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WO 2016/018887 Al 4 February 2016 (04.02.2016) P O P C T

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WO 2016/018887 Al 4 February 2016 (04.02.2016) 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 2016/018887 Al 4 February 2016 (04.02.2016) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 15/113 (2010.01) C12N 15/82 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US20 15/0424 15 KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (22) International Filing Date: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 28 July 2015 (28.07.2015) 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, (25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/030,430 29 July 2014 (29.07.2014) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (71) Applicant: MONSANTO TECHNOLOGY LLC TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, [US/US]; 800 North Lindbergh Boulevard, St. Louis, Mis DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, souri 63 167 (US). 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, (72) Inventors: CRAWFORD, Michael John; 800 North GW, KM, ML, MR, NE, SN, TD, TG). Lindbergh Boulevard, St. Louis, Missouri 63 167 (US). EADS, Brian Donovan; 800 North Lindbergh Boulevard, Published: St. Louis, Missouri 63 167 (US). — with international search report (Art. 21(3)) (74) Agents: QIN, Chunbo et al; Arnold & Porter LLP, 601 — before the expiration of the time limit for amending the Massachusetts Avenue, N.W., Washington, District of claims and to be republished in the event of receipt of Columbia 20001-3743 (US). amendments (Rule 48.2(h)) (81) Designated States (unless otherwise indicated, for every — with sequence listing part of description (Rule 5.2(a)) kind of national protection available): AE, AG, AL, AM, 00 00 00 © v o (54) Title: COMPOSITIONS AND METHODS FOR CONTROLLING INSECT PESTS (57) Abstract: Disclosed herein are polynucleotides, compositions, and methods for controlling insect pests, especially flea beetles, such as Phyllotreta spp. and Psylliodes spp., particularly in plants. More specifically, polynucleotides such as double-stranded RNA triggers and methods of use thereof for modifying the expression of genes in flea beetles. COMPOSITIONS AND METHODS FOR CONTROLLING INSECT PESTS CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS [0001] This application claims priority to U. S. Provisional Patent Application No. 62/030,430 filed 29 July 2014, which is incorporated by reference in its entirety herein. The sequence listing contained in the file "40-21_60779_0000_US_ST25.txt" (2098 kilobytes, created on 26 July 2014, filed with U. S. Provisional Patent Application No. 62/030,430 on 29 July 2014) is incorporated by reference in its entirety herein. The sequence listing contained in the file "40- 21_60779_0000_WO_ST25.txt" (2103 kilobytes, created on 8 July 2015) is filed herewith and is incorporated by reference in its entirety herein. FIELD [0002] Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants, compositions and polynucleotides useful in such methods, and plants having improved resistance to the invertebrate pests. More specifically, polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference are disclosed. Pest species of interest include flea beetles, such as Phyllotreta spp. and Psylliodes spp. BACKGROUND [0003] Commercial crops are often the targets of attack by invertebrate pests such as insects. Compositions for controlling insect infestations in plants have typically been in the form of chemical insecticides. However, there are several disadvantages to using chemical insecticides. For example, chemical insecticides are generally not selective, and applications of chemical insecticides intended to control insect pests in crop plants can exert their effects on non-target insects and other invertebrates as well. Chemical insecticides often persist in the environment and can be slow to degrade, thus potentially accumulating in the food chain. Furthermore the use of persistent chemical insecticides can result in the development of resistance in the target insect species. Thus there has been a long felt need for more environmentally friendly methods for controlling or eradicating insect infestation on or in plants, e. g., methods which are species-selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance management schemes. [0004] RNA interference (RNAi, RNA-mediated gene suppression) is an approach that shows promise for use in environmentally friendly pest control. In invertebrates, RNAi-based gene suppression was first demonstrated in nematodes (Fire etal, (1998) Nature, 391:806-811; Timmons & Fire (1998) Nature, 395:854). Subsequently, RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa, such as: root-knot nematodes (Meloidogyne spp.), see Huang et al. (2006) Proc. Natl. Acad. Sci. USA, 103:14302-14306, doi:10.1073/pnas.0604698103); cotton bollworm (Helicoverpa armigera), see Mao etal. (2007) Nature Biotechnol., 25:1307-1313, doi:10.1038/nbtl352; Western corn rootworm (Diabrotica virgifera LeConte), see Baum et al. (2007) Nature Biotechnol, 25:1322-1326, doi:10.1038/nbtl359; sugar beet cyst nematode (Heterodera schachtii), see Sindhu et al. (2008) J. Exp. Botany, 60:315-324, doi:10.1093/jxb/ern289; mosquito (Aedes aegypti), see Pridgeon et al. (2008) J. Med. Entomol, 45:414-420, doi: full/10.1603/0022-2585%282008%2945%5B414%3ATAADRK%5D2.0.CO%3B2; fruit flies (Drosophila melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta), see Whyard et al. (2009) Insect Biochem. Mol. Biol. , 39:824-832, doi:10.1016/j.ibmb.2009.09.00; diamondback moth (Plutella xylostella), see Gong et al. (2011) Pest Manag. Sc , 67: 514-520, doi:10.1002/ps.2086; green peach aphid (Myzus persicae), see Pitino et al. (2011) PLoS ONE, 6:e25709, doi:10.1371/journal.pone.0025709; brown planthopper (Nilaparvata lugens), see Li et al. (2011) Pest Manag. Scl, 67:852-859, doi:10.1002/ps.2124; and whitefly (Bemisia tabaci), see Upadhyay et al. (201 1) J. Bioscl, 36:153-161, doi:10.1007/sl2038-01 1- 9009-1. SUMMARY [0005] The present embodiments relate to control of insect species, especially flea beetle species that are economically or agriculturally important pests. The compositions and methods disclosed herein include recombinant polynucleotide molecules, such as recombinant DNA constructs for making transgenic plants resistant to infestation by insect species and polynucleotide agents, such as RNA "triggers", that are useful for controlling or preventing infestation by that insect species. Several embodiments described herein relate to a polynucleotide -containing composition (e. g., a composition containing a dsRNA trigger for suppressing a target gene) that is topically applied to an insect species or to a plant, plant part, or seed to be protected from infestation by an insect species. Other embodiments relate to methods for selecting preferred insect target genes that are more likely to be effective targets for RNAi-mediated control of an insect species. [0006] Several embodiments relate to a method for controlling an insect infestation of a plant including contacting the insect with a dsRNA including at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100%> complementarity with a fragment of a target gene of the insect. In some embodiments, the dsRNA includes at least one segment of 2 1 contiguous nucleotides with a sequence of 100%> complementarity with a fragment of a target gene of the insect. In some embodiments, the target gene is selected from the group consisting of Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpS21, RpS4, Rpn2, Rpn3, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V- ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments, the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the dsRNA includes an RNA strand with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1724 or a fragment thereof. In some embodiments, the dsRNA includes an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1724. In various embodiments, the contacting of the insect is by oral delivery, or by non-oral contact, e. g., by absorption through the cuticle, or through a combination of oral and non-oral delivery.
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