for production of PIP-1 polypeptides in amino acid sequences deduced from the full-length nucleic host cells when operably linked to suitable promoter, tran acid sequences disclosed herein, and amino acid sequences 10 Scription termination and/or polyadenylation sequences. that are shorter than the full-length sequences, either due to Such polynucleotides are also useful as probes for isolating the use of an alternate downstream start site or due to homologous or Substantially homologous polynucleotides processing that produces a shorter protein having pesticidal that encode PIP-1 polypeptides or related proteins. activity. Processing may occur in the organism after the One source of polynucleotides that encode PIP-1 poly protein is expressed in or in the pest after ingestion of the 15 peptides or related proteins is a Pseudomonas chlororaphis protein. strain which contains the PIP-1A polynucleotide of SEQID Thus, provided herein are novel isolated or recombinant NO: 1 encoding the PIP-1A polypeptide of SEQID NO: 2. nucleic acid sequences encoding polypeptides that confer This polynucleotide sequence was isolated from a pesticidal activity. Also provided are the amino acid Pseudomonas chlororaphis host and is thus suitable for sequences of PIP-1 polypeptides. The protein resulting from expression of the encoded PIP-1A polypeptide in other translation of these PIP-1 polypeptide genes allows cells to bacterial hosts. For example, SEQID NO: 1 can be used to control or kill pests that ingest it. express the PIP-1A protein in bacterial hosts that include but Bacterial Strains are not limited to an Agrobacterium, an Alcaligenes, a One aspect of the invention pertains to bacterial Strains Bacillus, an Escherichia, a Salmonella, a Pseudomonas and that are capable of expressing a PIP-1 polypeptide. In some 25 a Rhizobium bacterial host cells. The polynucleotides are embodiments the bacterial strain is a Pseudomonas chloro also useful as probes for isolating homologous or Substan raphis strain. In some embodiments the bacterial strain is a tially homologous polynucleotides that encode PIP-1 poly biologically pure culture of a Pseudomonas chlororaphis peptides or related proteins. Such probes can be used to strain SS44C4, deposited on Dec. 1, 2011 under Accession identify homologous or Substantially homologous poly Number NRRLB-50613 with the Agricultural Research Ser 30 nucleotides derived from Pseudomonas or other bacterial vice Culture Collection (NRRL). In some embodiments the strains. bacterial strain is a biologically pure culture of a Pseudomo Polynucleotides that encode a PIP-1 polypeptide can also nas chlororaphis strain having a 16S ribosomal DNA having be synthesized de novo from a PIP-1 polypeptide sequence. at least about 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, The sequence of the polynucleotide gene can be deduced 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 35 from a PIP-1A polypeptide sequence through use of the 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, genetic code. Computer programs such as "BackTranslate 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% (GCGTM Package. Acclerys, Inc. San Diego, Calif.) can be or 99.9% sequence identity compared to SEQ ID NO: 216. used to convert a peptide sequence to the corresponding Nucleic Acid Molecules, and Variants and Fragments nucleotide sequence encoding the peptide. Examples of Thereof 40 PIP-1 polypeptide sequences that can be used to obtain Another aspect of the invention pertains to isolated or corresponding nucleotide encoding sequences include, but recombinant nucleic acid molecules comprising nucleic acid are not limited to, the PIP-1 polypeptide sequence of SEQ sequences encoding PIP-1 polypeptides and polypeptides or ID NO: 2. Furthermore, synthetic PIP-1A polynucleotide biologically active portions thereof, as well as nucleic acid sequences of the invention can be designed so that they will molecules sufficient for use as hybridization probes to 45 be expressed in plants. U.S. Pat. No. 5,500,365 describes a identify nucleic acid molecules encoding proteins with method for synthesizing plant genes to improve the expres regions of sequence homology. As used herein, the term sion level of the protein encoded by the synthesized gene. “nucleic acid molecule' is intended to include DNA mol This method relates to the modification of the structural gene ecules (e.g., recombinant DNA, cDNA, genomic DNA, sequences of the exogenous transgene, to cause them to be plastid DNA, mitochondrial DNA) and RNA molecules 50 more efficiently transcribed, processed, translated and (e.g., mRNA) and analogs of the DNA or RNA generated expressed by the plant. Features of genes that are expressed using nucleotide analogs. The nucleic acid molecule can be well in plants include elimination of sequences that can single-stranded or double-stranded, but preferably is double cause undesired intron splicing or polyadenylation in the stranded DNA. coding region of a gene transcript while retaining Substan An "isolated' or “recombinant nucleic acid molecule (or 55 tially the amino acid sequence of the toxic portion of the DNA) is used herein to refer to a nucleic acid sequence (or insecticidal protein. A similar method for obtaining DNA) that is no longer in its natural environment, for enhanced expression of transgenes in monocotyledonous example in an in vitro or in a recombinant bacterial or plant plants is disclosed in U.S. Pat. No. 5,689,052. host cell. In some embodiments, an "isolated' or “recom In some embodiments the nucleic acid molecule encoding binant nucleic acid is free of sequences (preferably protein 60 a PIP-1 polypeptide is a polynucleotide having the sequence encoding sequences) that naturally flank the nucleic acid set forth in SEQID NO: 1, 3 or 331 and variants, fragments (i.e., sequences located at the 5' and 3' ends of the nucleic and complements thereof. By “complement' is intended a acid) in the genomic DNA of the organism from which the nucleic acid sequence that is sufficiently complementary to nucleic acid is derived. For purposes of the disclosure, a given nucleic acid sequence Such that it can hybridize to "isolated' or “recombinant' when used to refer to nucleic 65 the given nucleic acid sequence to thereby form a stable acid molecules excludes isolated chromosomes. For duplex. In some embodiments the nucleic acid molecule example, in various embodiments, the recombinant nucleic encoding a PIP-1 polypeptide is a nucleic acid molecule US 9,688,730 B2 31 32 having the sequence set forth in SEQ ID NO: 1, 3 or 331. Pro or Glu; Xaa at position 228 is Seror Gly: Xaa at position The corresponding amino acid sequences for the insecticidal 229 is Lys or Asn; Xaa at position 231 is Ile or Val: Xaa at protein encoded by these nucleic acid sequences are set forth position 232 is Ala, Thr or Glu; and Xaa at position 251 is in SEQ ID NO: 2, 4 and 332. Gly, Ser or Glu; Xaa at position 254 is Ser or Asn; Xaa at In some embodiments the nucleic acid molecule encoding position 258 is Seror Arg; Xaa at position 265 is Asn or Asp; a PIP-1 polypeptide is a polynucleotide having a nucleotide and Xaa at position 266 is Asp or Asn; and wherein, 1 to 28 sequence encoding a polypeptide comprising an amino acid amino acids are optionally deleted from the N-terminus of sequence having at least 80% identity, to the amino acid the polypeptide. sequence of SEQ ID NO: 2, SEQID NO:4 or SEQ ID NO: In some embodiments the nucleic acid molecule encoding 332, wherein the polypeptide has pesticidal activity. In some 10 a PIP-1 polypeptide is a polynucleotide having a nucleotide embodiments the nucleic acid molecule encoding a PIP-1 sequence encoding a polypeptide comprising an amino acid polypeptide is a polynucleotide having a nucleotide sequence of a sequence of SEQ ID NO: 212, wherein Xaa sequence encoding a polypeptide comprising an amino acid at position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr: sequence having at least 80% identity, to the amino acid Xaa at position 6 is Glu or Gly: Xaa at position 8 is Ser, Gly sequence of SEQ ID NO: 2, wherein the polypeptide has 15 or ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at pesticidal activity. In some embodiments the nucleic acid position 20 is Leu or Val: Xaa at position 21 is Lys, Ser or molecule encoding a PIP-1 polypeptide is a polynucleotide ASn; Xaa at position 22 is Ser, Lys or Arg; Xaa at position having a nucleotide sequence encoding a polypeptide com 24 is Gln or Ala; Xaa at position 25 is Gly or Ala; Xaa at prising an amino acid sequence having at least 80% identity, position 26 is Ser or Asn; Xaa at position 27 is Leu, Thr or to the amino acid sequence of SEQ ID NO: 4, wherein the Ala; Xaa at position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, polypeptide has pesticidal activity. In some embodiments Met, Asp, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 the nucleic acid molecule encoding a PIP-1 polypeptide is a is Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position polynucleotide having a nucleotide sequence encoding a 36 is Ala, Ser or Val: Xaa at position 38 is Asn, Arg or Ser; polypeptide comprising an amino acid sequence having at Xaa at position 42 is Phe or Tyr; Xaa at position 43 is Pro, least 80% identity, to the amino acid sequence of SEQ ID 25 Met, Gly, Gln, Ser, Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, NO: 332, wherein the polypeptide has pesticidal activity. Phe, Trp, Glu or Cys; Xaa at position 46 is Arg, Lys or His: In some embodiments the nucleic acid molecule encoding Xaa at position 48 is Gly or Asp; Xaa at position 49 is Phe, a PIP-1 polypeptide is a polynucleotide having a nucleotide Tyr or Leu; Xaa at position 53 is Ser or Gly; Xaa at position sequence encoding a polypeptide comprising an amino acid 58 is Tyr or Phe: Xaa at position 60 is Ala or Ser; Xaa at sequence of (SEQ ID NO: 211), wherein Xaa at position 2 30 position 63 is Glin or Lys: Xaa at position 66 is Trp, Tyr, Phe, is Pro or Thr; Xaa at position 8 is Ser, Gly or ASn; Xaa at Arg, Lys, His, Ile, Val or Ser; Xaa at position 77 is Phe or position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu or Tyr; Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at position Ala, Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, 22 is Ser, Lys or Arg; Xaa at position 24 is Gln or Ala; Xaa Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa at at position 25 is Gly or Ala Xaa at position 26 is Seror ASn; 35 position 97 is Met or Val; Xaa at position 98 is Asp or Glu; Xaa at position 27 is Leu, Thr or Ala; Xaa at position 30 is Xaa at position 105 is Gln or ASn; Xaa at position 107 is Thr Ala or Ile: Xaa at position 35 is Phe or Leu; Xaa at position or Ile: Xaa at position 108 is Glin or Thr; Xaa at position 110 36 is Ala, Ser or Val: Xaa at position 38 is Asn, Arg or Ser; is Arg or Leu, Xaa at position 120 is Lys, Arg or Glin; Xaa Xaa at position 42 is Phe or Tyr, Xaa at position 46 is Arg, at position 121 is Thr or Ser; Xaa at position 123 is Thr or Lys or His; Xaa at position 48 is Gly or Asp; Xaa at position 40 Glu; Xaa at position 125 is Asn or Ser; Xaa at position 127 49 is Phe or Tyr; Xaa at position 53 is Ser or Gly; Xaa at is Ser, Asn. Thr or Lys; Xaa at position 134 is Gly or Ala; position 58 is Tyr or Phe: Xaa at position 60 is Ala or Ser; Xaa at position 135 is Ser, Asin or Lys; Xaa at position 137 Xaa at position 63 is Glin or Lys; Xaa at position 77 is Phe is Asp or Gly: Xaa at position 141 is Val or Ile: Xaa at or Tyr; Xaa at position 97 is Met or Val: Xaa at position 98 position 142 is Gly or Asp; Xaa at position 144 is Asp or is Asp or Glu; Xaa at position 105 is Glin or ASn; Xaa at 45 Glu; Xaa at position 147 is Ile, Thr or Val; Xaa at position position 107 is Thr or Ile: Xaa at position 108 is Glin or Thr: 150 is Seror Thr; Xaa at position 151 is Asn, Arg or Ser; Xaa Xaa at position 110 is Arg or Leu, Xaa at position 120 is LyS, at position 160 is Thr or Ser; Xaa at position 162 is Ser or Argor Glin; Xaa at position 121 is Thr or Ser; Xaa at position Thr; Xaa at position 163 is Asn, Asp or Glu; Xaa at position 123 is Thr or Glu; Xaa at position 125 is Asn or Ser; Xaa at 164 is Ser or Thr; Xaa at position 166 is Glin or Glu; Xaa at position 127 is Ser, Asn. Thr or Lys; Xaa at position 134 is 50 position 167 is Leu or Met; Xaa at position 168 is Thr, Lys Gly or Ala; Xaa at position 135 is Ser, Asn or Lys; Xaa at or Ala; Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn. position 137 is Asp or Gly: Xaa at position 141 is Val or Ile: Asp, Ser or Ala; Xaa at position 172 is Thr, Gly. His, Phe, Xaa at position 142 is Gly or Asp; Xaa at position 144 is Asp Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; or Glu; Xaa at position 147 is Ile, Thr or Val: Xaa at position Xaa at position 173 is Phe, Gly. His, Leu, Ala, Arg, Asn. Cys, 150 is Seror Thr; Xaa at position 151 is Asn, Arg or Ser; Xaa 55 Lys, Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, at position 160 is Thr or Ser; Xaa at position 162 is Ser or Gly, Arg, ASn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, Thr; Xaa at position 163 is Asn, Asp or Glu, Xaa at position Ser. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, 164 is Ser or Thr; Xaa at position 166 is Glin or Glu; Xaa at Lys, Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu position 167 is Leu or Met; Xaa at position 168 is Thr, Lys or Cys; Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at or Ala; Xaa at position 174 is Ile, Val or Met; Xaa at position 60 position 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, 175 is Val or Ile: Xaa at position 180 is Met or Leu; Xaa at Ser or Lys; Xaa at position 179 is Val, Phe, Thr, Ile, Cys, position 191 is Arg or Lys; Xaa at position 194 is Gly or Ala; Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, Leu, Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at 65 Lys: Xaa at position 182 is Tyr, Phe, Met or His: Xaa at position 220 is ASnor Arg; Xaa at position 221 is Seror Lys; position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Xaa at position 222 is Thr or Arg; Xaa at position 226 is Asp, Ser, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at US 9,688,730 B2 33 34 position 194 is Gly or Ala; Xaa at position 195 is Asn or Tyr; Ser; Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn position 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 Ser, Asn. Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at Gly or Ala; Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or position 213 is Tyr or Phe: Xaa at position 220 is Asn or Arg; Lys: Xaa at position 137 is Asp, Gly, Glu or Ala; Xaa at Xaa at position 221 is Ser or Lys; Xaa at position 222 is Thr position 141 is Val, Ile or Leu; Xaa at position 142 is Gly, or Arg; Xaa at position 226 is Asp, Pro or Glu, Xaa at Asp, Ala or Glu, Xaa at position 144 is Asp or Glu, Xaa at position 228 is Seror Gly: Xaa at position 229 is Lys or ASn; position 147 is Ile, Thr, Val, Leu, Met or Ser; Xaa at position Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, 150 is Ser or Thr; Xaa at position 151 is Asn, Arg, Ser, Gln, Thr or Glu, Xaa at position 240 is Gln, Arg, Ala, Val, Glu, 10 Lys or Thr; Xaa at position 160 is Thr or Ser; Xaa at position Met, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu: 162 is Ser or Thr; Xaa at position 163 is Asn., Asp, Glu or Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Gln; Xaa at position 164 is Ser or Thr; Xaa at position 166 Tyr, Met, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa is Gln, Glu, Asp or Asn; Xaa at position 167 is Leu, Met, Ile, at position 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Val: Xaa at position 168 is Thr, Lys, Ala, Ser, Arg or Gly; Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at 15 Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn., Asp, Ser position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; or Ala; Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaa at or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Glu, Leu, position 173 is Phe, Gly, His, Leu, Ala, Arg, ASn, Cys, Lys, Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Ile, Gln, Tyr Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, Gly, or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Arg, Asn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser, Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, or Lys, Xaa at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu or Cys: His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at position or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser, 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, Seror Lys: His, Cys, Leu, Trp, Ile, Asp, Gly or Ala; Xaa at position 249 25 Xaa at position 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ala or Glin; Xaa at position 180 is Met, Leu: Pro, Trp, Asn. Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; Xaa at position is Gly, Ser or Glu; Xaa at position 254 is Ser or ASn; Xaa at 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or Lys; Xaa at position 258 is Ser or Arg; Xaa at position 259 is Phe, Trp, position 182 is Tyr, Phe, Met or His; Xaa at position 183 is Tyr, Cys, Met, Leu, Val, Ile or His: Xaa at position 265 is 30 Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, Gln or Leu: ASn or Asp; and Xaa at position 266 is Asp or ASn. Xaa at position 191 is Arg or Lys; Xaa at position 194 is Gly In some embodiments the nucleic acid molecule encoding or Ala; Xaa at position 195 is Asn. Tyr, Gln or Trp; Xaa at a PIP-1 polypeptide is a polynucleotide having a nucleotide position 200 is Asn. Ser. Thr or Glin; Xaa at position 203 is sequence encoding a polypeptide comprising an amino acid Asin or Glin; Xaa at position 204 is Thr, Ala, Ser or Gly: Xaa sequence of a sequence of SEQID NO: 213 wherein Xaa at 35 at position 206 is Gly, Asp, Ala or Glu; Xaa at position 209 position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, Thr, is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: Xaa Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp or at position 220 is ASn, Arg, Gln or Lys, Xaa at position 221 Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at is Ser, Lys, Thr or Arg; Xaa at position 222 is Thr, Arg, Ser position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at Val, Ile or Met; Xaa at position 21 is Lys, Ser, Asn, Arg, Thr 40 position 228 is Ser or Gly; Xaa at position 229 is Lys, Asn. or Glin; Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at Arg or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa position 24 is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly at position 232 is Ala, Thr, Ser, Gly, Asp or Glu; Xaa at or Ala; Xaa at position 26 is Ser, Asn. Thr or Glin; Xaa at position 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, position 27 is Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, 45 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala, Ile, Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa at position 242 is Leu, Val or Met; Xaa at position 35 is Phe, Leu, Ile, Val or Asn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, Met; Xaa at position 36 is Ala, Ser, Thr, Val, Ile or Leu; Xaa Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 is at position 38 is Asn, Arg, Ser, Gln, Lys or Thr; Xaa at Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; Xaa at position position 42 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at 50 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, Val, Leu, position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, LyS, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at position 46 Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or Asn; Xaa is Arg, Lys or His; Xaa at position 48 is Gly, Asp, Ala or Glu; at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys; Xaa at position 53 is Ser, Gly, Ala or Thr; Xaa at position 58 is 55 at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, Lys, Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Thr; Xaa Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr or Cys; Xaa at position 63 is Gln, Lys, ASn or Arg; Xaa at position 66 is at position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, Tyr, Phe, Arg, Lys. His, Ile, Val or Ser; Xaa at position Trp, Ile, Asp, Gly or Ala; Xaa at position 249 is ASn, LyS, 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at position 89 Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys 60 Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 is Gly, or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Ser. Thr, Ala, Asp or Glu; Xaa at position 254 is Ser, Asn. Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, Val, Thr or Gln; Xaa at position 258 is Ser, Arg, Thr or Lys: Xaa Leu or Ile: Xaa at position 98 is Asp or Glu; Xaa at position at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, Leu His; Xaa at position 265 is Asn, Asp, Gln or Glu; and Xaa or Val: Xaa at position 108 is Gln, Thr, Ser or Asn; Xaa at 65 at position 266 is Asp, ASn, Gln or Glu. position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at position In some embodiments the nucleic acid molecules encode 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thr or a PIP-1 polypeptide having a nucleotide sequence encoding US 9,688,730 B2 35 36 a polypeptide comprising one or more amino acid motifs 211, amino acids 64-79 of SEQID NO: 212 or amino acids selected from i) amino acids 64-79 of SEQID NO: 2, amino 64-79 of SEQID NO: 213, ii) amino acids 149-159 of SEQ acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ ID NO: 2, amino acids 149-159 of SEQID NO: 211, amino ID NO: 212 or amino acids 64-79 of SEQ ID NO: 213, ii) acids 149-159 of SEQ ID NO: 212 or amino acids 149-159 amino acids 149-159 of SEQ ID NO: 2, amino acids of SEQ ID NO: 213, iii) amino acids 171-183 of SEQ ID 149-159 of SEQ ID NO: 211, amino acids 149-159 of SEQ NO: 2, amino acids 171-183 of SEQ ID NO: 211, amino ID NO: 212 or amino acids 149-159 of SEQID NO: 213, iii) acids 171-183 of SEQ ID NO: 212 or amino acids 171-183 amino acids 171-183 of SEQ ID NO: 2, amino acids of SEQ ID NO: 213, and iv) amino acids 240-249 of SEQ 171-183 of SEQ ID NO: 211, amino acids 171-183 of SEQ ID NO: 2, amino acids 240-249 of SEQID NO: 211, amino ID NO: 212 or amino acids 171-183 of SEQ ID NO: 213, 10 and iv) amino acids 240-249 of SEQID NO: 2, amino acids acids 240-249 of SEQ ID NO: 212 or amino acids 240-249 240-249 of SEQID NO: 211, amino acids 240-249 of SEQ of SEQ ID NO: 213. ID NO: 212 or amino acids 240-249 of SEQID NO: 213. In In some embodiments the nucleic acid molecules encode Some embodiments the nucleic acid molecules encode a a PIP-1 polypeptide having a nucleotide sequence encoding PIP-1 polypeptide having a nucleotide sequence encoding a 15 a polypeptide comprising an amino acid sequence having at polypeptide comprising an amino acid as represented by least 80% identity to the amino acid sequence set forth in positions 171-183 of SEQID NO: 213 wherein at least one SEQ ID NO: 2 and wherein the polypeptide comprises one amino acid at positions 171-183 of SEQID NO: 213 are not or more amino acid motifs selected from i) amino acids identical to amino acids at positions 171-183 of SEQID NO: 64-79 of SEQID NO: 2, amino acids 64-79 of SEQID NO: 6. 211, amino acids 64-79 of SEQID NO: 212 or amino acids In some embodiments the nucleic acid molecules encode 64-79 of SEQID NO: 213, ii) amino acids 149-159 of SEQ a PIP-1 polypeptide having a nucleotide sequence encoding ID NO: 2, amino acids 149-159 of SEQID NO: 211, amino a polypeptide comprising an amino acid sequence having at acids 149-159 of SEQ ID NO: 212 or amino acids 149-159 least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, of SEQ ID NO: 213, iii) amino acids 171-183 of SEQ ID 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 25 NO: 2, amino acids 171-183 of SEQ ID NO: 211, amino 99% or greater identity to the amino acid sequence set forth acids 171-183 of SEQ ID NO: 212 or amino acids 171-183 in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO.4 and of SEQ ID NO: 213, and iv) amino acids 240-249 of SEQ wherein the polypeptide comprises one or more amino acid ID NO: 2, amino acids 240-249 of SEQID NO: 211, amino motifs selected from i) amino acids 64-79 of SEQ ID NO: acids 240-249 of SEQ ID NO: 212 or amino acids 240-249 2, amino acids 64-79 of SEQID NO: 211, amino acids 64-79 30 of SEQ ID NO: 213. of SEQ ID NO: 212 or amino acids 64-79 of SEQ ID NO: In some embodiments the nucleic acid molecules encode 213, ii) amino acids 149-159 of SEQID NO: 2, amino acids a PIP-1 polypeptide having a nucleotide sequence encoding 149-159 of SEQ ID NO: 211, amino acids 149-159 of SEQ a polypeptide comprising an amino acid sequence having at ID NO: 212 or amino acids 149-159 of SEQID NO: 213, iii) least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, amino acids 171-183 of SEQ ID NO: 2, amino acids 35 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 171-183 of SEQ ID NO: 211, amino acids 171-183 of SEQ 99% or greater identity to the amino acid sequence set forth ID NO: 212 or amino acids 171-183 of SEQ ID NO: 213, in SEQ ID NO: 2, and wherein the polypeptide comprises and iv) amino acids 240-249 of SEQID NO: 2, amino acids one or more amino acid motifs selected from i) amino acids 240-249 of SEQID NO: 211, amino acids 240-249 of SEQ 64-79 of SEQ ID NO: 2 or amino acids 64-79 of SEQ ID ID NO: 212 or amino acids 240-249 of SEQ ID NO: 213. 40 NO: 213, ii) amino acids 149-159 of SEQID NO: 2 or amino In some embodiments the nucleic acid molecules encode acids 149-159 of SEQID NO: 213, iii) amino acids 171-183 a PIP-1 polypeptide having a nucleotide sequence encoding of SEQ ID NO: 2 or amino acids 171-183 of SEQ ID NO: a polypeptide comprising an amino acid sequence having at 213 and iv) amino acids 240-249 of SEQID NO: 2 or amino least 80% identity to the amino acid sequence set forth in acids 240-249 of SEQID NO: 213. SEQID NO: 2, SEQID NO: 6 or SEQID NO.4 and wherein 45 In some embodiments the nucleic acid molecules encode the polypeptide comprises one or more amino acid motifs a PIP-1 polypeptide having a nucleotide sequence encoding selected from i) amino acids 64-79 of SEQID NO: 2, amino a polypeptide comprising an amino acid sequence having at acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ least 80% identity to the amino acid sequence set forth in ID NO: 212 or amino acids 64-79 of SEQ ID NO: 213, ii) SEQID NO: 2, and wherein the polypeptide comprises one amino acids 149-159 of SEQ ID NO: 2, amino acids 50 or more amino acid motifs selected from i) amino acids 149-159 of SEQ ID NO: 211, amino acids 149-159 of SEQ 64-79 of SEQ ID NO: 2 or amino acids 64-79 of SEQ ID ID NO: 212 or amino acids 149-159 of SEQID NO: 213, iii) NO: 213, ii) amino acids 149-159 of SEQID NO: 2 or amino amino acids 171-183 of SEQ ID NO: 2, amino acids acids 149-159 of SEQID NO: 213, iii) amino acids 171-183 171-183 of SEQ ID NO: 211, amino acids 171-183 of SEQ of SEQ ID NO: 2 or amino acids 171-183 of SEQ ID NO: ID NO: 212 or amino acids 171-183 of SEQ ID NO: 213, 55 213 and iv) amino acids 240-249 of SEQID NO: 2 or amino and iv) amino acids 240-249 of SEQID NO: 2, amino acids acids 240-249 of SEQID NO: 213. 240-249 of SEQID NO: 211, amino acids 240-249 of SEQ In some embodiments exemplary nucleic acid molecules ID NO: 212 or amino acids 240-249 of SEQ ID NO: 213. encode a PIP-1 polypeptide of SEQID NO: 101, 102, 103, In some embodiments the nucleic acid molecules encode 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, a PIP-1 polypeptide having a nucleotide sequence encoding 60 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, a polypeptide comprising an amino acid sequence having at 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 204, 206, 208,211, 212, 213, 214, 245, 246, 247, 248, 249, 99% or greater identity to the amino acid sequence set forth 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260,261, in SEQ ID NO: 2 and wherein the polypeptide comprises 65 262, 263,264, 265, 266, 267, 268, 269,298, 299, 300, 301, one or more amino acid motifs selected from i) amino acids 302,303, 304,305, 306, 307, 308, 309, 310,311, 312,313, 64-79 of SEQID NO: 2, amino acids 64-79 of SEQID NO: 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,324, and US 9,688,730 B2 37 38 325 as well as amino acid Substitutions, amino acid dele polypeptide will only be expressed if all required fragments tions, amino acid insertions and fragments thereof and are expressed in an environment that permits splicing pro combinations thereof. cesses to generate functional product. In another example, In some embodiments exemplary nucleic acid molecules introduction of one or more insertion sequences into a encode a PIP-1 polypeptide of SEQID NO: 101, 102, 103, polynucleotide can facilitate recombination with a low 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, homology polynucleotide; use of an intron or intein for the 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, insertion sequence facilitates the removal of the intervening 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, sequence, thereby restoring function of the encoded variant. 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, Nucleic acid molecules that are fragments of these nucleic 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 10 acid sequences encoding PIP-1 polypeptides are also encom 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, passed by the embodiments. By “fragment' is intended a 262, 263,264, 265, 266, 267, 268, and 269 as well as amino portion of the nucleic acid sequence encoding a PIP-1 acid substitutions, deletions, insertions and fragments polypeptide. A fragment of a nucleic acid sequence may thereof and combinations thereof. encode a biologically active portion of a PIP-1 polypeptide In some embodiments exemplary nucleic acid molecules 15 or it may be a fragment that can be used as a hybridization comprise a sequence set forth in SEQID NO: 152, 153, 154, probe or PCR primer using methods disclosed below. 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, Nucleic acid molecules that are fragments of a nucleic acid 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, sequence encoding a PIP-1 polypeptide comprise at least 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 190, about 50, 100, 200, 300, 400, 500, 600 or 700, contiguous 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, nucleotides or up to the number of nucleotides present in a 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, 228, full-length nucleic acid sequence encoding a PIP-1 polypep 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, 240, tide disclosed herein, depending upon the intended use. By 241, 242, 243, 244, 270, 271, 272,273, 274, 275,276, 277, “contiguous nucleotides is intended nucleotide residues 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, that are immediately adjacent to one another. Fragments of 290, 291, 292, 293, 294, 295, 296, and 297 as well as 25 the nucleic acid sequences of the embodiments will encode variants and fragments thereof encoding PIP-1 polypeptides. protein fragments that retain the biological activity of the In some embodiments exemplary nucleic acid molecules PIP-1 polypeptide and, hence, retain insecticidal activity. As comprise a sequence set forth in SEQID NO: 152, 153, 154, used herein, the term "pesticidal activity” refers to activity 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, of an organism or a Substance (Such as, for example, a 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 30 protein) that can be measured by, but is not limited to, pest 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 190, mortality, pest weight loss, pest repellency, and other behav 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, ioral and physical changes of a pest after feeding and 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, 228, exposure for an appropriate length of time. Thus, an organ 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, 240, ism or Substance having pesticidal activity adversely 241, 242, 243, and 244 as well as variants and fragments 35 impacts at least one measurable parameter of pest fitness. thereof encoding PIP-1 polypeptides. For example, "pesticidal proteins’ are proteins that display In some embodiments the nucleic acid molecules encode pesticidal activity by themselves or in combination with a PIP-1 polypeptide of Table 4, Table 6, Table 9, Table 12, other proteins. As used herein, the term “insecticidal activ Table 13, Table 14 and/or Table 16, combinations of the ity refers to activity of an organism or a Substance (such as, amino acid Substitutions thereof and deletions and/or inser 40 for example, a protein) that can be measured by, but is not tions thereof. limited to, insect mortality, insect weight loss, insect repel Also provided are nucleic acid molecules that encode lency, and other behavioral and physical changes of an insect transcription and/or translation products that are Subse after feeding and exposure for an appropriate length of time. quently spliced to ultimately produce functional PIP-1 poly Thus, an organism or Substance having insecticidal activity peptide. Splicing can be accomplished in vitro or in vivo, 45 adversely impacts at least one measurable parameter of and can involve cis- or trans-splicing. The Substrate for insect fitness. For example, “insecticidal proteins are pro splicing can be polynucleotides (e.g., RNA transcripts) or teins that display insecticidal activity by themselves or in polypeptides. An example of cis-splicing of a polynucleotide combination with other proteins. is where an intron inserted into a coding sequence is As used herein, the term "pesticidally effective amount removed and the two flanking exon regions are spliced to 50 connotes a quantity of a Substance or organism that has generate a PIP-1 polypeptide encoding sequence. An pesticidal activity when present in the environment of a pest. example of trans splicing would be where a polynucleotide For each Substance or organism, the pesticidally effective is encrypted by separating the coding sequence into two or amount is determined empirically for each pest affected in a more fragments that can be separately transcribed and then specific environment. Similarly, an “insecticidally effective spliced to form the full-length pesticidal encoding sequence. 55 amount may be used to refer to a "pesticidally effective The use of a splicing enhancer sequence, which can be amount when the pest is an insect pest. introduced into a construct, can facilitate splicing either in By “retains activity” is intended that the PIP-1A polypep cis or trans-splicing of polypeptides (U.S. Pat. Nos. 6,365, tide has at least about 10%, at least about 30%, at least about 377 and 6,531.316). Thus, in some embodiments the poly 50%, at least about 70%, 80%, 90%. 95% or higher of the nucleotides do not directly encode a full-length PIP-1 poly 60 insecticidal activity compared to the full-length PIP-1A peptide, but rather encode a fragment or fragments of a polypeptide (SEQID NO:2). In one embodiment, the insec PIP-1 polypeptide. These polynucleotides can be used to ticidal activity is against a Lepidoptera species. In another express a functional PIP-1 polypeptide through a mechanism embodiment, the insecticidal activity is against a Hemiptera involving splicing, where splicing can occur at the level of species. polynucleotide (e.g., intron/exon) and/or polypeptide (e.g., 65 In some embodiments a fragment of a nucleic acid intein/extein). This can be useful, for example, in controlling sequence encoding a PIP-1 polypeptide encoding a biologi expression of pesticidal activity, since functional pesticidal cally active portion of a protein will encode at least about 15, US 9,688,730 B2 39 40 25, 30, 50, 75, 100, 125, 150, 175, 200 or 250, contiguous sequences homologous to pesticidal-like nucleic acid mol amino acids or up to the total number of amino acids present ecules. BLAST protein searches can be performed with the in a full-length PIP-1 polypeptide of the embodiments. In BLASTX program, score=50, wordlength=3, to obtain Some embodiments, the fragment is an N-terminal or a amino acid sequences homologous to pesticidal protein C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, molecules. To obtain gapped alignments for comparison 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino purposes, Gapped BLAST (in BLAST 2.0) can be utilized as acids relative to SEQ ID NO: 2, 3 or 4 or variants thereof, described in Altschul, et al., (1997) Nucleic Acids Res. e.g., by proteolysis, insertion of a start codon, deletion of the 25:3389. Alternatively, PSI-Blast can be used to perform an codons encoding the deleted amino acids with the concomi iterated search that detects distant relationships between tant insertion of a stop codon or by insertion of a stop codon 10 molecules. See, Altschul, et al., (1997) supra. When utilizing in the coding sequence. In some embodiments, the frag BLAST, Gapped BLAST, and PSI-Blast programs, the ments encompassed herein result from the removal of the default parameters of the respective programs (e.g., N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, BLASTX and BLASTN) can be used. Alignment may also 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, be performed manually by inspection. 33, 34 or more amino acids relative to SEQID NO: 2, 3 or 15 Another non-limiting example of a mathematical algo 4 or variants thereof, e.g., by proteolysis or by insertion of rithm utilized for the comparison of sequences is the Clust a start codon in the coding sequence. alW algorithm (Higgins, et al., (1994) Nucleic Acids Res. In some embodiments the PIP-1 polypeptides are encoded 22:4673-4680). ClustalW compares sequences and aligns by a nucleic acid sequence Sufficiently identical to the the entirety of the amino acid or DNA sequence and thus can nucleic acid sequence of SEQ ID NO: 1, 3 or 5. By provide data about the sequence conservation of the entire “sufficiently identical is intended an amino acid or nucleic amino acid sequence. The ClustalW algorithm is used in acid sequence that has at least about 50%, 55%, 60%. 65%, several commercially available DNA/amino acid analysis 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, software packages, such as the ALIGNX module of the 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Vector NTI Program Suite (Invitrogen Corporation, Carls 98%, 99% or greater sequence identity compared to a 25 bad, Calif.). After alignment of amino acid sequences with reference sequence using one of the alignment programs ClustalW, the percent amino acid identity can be assessed. A described herein using standard parameters. In some non-limiting example of a software program useful for embodiments the sequence homology identity is against the analysis of ClustalW alignments is GENEDOCTM. GENE full length sequence of the polynucleotide encoding a PIP-1 DOCTM (Karl Nicholas) allows assessment of amino acid (or polypeptide or against the full length sequence of a PIP-1 30 DNA) similarity and identity between multiple proteins. polypeptide. In some embodiments the PIP-1 polypeptide Another non-limiting example of a mathematical algorithm has at least about 50%, 55%, 60%. 65%, 70%, 75%, 80%, utilized for the comparison of sequences is the algorithm of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Myers and Miller, (1988) CABIOS 4:11-17. Such an algo 91%, 92%,93%, 94%.95%,96%,97%.98%, 99% or greater rithm is incorporated into the ALIGN program (version 2.0), sequence identity compared to SEQID NO: 2, SEQID NO: 35 which is part of the GCG Wisconsin Genetics Software 4, SEQ ID NO: 332 or SEQ ID NO: 6. One of skill in the Package, Version 10 (available from Accelrys, Inc., 9685 art will recognize that these values can be appropriately Scranton Rd., San Diego, Calif., USA). When utilizing the adjusted to determine corresponding identity of proteins ALIGN program for comparing amino acid sequences, a encoded by two nucleic acid sequences by taking into PAM120 weight residue table, a gap length penalty of 12 and account codon degeneracy, amino acid similarity, reading 40 a gap penalty of 4 can be used. frame positioning, and the like. Another non-limiting example of a mathematical algo To determine the percent identity of two amino acid rithm utilized for the comparison of sequences is the algo sequences or of two nucleic acids, the sequences are aligned rithm of Needleman and Wunsch, (1970) J. Mol. Biol. for optimal comparison purposes. The percent identity 48(3):443-453, used GAP Version 10 software to determine between the two sequences is a function of the number of 45 sequence identity or similarity using the following default identical positions shared by the sequences (i.e., percent parameters: % identity and % similarity for a nucleic acid identity=number of identical positions/total number of posi sequence using GAP Weight of 50 and Length Weight of 3 tions (e.g., overlapping positions)x100). In one embodi and the nwsgapdna.cmpii scoring matrix; % identity or % ment, the two sequences are the same length. In another similarity for an amino acid sequence using GAP weight of embodiment, the comparison is across the entirety of the 50 8 and length weight of 2, and the BLOSUM62 scoring reference sequence (e.g., across the entirety of SEQID NO: program. Equivalent programs may also be used. By 1, 331 or 3 or across the entirety of one of SEQ ID NO: 2, “equivalent program' is intended any sequence comparison 332 or 4). The percent identity between two sequences can program that, for any two sequences in question, generates be determined using techniques similar to those described an alignment having identical nucleotide residue matches below, with or without allowing gaps. In calculating percent 55 and an identical percent sequence identity when compared to identity, typically exact matches are counted. the corresponding alignment generated by GAP Version 10. The determination of percent identity between two The embodiments also encompass nucleic acid molecules sequences can be accomplished using a mathematical algo encoding variants of PIP-1 polypeptide. “Variants’ of the rithm. A non-limiting example of a mathematical algorithm PIP-1 polypeptide encoding nucleic acid sequences include utilized for the comparison of two sequences is the algo 60 those sequences that encode the PIP-1 polypeptides dis rithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. closed herein but that differ conservatively because of the USA 87:2264, modified as in Karlin and Altschul, (1993) degeneracy of the genetic code as well as those that are Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm Sufficiently identical as discussed above. Naturally occurring is incorporated into the BLASTN and BLASTX programs of allelic variants can be identified with the use of well-known Altschul, et al., (1990) J. Mol. Biol. 215:403. BLAST 65 molecular biology techniques, such as polymerase chain nucleotide searches can be performed with the BLASTN reaction (PCR) and hybridization techniques as outlined program, score=100, wordlength=12, to obtain nucleic acid below. Variant nucleic acid sequences also include syntheti US 9,688,730 B2 41 42 cally derived nucleic acid sequences that have been gener encode proteins with or which confer desirable properties. ated, for example, by using site-directed mutagenesis but Following diversification by one or more of the methods which still encode the PIP-1 polypeptides disclosed as herein or otherwise available to one of skill, any nucleic discussed below. acids that are produced can be selected for a desired activity The skilled artisan will further appreciate that changes can 5 or property, e.g. pesticidal activity or, such activity at a be introduced by mutation of the nucleic acid sequences desired pH, etc. This can include identifying any activity that thereby leading to changes in the amino acid sequence of the can be detected, for example, in an automated or automat encoded PIP-1 polypeptides, without altering the biological able format, by any of the assays in the art, see, e.g., activity of the proteins. Thus, variant nucleic acid molecules discussion of Screening of insecticidal activity, infra. A can be created by introducing one or more nucleotide 10 variety of related (or even unrelated) properties can be Substitutions, additions or deletions into the corresponding evaluated, in serial or in parallel, at the discretion of the nucleic acid sequence disclosed herein, such that one or practitioner. more amino acid substitutions, additions or deletions are Descriptions of a variety of diversity generating proce introduced into the encoded protein. Mutations can be dures for generating modified nucleic acid sequences, e.g., introduced by Standard techniques, such as site-directed 15 those coding for polypeptides having pesticidal activity or mutagenesis and PCR-mediated mutagenesis. Such variant fragments thereof, are found in the following publications nucleic acid sequences are also encompassed by the present and the references cited therein: Soong, et al., (2000) Nat invention. Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Tar Alternatively, variant nucleic acid sequences can be made geting 4:1-4; Ness et al. (1999) Nat Biotechnol 17:893-896; by introducing mutations randomly along all or part of the Chang et al. (1999) Nat Biotechnol 17:793-797; Minshull coding sequence, Such as by Saturation mutagenesis and the and Stemmer, (1999) Curr Opin Chem Biol 3:284-290; resultant mutants can be screened for ability to confer Christians, et al., (1999) Nat Biotechnol 17:259-264; Cra pesticidal activity to identify mutants that retain activity. meri, et al., (1998) Nature 391:288-291; Crameri, et al., Following mutagenesis, the encoded protein can be (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) expressed recombinantly, and the activity of the protein can 25 PNAS USA 94:4504-4509: Patten, et al., (1997) Curr Opin be determined using standard assay techniques. Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med The polynucleotides of the disclosure and fragments 2:100-103: Crameri, et al., (1996) Nat Biotechnol 14:315 thereof are optionally used as substrates for a variety of 319; Gates, et al., (1996).J Mol Biol 255:373-386; Stemmer, recombination and recursive recombination reactions, in (1996) “Sexual PCR and Assembly PCR” In: The Encyclo addition to standard cloning methods as set forth in, e.g., 30 pedia of Molecular Biology. VCH Publishers, New York. pp. Ausubel, Berger and Sambrook, i.e., to produce additional 447-457; Crameri and Stemmer, (1995) BioTechniques pesticidal polypeptide homologues and fragments thereof 18:194-195; Stemmer, et al., (1995) Gene, 164:49-53: Stem with desired properties. A variety of such reactions are mer, (1995) Science 270:1510; Stemmer, (1995) Bio/Tech known, including those developed by the inventors and their nology 13:549-553; Stemmer, (1994) Nature 370:389-391 co-workers. Methods for producing a variant of any nucleic 35 and Stemmer, (1994) PNAS USA 91: 10747-10751. acid listed herein comprising recursively recombining Such Mutational methods of generating diversity include, for polynucleotide with a second (or more) polynucleotide, thus example, site-directed mutagenesis (Ling, et al., (1997) Anal forming a library of variant polynucleotides are also Biochem 254(2): 157-178; Dale, et al., (1996) Methods Mol embodiments of the disclosure, as are the libraries produced, Biol 57:369-374; Smith, (1985) Ann Rev. Genet 19:423-462: the cells comprising the libraries, and any recombinant 40 Botstein and Shortle, (1985) Science 229:1193-1201; Carter, polynucleotide produces by Such methods. Additionally, (1986) Biochem J 237:1-7 and Kunkel, (1987) “The effi Such methods optionally comprise selecting a variant poly ciency of oligonucleotide directed mutagenesis' in Nucleic nucleotide from such libraries based on pesticidal activity, as Acids & Molecular Biology (Eckstein and Lilley, eds., is wherein such recursive recombination is done in vitro or Springer Verlag, Berlin)); mutagenesis using uracil contain in vivo. 45 ing templates (Kunkel, (1985) PNAS USA 82:488-492; Kun A variety of diversity generating protocols, including kel, et al., (1987) Methods Enzymol 154:367-382 and Bass, nucleic acid recursive recombination protocols are available et al., (1988) Science 242:240-245); oligonucleotide-di and fully described in the art. The procedures can be used rected mutagenesis (Zoller and Smith, (1983) Methods separately, and/or in combination to produce one or more Enzymol 100:468-500; Zoller and Smith, (1987) Methods variants of a nucleic acid or set of nucleic acids, as well as 50 Enzymol 154:329-350; Zoller and Smith, (1982) Nucleic variants of encoded proteins. Individually and collectively, Acids Res 10:6487-6500), phosphorothioate-modified DNA these procedures provide robust, widely applicable ways of mutagenesis (Taylor, et al., (1985) NuclAcids Res 13:8749 generating diversified nucleic acids and sets of nucleic acids 8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (including, e.g., nucleic acid libraries) useful, e.g., for the (1985); Nakamaye and Eckstein (1986) Nucl Acids Res engineering or rapid evolution of nucleic acids, proteins, 55 14:9679–9698; Sayers, et al., (1988) Nucl Acids Res 16:791 pathways, cells and/or organisms with new and/or improved 802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); characteristics. mutagenesis using gapped duplex DNA (Kramer, et al., While distinctions and classifications are made in the (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, course of the ensuing discussion for clarity, it will be (1987) Methods Enzymol 154:350-367; Kramer, et al., appreciated that the techniques are often not mutually exclu 60 (1988) Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl sive. Indeed, the various methods can be used singly or in Acids Res 16:6987-6999). combination, in parallel or in series, to access diverse Additional suitable methods include point mismatch sequence variants. repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis The result of any of the diversity generating procedures using repair-deficient host strains (Carter, et al., (1985) Nucl described herein can be the generation of one or more 65 Acids Res 13:4431-4443 and Carter, (1987) Methods in nucleic acids, which can be selected or screened for nucleic Enzymol 154:382-403), deletion mutagenesis (Eghtedarza acids with or which confer desirable properties or that deh and Henikoff, (1986) Nucl Acids Res 14:5115), restric US 9,688,730 B2 43 44 tion-selection and restriction-purification (Wells, et al., PSEEN3174 (SEQID NO: 6), PIP-1B (SEQ ID NO: 4) and (1986) Phil Trans RSoc Lond A317:415-423), mutagenesis PIP-1C (SEQ ID NO: 332) proteins using Western blotting by total gene synthesis (Nambiar, et al., (1984) Science and/or ELISA methods. This type of assays can be per 223:1299-1301: Sakamar and Khorana, (1988) Nucl Acids formed in a high throughput fashion. Positive samples can Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 be further analyzed by various techniques such as antibody and Grundstrom, et al., (1985) Nucl Acids Res 13:3305 based protein purification and identification. Methods of 3316), double-strand break repair (Mandecki, (1986) PNAS generating antibodies are well known in the art as discussed USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech infra. 4:450-455). Additional details on many of the above meth Alternatively, mass spectrometry based protein identifi ods can be found in Methods Enzymol Volume 154, which 10 cation method can be used to identify homologs of PIP-1A also describes useful controls for trouble-shooting problems (SEQID NO: 2) using protocols in the literatures (Patterson, with various mutagenesis methods. (1998), 10(22): 1-24, Current Protocol in Molecular Biology Additional details regarding various diversity generating published by John Wiley & Son Inc). Specifically, LC-MS/ methods can be found in the following US Patents, PCT MS based protein identification method is used to associate Publications and Applications and EPO Publications: U.S. 15 the MS data of given cell lysate or desired molecular weight Pat. No. 5,723,323, U.S. Pat. No. 5,763,192, U.S. Pat. No. enriched samples (excised from SDS-PAGE gel of relevant 5,814,476, U.S. Pat. No. 5,817,483, U.S. Pat. No. 5,824,514, molecular weight bands to PIP-1A protein) with sequence U.S. Pat. No. 5,976,862, U.S. Pat. No. 5,605,793, U.S. Pat. information of PIP-1A (SEQ ID NO: 2) and its homologs. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. No. Any match in peptide sequences indicates the potential of 5,834,252, U.S. Pat. No. 5,837,458, WO 1995/22625, WO having the homologous proteins in the samples. Additional 1996/33207, WO 1997/20078, WO 1997/35966, WO 1999/ techniques (protein purification and molecular biology) can 41402, WO 1999/41383, WO 1999/41369, WO 1999/41368, be used to isolate the protein and identify the sequences of EP 752008, EP 0932670, WO 1999/23107, WO 1999/ the homologs. 21979, WO 1998/31837, WO 1998/27230, WO 1998/27230, In hybridization methods, all or part of the pesticidal WO 2000/00632, WO 2000/09679, WO 1998/42832, WO 25 nucleic acid sequence can be used to screen cDNA or 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/ genomic libraries. Methods for construction of such cDNA 42727, WO 2000/18906, WO 2000/04190, WO 2000/42561, and genomic libraries are generally known in the art and are WO 2000/42559, WO 2000/42560, WO 2001/23401, and disclosed in Sambrook and Russell. (2001), supra. The PCT/USO1/06775. so-called hybridization probes may be genomic DNA frag The nucleotide sequences of the embodiments can also be 30 ments, cDNA fragments, RNA fragments or other oligo used to isolate corresponding sequences from other organ nucleotides, and may be labeled with a detectable group isms, particularly other bacteria, particularly a Pseudomonas such as 32P or any other detectable marker, such as other species and more particularly a Pseudomonas chlororaphis radioisotopes, a fluorescent compound, an enzyme or an strain. In this manner, methods such as PCR, hybridization enzyme co-factor. Probes for hybridization can be made by and the like can be used to identify Such sequences based on 35 labeling synthetic oligonucleotides based on the known their sequence homology to the sequences set forth herein. PIP-1 polypeptide-encoding nucleic acid sequence disclosed Sequences that are selected based on their sequence identity herein. Degenerate primers designed on the basis of con to the entire sequences set forth herein or to fragments served nucleotides or amino acid residues in the nucleic acid thereof are encompassed by the embodiments. Such sequence or encoded amino acid sequence can additionally sequences include sequences that are orthologs of the dis 40 be used. The probe typically comprises a region of nucleic closed sequences. The term “orthologs’ refers to genes acid sequence that hybridizes under stringent conditions to derived from a common ancestral gene and which are found at least about 12, at least about 25, at least about 50, 75, 100, in different species as a result of speciation. Genes found in 125, 150, 175 or 200 consecutive nucleotides of nucleic acid different species are considered orthologs when their nucleo sequence encoding a PIP-1 polypeptide of the disclosure or tide sequences and/or their encoded protein sequences share 45 a fragment or variant thereof. Methods for the preparation of substantial identity as defined elsewhere herein. Functions probes for hybridization are generally known in the art and of orthologs are often highly conserved among species. are disclosed in Sambrook and Russell, (2001), supra, herein In a PCR approach, oligonucleotide primers can be incorporated by reference. designed for use in PCR reactions to amplify corresponding For example, an entire nucleic acid sequence, encoding a DNA sequences from cDNA or genomic DNA extracted 50 PIP-1 polypeptide, disclosed herein or one or more portions from any organism of interest. Methods for designing PCR thereof, may be used as a probe capable of specifically primers and PCR cloning are generally known in the art and hybridizing to corresponding nucleic acid sequences encod are disclosed in Sambrook, et al., (1989) Molecular Clon ing PIP-1 polypeptide-like sequences and messenger RNAS. ing: A Laboratory Manual (2d ed., Cold Spring Harbor To achieve specific hybridization under a variety of condi Laboratory Press, Plainview, N.Y.), hereinafter “Sambrook”. 55 tions, such probes include sequences that are unique and are See also, Innis, et al., eds. (1990) PCR Protocols: A Guide preferably at least about 10 nucleotides in length or at least to Methods and Applications (Academic Press, New York); about 20 nucleotides in length. Such probes may be used to Innis and Gelfand, eds. (1995) PCR Strategies (Academic amplify corresponding pesticidal sequences from a chosen Press, New York); and Innis and Gelfand, eds. (1999) PCR organism by PCR. This technique may be used to isolate Methods Manual (Academic Press, New York). Known 60 additional coding sequences from a desired organism or as methods of PCR include, but are not limited to, methods a diagnostic assay to determine the presence of coding using paired primers, nested primers, single specific primers, sequences in an organism. Hybridization techniques include degenerate primers, gene-specific primers, vector-specific hybridization screening of plated DNA libraries (either primers, partially-mismatched primers, and the like. plaques or colonies; see, for example, Sambrook, et al., To identify potential PIP-1 polypeptides from bacterial 65 (1989) Molecular Cloning: A Laboratory Manual (2d ed., collections, the bacterial cell lysates can be screened with Cold Spring Harbor Laboratory Press, Cold Spring Harbor, antibodies generated against PIP-1A (SEQ ID NO: 2), N.Y.). US 9,688,730 B2 45 46 Hybridization of Such sequences may be carried out under described. If the desired degree of mismatching results in a stringent conditions. By "stringent conditions' or 'stringent Tm of less than 45° C. (aqueous solution) or 32° C. hybridization conditions” is intended conditions under (formamide solution), it is preferred to increase the SSC which a probe will hybridize to its target sequence to a concentration so that a higher temperature can be used. An detectably greater degree than to other sequences (e.g., at extensive guide to the hybridization of nucleic acids is found least 2-fold over background). Stringent conditions are in Tijssen, (1993) Laboratory Techniques in Biochemistry sequence-dependent and will be different in different cir and Molecular Biology—Hybridization with Nucleic Acid cumstances. By controlling the Stringency of the hybridiza Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al., tion and/or washing conditions, target sequences that are eds. (1995) Current Protocols in Molecular Biology, Chapter 100% complementary to the probe can be identified (ho 10 2 (Greene Publishing and Wiley-Interscience, New York). mologous probing). Alternatively, stringency conditions can See, Sambrook, et al., (1989) Molecular Cloning: A Labo be adjusted to allow some mismatching in sequences so that ratory Manual (2d ed., Cold Spring Harbor Laboratory lower degrees of similarity are detected (heterologous prob Press, Cold Spring Harbor, N.Y.). ing). Generally, a probe is less than about 1000 nucleotides Proteins and Variants and Fragments Thereof in length, preferably less than 500 nucleotides in length. 15 Pseudomonas Insecticidal Protein-1 (PIP-1) polypeptides Typically, stringent conditions will be those in which the are also encompassed by the disclosure. By "Pseudomonas salt concentration is less than about 1.5 M Naion, typically Insecticidal Protein-1”, “PIP-1 polypeptide' or “PIP-1 pro about 0.01 to 1.0 M Na ion concentration (or other salts) at tein’ is intended a polypeptide that retains insecticidal pH 7.0 to 8.3 and the temperature is at least about 30°C. for activity against one or more insect pests of the Lepidoptera short probes (e.g., 10 to 50 nucleotides) and at least about and/or Hemiptera orders compared to, and including, the 60° C. for long probes (e.g., greater than 50 nucleotides). protein of SEQID NO: 2, and is sufficiently homologous to, Stringent conditions may also be achieved with the addition and includes, the protein of SEQ ID NO: 2. A variety of of destabilizing agents such as formamide. Exemplary low PIP-1 polypeptides are contemplated. One source of poly stringency conditions include hybridization with a buffer peptides that encode a PIP-1 polypeptide or related proteins solution of 30 to 35% formamide, 1 M NaCl, 1% SDS 25 is a Pseudomonas chlororaphis strain which comprises the (sodium dodecyl sulphate) at 37° C., and a wash in 1x to polynucleotide of SEQ ID NO: 1 encoding the PIP-1 poly 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 peptide of SEQ ID NO: 2. to 55° C. Exemplary moderate stringency conditions include As used herein, the terms “protein,” “peptide molecule' hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% or “polypeptide' includes any molecule that comprises five SDS at 37° C., and a wash in 0.5x to 1XSSC at 55 to 60° C. 30 or more amino acids. It is well known in the art that protein, Exemplary high Stringency conditions include hybridization peptide or polypeptide molecules may undergo modifica in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash tion, including post-translational modifications, such as, but in 0.1xSSC at 60 to 65° C. Optionally, wash buffers may not limited to, disulfide bond formation, glycosylation, comprise about 0.1% to about 1% SDS. Duration of hybrid phosphorylation or oligomerization. Thus, as used herein, ization is generally less than about 24 hours, usually about 35 the terms “protein,” “peptide molecule' or “polypeptide' 4 to about 12 hours. includes any protein that is modified by any biological or Specificity is typically the function of post-hybridization non-biological process. The terms "amino acid and "amino washes, the critical factors being the ionic strength and acids’ refer to all naturally occurring L-amino acids. temperature of the final wash solution. For DNA-DNA A “recombinant protein' is used to refer to a protein that hybrids, the Tm can be approximated from the equation of 40 is no longer in its natural environment, for example in vitro Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: or in a recombinant bacterial or plant host cell. A PIP-1 Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)- polypeptide that is substantially free of cellular material 500/L: where M is the molarity of monovalent cations, '% includes preparations of protein having less than about 30%, GC is the percentage of guanosine and cytosine nucleotides 20%, 10% or 5% or less (by dry weight) of non-pesticidal in the DNA,% form is the percentage of formamide in the 45 protein (also referred to herein as a “contaminating pro hybridization solution, and L is the length of the hybrid in tein'). base pairs. The Tm is the temperature (under defined ionic “Fragments’ or “biologically active portions” include strength and pH) at which 50% of a complementary target polypeptide fragments comprising amino acid sequences sequence hybridizes to a perfectly matched probe. Tm is sufficiently identical to a PIP-1 polypeptide and that exhibit reduced by about 1° C. for each 1% of mismatching; thus, 50 insecticidal activity. "Fragments’ or “biologically active Tm, hybridization, and/or wash conditions can be adjusted portions' include polypeptide fragments comprising amino to hybridize to sequences of the desired identity. For acid sequences sufficiently identical to the amino acid example, if sequences with 90% identity are sought, the Tm sequence set forth in SEQID NO: 2, 4, 332 and 6 including can be decreased 10° C. Generally, stringent conditions are but not limited to SEQ ID NO: 204, 206 and 208 and that selected to be about 5° C. lower than the thermal melting 55 exhibit insecticidal activity. A biologically active portion of point (Tm) for the specific sequence and its complement at a PIP-1 polypeptide can be a polypeptide that is, for a defined ionic strength and pH. However, severely stringent example, 10, 25, 50, 100, 150, 200, 250 or more amino acids conditions can utilize a hybridization and/or wash at 1, 2, 3 in length. Such biologically active portions can be prepared or 4°C. lower than the thermal melting point (Tm); mod by recombinant techniques and evaluated for insecticidal erately stringent conditions can utilize a hybridization and/or 60 activity. As used here, a fragment comprises at least 8 wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting contiguous amino acids of a PIP-1 polypeptide. In some point (Tm); low stringency conditions can utilize a hybrid embodiments a fragment comprises at least 8 contiguous ization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than amino acids of SEQ ID NO: 2 or 4. In some embodiments the thermal melting point (Tm). Using the equation, hybrid a fragment comprises at least 8 contiguous amino acids of ization and wash compositions, and desired Tm, those of 65 SEQID NO: 2. In some embodiments a fragment comprises ordinary skill will understand that variations in the strin at least 8 contiguous amino acids of SEQ ID NO: 4. The gency of hybridization and/or wash Solutions are inherently embodiments encompass other fragments, however, such as US 9,688,730 B2 47 48 any fragment in the protein greater than about 10, 20, 30, 50. comprises an amino acid sequence having at least 80% 100, 150, 200, 250 or more amino acids. identity to the amino acid sequence of SEQ ID NO: 2, In Some embodiments, the fragment is an N-terminal wherein the polypeptide has insecticidal activity. and/or a C-terminal truncation of at least about 1, 2, 3, 4, 5, In some embodiments a PIP-1 polypeptide comprises an 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acid sequence of SEQ ID NO: 211, wherein Xaa at amino acids relative to SEQ ID NO: 2 or 4 or variants position 2 is Pro or Thr; Xaa at position 8 is Ser, Gly or Asn; thereof e.g., by proteolysis, by insertion of a start codon, by Xaa at position 19 is Asp, Glu or Cys: Xaa at position 20 is deletion of the codons encoding the deleted amino acids and Leu or Val; Xaa at position 21 is Lys, Ser or ASn; Xaa at concomitant insertion of a start codon and/or insertion of a position 22 is Ser, Lys or Arg; Xaa at position 24 is Glin or stop codon. In some embodiments, the fragments encom 10 passed herein result from the removal of the N-terminal 1, Ala; Xaa at position 25 is Gly or Ala Xaa at position 26 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, Ser or Asn; Xaa at position 27 is Leu, Thr or Ala; Xaa at 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or position 30 is Ala or Ile: Xaa at position 35 is Phe or Leu: more amino acids relative to SEQ ID NO: 2 or 4, and Xaa at position 36 is Ala, Ser or Val: Xaa at position 38 is variants thereof (e.g., SEQ ID NO. 204, 206, 208 and 330), 15 Asn, Arg or Ser; Xaa at position 42 is Phe or Tyr; Xaa at e.g., by proteolysis or by insertion of a start codon, by position 46 is Arg, Lys or His; Xaa at position 48 is Gly or deletion of the codons encoding the deleted amino acids and Asp; Xaa at position 49 is Phe or Tyr; Xaa at position 53 is concomitant insertion of a start codon. In particular embodi Ser or Gly; Xaa at position 58 is Tyr or Phe, Xaa at position ments the proteolytic cleavage site is between Ser34 and 60 is Ala or Ser; Xaa at position 63 is Glin or Lys; Xaa at Asn35 of SEQ ID NO: 2 or variants thereof. In some position 77 is Phe or Tyr; Xaa at position 97 is Met or Val; embodiments the truncation is of the first 34 amino acids of Xaa at position 98 is Asp or Glu; Xaa at position 105 is Gln SEQID NO: 2 resulting in a PIP-1 polypeptide from amino or ASn; Xaa at position 107 is Thr or Ile: Xaa at position 108 acids 35-271 of SEQ ID NO: 2. It is well known in the art is Gln or Thr; Xaa at position 110 is Arg or Leu; Xaa at that polynucleotides encoding the truncated PIP-1 polypep position 120 is Lys, Arg or Glin; Xaa at position 121 is Thr tides can be engineered to add a start codon at the N-ter 25 or Ser; Xaa at position 123 is Thr or Glu; Xaa at position 125 minus Such as ATG encoding methionine or methionine is Asn or Ser; Xaa at position 127 is Ser, Asn. Thr or Lys: followed by an alanine. It is also well known in the art that Xaa at position 134 is Gly or Ala; Xaa at position 135 is Ser, depending on what host the PIP-1 polypeptide is expressed Asin or Lys; Xaa at position 137 is Asp or Gly; Xaa at in the methionine may be partially of completed processed position 141 is Val or Ile: Xaa at position 142 is Gly or Asp; off. 30 In some embodiments fragments, biologically active por Xaa at position 144 is Asp or Glu, Xaa at position 147 is Ile, tions of SEQID NO: 2 or 4, including but not limited to SEQ Thr or Val: Xaa at position 150 is Seror Thr; Xaa at position ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 151 is Asn, Arg or Ser; Xaa at position 160 is Thr or Ser; Xaa 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, at position 162 is Ser or Thr; Xaa at position 163 is Asn., Asp 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 35 or Glu; Xaa at position 164 is Seror Thr; Xaa at position 166 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, is Gln or Glu, Xaa at position 167 is Leu or Met; Xaa at 147, 148, 149, 150, 151, 204, 206, 208, 211, 212, 213, 214, position 168 is Thr, Lys or Ala; Xaa at position 174 is Ile, Val 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, or Met; Xaa at position 175 is Val or Ile: Xaa at position 180 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, is Met or Leu; Xaa at position 191 is Arg or Lys: Xaa at and 269, as well as amino acid substitutions, deletions 40 position 194 is Gly or Ala; Xaa at position 200 is Asn or Ser; and/or insertions thereof are also provided, and may be used Xaa at position 203 is ASnor Gln; Xaa at position 204 is Thr to practice the methods of the disclosure. or Ala; Xaa at position 206 is Gly or Asp; Xaa at position By variants is intended proteins or polypeptides having an 209 is Leu or Val: Xaa at position 220 is Asn or Arg; Xaa at amino acid sequence that is at least about 50%, 55%, 60%, position 221 is Ser or Lys: Xaa at position 222 is Thr or Arg; 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 45 Xaa at position 226 is Asp, Pro or Glu; Xaa at position 228 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, is Ser or Gly; Xaa at position 229 is Lys or Asn; Xaa at 97%, 98% or 99% identical to the parental amino acid position 231 is Ile or Val: Xaa at position 232 is Ala, Thr or sequence. In some embodiments a PIP-1 polypeptide has at Glu; and Xaa at position 251 is Gly, Ser or Glu; Xaa at least about 60%, 65%, about 70%, 75%, at least about 80%, position 254 is Ser or Asn; Xaa at position 258 is Ser or Arg; 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 50 Xaa at position 265 is ASn or Asp; and Xaa at position 266 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% is Asp or ASn; and amino acid deletions, amino acid inser identity across the entire length of the amino acid sequence tions, and fragments thereof, and combinations thereof. of SEQID NO: 2, SEQ ID NO: 332 or SEQ ID NO: 4. In In some embodiments a PIP-1 polypeptide comprises an some embodiments a PIP-1 polypeptide has at least about amino acid sequence of SEQ ID NO: 211 having 1, 2, 3, 4, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 55 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, identity across the entire length of the amino acid sequence 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, of SEQ ID NO: 2. 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, in any In some embodiments a PIP-1 polypeptide comprises an combination, at residues designated by Xaa in SEQID NO: amino acid sequence having at least 80% identity, to the 60 211 compared to the native amino acid at the corresponding amino acid sequence of SEQID NO: 2, SEQID NO: 332 or position of SEQ ID NO: 2. SEQ ID NO:4, wherein the polypeptide has insecticidal In some embodiments a PIP-1 polypeptide comprises an activity. In some embodiments a PIP-1 polypeptide com amino acid sequence of SEQ ID NO: 211 having 1, 2, 3, 4, prises an amino acid sequence having at least 80% identity 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, to the amino acid sequence of SEQID NO: 2, SEQID NO: 65 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 332 or SEQ ID NO: 4, wherein the polypeptide has insec 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or ticidal activity. In some embodiments a PIP-1 polypeptide 54 amino acid Substitutions, in any combination, at residues US 9,688,730 B2 49 50 designated by Xaa in SEQ ID NO: 211 compared to the position 228 is Seror Gly: Xaa at position 229 is Lys or Asn; native amino acid at the corresponding position of SEQ ID Xaa at position 231 is Ile or Val: Xaa at position 232 is Ala, NO: 2. Thr or Glu, Xaa at position 240 is Gln, Arg, Ala, Val, Glu, In some embodiments a PIP-1 polypeptide comprises an Met, Gly, Asp, Trp, Asn. Thr, Ile, Ser, Phe, His, Cys or Leu: amino acid sequence of SEQ ID NO: 212, wherein Xaa at 5 Xaa at position 241 is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, position 2 is Pro or Thr; Xaa at position 3 is Ile or Thr; Xaa Tyr, Met, Asn. His, Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa at position 6 is Glu or Gly; Xaa at position 8 is Ser, Gly or at position 242 is ASn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, ASn; Xaa at position 19 is Asp, Glu or Cys; Xaa at position Trp, Gln, Thr, Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at 20 is Leu or Val: Xaa at position 21 is Lys, Ser or Asn; Xaa position 243 is Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; at position 22 is Ser, Lys or Arg; Xaa at position 24 is Gln 10 Xaa at position 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at position 25 is Gly or Ala; Xaa at position 26 or Ala; Xaa at position 245 is Met, Ala, Arg, Asp, Glu, Leu, is Ser or ASn; Xaa at position 27 is Leu, Thr or Ala; Xaa at Pro, Ser, Trp, Gly, Val, Lys, Phe, Cys, Thr. His, Ile, Gln, Tyr position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, or ASn; Xaa at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala or Ala, Trp, Gln, Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr Ile: Xaa at position 35 is Phe or Leu; Xaa at position 36 is 15 or Lys, Xaa at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, Ala, Ser or Val; Xaa at position 38 is Asn, Arg or Ser; Xaa His, Arg, Lys, Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr at position 42 is Phe or Tyr; Xaa at position 43 is Pro, Met, or Cys; Xaa at position 248 is Tyr, Val, Thr, Glu, Phe, Ser, Gly, Gln, Ser, Thr, Arg, Val, Leu, Lys, Asp, Ala, ASn, Phe, His, Cys, Leu, Trp, Ile, Asp, Gly or Ala; Xaa at position 249 Trp, Glu or Cys; Xaa at position 46 is Arg, Lys or His; Xaa is Asn. Lys, Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, at position 48 is Gly or Asp; Xaa at position 49 is Phe, Tyr Ser, Ile, Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 or Leu; Xaa at position 53 is Ser or Gly: Xaa at position 58 is Gly, Ser or Glu; Xaa at position 254 is Ser or ASn; Xaa at is Tyr or Phe; Xaa at position 60 is Ala or Ser; Xaa at position 258 is Ser or Arg; Xaa at position 259 is Phe, Trp, position 63 is Glin or Lys: Xaa at position 66 is Trp, Tyr, Phe, Tyr, Cys, Met, Leu, Val, Ile or His: Xaa at position 265 is Arg, Lys, His, Ile, Val or Ser; Xaa at position 77 is Phe or ASn or Asp, and Xaa at position 266 is Asp or ASn; and Tyr; Xaa at position 89 is Pro, Leu, Gly, Arg, Thr, Ser, Met, 25 amino acid deletions, amino acid insertions, and fragments Ala, Ile, Asn. Val, Cys or Lys; Xaa at position 93 is Tyr, Cys, thereof, and combinations thereof. Trp, Val, Asp, Asn. Ile, Leu, Met, Phe, Ala or Thr; Xaa at In some embodiments a PIP-1 polypeptide comprises an position 97 is Met or Val; Xaa at position 98 is Asp or Glu; amino acid sequence of SEQ ID NO: 212 having 1, 2, 3, 4, Xaa at position 105 is Gln or Asn; Xaa at position 107 is Thr 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or Ile: Xaa at position 108 is Glin or Thr; Xaa at position 110 30 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, is Arg or Leu, Xaa at position 120 is Lys, Arg or Glin; Xaa 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, at position 121 is Thr or Ser; Xaa at position 123 is Thr or 55, 56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, Glu; Xaa at position 125 is Asn or Ser; Xaa at position 127 71, 72,73, 74, 75, 76, 77,78, 79,80, 81, 82, 83, 84, 85, 86, is Ser, Asn. Thr or Lys: Xaa at position 134 is Gly or Ala; 87, 88 or 89 amino acid substitutions, in any combination, Xaa at position 135 is Ser, Asn or Lys; Xaa at position 137 35 at residues designated by Xaa in SEQID NO: 212 compared is Asp or Gly; Xaa at position 141 is Val or Ile: Xaa at to the native amino acid at the corresponding position of position 142 is Gly or Asp; Xaa at position 144 is Asp or SEQ ID NO: 2. Glu; Xaa at position 147 is Ile, Thr or Val: Xaa at position In some embodiments a PIP-1 polypeptide comprises an 150 is Seror Thr; Xaa at position 151 is Asn, Arg or Ser; Xaa amino acid sequence of SEQ ID NO: 212 having 1, 2, 3, 4, at position 160 is Thr or Ser; Xaa at position 162 is Ser or 40 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Thr; Xaa at position 163 is Asn, Asp or Glu, Xaa at position 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 164 is Ser or Thr; Xaa at position 166 is Glin or Glu; Xaa at 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or position 167 is Leu or Met; Xaa at position 168 is Thr, Lys 54 amino acid Substitutions, in any combination, at residues or Ala; Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn. designated by Xaa in SEQ ID NO: 212 compared to the Asp, Ser or Ala; Xaa at position 172 is Thr, Gly. His, Phe, 45 native amino acid at the corresponding position of SEQ ID Glu, Arg, Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; NO: 2. Xaa at position 173 is Phe, Gly. His, Leu, Ala, Arg, Asn. Cys, In some embodiments a PIP-1 polypeptide comprises an Lys, Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, amino acid sequence of SEQ ID NO: 213 wherein Xaa at Gly, Arg, ASn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, position 2 is Pro, Thr or Ser; Xaa at position 3 is Ile, Thr, Ser. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, 50 Leu, Val, Met or Ser; Xaa at position 6 is Glu, Gly, Asp or Lys, Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu Ala; Xaa at position 8 is Ser, Gly, Asn. Thr or Glin; Xaa at or Cys; Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at position 19 is Asp, Glu or Cys; Xaa at position 20 is Leu, position 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, Val, Ile or Met; Xaa at position 21 is Lys, Ser, Asn, Arg, Thr Ser or Lys; Xaa at position 179 is Val, Phe, Thr, Ile, Cys, or Glin; Xaa at position 22 is Ser, Lys, Arg or Thr, Xaa at Leu, Met, Ser, Ala or Glin; Xaa at position 180 is Met, Leu, 55 position 24 is Gln, Gly, Asn or Ala; Xaa at position 25 is Gly Pro, Trp, Asn., Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; or Ala; Xaa at position 26 is Ser, Asn. Thr or Glin; Xaa at Xaa at position 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or position 27 is Leu, Thr, Ala, Ser, Ile, Val or Met; Xaa at Lys; Xaa at position 182 is Tyr, Phe, Met or His: Xaa at position 28 is Arg, Ser, Lys, Thr, Val, Gly, Ala, Met, Asp, position 183 is Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Trp, Pro, Leu, His, Cys or Glin; Xaa at position 30 is Ala, Ile, Ser, Gln or Leu; Xaa at position 191 is Arg or Lys; Xaa at 60 Leu, Val or Met; Xaa at position 35 is Phe, Leu, Ile, Val or position 194 is Gly or Ala; Xaa at position 195 is Asn or Tyr; Met; Xaa at position 36 is Ala, Ser, Thr, Val, Ile or Leu; Xaa Xaa at position 200 is Asn or Ser; Xaa at position 203 is Asn at position 38 is Asn, Arg, Ser, Gln, Lys or Thr; Xaa at or Glin; Xaa at position 204 is Thror Ala; Xaa at position 206 position 42 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at is Gly or Asp; Xaa at position 209 is Leu or Val: Xaa at position 43 is Pro, Met, Gly, Gln, Ser. Thr, Arg, Val, Leu, position 213 is Tyr or Phe: Xaa at position 220 is Asn or Arg; 65 LyS, Asp, Ala, ASn, Phe, Trp, Glu or Cys; Xaa at position 46 Xaa at position 221 is Ser or Lys; Xaa at position 222 is Thr is Arg, Lys or His; Xaa at position 48 is Gly, Asp, Ala or Glu; or Arg; Xaa at position 226 is Asp, Pro or Glu, Xaa at Xaa at position 49 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa US 9,688,730 B2 51 52 at position 53 is Ser, Gly, Ala or Thr; Xaa at position 58 is at position 247 is ASn, Leu, Asp, Tyr, Ala, Phe, His, Arg, Lys, Tyr or Phe: Xaa at position 60 is Ala, Ser, Gly or Thr; Xaa Gln, Gly, Val, Ile, Ser, Glu, Pro, Met, Trp, Thr or Cys; Xaa at position 63 is Gln, Lys, ASn or Arg; Xaa at position 66 is at position 248 is Tyr, Val, Thr, Glu, Phe, Ser. His, Cys, Leu, Trp, Tyr, Phe, Arg, Lys. His, Ile, Val or Ser; Xaa at position Trp, Ile, Asp, Gly or Ala; Xaa at position 249 is ASn, LyS, 77 is Phe, Tyr, Trp, Leu, Ile, Val or Met; Xaa at position 89 Val, Gly, Met, Asp, Cys, Phe, Arg, Glu, Trp, Tyr, Ser, Ile, is Pro, Leu, Gly, Arg, Thr, Ser, Met, Ala, Ile, Asn. Val, Cys Thr, Pro, Leu, Ala, His or Glin; Xaa at position 251 is Gly, or Lys; Xaa at position 93 is Tyr, Cys, Trp, Val, Asp, Asn. Ile, Ser. Thr, Ala, Asp or Glu; Xaa at position 254 is Ser, Asn. Leu, Met, Phe, Ala or Thr; Xaa at position 97 is Met, Val, Thr or Gln; Xaa at position 258 is Ser, Arg, Thr or Lys: Xaa Leu or Ile: Xaa at position 98 is Asp or Glu; Xaa at position at position 259 is Phe, Trp, Tyr, Cys, Met, Leu, Val, Ile or 105 is Glin or Asn; Xaa at position 107 is Thr, Ile, Ser, Leu 10 His; Xaa at position 265 is Asn, Asp, Gln or Glu; and Xaa or Val: Xaa at position 108 is Gln, Thr, Ser or Asn; Xaa at at position 266 is Asp, ASn, Gln or Glu; and amino acid position 110 is Arg, Leu, Lys, Ile, Val or Met; Xaa at position deletions, amino acid insertions and fragments thereof, and 120 is Lys, Arg, Gln or Asn; Xaa at position 121 is Thr or combinations thereof. Ser; Xaa at position 123 is Thr, Glu, Ser or Asp; Xaa at In some embodiments a PIP-1 polypeptide comprises an position 125 is Asn. Ser, Gln or Thr; Xaa at position 127 is 15 amino acid sequence of SEQ ID NO: 213 having 1, 2, 3, 4, Ser, Asn. Thr, Gln, Lys, Ser or Arg; Xaa at position 134 is 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Gly or Ala; Xaa at position 135 is Ser, Asn. Thr, Gln, Arg or 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, Lys; Xaa at position 137 is Asp, Gly, Glu or Ala; Xaa at 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, position 141 is Val, Ile or Leu; Xaa at position 142 is Gly, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, Asp, Ala or Glu, Xaa at position 144 is Asp or Glu, Xaa at 71, 72,73, 74, 75, 76, 77,78, 79,80, 81, 82, 83, 84, 85, 86, position 147 is Ile, Thr, Val, Leu, Met or Ser; Xaa at position 87, 88 or 89 amino acid substitutions, in any combination, 150 is Ser or Thr; Xaa at position 151 is Asn, Arg, Ser, Gln, at residues designated by Xaa in SEQID NO: 213 compared Lys or Thr; Xaa at position 160 is Thr or Ser; Xaa at position to the native amino acid at the corresponding position of 162 is Ser or Thr; Xaa at position 163 is Asn., Asp, Glu or SEQ ID NO: 2. Gln; Xaa at position 164 is Ser or Thr; Xaa at position 166 25 In some embodiments a PIP-1 polypeptide comprises an is Gln, Glu, Asp or Asn; Xaa at position 167 is Leu, Met, Ile, amino acid sequence of SEQ ID NO: 213 having 1, 2, 3, 4, Val: Xaa at position 168 is Thr, Lys, Ala, Ser, Arg or Gly; 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Xaa at position 171 is Gly, Leu, Gln, Met, Cys, Asn, Asp, Ser 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, or Ala; Xaa at position 172 is Thr, Gly. His, Phe, Glu, Arg, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or Ser, Asn. Ile, Trp, Lys, Gln, Cys, Val, Ala or Met; Xaa at 30 54 amino acid Substitutions, in any combination, at residues position 173 is Phe, Gly. His, Leu, Ala, Arg, ASn, Cys, LyS, designated by Xaa in SEQ ID NO: 213 compared to the Trp, Thr, Ser, Tyr or Met; Xaa at position 174 is Ile, Val, Gly, native amino acid at the corresponding position of SEQ ID Arg, Asn, Ala, Gln, Met, Cys, Leu, Phe, Tyr, Lys, Glu, Ser, NO: 2. His or Thr; Xaa at position 175 is Val, Ile, Ala, Cys, Glu, Lys, In some embodiments a PIP-1 polypeptide comprises one Leu or Met; Xaa at position 176 is Tyr, Met, Phe, Leu or Cys: 35 or more amino acid motifs selected from i) an amino acid Xaa at position 177 is Gln, Ile, Met or Pro; Xaa at position motif represented by amino acids at positions 64-79 of SEQ 178 is Val, Cys, Thr, Pro, Ala, Met, Gln, Phe, Ile, Seror Lys: ID NO: 2, amino acids 64-79 of SEQ ID NO: 211, amino Xaa at position 179 is Val, Phe, Thr, Ile, Cys, Leu, Met, Ser, acids 64-79 of SEQ ID NO: 212 or amino acids 64-79 of Ala or Glin; Xaa at position 180 is Met, Leu: Pro, Trp, Asn. SEQ ID NO: 213, ii) an amino acid motif represented by Tyr, Gly, Gln, Ala, Val, Phe, Ile, Cys or Ser; Xaa at position 40 amino acids at positions 149-159 of SEQ ID NO: 2, amino 181 is Val, Ala, Leu, Trp. Cys, Thr, Ile or Lys; Xaa at acids 149-159 of SEQID NO: 211, amino acids 149-159 of position 182 is Tyr, Phe, Met or His; Xaa at position 183 is SEQ ID NO: 212 or amino acids 149-159 of SEQ ID NO: Ala, Met, Val, Thr, Asp, Gly, Cys, Ile, Phe, Ser, Gln or Leu: 213, iii) an amino acid motif represented by amino acids at Xaa at position 191 is Arg or Lys; Xaa at position 194 is Gly positions 171-183 of SEQ ID NO: 2, amino acids 171-183 or Ala; Xaa at position 195 is Asn. Tyr, Gln or Trp; Xaa at 45 of SEQ ID NO: 211, amino acids 171-183 of SEQ ID NO: position 200 is Asn. Ser. Thr or Glin; Xaa at position 203 is 212 or amino acids 171-183 of SEQID NO: 213, and iv) an Asin or Glin; Xaa at position 204 is Thr, Ala, Ser or Gly; Xaa amino acid motif represented by amino acids at positions at position 206 is Gly, Asp, Ala or Glu; Xaa at position 209 240-249 of SEQID NO: 2, amino acids 240-249 of SEQID is Leu, Val, Ile or Met; Xaa at position 213 is Tyr or Phe: Xaa NO: 211, amino acids 240-249 of SEQID NO: 212 or amino at position 220 is ASn, Arg, Gln or Lys, Xaa at position 221 50 acids 240-249 of SEQ ID NO: 213. In some embodiments is Ser, Lys, Thr or Arg; Xaa at position 222 is Thr, Arg, Ser the PIP-1 polypeptide comprises an amino acid as repre or Lys; Xaa at position 226 is Asp, Pro, Glu or Glin; Xaa at sented by positions 171-183 of SEQID NO: 213 wherein at position 228 is Ser or Gly; Xaa at position 229 is Lys, Asn. least one amino acid at positions 171-183 of SEQ ID NO: Arg or Glin; Xaa at position 231 is Ile, Val, Leu or Met; Xaa 213 are not identical to amino acids at positions 171-183 of at position 232 is Ala, Thr, Ser, Gly, Asp or Glu; Xaa at 55 SEQ ID NO: 6. position 240 is Gln, Arg, Ala, Val, Glu, Met, Gly, Asp, Trp, In some embodiments a PIP-1 polypeptide comprises an Asn. Thr, Ile, Ser, Phe, His, Cys or Leu; Xaa at position 241 amino acid sequence having at least 80% identity to the is Arg, Lys, Glu, Gln, Ser, Ile, Val, Asp, Tyr, Met, Asn. His, amino acid sequence set forth in SEQ ID NO: 2, SEQ ID Pro, Gly, Leu, Phe, Thr, Ala or Cys; Xaa at position 242 is NO: 332 or SEQID NO. 4 and comprises one or more amino Asn, Ala, Arg, Lys, His, Ser, Cys, Glu, Pro, Trp, Gln, Thr, 60 acid motifs selected from i) an amino acid motif represented Phe, Tyr, Met, Asp, Gly, Leu or Val: Xaa at position 243 is by amino acids at positions 64-79 of SEQ ID NO: 2, amino Val, Leu, Ala, Thr, Gly, Cys, Ile, Ser or Met; Xaa at position acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ 244 is Leu, Val, Phe, Ile, Met, Gln, Cys, Trp or Ala; Xaa at ID NO: 212 or amino acids 64-79 of SEQ ID NO: 213, ii) position 245 is Met, Ala, Arg, Asp, Glu, Leu, Pro, Ser, Trp, an amino acid motif represented by amino acids at positions Gly, Val, Lys, Phe, Cys, Thr, His, Ile, Gln, Tyr or Asn; Xaa 65 149-159 of SEQID NO: 2, amino acids 149-159 of SEQID at position 246 is Glu, Asp, Tyr, Gly, Arg, Val, Ala, Trp, Gln, NO: 211, amino acids 149-159 of SEQID NO: 212 or amino Ser, Asn. Ile Leu, Met, Cys, Pro. His, Phe, Thr or Lys; Xaa acids 149-159 of SEQID NO: 213, iii) an amino acid motif US 9,688,730 B2 53 54 represented by amino acids at positions 171-183 of SEQID In some embodiments a PIP-1 polypeptide includes vari NO: 2, amino acids 171-183 of SEQ ID NO: 211, amino ants where an amino acid that is part of a proteolytic acids 171-183 of SEQ ID NO: 212 or amino acids 171-183 cleavage site is changed to another amino acid to eliminate of SEQID NO: 213, and iv) an amino acid motif represented or alter the proteolytic cleavage at that site. In some embodi by amino acids at positions 240-249 of SEQ ID NO: 2, ments the proteolytic cleavage is by a protease in the insect amino acids 240-249 of SEQ ID NO: 211, amino acids gut. In other embodiments the proteolytic cleavage is by a 240-249 of SEQID NO: 212 or amino acids 240-249 of SEQ plant protease in the transgenic plant. ID NO 213. In some embodiments exemplary PIP-1 polypeptides are In some embodiments a PIP-1 polypeptide comprises an the polypeptides shown in Table 4, Table 6, Table 9, Table amino acid sequence having at least 80% identity to the 10 12, Table 13, Table 14 and/or Table 16 and combinations of the amino substitutions thereofas well as deletions, and or amino acid sequence set forth in SEQ ID NO: 2, SEQ ID insertions and fragments thereof. NO: 332 or SEQID NO. 4 and comprises one or more amino In some embodiments a PIP-1 polypeptide does not have acid motifs selected from i) an amino acid motif represented the amino acid sequence of SEQ ID NO: 4. In some by amino acids at positions 64-79 of SEQ ID NO: 2, amino 15 embodiments a PIP-1 polypeptide does not have the amino acids 64-79 of SEQID NO: 211, amino acids 64-79 of SEQ acid sequence of SEQ ID NO: 6. ID NO: 212 or amino acids 64-79 of SEQ ID NO: 213, ii) In some embodiments a PIP-1 polypeptide has a calcu an amino acid motif represented by amino acids at positions lated molecular weight of between about 15 kD and about 35 149-159 of SEQID NO: 2, amino acids 149-159 of SEQID kD, between about 19 kD and about 35 kD, between about NO: 211, amino acids 149-159 of SEQID NO: 212 or amino 21 kD and about 35 kD, between about 23 kD and about 35 acids 149-159 of SEQID NO: 213, iii) an amino acid motif kD, between about 25 kD and about 32 kD, between about represented by amino acids at positions 171-183 of SEQID 27 kD and about 32 kD, between about 28 kD and about 32 NO: 2, amino acids 171-183 of SEQ ID NO: 211, amino kD, between about 29 kD and about 32 kD, between about acids 171-183 of SEQ ID NO: 212 or amino acids 171-183 30 kD and about 31 kD or about 30.5 kD. of SEQ ID NO: 213, and iv) amino acids 240-249 of SEQ 25 In some embodiments a PIP-1 polypeptide is encoded by ID NO: 2, amino acids 240-249 of SEQID NO: 211, amino a nucleic acid molecule that hybridizes under stringent acids 240-249 of SEQ ID NO: 212 or amino acids 240-249 conditions to the nucleic acid molecule of SEQID NO: 1 or of SEQ ID NO: 213. 3. Variants include polypeptides that differ in amino acid In some embodiments the amino acid motifs represented sequence due to mutagenesis. Variant proteins encompassed by i) amino acids 64-79 of SEQ ID NO: 2, amino acids 30 by the disclosure are biologically active, that is they con 64-79 of SEQ ID NO: 211, amino acids 64-79 of SEQ ID tinue to possess the desired biological activity (i.e. pesticidal NO: 212 or amino acids 64-79 of SEQID NO: 213, ii) amino activity) of the native protein. By “retains activity” is acids 149-159 of SEQ ID NO: 2, amino acids 149-159 of intended that the variant will have at least about 30%, at least SEQID NO: 211, amino acids 149-159 of SEQID NO: 212 about 50%, at least about 70% or at least about 80% of the or amino acids 149-159 of SEQID NO: 213, iii) amino acids 35 insecticidal activity of the native protein. In some embodi 171-183 of SEQID NO: 2, amino acids 171-183 of SEQID ments, the variants may have improved activity over the NO: 211, amino acids 171-183 of SEQID NO: 212 or amino native protein. acids 171-183 of SEQ ID NO: 213, and iv) amino acids Bacterial genes quite often possess multiple methionine 240-249 of SEQID NO: 2, amino acids 240-249 of SEQID initiation codons in proximity to the start of the open reading NO: 211, amino acids 240-249 of SEQID NO: 212 or amino 40 frame. Often, translation initiation at one or more of these acids 240-249 of SEQ ID NO: 213, the amino acid motif start codons will lead to generation of a functional protein. may optional have a deletion of one or more amino acids These start codons can include ATG codons. For example, within the motif, a insertion of one or more amino acids SEQ ID NO: 215 represent alternate start site protein within the motif or combinations thereof. encoded by SEQ ID NO: 1. However, bacteria such as In some embodiments exemplary PIP-1 polypeptides are 45 Bacillus sp. also recognize the codon GTG as a start codon, encoded by the polynucleotide sequence set forth in SEQID and proteins that initiate translation at GTG codons contain NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, a methionine at the first amino acid. On rare occasions, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, translation in bacterial systems can initiate at a TTG codon, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, though in this event the TTG encodes a methionine. Fur 197, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 50 thermore, it is not often determined a priori which of these 199, 200, 201, 202, 203, 205, 207, 220, 221, 222, 223, 224, codons are used naturally in the bacterium. Thus, it is 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, understood that use of one of the alternate methionine 237, 238,239, 240, 241, 242, 243, 244, 270, 271, 272, 273, codons may also lead to generation of pesticidal proteins. 274, 275,276, 277,278, 279, 280, 281, 282,283, 284, 285, These pesticidal proteins are encompassed in the present 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, and 55 disclosure and may be used in the methods of the present 297 as well as variants and fragments thereof encoding disclosure. It will be understood that, when expressed in PIP-1 polypeptides. plants, it will be necessary to alter the alternate start codon In some embodiments exemplary nucleic acid molecules to ATG for proper translation. comprise a sequence set forth in SEQID NO: 152, 153, 154, In another aspect the PIP-1 polypeptide may be expressed 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 60 as a precursor protein with an intervening sequence that 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, catalyzes multi-step, post translational protein splicing. Pro 179, 180, 181, 182, 183, 184, 185, 186, 197, 188, 189, 190, tein splicing involves the excision of an intervening 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, sequence from a polypeptide with the concomitant joining of 203, 205, 207, 220, 221, 222, 223, 224, 225, 226, 227, 228, the flanking sequences to yield a new polypeptide (Chong, 229, 230, 231, 232, 233,234, 235, 236, 237,238, 239, 240, 65 et al., (1996) J. Biol. Chem. 271:22159-22168). This inter 241, 242, 243, and 244 as well as variants and fragments vening sequence or protein splicing element, referred to as thereof encoding PIP-1 polypeptides. inteins, which catalyze their own excision through three US 9,688,730 B2 55 56 coordinated reactions at the N-terminal and C-terminal intein coding sequence is linked to the 5' end of the second splice junctions: an acyl rearrangement of the N-terminal fragment coding for the C-terminal part of the PIP-1 poly cysteine or Serine; a transesterification reaction between the peptide. two termini to form a branched ester or thioester interme In general, the trans-splicing partners can be designed diate and peptide bond cleavage coupled to cyclization of 5 using any split intein, including any naturally-occurring or the intein C-terminal asparagine to free the intein (Evans, et artificially-split split intein. Several naturally-occurring split al., (2000).J. Biol. Chem. 275:9091-9094. The elucidation of inteins are known, for example: the split intein of the DnaB. the mechanism of protein splicing has led to a number of gene of Synechocystis sp. PCC6803 (see, Wu, et al., (1998) intein-based applications (Comb, et al., U.S. Pat. No. 5,496, Proc Natl AcadSci USA 95(16): 9226-31 and Evans, et al., 10 (2000) J Biol Chem 275(13):9091-4 and of the DnaB gene 714; Comb, et al., U.S. Pat. No. 5,834,247; Camarero and from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett Muir, (1999).J. Amer: Chem. Soc. 121:5597-5598; Chong, et 580(7):1853-8). Non-split inteins have been artificially split al., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic in the laboratory to create new split inteins, for example: the Acids Res. 26:5109-5115; Chong, et al., (1998) J. Biol. artificially split Ssp DnaB intein (see, Wu, et al., (1998) Chem. 273:10567-10577; Cotton, et al., (1999).J. Am. Chem. 15 Biochim Biophy's Acta 1387:422-32) and split Sce VMA Soc. 121:1100-1101: Evans, et al., (1999) J. Biol. Chem. intein (see, Brenzel, et al., (2006) Biochemistry 45(6): 1571 274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 8) and an artificially split fungal mini-intein (see, Elleuche, 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256 et al., (2007) Biochem Biophy's Res Commun 355(3):830-4). 2264: Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; There are also intein databases available that catalogue Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172: known inteins (see, for example the online-database avail Mathys, et al., (1999) Gene 231: 1-13; Mills, et al., (1998) able at: bioinformatics.weizmann.ac.il/pietro/inteins/In Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, et al., teinstable.html, which can be accessed on the world-wide (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et web using the “www’ prefix). al., (1999) Biochemistry 38:16040-16044: Otomo, et al., Naturally-occurring non-split inteins may have endonu (1999) J. Biolmol. NMR 14:105-114; Scott, et al., (1999) 25 clease or other enzymatic activities that can typically be Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov and removed when designing an artificially-split split intein. Muir, (1998) J. Biol. Chem. 273:16205-16209; Shingle Such mini-inteins or minimized split inteins are well known decker, et al., (1998) Gene 207:187-195; Southworth, et al., in the art and are typically less than 200 amino acid residues (1998) EMBO J. 17:918-926: Southworth, et al., (1999) long (see, Wu, et al., (1998) Biochim Biophys Acta 1387: 30 422-32). Suitable split inteins may have other purification Biotechniques 27:110-120: Wood, et al., (1999) Nat. Bio enabling polypeptide elements added to their structure, technol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. provided that such elements do not inhibit the splicing of the Sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Bio split intein or are added in a manner that allows them to be phys Acta 1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. removed prior to splicing. Protein splicing has been reported Sci. USA 96:388-393: Yamazaki, et al., (1998).J. Am. Chem. 35 using proteins that comprise bacterial intein-like (BIL) Soc. 120:5591-5592). For the application of inteins in plant domains (see, Amitai, et al., (2003) Mol Microbiol 47:61-73) transgenes see Yang, J, et al., (Transgene Res 15:583-593 and hedgehog (Hog) auto-processing domains (the latter is (2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56:375 combined with inteins when referred to as the Hog/intein 392, (2005)). superfamily or HINT family (see, Dassa, et al., (2004) J Biol In another aspect the PIP-1 polypeptide may be encoded 40 Chem. 27932001-7) and domains such as these may also be by two separate genes where the intein of the precursor used to prepare artificially-split inteins. In particular, non protein comes from the two genes, referred to as a split splicing members of Such families may be modified by intein and the two portions of the precursor are joined by a molecular biology methodologies to introduce or restore peptide bond formation. This peptide bond formation is splicing activity in Such related species. Recent studies accomplished by intein-mediated trans-splicing. For this 45 demonstrate that splicing can be observed when a N-termi purpose, a first and a second expression cassette comprising nal split intein component is allowed to react with a C-ter the two separate genes further code for inteins capable of minal split intein component not found in nature to be its mediating protein trans-splicing. By trans-splicing, the pro "partner'; for example, splicing has been observed utilizing teins and polypeptides encoded by the first and second partners that have as little as 30 to 50% homology with the fragments may be linked by peptide bond formation. Trans 50 “natural splicing partner (see, Dassa, et al., (2007) Bio splicing inteins may be selected from the nucleolar and chemistry 46(1):322-30). Other such mixtures of disparate organellar genomes of different organisms including eukary split intein partners have been shown to be unreactive one otes, archaebacteria and eubacteria. Inteins that may be used with another (see, Brenzel, et al., 2006 Biochemistry 45(6): for are listed at neb.com/neb/inteins.html, which can be 1571-8). However, it is within the ability of a person skilled accessed on the world-wide web using the “www’ prefix). 