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WO 2019/067496 Al 04 April 2019 (04.04.2019) W 1P O PCT

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WO 2019/067496 Al 04 April 2019 (04.04.2019) W 1P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/067496 Al 04 April 2019 (04.04.2019) W 1P O PCT (51) International Patent Classification: C07K 14/00 (2006.01) A01N 63/00 (2006.01) C12N 15/63 (2006.01) (21) International Application Number: PCT/US20 18/052788 (22) International Filing Date: 26 September 2018 (26.09.2018) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/563,228 26 September 2017 (26.09.2017) US (71) Applicant: DOW AGROSCIENCES LLC [US/US]; 9330 Zionsville Road, Indianapolis, IN 46268 (US). (72) Inventors: ZACK, Marc D.; 9330 Zionsville Road, Indi¬ anapolis, IN 46268 (US). SOPKO, Megan; 9330 Zionsville Road, Indianapolis, IN 46268 (US). HASLER, James M.; 9330 Zionsville Road, Indianapolis, IN 46268 (US). (74) Agent: RIVAS, Marcos; Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available) : ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) — with sequence listing part of description (Rule 5.2(a)) (54) Title: CHIMERIC INSECTICIDAL PROTEINS (57) Abstract: IRDIG35563 vegetative insecticidal toxins, polynucleotides encoding such toxins, use of such toxins to control pests, o and transgenic plants that produce such toxins are disclosed. The invention includes IRDIG35563 variants, fragments and analogs. CHIMERIC INSECTICIDAL PROTEINS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This Application claims the benefit of priority to U.S. Provisional Application No. 62/563,228 filed on September 26, 2017, which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "80144_WO_PCT_Sequence_Listing_ST25" created on September 25, 2018, and having a size of 17 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety. FIELD OF THE INVENTION [0003] This invention generally relates to the field of molecular biology. More specifically the invention concerns new insecticidal protein toxins developed from vegetative insecticidal protein toxins found in Bacillus thuringiensis and their use to control insects. BACKGROUND OF INVENTION [0004] Insects and other pests cost farmers billions of dollars annually in crop losses and expense to keep these pests under control. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs. [0005] Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect populations. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control agents. Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like delta-endotoxins, have also been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. The genes coding for some of these delta-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of insecticidal toxins, such as Bacillus thuringiensis delta-endotoxins, in transgenic plants have provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents. [0006] The soil microbe Bacillus thuringiensis is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. Bacillus thuringiensis continues to be the leading source of novel insecticidal proteins for development of plant incorporated pesticides. In the North American maize insect resistance market, Spodoptera frugiperda (fall armyworm "FAW"), Ostrinia nubialis Hiibner (European corn borer "ECB"), and Helicoverpa zea Boddie (corn earworm "CEW") are key driver pests, although there are other key insect pests in other geographies (e.g. Helicoverpa armigera (cotton bollworm "CBW" or corn earworm "CEW")) and additional secondary, but important insect pest species. Bt toxins represent over 90% of the bioinsecticide products commercially marketed and essentially the entire source of genes for transgenic crops that have been developed to provide resistance to insect feeding. Bt bacteria produce insecticidal delta-endotoxins including Crystal (Cry), Cytotoxin (Cyt), and Vegetative Insecticidal Protein (VIP) toxins, depending on their gene and protein structure. Cry toxins are produced during spore formation as insoluble crystal proteins. VIP toxins, on the other hand, are produced as soluble proteins during the vegetative stage of Bt bacterial growth. VIP proteins are distinct from Cry proteins in their structure, but share the property with Cry toxins of being pore formers acting on cells located in the insect midgut. The classification of VIP protein was previously based on their target insect types. Nomenclature is currently employed that systematically classifies the VIP genes based upon amino acid sequence homology rather than upon insect specificities. Crickmore, N., Baum, J., Bravo, A., Lereclus, D., Narva, K., Sampson, K., Schnepf, E., Sun, M. and Zeigler, D.R. "Bacillus thuringiensis toxin nomenclature" (2016); http://www.btnomenclature.info/; and http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html [0007] The continued use of chemical and biological agents to control insect pests heightens the chance for insects to develop resistance to such control measures. Also, the high selectivity of biological control agents often results in only a few specific insect pests being controlled by each agent. Despite the success of ECB-resistant transgenic corn, the possibility of the development of resistant insect populations threatens the long-term durability of Cry proteins in ECB control and creates the need to discover and develop new Cry or other types of biological control agents to control ECB and other pests. [0008] There remains a need to discover and develop new and effective pest control agents that provide an economic benefit to farmers and that are environmentally acceptable. Particularly needed are control agents targeted to a wide spectrum of economically important insect pests that efficiently control insect populations that are, or could become, resistant to existing insect control agents and those with equal to or increased potency compared to current control agents. BRIEF SUMMARY OF THE INVENTION [0009] The present invention provides insecticidal chimeric VIP toxins, including the protein toxin designated as IRDIG35563 (SEQ ID NO:2) that was constructed based on the first 613 amino acid residues of VIP3AM and a C-terminal portion of another VIP toxin, variants of IRDIG35563, nucleic acids encoding these chimeric toxins, methods of controlling pests using the toxins, methods of producing the toxins in transgenic host cells, and transgenic plants that express the toxins. The invention further provides a recombinant nucleic acid construct comprising one or more heterologous regulatory elements that drive expression of a nucleic acid sequence encoding SEQ ID NO:2 and nucleic acid sequences chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:4. In another embodiment, the invention provides nucleic acid constructs comprising a nucleotide sequence that encodes
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