WO 2015/010026 A2 22 January 2015 (22.01.2015) P O P C T
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(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 2015/010026 A2 22 January 2015 (22.01.2015) P O P C T (51) International Patent Classification: (74) Agents: LANSBERY, Kristan et al; 555 12th Street, C12N 15/82 (2006.01) NW, Washington, District of Columbia 20004-1206 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 14/047204 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) Date: International Filing BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 18 July 2014 (18.07.2014) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (26) Publication Language: English MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (30) Priority Data: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 61/856,137 19 July 2013 (19.07.2013) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 61/899,000 1 November 2013 (01. 11.2013) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, 61/980,800 17 April 2014 (17.04.2014) ZW. (71) Applicant: MONSANTO TECHNOLOGY LLC (84) Designated States (unless otherwise indicated, for every [US/US]; 800 North Lindbergh Boulevard, Mail Zone kind of regional protection available): ARIPO (BW, GH, E1NA, St. Louis, Missouri 63 167 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: BEATTIE, Jodi Lynn; 800 North Lindbergh TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (US). CRAWFORD, Michael John; 800 North Lindbergh MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (US). EADS, Brian Donovan; 800 North Lindbergh KM, ML, MR, NE, SN, TD, TG). Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 (US). FLAGEL, Lex Evan; 800 North Lindbergh Published: Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 — without international search report and to be republished (US). KAPOOR, Mahak; 800 North Lindbergh upon receipt of that report (Rule 48.2(g)) Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 — with sequence listing part of description (Rule 5.2(a)) (US). TAYLOR, Christina Marie; 800 North Lindbergh Boulevard, Mail Zone E1NA, St. Louis, Missouri 631 67 (US). < o (54) Title: COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA © (57) Abstract: Disclosed herein are methods of controlling insect pests, in particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and con - structs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with improved resistance to infestation by Leptinotarsa spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of Leptinotarsa spp. COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS [0001] This application claims priority to U. S. Provisional Patent Application No. 61/856,137 filed 19 July 2013, U. S. Provisional Patent Application No. 61/899,000 filed 1 November 2013, and U. S. Provisional Patent Application No. 61/980,800 filed 17 April 2014, which are incorporated by reference in their entirety herein. The sequence listings contained in the files "40-21_60191_A.t (2,291 kilobytes, created on 19 July 2013, filed with U. S. Provisional Patent Application No. 61/856,137 on 19 July 2013), "40-21_60191_0001US_ST25.txt" (2,322 kilobytes, created on 30 October 2013, filed with U. S. Provisional Patent Application No. 61/899,000 on 1 November 2013), and "40- 21_60191_0002_US_ST25.t (2,338 kilobytes, created on 17 April 2014, filed with U. S. Provisional Patent Application No. 61/980,800 on 17 April 2014) are incorporated by reference in their entirety herein. The sequence listing contained in the file "40-21_60191_0003_ST25_new.t bytes, created on 17 July 2014) is filed herewith and incorporated by reference in its entirety herein. FIELD [0002] Methods for controlling invertebrate pest infestations, particularly in plants, as well as compositions, polynucleotides, and recombinant DNA constructs useful in such methods are disclosed. More specifically, this invention is related to polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference. Pest species of interest include Leptinotarsa species, especially those that infest crop plants. 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, i. e., 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 another approach used for pest control. In invertebrates, RNAi-based gene suppression was first demonstrated in nematodes (Fire et al, (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. [0005] Leptinotarsa spp. form a genus including a number of species that infest commercially important plants, including many solanaceous plants (e. g., potato, tomato, eggplant, peppers, tobacco, and petunia). For example, Leptinotarsa decemlineata (Colorado potato beetle, CPB) is an early- to mid-season pest affecting solanaceous plants such as potato. Colorado potato beetles primarily feed on above-ground portions of the plant, and defoliation leads to lower tuber yields.Methods and compositions for controlling insect pests, in particular Leptinotarsa spp. which infest crop plants are desired. SUMMARY [0006] The present embodiments are related to control of Leptinotarsa species, especially those that are economically or agriculturally important pests. In various embodiments, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa behrensi, Leptinotarsa collinsi, Leptinotarsa decemlineata (Colorado potato beetle), Leptinotarsa dejecta, Leptinotarsa haldemani (Haldeman's green potato beetle), Leptinotarsa heydeni, Leptinotarsajuncta (false potato beetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsa peninsularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsa tlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica. In specific embodiments, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa decemlineata (Colorado potato beetle), Leptinotarsajuncta (false potato beetle), Leptinotarsa haldemani (Haldeman's green potato beetle), and Leptinotarsa lineolata (burrobrush leaf beetle). [0007] The compositions and methods described herein include recombinant polynucleotide molecules, such as recombinant DNA constructs for making transgenic plants resistant to infestation by Leptinotarsa species, and single- or double-stranded DNA or RNA molecules, referred to herein as "triggers", that are useful for controlling or preventing infestation of a plant by that Leptinotarsa species. In some embodiments, polynucleotide triggers are provided as topically applied agents for controlling or preventing infestation of a plant by a Leptinotarsa species. In some embodiments, solanaceous plants with improved resistance to infestation by Leptinotarsa species, such as transgenic solanaceous plants (including seeds or propagatable parts such as tubers) expressing a polynucleotide trigger are provided. In some embodiments, solanaceous plants (including seeds or propagatable parts such as tubers) that have been topically treated with a composition comprising a polynucleotide trigger (e. g., solanaceous plants that have been sprayed with a solution of dsRNA molecules) are provided. Also provided are polynucleotide-containing compositions that are topically applied to a Leptinotarsa species or to a plant, plant part, or seed to be protected from infestation by a Leptinotarsa species. [0008]