55 in the relevant art to determine whether a particular pair of The nucleotide sequence coding for an intein may be split polypeptides is able to associate with each other to provide into a 5' and a 3' part that code for the 5' and the 3' part of a functional intein, using routine methods and without the the intein, respectively. Sequence portions not necessary for exercise of inventive skill. intein splicing (e.g., homing endonuclease domain) may be In another aspect the PIP-1 polypeptide is a circular deleted. The intein coding sequence is split such that the 5' 60 permuted variant. In certain embodiments the PIP-1 poly and the 3' parts are capable of trans-splicing. For selecting peptide is a circular permuted variant of the polypeptide of a Suitable splitting site of the intein coding sequence, the SEQ ID NO: 2, 4, 101, 102, 103, 104, 105, 106, 107, 108, considerations published by Southworth, et al., (1998) 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, EMBO.J. 17:918-926 may be followed. In constructing the 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, first and the second expression cassette, the 5' intein coding 65 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, sequence is linked to the 3' end of the first fragment coding 145, 146, 147,148, 149, 150, 151, 204, 206, 208, 211, 212, for the N-terminal part of the PIP-1 polypeptide and the 3' 213, 214, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, US 9,688,730 B2 57 58 255, 256, 257, 258, 259, 260,261, 262, 263,264, 265, 266, steric strain. Those skilled in the analysis of protein struc 267, 268, 269,298, 299, 300, 301, 302,303, 304, 305, 306, tural information will recognize that using the distance 307, 308,309, 310, 311, 312,313, 314, 315, 316, 317, 318, between the chain ends, defined as the distance between the 319, 320, 321, 322, 323, 324, 325, and 332. In certain c-alpha carbons, can be used to define the length of the embodiments the PIP-1 polypeptide is a circular permuted sequence to be used or at least to limit the number of variant of the polypeptide of SEQ ID NO: 2, 4, 101, 102, possibilities that must be tested in an empirical selection of 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, linkers. They will also recognize that it is sometimes the case 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, that the positions of the ends of the polypeptide chain are 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, ill-defined in structural models derived from X-ray diffrac 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 10 tion or nuclear magnetic resonance spectroscopy data, and 151, 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, that when true, this situation will therefore need to be taken 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, into account in order to properly estimate the length of the 261, 262, 263, 264, 265, 266, 267, 268, 269, and 332. linker required. From those residues whose positions are The development of recombinant DNA methods has made well defined are selected two residues that are close in it possible to study the effects of sequence transposition on 15 sequence to the chain ends, and the distance between their protein folding, structure and function. The approach used in c-alpha carbons is used to calculate an approximate length creating new sequences resembles that of naturally occur for a linker between them. Using the calculated length as a ring pairs of proteins that are related by linear reorganization guide, linkers with a range of number of residues (calculated of their amino acid sequences (Cunningham, et al., (1979) using 2 to 3.8 A per residue) are then selected. These linkers Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and may be composed of the original sequence, shortened or Erfle, (1990) J. Bacteriol. 172:3837-3841; Schimming, et lengthened as necessary, and when lengthened the additional al., (1992) Eur: J. Biochem. 204:13-19: Yamiuchi and Mina residues may be chosen to be flexible and hydrophilic as mikawa, (1991) FEBS Lett. 260:127-130; MacGregor, et al., described above; or optionally the original sequence may be (1996) FEBS Lett. 378:263-266). The first in vitro applica Substituted for using a series of linkers, one example being tion of this type of rearrangement to proteins was described 25 the Gly-Gly-Gly-Ser cassette approach mentioned above; or by Goldenberg and Creighton (J. Mol. Biol. 165:407-413, optionally a combination of the original sequence and new 1983). In creating a circular permuted variant a new N-ter sequence having the appropriate total length may be used. minus is selected at an internal site (breakpoint) of the Sequences of pesticidal polypeptides capable of folding to original sequence, the new sequence having the same order biologically active states can be prepared by appropriate of amino acids as the original from the breakpoint until it 30 selection of the beginning (amino terminus) and ending reaches an amino acid that is at or near the original C-ter (carboxyl terminus) positions from within the original poly minus. At this point the new sequence is joined, either peptide chain while using the linker sequence as described directly or through an additional portion of sequence above. Amino and carboxyl termini are selected from within (linker), to an amino acid that is at or near the original a common stretch of sequence, referred to as a breakpoint N-terminus and the new sequence continues with the same 35 region, using the guidelines described below. A novel amino sequence as the original until it reaches a point that is at or acid sequence is thus generated by selecting amino and near the amino acid that was N-terminal to the breakpoint carboxyl termini from within the same breakpoint region. In site of the original sequence, this residue forming the new many cases the selection of the new termini will be such that C-terminus of the chain. The length of the amino acid the original position of the carboxyl terminus immediately sequence of the linker can be selected empirically or with 40 preceded that of the amino terminus. However, those skilled guidance from structural information or by using a combi in the art will recognize that selections of termini anywhere nation of the two approaches. When no structural informa within the region may function, and that these will effec tion is available, a small series of linkers can be prepared for tively lead to either deletions or additions to the amino or testing using a design whose length is varied in order to span carboxyl portions of the new sequence. It is a central tenet a range from 0 to 50 A and whose sequence is chosen in 45 of molecular biology that the primary amino acid sequence order to be consistent with surface exposure (hydrophilicity, of a protein dictates folding to the three-dimensional struc Hopp and Woods, (1983) Mol. Immunol. 20:483-489; Kyte ture necessary for expression of its biological function. and Doolittle, (1982) J. Mol. Biol. 157:105-132; solvent Methods are known to those skilled in the art to obtain and exposed surface area, Lee and Richards, (1971).J. Mol. Biol. interpret three-dimensional structural information using 55:379-400) and the ability to adopt the necessary confor 50 X-ray diffraction of single protein Crystals or nuclear mag mation without deranging the configuration of the pesticidal netic resonance spectroscopy of protein Solutions. Examples polypeptide (conformationally flexible; Karplus and Schulz, of structural information that are relevant to the identifica (1985) Naturwissenschaften 72:212-213. Assuming an aver tion of breakpoint regions include the location and type of age of translation of 2.0 to 3.8 A per residue, this would protein secondary structure (alpha and 3-10 helices, parallel mean the length to test would be between 0 to 30 residues, 55 and anti-parallel beta sheets, chain reversals and turns, and with 0 to 15 residues being the preferred range. Exemplary loops; Kabsch and Sander, (1983) Biopolymers 22:2577 of Such an empirical series would be to construct linkers 2637; the degree of solvent exposure of amino acid residues, using a cassette sequence Such as Gly-Gly-Gly-Ser repeated the extent and type of interactions of residues with one n times, where n is 1, 2, 3 or 4. Those skilled in the art will another (Chothia, (1984) Ann. Rev. Biochem, 53:537-572) recognize that there are many such sequences that vary in 60 and the static and dynamic distribution of conformations length or composition that can serve as linkers with the along the polypeptide chain (Alber and Mathews, (1987) primary consideration being that they be neither excessively Methods Enzymol. 154:511-533). In some cases additional long nor short (cf., Sandhu, (1992) Critical Rev. Biotech. information is known about solvent exposure of residues; 12:437-462); if they are too long, entropy effects will likely one example is a site of post-translational attachment of destabilize the three-dimensional fold, and may also make 65 carbohydrate which is necessarily on the surface of the folding kinetically impractical, and if they are too short, they protein. When experimental structural information is not will likely destabilize the molecule because of torsional or available or is not feasible to obtain, methods are also US 9,688,730 B2 59 60 available to analyze the primary amino acid sequence in 252,253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, order to make predictions of protein tertiary and secondary 264. 265, 266, 267. 268, 269, 332, and active fragments structure, Solvent accessibility and the occurrence of turns thereof. and loops. Biochemical methods are also sometimes appli In another aspect fusion proteins are provided comprising cable for empirically determining surface exposure when 5 a PIP-1 polypeptide and a second pesticidal polypeptide direct structural methods are not feasible; for example, using Such a Cry protein. Methods for design and construction of the identification of sites of chain scission following limited fusion proteins (and polynucleotides encoding same) are proteolysis in order to infer surface exposure (Gentile and known to those of skill in the art. Polynucleotides encoding Salvatore, (1993) Eur: J. Biochem. 218:603-621). Thus a PIP-1 polypeptide may be fused to signal sequences which 10 will direct the localization of the PIP-1 polypeptide to using either the experimentally derived structural informa particular compartments of a prokaryotic or eukaryotic cell tion or predictive methods (e.g., Srinivisan and Rose, (1995) and/or direct the secretion of the PIP-1 polypeptide of the Proteins. Struct., Funct. & Genetics 22:81-99) the parental embodiments from a prokaryotic or eukaryotic cell. For amino acid sequence is inspected to classify regions accord example, in E. coli, one may wish to direct the expression of ing to whether or not they are integral to the maintenance of 15 the protein to the periplasmic space. Examples of signal secondary and tertiary structure. The occurrence of sequences or proteins (or fragments thereof) to which the sequences within regions that are known to be involved in PIP-1 polypeptide may be fused in order to direct the periodic secondary structure (alpha and 3-10 helices, paral expression of the polypeptide to the periplasmic space of lel and anti-parallel beta sheets) are regions that should be bacteria include, but are not limited to, the pelB signal avoided. Similarly, regions of amino acid sequence that are 20 sequence, the maltose binding protein (MBP) signal observed or predicted to have a low degree of solvent sequence, MBP, the ompA signal sequence, the signal exposure are more likely to be part of the so-called hydro sequence of the periplasmic E. coli heat-labile enterotoxin phobic core of the protein and should also be avoided for B-subunit, and the signal sequence of alkaline phosphatase. selection of amino and carboxyl termini. In contrast, those Several vectors are commercially available for the construc regions that are known or predicted to be in Surface turns or 25 tion of fusion proteins which will direct the localization of loops, and especially those regions that are known not to be a protein, Such as the pMAL series of vectors (particularly required for biological activity, are the preferred sites for the pMAL-p series) available from New England Biolabs. In location of the extremes of the polypeptide chain. Continu a specific embodiment, the PIP-1 polypeptide may be fused ous stretches of amino acid sequence that are preferred to the pelB pectate lyase signal sequence to increase the based on the above criteria are referred to as a breakpoint 30 efficiency of expression and purification of Such polypep region. Polynucleotides encoding circular permuted PIP-1 tides in Gram-negative bacteria (see, U.S. Pat. Nos. 5,576, polypeptides with new N-terminus/C-terminus which con 195 and 5,846,818). Plant plastid transit peptide/polypeptide tain a linker region separating the original C-terminus and fusions are well known in the art (see, U.S. Pat. No. N-terminus can be made essentially following the method 7,193,133). Apoplast transit peptides such as rice or barley described in Mullins, et al., (1994) J. Am. Chem. Soc. 35 alpha-amylase secretion signal are also well known in the 116:5529-5533. Multiple steps of polymerase chain reaction art. The plastid transit peptide is generally fused N-terminal (PCR) amplifications are used to rearrange the DNA to the polypeptide to be targeted (e.g., the fusion partner). In sequence encoding the primary amino acid sequence of the one embodiment, the fusion protein consists essentially of protein. Polynucleotides encoding circular permuted PIP-1 the peptide transit plastid and the PIP-1 polypeptide to be polypeptides with new N-terminus/C-terminus which con- 40 targeted. In another embodiment, the fusion protein com tain a linker region separating the original C-terminus and prises the peptide transit plastid and the polypeptide to be N-terminus can be made based on the tandem-duplication targeted. In Such embodiments, the plastid transit peptide is method described in Horlick, et al., (1992) Protein Eng. preferably at the N-terminus of the fusion protein. However, 5:427-431. Polymerase chain reaction (PCR) amplification additional amino acid residues may be N-terminal to the of the new N-terminus/C-terminus genes is performed using 45 plastid transit peptide providing that the fusion protein is at a tandemly duplicated template DNA. least partially targeted to a plastid. In a specific embodiment, In another aspect fusion proteins are provided that include the plastid transit peptide is in the N-terminal half, N-ter within its amino acid sequence an amino acid sequence minal third or N-terminal quarter of the fusion protein. Most comprising a PIP-1 polypeptide including but not limited to or all of the plastid transit peptide is generally cleaved from the polypeptide of SEQ ID NO: 2, 4, 101, 102, 103, 104, 50 the fusion protein upon insertion into the plastid. The 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, position of cleavage may vary slightly between plant spe 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, cies, at different plant developmental stages, as a result of 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, specific intercellular conditions or the particular combina 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 204, tion of transit peptide/fusion partner used. In one embodi 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, 55 ment, the plastid transit peptide cleavage is homogenous 251, 252,253,254, 255, 256, 257, 258, 259, 260, 261, 262, Such that the cleavage site is identical in a population of 263,264, 265, 266, 267, 268,269,298, 299, 300, 301, 302, fusion proteins. In another embodiment, the plastid transit 303, 304,305, 306, 307, 308,309, 310, 311, 312, 313, 314, peptide is not homogenous, such that the cleavage site varies 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, and by 1-10 amino acids in a population of fusion proteins. The 332. 60 plastid transit peptide can be recombinantly fused to a In some embodiments fusion proteins comprises a PIP-1 second protein in one of several ways. For example, a polypeptide of SEQ ID NO: 2, 4, 101, 102, 103, 104, 105, restriction endonuclease recognition site can be introduced 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, into the nucleotide sequence of the transit peptide at a 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, position corresponding to its C-terminal end and the same or 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 65 a compatible site can be engineered into the nucleotide 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 204, 206, sequence of the protein to be targeted at its N-terminal end. 208, 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, Care must be taken in designing these sites to ensure that the US 9,688,730 B2 61 62 coding sequences of the transit peptide and the second In some embodiments the linkers comprise sequences protein are kept “in frame' to allow the synthesis of the selected from the group of formulas: (Gly Ser), (Gly-Ser), desired fusion protein. In some cases, it may be preferable (GlysSer), (Gly, Ser), or (AlaGlySer), where n is an integer. to remove the initiator methionine codon of the second One example of a highly-flexible linker is the (GlySer)-rich protein when the new restriction site is introduced. The spacer region present within the pill protein of the filamen introduction of restriction endonuclease recognition sites on tous bacteriophages, e.g., bacteriophages M13 or fa both parent molecules and their Subsequent joining through (Schaller, et al., 1975). This region provides a long, flexible recombinant DNA techniques may result in the addition of spacer region between two domains of the pill Surface one or more extra amino acids between the transit peptide protein. Also included are linkers in which an endopeptidase and the second protein. This generally does not affect 10 recognition sequence is included. Such a cleavage site may targeting activity as long as the transit peptide cleavage site be valuable to separate the individual components of the remains accessible and the function of the second protein is fusion to determine if they are properly folded and active in not altered by the addition of these extra amino acids at its vitro. Examples of various endopeptidases include, but are N-terminus. Alternatively, one skilled in the art can create a not limited to, Plasmin, Enterokinase, Kallikerin, Urokinase, precise cleavage site between the transit peptide and the 15 Tissue Plasminogen activator, clostripain, Chymosin, Col second protein (with or without its initiator methionine) lagenase, Russell's Viper Venom Protease, Postproline using gene synthesis (Stemmer, et al., (1995) Gene 164:49 cleavage enzyme, V8 protease. Thrombin and factor Xa. In 53) or similar methods. In addition, the transit peptide fusion Some embodiments the linker comprises the amino acids can intentionally include amino acids downstream of the EEKKN from the multi-gene expression vehicle (MGEV), cleavage site. The amino acids at the N-terminus of the which is cleaved by vacuolar proteases as disclosed in US mature protein can affect the ability of the transit peptide to 2007/0277263. In other embodiments, peptide linker seg target proteins to plastids and/or the efficiency of cleavage ments from the hinge region of heavy chain immunoglobu following protein import. This may be dependent on the lins IgG, IgA, IgM, Ig) or IgE provide an angular relation protein to be targeted. See, e.g., Comai, et al., (1988).J. Biol. ship between the attached polypeptides. Especially useful Chem. 263(29): 15104-9. 25 are those hinge regions where the cysteines are replaced In some embodiments fusion proteins are provide com with serines. Preferred linkers of the present invention prising a PIP-1 polypeptide, a pesticidal protein such as a include sequences derived from murine IgG gamma 2b Cry protein, and an amino acid linker. hinge region in which the cysteines have been changed to In some embodiments fusion proteins are provided rep serines. The fusion proteins are not limited by the form, size resented by a formula selected from the group consisting of 30 or number of linker sequences employed and the only R-L-R, R-L-R, R-R or R-R' requirement of the linker is that functionally it does not where R' is a PIP-1 polypeptide, R is a pesticidal protein interfere adversely with the folding and function of the with a different but complementary activity to the PIP-1 individual molecules of the fusion. polypeptide, including but not limited to Cry proteins; a In another aspect chimeric PIP-1 polypeptide are provided polypeptide that increases the solubility and/or stability of 35 that are created through joining two or more portions of the PIP-1 polypeptide; or a transit peptide or leader genes, which originally encoded separate insecticidal pro sequence. The R' polypeptide is fused either directly or teins from different species, to create a chimeric gene. The through a linker segment to the R polypeptide. The term translation of the chimeric gene results in a single chimeric “directly defines fusions in which the polypeptides are pesticidal polypeptide with regions, motifs or domains joined without a peptide linker. Thus L represents a chemical 40 derived from each of the original polypeptides. In certain bound or polypeptide segment to which both R" and Rare embodiments the chimeric protein comprises portions, fused in frame, most commonly L is a linear peptide to motifs or domains of PIP-1A (SEQID NO: 2) and orthologs which R' and R are bound by amide bonds linking the PSEEN3174 (SEQ ID NO: 6), PIP-1C (SEQ ID NO:332), carboxy terminus of R' to the amino terminus of L and and PIP-1B (SEQ ID NO: 4) in any combination. In certain carboxy terminus of L to the amino terminus of R. By 45 embodiments the chimeric insecticidal polypeptide includes “fused in frame' is meant that there is no translation but not limited to the polypeptides of SEQID NO: 101, 102, termination or disruption between the reading frames of R' 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, and R. The linking group (L) is generally a polypeptide of 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, between 1 and 500 amino acids in length. The linkers joining 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, the two molecules are preferably designed to (1) allow the 50 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, two molecules to fold and act independently of each other, 151, 204, 206, 208, 211, 212, 213, 214, 245, 246, 247, 248, (2) not have a propensity for developing an ordered second 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, ary structure which could interfere with the functional 261, 262, 263, 264, 265, 266, 267, 268, 269, and 332. domains of the two proteins, (3) have minimal hydrophobic It is recognized that DNA sequences may be altered by or charged characteristic which could interact with the 55 various methods, and that these alterations may result in functional protein domains and (4) provide steric separation DNA sequences encoding proteins with amino acid of R' and R such that R' and R could interact simultane sequences different than that encoded by the wild-type (or ously with their corresponding receptors on a single cell. native) pesticidal protein. These proteins may be altered in Typically surface amino acids in flexible protein regions various ways including amino acid substitutions, deletions, include Gly, Asn and Ser. Virtually any permutation of 60 truncations, and insertions of one or more amino acids, amino acid sequences containing Gly, ASn and Ser would be including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, expected to satisfy the above criteria for a linker sequence. 4045, 50, about 55, 60, 65,70, 75, 80, 85,90, 100, 105, 110, Other neutral amino acids, such as Thr and Ala, may also be 115, 120, 125, 130, 135, 140, 145, 150, 155 or more amino used in the linker sequence. Additional amino acids may also acid substitutions, deletions and/or insertions or combina be included in the linkers due to the addition of unique 65 tions thereof compared to SEQID NO: 2 or 4 including but restriction sites in the linker sequence to facilitate construc not limited to SEQ ID NO: 101, 102, 103, 104, 105, 106, tion of the fusions. 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, US 9,688,730 B2 63 64 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, appropriate amino acid substitutions that do not affect bio 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, logical activity of the protein of interest may be found in the 143, 144, 145, 146, 147, 148, 149, 150, 151, 204, 206, 208, model of Dayhoff, et al., (1978) Atlas of Protein Sequence 211, 212, 213, 214, 245, 246, 247, 248, 249, 250, 251, 252, and Structure (Natl. Biomed. Res. Found. Washington, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, D.C.), herein incorporated by reference. 265, 266, 267, 268, 269, and 332. In some embodiments a In making Such changes, the hydropathic index of amino PIP-1 polypeptide comprises the deletion of 1, 2, 3, 4, 5, 6, acids may be considered. The importance of the hydropathic 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, amino acid index in conferring interactive biologic function 24, 25, 26, 27, 28 or more amino acids from the N-terminus on a protein is generally understood in the art (Kyte and of the PIP-1 polypeptide relative to the amino acid position 10 Doolittle, (1982) J Mol Biol. 157(1):105-32). It is accepted of SEQ ID NO: 2. Methods for such manipulations are that the relative hydropathic character of the amino acid generally known in the art. For example, amino acid contributes to the secondary structure of the resultant pro sequence variants of a PIP-1 polypeptide can be prepared by tein, which in turn defines the interaction of the protein with mutations in the DNA. This may also be accomplished by other molecules, for example, enzymes, Substrates, recep one of several forms of mutagenesis and/or in directed 15 tors, DNA, antibodies, antigens and the like. evolution. In some aspects, the changes encoded in the It is known in the art that certain amino acids may be amino acid sequence will not substantially affect the func Substituted by other amino acids having a similar hydro tion of the protein. Such variants will possess the desired pathic index or score and still result in a protein with similar pesticidal activity. However, it is understood that the ability biological activity, i.e., still obtain a biological functionally of a PIP-1 polypeptide to confer pesticidal activity may be equivalent protein. Each amino acid has been assigned a improved by the use of Such techniques upon the composi hydropathic index on the basis of its hydrophobicity and tions of this disclosure. charge characteristics (Kyte and Doolittle, ibid). These are: For example, conservative amino acid substitutions may isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala be made at one or more, predicted, nonessential amino acid nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala residues. A “nonessential amino acid residue is a residue 25 nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); that can be altered from the wild-type sequence of a PIP-1 tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine polypeptide without altering the biological activity. A "con (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); servative amino acid substitution' is one in which the amino asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In acid residue is replaced with an amino acid residue having making Such changes, the Substitution of amino acids whose a similar side chain. Families of amino acid residues having 30 hydropathic indices are within +2 is preferred, those which similar side chains have been defined in the art. These are within +1 are particularly preferred and those within families include: amino acids with basic side chains (e.g., +0.5 are even more particularly preferred. lysine, arginine, histidine); acidic side chains (e.g., aspartic It is also understood in the art that the substitution of like acid, glutamic acid); polar, negatively charged residues and amino acids can be made effectively on the basis of hydro their amides (e.g., aspartic acid, asparagine, glutamic, acid, 35 philicity. U.S. Pat. No. 4,554,101, states that the greatest glutamine; uncharged polar side chains (e.g., glycine, local average hydrophilicity of a protein, as governed by the asparagine, glutamine, serine, threonine, tyrosine, cysteine); hydrophilicity of its adjacent amino acids, correlates with a Small aliphatic, nonpolar or slightly polar residues (e.g., biological property of the protein. Alanine, serine, threonine, proline, glycine); nonpolar side As detailed in U.S. Pat. No. 4,554,101, the following chains (e.g., alanine, Valine, leucine, isoleucine, proline, 40 hydrophilicity values have been assigned to amino acid phenylalanine, methionine, tryptophan); large aliphatic, residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+ nonpolar residues (e.g., methionine, leucine, isoleucine, 0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2): valine, cystine); beta-branched side chains (e.g., threonine, glutamine (+0.2); glycine (O); threonine (-0.4); proline valine, isoleucine); aromatic side chains (e.g., tyrosine, (-0.5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); phenylalanine, tryptophan, histidine); large aromatic side 45 methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine chains (e.g., tyrosine, phenylalanine, tryptophan). (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan Amino acid Substitutions may be made in nonconserved (-3.4). regions that retain function. In general. Such substitutions Alternatively, alterations may be made to the protein would not be made for conserved amino acid residues or for sequence of many proteins at the amino or carboxy terminus amino acid residues residing within a conserved motif. 50 without substantially affecting activity. This can include where such residues are essential for protein activity. insertions, deletions or alterations introduced by modern Examples of residues that are conserved and that may be molecular methods, such as PCR, including PCR amplifi essential for protein activity include, for example, residues cations that alter or extend the protein coding sequence by that are identical between all proteins contained in an virtue of inclusion of amino acid encoding sequences in the alignment of similar or related toxins to the sequences of the 55 oligonucleotides utilized in the PCR amplification. Alterna embodiments (e.g., residues that are identical in an align tively, the protein sequences added can include entire pro ment of homologous proteins). Examples of residues that are tein-coding sequences, such as those used commonly in the conserved but that may allow conservative amino acid art to generate protein fusions. Such fusion proteins are often Substitutions and still retain activity include, for example, used to (1) increase expression of a protein of interest (2) residues that have only conservative substitutions between 60 introduce a binding domain, enzymatic activity or epitope to all proteins contained in an alignment of similar or related facilitate either protein purification, protein detection or toxins to the sequences of the embodiments (e.g., residues other experimental uses known in the art (3) target Secretion that have only conservative substitutions between all pro or translation of a protein to a Subcellular organelle. Such as teins contained in the alignment homologous proteins). the periplasmic space of Gram-negative bacteria, mitochon However, one of skill in the art would understand that 65 dria or chloroplasts of plants or the endoplasmic reticulum functional variants may have minor conserved or noncon of eukaryotic cells, the latter of which often results in served alterations in the conserved residues. Guidance as to glycosylation of the protein. US 9,688,730 B2 65 66 In some embodiments, the PIP-1 polypeptide comprises among natural homologues in this family (FIG. 1). In an amino acid sequence of SEQ ID NO: 2 having an amino example 9 Saturation mutagenesis was used to make and test acid substitutions compared to the native amino acid of SEQ most or all possible substitutions at each of 6 conserved ID NO: 2 at one or more residues selected from positions 2, residues. These mutants were tested for activity and a 3, 6, 8, 19, 20, 21, 22, 24, 25, 26, 27, 28, 30, 35, 36, 38, 42, 5 number of active Substitutions not present among the homo 43, 46, 48, 49, 53, 60, 63, 66, 77, 89, 93, 97,98, 105, 108, logues were identified providing an understanding of the 110, 120, 121, 123, 125, 127, 134, 135, 137, 141, 142, 144, functional constraints at these residues. In Example 10 four 147, 150, 151, 160, 162, 163, 164, 166, 167, 168, 171, 172, motifs were identified among the most conserved regions in 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 194, the alignment of SEQ ID NO: 2, 4 and 6. To further 195, 200, 203, 204, 209, 213, 220, 221, 222, 226, 228, 229, 10 characterize the functional constraints on these sequence 231, 232, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, motifs, they were compared to a set of three distant homo 251, 254, 258, 259, 265 and 266 of SEQ ID NO: 2. In logues (AECFG 592740 (SEQ ID NO: 12), Pput 1063 specific embodiments, the Substitution is an alanine for the (SEQID NO:8), and Pput 1064 (SEQID NO: 10) that have native amino acid at the recited position(s). Also encom no detectable insecticidal activity (FIG. 1). These homo passed are the nucleic acid sequence(s) encoding the variant 15 logues are deemed to fall within the same PFAM as SEQID protein or polypeptide. NO: 2, 4 and 6 and thus are likely to share the same overall Variant nucleotide and amino acid sequences of the dis fold. The sequences corresponding to these four motifs from closure also encompass sequences derived from mutagenic these distant homologues were swapped into the PIP-1A and recombinogenic procedures such as DNA shuffling. backbone. The data presented in Example 10 demonstrates With such a procedure, one or more different PIP-1 poly that these motifs are under relatively stringent functional peptide coding regions can be used to create a new PIP-1 constraints, as most of the motif Swaps from the distant polypeptide possessing the desired properties. In this man homologues resulted in loss of function. In Example 11 the ner, libraries of recombinant polynucleotides are generated functional constraints on two of these motifs were further from a population of related sequence polynucleotides com examined by performing Saturation mutagenesis on all resi prising sequence regions that have Substantial sequence 25 dues in motifs 3 and 4. identity and can be homologously recombined in vitro or in Antibodies vivo. For example, using this approach, sequence motifs Antibodies to a PIP-1 polypeptide of the embodiments or encoding a domain of interest may be shuffled between a to variants or fragments thereof, are also encompassed. pesticidal gene and other known pesticidal genes to obtain a Methods for producing antibodies are well known in the art new gene coding for a protein with an improved property of 30 (see, for example, Harlow and Lane, (1988) Antibodies: A interest, Such as an increased insecticidal activity. Strategies Laboratory Manual, Cold Spring Harbor Laboratory, Cold for such DNA shuffling are known in the art. See, for Spring Harbor, N.Y.: U.S. Pat. No. 4,196,265). example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA A kit for detecting the presence of a PIP-1 polypeptide, or 91:10747-10751; Stemmer, (1994) Nature 370:389-391; detecting the presence of a nucleotide sequence encoding a Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, 35 PIP-1 polypeptide, in a sample is provided. In one embodi et al., (1997).J. Mol. Biol. 272:336-347; Zhang, et al., (1997) ment, the kit provides antibody-based reagents for detecting Proc. Natl. Acad. Sci. USA 94:45.04-4509: Crameri, et al., the presence of a PIP-1 polypeptide in a tissue sample. In (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 another embodiment, the kit provides labeled nucleic acid and 5,837,458. probes useful for detecting the presence of one or more Domain Swapping or shuffling is another mechanism for 40 polynucleotides encoding PIP-1 polypeptide(s). The kit is generating altered PIP-1 polypeptides. Domains may be provided along with appropriate reagents and controls for swapped between PIP-1 polypeptides, resulting in hybrid or carrying out a detection method, as well as instructions for chimeric toxins with improved pesticidal activity or target use of the kit spectrum. Methods for generating recombinant proteins and Receptor Identification and Isolation testing them for pesticidal activity are well known in the art 45 Receptors to the PIP-1 polypeptide of the embodiments or (see, for example, Naimov, et al., (2001) Appl. Environ. to variants or fragments thereof, are also encompassed. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. Methods for identifying receptors are well known in the art Environ. Microbiol. 62:1537-1543; Ge, et al., (1991).J. Biol. (see, Hofmann, et. al., (1988) Eur: J. Biochem. 173:85-91; Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Gill, et al., (1995) J. Biol. Chem. 27277-27282) can be Chem. 265:20923-20930; Rang, et al., 91999) Appl. Envi 50 employed to identify and isolate the receptor that recognizes ron. Microbiol. 65:2918-2925). the PIP-1 polypeptides using the brush-border membrane Both DNA shuffling and site directed mutagenesis were vesicles from susceptible insects. In addition to the radio used to define polypeptide sequences that possess pesticidal active labeling method listed in the cited literatures, PIP-1 activity. In Example 8 DNA shuffling was used to generate polypeptide can be labeled with fluorescent dye and other a library of active variants by recombination of the diversity 55 common labels such as streptavidin. Brush-border mem present in PIP-1A (SEQ ID NO: 2) and PSEEN3174 (SEQ brane vesicles (BBMV) of susceptible insects such as soy ID NO: 6). The person skilled in the art will be able to use bean looper and Stink bugs can be prepared according to the comparisons to other proteins or functional assays to further protocols listed in the references and separated on SDS define motifs. High throughput screening can be used to test PAGE gel and blotted on suitable membrane. Labeled PIP-1 variations of those motifs to determine the role of specific 60 polypeptides can be incubated with blotted membrane of residues. Given that knowledge for several motifs, one can BBMV and labeled the PIP-1 polypeptides can be identified then define the requirements for a functional protein. with the labeled reporters. Identification of protein band(s) Knowledge of the motifs allows the skilled artisan to design that interact with the PIP-1 polypeptides can be detected by sequence variations that would not impact function. N-terminal amino acid gas phase sequencing or mass spec This line of investigation was pursued in Examples 9-11. 65 trometry based protein identification method (Patterson, Alignment of homologues of SEQ ID NO: 2, 4 and 6 (1998) 10(22):1-24, Current Protocol in Molecular Biology allowed identification of residues that are highly conserved published by John Wiley & Son Inc). Once the protein is US 9,688,730 B2 67 68 identified, the corresponding gene can be cloned from or heterologous to the host organism and/or to the sequence genomic DNA or cDNA library of the susceptible insects of the embodiments. Additionally, the promoter may be the and binding affinity can be measured directly with the PIP-1 natural sequence or alternatively a synthetic sequence. The polypeptides. Receptor function for insecticidal activity by term “foreign' as used herein indicates that the promoter is the PIP-1 polypeptides can be verified by accomplished by not found in the native organism into which the promoter is RNAi type of gene knock out method (Rajagopal, et al., introduced. Where the promoter is “foreign” or "heterolo (2002) J. Biol. Chem. 277:46849-46851). gous' to the sequence of the embodiments, it is intended that Nucleotide Constructs, Expression Cassettes and Vectors the promoter is not the native or naturally occurring pro The use of the term “nucleotide constructs' herein is not moter for the operably linked sequence of the embodiments. intended to limit the embodiments to nucleotide constructs 10 As used herein, a chimeric gene comprises a coding comprising DNA. Those of ordinary skill in the art will sequence operably linked to a transcription initiation region recognize that nucleotide constructs particularly polynucle that is heterologous to the coding sequence. Where the otides and oligonucleotides composed of ribonucleotides promoter is a native or natural sequence, the expression of and combinations of ribonucleotides and deoxyribonucle the operably linked sequence is altered from the wild-type otides may also be employed in the methods disclosed 15 expression, which results in an alteration in phenotype. herein. The nucleotide constructs, nucleic acids, and nucleo In some embodiments the DNA construct may also tide sequences of the embodiments additionally encompass include a transcriptional enhancer sequence. As used herein, all complementary forms of Such constructs, molecules and the term an "enhancer refers to a DNA sequence which can sequences. Further, the nucleotide constructs, nucleotide stimulate promoter activity and may be an innate element of molecules and nucleotide sequences of the embodiments the promoter or a heterologous element inserted to enhance encompass all nucleotide constructs, molecules and the level or tissue-specificity of a promoter. Various enhanc sequences which can be employed in the methods of the ers are known in the art including for example, introns with embodiments for transforming plants including, but not gene expression enhancing properties in plants (US Patent limited to, those comprised of deoxyribonucleotides, ribo Application Publication Number 2009/0144863, the ubiq nucleotides and combinations thereof. Such deoxyribo 25 uitin intron (i.e., the maize ubiquitin intron 1 (see, for nucleotides and ribonucleotides include both naturally example, NCBI sequence S94464)), the omega enhancer or occurring molecules and synthetic analogues. The nucleo the omega prime enhancer (Gallie, et al., (1989) Molecular tide constructs, nucleic acids, and nucleotide sequences of Biology of RNA ed. Cech (Liss, New York) 237-256 and the embodiments also encompass all forms of nucleotide Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S constructs including, but not limited to, single-stranded 30 enhancer (see, e.g., Benfey, et al., (1990) EMBO.J. 9:1685 forms, double-stranded forms, hairpins, stem-and-loop 96) and the enhancers of U.S. Pat. No. 7,803,992 may also structures and the like. be used, each of which is incorporated by reference. The A further embodiment relates to a transformed organism above list of transcriptional enhancers is not meant to be Such as an organism selected from plant and insect cells, limiting. Any appropriate transcriptional enhancer can be bacteria, yeast, baculovirus, protozoa, nematodes and algae. 35 used in the embodiments. The transformed organism comprises a DNA molecule of The termination region may be native with the transcrip the embodiments, an expression cassette comprising the tional initiation region, may be native with the operably DNA molecule or a vector comprising the expression cas linked DNA sequence of interest, may be native with the sette, which may be stably incorporated into the genome of plant host or may be derived from another source (i.e., the transformed organism. 40 foreign or heterologous to the promoter, the sequence of The sequences of the embodiments are provided in DNA interest, the plant host or any combination thereof). constructs for expression in the organism of interest. The Convenient termination regions are available from the construct will include 5' and 3' regulatory sequences oper Ti-plasmid of A. tumefaciens, such as the octopine synthase ably linked to a sequence of the embodiments. The term and nopaline synthase termination regions. See also, Guer “operably linked as used herein refers to a functional 45 ineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proud linkage between a promoter and a second sequence, wherein foot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes the promoter sequence initiates and mediates transcription of Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261 the DNA sequence corresponding to the second sequence. 1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., Generally, operably linked means that the nucleic acid (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., sequences being linked are contiguous and where necessary 50 (1987) Nucleic Acid Res. 15:9627-9639. to join two protein coding regions in the same reading frame. Where appropriate, a nucleic acid may be optimized for The construct may additionally contain at least one addi increased expression in the host organism. Thus, where the tional gene to be cotransformed into the organism. Alterna host organism is a plant, the synthetic nucleic acids can be tively, the additional gene(s) can be provided on multiple synthesized using plant-preferred codons for improved DNA constructs. 55 expression. See, for example, Campbell and Gowri, (1990) Such a DNA construct is provided with a plurality of Plant Physiol. 92:1-11 for a discussion of host-preferred restriction sites for insertion of the PIP-1 polypeptide gene codon usage. For example, although nucleic acid sequences sequence to be under the transcriptional regulation of the of the embodiments may be expressed in both monocotyle regulatory regions. The DNA construct may additionally donous and dicotyledonous plant species, sequences can be contain selectable marker genes. 60 modified to account for the specific codon preferences and The DNA construct will generally include in the 5' to 3' GC content preferences of monocotyledons or dicotyledons direction of transcription: a transcriptional and translational as these preferences have been shown to differ (Murray et al. initiation region (i.e., a promoter), a DNA sequence of the (1989) Nucleic Acids Res. 17:477-498). Thus, the maize embodiments and a transcriptional and translational termi preferred codon for a particular amino acid may be derived nation region (i.e., termination region) functional in the 65 from known gene sequences from maize. Maize codon organism serving as a host. The transcriptional initiation usage for 28 genes from maize plants is listed in Table 4 of region (i.e., the promoter) may be native, analogous, foreign Murray, et al., Supra. Methods are available in the art for US 9,688,730 B2 69 70 synthesizing plant-preferred genes. See, for example, U.S. portion of the transit peptide, and contains all the informa Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al., tion for targeting to the lumen. Recent research in proteom (1989) Nucleic Acids Res. 17:477-498, herein incorporated ics of the higher plant chloroplast has achieved in the by reference. identification of numerous nuclear-encoded lumen proteins Additional sequence modifications are known to enhance 5 (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et gene expression in a cellular host. These include elimination al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. of sequences encoding spurious polyadenylation signals, Biophys Acta 1503:350-356, 2001), the lumen targeting exon-intron splice site signals, transposon-like repeats, and signal peptide of which can potentially be used in accor other well-characterized sequences that may be deleterious dance with the present invention. About 80 proteins from to gene expression. The GC content of the sequence may be 10 Arabidopsis, as well as homologous proteins from spinach adjusted to levels average for a given cellular host, as and garden pea, are reported by Kieselbach et al., Photo calculated by reference to known genes expressed in the host synthesis Research, 78:249-264, 2003. In particular, table 2 cell. The term "host cell as used herein refers to a cell which of this publication, which is incorporated into the descrip contains a vector and Supports the replication and/or expres tion herewith by reference, discloses 85 proteins from the sion of the expression vector is intended. Host cells may be 15 chloroplast lumen, identified by their accession number (see prokaryotic cells such as E. coli or eukaryotic cells such as also US Patent Application Publication 2009/09044298). In yeast, insect, amphibian or mammalian cells or monocoty addition, the recently published draft version of the rice ledonous or dicotyledonous plant cells. An example of a genome (Goffetal, Science 296:92-100, 2002) is a suitable monocotyledonous host cell is a maize host cell. When Source for lumen targeting signal peptide which may be used possible, the sequence is modified to avoid predicted hairpin 20 in accordance with the present invention. secondary mRNA structures. Suitable chloroplast transit peptides (CTP) are well The expression cassettes may additionally contain 5' known to one skilled in the art including chimeric CTPs leader sequences. Such leader sequences can act to enhance comprising but not limited to, an N-terminal domain, a translation. Translation leaders are known in the art and central domain or a C-terminal domain from a CTP from include: picornavirus leaders, for example, EMCV leader 25 Oryza sativa 1-deoxy-D xylulose-5-Phosphate Synthase (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et Oryza sativa-Superoxide dismutase Oryza sativa-Soluble al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130): starch synthase oryza sativa-NADP-dependent Malic acid potyvirus leaders, for example, TEV leader (Tobacco Etch enzyme Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV Aldolase 2 oryza sativa-L-AScorbate peroxidase 5 oryza leader (Maize Dwarf Mosaic Virus), human immunoglobu- 30 sativa-Phosphoglucan water dikinase, Zea Mays lin heavy-chain binding protein (BiP) (Maceak, et al., ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate (1991) Nature 353:90-94); untranslated leader from the coat dehydrogenase, Zea Mays Thioredoxin M-type US Patent protein mRNA of alfalfa mosaic virus (AMV RNA 4) Application Publication 2012/0304336). (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic The PIP-1 polypeptide gene to be targeted to the chloro virus leader (TMV) (Gallie, et al., (1989) in Molecular 35 plast may be optimized for expression in the chloroplast to Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) account for differences in codon usage between the plant and maize chlorotic mottle virus leader (MCMV) (Lommel, nucleus and this organelle. In this manner, the nucleic acids et al., (1991) Virology 81:382-385). See also, Della-Cioppa, of interest may be synthesized using chloroplast-preferred et al., (1987) Plant Physiol. 84:965-968. Such constructs codons. See, for example, U.S. Pat. No. 5,380,831, herein may also contain a 'signal sequence' or "leader sequence 40 incorporated by reference. to facilitate co-translational or post-translational transport of In preparing the expression cassette, the various DNA the peptide to certain intracellular structures such as the fragments may be manipulated so as to provide for the DNA chloroplast (or other plastid), endoplasmic reticulum or sequences in the proper orientation and, as appropriate, in Golgi apparatus. the proper reading frame. Toward this end, adapters or By "signal sequence' is intended a sequence that is 45 linkers may be employed to join the DNA fragments or other known or Suspected to result in cotranslational or post manipulations may be involved to provide for convenient translational peptide transport across the cell membrane. In restriction sites, removal of superfluous DNA, removal of eukaryotes, this typically involves secretion into the Golgi restriction sites or the like. For this purpose, in vitro muta apparatus, with Some resulting glycosylation. Insecticidal genesis, primer repair, restriction, annealing, resubstitutions, toxins of bacteria are often synthesized as protoxins, which 50 e.g., transitions and transversions, may be involved. are protolytically activated in the gut of the target pest A number of promoters can be used in the practice of the (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments. The promoters can be selected based on the embodiments, the signal sequence is located in the native desired outcome. The nucleic acids can be combined with sequence or may be derived from a sequence of the embodi constitutive, tissue-preferred, inducible or other promoters ments. By “leader sequence' is intended any sequence that 55 for expression in the host organism. Suitable constitutive when translated, results in an amino acid sequence Sufficient promoters for use in a plant host cell include, for example, to trigger co-translational transport of the peptide chain to a the core promoter of the Rsyn? promoter and other consti Subcellular organelle. Thus, this includes leader sequences tutive promoters disclosed in WO 1999/43838 and U.S. Pat. targeting transport and/or glycosylation by passage into the No. 6,072,050; the core CaMV 35S promoter (Odell, et al., endoplasmic reticulum, passage to vacuoles, plastids includ- 60 (1985) Nature 313:810-812); rice actin (McElroy, et al., ing chloroplasts, mitochondria and the like. Nuclear-en (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., coded proteins targeted to the chloroplast thylakoid lumen (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., compartment have a characteristic bipartite transit peptide, (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., composed of a stromal targeting signal peptide and a lumen (1991) Theor: Appl. Genet. 81:581-588); MAS (Velten, et targeting signal peptide. The stromal targeting information is 65 al., (1984) EMBO.J. 3:2723-2730); ALS promoter (U.S. Pat. in the amino-proximal portion of the transit peptide. The No. 5,659,026) and the like. Other constitutive promoters lumen targeting signal peptide is in the carboxyl-proximal include, for example, those discussed in U.S. Pat. Nos. US 9,688,730 B2 71 72 5,608, 149; 5,608,144; 5,604,121 : 5,569,597; 5,466,785; Tissue-preferred promoters can be utilized to target 5,399,680: 5,268,463; 5,608,142 and 6,177,611. enhanced PIP-1 polypeptide expression within a particular Depending on the desired outcome, it may be beneficial to plant tissue. Tissue-preferred promoters include those dis express the gene from an inducible promoter. Of particular cussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; interest for regulating the expression of the nucleotide Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; sequences of the embodiments in plants are wound-induc Hansen, et al., (1997) Mol. Gen. Genet. 254(3):337-343: ible promoters. Such wound-inducible promoters, may Russell, et al., (1997) Transgenic Res. 6(2): 157-168; Rine respond to damage caused by insect feeding, and include hart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Camp, et al., (1996) Plant Physiol. 112(2):525-535: 10 Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Rev. Phytopath. 28:425-449: Duan, et al., (1996) Nature Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778: Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, 5,428, 148; win1 and win2 (Stanford, et al., (1989) Mol. Gen. et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et Genet. 215:200-208); systemin (McGurl, et al., (1992) Sci al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and ence 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant 15 Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such Mol. Biol. 22:783-792: Eckelkamp, et al., (1993) FEBS promoters can be modified, if necessary, for weak expres Letters 323:73-76); MPI gene (Corderok, et al., (1994) Plant S1O. J. 6(2): 141-150) and the like, herein incorporated by refer Leaf-preferred promoters are known in the art. See, for CCC. example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Additionally, pathogen-inducible promoters may be Kwon, et al., (1994) Plant Physiol. 105:357-67: Yamamoto, employed in the methods and nucleotide constructs of the et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et embodiments. Such pathogen-inducible promoters include al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant those from pathogenesis-related proteins (PR proteins), Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) which are induced following infection by a pathogen; e.g., Proc. Natl. Acad. Sci. USA 90(20):9586-9590. PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, 25 Root-preferred or root-specific promoters are known and etc. See, for example, Redolfi, et al., (1983) Neth, J. Plant can be selected from the many available from the literature Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4:645 or isolated de novo from various compatible species. See, 656 and Van Loon, (1985) Plant Mol. Virol. 4:111-116. See for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207 also, WO 1999/43819, herein incorporated by reference. 218 (Soybean root-specific glutamine synthetase gene); Of interest are promoters that are expressed locally at or 30 Keller and Baumgartner, (1991) Plant Cell3(10): 1051-1061 near the site of pathogen infection. See, for example, (root-specific control element in the GRP 1.8 gene of French Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 et al., (1989) Molecular Plant-Microbe Interactions 2:325 (root-specific promoter of the mannopine synthase (MAS) 331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA gene of Agrobacterium tumefaciens) and Miao, et al., (1991) 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 35 Plant Cell 3(1):11-22 (full-length cDNA clone encoding 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA cytosolic glutamine synthetase (GS), which is expressed in 93:14972-14977. See also, Chen, et al., (1996) Plant J. roots and root nodules of soybean). See also, Bogusz, et al., 10:955-966: Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA (1990) Plant Cell 207):633-641, where two root-specific 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201; promoters isolated from hemoglobin genes from the nitro Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 40 gen-fixing nonlegume Parasponia andersonii and the 5,750,386 (nematode-inducible) and the references cited related non-nitrogen-fixing nonlegume Trema tomentosa are therein. Of particular interest is the inducible promoter for described. The promoters of these genes were linked to a the maize PRms gene, whose expression is induced by the 6-glucuronidase reporter gene and introduced into both the pathogen Fusarium moniliforme (see, for example, Cordero, nonlegume Nicotiana tabacum and the legume Lotus cor et al., (1992) Physiol. Mol. Plant Path. 41.189-200). 45 niculatus, and in both instances root-specific promoteractiv Chemical-regulated promoters can be used to modulate ity was preserved. Leach and Aoyagi, (1991) describe their the expression of a gene in a plant through the application of analysis of the promoters of the highly expressed rolC and an exogenous chemical regulator. Depending upon the rolD root-inducing genes of Agrobacterium rhizogenes (see, objective, the promoter may be a chemical-inducible pro Plant Science (Limerick) 79(1):69-76). They concluded that moter, where application of the chemical induces gene 50 enhancer and tissue-preferred DNA determinants are disso expression or a chemical-repressible promoter, where appli ciated in those promoters. Teen, et al., (1989) used gene cation of the chemical represses gene expression. Chemical fusion to lacZ to show that the Agrobacterium T-DNA gene inducible promoters are known in the art and include, but are encoding octopine synthase is especially active in the epi not limited to, the maize ln 2-2 promoter, which is activated dermis of the root tip and that the TR2 gene is root specific by benzenesulfonamide herbicide safeners, the maize GST 55 in the intact plant and stimulated by wounding in leaf tissue, promoter, which is activated by hydrophobic electrophilic an especially desirable combination of characteristics for use compounds that are used as pre-emergent herbicides, and the with an insecticidal or larvicidal gene (see, EMBO.J. 8(2): tobacco PR-1a promoter, which is activated by salicylic 343-350). The TR1 gene fused to nptII (neomycin phos acid. Other chemical-regulated promoters of interest include photransferase II) showed similar characteristics. Additional steroid-responsive promoters (see, for example, the gluco 60 root-preferred promoters include the VfBNOD-GRP3 gene corticoid-inducible promoter in Schena, et al., (1991) Proc. promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759 Natl. Acad. Sci. USA 88: 10421-10425 and McNellis, et al., 772) and rolB promoter (Capana, et al., (1994) Plant Mol. (1998) Plant J. 14(2):247-257) and tetracycline-inducible Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876: and tetracycline-repressible promoters (see, for example, 5,750,386; 5,633,363; 5,459,252: 5,401,836; 5,110,732 and Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. 65 5,023,179. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by “Seed-preferred promoters include both “seed-specific' reference. promoters (those promoters active during seed development US 9,688,730 B2 73 74 Such as promoters of seed storage proteins) as well as 7:171-176); sulfonamide (Guerineau, et al., (1990) Plant 'seed-germinating promoters (those promoters active dur Mol. Biol. 15:127-136); bromoxynil (Stalker, et al., (1988) ing seed germination). See, Thompson, et al., (1989) Bio Science 242:419–423); glyphosate (Shaw, et al., (1986) Essays 10:108, herein incorporated by reference. Such seed Science 233:478-481 and U.S. patent application Ser. Nos. preferred promoters include, but are not limited to, Cim 1 10/004.357 and 10/427,692); phosphinothricin (DeBlock, et (cytokinin-induced message); cz 19B 1 (maize 19 kDa zein); al., (1987) EMBO.J. 6:2513-2518). Seegenerally, Yarranton, and milps (myo-inositol-1-phosphate synthase) (see, U.S. (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et Pat. No. 6.225,529, herein incorporated by reference). al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et Gamma-Zein and Glb-1 are endosperm-specific promoters. al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. For dicots, seed-specific promoters include, but are not 10 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku, K. D. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., and Goldberg, R. B. Plant Cell 1:1079-1093, 1989), bean (1987) Cell 49:603-612: Figge, et al., (1988) Cell 52:713 B-phaseolin, napin, B-conglycinin, glycinin 1, Soybean lec 722: Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA tin, cruciferin, and the like. For monocots, seed-specific 86:5400-5404: Fuerst, et al., (1989) Proc. Natl. Acad. Sci. promoters include, but are not limited to, maize 15 kDa Zein, 15 USA 86:2549-2553: Deuschle, et al., (1990) Science 248: 22 kDa Zein, 27 kDa Zein, g-Zein, waxy, shrunken 1, 480-483; Gossen, (1993) Ph.D. Thesis, University of shrunken 2, globulin 1, etc. See also, WO 2000/12733, Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA where seed-preferred promoters from endl and end2 genes 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. are disclosed; herein incorporated by reference. In dicots, 10:3343-3356: Zambretti, et al., (1992) Proc. Natl. Acad. seed specific promoters include but are not limited to seed Sci. USA 89:3952-3956; Bairn, et al., (1991) Proc. Natl. coat promoter from Arabidopsis, pFBAN; and the early seed Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) promoters from Arabidopsis, p26, p.63, and p63tr (U.S. Pat. Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, Nos. 7.294,760 and 7,847,153). A promoter that has “pre (1989) Topics Mol. Struc. Biol. 10:143-162: Degenkolb, et ferred expression in a particular tissue is expressed in that al., (1991) Antimicrob. Agents Chemother: 35:1591-1595: tissue to a greater degree than in at least one other plant 25 Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; tissue. Some tissue-preferred promoters show expression Bonin, (1993) Ph.D. Thesis, University of Heidelberg: Gos almost exclusively in the particular tissue. sen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Where low level expression is desired, weak promoters Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913 will be used. Generally, the term “weak promoter” as used 919; Hlavka, et al., (1985) Handbook of Experimental herein refers to a promoter that drives expression of a coding 30 Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and sequence at a low level. By low level expression at levels of Gill, et al., (1988) Nature 334:721-724. Such disclosures about 1/1000 transcripts to about 1/100,000 transcripts to are herein incorporated by reference. about 1/500,000 transcripts is intended. Alternatively, it is The above list of selectable marker genes is not meant to recognized that the term “weak promoters' also encom be limiting. Any selectable marker gene can be used in the passes promoters that drive expression in only a few cells 35 embodiments. and not in others to give a total low level of expression. Plant Transformation Where a promoter drives expression at unacceptably high The methods of the embodiments involve introducing a levels, portions of the promoter sequence can be deleted or polypeptide or polynucleotide into a plant. “Introducing is modified to decrease expression levels. intended to mean presenting to the plant the polynucleotide Such weak constitutive promoters include, for example 40 or polypeptide in Such a manner that the sequence gains the core promoter of the Rsyn? promoter (WO 1999/43838 access to the interior of a cell of the plant. The methods of and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, the embodiments do not depend on a particular method for and the like. Other constitutive promoters include, for introducing a polynucleotide or polypeptide into a plant, example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608, only that the polynucleotide or polypeptides gains access to 144; 5,604,121 : 5,569,597; 5,466,785; 5,399,680: 5,268, 45 the interior of at least one cell of the plant. Methods for 463; 5,608,142 and 6,177,611, herein incorporated by ref introducing polynucleotide or polypeptides into plants are CCC. known in the art including, but not limited to, stable trans The above list of promoters is not meant to be limiting. formation methods, transient transformation methods and Any appropriate promoter can be used in the embodiments. virus-mediated methods. Generally, the expression cassette will comprise a select 50 "Stable transformation' is intended to mean that the able marker gene for the selection of transformed cells. nucleotide construct introduced into a plant integrates into Selectable marker genes are utilized for the selection of the genome of the plant and is capable of being inherited by transformed cells or tissues. Marker genes include genes the progeny thereof. “Transient transformation' is intended encoding antibiotic resistance, such as those encoding neo to mean that a polynucleotide is introduced into the plant and mycin phosphotransferase II (NEO) and hygromycin phos 55 does not integrate into the genome of the plant or a poly photransferase (HPT), as well as genes conferring resistance peptide is introduced into a plant. By “plant' is intended to herbicidal compounds. Such as glufosinate ammonium, whole plants, plant organs (e.g., leaves, sterns, roots, etc.), bromoxynil, imidazolinones and 2,4-dichlorophenoxyac seeds, plant cells, propagules, embryos and progeny of the etate (2,4-D). Additional examples of suitable selectable same. Plant cells can be differentiated or undifferentiated marker genes include, but are not limited to, genes encoding 60 (e.g. callus, Suspension culture cells, protoplasts, leaf cells, resistance to chloramphenicol (Herrera Estrella, et al., root cells, phloem cells, and pollen). (1983) EMBO.J. 2:987-992); methotrexate (Herrera Estrella, Transformation protocols as well as protocols for intro et al., (1983) Nature 303:209-213 and Meijer, et al., (1991) ducing nucleotide sequences into plants may vary depending Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., on the type of plant or plant cell, i.e., monocot or dicot, (1987) Mol. Gen. Genet. 210:86-91); spectinomycin 65 targeted for transformation. Suitable methods of introducing (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131 nucleotide sequences into plant cells and Subsequent inser 137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. tion into the plant genome include microinjection (Cross US 9,688,730 B2 75 76 way, et al., (1986) Biotechniques 4:320-334), electropora desired genomic location is achieved using a site-specific tion (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA recombination system. See, for example, WO 1999/25821, 83:5602-5606), Agrobacterium-mediated transformation WO 1999/25854, WO 1999/25840, WO 1999/25855 and (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene trans WO 1999/25853, all of which are herein incorporated by fer (Paszkowski, et al., (1984) EMBO.J. 3:2717-2722) and reference. Briefly, the polynucleotide of the embodiments ballistic particle acceleration (see, for example, U.S. Pat. can be contained in transfer cassette flanked by two non Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782: identical recombination sites. The transfer cassette is intro Tomes, et al., (1995) in Plant Cell, Tissue, and Organ duced into a plant have stably incorporated into its genome Culture. Fundamental Methods, ed. Gamborg and Phillips, a target site which is flanked by two non-identical recom (Springer-Verlag, Berlin) and McCabe, et al., (1988) Bio 10 technology 6:923-926) and Lecl transformation (WO 2000/ bination sites that correspond to the sites of the transfer 28058). For potato transformation see, Tu, et al., (1998) cassette. An appropriate recombinase is provided and the Plant Molecular Biology 37:829-838 and Chong, et al., transfer cassette is integrated at the target site. The poly (2000) Transgenic Research 9:71-78. Additional transfor nucleotide of interest is thereby integrated at a specific mation procedures can be found in Weissinger, et al., (1988) 15 chromosomal position in the plant genome. Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Par Plant transformation vectors may be comprised of one or ticulate Science and Technology 5:27-37 (onion); Christou, more DNA vectors needed for achieving plant transforma et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, tion. For example, it is a common practice in the art to utilize et al., (1988) Bio/Technology 6:923-926 (soybean): Finer plant transformation vectors that are comprised of more than and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 one contiguous DNA segment. These vectors are often (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319 referred to in the art as “binary vectors'. Binary vectors as 324 (soybean); Datta, et al., (1990) Biotechnology 8:736 well as vectors with helper plasmids are most often used for 740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA Agrobacterium-mediated transformation, where the size and 85:4305-4309 (maize): Klein, et al., (1988) Biotechnology complexity of DNA segments needed to achieve efficient 6:559-563 (maize): U.S. Pat. Nos. 5,240,855; 5,322,783 and 25 transformation is quite large, and it is advantageous to 5,324,646; Klein, et al., (1988) Plant Physiol. 91:440-444 separate functions onto separate DNA molecules. Binary (maize): Fromm, et al., (1990) Biotechnology 8:833-839 vectors typically contain a plasmid vector that contains the (maize): Hooykaas-Van Slogteren, et al., (1984) Nature cis-acting sequences required for T-DNA transfer (Such as (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); left border and right border), a selectable marker that is Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:53.45 30 engineered to be capable of expression in a plant cell, and a 5349 (Liliaceae); De Wet, et al., (1985) in The Experimental 'gene of interest' (a gene engineered to be capable of Manipulation of Ovule Tissues, ed. Chapman, et al., (Long expression in a plant cell for which generation of transgenic man, New York), pp. 197-209 (pollen); Kaeppler, et al., plants is desired). Also present on this plasmid vector are (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., sequences required for bacterial replication. The cis-acting (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated 35 sequences are arranged in a fashion to allow efficient transfer transformation); DHalluin, et al., (1992) Plant Cell 4:1495 into plant cells and expression therein. For example, the 1505 (electroporation); Li, et al., (1993) Plant Cell Reports selectable marker gene and the pesticidal gene are located 12:250-255 and Christou and Ford, (1995) Annals of Botany between the left and right borders. Often a second plasmid 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnol vector contains the trans-acting factors that mediate T-DNA ogy 14:745-750 (maize via Agrobacterium tumefaciens); all 40 transfer from Agrobacterium to plant cells. This plasmid of which are herein incorporated by reference. often contains the virulence functions (Virgenes) that allow In specific embodiments, the sequences of the embodi infection of plant cells by Agrobacterium, and transfer of ments can be provided to a plant using a variety of transient DNA by cleavage at border sequences and vir-mediated transformation methods. Such transient transformation DNA transfer, as is understood in the art (Hellens and methods include, but are not limited to, the introduction of 45 Mullineaux, (2000) Trends in Plant Science 5:446-451). the PIP-1 polypeptide or variants and fragments thereof Several types of Agrobacterium strains (e.g. LBA4404, directly into the plant or the introduction of the PIP-1 GV3101, EHA101, EHA105, etc.) can be used for plant polypeptide transcript into the plant. Such methods include, transformation. The second plasmid vector is not necessary for example, microinjection or particle bombardment. See, for transforming the plants by other methods such as micro for example, Crossway, et al., (1986) Mol Gen. Genet. 50 projection, microinjection, electroporation, polyethylene 202:179-185: Nomura, et al., (1986) Plant Sci. 44:53-58: glycol, etc. Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 In general, plant transformation methods involve trans and Hush, et al., (1994) The Journal of Cell Science 107: ferring heterologous DNA into target plant cells (e.g., imma 775-784, all of which are herein incorporated by reference. ture or mature embryos, Suspension cultures, undifferenti Alternatively, the PIP-1 polypeptide polynucleotide can be 55 ated callus, protoplasts, etc.), followed by applying a transiently transformed into the plant using techniques maximum threshold level of appropriate selection (depend known in the art. Such techniques include viral vector ing on the selectable marker gene) to recover the trans system and the precipitation of the polynucleotide in a formed plant cells from a group of untransformed cell mass. manner that precludes subsequent release of the DNA. Thus, Following integration of heterologous foreign DNA into transcription from the particle-bound DNA can occur, but 60 plant cells, one then applies a maximum threshold level of the frequency with which it is released to become integrated appropriate selection in the medium to kill the untrans into the genome is greatly reduced. Such methods include formed cells and separate and proliferate the putatively the use of particles coated with polyethylimine (PEI: Sigma transformed cells that survive from this selection treatment #P3143). by transferring regularly to a fresh medium. By continuous Methods are known in the art for the targeted insertion of 65 passage and challenge with appropriate selection, one iden a polynucleotide at a specific location in the plant genome. tifies and proliferates the cells that are transformed with the In one embodiment, the insertion of the polynucleotide at a plasmid vector. Molecular and biochemical methods can US 9,688,730 B2 77 78 then be used to confirm the presence of the integrated borne transgene by tissue-preferred expression of a nuclear heterologous gene of interest into the genome of the trans encoded and plastid-directed RNA polymerase. Such a sys genic plant. tem has been reported in McBride, et al., (1994) Proc. Natl. Explants are typically transferred to a fresh supply of the Acad. Sci. USA 91:7301-73.05. same medium and cultured routinely. Subsequently, the The embodiments further relate to plant-propagating transformed cells are differentiated into shoots after placing material of a transformed plant of the embodiments includ on regeneration medium Supplemented with a maximum ing, but not limited to, seeds, tubers, corms, bulbs, leaves, threshold level of selecting agent. The shoots are then and cuttings of roots and shoots. transferred to a selective rooting medium for recovering The embodiments may be used for transformation of any rooted shoot or plantlet. The transgenic plantlet then grows 10 plant species, including, but not limited to, monocots and into a mature plant and produces fertile seeds (e.g., Hiei, et dicots. Examples of plants of interest include, but are not al., (1994) The Plant Journal 6:271-282; Ishida, et al., limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. (1996) Nature Biotechnology 14:745-750). Explants are rapa, B. juncea), particularly those Brassica species useful typically transferred to a fresh Supply of the same medium as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza and cultured routinely. A general description of the tech 15 sativa), rye (Secale cereale), Sorghum (Sorghum bicolor, niques and methods for generating transgenic plants are Sorghum vulgare), millet (e.g., pearl millet (Pennisetum found in Ayres and Park, (1994) Critical Reviews in Plant glaucum), proso millet (Panicum miliaceum), foxtail millet Science 13:219-239 and Bommineni and Jauhar, (1997) (Setaria italica), finger millet (Eleusine Coracana)), Sun Maydica 42:107-120. Since the transformed material con flower (Helianthus annuus), safflower (Carthamus tincto tains many cells; both transformed and non-transformed rius), wheat (Triticum aestivum), soybean (Glycine max), cells are present in any piece of Subjected target callus or tobacco (Nicotiana tabacum), potato (Solanum tuberosum), tissue or group of cells. The ability to kill non-transformed peanuts (Arachis hypogaea), cotton (Gossypium bar cells and allow transformed cells to proliferate results in badense, Gossypium hirsutum), Sweet potato (Ipomoea transformed plant cultures. Often, the ability to remove batatus), cassava (Manihot esculenta), coffee (Coffea spp.), non-transformed cells is a limitation to rapid recovery of 25 coconut (Cocos nucifera), pineapple (Ananas comosus), transformed plant cells and Successful generation of trans citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea genic plants. (Camelia sinensis), banana (Musa spp.), avocado (Persea The cells that have been transformed may be grown into americana), fig (Ficus Casica), guava (Psidium guajava). plants in accordance with conventional ways. See, for mango (Mangifera indica), olive (Olea europaea), papaya example, McCormick, et al., (1986) Plant Cell Reports 30 (Carica papaya), cashew (Anacardium occidentale), maca 5:81-84. These plants may then be grown, and either polli damia (Macadamia integrifolia), almond (Prunus amygd nated with the same transformed strain or different strains alus), Sugar beets (Beta vulgaris), Sugarcane (Saccharum and the resulting hybrid having constitutive or inducible spp.), oats, barley, vegetables ornamentals and conifers. expression of the desired phenotypic characteristic identi Vegetables include tomatoes (Lycopersicon esculentum), fied. Two or more generations may be grown to ensure that 35 lettuce (e.g., Lactuca sativa), green beans (Phaseolus vul expression of the desired phenotypic characteristic is stably garis), lima beans (Phaseolus limensis), peas (Lathyrus maintained and inherited and then seeds harvested to ensure spp.), and members of the genus Cucumis Such as cucumber that expression of the desired phenotypic characteristic has (C. sativus), cantaloupe (C. cantalupensis), and muskmelon been achieved. (C. melo). Ornamentals include azalea (Rhododendron spp.), The nucleotide sequences of the embodiments may be 40 hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus provided to the plant by contacting the plant with a virus or rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daf viral nucleic acids. Generally, Such methods involve incor fodils (Narcissus spp.), petunias (Petunia hybrida), carna porating the nucleotide construct of interest within a viral tion (Dianthus caryophyllus), poinsettia (Euphorbia pull DNA or RNA molecule. It is recognized that the recombi cherrima), and chrysanthemum. Conifers that may be nant proteins of the embodiments may be initially synthe 45 employed in practicing the embodiments include, for sized as part of a viral polyprotein, which later may be example, pines such as loblolly pine (Pinus taeda), slash processed by proteolysis in vivo or in vitro to produce the pine (Pinus elliotil), ponderosa pine (Pinus ponderosa), desired PIP-1 polypeptide. It is also recognized that such a lodgepole pine (Pinus contorta) and Monterey pine (Pinus viral polyprotein, comprising at least a portion of the amino radiata); Douglas-fir (Pseudotsuga menziesii); Western acid sequence of a PIP-1 polypeptide of the embodiments, 50 hemlock (Tsuga Canadensis); Sitka spruce (Picea glauca); may have the desired pesticidal activity. Such viral poly redwood (Sequoia sempervirens); true firs such as silver fir proteins and the nucleotide sequences that encode for them (Abies amabilis) and balsam fir (Abies balsamea); and are encompassed by the embodiments. Methods for provid cedars such as Western red cedar (Thuja plicata) and Alaska ing plants with nucleotide constructs and producing the yellow-cedar (Chamaecyparis mootkatensis). Plants of the encoded proteins in the plants, which involve viral DNA or 55 embodiments include crop plants (for example, corn, alfalfa, RNA molecules are known in the art. See, for example, U.S. Sunflower, Brassica, soybean, cotton, safflower, peanut, Sor Pat. Nos. 5,889, 191; 5,889, 190; 5,866,785: 5,589,367 and ghum, wheat, millet, tobacco, etc.). Such as corn and Soy 5.316,931, herein incorporated by reference. bean plants. Methods for transformation of chloroplasts are known in Turfgrasses include, but are not limited to: annual blue the art. See, for example, Svab, et al., (1990) Proc. Natl. 60 grass (Poa annua); annual ryegrass (Lolium multiflorum); Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Canada bluegrass (Poa compressa); Chewings fescue (Fes Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, tuca rubra); colonial bentgrass (Agrostis tenuis); creeping (1993) EMBO.J. 12:601-606. The method relies on particle bentgrass (Agrostis palustris); crested wheatgrass (Agropy gun delivery of DNA containing a selectable marker and ron desertorum); fairway wheatgrass (Agropyron cristatum); targeting of the DNA to the plastid genome through homolo 65 hard fescue (Festuca longifolia); Kentucky bluegrass (Poa gous recombination. Additionally, plastid transformation pratensis); orchardgrass (Dactyls glomerata); perennial can be accomplished by transactivation of a silent plastid ryegrass (Lolium perenne); red fescue (Festuca rubra); US 9,688,730 B2 79 80 redtop (Agrostis alba); rough bluegrass (Poa trivialis); methods or through genetic engineering methods. These sheep fescue (Festuca ovina); Smooth bromegrass (Bromus methods include, but are not limited to, breeding individual inermis); tall fescue (Festuca arundinacea); timothy lines each comprising a polynucleotide of interest, trans (Phleum pratense); Velvet bentgrass (Agrostis canina); forming a transgenic plant comprising a gene disclosed weeping alkaligrass (Puccinelia distans); western wheat 5 herein with a Subsequent gene, and co-transformation of grass (Agropyron Smithii); Bermuda grass (Cynodon spp.); genes into a single plant cell. As used herein, the term St. Augustine grass (Stenotaphrum secundatum); Zoysia “stacked' includes having two or more traits present in the grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet same plant (e.g., both traits are incorporated into the nuclear grass (Axonopus affinis); centipede grass (Eremochloa Ophi genome, one trait is incorporated into the nuclear genome uroides): kikuyu grass (Pennisetum clandesinum); seashore 10 and one trait is incorporated into the genome of a plastid or paspalum (Paspalum vaginatum); blue gramma (Bouteloua both traits are incorporated into the genome of a plastid). In gracilis); buffalo grass (Buchloe dactyloids); Sideoats one non-limiting example, "stacked traits' comprise a gramma (Bouteloua curtipendula). molecular stack where the sequences are physically adjacent Plants of interest include grain plants that provide seeds of to each other. A trait, as used herein, refers to the phenotype interest, oil-seed plants, and leguminous plants. Seeds of 15 derived from a particular sequence or groups of sequences. interest include grain seeds, such as corn, wheat, barley, rice, Co-transformation of genes can be carried out using single Sorghum, rye, millet, etc. Oil-seed plants include cotton, transformation vectors comprising multiple genes or genes Soybean, safflower, Sunflower, Brassica, maize, alfalfa, carried separately on multiple vectors. If the sequences are palm, coconut, flax, castor, olive etc. Leguminous plants stacked by genetically transforming the plants, the poly include beans and peas. Beans include guar, locust bean, nucleotide sequences of interest can be combined at any fenugreek, soybean, garden beans, cowpea, mungbean, lima time and in any order. The traits can be introduced simul bean, fava bean, lentils, chickpea, etc. taneously in a co-transformation protocol with the poly Evaluation of Plant Transformation nucleotides of interest provided by any combination of Following introduction of heterologous foreign DNA into transformation cassettes. For example, if two sequences will plant cells, the transformation or integration of heterologous 25 be introduced, the two sequences can be contained in gene in the plant genome is confirmed by various methods separate transformation cassettes (trans) or contained on the Such as analysis of nucleic acids, proteins and metabolites same transformation cassette (cis). Expression of the associated with the integrated gene. sequences can be driven by the same promoter or by PCR analysis is a rapid method to screen transformed different promoters. In certain cases, it may be desirable to cells, tissue or shoots for the presence of incorporated gene 30 introduce a transformation cassette that will Suppress the at the earlier stage before transplanting into the soil (Sam expression of the polynucleotide of interest. This may be brook and Russell, (2001) Molecular Cloning: A Laboratory combined with any combination of other suppression cas Manual. Cold Spring Harbor Laboratory Press, Cold Spring settes or overexpression cassettes to generate the desired Harbor, N.Y.). PCR is carried out using oligonucleotide combination of traits in the plant. It is further recognized that primers specific to the gene of interest or Agrobacterium 35 polynucleotide sequences can be stacked at a desired vector background, etc. genomic location using a site-specific recombination sys Plant transformation may be confirmed by Southern blot tem. See, for example, WO 1999/25821, WO 1999/25854, analysis of genomic DNA (Sambrook and Russell, (2001) WO 1999/25840, WO 1999/25855 and WO 1999/25853, all supra). In general, total DNA is extracted from the trans of which are herein incorporated by reference. formant, digested with appropriate restriction enzymes, frac 40 In some embodiments the polynucleotides encoding the tionated in an agarose gel and transferred to a nitrocellulose PIP-1 polypeptides disclosed herein, alone or stacked with or nylon membrane. The membrane or “blot' is then probed one or more additional insect resistance traits can be stacked with, for example, radiolabeled 32P target DNA fragment to with one or more additional input traits (e.g., herbicide confirm the integration of introduced gene into the plant resistance, fungal resistance, virus resistance or stress tol genome according to standard techniques (Sambrook and 45 erance, disease resistance, male sterility, stalk strength, and Russell, (2001) supra). the like) or output traits (e.g., increased yield, modified In Northern blot analysis, RNA is isolated from specific starches, improved oil profile, balanced amino acids, high tissues of transformant, fractionated in a formaldehyde aga lysine or methionine, increased digestibility, improved fiber rose gel, and blotted onto a nylon filter according to standard quality, drought resistance, and the like). Thus, the poly procedures that are routinely used in the art (Sambrook and 50 nucleotide embodiments can be used to provide a complete Russell, (2001) supra). Expression of RNA encoded by the agronomic package of improved crop quality with the ability pesticidal gene is then tested by hybridizing the filter to a to flexibly and cost effectively control any number of radioactive probe derived from a pesticidal gene, by meth agronomic pests. ods known in the art (Sambrook and Russell. (2001) supra). Transgenes useful for stacking include but are not limited to: Western blot, biochemical assays and the like may be 55 1. Transgenes that Confer Resistance to Insects or Disease carried out on the transgenic plants to confirm the presence and that Encode: of protein encoded by the pesticidal gene by standard (A) Plant disease resistance genes. Plant defenses are procedures (Sambrook and Russell, 2001, Supra) using anti often activated by specific interaction between the product bodies that bind to one or more epitopes present on the PIP-1 of a disease resistance gene (R) in the plant and the product polypeptide. 60 of a corresponding avirulence (AVr) gene in the pathogen. A Stacking of Traits in Transgenic Plant plant variety can be transformed with cloned resistance gene Transgenic plants may comprise a stack of one or more to engineer plants that are resistant to specific pathogen insecticidal polynucleotides disclosed herein with one or strains. See, for example, Jones, et al., (1994) Science more additional polynucleotides resulting in the production 266:789 (cloning of the tomato Cf-9 gene for resistance to or Suppression of multiple polypeptide sequences. Trans 65 Cladosporium fulvum); Martin, et al., (1993) Science 262: genic plants comprising stacks of polynucleotide sequences 1432 (tomato Pto gene for resistance to Pseudomonas Syrin can be obtained by either or both of traditional breeding gae pv. tomato encodes a protein kinase); Mindrinos, et al., US 9,688,730 B2 81 82 (1994) Cell 78: 1089 (Arabidopsis RSP2 gene for resistance X54939), Cry1Ab10 (Accession # A29125), Cry1Ab11 (Ac to Pseudomonas Syringae), McDowell and Woffenden, cession if I12419), Cry1Ab12 (Accession # AF059670), (2003) Trends Biotechnol. 21(4):178-83 and Toyoda, et al., Cry1Ab13 (Accession if AF254640), Cry1Ab14 (Accession (2002) Transgenic Res. 11(6):567-82. A plant resistant to a # U94191), Cry1Ab15 (Accession # AF358861), Cry1Ab16 disease is one that is more resistant to a pathogen as (Accession # AF375608), Cry1Ab17 (Accession # compared to the wild type plant. AAT46415), Cry1Ab18 (Accession # AAQ88259), (B) Genes encoding a Bacillus thuringiensis protein, a Cry1Ab19 (Accession # AY847289), Cry1Ab20 (Accession derivative thereof or a synthetic polypeptide modeled # DQ241675), Cry1Ab21 (Accession # EF683163), thereon. See, for example, Geiser, et al., (1986) Gene Cry1Ab22 (Accession # ABW87320), Cry1Ab-like (Acces 48: 109, who disclose the cloning and nucleotide sequence of 10 sion # AF327924), Cry1Ab-like (Accession # AF327925), a Bt delta-endotoxin gene. Moreover, DNA molecules Cry1Ab-like (Accession if AF327926), Cry1Ab-like (Acces encoding delta-endotoxin genes can be purchased from sion # DQ781309), Cry1Ac1 (Accession # M11068), American Type Culture Collection (Rockville, Md.), for Cry1Ac2 (Accession ii M35524), Cry1Ac3 (Accession it example, under ATCCTM Accession Numbers 40098, 67136, X54159), Cry1Ac4 (Accession # MT3249), Cry1Ac5 (Ac 31995 and 31998. Other non-limiting examples of Bacillus 15 cession # M73248), Cry1Ac6 (Accession # U43606), thuringiensis transgenes being genetically engineered are Cry1Ac7 (Accession # U87793), Cry1Ac8 (Accession it given in the following patents and patent applications and U87397), Cry1Ac9 (Accession # U89872), Cry1Ac 10 (Ac hereby are incorporated by reference for this purpose: U.S. cession # AJO02514), Cry1Ac11 (Accession # AJ130970), Pat. Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; Cry1Ac12 (Accession if I12418), Cry1Ac13 (Accession it 6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, AF148644), Cry1Ac14 (Accession # AF492767), Cry1Ac15 6,642,030, 6,713,259, 6,893,826, 7,105,332; 7,179,965, (Accession if AY122057), Cry1Ac 16 (Accession it 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, AY730621), Cry1Ac17 (Accession # AY925090), 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862, Cry1Ac18 (Accession it DQ023296), Cry1Ac 19 (Accession 7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, # DQ195217), Cry1Ac20 (Accession it DQ285666), 7,790,846, 7,858,849, and WO 1991/14778; WO 1999/ 25 Cry1Ac21 (Accession it DQ062689), Cry1Ac22 (Accession 3.1248: WO 2001/12731: WO 1999/24581 and WO 1997/ # EU282379), Cry1Ac23 (Accession # AM949588), 4O162. Cry1Ac24 (Accession it ABLO 1535), Cry1Ad1 (Accession it Genes encoding pesticidal proteins may also be stacked M73250), Cry1Ad2 (Accession # A27531), Cry1Ae1 (Ac including but are not limited to: insecticidal proteins from cession # M65252), Cry1Af1 (Accession # U82003), Pseudomonas sp. such as PSEEN3174 (Monalysin, (2011) 30 Cry1Ag1 (Accession if AF081248), Cry1Ahl (Accession it PLOS Pathogens, 7:1-13), from Pseudomonas protegens AF28.1866), Cry1Ah2 (Accession it DQ269474), Cry1Ai1 strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (Accession # AY174873), Cry1A-like (Accession # (2008) Environmental Microbiology 10:2368-2386: Gen AF327927), Cry 1Ba1 (Accession # X06711), Cry 1Ba2 (Ac Bank Accession No. EU4001 57); from Pseudomonas Tai cession # X95704), Cry1Ba3 (Accession # AF368257), wanensis (Liu, et al., (2010).J. Agric. Food Chem. 58:12343 35 Cry 1Ba4 (Accession # AF363025), Cry 1Ba5 (Accession it 12349) and from Pseudomonas pseudoalcligenes (Zhang, et AB020894), Cry1 Ba6 (Accession # ABL60921), Cry1 Bb1 al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (Accession if L32020), Cry 1 Bc 1 (Accession it Z46442), (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecti Cry 1Bd1 (Accession it U70726), Cry1 Bd2 (Accession it cidal proteins from Photorhabdus sp. and Xenorhabdus sp. AY138457), Cry1Be1 (Accession # AF077326), Cry 1Be2 (Hinchliffe, et al., (2010) The Open Toxinology Journal 40 (Accession if AAO52387), Cry1 Bfl (Accession it 3:101-118 and Morgan, et al., (2001) Applied and Envir. AX189649), Cry1 Bf2 (Accession #AAQ52380), Cry1 Bg1 Micro. 67:2062-2069), U.S. Pat. No. 6,048,838, and U.S. (Accession # AY176063), Cry1Ca1 (Accession # X07518), Pat. No. 6,379,946; and Ö-endotoxins including, but not Cry 1 Ca2 (Accession if X13620), Cry 1Ca3 (Accession it limited to, the Cry1, Cry2, Cry3, Cry4, Crys, Cry6, Cry7. M73251), Cry1Ca4 (Accession if A27642), Cry1Ca5 (Ac Cry8, Cry9, Cry 10, Cry 11, Cry 12, Cry 13, Cry 14, Cry 15, 45 cession # X96682), Cry1Ca61 (Accession # AF215647), Cry16, Cry 17, Cry 18, Cry 19, Cry20, Cry21, Cry22, Cry23, Cry1Ca7 (Accession # AYO15492), Cry1Ca8 (Accession it Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, AF362020), Cry1Ca9 (Accession # AY078160), Cry1Ca10 Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, (Accession it AF540014), Cry1Ca11 (Accession it Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, AY955268), Cry1Cb1 (Accession # M97880), Cry1Cb2 Cry49, Cry 51 and Crys5 classes of 5-endotoxin genes and 50 (Accession # AY007686), Cry1Cb3 (Accession # the B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Mem EU679502), Cry1Cb-like (Accession # AAX63901), bers of these classes of B. thuringiensis insecticidal proteins Cry 1 Dal (Accession if X54160), Cry 1Da2 (Accession it include, but are not limited to Cry1Aa1 (Accession # Acces I76415), Cry1Db1 (Accession # Z22511), Cry1Db2 (Acces sion # M11250), Cry1Aa2 (Accession # M10917), Cry1Aa3 sion # AF358862), Cry1Dc1 (Accession # EF059913), (Accession # D00348), Cry1Aa4 (Accession if X13535), 55 Cry1 Ea1 (Accession # X53985), Cry1 Ea2 (Accession it Cry1Aa5 (Accession # D17518), Cry1Aao (Accession it X56144), Cry1 Ea3 (Accession # M73252), Cry1Ea-4 (Ac U43605), Cry1Aa7 (Accession # AF081790), Cry1Aa8 (Ac cession # U94323), Cry1 Eas (Accession # A15535), cession #126149), Cry1Aa9 (Accession if AB026261), Cry1 Eaé (Accession # AF202531), Cry1 Ea7 (Accession it Cry1Aa10 (Accession if AF154676), Cry1Aa11 (Accession AAW72936), Cry1 Ea8 (Accession # ABX11258), Cry1 Eb1 # Y09663), Cry1Aa12 (Accession # AF384211), Cry1Aa13 60 (Accession # MT3253), Cry1Fa1 (Accession it. M63897), (Accession it AF510713), Cry1Aa14 (Accession it Cry1 Fa2 (Accession # M73254), Cry1Fb1 (Accession it AY197341), Cry1Aa15 (Accession it DQ062690), Cry1Ab1 Z22512), Cry1Fb2 (Accession # AB012288), Cry1Fb3 (Ac (Accession # M13898), Cry1Ab2 (Accession # M12661), cession # AF062350), Cry1Fb4 (Accession # I73895), Cry1Ab3 (Accession if M15271), Cry1Ab4 (Accession it Cry1Fb5 (Accession # AF336114), Cry1Fb6 (Accession it D00117), Cry1Ab5 (Accession # X04698), Cry1Ab6 (Ac 65 EU679.500), Cry1Fb7 (Accession # EU6795.01), Cry1Ga1 cession # M37263), Cry1Ab7 (Accession #X13233), (Accession # Z22510), Cry1Ga2 (Accession # Y09326), Cry1Ab8 (Accession ii M16463), Cry1Ab9 (Accession it Cry1Gb1 (Accession it U70725), Cry 1Gb2 (Accession it

