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(12) Patent Application Publication (10) Pub. No.: US 2014/0007292 A1 Cerf Et Al US 2014.0007292A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0007292 A1 Cerf et al. (43) Pub. Date: Jan. 2, 2014 (54) NOVEL INSECTICIDAL PROTEINS AND Publication Classification METHODS FOR THEIR USE (51) Int. Cl. (71) Applicant: PIONEER HIBRED C07K I4/2 (2006.01) INTERNATIONAL INC, Johnston, IA GOIN33/68 (2006.01) (US) C07K 6/2 (2006.01) AOIN 43/50 (2006.01) (72) Inventors: David C. Cerf, Palo Alto, CA (US); A6II 45/06 (2006.01) James J. English, San Ramon, CA (US); (52) U.S. Cl. Carol A. Hendrick, Des Moines, IA CPC ................ C07K 14/21 (2013.01); A0IN 43/50 (US); Lu Liu, Palo Alto, CA (US); (2013.01); A61K 45/06 (2013.01); C07K Jarred K. Oral, San Carlos, CA (US); I6/1214 (2013.01); G0IN33/68 (2013.01) Philip A. Patten, Menlo Park, CA (US); USPC ....... 800/279; 536/23.7:435/320.1; 530/350; Barbara A. Rosen, Mountain View, CA 435/252.3; 435/252.34; 435/252.35:435/252.2: (US); Ute Schellenberger, Palo Alto, 435/254.11: 435/254.2:435/254.21; CA (US); Ingrid A. Udranszky, 435/252.33:435/252.31; 514/4.5; 424/94.6; Mountain View, CA (US); Jun-Zhi Wei, 424/94.61; 530/387.9; 800/302:436/501 Palo Alto, CA (US); Genhai Zhu, San (57) ABSTRACT Jose, CA (US) Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, (73) Assignee: Pioneer Hi Bred International Inc, the nucleic acid sequences are useful for preparing plants and Johnston, IA (US) microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and (21) Appl. No.: 13/792,861 seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for Subsequent trans (22) Filed: Mar 11, 2013 formation into organisms of interest, as probes for the isola tion of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling, inhibiting Related U.S. Application Data growth or killing lepidopteran, coleopteran, dipteran, fungal, (60) Provisional application No. 61/667,039, filed on Jul. 2, hemipteran, and nematode pest populations and for produc 2012. ing compositions with insecticidal activity. Patent Application Publication Jan. 2, 2014 Sheet 1 of 7 US 2014/0007292 A1 PIP-1A (1) PSEEN3174 (1) SK PIP-1B (1) S AECFG-592740 (1) NRocoERAVNIIDSKVEor Pput 1063 (1) Pput 1064 (1) BBBBBBBBBBBBBBBBB BBBBBBBBBBBB 100 PIP-1A (51) PSEEN3174 (51) PIP-1B (51) AECFG-592740 (34) Pput 1063 (21) Pput 1064 (25) DTTYGERCNYD PIP-1A (99) PSEEN3174 (99) PIP-1B (99) AECFG-592740 (84) TSR TKYEH, Pput 1063 (69) TYKTLSAGDCEIDLSRASG Pput 1064 (72) BBBBBBBBBBB 151 200 PIP-1A (149) PSEEN3174 (149) PIP-1B (149) AECFG-592740 (133) Pput 1063 (118) Pput 1064 (122) 201 250 PIP-1A (199) :--& PSEEN3174 (199) PIP-1B (199) AECFG-592740 (182) Pput 1063 (167) Pput 1064 (171) BBBBBBBB BBBBBBBB 251 PIP-1A (243) PSEEN3174 (243) PIP-1B (243) AECFG-592740 (227) Pput 1063 (211) FNLAYGSGDSGGSFNDQ&ASNRFLQ s Pput 1064 (221) FNSEKSPGRREFSV------- Patent Application Publication Jan. 2, 2014 Sheet 2 of 7 US 2014/0007292 A1 Fig. 2 Saturated mutagenesis 3.188 Reverse primer -> -H --> st Sphi Forwar primer BamHI 3188R PCR using 3188FI3188R to recover full length and clone to expression vector Transform to E. Coli host Pick 96 or more colonies for sequencing to recover variants Patent Application Publication Jan. 2, 2014 Sheet 4 of 7 US 2014/0007292 A1 Fig. 3B 66 O CGTTAAGTTG GA CGTGAAAT CCCCGGGCTC AACCTGGGAA. CTGCATCCAA AACTGGCGAG CGTTAAGTTG GA CGTGAAAG CCCCGGGCTC AACCTGGGAA CTGCATCCAA AACTGGCGAG 72 O CTAGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG CT AGAGTATG GTAGAGGGTG GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG 78O AAGGAACACC AG GGCGAAG GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC AAGGAACACC AG GGCGAAG GCGACCACCT GGACTGATAC GACACTGAG GTGCGAAAGC 8 AO GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC GTGGGGAGCA AACAGGATTA. GATACCCTGG TAGTCCACGC CGTAAA CGAT GTCAACTAGC 9 OO CGTTGGGAGC CT GAGCTCT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG CGTTGGAATC CT GAGATT TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG 96.O TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT TACGGCCGCA AGGTTAAAAC TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT 1 O2 O GTGGTTTAAT CGAAGCAAC GCGAAGAACC TTACCAGGCC PTGACATCCA ATGAACTTTC GTGGTTTAAT CGAAGCAAC GCGAAGAACC TTACCAGGCC TGACATGCA GAGAACTTTC 108 O CAGAGATGGA TTGGTGCCTT CGGGAACATT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CAGAGATGGA TTGGTGCCTT CGGGAACTCT GACACAGGTG CTGCATGGCT GTCGTCAGCT 114 O CGTGTCGTGA GA GTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA CGTGTCGTGA GAT GTTGGGT TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA 12 OO GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GCACGTTATG GTGGGCACTC TAAG GAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT Patent Application Publication Jan. 2, 2014 Sheet 5 of 7 US 2014/0007292 A1 Fig. 3C 126 O GACGTCAAGT CATCATGGCC CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC GACGTCAAGT CATCATGGCC CTTACCCCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC 1320 AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA. ATCCCATAAA ACCGATCGTA. GTCCGGATCG AGAGGGTTGC CAAGCCGCGA GGTGGAGCTA ATCTCACAAA ACCGATCGTA GTCCGGATCG 138O CAGTCTGCAA CTCGACTGCG TGAAGTCGGA. ATCGCTAGTA ATCGCGAATC AGAATGTCGC CAGTCTGCAA CTCGACTGCG TGAAGTCGGA ATCGCTAGTA ATCGCAAATC AGAATGTTGC 144 O GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC GGTGAATACG TTCCCGGGCC TTGTACACAC CGCCCGT CAC ACCATGGGAG TGGGTTGCAC 15 OO CAGAAGTAGC TAGTCTAACC TTCGGGAGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG CAGAAGTAGC TAGTCTAACC TTCGGGGGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG 15 6.O TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GATCAC CTCC TTAATCGACG TGAAGTCGTA ACAAGGTAGC CGTAGGGGAA CCTGCGGCTG GAT CACCTCC TT 162O ACATCAGCTG CTTCATAAGC TCCCACACGA ATTGCTTGAT TCATTGAAGA AGACGATTGG 1680 GTCTGTAGCT CAGTTGGTTA GAGCGCACCC CTGATAAGGG TGAGGTCGGC AGTTCGAATC 17 OO TGCCCAGACC CACCAATTAC S EO ID NO : 21 6 P. chlororaphis SS 44C4 16S-rDNA SEO ID NO: 217 P. entomophila-L48 16S-rDNA Patent Application Publication Jan. 2, 2014 Sheet 6 of 7 US 2014/0007292 A1 Fig. 4 Plate 1 £3.u.)u.993. US 2014/0007292 A1 Jan. 2, 2014 NOVEL, INSECTICDAL PROTEINS AND against insect pests, e.g., insecticidal proteins which are METHODS FOR THEIR USE active against a variety of insects in the order Lepidoptera and the order Hemiptera including but not limited to species CROSS REFERENCE belonging to the family Pentatomidae, the family Plataspidae 0001. This utility application claims the benefit U.S. Pro and the family Cydnidae. In addition, there remains a need for visional Application No. 61/667,039, filed Jul. 2, 2012, which biopesticides having activity against a variety of insect pests is incorporated herein by reference in its entirety. that have developed resistance to existing pesticides. REFERENCE TO SEQUENCE LISTING SUMMARY OF THE INVENTION SUBMITTED ELECTRONICALLY 0008 Compositions and methods for conferring pesticidal 0002 The official copy of the sequence listing is submitted activity to bacteria, plants, plant cells, tissues and seeds are electronically via EFS-Web as an ASCII formatted sequence provided. Compositions include nucleic acid molecules listing with a file named "4208 sequence listing..txt created encoding sequences for pesticidal and insecticidal polypep on Mar. 4, 2013, and having a size of 471 kilobytes and is filed tides, vectors comprising those nucleic acid molecules, and concurrently with the specification. The sequence listing con host cells comprising the vectors. Compositions also include tained in this ASCII formatted document is part of the speci the pesticidal polypeptide sequences and antibodies to those fication and is herein incorporated by reference in its entirety. polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and FIELD OF THE INVENTION expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be syn 0003. This disclosure relates to the field of molecular biol thetic sequences that have been designed for expression in an ogy. Provided are novel genes that encode pesticidal proteins. organism including, but not limited to, a microorganism or a These pesticidal proteins and the nucleic acid sequences that plant. Compositions also comprise transformed bacteria, encode them are useful in preparing pesticidal formulations plants, plant cells, tissues and seeds. and in the production of transgenic pest-resistant plants. 0009. In particular, isolated or recombinant nucleic acid molecules are provided encoding Pseudomonas Insecticidal BACKGROUND OF THE INVENTION Protein-1 (PIP-1) polypeptides including amino acid substi 0004 Biological control of insect pests of agricultural sig tutions, amino aciddeletions, amino acid insertions, and frag nificance using a microbial agent, Such as fungi, bacteria or ments thereof, and combinations thereof. Additionally, amino another species of insect affords an environmentally friendly acid sequences corresponding to the PIP-1 polypeptides are and commercially attractive alternative to synthetic chemical encompassed. Provided are an isolated or recombinant pesticides. Generally speaking, the use of biopesticides pre nucleic acid molecule capable of encoding a PIP-1 polypep sents a lower risk of pollution and environmental hazards, and tide of SEQID NO: 2, 101, 102, 103,104,105,106, 107, 108, biopesticides provide greater target specificity than is
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