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