US 9,688,730 B2 85 86 AB023293), Cry28Aa1 (Accession # AF132928), ing but not limited to an engineered hybrid insecticidal Cry28Aa2 (Accession # AF285775), Cry29Aa1 (Accession protein (eHIP) created by fusing unique combinations of # AJ251977), Cry30Aa1 (Accession # AJ251978), variable regions and conserved blocks of at least two dif Cry30Ba1 (Accession # BAD00052), Cry30Ca1 (Accession ferent Cry proteins (US Patent Application Publication # BAD67157), Cry30Da1 (Accession # EF095955), Cry30 5 Number 2010/0017914); a Cry4 protein; a Crys protein; a Db1 (Accession it BAE80088), Cry30Ea1 (Accession # Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, EU503140), Cry30Fa1 (Accession # EU751609), Cry30Ga1 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378.499 and (Accession it EU882064), Cry31 Aa1 (Accession it 7.462.760; a Cry9 protein such as such as members of the AB031065), Cry31Aa2 (Accession # AY081052), Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families: Cry31 Aa3 (Accession if AB250922), Cry31Aa4 (Accession 10 a Cry 15 protein of Naimov, et al., (2008) Applied and # AB274826), Cry31Aa5 (Accession # AB274827), Environmental Microbiology 74.71.45-7151; a Cry22, a Cry31Ab1 (Accession # AB250923), Cry31Ab2 (Accession Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 # AB274825), Cry31 Ac1 (Accession # AB276125), and 6,340,593: a CryET33 and CryET34 protein of U.S. Pat. Cry32Aa1 (Accession # AY008143), Cry32Ba1 (Accession Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 # BAB78601), Cry32Ca1 (Accession # BAB78602), 15 and 7,504,229; a CryET33 and CryET34 homologs of US Cry32Dal (Accession it. BAB78603), Cry33Aa1 (Accession Patent Publication Number 2006/0191034, 2012/0278954, # AAL26871), Cry34Aa1 (Accession # AAG50341), and PCT Publication Number WO 2012/139004: a Cry34Aa2 (Accession if AAK64560), Cry34Aa3 (Accession Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 # AY536899), Cry34Aa-4 (Accession # AY536897), and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry Cry34Ab1 (Accession if AAG41671), Cry34Ac1 (Accession binary toxin; a TIC901 or related toxin: TIC807 of US # AAG50118), Cry34Ac2 (Accession # AAK64562), 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, Cry34Ac3 (Accession # AY536896), Cry34Ba1 (Accession TIC127, TIC128 of PCT US 2006/033867; AXMI-027, # AAK64565), Cry34Ba2 (Accession # AY536900), AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757: Cry34Ba3 (Accession # AY536898), Cry35Aa1 (Accession AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. # AAG50342), Cry35Aa2 (Accession #AAK64561), 25 No. 7,923,602: AXMI-018, AXMI-020, and AXMI-021 of Cry35Aa3 (Accession # AY536895), Cry35Aa4 (Accession WO 2006/083891: AXMI-010 of WO 2005/038032: AXMI # AY536892), Cry35Ab1 (Accession # AAG41672), 003 of WO 2005/021585; AXMI-008 of US 2004/0250311; Cry35Ab2 (Accession # AAK64563), Cry35Ab3 (Acces AXMI-006 of US 2004/0216186: AXMI-007 of US 2004/ sion # AY536891), Cry35Ac1 (Accession # AAG50117), 0210965; AXMI-009 of US 2004/0210964: AXMI-014 of Cry35Ba1 (Accession #AAK64566), Cry35Ba2 (Accession 30 US 2004/0197917; AXMI-004 of US 2004/0197916: # AY536894), Cry35Ba3 (Accession # AY536893), AXMI-028 and AXMI-029 of WO 2006/119457; AXMI Cry36Aal (Accession #AAK64558), Cry37Aal (Accession 007, AXMI-008, AXMI-0080r12, AXMI-009, AXMI-014 # AAF76376), Cry38Aa1 (Accession # AAK64559), and AXMI-004 of WO 2004/074462: AXMI-150 of U.S. Cry39Aa1 (Accession it BAB72016), Cry40Aa1 (Accession Pat. No. 8,084,416: AXMI-205 of US20110023184: AXMI # BAB72018), Cry40Ba1 (Accession # BAC77648), 35 011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, Cry40Ca1 (Accession # EU381045), Cry40Dal (Accession AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI # EU596478), Cry41Aa1 (Accession # AB116649), 034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and Cry41 Ab1 (Accession it AB 116651), Cry42Aa1 (Accession AXMI-064 of US 2011/0263488; AXMI-R1 and related # AB116652), Cry43Aa1 (Accession # AB115422), proteins of US 2010/0197592: AXMI221Z, AXMI222z, Cry43Aa2 (Accession if AB176668), Cry43 Ba1 (Accession 40 AXMI223Z, AXMI224Z and AXMI225Z of WO 2011/ # AB115422), Cry43-like (Accession # AB115422), 103248: AXMI218, AXMI219, AXMI220, AXMI226, Cry44Aa (Accession it BAD08532), Cry45Aa (Accession it AXMI227, AXMI228, AXMI229, AXMI230, and BAD22577), Cry46Aa (Accession # BAC79010), AXMI231 of WO11/103,247; AXMI-115, AXMI-113, Cry46Aa2 (Accession if BAG68906), Cry46 Ab (Accession AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. # BAD35170), Cry47Aa (Accession # AY950229), 45 8,334431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, Cry48Aa (Accession if AJ841948), Cry48Aa2 (Accession it and AXMI-045 of US 2010/0298211: AXMI-066 and AM237205), Cry48Aa3 (Accession # AM237206), AXMI-076 of US2009014.4852: AXMI128, AXMI130, Cry48Ab (Accession # AM237207), Cry48Ab2 (Accession AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, # AM237208), Cry49Aa (Accession # AJ841948), AXMI 143, AXMI144, AXMI146, AXMI148, AXMI149, Cry49Aa2 (Accession it AM237201), Cry49Aa3 (Accession 50 AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, # AM237203), Cry49Aa4 (Accession # AM237204), AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, Cry49Ab1 (Accession # AM237202), CrysOAa1 (Accession AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, # AB253419), Cry51Aa1 (Accession # DQ836184), AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, Cry52Aa1 (Accession # EF613489), Crys3Aa1 (Accession AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, # EF633476), Crys4Aa1 (Accession # EU339367), 55 AXMI 182, AXMI 188, Crys5Aa1 (Accession # EU121521), Cry55Aa2 (Accession AXIMI189 AXMI079, # AAE33526). AXMI080, AXMI092, Examples of 8-endotoxins also include but are not limited AXMI096, AXMI100, to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858, AXMI101, AXMI107, 849; a DIG-3 or DIG-11 toxin (N-terminal deletion of 60 AXMI108, AXMI112, C.-helix 1 and/or C-helix 2 variants of Cry proteins such as AXMI114, AXMI119, Cry1A) of U.S. Pat. Nos. 8.304,604 and 8,304,605, Cry 1B AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, of U.S. patent application Ser. No. 10/525,318; Cry1C of AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, 6.218, 188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982: 65 AXMI137 of US 2010/0005543; Cry proteins such as 6,962,705 and 6,713,063): a Cry2 protein such as Cry2Ab Cry1A and Cry3A having modified proteolytic sites of U.S. protein of U.S. Pat. No. 7,064.249); a Cry3A protein includ Pat. No. 8.319,019; and a Cry1Ac, Cry2Aa and Cry1Ca US 9,688,730 B2 87 88 toxin protein from Bacillus thuringiensis strain VBTS 2528 Hammock, et al., (1990) Nature 344:458, of baculovirus of US Patent Application Publication Number 2011/ expression of cloned juvenile hormone esterase, an inacti 0064710. Other Cry proteins are well known to one skilled vator of juvenile hormone. in the art (see, Crickmore, et al., “Bacillus thuringiensis (D) A polynucleotide encoding an insect-specific peptide toxin nomenclature' (2011), at lifesci.sussex.ac.uk/home/ which, upon expression, disrupts the physiology of the Neil Crickmore/Bt? which can be accessed on the world affected pest. For example, see the disclosures of Regan, wide web using the “www’ prefix). The insecticidal activity (1994).J. Biol. Chem. 269:9 (expression cloning yields DNA of Cry proteins is well known to one skilled in the art (for coding for insect diuretic hormone receptor); Pratt, et al., review, see, van Frannkenhuyzen, (2009) J. Invert. Path. (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allosta 101:1-16). The use of Cry proteins as transgenic plant traits 10 tin is identified in Diploptera puntata); Chattopadhyay, et is well known to one skilled in the art and Cry-transgenic al., (2004) Critical Reviews in Microbiology 30(1):33-54; plants including but not limited to Cry1Ac, Cry1Ac-- Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini and Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, Cry1 F+ Grossi-de-Sa (2002) Toxicon 40(11): 1515-1539; Ussuf, et Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, al., (2001) Curr Sci. 80(7):847-853 and Vasconcelos and 15 Oliveira (2004) Toxicon 44(4):385-403. See also, U.S. Pat. Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have No. 5,266,317 to Tomalski, et al., who disclose genes received regulatory approval (see, Sanahuja, (2011) Plant encoding insect-specific toxins. Biotech Journal 9:283-300 and the CERA (2010) GM Crop (E) A polynucleotide encoding an enzyme responsible for Database Center for Environmental Risk Assessment a hyperaccumulation of a monterpene, a sesquiterpene, a (CERA), ILSI Research Foundation, Washington D.C. at steroid, hydroxamic acid, a phenylpropanoid derivative or cera-gmc.org/index.php?action gm crop database which another non-protein molecule with insecticidal activity. can be accessed on the world-wide web using the “www. (F) A polynucleotide encoding an enzyme involved in the prefix). More than one pesticidal proteins well known to one modification, including the post-translational modification, skilled in the art can also be expressed in plants such as of a biologically active molecule; for example, a glycolytic Vip3Ab & Cry1 Fa (US2012/0317682), Cry 1BE & Cry1F 25 enzyme, a proteolytic enzyme, a lipolytic enzyme, a nucle (US2012/0311746), Cry1CA & Cry1AB (US2012/ ase, a cyclase, a transaminase, an esterase, a hydrolase, a 0311745), Cry1F & CryCa (US2012/0317681), Cry1DA & phosphatase, a kinase, a phosphorylase, a polymerase, an Cry1BE (US2012/0331590), Cry 1DA & Cry1 Fa (US2012/ elastase, a chitinase and a glucanase, whether natural or 0331589), Cry1AB & Cry 1BE (US2012/0324606), and synthetic. See, PCT Application WO 1993/02197 in the Cry1 Fa & Cry2Aa, Cry1 I or Cry1E (US2012/0324605). 30 name of Scott, et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain Pesticidal proteins also include insecticidal lipases including chitinase-encoding sequences can be obtained, for example, lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and cho from the ATCC under Accession Numbers 39637 and 67152. lesterol oxidases such as from Streptomyces (Purcell et al. See also, Kramer, et al., (1993) Insect Biochem. Molec. Biol. (1993) Biochem Biophy's Res Commun 15:1406-1413). Pes 35 23:691, who teach the nucleotide sequence of a cDNA ticidal proteins also include VIP (vegetative insecticidal encoding tobacco hookworm chitinase and Kawaleck, et al., proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,279, (1993) Plant Molec. Biol. 21:673, who provide the nucleo 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the tide sequence of the parsley ubi4-2 polyubiquitin gene and like. Other VIP proteins are well known to one skilled in the U.S. Pat. Nos. 6,563,020; 7,145,060 and 7,087,810. art (see, lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/ 40 (G) A polynucleotide encoding a molecule that stimulates Vip.html which can be accessed on the world-wide web signal transduction. For example, see the disclosure by using the “www’ prefix). Pesticidal proteins also include Botella, et al., (1994) Plant Molec. Biol. 24:757, of nucleo toxin complex (TC) proteins, obtainable from organisms tide sequences for mung bean calmodulin cDNA clones and such as Xenorhabdus, Photorhabdus and Paenibacillus (see, Griess, et al., (1994) Plant Physiol. 104:1467, who provide U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins 45 the nucleotide sequence of a maize calmodulin cDNA clone. have “stand alone' insecticidal activity and other TC pro (H) A polynucleotide encoding a hydrophobic moment teins enhance the activity of the stand-alone toxins produced peptide. See, PCT Application WO 1995/16776 and U.S. by the same given organism. The toxicity of a 'stand-alone” Pat. No. 5,580,852 disclosure of peptide derivatives of TC protein (from Photorhabdus, Xenorhabdus or Paeniba Tachyplesin which inhibit fungal plant pathogens) and PCT cillus, for example) can be enhanced by one or more TC 50 Application WO 1995/18855 and U.S. Pat. No. 5,607.914 protein “potentiators' derived from a source organism of a (teaches synthetic antimicrobial peptides that confer disease different genus. There are three main types of TC proteins. resistance). As referred to herein, Class A proteins (“Protein A') are (I) A polynucleotide encoding a membrane permease, a stand-alone toxins. Class B proteins (“Protein B) and Class channel former or a channel blocker. For example, see the C proteins (“Protein C) enhance the toxicity of Class A 55 disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of proteins. Examples of Class A proteins are TcbA, TcdA, heterologous expression of a cecropin-beta lytic peptide XptA1 and XptA2. Examples of Class B proteins are TcaG, analog to render transgenic tobacco plants resistant to TcdB, XptB1Xb and XptC1 Wi. Examples of Class C pro Pseudomonas Solanacearum. teins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins (J) A gene encoding a viral-invasive protein or a complex also include spider, Snake and Scorpion venom proteins. 60 toxin derived therefrom. For example, the accumulation of Examples of spider venom peptides include but are not viral coat proteins in transformed plant cells imparts resis limited to lycotoxin-1 peptides and mutants thereof (U.S. tance to viral infection and/or disease development effected Pat. No. 8,334,366). by the virus from which the coat protein gene is derived, as (C) A polynucleotide encoding an insect-specific hormone well as by related viruses. See, Beachy, et al., (1990) Ann. or pheromone such as an ecdysteroid and juvenile hormone, 65 Rev. Phytopathol. 28:451. Coat protein-mediated resistance a variant thereof, a mimetic based thereon or an antagonist has been conferred upon transformed plants against alfalfa or agonist thereof. See, for example, the disclosure by mosaic virus, cucumber mosaic virus, tobacco streak virus, US 9,688,730 B2 89 90 potato virus X, potato virus Y. tobacco etch virus, tobacco (V) Genes that confer resistance to Brown Stem Rot, such rattle virus and tobacco mosaic virus. Id. as described in U.S. Pat. No. 5,689,035 and incorporated by (K) A gene encoding an insect-specific antibody or an reference for this purpose. immunotoxin derived therefrom. Thus, an antibody targeted (W) Genes that confer resistance to Colletotrichum, such to a critical metabolic function in the insect gut would as described in US Patent Application Publication US 2009/ inactivate an affected enzyme, killing the insect. Cf. Taylor, 0035765 and incorporated by reference for this purpose. et al., Abstract #497, SEVENTH INTLSYMPOSIUM ON This includes the Rcg locus that may be utilized as a single MOLECULAR PLANT-MICROBE INTERACTIONS (Ed locus conversion. inburgh, Scotland, 1994) (enzymatic inactivation in trans 2. Transgenes that Confer Resistance to a Herbicide, for 10 Example: genic tobacco via production of single-chain antibody frag (A) A polynucleotide encoding resistance to a herbicide ments). that inhibits the growing point or meristem, Such as an (L) A gene encoding a virus-specific antibody. See, for imidazolinone or a Sulfonylurea. Exemplary genes in this example, Tavladoraki, et al., (1993) Nature 366:469, who category code for mutant ALS and AHAS enzyme as show that transgenic plants expressing recombinant anti 15 described, for example, by Lee, et al., (1988) EMBO J. body genes are protected from virus attack. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, (M) A polynucleotide encoding a developmental-arrestive respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; protein produced in nature by a pathogen or a parasite. Thus, 5,141,870; 5,767,361; 5,731, 180; 5,304,732; 4,761,373; fungal endo alpha-1,4-D-polygalacturonases facilitate fun 5,331,107: 5,928,937 and 5,378,824; U.S. patent application gal colonization and plant nutrient release by Solubilizing Ser. No. 11/683,737 and International Publication WO 1996/ plant cell wall homo-alpha-1,4-D-galacturonase. See, Lamb, 33270. et al., (1992) Bio/Technology 10:1436. The cloning and (B) A polynucleotide encoding a protein for resistance to characterization of a gene which encodes a bean endopoly Glyphosate (resistance imparted by mutant 5-enolpyruv1-3- galacturonase-inhibiting protein is described by Toubart, et phosphikimate synthase (EPSP) and aroA genes, respec al., (1992) Plant J. 2:367. 25 tively) and other phosphono compounds such as glufosinate (N) A polynucleotide encoding a developmental-arrestive (phosphinothricin acetyltransferase (PAT) and Streptomyces protein produced in nature by a plant. For example, Loge hygroscopicus phosphinothricin acetyl transferase (bar) mann, et al., (1992) Bio/Technology 10:305, have shown that genes) and pyridinoxy or phenoxy proprionic acids and transgenic plants expressing the barley ribosome-inactivat cyclohexones (ACCase inhibitor-encoding genes). See, for ing gene have an increased resistance to fungal disease. 30 example, U.S. Pat. No. 4,940,835 to Shah, et al., which (O) Genes involved in the Systemic Acquired Resistance discloses the nucleotide sequence of a form of EPSPS which (SAR) Response and/or the pathogenesis related genes. can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, Barry, et al., also describes genes encoding EPSPS enzymes. (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich, See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 (2003) Cell 113(7):815-6. 35 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783: 4,971,908: (P) Antifungal genes (Cornelissen and Melchers, (1993) 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; Pl. Physiol. 101:709-712 and Parijs, et al., (1991) Planta 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 183:258-264 and Bushnell, et al., (1998) Can. J. of Plant 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and 5,491, Path. 2002): 137-149. Also see, U.S. application Ser. Nos. 288 and International Publications EP 1173580; WO 2001/ 09/950,933; 11/619,645; 11/657,710; 11/748,994; 11/774, 40 66704; EP 1173581 and EP 1173582, which are incorporated 121 and U.S. Pat. Nos. 6,891,085 and 7.306,946. LysM herein by reference for this purpose. Glyphosate resistance Receptor-like kinases for the perception of chitin fragments is also imparted to plants that express a gene encoding a as a first step in plant defense response against fungal glyphosate oxidoreductase enzyme as described more fully pathogens (US 2012/0110696). in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are (Q) Detoxification genes, such as for fumonisin, beau 45 incorporated herein by reference for this purpose. In addition vericin, moniliformin and Zearalenone and their structurally glyphosate resistance can be imparted to plants by the over related derivatives. For example, see, U.S. Pat. Nos. 5,716. expression of genes encoding glyphosate N-acetyltrans 820; 5,792,931; 5,798.255; 5,846,812: 6,083,736: 6,538, ferase. See, for example, U.S. Pat. Nos. 7.462.481; 7,405, 177; 6,388,171 and 6,812,380. 074 and US Patent Publication Number US 2008/0234130). (R) A polynucleotide encoding a CyStatin and cysteine 50 A DNA molecule encoding a mutant aroA gene can be proteinase inhibitors. See, U.S. Pat. No. 7,205.453. obtained under ATCC Accession Number 39256 and the (S) Defensin genes. See, WO 2003/0008.63 and U.S. Pat. nucleotide sequence of the mutant gene is disclosed in U.S. Nos. 6,911,577; 6,855,865; 6,777,592 and 7,238,781. Pat. No. 4,769,061 to Comai. European Patent Application (T) Genes conferring resistance to nematodes. See, e.g., Number 0 333 033 to Kumada, et al., and U.S. Pat. No. PCT Application Number WO 1996/30517; PCT Applica 55 4.975,374 to Goodman, et al. disclose nucleotide sequences tion Number WO 1993/19181, WO 2003/033651 and of glutamine synthetase genes which confer resistance to Urwin, et al., (1998) Planta 204:472-479, Williamson, herbicides such as L-phosphinothricin. The nucleotide (1999) Curr Opin Plant Bio. 2(4):327-31; U.S. Pat. Nos. sequence of a phosphinothricin-acetyl-transferase gene is 6.284,948 and 7.301,069 and miR164 genes (WO 2012/ provided in European Patent No. 0 242 246 and 0 242 236 058266). 60 to Leemans, et al. De Greef, et al., (1989) Bio/Technology (U) Genes that confer resistance to Phytophthora Root 7:61, describe the production of transgenic plants that Rot, such as the Rps 1. Rps 1-a, Rps 1-b, Rps 1-c. Rps 1-d, express chimeric bar genes coding for phosphinothricin Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c. Rps 4, acetyl transferase activity. See also, U.S. Pat. Nos. 5,969. Rps 5, Rps 6, Rps 7 and other Rps genes. See, for example, 213: 5489,520; 5,550,318; 5,874,265; 5,919,675; 5,561, Shoemaker, et al., Phytophthora Root Rot Resistance Gene 65 236; 5,648,477; 5,646,024; 6,177,616 B1 and 5,879,903, Mapping in Soybean, Plant Genome IV Conference, San which are incorporated herein by reference for this purpose. Diego, Calif. (1995). Exemplary genes conferring resistance to phenoxy propri US 9,688,730 B2 91 92 onic acids and cyclohexones, such as Sethoxydim and 3. Transgenes that Confer or Contribute to an Altered haloxyfop, are the Acc1-S1, Acc1-52 and Acc1-53 genes Grain Characteristic, described by Marshall, et al., (1992) Theor: Appl. Genet. Such as: 83:435. (A) Altered fatty acids, for example, by (C) A polynucleotide encoding a protein for resistance to (1) Down-regulation of stearoyl-ACP to increase stearic herbicide that inhibits photosynthesis, such as a triazine acid content of the plant. See, KnultZon, et al., (1992) Proc. (psbA and gS+genes) and a benzonitrile (nitrilase gene). Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 (Genes Przibilla, et al., (1991) Plant Cell 3:169 describe the trans to Alter Lipid Profiles in Corn), (2) Elevating oleic acid via FAD-2 gene modification formation of Chlamydomonas with plasmids encoding 10 mutant psbA genes. Nucleotide sequences for nitrilase genes and/or decreasing linolenic acid via FAD-3 gene modifica are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA tion (see, U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 molecules containing these genes are available under ATCC and WO 1993/11245), Accession Numbers 53435, 67441 and 67442. Cloning and (3) Altering conjugated linolenic or linoleic acid content, expression of DNA coding for a glutathione S-transferase is 15 such as in WO 2001/12800, described by Hayes, et al., (1992) Biochem. J. 285: 173. (4) Altering LEC1, AGP, Dek1, Superall, mill ps, and various Ipa genes such as Ipal, Ipa3, hpt or hggt. For (D) A polynucleotide encoding a protein for resistance to example, see, WO 2002/42424, WO 1998/22604, WO 2003/ Acetohydroxy acid synthase, which has been found to make 011015, WO 2002/057439, WO 2003/011015, U.S. Pat. plants that express this enzyme resistant to multiple types of Nos. 6,423,886, 6,197.561, 6,825,397 and US Patent Appli herbicides, has been introduced into a variety of plants (see, cation Publication Numbers US 2003/0079247, US 2003/ e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other 0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. genes that confer resistance to herbicides include: a gene Sci. 92:5620-5624. encoding a chimeric protein of rat cytochrome P4507A1 and (5) Genes encoding delta-8 desaturase for making long yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et 25 chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 al., (1994) Plant Physiol 106:17), genes for glutathione and 8.338,152), delta-9 desaturase for lowering saturated reductase and superoxide dismutase (Aono, et al., (1995) fats (U.S. Pat. No. 8,063,269), Primula A6-desaturase for Plant Cell Physiol 36:1687, and genes for various phospho improving omega-3 fatty acid profiles. transferases (Datta, et al., (1992) Plant Mol Biol. 20:619). (6) Isolated nucleic acids and proteins associated with (E) A polynucleotide encoding resistance to a herbicide 30 lipid and Sugar metabolism regulation, in particular, lipid targeting Protoporphyrinogen oxidase (protox) which is metabolism protein (LMP) used in methods of producing necessary for the production of chlorophyll. The protox transgenic plants and modulating levels of seed storage enzyme serves as the target for a variety of herbicidal compounds including lipids, fatty acids, starches or seed compounds. These herbicides also inhibit growth of all the storage proteins and use in methods of modulating the seed 35 size, seed number, seed weights, root length and leaf size of different species of plants present, causing their total plants (EP2404499). destruction. The development of plants containing altered (7) Altering expression of a High-Level Expression of protox activity which are resistant to these herbicides are Sugar-Inducible 2 (HSI2) protein in the plant to increase or described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and decrease expression of HSI2 in the plant. Increasing expres 5,767,373 and International Publication WO 2001/12825. 40 sion of HSI2 increases oil content while decreasing expres (F) The aad-1 gene (originally from Sphingobium herbi sion of HSI2 decreases abscisic acid sensitivity and/or cidovorans) encodes the aryloxyalkanoate dioxygenase increases drought resistance (US 2012/0066794). (AAD-1) protein. The trait confers tolerance to 2,4-dichlo (8) Expression of cytochrome b5 (Cb5) alone or with rophenoxyacetic acid and aryloxyphenoxypropionate (com FAD2 to modulate oil content in plant seed, particularly to monly referred to as “fop herbicides such as quizalofop) 45 increase the levels of omega-3 fatty acids and improve the herbicides. The aad-1 gene, itself, for herbicide tolerance in ratio of omega-6 to omega-3 fatty acids (US Patent Appli plants was first disclosed in WO 2005/107437 (see also, US cation Publication Number 2011/0191904). 2009/0093366). The aad-12 gene, derived from Delfia (9) Nucleic acid molecules encoding wrinkled 1-like poly acidovorans, which encodes the aryloxyalkanoate dioxy peptides for modulating sugar metabolism (U.S. Pat. No. genase (AAD-12) protein that confers tolerance to 2,4- 50 8,217.223). dichlorophenoxyacetic acid and pyridyloxyacetate herbi B) Altered phosphorus content, for example, by the cides by deactivating several herbicides with an (1) Introduction of a phytase-encoding gene would aryloxyalkanoate moiety, including phenoxy auxin (e.g., enhance breakdown of phytate, adding more free phosphate 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluoroxy to the transformed plant. For example, see Van Hartings pyr, triclopyr). 55 veldt, et al., (1993) Gene 127:87, for a disclosure of the (G) A polynucleotide encoding a herbicide resistant nucleotide sequence of an Aspergillus niger phytase gene. dicamba monooxygenase disclosed in US Patent Applica (2) Modulating a gene that reduces phytate content. In tion Publication 2003/0135879 for imparting dicamba tol maize, this, for example, could be accomplished, by cloning erance; and then re-introducing DNA associated with one or more of 60 the alleles, such as the LPA alleles, identified in maize (H) A polynucleotide molecule encoding bromoxynil nit mutants characterized by low levels of phytic acid, such as rilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for impart in WO 2005/113778 and/or by altering inositol kinase ing bromoxynil tolerance; activity as in WO 2002/059324, US 2003/0009011, WO (I) A polynucleotide molecule encoding phytoene (crt1) 2003/027243, US 2003/0079247, WO 1999/05298, U.S. described in Misawa, et al., (1993) Plant J. 4:833-840 and 65 Pat. No. 6, 197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. in Misawa, et al., (1994) Plant J. 6:481-489 for norflurazon 6,391,348, WO 2002/059324, US 2003/0079247, WO 1998/ tolerance. 45448, WO 1999/55882, WO 2001/04147. US 9,688,730 B2 93 94 (C) Altered carbohydrates effected, for example, by alter Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. In ing a gene for an enzyme that affects the branching pattern addition to these methods, Albertsen, et al., U.S. Pat. No. of starch or, a gene altering thioredoxin Such as NTR and/or 5.432,068, describe a system of nuclear male sterility which TRX (see, (see, U.S. Pat. No. 6,531,648 which is incorpo includes: identifying a gene which is critical to male fertil rated by reference for this purpose) and/or a gamma Zein ity; silencing this native gene which is critical to male knock out or mutant such as cs27 or TUSC27 or en27 (see, fertility; removing the native promoter from the essential U.S. Pat. No. 6,858,778 and US 2005/0160488, US 2005/ male fertility gene and replacing it with an inducible pro 0204418; which are incorporated by reference for this moter, inserting this genetically engineered gene back into purpose). See, Shiroza, et al., (1988) J. Bacteriol. 170:810 the plant; and thus creating a plant that is male sterile (nucleotide sequence of Streptococcus mutant fructosyl 10 because the inducible promoter is not “on” resulting in the transferase gene), Steinmetz, et al., (1985) Mol. Gen. Genet. male fertility gene not being transcribed. Fertility is restored 200:220 (nucleotide sequence of Bacillus subtilis levansu by inducing or turning “on”, the promoter, which in turn crase gene), Pen, et al., (1992) Bio/Technology 10:292 allows the gene that confers male fertility to be transcribed. (production of transgenic plants that express Bacillus (A) Introduction of a deacetylase gene under the control licheniformis alpha-amylase), Elliot, et al., (1993) Plant 15 of a tapetum-specific promoter and with the application of Molec. Biol. 21:515 (nucleotide sequences of tomato inver the chemical N. Ac-PPT (WO 01/29237). tase genes), Sogaard, et al., (1993).J. Biol. Chem. 268:22480 (B) Introduction of various stamen-specific promoters (site-directed mutagenesis of barley alpha-amylase gene) (WO 1992/13956, WO 1992/13957). and Fisher, et al., (1993) Plant Physiol. 102:1045 (maize (C) Introduction of the barnase and the barstar gene (Paul, endosperm starch branching enzyme II), WO 1999/10498 et al., (1992) Plant Mol. Biol. 19:611-622). (improved digestibility and/or starch extraction through For additional examples of nuclear male and female modification of UDP-D-xylose 4-epimerase, Fragile 1 and 2. sterility systems and genes, see also, U.S. Pat. Nos. 5,859, Ref1, HCHL., C4H), U.S. Pat. No. 6,232,529 (method of 341; 6,297,426; 5,478,369; 5,824,524; 5,850,014; and producing high oil seed by modification of starch levels 6,265,640; all of which are hereby incorporated by refer (AGP)). The fatty acid modification genes mentioned herein 25 CCC. may also be used to affect starch content and/or composition 5. Genes that Create a Site for Site Specific DNA Integra through the interrelationship of the starch and oil pathways. tion. (D) Altered antioxidant content or composition, such as This includes the introduction of FRT sites that may be alteration of tocopherol or tocotrienols. For example, see, used in the FLP/FRT system and/or LOX sites that may be U.S. Pat. No. 6,787,683, US 2004/0034886 and WO 2000/ 30 used in the Cre/LOXp system. For example, see LyZnik, et al., 68393 involving the manipulation of antioxidant levels and (2003) Plant Cell Rep. 21:925-932 and WO 1999/25821, WO 2003/082899 through alteration of a homogentisate which are hereby incorporated by reference. Other systems geranylgeranyl transferase (hggt). that may be used include the Gin recombinase of phage Mu (E) Altered essential seed amino acids. For example, see, (Maeser, et al., (1991) Vicki Chandler, The Maize Handbook U.S. Pat. No. 6,127.600 (method of increasing accumulation 35 ch. 118 (Springer-Verlag 1994), the Pin recombinase of E. of essential amino acids in seeds), U.S. Pat. No. 6,080,913 coli (Enomoto, et al., 1983) and the R/RS system of the pSRi (binary methods of increasing accumulation of essential plasmid (Araki, et al., 1992). amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), 6. Genes that Affect Abiotic Stress Resistance WO 1999/40209 (alteration of amino acid compositions in Including but not limited to flowering, ear and seed seeds), WO 1999/29882 (methods for altering amino acid 40 development, enhancement of nitrogen utilization efficiency, content of proteins), U.S. Pat. No. 5,850,016 (alteration of altered nitrogen responsiveness, drought resistance or toler amino acid compositions in seeds), WO 1998/20133 (pro ance, cold resistance or tolerance, and salt resistance or teins with enhanced levels of essential amino acids), U.S. tolerance, and increased yield under stress. Pat. No. 5,885,802 (high methionine), U.S. Pat. No. 5,885, (A) For example, see, WO 2000/73475 where water use 801 (high threonine), U.S. Pat. No. 6,664,445 (plant amino 45 efficiency is altered through alteration of malate: U.S. Pat. acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (in Nos. 5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417, creased lysine and threonine), U.S. Pat. No. 6,441.274 (plant 428, 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO 2000/ tryptophan synthase beta subunit), U.S. Pat. No. 6,346,403 060089, WO 2001/026459, WO 2001/035725, WO 2001/ (methionine metabolic enzymes), U.S. Pat. No. 5,939,599 034726, WO 2001/035727, WO 2001/036444, WO 2001/ (high sulfur), U.S. Pat. No. 5,912,414 (increased methio 50 036597, WO 2001/036598, WO 2002/015675, WO 2002/ nine), WO 1998/56935 (plant amino acid biosynthetic 0.17430, WO 2002/077185, WO 2002/079403, WO 2003/ enzymes), WO 1998/45458 (engineered seed protein having 013227, WO 2003/013228, WO 2003/014327, WO 2004/ higher percentage of essential amino acids), WO 1998/ 031349, WO 2004/076638, WO 1998.09521; 42831 (increased lysine), U.S. Pat. No. 5,633,436 (increas (B) WO 199938977 describing genes, including CBF ing sulfur amino acid content), U.S. Pat. No. 5,559,223 55 genes and transcription factors effective in mitigating the (synthetic storage proteins with defined structure containing negative effects of freezing, high Salinity and drought on programmable levels of essential amino acids for improve plants, as well as conferring other positive effects on plant ment of the nutritional value of plants), WO 1996/01905 phenotype; (increased threonine), WO 1995/15392 (increased lysine), (C) US 2004/0148654 and WO 2001/36596 where US 2003/0163838, US 2003/0150014, US 2004/0068767, 60 abscisic acid is altered in plants resulting in improved plant U.S. Pat. No. 6,803,498, WO 2001/79516. phenotype such as increased yield and/or increased tolerance 4. Genes that Control Male-Sterility: to abiotic stress; There are several methods of conferring genetic male (D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. sterility available. Such as multiple mutant genes at separate 7,531,723 and 6,992,237 where cytokinin expression is locations within the genome that confer male sterility, as 65 modified resulting in plants with increased stress tolerance, disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, Such as drought tolerance, and/or increased yield. Also see, et al., and chromosomal translocations as described by WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S. US 9,688,730 B2 95 96 Pat. No. 6,084, 153, WO 200164898, U.S. Pat. No. 6, 177, increased tolerance to environmental stress as compared to 275 and U.S. Pat. No. 6,107,547 (enhancement of nitrogen a wild type variety of the plant (U.S. Pat. No. 8,097.769). utilization and altered nitrogen responsiveness); (B) Over-expression of maize Zinc finger protein gene (E) For ethylene alteration, see, US 2004/0128719, US 2003/0166197 and WO 2000/32761; (Zm-ZFP1) using a seed preferred promoter has been shown (F) For plant transcription factors or transcriptional regu to enhance plant growth, increase kernel number and total lators of abiotic stress, see e.g., US 2004/0098764 or US kernel weight per plant (US 2012/0079623). 2004/0078852; (C) Constitutive over-expression of maize lateral organ (G) Genes that increase expression of vacuolar pyrophos boundaries (LOB) domain protein (Zm-LOBDP1) has been phatase such as AVP1 (U.S. Pat. No. 8,058,515) for 10 shown to increase kernel number and total kernel weight per increased yield; nucleic acid encoding a HSFA4 or a HSFA5 plant (2012/0079622). (Heat Shock Factor of the class A4 or A5) polypeptides, an (D) Enhancing yield-related traits in plants by modulating oligopeptide transporter protein (OPT4-like) polypeptide; a expression in a plant of a nucleic acid encoding a VIM1 plastochron2-like (PLA2-like) polypeptide or a Wuschel (Variant in Methylation 1)-like polypeptide or a VTC2-like related homeobox 1-like (WOX1-like) polypeptide (US Pat 15 ent Application Publication Number US 2011/0283420): (GDP-L-galactose phosphorylase) polypeptide or a (H) Down regulation of polynucleotides encoding poly DUF1685 polypeptide or an ARF6-like (Auxin Responsive (ADP-ribose) polymerase (PARP) proteins to modulate pro Factor) polypeptide (WO 2012/038893). grammed cell death (U.S. Pat. No. 8,058,510) for increased (E) Modulating expression in a plant of a nucleic acid Vigor, encoding a Step 20-like polypeptide or a homologue thereof (I) Polynucleotide encoding DTP21 polypeptides for con gives plants having increased yield relative to control plants ferring drought resistance (US Patent Publication Number (EP2431472). US 2011/0277181); (J) Nucleotide sequences encoding ACC Synthase 3 (F) Genes encoding nucleoside diphosphatase kinase (ACS3) proteins for modulating development, modulating 25 (NDK) polypeptides and homologs thereof for modifying response to stress and modulating stress tolerance (US the plants root architecture (US Patent Application Publi Patent Pub. No. US20100287669). cation Number 2009/0064373). (K) Polynucleotides that encode proteins that confer a 8. Genes that Confer Plant Digestibility. drought tolerance phenotype (DTP) for conferring drought (A) Altering the level of xylan present in the cell wall of resistance (WO 2012/058528). 30 (L) Tocopherol cyclase (TC) genes for conferring drought a plant by modulating expression of Xylan synthase (U.S. and salt tolerance (US Patent Application Publication Num Pat. No. 8,173,866). ber 2012/0272352). In some embodiment the stacked trait may be a trait or (M) CAAX amino terminal family proteins for stress event that has received regulatory approval including but not tolerance (U.S. Pat. No. 8.338,661). 35 limited to the events in Table 1A-1F. (N) Mutations in the SAL1 encoding gene have increased stress tolerance, including increased drought resistant (US TABLE 1A Patent Application Publication Number 2010/0257633). (O) Expression of a nucleic acid sequence encoding a Triticum aestivum Wheat polypeptide selected from the group consisting of GRF 40 Event Company Description polypeptide, RAA1-like polypeptide, SYR polypeptide, AP2OSCL BASF Inc. Selection for a mutagenized version of the ARKL polypeptide, and YTP polypeptide increasing yield enzyme acetohydroxyacid synthase (AHAS), related traits (US Patent Application Publication Number also known as acetolactate synthase (ALS) or 2011/0061133). acetolactate pyruvate-lyase. (P) Modulating expression in a plant of a nucleic acid 45 AP602CL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), encoding a Class III Trehalose Phosphate Phosphatase (TPP) also known as acetolactate synthase (ALS) or polypeptide for enhancing yield-related traits in plants, acetolactate pyruvate-lyase. particularly increasing seed yield (US Patent Application BW255-2, BASF Inc. Selection for a mutagenized version of the Publication Number 2010/0024067). BW238-3 enzyme acetohydroxyacid synthase (AHAS), Other genes and transcription factors that affect plant 50 also known as acetolactate synthase (ALS) or growth and agronomic traits such as yield, flowering, plant acetolactate pyruvate-lyase. BWT BASF Inc. Tolerance to imidazolinone herbicides induced growth and/or plant structure, can be introduced or intro by chemical mutagenesis of the gressed into plants, see e.g., WO 1997/49811 (LHY), WO acetohydroxyacid synthase (AHAS) gene using 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No. Sodium azide. 6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/ 55 MON718OO Monsanto Glyphosate tolerant wheat variety produced by Company inserting a modified 5-enolpyruvylshikimate-3- 14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1), phosphate synthase (EPSPS) encoding gene WO 2000/44918 (VRN2), WO 1999/49064 (GI), WO 2000/ from the soil bacterium Agrobacterium 46358 (FR1), WO 1997/29123, U.S. Pat. No. 6,794,560, tumefaciens, strain CP4. U.S. Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and SWP965.001 Cyanamid Selection for a mutagenized version of the Rht), and WO 2004/076638 and WO 2004/031349 (tran 60 Crop enzyme acetohydroxyacid synthase (AHAS), Scription factors). Protection also known as acetolactate synthase (ALS) or 7. Genes that Confer Increased Yield acetolactate pyruvate-lyase. Teal 11A BASF Inc. Selection for a mutagenized version of the (A) A transgenic crop plant transformed by a 1-Amino enzyme acetohydroxyacid synthase (AHAS), Cyclopropane-1-Carboxylate Deaminase-like Polypeptide also known as acetolactate synthase (ALS) or (ACCDP) coding nucleic acid, wherein expression of the 65 acetolactate pyruvate-lyase. nucleic acid sequence in the crop plant results in the plants increased root growth, and/or increased yield, and/or US 9,688,730 B2 97 98 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 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 timefaciens. 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 gemmataiis) and soybean looper (Pseudoplitsia includens) via expression of the Cry1Ac encoding gene from B. thatringiensis. MON89788 Monsanto Company Glyphosate-tolerant soybean produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA (epsps) gene from Agrobacterium tunefaciens 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 9,688,730 B2 99 100 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 Saiva 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. LLRICEO6, LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicals). 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 hygroscopicals). PWC16 BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).

