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WO 2014/018853 Al 30 January 2014 (30.01.2014) P O P C T

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WO 2014/018853 Al 30 January 2014 (30.01.2014) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/018853 Al 30 January 2014 (30.01.2014) P O P C T (51) International Patent Classification: International, Inc., 7250 N.W. 62nd Avenue, Johnston, IA C12N 15/82 (2006.01) A01H 5/00 (2006.01) 5013 1-0552 (US). PATTEN, Phillip, A. [US/US]; c/o Pi C07K 14/225 (2006.01) A01N 63/02 (2006.01) oneer Hi-Bred International, Inc., 7250 N.W. 62nd Aven ue, Johnston, IA 5013 1-0552 (US). PEREZ-ORTEGA, (21) International Application Number: Claudia, D. [MX/US]; c/o Pioneer Hi-Bred International, PCT/US20 13/052254 Inc., 7250 N.W. 62nd Avenue, Johnston, IA 5013 1-0552 (22) International Filing Date: (US). SCHEPERS, Eric, J. [US/US]; c/o Pioneer Hi-Bred 26 July 2013 (26.07.2013) International, Inc., 7250 N.W. 62nd Avenue, Johnston, IA 5013 1-0552 (US). XIE, Weiping [US/US]; c/o Pioneer (25) Filing Language: English Hi-Bred International, Inc., 7250 N.W. 62nd Avenue, (26) Publication Language: English Johnston, IA 5013 1-0552 (US). YALPANI, Nasser [US/US]; c/o Pioneer Hi-Bred International, Inc., 7250 (30) Priority Data: N.W. 62nd Avenue, Johnston, IA 5013 1-0552 (US). 61/675,950 26 July 2012 (26.07.2012) US ZHAO, Jianzhou [CN/US]; c/o Pioneer Hi-Bred Interna 61/739,468 19 December 2012 (19. 12.2012) US tional, Inc., 7250 N.W. 62nd Avenue, Johnston, IA 5013 1- 61/779,457 13 March 2013 (13.03.2013) us 0552 (US). ZHONG, Xiaohong [US/US]; c/o Pioneer Hi- (71) Applicants: PIONEER HI-BRED INTERNATIONAL, Bred International, Inc., 7250 N.W. 62nd Avenue, John INC. [US/US]; 7100 N.W. 62nd Avenue, Johnston, IA ston, IA 5013 1-0552 (US). ZHU, Genhai [US/US]; c/o Pi 5013 1-1014 (US). E. I. DUPONT DE NEMOURS & oneer Hi-Bred International, Inc., 7250 N.W. 62nd Aven COMPANY [US/US]; 1007 Market Street, Wilmington, ue, Johnston, IA 501 31-0552 (US). DE 19898 (US). (74) Agent: BAUER, S., Christoper; Pioneer Hi-Bred Interna (72) Inventors; and tional, Inc., 7250 N.W. 62nd Avenue, Johnston, IA 5013 1- (71) Applicants : ALTIER, Daniel, J. [US/US]; c/o Pioneer 0552 (US). Hi-Bred International, Inc., 7250 N.W. 62nd Avenue, (81) Designated States (unless otherwise indicated, for every Johnston, IA 5013 1-0552 (US). BARRY, Jennifer, K. kind of national protection available): AE, AG, AL, AM, [US/US]; c/o Pioneer Hi-Bred International, Inc., 7250 AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, N.W. 62nd Avenue, Johnston, IA 501 31-0552 (US). BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, HENDRICK, Carol, A. [US/US]; c/o Pioneer Hi-Bred In DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, ternational, Inc., 7250 N.W. 62nd Avenue, Johnston, IA HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR, 5013 1-0552 (US). LIU, Lu [US/US]; c/o Pioneer Hi-Bred KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, [Continued on nextpage] (54) Title: NOVEL INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE Fig. 9 0 0 © (57) Abstract: Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expres - sion vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homolog- ¾ ous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Cole- opteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity. w o 2014/018853 Ai II II II I III IIII II III II II Hill III II I II MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, GW, KM, ML, MR, NE, SN, TD, TG). ZM, ZW. Published: (84) Designated States (unless otherwise indicated, for every — with international search report (Art. 21(3)) kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, — with sequence listing part of description (Rule 5.2(a)) UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, NOVEL INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY A sequence listing having the file name "4244PCT_sequence_listing.txt" created on June 13, 2013, and having a size of 1499 kilobytes is filed in computer readable form concurrently with the specification. The sequence listing is part of the specification and is herein incorporated by reference in its entirety. FIELD OF THE INVENTION This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants. BACKGROUND OF THE INVENTION Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad- spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops. Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect- control methods. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control. Accordingly, there remains a need for new pesticidal proteins with different ranges of insecticidal activity against insect pests, e.g., insecticidal proteins which are active against a variety of insects in the order Lepidoptera and the order Coleoptera including but not limited to insect pests that have developed resistance to existing insecticides. SUMMARY OF THE INVENTION Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds. In particular, isolated or recombinant nucleic acid molecules are provided encoding Alcaligenes Insecticidal Protein-1A and Alcaligenes Insecticidal Protein-1 B (AflP-1A and AflP-1 B) polypeptides including amino acid substitutions, deletions, insertions, and fragments thereof, and combinations thereof. Additionally, amino acid sequences corresponding to the AflP-1 A and AflP-1 B polypeptides are encompassed. Provided are an isolated or recombinant nucleic acid molecule capable of encoding an AflP-1A polypeptide of SEQ ID NO: 2 as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof. Also provided are an isolated or recombinant nucleic acid molecule capable of encoding an AflP-1 B polypeptide of SEQ ID NO: 4 as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. Also provided are isolated or recombinant AflP-1A polypeptides of SEQ ID NO: 2 as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof.
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