TABLE 1 F Zea maps L. Maize Event Company Description 176 Syngenta Seeds, Inc. Insect-resistant maize p roduced by inserting the Cry1Ab gene from Bacilius thuringiensis subsp. kurstaki. The genetic modification affords resistance to attack by he European corn borer (ECB). 3751IR Pioneer Hi-Bred Selection of Somaclona 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 produce by inserting genes encoding DNA adenine methylase and phosphinothricin acetyl transferase (PAT) from Escherichia coli and Streptomyces viridochromogenes, respectively. B16 (DLL25) Dekalb Genetics Glufosinate ammonium herbicide tolerant maize Corporation produced by inserting he gene encoding phosphinothricin acetyl transferase (PAT) from Streptomyces hygroscopicals. Syngenta Seeds, Inc. Insect-resistant and her bicide tolerant maize produced by inserting he Cry1Ab gene from Bacilius thiringiensis Subsp. ikurSiaki, and the phosphinothricin N-ace tyltransferase (PAT) encoding gene from S. viridochromogenes. BT11 x GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO 1-1) and GA21 (OECD unique identifier: MON-OOO2 1-9). BT11 x MIR162 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize pro uced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO 1-1) and MIR162 (OECD unique US 9,688,730 B2 101 102 TABLE 1 F-continued Zea mayS. L. Maize Event Company Description 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 that tringiensis subsp. ikirstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Resistance to other epidopteran pests, including H. zea, S.fugiperda, A. ipsilon, and S. albicosta, is derived rom MIR162, which contains the vip3Aa gene rom 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 Bacillus thiringiensis subsp. ikirstaki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mCry3A 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 Bacillus thiringiensis subsp. ikirstaki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mCry3A 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 that tringiensis war aizawai 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. 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. US 9,688,730 B2 103 104 TABLE 1 F-continued Zea mayS. L. Maize Event Company Description 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 kurSiaki 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. iiirstaki. 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 the bacterial version of a plant enzyme, 5-enolpyruvylshikimate-3- Inc. phosphate synthase (EPSPS). MON810 Monsanto Insect-resistant maize produced by inserting a truncated form of the Company Cry1Ab gene from Bacilius thuringiensis subsp. kurstaki 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). US 9,688,730 B2 105 106 TABLE 1 F-continued Zea mayS. L. Maize Event Company Description 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 Bacillus 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 that tringiensis 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 87.460 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 kumamotoensis 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 cross breeding 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 genes present in MON89043. Tolerance to glyphosate herbicide is derived from NK603. MON89034 x Monsanto Company and Mycogen StackedS insect resistant and herbicide tolerant TC1507 x Seeds co Dow AgroSciences LLC maize produced by conventional cross breeding 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 Male sterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilius amyloiquefaciens; PPT resistance was via PPT acetyltransferase (PAT). US 9,688,730 B2 107 108 TABLE 1 F-continued Zea mayS. L. Maize Event Company Description MS6 Bayer CropScience (Aventis Male sterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilius 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. Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid derived from conventional cross breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and MON810 (OECD identifier: MON-OO81O-6). Monsanto Company Stacked glufosinate ammonium and glyphosate herbicide tolerant maize hybrid derived from conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and T25 (OECD identifier: ACS-ZMOO3-2). 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 (cio 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).

Other events with regulatory approval are well known to peptides having insecticidal activity or agronomic traits as one skilled in the art and can be found at the Center for set forth Supra and optionally may further include one or Environmental Risk Assessment (cera-gmc.org/ more polynucleotides providing for gene silencing of one or '?action gm crop database, which can be accessed using 55 more target polynucleotides as discussed infra. the www prefix). “Suppression DNA construct” is a recombinant DNA Gene Silencing construct which when transformed or stably integrated into In some embodiments the stacked trait may be in the form the genome of the plant, results in 'silencing of a target of silencing of one or more polynucleotides of interest gene in the plant. The target gene may be endogenous or resulting in Suppression of one or more target pest polypep 60 transgenic to the plant. “Silencing,” as used herein with tides. In some embodiments the silencing is achieved respect to the target gene, refers generally to the Suppression through the use of a Suppression DNA construct. of levels of mRNA or protein/enzyme expressed by the In some embodiments one or more of the PIP-1, PIP-1A target gene, and/or the level of the enzyme activity or protein (SEQ ID NO: 2), PSEEN3174 (SEQ ID NO: 6), PIP-1C functionality. The term “suppression' includes lower, (SEQ ID NO: 332), and PIP-1B (SEQ ID NO: 4) polypep 65 reduce, decline, decrease, inhibit, eliminate and prevent. tides or fragments or variants thereof may be stacked with “Silencing or “gene silencing does not specify mechanism one or more polynucleotides encoding one or more poly and is inclusive, and not limited to, anti-sense, coSuppres US 9,688,730 B2 109 110 Sion, viral-Suppression, hairpin Suppression, stem-loop Sup Yet another variation includes using synthetic repeats to pression, RNAi-based approaches and small RNA-based promote formation of a stem in the stem-loop structure. approaches. Transgenic organisms prepared with Such recombinant DNA A Suppression DNA construct may comprise a region fragments have been shown to have reduced levels of the derived from a target gene of interest and may comprise all protein encoded by the nucleotide fragment forming the loop or part of the nucleic acid sequence of the sense Strand (or as described in PCT Publication Number WO 2002/00904. antisense Strand) of the target gene of interest. Depending RNA interference refers to the process of sequence upon the approach to be utilized, the region may be 100% specific post-transcriptional gene silencing in animals medi identical or less than 100% identical (e.g., at least 50% or ated by short interfering RNAs (siRNAs) (Fire, et al., (1998) any integer between 51% and 100% identical) to all or part 10 Nature 391:806). The corresponding process in plants is of the sense strand (or antisense Strand) of the gene of commonly referred to as post-transcriptional gene silencing interest. (PTGS) or RNA silencing and is also referred to as quelling Suppression DNA constructs are well-known in the art, in fungi. The process of post-transcriptional gene silencing are readily constructed once the target gene of interest is is thought to be an evolutionarily-conserved cellular defense selected, and include, without limitation, coSuppression con 15 mechanism used to prevent the expression of foreign genes structs, antisense constructs, viral-suppression constructs, and is commonly shared by diverse flora and phyla (Fire, et hairpin Suppression constructs, stem-loop Suppression con al., (1999) Trends Genet. 15:358). Such protection from structs, double-stranded RNA-producing constructs, and foreign gene expression may have evolved in response to the more generally, RNAi (RNA interference) constructs and production of double-stranded RNAs (dsRNAs) derived small RNA constructs such as siRNA (short interfering from viral infection or from the random integration of RNA) constructs and miRNA (microRNA) constructs. transposon elements into a host genome via a cellular “Antisense inhibition” refers to the production of anti response that specifically destroys homologous single sense RNA transcripts capable of Suppressing the expression stranded RNA of viral genomic RNA. The presence of of the target protein. dsRNA in cells triggers the RNAi response through a “Antisense RNA refers to an RNA transcript that is 25 mechanism that has yet to be fully characterized. complementary to all or part of a target primary transcript or The presence of long dsRNAs in cells stimulates the mRNA and that blocks the expression of a target isolated activity of a ribonuclease III enzyme referred to as dicer. nucleic acid fragment (U.S. Pat. No. 5,107,065). The Dicer is involved in the processing of the dsRNA into short complementarity of an antisense RNA may be with any part pieces of dsRNA known as short interfering RNAs (siRNAs) of the specific gene transcript, i.e., at the 5' non-coding 30 (Berstein, et al., (2001) Nature 409:363). Short interfering sequence, 3' non-coding sequence, introns or the coding RNAs derived from dicer activity are typically about 21 to sequence. about 23 nucleotides in length and comprise about 19 base “Cosuppression” refers to the production of sense RNA pair duplexes (Elbashir, et al., (2001) Genes Dev. 15:188). transcripts capable of Suppressing the expression of the Dicer has also been implicated in the excision of 21- and target protein. “Sense' RNA refers to RNA transcript that 35 22-nucleotide small temporal RNAs (stRNAs) from precur includes the mRNA and can be translated into protein within sor RNA of conserved structure that are implicated in a cell or in vitro. CoSuppression constructs in plants have translational control (Hutvagner, et al., (2001) Science 293: been previously designed by focusing on overexpression of 834). The RNAi response also features an endonuclease a nucleic acid sequence having homology to a native complex, commonly referred to as an RNA-induced silenc mRNA, in the sense orientation, which results in the reduc 40 ing complex (RISC), which mediates cleavage of single tion of all RNA having homology to the overexpressed Stranded RNA having sequence complementarity to the sequence (see, Vaucheret, et al. (1998) Plant J. 16:651-659 antisense strand of the siRNA duplex. Cleavage of the target and Gura, (2000) Nature 404:804-808). RNA takes place in the middle of the region complementary Another variation describes the use of plant viral to the antisense strand of the siRNA duplex (Elbashir, et al., sequences to direct the Suppression of proximal mRNA 45 (2001) Genes Dev. 15:188). In addition, RNA interference encoding sequences (PCT Publication WO 1998/36083). can also involve small RNA (e.g., miRNA) mediated gene Recent work has described the use of “hairpin' structures silencing, presumably through cellular mechanisms that that incorporate all or part, of an mRNA encoding sequence regulate chromatin structure and thereby prevent transcrip in a complementary orientation that results in a potential tion of target gene sequences (see, e.g., Allshire, (2002) “stem-loop” structure for the expressed RNA (PCT Publi 50 Science 297: 1818-1819; Volpe, et al., (2002) Science 297: cation Number WO 1999/53050). In this case the stem is 1833-1837; Jenuwein, (2002) Science 297:2215-2218; and formed by polynucleotides corresponding to the gene of Hall, et al., (2002) Science 297:2232–2237). As such, interest inserted in either sense or anti-sense orientation with miRNA molecules of the invention can be used to mediate respect to the promoter and the loop is formed by some gene silencing via interaction with RNA transcripts or polynucleotides of the gene of interest, which do not have a 55 alternately by interaction with particular gene sequences, complement in the construct. This increases the frequency of wherein Such interaction results in gene silencing either at coSuppression or silencing in the recovered transgenic the transcriptional or post-transcriptional level. plants. For review of hairpin Suppression see, Wesley, et al., Methods and compositions are further provided which (2003) Methods in Molecular Biology, Plant Functional allow for an increase in RNAi produced from the silencing Genomics. Methods and Protocols 236:273-286. 60 element. In Such embodiments, the methods and composi A construct where the stem is formed by at least 30 tions employ a first polynucleotide comprising a silencing nucleotides from a gene to be suppressed and the loop is element for a target pest sequence operably linked to a formed by a random nucleotide sequence has also effectively promoter active in the plant cell; and, a second polynucle been used for suppression (WO 1999/61632). otide comprising a Suppressor enhancer element comprising The use of poly-T and poly-A sequences to generate the 65 the target pest sequence or an active variant or fragment stem in the stem-loop structure has also been described (WO thereof operably linked to a promoter active in the plant cell. 2002/00894). The combined expression of the silencing element with US 9,688,730 B2 111 112 Suppressor enhancer element leads to an increased amplifi In specific embodiments, the combined expression of the cation of the inhibitory RNA produced from the silencing silencing element and the Suppressor enhancer element element over that achievable with only the expression of the increases the concentration of the inhibitory RNA in the silencing element alone. In addition to the increased ampli plant cell, plant, plant part, plant tissue or phloem over the fication of the specific RNAi species itself, the methods and 5 level that is achieved when the silencing element is compositions further allow for the production of a diverse expressed alone. population of RNAi species that can enhance the effective As used herein, an “increased level of inhibitory RNA ness of disrupting target gene expression. As such, when the comprises any statistically significant increase in the level of Suppressor enhancer element is expressed in a plant cell in RNAi produced in a plant having the combined expression combination with the silencing element, the methods and 10 when compared to an appropriate control plant. For composition can allow for the systemic production of RNAi example, an increase in the level of RNAi in the plant, plant throughout the plant; the production of greater amounts of part or the plant cell can comprise at least about a 1%, about RNAi than would be observed with just the silencing a 1%-5%, about a 5%-10%, about a 10%-20%, about a element construct alone; and, the improved loading of RNAi 20%-30%, about a 30%-40%, about a 40%-50%, about a into the phloem of the plant, thus providing better control of 15 50%-60%, about 60-70%, about 70%–80%, about a 80%- phloem feeding insects by an RNAi approach. Thus, the 90%, about a 90%-100% or greater increase in the level of various methods and compositions provide improved meth RNAi in the plant, plant part, plant cell or phloem when ods for the delivery of inhibitory RNA to the target organ compared to an appropriate control. In other embodiments, ism. See, for example, US 2009/0188008. the increase in the level of RNAi in the plant, plant part, As used herein, a 'suppressor enhancer element com plant cell or phloem can comprise at least about a 1 fold, prises a polynucleotide comprising the target sequence to be about a 1 fold-5 fold, about a 5 fold-10 fold, about a 10 Suppressed or an active fragment or variant thereof. It is fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40 fold, recognize that the Suppressor enhancer element need not be about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 identical to the target sequence, but rather, the Suppressor fold-70 fold, about 70 fold-80 fold, about a 80 fold-90 fold, enhancer element can comprise a variant of the target 25 about a 90 fold-100 fold or greater increase in the level of sequence, so long as the Suppressor enhancer element has RNAi in the plant, plant part, plant cell or phloem when Sufficient sequence identity to the target sequence to allow compared to an appropriate control. Examples of combined for an increased level of the RNAi produced by the silencing expression of the silencing element with Suppressor element over that achievable with only the expression of the enhancer element for the control of Stinkbugs and Lygus can silencing element. Similarly, the Suppressor enhancer ele 30 be found in US 2011/0301.223 and US 2009/01921.17. ment can comprise a fragment of the target sequence, Some embodiments relate to down-regulation of expres wherein the fragment is of sufficient length to allow for an sion of target genes in insect pest species by interfering increased level of the RNAi produced by the silencing ribonucleic acid (RNA) molecules. WO 2007/074405 element over that achievable with only the expression of the describes methods of inhibiting expression of target genes in silencing element. 35 invertebrate pests including Colorado potato beetle. WO It is recognized that multiple Suppressor enhancer ele 2005/110068 describes methods of inhibiting expression of ments from the same target sequence or from different target target genes in invertebrate pests including in particular sequences or from different regions of the same target Western corn rootworm as a means to control insect infes sequence can be employed. For example, the Suppressor tation. Furthermore, WO 2009/091864 describes composi enhancer elements employed can comprise fragments of the 40 tions and methods for the Suppression of target genes from target sequence derived from different region of the target insect pest species including pests from the Lygus genus. sequence (i.e., from the 3'UTR, coding sequence, intron, Nucleic acid molecules including RNAi for targeting the and/or 5'UTR). Further, the suppressor enhancer element vacuolar ATPase H subunit, useful for controlling a coleop can be contained in an expression cassette, as described teran pest population and infestation as described in US elsewhere herein, and in specific embodiments, the Suppres 45 Patent Application Publication 2012/0198586. WO 2012/ sor enhancer element is on the same or on a different DNA 055982 describes ribonucleic acid (RNA or double stranded vector or construct as the silencing element. The Suppressor RNA) that inhibits or down regulates the expression of a enhancer element can be operably linked to a promoter as target gene that encodes: an insect ribosomal protein Such as disclosed herein. It is recognized that the Suppressor the ribosomal protein L19, the ribosomal protein L40 or the enhancer element can be expressed constitutively or alter 50 ribosomal protein 527A; an insect proteasome subunit Such natively, it may be produced in a stage-specific manner as the Rpnó protein, the Pros 25, the Rpn2 protein, the employing the various inducible or tissue-preferred or devel proteasome beta 1 subunit protein or the Pros beta 2 protein; opmentally regulated promoters that are discussed else an insect B-coatomer of the COPI vesicle, the y-coatomer of where herein. the COPI vesicle, the B'-coatomer protein or the -coatomer In specific embodiments, employing both a silencing 55 of the COPI vesicle; an insect Tetraspanine 2 A protein element and the Suppressor enhancer element the systemic which is a putative transmembrane domain protein; an insect production of RNAi occurs throughout the entire plant. In protein belonging to the actin family such as Actin 5C, an further embodiments, the plant or plant parts of the invention insect ubiquitin-5E protein; an insect Sec23 protein which is have an improved loading of RNAi into the phloem of the a GTPase activator involved in intracellular protein trans plant than would be observed with the expression of the 60 port; an insect crinkled protein which is an unconventional silencing element construct alone and, thus provide better myosin which is involved in motor activity; an insect control of phloem feeding insects by an RNAi approach. In crooked neck protein which is involved in the regulation of specific embodiments, the plants, plant parts, and plant cells nuclear alternative mRNA splicing; an insect vacuolar of the invention can further be characterized as allowing for H+-ATPase G-subunit protein; and an insect Tbp-1 such as the production of a diversity of RNAi species that can 65 Tat-binding protein. US Patent Application Publications enhance the effectiveness of disrupting target gene expres 2012/029750 and 2012/0322660 describe an interfering S1O. ribonucleic acid (RNA or double stranded RNA) that func US 9,688,730 B2 113 114 tions upon uptake by an insect pest species to down-regulate chlororaphis), Erwinia spp., and Flavobacterium spp., and expression of a target gene in said insect pest, wherein the other Such organisms, including Agrobacterium tumefa RNA comprises at least one silencing element wherein the ciens, E. coli, Bacillus subtilis, and the like. silencing element is a region of double-stranded RNA Genes encoding the PIP-1 polypeptides of the embodi comprising annealed complementary Strands, one Strand of 5 ments can be introduced into microorganisms that multiply which comprises or consists of a sequence of nucleotides on plants (epiphytes) to deliver PIP-1 polypeptides to poten which is at least partially complementary to a target nucleo tial target pests. Epiphytes, for example, can be gram tide sequence within the target gene. US Patent Application positive or gram-negative bacteria. Publication 2012/0164205 describe potential targets for Root-colonizing bacteria, for example, can be isolated interfering double stranded ribonucleic acids for inhibiting 10 from the plant of interest by methods known in the art. invertebrate pests including: a Chd3 Homologous Sequence, Specifically, a Bacillus cereus Strain that colonizes roots can a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPase be isolated from roots of a plant (see, for example, Han Homologous Sequence, a EF1a Homologous Sequence, a delsman, et al., (1991) Appl. Environ. Microbiol. 56: 713 26S Proteosome Subunit p28 Homologous Sequence, a 718). Genes encoding the PIP-1 polypeptides of the embodi Juvenile Hormone Epoxide Hydrolase Homologous 15 ments can be introduced into a root-colonizing Bacillus Sequence, a Swelling Dependent Chloride Channel Protein cereus by standard methods known in the art. Homologous Sequence, a Glucose-6-Phosphate 1-Dehydro Genes encoding PIP-1 polypeptides can be introduced, for genase Protein Homologous Sequence, an Act 42A Protein example, into the root-colonizing Bacillus by means of Homologous Sequence, a ADP-Ribosylation Factor 1 electro transformation. Specifically, genes encoding the Homologous Sequence, a Transcription Factor IIB Protein PIP-1 polypeptides can be cloned into a shuttle vector, for Homologous Sequence, a Chitinase Homologous example, pHT3101 (Lerecius, et al., (1989) FEMS Micro Sequences, a Ubiquitin Conjugating Enzyme Homologous biol. Letts. 60:211-218. The shuttle vector pHT3101 con Sequence, a Glyceraldehyde-3-Phosphate Dehydrogenase taining the coding sequence for the particular PIP-1 poly Homologous Sequence, an Ubiquitin B Homologous peptide gene can, for example, be transformed into the Sequence, a Juvenile Hormone Esterase Homolog, and an 25 root-colonizing Bacillus by means of electroporation (Lere Alpha Tubuliln Homologous Sequence. cius, et al., (1989) FEMS Microbiol. Letts. 60:211-218). Use in Pesticidal Control Expression systems can be designed so that PIP-1 poly General methods for employing strains comprising a peptides are secreted outside the cytoplasm of gram-nega nucleic acid sequence of the embodiments or a variant tive bacteria, such as E. coli, for example. Advantages of thereof, in pesticide control or in engineering other organ 30 having PIP-1 polypeptides secreted are: (1) avoidance of isms as pesticidal agents are known in the art. See, for potential cytotoxic effects of the PIP-1 polypeptide example U.S. Pat. No. 5,039,523 and EP 0480762A2. expressed; and (2) improvement in the efficiency of purifi Microorganism hosts that are known to occupy the “phy cation of the PIP-1 polypeptide, including, but not limited to, tosphere' (phylloplane, phyllosphere, rhizosphere, and/or increased efficiency in the recovery and purification of the rhizoplana) of one or more crops of interest may be selected. 35 protein per volume cell broth and decreased time and/or These microorganisms are selected so as to be capable of costs of recovery and purification per unit protein. Successfully competing in the particular environment with PIP-1 polypeptides can be made to be secreted in E. coli, the wild-type microorganisms, provide for stable mainte for example, by fusing an appropriate E. coli signal peptide nance and expression of the gene expressing the PIP-1 to the amino-terminal end of the PIP-1 polypeptide. Signal polypeptide, and desirably, provide for improved protection 40 peptides recognized by E. coli can be found in proteins of the pesticide from environmental degradation and inac already known to be secreted in E. coli, for example the tivation. OmpA protein (Ghrayeb, et al., (1984) EMBOJ, 3:2437 Such microorganisms include bacteria, algae, and fungi. 2442). Omp A is a major protein of the E. coli outer mem Of particular interest are microorganisms such as bacteria, brane, and thus its signal peptide is thought to be efficient in e.g., Alcaligenes, Pseudomonas, Erwinia, Serratia, Kleb 45 the translocation process. Also, the Omp A signal peptide siella, Xanthomonas, Streptomyces, Rhizobium, Rho does not need to be modified before processing as may be dopseudomonas, Methylius, Agrobacterium, Acetobacter; the case for other signal peptides, for example lipoprotein Lactobacillus, Arthrobacter, Azotobacter; Leuconostoc, and signal peptide (Duffaud, et al., (1987) Meth. Enzymol. 153: Alcaligenes, fungi, particularly yeast, e.g., Saccharomyces, 492). Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodoto 50 PIP-1 polypeptides of the embodiments can be fermented rula, and Aureobasidium. Of particular interest are Such in a bacterial host and the resulting bacteria processed and phytosphere bacterial species as Alcaligenes faecalis, used as a microbial spray in the same manner that Bt strains Pseudomonas Syringae, Pseudomonas fluorescens, Serratia have been used as insecticidal sprays. In the case of a PIP-1 marcescens, Acetobacter xylinum, Agrobacteria, Rho polypeptide(s) that is secreted from Bacillus, the secretion dopseudomonas spheroides, Xanthomonas Campestris, 55 signal is removed or mutated using procedures known in the Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli art. Such mutations and/or deletions prevent secretion of the and Azotobacter vinelandii and phytosphere yeast species PIP-1 polypeptide(s) into the growth medium during the Such as Rhodotorula rubra, R. glutinis, R. marina, R. fermentation process. The PIP-1 polypeptides are retained aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, within the cell, and the cells are then processed to yield the Saccharomyces rosei, S. pretoriensis, S. cerevisiae, 60 encapsulated PIP-1 polypeptides. Any suitable microorgan Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, ism can be used for this purpose. Pseudomonas has been and Aureobasidium pollulans. Of particular interest are the used to express Bt toxins as encapsulated proteins and the pigmented microorganisms. Host organisms of particular resulting cells processed and sprayed as an insecticide interest include yeast, such as Rhodotorula spp., Aureoba (Gaertner, et al., (1993), in: Advanced Engineered Pesti sidium spp., Saccharomyces spp. (Such as S. cerevisiae), 65 cides, ed. Kim). Sporobolomyces spp., phylloplane organisms such as Alternatively, the PIP-1 polypeptides are produced by Pseudomonas spp. (Such as P. aeruginosa, Pfluorescens, P introducing a heterologous gene into a cellular host. Expres US 9,688,730 B2 115 116 sion of the heterologous gene results, directly or indirectly, environmental considerations, and/or frequency of applica in the intracellular production and maintenance of the pes tion and/or severity of pest infestation. ticide. These cells are then treated under conditions that The pesticide compositions described may be made by prolong the activity of the toxin produced in the cell when formulating either the bacterial cell, Crystal and/or spore the cell is applied to the environment of target pest(s). The 5 Suspension or isolated protein component with the desired resulting product retains the toxicity of the toxin. These agriculturally-acceptable carrier. The compositions may be naturally encapsulated PIP-1 polypeptides may then be formulated prior to administration in an appropriate means formulated in accordance with conventional techniques for Such as lyophilized, freeze-dried, desiccated or in an aque application to the environment hosting a target pest, e.g., ous carrier, medium or Suitable diluent. Such as Saline or 10 other buffer. The formulated compositions may be in the soil, water, and foliage of plants. See, for example EPA form of a dust or granular material or a Suspension in oil 0192319, and the references cited therein. (vegetable or mineral) or water or oil/water emulsions or as Pesticidal Compositions a wettable powder or in combination with any other carrier In some embodiments the active ingredients can be material Suitable for agricultural application. Suitable agri applied in the form of compositions and can be applied to the 15 cultural carriers can be solid or liquid and are well known in crop area or plant to be treated, simultaneously or in the art. The term "agriculturally-acceptable carrier covers Succession, with other compounds. These compounds can be all adjuvants, inert components, dispersants, Surfactants, fertilizers, weed killers, Cryoprotectants, surfactants, deter tackifiers, binders, etc. that are ordinarily used in pesticide gents, pesticidal Soaps, dormant oils, polymers, and/or time formulation technology; these are well known to those release or biodegradable carrier formulations that permit skilled in pesticide formulation. The formulations may be long-term dosing of a target area following a single appli mixed with one or more solid or liquid adjuvants and cation of the formulation. They can also be selective herbi prepared by various means, e.g., by homogeneously mixing, cides, chemical insecticides, Virucides, microbicides, amoe blending and/or grinding the pesticidal composition with bicides, pesticides, fungicides, bacteriocides, nematocides, Suitable adjuvants using conventional formulation tech molluscicides or mixtures of several of these preparations, if 25 niques. Suitable formulations and application methods are desired, together with further agriculturally acceptable car described in U.S. Pat. No. 6,468.523, herein incorporated by riers, Surfactants or application-promoting adjuvants cus reference. The plants can also be treated with one or more tomarily employed in the art of formulation. Suitable carri chemical compositions, including one or more herbicide, ers and adjuvants can be solid or liquid and correspond to the insecticides or fungicides. Exemplary chemical composi Substances ordinarily employed in formulation technology, 30 tions include: Fruits/Vegetables Herbicides: Atrazine, Bro e.g. natural or regenerated mineral Substances, solvents, macil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, dispersants, wetting agents, tackifiers, binders or fertilizers. Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Likewise the formulations may be prepared into edible Paraquat, PropyZamide, Sethoxydim, Butafenacil, Halosul “baits” or fashioned into pest “traps to permit feeding or furon, Indaziflam: Fruits/Vegetables Insecticides: Aldicarb, ingestion by a target pest of the pesticidal formulation. 35 Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Methods of applying an active ingredient or an agro Cypermethrin, Deltamethrin, Diazinon, Malathion, Abam chemical composition that contains at least one of the PIP-1 ectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cy polypeptides produced by the bacterial strains include leaf halothrin, Acequinocyl, Bifenazate, Methoxyfenozide, application, seed coating and soil application. The number Novaluron, Chromafenozide. Thiacloprid, Dinotefuran, Flu of applications and the rate of application depend on the 40 aCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen, intensity of infestation by the corresponding pest. Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, The composition may be formulated as a powder, dust, CyaZypyr, Spinoteram, Triflumuron, Spirotetramat, Imida pellet, granule, spray, emulsion, colloid, Solution or Such cloprid, Flubendiamide. Thiodicarb, Metaflumizone, Sul like, and may be prepared by Such conventional means as foxaflor, Cyflumetofen, Cyanopyrafen, Imidacloprid, Cloth desiccation, lyophilization, homogenation, extraction, filtra 45 ianidin, Thiamethoxam, Spinotoram, Thiodicarb, tion, centrifugation, sedimentation or concentration of a Flonicamid, Methiocarb, Emamectin-benzoate, lindoxacarb, culture of cells comprising the polypeptide. In all Such Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbu compositions that contain at least one such pesticidal poly tatin-oxid, Hexthiazox, Methomyl, 4-(6-Chlorpyridin-3- peptide, the polypeptide may be present in a concentration yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on; Fruits/ of from about 1% to about 99% by weight. 50 Vegetables Fungicides: Carbendazim, Chlorothalonil, Lepidopteran, dipteran, heteropteran, nematode, hemip EBDCs, Sulphur. Thiophanate-methyl, Azoxystrobin, tera or coleopteran pests may be killed or reduced in Cymoxanil, FluaZinam, Fosetyl, Iprodione, Kresoxim numbers in a given area by the methods of the disclosure or methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, may be prophylactically applied to an environmental area to Iprovalicarb, Trifloxystrobin, Fenhexamid, Oxpoconazole prevent infestation by a susceptible pest. Preferably the pest 55 fumarate, CyaZofamid, Fenamidone, Zoxamide, Picox ingests or is contacted with, a pesticidally-effective amount ystrobin, Pyraclostrobin, Cyflufenamid, Boscalid; Cereals of the polypeptide. By “pesticidally-effective amount” is Herbicides: Isoproturon, Bromoxynil, loxynil, Phenoxies, intended an amount of the pesticide that is able to bring Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenox about death to at least one pest or to noticeably reduce pest aprop, Florasulam, Fluoroxypyr. Metsulfuron, Triasulfuron, growth, feeding or normal physiological development. This 60 Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolin amount will vary depending on Such factors as, for example, afen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfu the specific target pests to be controlled, the specific envi ron. Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, ronment, location, plant, crop or agricultural site to be Sulfosulfuron, Pyrasulfotole, PyroxSulam, Flufenacet, treated, the environmental conditions, and the method, rate, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carben concentration, stability, and quantity of application of the 65 dazim, Chlorothalonil, AZOxystrobin, Cyproconazole, pesticidally-effective polypeptide composition. The formu Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim lations may also vary with respect to climatic conditions, methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Sime US 9,688,730 B2 117 118 conazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Prothioconazole, Fluoxastrobin: Cereals Insecticides: Dime Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spino thoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cyper toram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltame methrin, B-cyfluthrin, Bifenthrin, Imidacloprid, Clothiani thrin, B-Cyfluthrin, gamma and lambda Cyhalothrin, 4-(6- din, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Chlorpyridin-3-yl)methyl(2,2-difluorethyl)aminofuran-2 Clorphyriphos, Metamidophos. Oxidemethon-methyl, (5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Pirimicarb, Methiocarb. Maize Herbicides: Atrazine, Flonicamid. Thiodicarb, beta-Cyfluthrin; Soybean Fungi Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, cides: AZOxystrobin, Cyproconazole, Epoxiconazole, Flu (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, triafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Pro (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, 10 thioconazole, Tetraconazole; Sugarbeet Herbicides: Rimsulfuron, Sulcotrione, Foramsulfuron, ToprameZone, Chloridazon, Desmedipham, Ethiofumesate, Phenmed Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, ipham, Triallate, Clopyralid, Fluazifop, Lenacil, Met Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyri amitron, Quinmerac, Cycloxydim, TrifluSulfuron, Tepral fos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalo oxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, thrin, Tefluthrin, Terbufos. Thiamethoxam, Clothianidin, 15 Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Spiromesi?en, Flubendiamide, Triflumuron, Rynaxypyr. Dinetofuran, Deltamethrin, B-Cyfluthrin, gamma/lambda Deltamethrin, Thiodicarb, B-Cyfluthrin, Cypermethrin, Cyhalothrin, 4-(6-Chlorpyridin-3-yl)methyl(2,2-difluor Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirim ethyl)aminofuran-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxy phos, Ethiprole, CyaZypyr. Thiacloprid, Acetamiprid, pyr. Fipronil, Carbofuran; Canola Herbicides: Clopyralid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spiro Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, tetramat; Maize Fungicides: Fenitropan, Thiram, Prothio Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, conazole, Tebuconazole, Trifloxystrobin: Rice Herbicides: Clethodim, Tepraloxydim; Canola Fungicides: AZOX Butachlor, Propanil, AZimsulfuron, Bensulfuron, Cyhalo ystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, fop, Daimuron, FentraZamide, Imazosulfuron, Mefenacet, VincloZolin; Canola Insecticides: Carbofuran organophos Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, 25 phates, Pyrethroids. Thiacloprid, Deltamethrin, Imidaclo Thiobencarb, Indanofan, Flufenacet, FentraZamide, Halo prid, Clothianidin, Thiamethoxam, Acetamiprid, Dineto sulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, furan, B-Cyfluthrin, gamma and lambda Cyhalothrin, tau Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pre Fluvaleriate, Ethiprole, Spinosad, Spinotoram, tilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenox Flubendiamide, Rynaxypyr, Cyazypyr, 4-(6-Chlorpyridin aprop, Pyrimisulfan; Rice Insecticides: Diazinon, Fenitro 30 3-yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on. thion, Fenobucarb, Monocrotophos, Benfuracarb, In some embodiments the herbicide is Atrazine, Bromacil, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isopro Diuron, Chlorsulfuron, Metsulfuron, Thifensulfuron carb. Thiacloprid, Chromafenozide. Thiacloprid, Dinote Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, furan, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr. Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumiox Deltamethrin, Acetamiprid. Thiamethoxam, CyaZypyr, 35 azin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-meto Spinosad, Spinotoram, Emamectin-Benzoate, Cyper lachlor, Hexazinne or combinations thereof. methrin, Chlorpyriphos, Cartap, Methamidophos, Etofen In some embodiments the insecticide is Esfenvalerate, prox. Triazophos, 4-(6-Chlorpyridin-3-yl)methyl(2,2-dif Chlorantraniliprole, Methomyl, lindoxacarb, Oxamyl or luorethyl)aminofuran-2(5H)-on, Carbofuran, Benfuracarb: combinations thereof. Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Car 40 Pesticidal and Insecticidal Activity propamid, Edifenphos, FerimZone, probenfos, Isoprothi “Pest includes but is not limited to, insects, fungi, olane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole, bacteria, nematodes, mites, ticks, and the like. Insect pests Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tia include insects selected from the orders Coleoptera, Diptera, dinil; Cotton Herbicides: Diuron, Fluometuron, MSMA, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Oxyfluorfen, Prometryn, Trifluralin, CarfentraZone, 45 Hemiptera orthroptera, Thysanoptera, Dermaptera, Isoptera, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepi Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, doptera, and Hemiptera. Tepraloxydim, Glufosinate, Flumioxazin, ThidiaZuron; Cot Those skilled in the art will recognize that not all com ton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cyper pounds are equally effective againstall pests. Compounds of methrin, Deltamethrin, Malathion, Monocrotophos, Abam 50 the embodiments display activity against insect pests, which ectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, may include economically important agronomic, forest, Indoxacarb, Lambda-Cyhalothrin, Spinosad. Thiodicarb, greenhouse, nursery ornamentals, food and fiber, public and Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, animal health, domestic and commercial structure, house Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, hold and stored product pests. Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, 55 Larvae of the order Lepidoptera include, but are not Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spino limited to, armyworms, cutworms, loopers, and heliothines toram, gamma Cyhalothrin, 4-(6-Chlorpyridin-3-yl) in the family Noctuidae Spodoptera frugiperda JE Smith methyl(2,2-difluorethyl)aminofuran-2(5H)-on, Thiodi (fall armyworm): S. exigua Hübner (beet armyworm): S. carb, Avermectin, Flonicamid, Pyridalyl, Spiromesi?en, litura Fabricius (tobacco cutworm, cluster caterpillar): Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton 60 Mamestra configurata Walker (bertha armyworm); M. bras Fungicides: Etridiazole, Metalaxyl, Quintozene: Soybean sicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron (black cutworm); A. Orthogonia Morrison (western cut Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Flu worm); A. subterranea Fabricius (granulate cutworm); Ala azifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, bama argillacea Hübner (cotton leaf worm); Trichoplusia ni (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, 65 Hübner (cabbage looper); Pseudoplusia includens Walker Glufosinate: Soybean Insecticides: Lambda-cyhalothrin, (soybean looper); Anticarsia gemmatalis Hübner (velvet Methomyl, Parathion. Thiocarb. Imidacloprid, Clothianidin, bean caterpillar); Hypena scabra Fabricius (green clover US 9,688,730 B2 119 120 worm); Heliothis virescens Fabricius (tobacco budworm): lycopersicella Walsingham (tomato pinworm); Lambdina Pseudaletia unipuncta Haworth (armyworm); Athetis min fiscellaria fiscellaria Hulst (Eastern hemlock looper): L. dara Barnes and Mcdunnough (rough skinned cutworm): fiscellaria lugubrosa Hulst (Western hemlock looper); Leu Euxoa messoria Harris (darksided cutworm); Earias insu coma salicis Linnaeus (satin moth); Lymantria dispar Lin lana Boisduval (spiny bollworm); E. Vittella Fabricius (spot 5 naeus (gypsy moth); Manduca quinquemaculata Haworth ted bollworm); Helicoverpa armigera Hübner (American (five spotted hawk moth, tomato hornworm); M. sexta bollworm); H. zea Boddie (corn earworm or cotton boll Haworth (tomato hornworm, tobacco hornworm): worm); Melanchra picta Harris (Zebra caterpillar); Egira Operophtera brumata Linnaeus (winter moth); Paleacrita (Xvlomyges) curialis Grote (citrus cutworm); borers, case vernata Peck (spring cankerworm); Papilio Cresphontes bearers, webworms, coneworms, and skeletonizers from the 10 Cramer (giant Swallowtail orange dog); Phryganidia Cali family Pyralidae Ostrinia nubilalis Hübner (European corn fornica Packard (California oakworm); Phyllocnistis citrella borer); Amyelois transitella Walker (naval orangeworm): Stainton (citrus leafminer); Phyllonorycter blancardella Anagasta kuehniella Zeller (Mediterranean flour moth); Fabricius (spotted tentiform leafminer); Pieris brassicae Cadra cautella Walker (almond moth); Chilo suppressalis Linnaeus (large white butterfly); P rapae LinnaeuS (Small Walker (rice stem borer); C. partellus, (sorghum borer); 15 white butterfly); P napi Linnaeus (green veined white Corcyra cephalonica Stainton (rice moth); Crambus caligi butterfly): Platyptilia carduidactyla Riley (artichoke plume nosellus Clemens (corn root webworm): C. teterrellus moth); Plutella xylostella LinnaeuS (diamondback moth); Zincken (bluegrass webworm): Cnaphalocrocis medinalis Pectinophora gossypiella Saunders (pink bollworm); Pontia Guenee (rice leaf roller); Desmia fineralis Hübner (grape protodice Boisduval & Leconte (Southern cabbageworm): leaffolder); Diaphania hyalinata Linnaeus (melon worm): 20 Sabulodes aegrotata Guenee (omnivorous looper); Schizura D. mitidalis Stoll (pickleworm); Diatraea grandiosella Dyar concinna J. E. Smith (red humped caterpillar); Sitotroga (Southwestern corn borer), D. Saccharalis Fabricius (Surg cerealella Olivier (Angoumois grain moth); Thaumetopoea arcane borer); Eoreuma loftini Dyar (Mexican rice borer); pityocampa Schiffermuller (pine processionary caterpillar); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria Tineola bisselliella Hummel (webbing clothesmoth); Tuta mellonella Linnaeus (greater wax moth); Herpetogramma 25 absoluta Meyrick (tomato leafminer); Yponomeuta padella licarsisalis Walker (sod webworm); Homoeosoma electel Linnaeus (ermine moth); Heliothis subflexia Guenee; Mala lum Hulst (Sunflower moth); Elasmopalpus lignosellus COSOma spp. and Orgvia spp. Zeller (lesser cornstalk borer); Achroia grisella Fabricius Of interest are larvae and adults of the order Coleoptera (lesser wax moth); Loxostege Sticticalis Linnaeus (beet including weevils from the families Anthribidae, Bruchidae, webworm); Orthaga thyrisalis Walker (tea tree web moth); 30 and Curculionidae (including, but not limited to: Anthono Maruca testulalis Geyer (bean pod borer); Plodia interpunc mus grandis Boheman (boll weevil); Lissorhoptrus Oryzo tella Hibner (Indian meal moth); Scirpophaga incertulas philus Kuschel (rice water weevil); Sitophilus granarius Walker (yellow stem borer); Udea rubigalis Guenee (celery Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); leaftier); and leafrollers, budworms, seed worms, and fruit Hypera punctata Fabricius (clover leaf weevil); Cylindro worms in the family Tortricidae Acleris gloverana Walsing 35 copturus adspersus LeConte (Sunflower stem weevil); Smi ham (Western blackheaded budworm); A. variana Fernald cronyx fulvus LeConte (red sunflower seed weevil); S. (Eastern blackheaded budworm); Archips argyrospilla SOrdidus LeConte (gray Sunflower seed weevil); Sphenopho Walker (fruit tree leaf roller); A. rosana Linnaeus (European rus maidis Chittenden (maize billbug)); flea beetles, cucum leaf roller); and other Archips species, Adoxophyes Orana ber beetles, rootworms, leaf beetles, potato beetles, and Fischer von Rosslerstamm (summer fruit tortrix moth); 40 leafminers in the family Chrysomelidae (including, but not Cochylis hospes Walsingham (banded sunflower moth); limited to: Leptinotarsa decemlineata Say (Colorado potato Cydia latiferreana Walsingham (filbertworm): C. pomonella beetle); Diabrotica virgifera virgifera LeConte (western Linnaeus (coding moth); Platynota flavedana Clemens (var corn rootworm): D. barberi Smith & Lawrence (northern iegated leafroller); P Stultana Walsingham (omnivorous corn rootworm); D. undecimpunctata howardi Barber leafroller); Lobesia botrana Denis & Schiffermuller (Euro 45 (Southern corn rootworm); Chaetocnema pullicaria pean grape vine moth); Spilonota ocellana Denis & Schif Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze fermuller (eyespotted bud moth); Endopiza viteana Clemens (corn flea beetle); Colaspis brunnea Fabricius (grape colas (grape berry moth); Eupoecilia ambiguella Hübner (vine pis); Oulema melanopus Linnaeus (cereal leaf beetle); Z'go moth); Bonagota salubricola Meyrick (Brazilian apple lea gramma exclamationis Fabricius (sunflower beetle)); beetles froller); Grapholita molesta Busck (oriental fruit moth); 50 from the family Coccinellidae (including, but not limited to: Suleima helianthana Riley (sunflower bud moth); Argyro Epilachna varivestis Mulsant (Mexican bean beetle)); cha taenia spp., Choristoneura spp. fers and other beetles from the family Scarabaeidae (includ Selected other agronomic pests in the order Lepidoptera ing, but not limited to: Popillia japonica Newman (Japanese include, but are not limited to. Alsophila pometaria Harris beetle); Cyclocephala borealis Arrow (northern masked (fall cankerworm); Anarsia lineatella Zeller (peach twig 55 chafer, white grub); C. immaculate Olivier (southern borer); Anisota Senatoria J. E. Smith (orange striped oak masked chafer, white grub); Rhizotrogus maialis Razou worm); Antheraea pernyi Guérin-Meneville (Chinese Oak mowsky (European chafer); Phyllophaga crimita Burmeister Tussah Moth); Bombyx mori Linnaeus (Silkworm); Buccu (white grub): Ligyrus gibbosus De Geer (carrot beetle)); latrix thurberiella Busck (cotton leaf perforator); Collas carpet beetles from the family Dermestidae; wireworms eurytheme Boisduval (alfalfa caterpillar); Datana inte 60 from the family Elateridae, Eleodes spp., Melanotus spp.; gerrima Grote & Robinson (walnut caterpillar); Dendroli Conoderus spp., Limonius spp.; Agriotes spp., Ctenicera mus Sibiricus Tschetwerikov (Siberian silk moth), Ennomos spp., Aeolus spp., bark beetles from the family Scolytidae subsignaria Hübner (elm spanworm): Erannis tiliaria Harris and beetles from the family Tenebrionidae. (linden looper); Euproctis chrysorrhoea Linnaeus (brown Adults and immatures of the order Diptera are of interest, tail moth); Harrisina americana Guérin-Meneville (grape 65 including leafminers Agromyza parvicornis Loew (corn leaf skeletonizer); Hemileuca Oliviae Cockrell (range cater blotch leafminer); midges (including, but not limited to: pillar); Hyphantria cunea Drury (fall webworm); Keiferia Contarinia sorghicola Coquillett (Sorghum midge); May US 9,688,730 B2 121 122 etiola destructor Say (Hessian fly); Sitodiplosis mosellana delphax striatellus Fallen (smaller brown planthopper); Géhin (wheat midge); Neolasioptera murtfeldtiana Felt, Macrolestes quadrilineatus Forbes (aster leafhopper); (sunflower seed midge)); fruit flies (Tephritidae), Oscinella Nephotettix cinticeps Uhler (green leafhopper); N. nigropic frit Linnaeus (fruit flies); maggots (including, but not limited tus Stal (rice leafhopper); Nilaparvata lugens Stal (brown to: Delia platura Meigen (seedcorn maggot); D. coarctata planthopper); Peregrinus maidis Ashmead (corn planthop Fallen (wheat bulb fly); and other Delia spp., Meromyza per); Sogatella fircifera Horvath (white-backed planthop americana Fitch (wheat stem maggot); Musca domestica per); Sogatodes Orizicola Muir (rice delphacid), Tiphlocyba Linnaeus (house flies); Fannia canicularis Linnaeus, F. pomaria McAtee (white apple leafhopper); Erythroneoura femoralis Stein (lesser house flies); Stomoxys calcitrans spp. (grape leafhoppers); Magicicada Septendecim Linnaeus Linnaeus (stable flies)); face flies, horn flies, blow flies, 10 Chrysomya spp., Phormia spp.; and other muscoid fly pests, (periodical cicada); Icerya purchasi Maskell (cottony cush horse flies Tabanus spp.; bot flies Gastrophilus spp., Oestrus ion scale); Ouadraspidiotus perniciosus Comstock (San spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Jose scale); Planococcus citri Risso (citrus mealybug); Melophagus Ovinus Linnaeus (keds); and other Brachycera, Pseudococcus spp. (other mealybug complex); Cacopsylla mosquitoes Aedes spp.; Anopheles spp., Culex spp.; black 15 pyricola Foerster (pear psylla); Trioza diospyri Ashmead flies Prosimulium spp., Simulium spp.; biting midges, sand (persimmon psylla). flies, Sciarids, and other Nematocera. Agronomically important species of interest from the Included as insects of interest are adults and nymphs of order Hemiptera include, but are not limited to: Acrosternum the orders Hemiptera and Homoptera such as, but not limited hilare Say (green Stink bug); Anasa tristis De Geer (squash to, adelgids from the family Adelgidae, plant bugs from the bug); Blissus leucopterus leucopterus Say (chinch bug); family Miridae, cicadas from the family Cicadidae, leafhop Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis pers, Empoasca spp.; from the family Cicadellidae, plan modesta Distant (tomato bug); Dysdercus suturellus Her thoppers from the families Cixiidae, Flatidae, Fulgoroidea, rich-Schäffer (cotton stainer); Euschistus servus Say (brown lssidae and Delphacidae, treehoppers from the family Mem stink bug); E. variolarius Palisot de Beauvois (one-spotted bracidae, psyllids from the family Psyllidae, whiteflies from 25 Stink bug); Graptostethus spp. (complex of seed bugs); the family Aleyrodidae, aphids from the family Aphididae, Leptoglossus Corculus Say (leaf-footed pine seed bug); phylloxera from the family Phylloxeridae, mealybugs from Lygus lineolaris Palisot de Beauvois (tarnished plant bug); the family Pseudococcidae, scales from the families Aster L. Hesperus Knight (Western tarnished plant bug); L. prat olecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococ ensis Linnaeus (common meadow bug); L. rugulipennis cidae ortheziidae, Phoenicococcidae and Margarodidae, lace 30 Poppius (European tarnished plant bug); Lygocoris pabuli bugs from the family Tingidae, Stink bugs from the family nus Linnaeus (common green capsid); Nezara viridula Lin Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs naeus (southern green stink bug); Oebalus pugnax Fabricius from the family Lygaeidae, Spittlebugs from the family (rice Stink bug). Oncopeltus fasciatus Dallas (large milk Cercopidae squash bugs from the family Coreidae, and red weed bug); Pseudatomoscelis seriatus Reuter (cotton flea bugs and cotton stainers from the family Pyrrhocoridae. 35 hopper). Agronomically important members from the order Furthermore, embodiments may be effective against Homoptera further include, but are not limited to: Acyrthiisi Hemiptera such, Calocoris norvegicus Gmelin (Strawberry phon pisum Harris (pea aphid), Aphis craccivora Koch bug); Orthops campestris Linnaeus; Plesiocoris rugicollis (cowpea aphid); A. fabae Scopoli (black bean aphid); A. Fallen (apple capsid), Cyrtopeltis modestus Distant (tomato gossypii Glover (cotton aphid, melon aphid); A. maidiradi 40 bug); Cyrtopeltis notatus Distant (Suckfly); Spanagonicus cis Forbes (corn root aphid); A. pomi De Geer (apple aphid); albofasciatus Reuter (whitemarked fleahopper); Diaphno A. spiraecola Patch (spirea aphid); Aulacorthum Solani coris chlorionis Say (honeylocust plant bug); Labopidicola Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii allii Knight (onion plant bug); Pseudatomoscelis seriatus Cockerell (strawberry aphid); Diuraphis noxia Kurdumov/ Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid Mordvilko (Russian wheat aphid); Dysaphis plantaginea 45 plant bug); Poecilocapsus lineatus Fabricius (four-lined Paaserini (rosy apple aphid); Eriosoma lanigerum Haus plant bug), Nysius ericae Schilling (false chinch bug); mann (woolly apple aphid); Brevicoryne brassicae Linnaeus Nysius raphanus Howard (false chinch bug); Nezara (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum viridula Linnaeus (Southern green Stink bug); Eurygaster aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Meto spp.; Coreidae spp., Pyrrhocoridae spp., Tinidae spp., Blos polophium dirrhodium Walker (cereal aphid); Macrosiphum 50 tomatidae spp.; Reduviidae spp.; and Cimicidae spp. euphorbiae Thomas (potato aphid), Myzus persicae Sulzer Also included are adults and larvae of the order Acari (peach-potato aphid, green peach aphid); Nasonovia ribis (mites) such as Aceria to Sichella Keifer (wheat curl mite); nigri Mosley (lettuce aphid); Pemphigus spp. (root aphids Petrobia latens Müller (brown wheat mite); spider mites and and gall aphids); Rhopalosiphum maidis Fitch (corn leaf red mites in the family Tetranychidae, Panonychus ulmi aphid): R. padi Linnaeus (bird cherry-oat aphid); Schizaphis 55 Koch (European red mite); Tetranychus urticae Koch (two graminum Rondani (greenbug); Sipha flava Forbes (yellow spotted spider mite); (T. mcdanieli McGregor (McDaniel Sugarcane aphid); Sitobion avenae Fabricius (English grain mite); T cinnabarinus Boisduval (carmine spider mite); T. aphid); Therioaphis maculata Buckton (spotted alfalfa turkestani Ugarov & Nikolski (strawberry spider mite); flat aphid); Toxoptera aurantii Boyer de Fonscolombe (black mites in the family Tenuipalpidae, Brevipalpus lewisi citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); 60 McGregor (citrus flat mite); rust and bud mites in the family Adelges spp. (adelgids); Phylloxera devastatrix Pergande Eriophyidae and other foliar feeding mites and mites impor (pecan phylloxera); Bemisia tabaci Gennadius (tobacco tant in human and animal health, i.e. dust mites in the family whitefly, sweetpotato whitefly); B. argentifolii Bellows & Epidermoptidae, follicle mites in the family Demodicidae, Perring (silverleaf whitefly); Dialeurodes citri Ashmead grain mites in the family Glycyphagidae, ticks in the order (citrus whitefly); Trialeurodes abutiloneus (bandedwinged 65 Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus whitefly) and T. vaporariorum Westwood (greenhouse Neumann (Australian paralysis tick); Dermacentor variabi whitefly); Empoasca fabae Harris (potato leafhopper); Lao lis Say (American dog tick); Amblyomma americanum Lin US 9,688,730 B2 123 124 naeus (lone startick); and scab and itch mites in the families acilbenzolar-5-methyl, avermectin, amitrol, azaconazole, Psoroptidae, Pyemotidae, and Sarcoptidae. aZospirillum, azadirachtin, azoxystrobin, bacillus spp. (in Insect pests of the order Thysanura are of interest. Such as cluding one or more of cereus, firmus, megaterium, pumilis, Lepisma saccharina Linnaeus (silverfish); Thermobia sphaericus, Subtilis and/or thuringiensis), bradyrhizobium domestica Packard (firebrat). 5 spp. (including one or more of betae, canariense, elkanii, Additional arthropod pests covered include: spiders in the iriomotense, japonicum, liaonigense, pachyrhizi and/or order Araneae such as Loxosceles reclusa Gertsch & Mulaik yuanmingense), captan, carboxin, chitosan, clothianidin, (brown recluse spider); and the Latrodectus mactans Fab copper, cyaZypyr, difenoconazole, etidiazole, fipronil, flu ricius (black widow spider); and centipedes in the order dioxonil, fluoxastrobin, fluguinconazole, flurazole, flux Scutigeromorpha Such as Scutigera Coleoptrata Linnaeus 10 ofenim, harpin protein, imazalil, imidacloprid, ipconazole, (house centipede). isoflavenoids, lipo-chitooligosaccharide, mancoZeb, manga Insect pest of interest include the Superfamily of stink nese, maneb, mefenoxam, metalaxyl, metconazole, bugs and other related insects including but not limited to myclobutanil, PCNB. penflufen, penicillium, penthiopyrad, species belonging to the family Pentatomidae (Nezara permethrine, picoxystrobin, prothioconazole, pyra viridula, Halyomorpha haly's, Piezodorus guildini, Euschis- 15 clostrobin, rynaxypyr, S-metolachlor, Saponin, sedaxane, tus servus, Acrosternum hilare, Euschistus heros, Euschistus TCMTB, tebuconazole, thiabendazole, thiamethoxam, thio tristigmus, Acrosternum hilare, Dichelops fircatus, Dich carb, thiram, tolclofoS-methyl, triadimenol, trichoderma, elops melacanthus, and Bagrada hilaris (Bagrada Bug)), trifloxystrobin, triticonazole and/or zinc. PCNB seed coat the family Plataspidae (Megacopta cribraria Bean refers to EPA registration number 002935.00419, containing plataspid), and the family Cydnidae (Scaptocoris cas- 20 quintozen and terrazole. TCMTB refers to 2-(thiocyanom tanea—Root Stink bug); and Lepidoptera species including ethylthio) benzothiazole. but not limited to: diamond-back moth, e.g., Helicoverpa Seed varieties and seeds with specific transgenic traits zea Boddie; soybean looper, e.g., Pseudoplusia includens may be tested to determine which seed treatment options and Walker, and Velvet bean caterpillar e.g., Anticarsia gem application rates may complement such varieties and trans matalis Hübner. 25 genic traits in order to enhance yield. For example, a variety Methods for measuring pesticidal activity are well known with good yield potential but head Smut Susceptibility may in the art. See, for example, Czapla and Lang, (1990) J. benefit from the use of a seed treatment that provides Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Bio protection against head Smut, a variety with good yield chem. J. 252: 199-206; Marrone, et al., (1985) J. of Eco potential but cyst nematode susceptibility may benefit from nomic Entomology 78:290–293 and U.S. Pat. No. 5,743,477, 30 the use of a seed treatment that provides protection against all of which are herein incorporated by reference in their cyst nematode, and so on. Likewise, a variety encompassing entirety. Generally, the protein is mixed and used in feeding a transgenic trait conferring insect resistance may benefit assays. See, for example Marrone, et al., (1985) J. of from the second mode of action conferred by the seed Economic Entomology 78:290–293. Such assays can include treatment, a variety encompassing a transgenic trait confer contacting plants with one or more pests and determining the 35 ring herbicide resistance may benefit from a seed treatment plant’s ability to survive and/or cause the death of the pests. with a safener that enhances the plants resistance to that Nematodes include parasitic nematodes such as root-knot, herbicide, etc. Further, the good root establishment and early cyst, and lesion nematodes, including Heterodera spp., emergence that results from the proper use of a seed treat Meloidogyne spp., and Globodera spp.; particularly mem ment may result in more efficient nitrogen use, a better bers of the cyst nematodes, including, but not limited to. 40 ability to withstand drought and an overall increase in yield Heterodera glycines (soybean cyst nematode); Heterodera potential of a variety or varieties containing a certain trait Schachtii (beet cyst nematode); Heterodera avenae (cereal when combined with a seed treatment. cyst nematode); and Globodera rostochiensis and Glo Methods for Inhibiting Growth or Killing an Insect Pest and bodera pailida (potato cyst nematodes). Lesion nematodes Controlling an Insect Population include Pratylenchus spp. 45 In some embodiments methods are provided for inhibiting Seed Treatment growth or killing an insect pest, comprising contacting the To protect and to enhance yield production and trait insect pest with an insecticidally-effective amount of a technologies, seed treatment options can provide additional recombinant PIP-1 polypeptide. In some embodiments crop plan flexibility and cost effective control against methods are provided for inhibiting growth or killing an insects, weeds and diseases. Seed material can be treated, 50 insect pest, comprising contacting the insect pest with an typically Surface treated, with a composition comprising insecticidally-effective amount of a recombinant pesticidal combinations of chemical or biological herbicides, herbicide protein of SEQ ID NO: 6 or a variant thereof. safeners, insecticides, fungicides, germination inhibitors and In some embodiments methods are provided for control enhancers, nutrients, plant growth regulators and activators, ling an insect pest population, comprising contacting the bactericides, nematocides, avicides and/or molluscicides. 55 insect pest population with an insecticidally-effective These compounds are typically formulated together with amount of a recombinant PIP-1 polypeptide. In some further carriers, Surfactants or application-promoting adju embodiments methods are provided for controlling an insect vants customarily employed in the art of formulation. The pest population, comprising contacting the insect pest popu coatings may be applied by impregnating propagation mate lation with an insecticidally-effective amount of a recombi rial with a liquid formulation or by coating with a combined 60 nant pesticidal protein of SEQID NO: 6 or a variant thereof. wet or dry formulation. Examples of the various types of As used herein, by “controlling a pest population' or “con compounds that may be used as seed treatments are provided trols a pest' is intended any effect on a pest that results in in The Pesticide Manual: A World Compendium, C. D. S. limiting the damage that the pest causes. Controlling a pest Tomlin Ed., Published by the British Crop Production Coun includes, but is not limited to, killing the pest, inhibiting cil, which is hereby incorporated by reference. 65 development of the pest, altering fertility or growth of the Some seed treatments that may be used on crop seed pest in Such a manner that the pest provides less damage to include, but are not limited to, one or more of abscisic acid, the plant, decreasing the number of offspring produced, US 9,688,730 B2 125 126 producing less fit pests, producing pests more Susceptible to geometric planting patterns in the fields and in-bag seed predator attack or deterring the pests from eating the plant. mixtures, as discussed further by Roush. In some embodiments methods are provided for control In some embodiments the PIP-1 polypeptides of the ling an insect pest population resistant to a pesticidal protein, disclosure are useful as an insect resistance management comprising contacting the insect pest population with an 5 strategy in combination (i.e., pyramided) with other pesti insecticidally-effective amount of a recombinant PIP-1 poly cidal proteins include but are not limited to Bt toxins, peptide. In some embodiments methods are provided for Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, controlling an insect pest population resistant to a pesticidal and the like. protein, comprising contacting the insect pest population Provided are methods of controlling Lepidoptera and/or with an insecticidally-effective amount of a recombinant 10 pesticidal protein of SEQ ID NO: 6 or a variant thereof. Hemiptera insect infestation(s) in a transgenic plant that In some embodiments methods are provided for protect promote insect resistance management, comprising express ing a plant from an insect pest, comprising expressing in the ing in the plant at least two different insecticidal proteins plant or cell thereof a recombinant PIP-1 polypeptide. In having different modes of action. Some embodiments methods are provided for protecting a 15 In some embodiments the methods of controlling Lepi plant from an insect pest, comprising expressing in the plant doptera and/or Hemiptera insect infestation in a transgenic or cell thereof a recombinant pesticidal protein of SEQ ID plant and promoting insect resistance management the at NO: 6 or variants thereof. least one of the insecticidal proteins comprise a PIP-1 Insect Resistance Management (IRM) Strategies polypeptide insecticidal to insects in the order Lepidoptera Expression of B. thuringiensis Ö-endotoxins in transgenic and/or Hemiptera. corn plants has proven to be an effective means of control In some embodiments the methods of controlling Lepi ling agriculturally important insect pests (Perlak, et al., doptera and/or Hemiptera insect infestation in a transgenic 1990; 1993). However, insects have evolved that are resis plant and promoting insect resistance management the at tant to B. thuringiensis Ö-endotoxins expressed in transgenic least one of the insecticidal proteins comprises a protein of plants. Such resistance, should it become widespread, would 25 SEQ ID NO: 6 or variants thereof, insecticidal to insects in clearly limit the commercial value of germplasm containing the order Lepidoptera and/or Hemiptera. genes encoding Such B. thuringiensis Ö-endotoxins. In some embodiments the methods of controlling Lepi One way to increasing the effectiveness of the transgenic doptera and/or Hemiptera insect infestation in a transgenic insecticides against target pests and contemporaneously plant and promoting insect resistance management comprise reducing the development of insecticide-resistant pests is to 30 expressing in the transgenic plant a PIP-1 polypeptide and a use provide non-transgenic (i.e., non-insecticidal protein) Cry protein insecticidal to insects in the order Lepidoptera refuges (a section of non-insecticidal crops/corn) for use and/or Hemiptera having different modes of action. with transgenic crops producing a single insecticidal protein In some embodiments the methods of controlling Lepi active against target pests. The United States Environmental doptera and/or Hemiptera insect infestation in a transgenic Protection Agency (epa.gov/oppbppdl/biopesticides/pips/ 35 plant and promoting insect resistance management comprise bt corn refuge 2006.htm, which can be accessed using the in the transgenic plant a protein of SEQID NO: 6 or variants www prefix) publishes the requirements for use with trans thereof and a Cry protein insecticidal to insects in the order genic crops producing a single Bt protein active against Lepidoptera and/or Hemiptera having different modes of target pests. In addition, the National Corn Growers Asso action. ciation, on their website: (incga.com/insect-resistance-man 40 Also provided are methods of reducing likelihood of agement-fact-sheet-bt-corn, which can be accessed using the emergence of Lepidoptera and/or Hemiptera insect resis www prefix) also provides similar guidance regarding ref tance to transgenic plants expressing in the plants insecti uge requirements. Due to losses to insects within the refuge cidal proteins to control the insect species, comprising area, larger refuges may reduce overall yield. expression of a PIP-1 polypeptide insecticidal to the insect Another way of increasing the effectiveness of the trans 45 species in combination with a second insecticidal protein to genic insecticides against target pests and contemporane the insect species having different modes of action. ously reducing the development of insecticide-resistant Also provided are methods of reducing likelihood of pests would be to have a repository of insecticidal genes that emergence of Lepidoptera and/or Hemiptera insect resis are effective against groups of insect pests and which tance to transgenic plants expressing in the plants insecti manifest their effects through different modes of action. 50 cidal proteins to control the insect species, comprising Expression in a plant of two or more insecticidal com expression of a protein of SEQID NO: 6 or variants thereof, positions toxic to the same insect species, each insecticide insecticidal to the insect species in combination with a being expressed at efficacious levels would be another way second insecticidal protein to the insect species having to achieve control of the development of resistance. This is different modes of action. based on the principle that evolution of resistance against 55 Also provided are means for effective Lepidoptera and/or two separate modes of action is far more unlikely than only Hemiptera insect resistance management of transgenic one. Roush for example, outlines two-toxin strategies, also plants, comprising co-expressing at high levels in the plants called "pyramiding or 'stacking.” for management of two or more insecticidal proteins toxic to Lepidoptera and/or insecticidal transgenic crops. (The Royal Society. Phil. Hemiptera insects but each exhibiting a different mode of Trans. R. Soc. Lond. B. (1998) 353:777-1786). Stacking or 60 effectuating its inhibiting growth or killing activity, wherein pyramiding of two different proteins each effective against the two or more insecticidal proteins comprise a PIP-1 the target pests and with little or no cross-resistance can polypeptide and a Cry protein. Also provided are means for allow for use of a smaller refuge. The U.S. Environmental effective Lepidoptera and/or Hemiptera insect resistance Protection Agency requires significantly less (generally 5%) management of transgenic plants, comprising co-expressing structured refuge of non-Bt corn be planted than for single 65 at high levels in the plants two or more insecticidal proteins trait products (generally 20%). There are various ways of toxic to Lepidoptera and/or Hemiptera insects but each providing the IRM effects of a refuge, including various exhibiting a different mode of effectuating its inhibiting US 9,688,730 B2 127 128 growth or activity, wherein the two or more insecticidal 3 g/L, Dextrose 2.5g/L, Sodium Chloride 5 g/L. K2HPO4 proteins comprise a protein of SEQ ID NO: 6 or variants 2.5 g/L) and cultured overnight at 30° C. This insecticidal thereof and a Cry protein. activity exhibited heat and proteinase sensitivity indicating In addition, methods are provided for obtaining regulatory proteinaceous nature. approval for planting or commercialization of plants Lygus (Lygus hesperus) bioassays were conducted using expressing proteins insecticidal to insects in the order Lepi the cell lysate samples mixed with insect diet (Bio-Serv doptera and/or Hemiptera, comprising the step of referring F964.4B) in each well of a 96 well bioassay plate (BD to. Submitting or relying on insect assay binding data show FalconTM 353910). A variable number of Lygus hesperus ing that the PIP-1 polypeptide does not compete with second instar nymphs (2 to 7) were placed into each well of binding sites for Cry proteins in Such insects. In addition, 10 a 96 well plate. The assay was run four days at 25° C. and methods are provided for obtaining regulatory approval for then was scored for insect mortality and stunting of insect planting or commercialization of plants expressing proteins growth. A series of concentrations of the purified protein insecticidal to insects in the order Lepidoptera and/or sample was assayed against those insects and concentrations Hemiptera, comprising the step of referring to, Submitting or for 50% mortality (LC50) or inhibition of 50% of the relying on insect assay binding data showing that the protein 15 individuals (1050) were calculated. The Lygus bioassay of SEQ ID NO: 6 or variant thereof does not compete with results for PIP-1A is shown in Table 2. binding sites for Cry proteins in Such insects. Genomic DNA was extracted with a Sigma Bacterial Methods for Increasing Plant Yield Genomic DNA Extraction Kit (Cat if NA2110-KT, Sigma Methods for increasing plant yield are provided. The Aldrich, PO Box 14508, St. Louis, Mo. 63178) according to methods comprise providing a plant or plant cell expressing the manufactures instructions. The DNA concentration was a polynucleotide encoding the pesticidal polypeptide determined using a NanoDrop Spectrophotometer (Thermo sequence disclosed herein and growing the plant or a seed Scientific, 3411 Silverside Road, Bancroft Building, Suite thereof in a field infested with a pest against which the 100, Wilmington, Del. 19810) and the genomic DNA was polypeptide has pesticidal activity. In some embodiments, diluted to 40 ng/ul with sterile water. A 25 ul PCR reaction the polypeptide has pesticidal activity against a lepi 25 was set up by combining 80 ng genomic DNA, 2 ul (5 uM) dopteran, coleopteran, dipteran, hemipteran or nematode 16S ribosomal DNA primers TACCTTGTTACGACTT pest, and the field is infested with a lepidopteran, (SEQ ID NO: 209) and AGAGTTTGATCMTGGCTCAG hemipteran, coleopteran, dipteran or nematode pest. (SEQ ID NO: 210), 1 ul 10 cmM dNTP, 1x PhusionTM HF As defined herein, the "yield' of the plant refers to the buffer, and 1 unit of PhusionTM High-Fidelity DNA Poly quality and/or quantity of biomass produced by the plant. By 30 merase (New England Biolabs, Cat #M0530L, 240 County “biomass is intended any measured plant product. An Road, Ipswich, Mass. 01938-2723). The PCR reaction was increase in biomass production is any improvement in the run in MJ Research PTC-200 Thermo Cycler (Bio-Rad yield of the measured plant product. Increasing plant yield Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, has several commercial applications. For example, increas Calif., 94547, USA) with the following program: 96° C. 1 ing plant leaf biomass may increase the yield of leafy 35 min; 30 cycles of 96° C. 15 seconds, 52° C. 2 minutes and vegetables for human or animal consumption. Additionally, 72°C. 2 minutes; 72° C. 10 minutes; and hold on 4°C. The increasing leaf biomass can be used to increase production PCR products were purified with QiaGuickR DNA purifi of plant-derived pharmaceutical or industrial products. An cation Kit (Cat #28104, QIAGENR, Inc., 27220 Turnberry increase in yield can comprise any statistically significant Lane, Valencia, Calif. 91355). The purified PCR sample was increase including, but not limited to, at least a 1% increase, 40 DNA sequenced and the resulting 16S ribosomal DNA at least a 3% increase, at least a 5% increase, at least a 10% sequence was BLAST searched against the NCBI database increase, at least a 20% increase, at least a 30%, at least a which indicated that SS44C4 is a Pseudomonas chlorora 50%, at least a 70%, at least a 100% or a greater increase in phis strain. The Pseudomonas chlororaphis strain SS44C4 yield compared to a plant not expressing the pesticidal was deposited on Dec. 1, 2011 under accession if NRRLB Sequence. 45 50613 with the Agricultural Research Service Culture Col In specific methods, plant yield is increased as a result of lection (NRRL), 1815 North University Street, Peoria, Ill. improved pest resistance of a plant expressing a PIP-1 61604, (nrrl.ncaurusda.gov, which can be accessed on the polypeptide disclosed herein. Expression of the PIP-1 poly world-wide web using the “www’ prefix). peptide results in a reduced ability of a pest to infest or feed The cell pellet of an overnight culture from a single on the plant, thus improving plant yield. 50 colony of SS44C4 grown in LB Broth at 30°C. was lyzed The following examples are offered by way of illustration in a French Press at ~20,000 psi in a single pass after and not by way of limitation. resuspension with PBS buffer. The lysis product was cen trifuged and the soluble fraction retained and stored at 4°C. EXPERIMENTALS overnight to allow insoluble chlororaphin products to pre 55 cipitate. The remaining Supernatant was filtered sequentially Example 1 through 25 um, 8 um, 5um, 1.2 um and 0.45 um filters to remove the majority of the Crystalline material. The soluble Identification of an Insecticidal Protein Active cell lysate was adjusted to 1.2 M ammonium Sulfate and Against Lygus from Strain SS44C4 loaded onto an Ether column (ToyopearlTM Ether-650S, 60 Tosoh Bioscience LLC, 3604 Horizon Drive, Suite 100, The Lygus active protein PIP-1A was identified by protein King of Prussia, Pa. 19406) of appropriate size. A linear purification, N-terminal amino acid sequencing, PCR clon gradient was run from 1.2 Mammonium sulfate to 0.6 M ing from Pseudomonas chlororaphis strain SS44C4 as fol ammonium sulfate over 15 column volumes. The elution lows: peak fractions containing protein of interest were then Insecticidal activity against Lygus (Lygus hesperus) was 65 concentrated via a spin concentrator. The concentrate was observed from a cell lysate of SS44C4 grown in Trypticase then buffer exchanged into 25 mM Tris pH 8 to remove soy medium (Tryptone 17 g/L, enzymatic digest of soy meal ammonium sulfate using a 7000 MWCO ZebaTM desalting US 9,688,730 B2 129 130 column (Thermo Fisher Scientific Inc., 747 Meridian Rd, were subcloned into an E. coli expression vector pMALTM Rockford, Ill. 61101). The concentrated and desalted protein (New England Biolabs, 240 County Road, Ipswich, Mass. was then loaded onto a MonoCTM column (cat #17-5166-01, 01938-2723) having a 6xHis tag added to the Maltose GE Healthcare). Optimum elution and purity was achieved Binding Protein and transformed into E. coli for recombi by application of a linear gradient from 0 to 400 mM NaCl. 5 The active fraction pool from the Mono OTM purification nant protein expression. E. coli cells transformed with the was subjected to N-terminal sequencing. The protein pool expression constructs were grown overnight at 37° C. with was run on SDS-PAGE, transferred to a PVDF membrane, carbenicillin selection and then inoculated to a fresh 2XYT and stained with CoomassieTM Blue dye. Four bands were medium (1:250) and further grown to ODoo -0.8. IPTG was present on the membrane. All were successfully identified then added and the cells were grown further at 37° C. for by N-terminal sequencing with a single sequence per band. 10 another 6 hours or transferred to 16°C. for overnight growth The N-terminal amino acid sequence of two protein bands to induce protein expression. The E. coli expressed proteins were BLAST searched against the NCBI database and a were purified either by Amylose resin (New England Bio hypothetical protein (PSEEN3174) from a genome sequence labs, 240 County Road, Ipswich, Mass. 01938-2723) or of Pseudomonas entomophila (Vodovar, Netal. (2006) Nat. Ni-NTAagarose (Cat. No. K950-01, Invitrogen, 3175 Staley Biotechnol. 24 (6), 673-679) was identified as a homology 15 match (FIG. 1). The PSEEN3174 gene, was cloned by PCR Road, Grand Island, N.Y. 14072), according to the manu using primers ATACATATGACGATCAAGGAAGAGCTG facturer's protocols. (SEQ ID NO: 13) and TTGGATCCTCAATAACGGCGAT GAGGATCGTTGTAG (SEQ ID NO: 14). PCR with the Example 3 cloning primers (SEQ ID NO: 13 and 14) was performed against the SS44C4 genomic DNA preparation, and a band Lepidoptera and Coleoptera Assays with Purified of the expected molecular weight was isolated. Proteins The resulting PCR product was DNA sequenced and coupled with MS/MS spectra from in-gel digests showed Insecticidal activity bioassay Screens were conducted on this gene product having the DNA sequence of SEQID NO: 25 the cell lysate to evaluate the effects of the insecticidal 1 encoding a protein designated herein as “PIP-1A, having proteins on a variety of Lepidoptera species (European corn the amino acid sequence of SEQID NO: 2. The PSEEN3174 borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), gene has the DNA sequence set forth in SEQID NO: 5 and black cutworm (Agrotis ipsilon), fall armyworm (Spodop encodes an amino acid sequence having the amino acid tera frugiperda), Soybean looper (Pseudoplusia includens) sequence set forth in SEQID NO: 6. Using the PIP-1A (SEQ 30 and Velvet bean caterpillar (Anticarsia gemmatalis)), a ID NO: 2) and PSEEN3174 (SEQ ID NO: 6) sequence Coleoptera specie (Western corn rootworm (Diabrotica vir information another homologous gene, SPBB 340380 (an gifera) notated as a hypothetical protein from Dendroctonus fron Lepidoptera feeding assays were conducted on an artifi talis Bacterial community), was identified by BLAST search cial diet containing the cell lysates of bacterial strains in a 96 from the Department of Energy Joint Genomic Institute 35 well plate set up. The cell lysate was incorporated with the website (gi.doe.gov/, which can be accessed on the world Lepidopteran-specific artificial diet in a ratio of 1:2 cell wide web using the “www” prefix). The SPBB 340380 lysate to diet mixture. Neonate larvae were placed in each coding sequence was generated by back translation of pro well to feed ad libitum for 5 days. Results were expressed as tein sequence using PSEEN3174 (SEQ ID NO: 5) codon positive for larvae reactions such as stunting and or mortal usage and the gene was synthesized. The SPBB 340380 40 ity. Results were expressed as negative if the larvae were coding sequence has the DNA sequence set forth in SEQID similar to the negative control that is feeding diet to which NO: 3 and encodes an amino acid sequence, designated the above buffer only has been applied. Cell lysates was herein as “PIP-1B, having the amino acid sequence set assayed on European corn borer (Ostrinia nubilalis), corn forth in SEQ ID NO: 4. earworm (Helicoverpa zea), black cutworm (Agrotis ipsi 45 lon), fall armyworm (Spodoptera frugiperda), Soybean Example 2 looper (Pseudoplusia includens) and Velvet bean caterpillar (Anticarsia gemmatalis). A series of concentrations of the E. coli Expression of PIP-1A, PSEEN3174 and purified protein sample was assayed against those insects PIP-1B and concentrations for 50% mortality (LC50) or inhibition 50 of 50% of the individuals (IC50) were calculated. The The three coding sequences, PIP-1A (SEQ ID NO: 1); insecticidal activity for PIP-1A and PSEEN3174 are shown PSEEN3174 (SEQ ID NO. 5): & PIP-1B (SEQ ID NO:3), in Table 2. TABLE 2 PIP-1A (SEQ ID NO. 2 PSEEN3174 (SEQ ID 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% not tested 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/cm2 LC-50 US 9,688,730 B2 131 132 TABLE 2-continued PIP-1A (SEQ ID NO. 2 PSEEN3174 (SEQ ID NO: 6 Insect dose effect dose effect 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

Coleoptera feeding assays were conducted on an artificial nymphs were placed in polystyrene Petri dishes (100 diet containing the cell lysates of bacterial strains. The cell mmx20 mm) lined with moist Whatman(R) filter paper (100 lysate was incorporated with the coleopteran-specific artifi mm diameter). The bioassay was incubated at 25°C. for four cial diet in a ratio of 1:5 cell lysate to diet mixture. Western 25 days. The bioassay was scored for insect mortality and corn rootworm (Diabrotica virgifera) neonate larvae were placed in each well to feed ad libitum for 5 days. Results stunting of growth. To generate IC50 or LC50 data, a series were expressed as positive for larvae reactions such as of concentrations of purified proteins were assayed against stunting and or mortality. Results were expressed as negative insects and the concentration at which 50% of insects if the larvae were similar to the negative control that is experienced severe damage was the IC50 and the concen feeding diet to which the above buffer only has been applied. 30 tration at which 50% of insects were dead was the LC50. A series of concentrations of the purified protein sample was The results for PIP-1A (SEQ ID NO: 2) and PSEEN3174 assayed against those insects and concentrations for 50% (SEQ ID NO: 6) are shown in Table 2. mortality (LC50) or inhibition of 50% of the individuals (1050) were calculated. The results for PIP-1A and Example 6 PSEEN3174 are shown in Table 2. 35 Colorado Potato Beetle (Leptinotarsa decemlineata) Example 4 Bioassay with Purified Proteins Aphid Oral Feeding Assays with Purified Proteins 20 ul of cell lysate samples were mixed with 75 ul of Membrane feeding assays as described (Li, et al., (2011) 40 modified Coleopteran diet (Bio-Serv F9800B) in each well Journal of Invertebrate Pathology 107:69-78) were used to of a 96 well bioassay plate (BD FalconTM 353910) and assess the toxicity of PIP-1A and PSEEN3174, formulated allowed to Solidify. A single neonate larva was placed in in PBS pH 7.4. Briefly, the individual proteins were mixed each well and the plate sealed with a Mylar covering. Holes with filter-sterilized complete artificial diet as described in were punched in the Mylar sheet and the plate incubated at Febvay, et al., (1988), Can. J. Zool. 66:2449-2453) to a final 45 25° C. for four days. The bioassay was scored for insect concentration of up to 1250 micrograms/ml. This diet (100 mortality and stunting of growth. The results for PIP-1A ul) was placed on stretched parafilm pulled tightly across a (SEQ ID NO: 2) and PSEEN3174 (SEQ ID NO: 6) are 3 cm cell culture plate with a 1 cm hole on one side of the shown in Table 2. plate. A second layer of stretched parafilm was applied to form a thin film of diet exposed to aphids through the 1 cm 50 Example 7 hole. Around 30 second instar pea or green peach aphids were transferred to each plate, with three replicates for each Cross-Resistance Test in Diamondback Moth toxin. The same number of aphids were fed on diet only, as (Plutella xylostella) with Purified Proteins a control treatment. All plates were incubated at 24°C. with an 18:6 light:dark photoperiod. Mortality was scored every 55 A diet overlay assay similar to Wang, et al., ((2007) Appl. 24 hours and dead aphids were removed. The artificial diet Environ. Microbiol. 73:1199-1207) was used for testing the was replaced every 3 days. Data were analyzed by one-way LC50 and IC50 of the sample on susceptible and Cry1A ANOVA. The results for PIP-1A (SEQ ID NO: 2) and resistant diamondback moth (DBM, Plutella xylostella). For PSEEN3174 (SEQ ID NO: 6) are shown in Table 2. neonate bioassays, an aliquot of PIP-1A (SEQ ID NO: 2) Example 5 60 sample solution was applied to the surface (-7 cm) of 5 ml artificial diet (Southland Products Inc.) in a 30-ml insect Southern Green Stinkbug (Nezara viridula) and rearing cup. Each bioassay included seven 2x consecutive Brown Marmorated Stinkbug (Halvomorpha haly) dilutions from 500 ng/cm of the PIP-1A (SEQ ID NO: 2) Bioassay with Purified Proteins sample and the negative control, with three replications for 65 each concentration. The PIP-1A (SEQ ID NO: 2) protein 40 ul of the cell lysate samples were mixed with 360 ul of dilutions were prepared by mixing PIP-1A protein (SEQ ID the diet (Bio-Serv F964.4B). 10 to 15 newly molted instar NO: 2) with appropriate amount of PBS buffer solution US 9,688,730 B2 133 134 (Fisher Scientific Inc). Neonate larvae (<24 h after hatch) sized. The DNA sequence identity between those two genes were placed in each assaying cup. Mortality and larval was increased from 78% to 87% after the modification. To growth inhibition (defined as inhibition if larvae did not perform the classic gene family shuffling, random DNA enter second instar within 4 days) by each sample were fragments of both PIP-1A Synth (SEQ ID NO: 15) and scored after 4 days of feeding on the treated diet at 27°C. PSEEN3174 (SEQ ID NO: 5) were generated by limited Concentrations for 50% mortality (LC50) or inhibition of nuclease digestion. DNA fragments with molecular weights 50% of the individuals (IC50) were calculated based on of 50 to 200 base pairs of both genes were recovered from probit analysis. The results (Table 3) showed no cross agarose gel. The isolated DNA fragments were assembled on resistance (resistance ratio<2) for PIP-1A (SEQ ID NO: 2) a thermo cycler with polymerase and rescued by cloning to Cry1A in diamondback moth. 10 primers franking both termini. The libraries were cloned as Maltose-Binding-Protein fusions into pMAL(R)-c2x (NEB) TABLE 3 and transformed into E. coli cells. Approximately 5000 clones from the shuffled libraries were screened in the Lygus DBM strain LCIC ng/cm 95% FL Resistance Ratio assay and approximately 1000 clones expressed a polypep Susceptible LC 122.5 80.8-172.3 1.O 15 tide at significant levels and were active as clear cell lysates IC 66.71 42.2O-98.21 1.O in the Lygus bioassay. Lygus bioassays were conducted using Cry1A-Res LC 205.3 145.7-285.1 1.7 the cell lysates at 100 ppm concentration of the PIP-1 IC 59.94 36.90-88.64 O.90 polypeptide. The concentrations of PIP-1 polypeptides were estimated using densitometry method of SDS-PAGE with BSA as standard using program Phoretix ID (Totall ab Ltd Example 8 Keel House, Garth Heads, Newcastle upon Tyne NE1 2JE). Of the active clones, 50 were DNA sequenced (SEQ ID Creation and Identification of PIP-1A Variants NOS: 152-202) and the amino acid sequence (SEQID NOS: 101-151) of the encoded PIP-1 polypeptide was determined. Libraries of modified PIP-1A polynucleotides were gen 25 Table 4 shows the percent homology of the PIP-1 polypep erated using recursive sequence recombination methods tides (SEQ ID NOs: 101-151) to PIP-1A (SEQ ID NO: 2). (Crameri, et al., (1998) Nature. 391:288-291; Stemmer, For each of the sequences in Table 4 only those positions and (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Ness, the corresponding amino acids where PIP-1A (SEQID NO: et. al., (2002) Nature Biotechnology 20:1251-1255), also 2), PSEEN3174 (SEQID NO: 6) and the PIP-1 polypeptide known as gene shuffling methods. To increase the crossover 30 differ are shown. Amino acid substitutions were also iden points between the two genes, codons of PIP-1A (SEQ ID tified at positions 3, 6, 49, 213, 249 (shaded) of PIP-1A NO: 1) were modified using the codon usage of PSEEN3174 (SEQID NO: 2) which arent the corresponding amino acid (SEQID NO: 5) as the template while the protein sequences of PSEEN3174 (SEQID NO: 6). These results demonstrate are not changed. The modified PIP-1A coding sequence was a diverse set of PIP-1A polypeptide variants that have named as PIP-1A Synth (SEQ ID NO: 15) and was synthe insecticidal activity. TABLE 4

% Identity Sequence Name PIP-1A (SEQID NO: 2)

D D0274322 SEQI 48 D D0274327 SEQ 51 D0274236 SEQE : 04 US 9,688,730 B2 135 136 TABLE 4-continued

US 9,688,730 B2 139 140 TABLE 4-continued

% Identity to PIP- 21 22 24 25 Sequence Name 1A PIP-1A (SEQ ID NO: 2) Q G D0274266 SEQID NO: 124 DO274283 SEOIDQ NO: 3 89 D D0274290 SEQID NO: 134 89 A D D0274233 S EQ DNO: O1 88 EQ 20 88 EQ 29

D DO27428O SEOIDEQ NO: 130 88 D D0274306 SEQID NO: 142 88 D DO274316 SEOIDEQ NO: 46 88 D D0274323 SEQ 49 88 D DO274239 SEQ O6 7

EQ 41 87 32 AAAAAA AAAAA

V D D D EQ 10 82 V DO274313 SEOIDQ NO: 145 82 V PSEEN3174 (SEQID NO: 6) 79 vs

% Identity syne to PIP- 98 105 108110 120 12 1 1 23 127 134 135 150 151 160 164

D D0274266 89 D D0274283 89 V N T S E S N S T T D D0274290 89 V E T D D0274233 88 Y s Es NNAN AN ET TD D D0274259 88 Ss EEs N D D0274278 88 Y | V | E N T L R. S E US 9,688,730 B2 141 142 TABLE 4 -continued

US 9,688,730 B2 143 144 TABLE 4-continued

D D02743.02 M R PD N D D0274240 D D0274252 D D0274249 D D0274255 M D D0274243 E M K N D D0274281 D D0274313 PSEEN3174 K

Example 9 The protein sequence alignment of three active homologs, 2O PIP-1A (SEQID NO: 2), PSEEN3174 (SEQ ID NO: 6) and Identification of Amino Acid Positions Affecting PIP-1B (SEQ ID NO: 4), and three inactive orthologs, the Protein Stability and Function AECFG 592740 (SEQ ID NO: 12), Pput 1063 (SEQ ID NO: 8) and Pput 1064 (SEQ ID NO: 10) is shown in FIG. 1. Secondary structure features of PIP-1A (SEQ ID NO: 2) BLAST searching the Department of Energy Joint were obtained using program Garnier (EMBOSS Explorer) Genomic Institute website (www.jgi.doe.gov/) and NCBI 25 (Garnier, et al., (1978) J. Mol. Biol. 120:97-120) and database using the PIP-1A (SEQID NO: 2) and PSEEN3174 selected structure features shown above the alignment of (SEQ ID NO: 6) sequence revealed information regarding FIG. 1. Six positions (P43, W66, P89, Y93, Y176, F259 of three additional genes having lower homology: SEQ ID NO: 2) were selected for saturated mutagenesis AECFG 592740 (2035954615 annotated as a hypotheti analysis. Mutants were generated using degenerate oligos cal protein Acromyrmex echination fungus garden), 30 (Table 5) for each site using sewing and rescuing PCR Pput 1063 (Accession # ABQ77224; Gene ID:5191350– strategy of two overlapping fragments of N-terminus (no annotated as a hypothetical protein Pseudomonas putida mutation) and C-terminus (with mutations) gene) for each F1) and Pput 1064 (Accession # ABQ77225; Gene site using sewing and rescuing PCR strategy of two over ID:5191351—annotated as a hypothetical protein lapping fragments of N-terminus (no mutation) and C-ter Pseudomonas putida F1). The AECFG 592740 coding minus (with mutations) gene) as illustrated in FIG. 2. The sequence has the DNA sequence set forth in SEQID NO: 11 rescued mutant libraries were cloned into the Maltose and encodes a polypeptide, having the amino acid sequence Binding-Protein fusions of pMAL(R)-c2x (NEB). Individual set forth in SEQID NO: 12. The Pput 1063 coding sequence mutations were identified by sequencing 96 clones of each has the DNA sequence set forth in SEQ ID NO: 7 and library. The respective variant proteins were expressed in E. encodes the polypeptide set forth in SEQ ID NO: 8. The coli, and cell lysates tested in the Lygus assay as described Pput 1064 coding sequence has the DNA sequence set forth previously for PIP-1A (SEQ ID NO: 2). Table 6 shows for in SEQ ID NO: 9 and encodes the polypeptide set forth in each mutated position the amino acid Substitutions identified SEQ ID NO: 10. The AECFG 592740 (SEQ ID NO: 11), those Substitutions that expressed soluble protein, and those Pput 1063 (SEQ ID NO: 7), and Pput 1064 (SEQ ID NO: Substitutions that were active in the Lygus assay and/or the 9) genes were synthesized, the respective proteins were 45 Soybean looper assay with a minimal score of 4 or greater expressed as Maltose binding protein fusions in E. coli, and out of total maximal score of 8. The expression and activity cell lysates were tested in the Lygus assay as described of the substitutions in parenthesis were not determined. previously for PIP-1A (SEQ ID NO: 2). The Substitutions indicated with an “” had significantly reduced AECFG 592740 (SEQ ID NO: 12), Pput 1063 (SEQ ID soluble expression. This data demonstrate that the amino NO: 8), and Pput 1064 (SEQ ID NO: 8) proteins were 50 substitutions indicated in Table 6 as "Active mutants' can be inactive in the Lygus assay. made while retaining activity. TABLE 5 Oligo Residue name Sequence

P43 P43 GAACTGATCGAAGTTGCCAGC SEO ID NO: 16 P43 f1 GCTGGCAACTTCGATCAGTTCCDNACTAAGCGTGGTGGCTTTGC SEO ID NO: 17 GCTGGCAACTTCGATCAGTTCDNNACTAAGCGTGGTGGCTTTGC SEQ ID NO: 18

GCAGCCTTGCTTGGGCGCGCTG SEO ID NO : 19 CAGCGCGCCCAAGCAAGGCTGCTNGTAGATGGCATTACCGTCTACG SEQ ID NO: 2O CAGCGCGCCCAAGCAAGGCTGCWNNGTAGATGGCATTACCGTCTACG SEQ ID NO: 21 US 9,688,730 B2 145 146 TABLE 5 - continued Oligo Residue ale Sequence P89 GCGAGTGTAGGTGCCCCAATT SEO ID NO : 22 AA TGGGGCACCTACACTCGCNNKGTCTTTGCCTACCTGCAGTACATG SEQ D NO: 23

GGCAAAGACCGGGCGAGTGTAGG SEQ ID NO: 24 CC ACACTCGCCCGGTCTTTGCCTBBCTGCAGTACATGGACACCATT SEQ D NO 25 CC ACACTCGCCCGGTCTTTGCCWNNCTGCAGTACATGGACACCATT SEQ D NO: 26

Y176 Y178 CACGATGAAGGTGCCAGGACC SEQ ID NO: 27 Y178f 1 CCTGGCACCTTCATCGTGTBBCAGGTTGTTATGGTTTATGC SEQ D NO: 28 CCTGGCACCTTCATCGTGWNNCAGGTTGTTATGGTTTATGC SEO ID NO: 29

F259 ACTGAAGTGCCCACTATTGCTG SEO ID NO : 30 CAGCAATAGTGGGCACTTCAGTTWNGACTGGAGCGCCTACAACGATC SEO ID NO: 31 CAGCAATAGTGGGCACTTCAGTWNNGACTGGAGCGCCTACAACGATC SEO ID NO: 32

TABLE 6 25 TABLE 6-continued

Soluble Soluble Identified expressed Lygus Active SBL Active Identified expressed Lygus Active SBL Active Residue mutations Mutants mutants mutants Residue mutations Mutants mutants mutants P43 M, G, Q, S, MG, Q, S, MG, Q, S, MG, Q, S, T, so F259 W, C, A, D, W, Y, F, M, W, Y, C, M, W, M, L, V, I, T. R. V. L., T, R*, V, T. R. V. L. V. L., K, D, A, M. Y., L, V, I, H Y, K, D, A, N, L., K, D, A, K, D, A, N. N. F. W. E., NT I. G. F. W. E., C, N, F, W, E, C F. W. E., C, C, Y soy (I), (Y), Y, F, H, Y, F, K, R, V. S 35 K*, R*, M*, H, I Example 10 L*, A., I, C, V, S Identification of Motifs for Insecticidal Activity K, A, C, L, L. G., R., T. V, C, Four conserved motifs, amino acids 64-79 of SEQID NO: CY. S.M.A. 40 2 (motif 1), amino acids 149-159 of SEQ ID NO: 2 (motif s so , N, V, C, K 2), amino acids 171-183 of SEQ ID NO: 2 (motif 3), and s amino acids 240-249 of SEQ ID NO: 2 (motif 4) (motifs W, M, F, C, W. V. M, W, V, D, N, L, underlined in FIG. 1) of active proteins (PIP-1A (SEQ ID C* V: , T: L. I. F. A. T IF NO: 2), PSEEN3174 (SEQ ID NO: 6) and PIP-1B (SEQ ID L.I.A. 3 as as a ks 9 a. s. 4s NO: 4) were selected to determine their roles for insecticidal W. E., I, G, functions. For each selected motif, amino acids 64-79 of S. P. F SEQ ID NO: 2, amino acids 149-159 of SEQ ID NO: 2, Y176 S, W, V, T, M, F, L*, M, F, L, M. L., C amino acids 171-183 of SEQ ID NO: 2, and amino acids M, R, Q, L, C*, A*, w; 240-249 of SEQID NO: 2, the sequence was replaced with N, D, C, A, 50 corresponding sequences from three distantly related but E. G. F., I, functionally inactive proteins AECFG 592740 (SEQ ID P. (H), (K) NO: 12), Pput 1063 (SEQ ID NO: 8), and Pput 1064 (SEQ ID NO: 10) respectively (Table 7 shows the % identity). 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 US 9,688, 730 B2 147 148 The chimeras were generated using a sewing PCR strat shows the amino acid sequence for each of the four motifs egy with fragments of N-terminus and C-terminus of the (underlined in FIG. 1) from PIP-1A and the corresponding wild type PIP-1A with overlapping oligonucleotides (Table 8) coding for the replaced sequence of inactive proteins. amino acid sequence based on the alignment (FIG. 1) with The rescued PCR products containing the replacements AECFG 592740 (SEQ ID NO:12), Pput 1063 (SEQ ID were cloned into the pMAL expression vector as described NO: 8), and Pput 1064 (SEQ ID NO: 10) that were substi above for PIP-1A. The resulting chimeras were expressed tuted. In Table 9 the differences between the respective and functionally tested in Lygus insect bioassays. Table 9 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 1063MotF GAAACCGTCTACGGTGGCTTCGGTTTCCCCAAGCAGAATTGGGGCACCTAC 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 AGTGAAACCGGTCTGGTTCGGAGACGTTCAGCAATAGCACTCAATTG 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 Pput 1064ydt 4R. CTGGTTGAACAACACAGCCTGGTTAACAGTATCCCAATCCAGCGGC 1064 SEO ID NO. 55 1064ydt 4F 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 SEO ID NO: 2 SEO ID NO: 12 Pput 1063 1063Mot1R GCWIEGETWYGGFGFP No No 1063Mot1F a.a. 34 - 49 of SEO ID NO: 8 US 9,688,730 B2 149 150 TABLE 9- continued

Soluble Replaced PIP-1A WT amino Amino acids protein Motif from Oligos acid sequence replaced expressed Activity 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 18 - 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 55-167 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 GTIMVYOVHMVYA 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

Table 9 also indicates if the resulting proteins were 3s selected to further define the roles of the regions in insec soluble when expressed as a MAL fusion in E. coli. and were ticidal functions. To further define the permitted sequence active in the Lygus assay. variation within those two selected motifs, Saturated muta As indicated in Table 9, all but one of these chimeras had genesis was designed for each position of the motifs using reduced expression of soluble protein and was inactive in the the mutagenesis oligonucleotides as shown in Tables 10 and bioassay indicating that these four motifs have functional 40 11 for motifs 3 and 4 respectively. The variants were constraints. generated using a similar strategy as described in Example Example 11 9. Tables 12 and 13 show for each mutated position the amino acid substitutions identified, those substitutions that Saturated Mutagenesis of Motifs to Define expressed soluble protein, and those substitutions that were Sequence Variations that Retain Insecticidal 45 active in the Lygus assay and/or the Soybean looper assay Activity with a minimal score of 4 or greater out of total maximal score of 8. This data demonstrate that the amino substitu Two motifs, amino acids 171 to 183 (motif3) and amino tions indicated in Tables 12 and 13 as "Active mutants' can acids 240 to 249 (motif 4) of PIP-1A (SEQ ID NO: 2) were be made while retaining activity. TABL E 1O

Amino acid Position of PIP-1A Motif (SEQ ID Oligo NO: 2) ale Sequence

3 G171 G173R AGGACCAGTCAATTGAGTGCT SEO ID NO: 57 G173 AGCACTCAATTGACTGGTCCTNNKACCTTCATCGTGTATCAGGT SEO ID NO: 58

T172 T174R GCCAGGACCAGTCAATTGAGT SEO ID NO. 59 T174F ACT CAATTGACTGGTCCTGGCNNKTTCATCGTGTATCAGGTTG SEO ID NO: 60

US 9,688,730 B2 155 156 TABLE 13-continued Identified Soluble expressed Position mutations mutants Active mutants 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 of the protein is defined with the variant still retains detectible insecticidal activity. Transient Expression and Insect Bioassay on 25 Transient Leaf Tissues Example 14 Both PIP-1A (SEQ ID NO: 2) and PSEEN3174 (SEQ ID N-Terminal Truncation Variants NO: 6) as MBP fusions and alone were cloned into a transient expression vector under control of a viral promoter 30 The PIP-1A (SEQID NO: 2), PSEEN3174 (SEQ ID NO: pDMMV (Day, et al., (1999) Plant Mol. Biol. 40.771-782). 6) and PIP-1B (SEQ ID NO: 4) proteins were digested with The agro-infiltration method of introducing an Agrobacte a limited Trypsin digestion (1 part of Trypsin vs. 100 parts rium cell Suspension to plant cells of intact tissues so that of purified protein). The resulting N-terminal trypsin trun reproducible infection and Subsequent plant derived trans cated variants, PIP-1AT1 (SEQ ID NO. 204), gene expression may be measured or studied is well known 35 PSEEN3174T1 (SEQID NO: 206), PIP-1BT1 (SEQID NO: in the art (Kapila, et. al., (1997) Plant Science 122:101-108). 208), have amino acids 1-28 deleted compared to the respec Briefly, young plantlets of Phaseolus vulgaris or Glycine tive full length proteins by N-terminal Amino Acid sequenc max, were agro-infiltrated with normalized bacterial cell ing. The PIP-1AT1 (SEQ ID NO: 204), PSEEN3174T1 cultures of test and control Strains. Leaf discs were generated (SEQ ID NO: 206), PIP-1BT1 (SEQ ID NO. 208) were and infested with 3 neonates of both Soy Bean Looper (SBL) 40 assayed in the Lygus assay and found to have Substantially (Pseudoplusia includes) or Velvet bean caterpillar (VBC) the same activity as the respective full length proteins. (Velvet Anticarsia gemmatalis) with two control leaf discs generated with Agrobacterium only. The consumption of Example 15 green leaf tissues was scored after two days infestation. The transiently expressed PIP-1A (SEQ ID NO: 2) and 45 Proteolytic Cleavage Site Variants PSEEN3174 (SEQ ID NO: 6) protected leaf discs from The arginine (R) at position 28 of PIP-1A was mutated to consumption by the infested SBL and VBC insects while the alter the trypsin cleavage site. The variants were generated total green tissue consumption was observed for the two using a similar strategy as described in Example 9 using the negative controls. Transient protein expressions of both saturation mutagenesis primers R28R (SEQ ID NO: 218), PIP-1A (SEQ ID NO: 2) and PSEEN3174 (SEQ ID NO: 6) 50 and R28F (SEQ ID NO: 219). Table 14 shows the amino were confirmed by Mass spectrometry based protein iden acid substitutions identified, those substitutions that tification method using extracted protein lysates from infil expressed soluble protein, and those substitutions that were trate leaf tissues (Patterson, (1998) 10(22):1-24, Current active in the Lygus assay with a minimal score of 4 or greater Protocol in Molecular Biology published by John Wiley & out of total maximal score of 8. This data demonstrate that Son Inc). 55 the amino substitutions indicated in Table 14 as 'Active Example 13 mutants' can be made to eliminate a proteolytic cleavage site while retaining activity. Defined Protein Sequences of Fragments Retaining Activity 60 TABLE 1.4 Identified Soluble expressed Lygus Active A series of truncated variants of PIP-1A (SEQID NO: 2) Position mutations Mutants mutants are generated in 5 amino acid increments from both ends by R28 S. K., T. V. G, A, S, K, T, V, G, A, S, K, T. V. G., PCR cloning for the first and/or last 30 amino acids. The M, D, W, P, L, H, M, D, W, P, L, H, A, M., D, W, truncated genes are cloned to the same expression system as 65 C, Q, C, Q, L, H, C, Q, listed above. Recombinant proteins of those truncated ver sions of PIP-1A are assayed with insects and minimal length US 9,688,730 B2 157 158 Example 16 residues 240-249 of the PIP1A polypeptides of SEQID NO: 245, SEQID NO: 246, SEQID NO: 247, SEQID NO: 248, Multiple Residue Motif 4 PIP-1A Variants SEQID NO. 249, SEQID NO: 250, SEQID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID To further explore the role of motif 4 (amino acids 240 to NO: 255, SEQID NO: 256, SEQ ID NO: 257, SEQID NO: 258, SEQID NO: 259, SEQID NO: 260, SEQID NO: 261, 249 of PIP-1A (SEQ ID NO: 2), a series of variants were SEQID NO: 262, SEQID NO: 263, SEQID NO: 264, SEQ generated with multiple amino acid Substitutions in motif 4. ID NO. 265, SEQ ID NO. 266, SEQ ID NO: 267, SEQ ID The variants were generated using a similar mutagenesis NO: 268, and SEQ ID NO: 269, which are shown in Table strategy as described in Example 9 using the mutagenesis 16. The motif 4 amino acid substitutions compared to primer Motif 4-Comb-F CCGCTGGATTGGGATACTGTT 10 PIP-1A (SEQ ID NO: 2) are indicated in bold and under VWWNGCHAYDTTWTKDTKGRKNAYTWTNAYCCA lining. GGCAGC AATAGTGGGCACTTC (SEQ ID NO: 326) paired with primer 3188R GGATGTGCTGCAAGGCGAT TABL E 16 TAAG (SEQ ID NO: 327) and Comb-R AACAGTATC CCAATCCAGCGG (SEQ ID NO:328) paired with 3188F 15 of Soluble CAGACTGTCGATGAAGCCCTGAAAG (SEQ ID NO: Variant Amino acids seq. mutations expression 329). The mutagenesis primer Motif 4-Comb-F was PIP-1A ORNVLMENYN O Yes designed to be partially degenerate at residues 240-249 of (a.a. 240-249 PIP-1A (SEQ ID NO: 2) resulting in selected amino acid of SEQ ID NO: 2) substitutions at each residues. Table 15 shows the degenerate 20 1A2 NSYWLLDYYY 7 Yes codon encoding each of residues 240-249 and the possible SEQ ID (a.a. 240-249 resulting amino acids. In Table 15 the native amino acid is NO: 245 of SEQ ID NO: 245) indicated in bold and underlining. TABLE 1.5 Degenerate Residue codon Degeneracy Resulting amino acids* 24 O WWW W = A G OR C Gln, 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 Val, Ile and Phe

244 WTK W = A OR 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 T

248 TWT W = A OR Tyr and Phe 249 NAY N = G, A, T OR C Asn., Asp, Tyr and His Y = C OR T

The resulting polynucleotides encoding the PIP-1A vari- TABLE 16 - continued ant polypeptides were expressed as MBP fusions in E. coli and screened as cleared lysates in a 96 well format (3 plates) # of Soluble for Lygus insecticidal activity as described in Example 1 and 55 V.'" " '' . mutations expression scored for activity on a scale of 0 to 8 (see FIG. 4). The clones encoding the variant PIP-polypeptides having Lygus 1E3 NCYIFMEYYD 7 Yes insecticidal activity ranging from 4 to 8 were DNA SEQ ID (a.a. 240-249 sequenced (SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 246 of SEQ ID NO: 246) NO: 222, SEQID NO: 223, SEQID NO: 224, SEQ ID NO: 60 225, SEQID NO: 226, SEQID NO: 227, SEQID NO: 228, ID NYPEP. 7 Yes SEQID NO: 229, SEQID NO. 230, SEQID NO. 231, SEQ NO: 247 of SEO ID NO: 247) ID NO. 232, SEQ ID NO. 233, SEQ ID NO. 234, SEQ ID NO: 235, SEQID NO: 236, SEQID NO. 237, SEQID NO: 1B9 QCNVLFDNFH 5 Yes 238, SEQID NO: 239, SEQID NO: 240, SEQID NO: 241, 65 seo ID (a.a. 240-249 SEQID NO: 242, SEQID NO: 243, and SEQID NO: 244) NO: 248 of SEQ ID NO: 248) to determine the identity of the amino acid substitutions at US 9,688,730 B2 159 160 TABLE 16- continued TABLE 16- continued

of Soluble of Soluble Variant Amino acids seq. mutations expression Variant Amino acids seq. mutations expression 3F1 RCNVLMGDFD 6 Yes 1C10 QGYVLVDNFN 5 Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 NO: 264 of SEQ ID NO: 264) NO: 249 of SEQ ID NO: 249) 3F2 IGNVMVGDFD 8 Yes 1A11 NRYVFFGNYD 6 Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 10 NO: 265 of SEQ ID NO: 265) NO: 25 O of SEO ID NO: 25O) 3F6 QCYVLIENFH 5 Yes 2A2 QCNIMIGYFD 8 Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 NO: 266 of SEQ ID NO: 266) NO: 251 of SEQ ID NO: 251) 15 3F12 vcNVLMEHFY 5 Yes 2G1. QGNVLMENYN 1. Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 NO: 267 of SEQ ID NO: 267) NO: 252 of SEQ ID NO: 252) 3G7 VRNVFFDYFD 7 Yes 2C7 VSNILVGNFN 6 Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 NO: 268 of SEQ ID NO: 268) NO: 253 of SEO ID NO: 253) 3F4 WSYILFDNFH 8 Yes 2E1 NRHVLVDNFY 5 Yes SEQ ID (a.a. 240-249 SEO ID (a.a. 240-249 NO: 269 of SEQ ID NO: 269) NO: 254 of SEQ ID NO: 254)

2E12 WSNVLIDDFD 7 Yes 25 The clones encoding the variant PIP-1A polypeptides SEO ID (a.a. 240-249 having Lygus insecticidal activity ranging from 0 to 4 were NO: 255 of SEQ ID NO: 255) DNA sequenced (SEQID NO: 270, SEQID NO: 271, SEQ 2F4. VSHVMMEDYD 6 Yes ID NO. 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID SEO ID (a.a. 240-249 NO: 275, SEQID NO: 276, SEQ ID NO: 277, SEQID NO: NO: 256 of SEQ ID NO: 256) 30 278, SEQID NO: 279, SEQID NO: 280, SEQID NO. 281, SEQID NO: 282, SEQID NO: 283, SEQID NO: 284, SEQ 2F8 NSHILWGNYD 7 Yes ID NO: 285, SEQID NO: 286, SEQID NO: 287, SEQID SEO ID (a.a. 240-249 NO: 288, SEQID NO: 289, SEQ ID NO: 290, SEQID NO: NO: 257 of SEQ ID NO: 257) 291, SEQID NO. 292, SEQID NO: 293, SEQID NO: 294, 2G5 NSYVMIENFY 7 Yes 35 SEQID NO: 295, SEQID NO: 296, and SEQID NO:297), SEO ID (a.a. 240-249 to determine the identity of the amino acid substitutions at NO: 258 of SEO ID NO: 258) residues 240-249 of the PIP-1A polypeptides SEQ ID NO: 2G6 NCNIIMENYD 5 Yes 298, SEQID NO: 299, SEQID NO:300, SEQID NO:301, SEO ID (a.a. 240-249 SEQID NO:302, SEQID NO:303, SEQID NO:304, SEQ NO: 259 of SEO ID NO: 259) 40 ID NO:305, SEQ ID NO:306, SEQ ID NO: 307, SEQ ID 3A2 IRYIFIDNFD 8 Yes NO:308, SEQID NO:309, SEQ ID NO:310, SEQID NO: SEO ID (a.a. 240-249 311, SEQ ID NO:312, SEQID NO:313, SEQ ID NO:314, NO: 26 O of SEQ ID NO: 260) SEQID NO:315, SEQID NO:316, SEQID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO: 320, SEQ ID 3A1O VRNVLVENYH 3 Yes NO:321, SEQID NO:322, SEQ ID NO:323, SEQID NO: SEO ID (a.a. 240-249 45 324, and SEQ ID NO: 325, which are shown in Table 17. NO: 261 of SEQ ID NO: 261) Protein expression analysis by SDS-PAGE (data not shown) 3C7 QRYVLIDNFY 5 Yes revealed that the variant proteins with Lygus insecticidal SEO ID (a.a. 240-249 activity from 0 to 4 affect soluble expression (protein folding NO: 262 of SEQ ID NO: 262) 50 and solubility) in E. coli with the proteins accumulating as 3E3 LSHFMLGNFN 8 Yes insoluble fraction of the cleared lysate. The loss of activity SEO ID (a.a. 240-249 from the multiple substitutions in motif 4 appears to be from NO: 263 of SEQ ID NO: 263) the lack of soluble expressed proteins in the E. coli expres sion system. Motif 4 appears to be tolerant to multiple amino acid Substitution while remaining active. TABL E 17

of Soluble Variants Amino acids seq. mutations expression PIP-1A ORNVLMENYN (a.a. 240-249 O yes of SEQ ID NO: 2)

HsyWFDNYN (a.a. 240-249 6 No SEO ID NO: 298 of SEQ ID NO : 298)

VCNFFFGDFD (a.a. 240-249 9 No SEO ID NO : 299 of SEQ ID NO : 299)

US 9,688,730 B2

TABLE 1.7-continued

of Soluble Variants Amino acids seq. mutations expression 1E12 IGHFMLDYYH (a.a. 240-249 9 No SEO ID NO: 324 of SEQ ID NO: 324)

1G12 ICYVMVGNYH (a.a. 240-249 7 No SEO ID NO: 325 of SEO ID NO : 325)

Example 17 for 4 hours and then aligned within the 2.5 cm target Zone in preparation for bombardment. Identification of an Insecticidal Protein Active Against Lygus from Strain JH19887-2 15 Preparation of DNA A Blast search of a proprietary genomic contig library of A plasmid vector comprising a nucleotide sequence (e.g., a Pseudomonas Protegens strain JH19887-2 against the SEQID NO: 1) operably linked to an ubiquitin promoter is PIP-1 polynucleotide sequence of SEQID NO: 1 identified made. For example, a Suitable transformation vector com a polynucleotide of SEQID NO: 331, encoding a polypep prises a UBI1 promoter from Zea mays, a 5' UTR from UBI1 tide of SEQ ID NO: 332 (herein referred to as PIP-1C) and a UBI1 intron, in combination with a Pin terminator. having 82% sequence identity to PIP-1A (SEQ ID NO: 2). The vector additionally contains a PAT selectable marker Table 18 shows the % sequence identity between PIP-1C gene driven by a CAMV35S promoter and includes a (SEQ ID NO: 332) and PIP-1A (SEQ ID NO: 2), PIP-1B CAMV35S terminator. Optionally, the selectable marker can (SEQ ID NO: 4), and PSEEN3174 (SEQ ID NO: 6). FIG.5 25 reside on a separate plasmid. A DNA molecule comprising shows the sequence alignment of PIP-1A (SEQ ID NO: 2), a toxin nucleotide sequence as well as a PAT selectable PIP-1B (SEQ ID NO: 4), PIP-1C (SEQ ID NO: 332) and marker is precipitated onto 1.1 um (average diameter) tung PSEEN3174 (SEQ ID NO: 6). PIP-1C was expressed in E. Sten pellets using a CaCl2 precipitation procedure as follows: coli in the same way as PIP-1A. The purified PIP-1C was 100 uL prepared tungsten particles in water assayed against Soybean looper (SBL) and lygus in diet 30 10 uL (1 lug) DNA in Tris EDTA buffer (1 lug total DNA) based assays. PIP-1C (SEQ ID NO: 332) demonstrated 100 uL 2.5 M CaCl, killing activity against both SBL and lygus demonstrating 10 uL 0.1 M spermidine insecticidal spectrum similar to PIP-1A (SEQ ID NO: 2). Each reagent is added sequentially to a tungsten particle 35 suspension, while maintained on the multitube vortexer. The TABLE 1.8 final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipi PSEEN3174 PIP-1C PIP-1A PIP-1B SEQ ID SEQ ID tation period, the tubes are centrifuged briefly, liquid SEQ ID NO: 2 SEQ ID NO: 4 NO: 6 NO: 332 removed, washed with 500 mL 100% ethanol, and centri 40 fuged for 30 seconds. Again the liquid is removed, and 105 PIP-1A 93% 79% 82% PIP-1B 79% 84% uL 100% ethanol is added to the final tungsten particle PSEEN3174 80% pellet. For particle gun bombardment, the tungsten/DNA PIP-1C particles are briefly sonicated and 10 uI spotted onto the center of each macrocarrier and allowed to dry about 2 45 minutes before bombardment. Example 18 Particle Gun Treatment The sample plates are bombarded at level #4 in particle Transformation of Maize by Particle Bombardment gun iHE34-1 or iHE34-2. All samples receive a single shot and Regeneration of Transgenic Plants at 650 PSI, with a total often aliquots taken from each tube 50 of prepared particles/DNA. Immature maize embryos from greenhouse donor plants Subsequent Treatment are bombarded with a DNA molecule containing the toxin Following bombardment, the embryos are kept on 560Y nucleotide sequence (e.g., SEQ ID NO: 1) operably linked medium for 2 days, then transferred to 560R selection to an ubiquitin promoter and the selectable marker gene PAT medium containing 3 mg/liter Bialaphos, and Subcultured (Wohleben, et al., (1988) Gene 70: 25-37), which confers 55 every 2 weeks. After approximately 10 weeks of selection, resistance to the herbicide Bialaphos. Alternatively, the selection-resistant callus clones are transferred to 288J selectable marker gene is provided on a separate DNA medium to initiate plant regeneration. Following Somatic molecule. Transformation is performed as follows. Media embryo maturation (2-4 weeks), well-developed somatic recipes follow below. embryos are transferred to medium for germination and 60 transferred to the lighted culture room. Approximately 7-10 Preparation of Target Tissue days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets The ears are husked and surface sterilized in 30% CLO are well established. Plants are then transferred to inserts in ROXTM bleach plus 0.5% Micro detergent for 20 minutes, flats (equivalent to 2.5" pot) containing potting soil and and rinsed two times with sterile water. The immature 65 grown for 1 week in a growth chamber, Subsequently grown embryos are excised and placed embryo axis side down an additional 1-2 weeks in the greenhouse, then transferred (scutellum side up), 25 embryos per plate, on 560Y medium to classic 600 pots (1.6 gallon) and grown to maturity. Plants US 9,688,730 B2 165 166 are monitored and scored for expression of the toxin by mants (step 3: resting step). The immature embryos can be assays known in the art or as described above. cultured on solid medium with antibiotic, but without a Bombardment and Culture Media selecting agent, for elimination of Agrobacterium and for a Bombardment medium (560Y) comprises 4.0 g/L N6 resting phase for the infected cells. Next, inoculated basal salts (SIGMA C-1416), 1.0 mL/L Eriksson’s Vitamin 5 embryos are cultured on medium containing a selective Mix (1000xSIGMA-1511), 0.5 mg/L thiamine HCl, 120.0 agent and growing transformed callus is recovered (step 4: g/L Sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline (brought the selection step). The immature embryos are cultured on to volume with deionized HO following adjustment to pH Solid medium with a selective agent resulting in the selective 5.8 with KOH); 2.0 g/L GelriteTM (added after bringing to growth of transformed cells. The callus is then regenerated volume with d1 HO); and 8.5 mg/L silver nitrate (added 10 into plants (step 5: the regeneration step), and calli grown on after sterilizing the medium and cooling to room tempera selective medium can be cultured on solid medium to ture). Selection medium (560R) comprises 4.0 g/L N6 basal 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 H2O 15 following adjustment to pH 5.8 with KOH); 3.0 g/L Gel Transformation of Soybean Embryos riteTM (added after bringing to volume with dl H2O); and 0.85 mg/L silver nitrate and 3.0 mg/L Bialaphos (both added Soybean embryos are bombarded with a plasmid contain after sterilizing the medium and cooling to room tempera ing a nucleotide sequence (e.g., SEQ ID NO: 1) operably ture). linked to a pin I promoter as follows. To induce Somatic Plant regeneration medium (288J) comprises 4.3 g/L MS embryos, cotyledons, 3-5 mm in length dissected from salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock Surface-sterilized, immature seeds of an appropriate Soybean solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0.10 cultivar are cultured in the light or dark at 26° C. on an g/L pyridoxine HCl, and 0.40 g/L Glycine brought to volume appropriate agar medium for six to ten weeks. Somatic with polished D-1HO) (Murashige and Skoog, (1962) 25 embryos producing secondary embryos are then excised and Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L placed into a Suitable liquid medium. After repeated selec Zeatin, 60 g/L Sucrose, and 1.0 mL/L of 0.1 mM abscisic acid tion for clusters of Somatic embryos that multiplied as early, (brought to volume with polished d1 HO after adjusting to globular-staged embryos, the Suspensions are maintained as pH 5.6): 3.0 g/L GelriteTM (added after bringing to volume described below. with dl HO); and 1.0 mg/L indoleacetic acid and 3.0 mg/L 30 Soybean embryogenic suspension cultures can be main Bialaphos (added after Sterilizing the medium and cooling to tained in 35 mL liquid media on a rotary shaker, 150 rpm, 60 C). at 26° C. with florescent lights on a 16:8 hour day/night Hormone-free medium (272V) comprises 4.3 g/L MS schedule. Cultures are subcultured every two weeks by salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock inoculating approximately 35 mg of tissue into 35 mL of solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, 35 liquid medium. 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to Soybean embryogenic Suspension cultures may then be volume with polished d1 HO), 0.1 g/L myo-inositol, and transformed by the method of particle gun bombardment 40.0 g/L sucrose (brought to volume with polished dl HO (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Pat. after adjusting pH to 5.6); and 6 g/L Bacto-agar (added after No. 4,945,050). A Du Pont Biolistic PDS1000/HE instru bringing to volume with polished d1 HO), sterilized and 40 ment (helium retrofit) can be used for these transformations. cooled to 60° C. A selectable marker gene that can be used to facilitate Soybean transformation is a transgene composed of the 35S Example 19 promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), the hygromycin phosphotrans Agrobacterium-Mediated Transformation of Maize 45 ferase gene from plasmid plR225 (from E. coli; Gritz, et al., and Regeneration of Transgenic Plants (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agro For Agrobacterium-mediated transformation of maize bacterium tumefaciens. The expression cassette comprising with a toxin nucleotide sequence (e.g., SEQ ID NO: 1), the a toxin nucleotide sequence (e.g., SEQ ID NO: 1) operably method of Zhao can be used (U.S. Pat. No. 5,981,840 and 50 linked to the pin II promoter can be isolated as a restriction PCT Patent Publication Number WO 1998/32326; the con fragment. This fragment can then be inserted into a unique tents of which are hereby incorporated by reference). restriction site of the vector carrying the marker gene. Briefly, immature embryos are isolated from maize and the To 50 LL of a 60 mg/mL 1 Lim gold particle Suspension is embryos contacted with a suspension of Agrobacterium added (in order): 5 LL DNA (1 lug/uL), 20 uL spermidine under conditions whereby the bacteria are capable of trans 55 (0.1M), and 50 uL CaCl (2.5M). The particle preparation is ferring the nucleotide sequence (e.g. SEQ ID NO: 1) to at then agitated for three minutes, spun in a microfuge for 10 least one cell of at least one of the immature embryos (step seconds and the supernatant removed. The DNA-coated 1: the infection step). In this step the immature embryos can particles are then washed once in 400 uL 70% ethanol and be immersed in an Agrobacterium suspension for the initia resuspended in 40 uL of anhydrous ethanol. The DNA/ tion of inoculation. The embryos are co-cultured for a time 60 particle Suspension can be Sonicated three times for one with the Agrobacterium (step 2: the co-cultivation step). The second each. Five microliters of the DNA-coated gold immature embryos can be cultured on solid medium follow particles are then loaded on each macro carrier disk. ing the infection step. Following this co-cultivation period Approximately 300-400 mg of a two-week-old suspen an optional “resting step is contemplated. In this resting sion culture is placed in an empty 60x15 mm petri dish and step, the embryos are incubated in the presence of at least 65 the residual liquid removed from the tissue with a pipette. one antibiotic known to inhibit the growth of Agrobacterium For each transformation experiment, approximately 5-10 without the addition of a selective agent for plant transfor plates of tissue are normally bombarded. Membrane rupture US 9,688,730 B2 167 168 pressure is set at 1100 psi, and the chamber is evacuated to embodiments of, and examples for, the invention are a vacuum of 28 inches mercury. The tissue is placed approxi described herein for illustrative purposes, various equivalent mately 3.5 inches away from the retaining screen and modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The bombarded three times. Following bombardment, the tissue teachings provided herein of the invention can be applied to can be divided in half and placed back into liquid and other purposes, other than the examples described above. cultured as described above. The invention may be practiced in ways other than those Five to seven days post bombardment the liquid media particularly described in the foregoing description and may be exchanged with fresh media, and eleven to twelve examples. Numerous modifications and variations of the days post-bombardment with fresh media containing 50 invention are possible in light of the above teachings and, mg/mL hygromycin. This selective media can be refreshed 10 therefore, are within- 0 the scope of the appended claims. weekly. Seven to eight weeks post-bombardment, green, These and other changes may be made to the invention in transformed tissue may be observed growing from untrans- light of the above detailed description. In general, in the formed, necrotic embryogenic clusters. Isolated green tissue following claims, the terms used should not be construed to is removed and inoculated into individual flasks to generate 15 limit the invention to the specific embodiments disclosed in new, clonally propagated, transformed embryogenic Suspen- the specification and the claims. sion cultures. Each new line may be treated as an indepen- Certain teachings related to PIP polynucleotides and poly dent transformation event. These suspensions can then be peptides were disclosed in U.S. Provisional patent applica Subcultured and maintained as clusters of immature embryos tion No. 61/667,039, filed Jul. 2, 2012, the disclosure of or regenerated into whole plants by maturation and germi- 2O which is herein incorporated by reference in its entirety. nation of individual Somatic embryos. The entire disclosure of each document cited (including All publications and patent applications mentioned in the patents, patent applications, journal articles, abstracts, specification are indicative of the level of skill of those manuals, books, or other disclosures) in the Background of skilled in the art to which this disclosure pertains. All the Invention, Detailed Description, and Examples is herein publications and patent applications are herein incorporated 25 incorporated by reference in their entireties. by reference to the same extent as if each individual publi- The above examples are put forth so as to provide those cation or patent application was specifically and individually of ordinary skill in the art with a complete disclosure and indicated to be incorporated by reference. description of how to make and use the Subject invention, Although the foregoing disclosure has been described in and are not intended to limit the scope of what is regarded Some detail by way of illustration and example for purposes so as the invention. Efforts have been made to ensure accuracy of clarity of understanding, it will be obvious that certain with respect to the numbers used (e.g. amounts, temperature, changes and modifications may be practiced within the concentrations, etc.) but some experimental errors and Scope of the appended claims. deviations should be allowed for. Unless otherwise indi The above description of various illustrated embodiments cated, parts are parts by weight, molecular weight is average of the invention is not intended to be exhaustive or to limit molecular weight; temperature is in degrees centigrade; and the invention to the precise form disclosed. While specific pressure is at or near atmospheric.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 332

<21 Os SEQ ID NO 1 &211s LENGTH: 816 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas chlororaphis <4 OOs SEQUENCE: 1 atgc.cgatca aggaagagct gagc.ca.gc.ct caaagt catt catcgaact tacgacctg 60

aaaagtgagc aaggaagttct cogcogcc.gct ttgacatcca actittgctgg caactt.cgat 12O cagttcc caa ctaag.cgtgg togctttgcg atcgacagct acctgctgga ttacagcgcg 18O

Cccaa.gcaag gCtgctgggt agatggcatt accgt.ctacg gtgacatctt tatcggcaag 24 O

cagaattggg gcaccitacac togc.ccggit c tittgcct acc tdcagtacat ggacac catt 3 OO

to catt.ccgc agcaggtgac acagact cqc agct atcagt tacta aggg acacaccalaa 360 acgttcacga ccalatgtcag cqccaaatac agcgttggag gtag tattga catcgt caac 42O

gtcggttcgg at atctoraat tigatt Cagt aa.ca.gtgaat Cotggit ctac tacgcagacg 48O ttcagdaata gcact caatt gactggit cot ggcacct tca togtgitatica ggttgttatg 54 O

gtt tatgcgc acaacgc.cac ttctg.cgggc aggcagaatg gtaatgcctt cqcctacaac 6 OO aagaccalata citgtcggctic goggctggac ttgtactatt tdtctgc.cat cact cagaac 660

agtacggtca ttgtcgatt C cagdaaggcc atcgc.gc.cgc tiggattggga tactgttcag 72O US 9,688,730 B2 169 170 - Continued 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 Phe Ser Asn Ser Thr Gln Lieu. Thr Gly Pro Gly Thr Phe Ile Val Tyr 1.65 17O 17s Glin Val Val Met 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 Asn 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 Asp 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 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 3 &211s LENGTH: 816 &212s. TYPE: DNA <213> ORGANISM: Dendroctonus frontalis

<4 OOs, SEQUENCE: 3 atgaccatca aggaggagct galaccagc.cg cagagccata gcatcgagct ggacgacctg 6 O alacagcgagc agggcaacgc cc.gc.gc.catc Ctgaccagda actitcgc.cgg cagct tcgac 12 O

Cagttc.ccga C caa.gc.gcgg C9gctacgcc atcgacagot acctgctgga ctacagcgc.c 18O ccgaag Cagg gctgctgggt ggacggcatc accgtgtacg gcgacat citt catcggcaa.g 24 O US 9,688,730 B2 171 172 - Continued

Cagaactggg gcacct acac cc.gc.ccggtg titcgcct acc tgcagtacat gga caccatc agcatc.ccgc agcaggtgac ccagaccc.gc agctaccagc tgacCaaggg c cataccaag 360 acct tcacca Ccagcgtgac cgc.caagtac agcgtgggcg gcago atcgg catcgtgaac gtgggcagcg a catcagogt gggct tcagc agcagcgaga gctggagcac cacccagacc ttcagcgaga gcacccagct ggc.cggc.ccg ggcacct tca tcqtgtacca ggtggtgctg 54 O gtgtacgc.cc ataacgc.cac Cagcgc.cggc cgc.ca.gaacg gcaacgc.ctt cgc.ctacaac alagacccaga CC9tgggcag cc.gc.ctggac ctgtact acc tgagcgc.cat cacccagaac 660 agcaccgtga tCgtggaga.g Cagcaaggcc atcgc.ccc.gc tggactggga Caccgtgcag 72 O cgcaacgtgc tgatggagaa ctaca accc.g agcagcaa.ca gcggc cattt cagctitcgac tggagcgc.ct acaacgaccc gcatcgc.cgc tactaa 816

<210s, SEQ I D NO 4 &211s LENGT H: 271 212. TYPE : PRT <213> ORGANISM: Dendroctonus frontalis

<4 OOs, SEQUENCE: 4 Met Thir Ile Lys Glu Glu Lieu. Asn Gln Pro Glin Ser His Ser Ile Glu 1. 5 1O 15

Lieu. Asp Asp Lieu. Asn. Ser Glu Glin Gly Asn Ala Arg Ala Ile Lieu. Thir 25 3O

Ser Asn. Phe Ala Gly Ser Phe Asp Glin Phe Pro Thir Lys Arg Gly Gly 35 4 O 45

Tyr Ala Ile Asp Ser Tyr Lieu. Lieu. Asp Tyr Ser Ala Pro Llys Gln Gly SO 55 6 O

Cys Trp Val Asp Gly Ile Thr Val Ile Phe Ile Gly Lys 65 8O

Glin Asn Trp Gly Thr Tyr Thr Arg Pro Wall Phe Ala Tyr Lieu Gln Tyr 85 90 95

Met Asp Thr Ile Ser Ile Pro Glin Glin Wall. Thir Gln Thr Arg Ser Tyr 105 11 O

Glin Lieu. Thir Lys Gly His Thr Lys Thir Phe Thir Thir Ser Wall Thir Ala 115 12 O 125 Lys Tyr Ser Val Gly Gly Ser Ile Gly Ile Val Asn Val Gly Ser Asp 13 O 135 14 O

Ile Ser Wall Gly Phe Ser Ser Ser Glu Ser Trp Ser Thir Thr Gn. Thir 145 150 155 160

Phe Ser Glu Ser Thr Glin Leu Ala Gly Pro Gly Thir Phe Ile Val Tyr 1.65 17O 17s

Glin Wal Wall Lieu Val Tyr Ala His Asn Ala Thr Ser Ala Gly Arg Glin 18O 185 19 O Asn Gly Asn Ala Phe Ala Tyr Asn Lys Thr Glin Thr Val Gly Ser Arg 195 2O5

Lieu. Asp Lieu. Tyr Tyr Lieu Ser Ala Ile Thr Glin Asn. Ser Thr Wall Ile 21 O 215 22O

Wall Glu Ser Ser Lys Ala Ile Ala Pro Lieu. Asp Trp Asp Thr Wall Glin 225 23 O 235 24 O

Arg Asn. Wall Leu 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 26 O 265 27 O US 9,688,730 B2 173 174 - Continued

<210s, SEQ ID NO 5 &211s LENGTH: 816 212. TYPE : DNA <213> ORGANISM: Pseudomonas entomophila <4 OOs, SEQUENCE: 5 atgacgat.ca aggaagagct gggc.ca.gc.ct caaagcc att cgatcgaact ggacgaggtg 6 O agcaaggagg cc.gcaagtac gC9ggcc.gc.g ttgact tcca acctgtctgg cc.gctitcgac 12 O

Cagtaccc.ga cCaagaaggg cgactittgcg atcgatggitt atttgctgga ctacagotca 18O

Cccaa.gcaag gttgctgggit ggacggitatic actgtctato gcqatat cta catcggcaa.g 24 O

Cagaactggg gcacttatac cc.gc.ccggtg tittgcct acc tacagtatgt ggaalaccatc 3OO to Catt CCaC agaatgtgac gaccaccctic agctato agc tgacCaaggg gcatacccgt. 360 t cct tcgaga c cagtgtcaa cgc.caagtac agcgttggcg c caacataga tat cqtcaac gtgggttcgg agattt coac cgggtttacc cgcagcgagt cctggtc. cac cacgcagtcg ttcaccgata ccaccgagat galaggggc.ca gggacgttcg to atttacca ggtC9tgctg 54 O gtgtatgcgc acaacgc.cac CtcggCaggg cggcagaatg c caatgc citt cgc.ctacagc aaaacc cagg Cagtgggctic gC9ggtggac ttgtact act tgtcggc cat tacccagcgc 660 aagcgggt ca tcqttic cqtc gag caatgcc gtcacgc.cgc tggactggga tacggtgcaa. 72 O cgcaacgtgc tgatggaaaa Ctaca accoa. ggcagta aca gcgga cactt cagctitcgac tggagtgc ct acaacgatcc t catcgc.cgt. tattga 816

<210s, SEQ ID NO 6 &211s LENGTH: 271 212. TYPE PRT <213s 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 Thir Arg Ala Ala Lieu. Thir 25

Ser Asn Lieu. Ser Gly Arg Phe Asp Glin Tyr Pro Thir Lys Llys Gly Asp 35 4 O 45

Phe Ala Ile Asp Gly Tyr Lieu. Lieu. Asp Ser Ser Pro Llys Gln Gly SO 55 6 O

Cys Trp Wall Asp Gly Ile Thir Wall Gly Asp Ile Ile Gly 65 70

Glin Asn Trp Gly Thir Tyr Thir Arg Pro Wall Phe Ala Luell Glin Tyr 85 90 95

Wall Glu Thir Ile Ser Ile Pro Glin Asn Wall Thir Thir Thir Luell Ser 105 11 O

Glin Luell Thir Gly His Thir Arg Ser Phe Glu Thir Ser Wall Asn Ala 115 12 O 125

Tyr Ser Wall Gly Ala Asn Ile Asp Ile Wall Asn Wall Gly Ser Glu 13 O 135 14 O

Ile Ser Thir Gly Phe Thir Arg Ser Glu Ser Trp Ser Thir Thir Glin Ser 145 150 155 160

Phe Thir Asp Thir Thir Glu Met Gly Pro Gly Thir Phe Wall Ile 1.65 17O 17s

Glin Wall Wall Luell Wall Tyr Ala His Asn Ala Thir Ser Ala Gly Arg Glin 18O 185 19 O US 9,688,730 B2 175 176 - Continued

Asn Ala Asn Ala Phe Ala Tyr Ser Lys Thr Glin Ala Val Gly Ser Arg 195

Val Asp Lieu. Tyr Tyr Lieu. Ser Ala Ile Thr Glin Arg Lys Arg Wall Ile 21 O 215 22O

Wall Pro Ser Ser Asn Ala Wall. Thir Pro Lieu. Asp Trp Asp Thr Wall Glin 225 23 O 235 24 O

Arg Asn. Wall Leu 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 26 O 265 27 O

<210s, SEQ I D NO 7 &211s LENGT H: 720 212. TYPE : DNA <213> ORGANISM: Pseudomonas puti da <4 OO > SEQUENCE: 7 atgacaggct tcgagcgttt gtcacccgat gcgttcc.ccg ttittaaacgg tt catacctg 6 O attgaaaggt acctgcticag cacggacgag titt catcctg gatgttggat agalaggtgaa 12 O accotctacg gtgggtttgg gtttcctt.ca ggaaaaaaga agg tattgac cc.gc.ccggitt 18O titcgcc tact tcqactacgt. gggcaccitat aaaac attaa. gtgctggaga ctgtgaaatt 24 O gatctgtc.cc gtgc.ca.gtgg gcatgaggto tggitttgcac atgatgc.cga aggcttittct 3OO gcgc.cgagtg gaattgggct ggtaa.gcgta aagtcagatc tgctict cc.gg ctgct ctdcc 360 gaagagtggC ggc.cgittatc atcggttggg Catac Citgc gC9tagcggg agctgaatgc tatgtggc ct accagttgaa actggtctat gcgcattggg taaaacaggg cgatgcc.ca.g tgct Ctgagc tgttcaaggt acagc.ccgtg cgtgtgcaag gcgacaacaa aggcgttitt C 54 O ttcott tott cc.gtggccac agacctgatg tggg taggac atggttcgga talacaccalaa. gcqc caat at cacgacaggc gttatat cac citgat attca atc.ttgctta tgg.cgcagog 660

gctggagttt taatgat cag accoct tcct gcaat attga 72 O

<210s, SEQ I D NO 8 &211s LENGT H: 239 212. TYPE : PRT <213> ORGANISM: Pseudomonas puti da <4 OOs, SEQUENCE: 8

Met Thr Gly Phe Glu Arg Lieu. Ser Pro Asp Ala Phe Pro Wall Luell Asn 1. 5 1O 15

Gly Ser Tyr Lieu. Ile Glu Arg Tyr Lieu. Luell Ser Thir Asp Glu Phe His 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 Llys Llys Llys Val Lieu Thr Arg Pro Wall Phe Ala Tyr Phe SO 55 6 O

Asp Tyr Val Gly Thr Tyr Lys 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. Wall Ser Wall Llys Ser 105 11 O

Asp Lieu. Lieu. Ser Gly Cys Ser Ala Glu Glu Trp Arg Pro Lieu Ser Ser 115 12 O 125 US 9,688,730 B2 177 178 - Continued

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 Val 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 Wall Ala Thr Asp Lieu Met Trp Val 18O 185 19 O

Gly His Gly Ser Asp Asn. Thir Lys Ala Pro Ile Ser Arg Glin Ala Lieu 195 2O5

Tyr His Leu Ile Phe Asn Lieu Ala Tyr Gly Ala Ala Gly Asp Ala Gly 21 O 215 22O

Trp Ser Phe Asn Asp Glin Ala Ala Ser Asn Arg Phe Leul Glin 225 23 O 235

<210s, SEQ I D NO 9 &211s LENGT H: 729 212. TYPE : DNA <213> ORGANISM: Pseudomonas puti da <4 OOs, SEQUENCE: 9 atgaggltd at attcaatgag cgatttgatg aatgaaatca gcc.gg taccC Cctgaaaaga 6 O gggit ctitt.cg aaatcgagca gtacctgata ggtgat cagt tgcatgc.cgg ttgctgggtg 12 O gatgc.cgata ccaccitacgg tgatgtcagg tgtggtaact atgactgggc cacttacacg 18O

CggC Cagt ct ttgctitat ct gcaa.catgtg gcc acgatac gatcCaatgt gcaaacggaa 24 O cacgagcgcg aagtgg tagt ttgcgagggc ttcagcaaga gtttct coca agg.cgt.cgag 3OO tttalagg tag gctitctctgc tgactitcggc ccggcgaatg cgaacgctga act cact gcc 360 atgtttitcgg tittctgaaac ggtgagcggt tcggagt caa c caag.cgctic attgaga.gtg aagggcgatg ggaccatcat ggtgt at Caa gtgcacatgg tctacgc.gca ccacatgaca tcc.gctggcg tgcttgctgg atacgtaccc tataccalaga gct cagatat attcaatgac 54 O gatggg.cggc tggtgcggca gga catcacg atgctitt cat cggtggtttg cgatcagctt gttc.cggit Ca ggaatgaaaa atcaataaag cct ctacct ggagt Caggit talaccaa.gc.g 660 gtc.ttgttca atcaatttga gaaag.cgc.ca ggtgc.ca.gac gctggactitt tgatttctic g 72 O gtattittga 729

<210s, SEQ I D NO 10 &211s LENGT H: 242 212. TYPE : PRT &213s ORGAN ISM: Pseudomonas puti da <4 OOs, SEQUENCE: 10

Met Arg Ser Tyr Ser Met Ser Asp Luell Met Asn Glu Ile Ser Arg Tyr 1. 5 1O 15

Pro Lieu Lys Arg Gly Ser Phe Glu Ile Glu Glin Tyr Lieu. Ile Gly Asp 25 3O

Glin Lieu. His Ala Gly Cys Trp Val Asp Ala Asp Thr Thr Tyr Gly Asp 35 4 O 45

Val Arg Cys Gly Asn Tyr Asp Trp Ala Thr Tyr Thr Arg Pro Wall Phe SO 55 6 O

Ala Tyr Lieu. Gln His Wall Ala Thr Ile Arg Ser Asn. Wall Glin Thr Glu 65 70 7s 8O US 9,688,730 B2 179 180 - Continued

His Glu Arg Glu Val Val Val Cys Glu Gly Phe Ser Lys Ser Phe Ser 85 90 95

Gln Gly Val Glu Phe Llys Val Gly Phe Ser Ala Asp Phe Gly Pro Ala 105 11 O

Asn Ala Asn Ala Glu Lieu. Thir Ala Met Phe Ser Wall Ser Glu Thir Wall 115 12 O 125

Ser Gly Ser Glu Ser Thr Lys Arg Ser Luell Arg Val Lys Gly Asp Gly 135 14 O

Thir Ile Met Val Tyr Glin Val His Met Wall Tyr Ala His His Met Thr 145 150 155 160

Ser Ala Gly Val Lieu Ala Gly Tyr Wall Pro Thr Lys Ser Ser Asp 1.65 17O 17s

Ile Phe Asn Asp Asp Gly Arg Lieu. Wall Arg Glin Asp Ile Thr Met Lieu. 18O 185 19 O

Ser Ser Wall Val Cys Asp Glin Lieu. Wall Pro Wall Arg Asn. Glu Llys Ser 195 2O5

Ile Llys Pro Lieu. Thir Trp Ser Glin Wall Asn Glin Ala Wall Lieu. Phe Asn 21 O 215 22O

Glin Phe Glu Lys Ala Pro Gly Ala Arg Arg Trp Thr Phe Asp Phe Ser 225 23 O 235 24 O

Wall Phe

<210s, SEQ I D NO 11 &211s LENGT H: 771 212. TYPE : DNA <213> ORGANISM: Acromyrmex echinatior <4 OOs, SEQUENCE: 11 atgtcagtaa accotcaatg cCaggagcgt. gcagtgaata taatcgatag caaagttgtt 6 O gaggagatta gttatatgcc ggagaalacac gg tagttacg aaattgataa ctatttgctic 12 O ggcgaalaccg ggaagttctgct taatc.ccggc tgctggg tac gaggcgggac catatatggg 18O gacatgtgga tctggalacca galactgggga acctacagcg taccggtgtt tgcctacctt 24 O gaacatgtgc agacggttcg tataccaaac gcgaccalaat acact cacgc cgttgaggitt 3OO acggalagggit t cagotcatc tgttacccaa actitcagagg tcgagctgtc. tgtaggcggc 360 ggatt.cgtgg cgctaggcgc tggaggggtg aagct ct cita gcagttatac cgaaggcgtt

Catggat.cga acaagcgitat ggaga cattt gagatticagg ggc.cggggat ttata actt C tat caaatgc acatggittitt tgcgcacaag gctacatctg caggc catct gaatgagctg 54 O titccagtatt Cccaagtggc cacgaatgaa agcgggcggg aggatttgtg titt cott cacci tctatagdaa ctgacactgt cgt.gc.cggtc gcggc.cgatt citt cataac gcc actgggit 660 tggcatgaga tccalaagggc tgtgctgatg gacaattaca aggct tcgga Caatagtggc 72 O

Cactggctgt tcc attctag cgcataccat cggc.ccggitt cgc.gctattg a. 771

<210s, SEQ I D NO 12 &211s LENGT H: 255 212. TYPE : PRT <213> ORGANISM: Acromyrmex echinatior

<4 OOs, SEQUENCE: 12 Met Ser Val Asn Arg Glin Cys Glin Glu Arg Ala Val Asn. Ile Ile Asp 1. 5 15 Ser Llys Val Glu Glin Ile Ser Tyr Met Pro Glu Lys His Gly Ser Tyr 25 US 9,688,730 B2 181 182 - Continued

Glu Ile Asp Asn Tyr Lell Lieu. Gly Glu Thr Gly Llys Ser Lieu. Asn Pro 35 4 O 45

Gly Cys Trp Wall Arg Gly Gly. Thir Ile Tyr Gly Asp Met Trp Ile Trp SO 55 6 O

Asn Glin Asn Trp Gly Thir Tyr Ser Val Pro Val Phe Ala Tyr Lieu Glu 65 70 7s

His Wall Glin Thir Wall Arg Ile Pro Asn Ala Thr Lys Tyr Thr His Ala 85 90 95

Wall Glu Wall Thir Glu Gly Phe Ser Ser Ser Wall. Thir Glin. Thir Ser Glu 105 11 O

Wall Glu Luell Ser Wall Gly Gly Gly Phe Val Ala Lieu. Gly Ala Gly Gly 115 12 O 125

Wall Lys Luell Ser Ser Ser Tyr Thr Glu Gly Val His Gly Ser Asn 13 O 135 14 O

Arg Met Glu Thir Phe Glu Ile Glin Gly Pro Gly Ile Tyr Asn Phe Tyr 145 150 155 160

Glin Met His Met Wall Phe Ala His Lys Ala Thir Ser Ala Gly His Luell 1.65 17O 17s

Asn Glu Luell Phe Glin Tyr Ser Glin Val Ala Thr Asn Glu Ser Gly Arg 18O 185 19 O

Glu Asp Luell Phe Lell Thir Ser Ile Ala Thr Asp Thr Val Val Pro 195 2OO 2O5

Wall Ala Ala Asp Ser Ser Ile Thr Pro Leu Gly Trp His Glu Ile Glin 21 O 215 22O

Arg Ala Wall Luell Met Asp Asn Tyr Lys Ala Ser Asp Asn. Ser Gly His 225 23 O 235 24 O

Trp Luell Phe His Ser Ser Ala Tyr His Arg Pro Gly Ser Arg Tyr 245 250 255

SEQ ID NO 13 LENGTH: 27 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: PCR primer

<4 OOs, SEQUENCE: 13 atacatatga catcaagga agagctg 27

SEQ ID NO 14 LENGTH: 36 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: PCR primer

SEQUENCE: 14 ttggat.cctic aataacggcg atgaggat.cg ttgtag 36

SEO ID NO 15 LENGTH: 816 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: modified codons

SEQUENCE: 15 atgcct at ca aggaagagct gagcc agcct caaagccatt catcgaact ggacgatctg 6 O