US 2015O143580A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0143580 A1 Beattie et al. (43) Pub. Date: May 21, 2015 (54) COMPOSITIONS AND METHODS FOR filed on Nov. 1, 2013, provisional application No. CONTROLLING LEPTINOTARSA 61/856,137, filed on Jul 19, 2013. (71) Applicant: MONSANTO TECHNOLOGY LLC, Publication Classification St. Louis, MO (US) (51) Int. Cl. (72) Inventors: Jodi Lynn Beattie, Wentzville, MO CI2N 5/82 (2006.01) (US); Michael John Crawford, AOIN57/6 (2006.01) Chesterfield, MO (US); Brian Donovan CI2N IS/II3 (2006.01) Eads, Ballwin, MO (US); Lex Evan (52) U.S. Cl. Flagel, St. Louis, MO (US); Mahak CPC .......... CI2N 15/8286 (2013.01); C12N 15/I 13 Kapoor, Chesterfield, MO (US); (2013.01); C12N 15/8218 (2013.01); A0IN Christina Marie Taylor, Chesterfield, 57/16 (2013.01); C12N 23 10/14 (2013.01) MO (US) (57) ABSTRACT Assignee: (73) MONSANTO TECHNOLOGY LLC, Disclosed herein are methods of controlling insect pests, in St. Louis, MO (US) particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also (21) Appl. No.: 14/335,135 disclosed are polynucleotides and recombinant DNA mol ecules and constructs useful in Such methods, insecticidal (22) Filed: Jul.18, 2014 compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with Related U.S. Application Data improved resistance to infestation by Leptinotarsa spp. Fur (60) Provisional application No. 61/980,800, filed on Apr. ther disclosed are methods of selecting target genes for 17, 2014, provisional application No. 61/899,000, RNAi-mediated silencing and control of Leptinotarsa spp. US 2015/O 143580 A1 May 21, 2015 COMPOSITIONS AND METHODS FOR binant nucleic acid techniques has been reported in a number CONTROLLINGLEPTINOTARSA of species, including agriculturally or economically impor tant pests from various insect and nematode taxa. CROSS-REFERENCE TO RELATED 0005 Leptinotarsa spp. form a genus including a number APPLICATIONS AND INCORPORATION OF of species that infest commercially important plants, includ SEQUENCE LISTINGS ing many Solanaceous plants (e.g., potato, tomato, eggplant, 0001. This application claims priority to U.S. Provisional peppers, tobacco, and petunia). For example, Leptinotarsa Patent Application No. 61/856,137 filed 19 Jul. 2013, U.S. decemlineata (Colorado potato beetle, CPB) is an early- to Provisional Patent Application No. 61/899,000 filed 1 Nov. mid-season pest affecting Solanaceous plants such as potato. 2013, and U.S. Provisional Patent Application No. 61/980, Colorado potato beetles primarily feed on above-ground por 800 filed 17 Apr. 2014, which are incorporated by reference in tions of the plant, and defoliation leads to lower tuber yields. their entirety herein. The sequence listings contained in the Methods and compositions for controlling insect pests, in files “40-21 60191 A.txt” (2,291 kilobytes, created on 19 particular Leptinotarsa spp. which infest crop plants are Jul. 2013, filed with U.S. Provisional Patent Application No. desired. 61/856,137 on 19 Jul. 2013), “40-21 60191 0001 US ST25.txt” (2.322 kilobytes, created on 30 Oct. 2013, filed SUMMARY with U.S. Provisional Patent Application No. 61/899,000 on 1 0006. The present embodiments are related to control of Nov. 2013), and “40-21 60191 0002 US ST25.txt Leptinotarsa species, especially those that are economically (2.338 kilobytes, created on 17 Apr. 2014, filed with U.S. or agriculturally important pests. In various embodiments, Provisional Patent Application No. 61/980,800 on 17 Apr. the Leptinotarsa species is at least one selected from the 2014) are incorporated by reference in their entirety herein. group consisting of Leptinotarsa behrensi, Leptinotarsa col The sequence listing contained in the file “40-2160191 linsi, Leptinotarsa decemlineata (Colorado potato beetle), 0003 ST25 new.txt” (2,532,856 bytes, created on 17 Jul. Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman’s 2014) is filed herewith and incorporated by reference in its green potato beetle), Leptinotarsa heydeni, Leptinotarsa entirety herein. juncta (false potato beetle), Leptinotarsa lineolata (burro brush leaf beetle), Leptinotarsa peninsularis, Leptinotarsa FIELD rubiginosa, Leptinotarsa texana, Leptinotarsa tilascalana, 0002 Methods for controlling invertebrate pest infesta Leptinotarsa tumamoca, and Leptinotarsa typographica. In tions, particularly in plants, as well as compositions, poly specific embodiments, the Leptinotarsa species is at least one nucleotides, and recombinant DNA constructs useful in such selected from the group consisting of Leptinotarsa decemlin methods are disclosed. More specifically, this invention is eata (Colorado potato beetle), Leptinotarsa juncta (false related to polynucleotides and methods of use thereof for potato beetle), Leptinotarsa haldemani (Haldeman's green modifying the expression of genes in an insect pest, particu potato beetle), and Leptinotarsa lineolata (burrobrush leaf larly through RNA interference. Pest species of interest beetle). include Leptinotarsa species, especially those that infest crop 0007. The compositions and methods described herein plants. include recombinant polynucleotide molecules. Such as recombinant DNA constructs for making transgenic plants BACKGROUND resistant to infestation by Leptinotarsa species, and single- or double-stranded DNA or RNA molecules, referred to herein 0003 Commercial crops are often the targets of attack by as “triggers', that are useful for controlling or preventing invertebrate pests such as insects. Compositions for control infestation of a plant by that Leptinotarsa species. In some ling insect infestations in plants have typically been in the embodiments, polynucleotide triggers are provided as topi form of chemical insecticides. However, there are several cally applied agents for controlling or preventing infestation disadvantages to using chemical insecticides. For example, of a plant by a Leptinotarsa species. In some embodiments, chemical insecticides are generally not selective, and appli Solanaceous plants with improved resistance to infestation by cations of chemical insecticides intended to control insect Leptinotarsa species, such as transgenic Solanaceous plants pests in crop plants can exert their effects on non-target (including seeds or propagatable parts such as tubers) insects and other invertebrates as well. Chemical insecticides expressing a polynucleotide trigger are provided. In some often persist in the environment and can be slow to degrade, embodiments, Solanaceous plants (including seeds or propa thus potentially accumulating in the food chain. Furthermore gatable parts such as tubers) that have been topically treated the use of persistent chemical insecticides can result in the with a composition comprising a polynucleotide trigger (e.g., development of resistance in the target insect species. Thus Solanaceous plants that have been sprayed with a solution of there has been a long felt need for more environmentally dsRNA molecules) are provided. Also provided are poly friendly methods for controlling or eradicating insect infes nucleotide-containing compositions that are topically applied tation on or in plants, i.e., methods which are species-selec to a Leptinotarsa species or to a plant, plant part, or seed to be tive, environmentally inert, non-persistent, and biodegrad protected from infestation by a Leptinotarsa species. able, and that fit well into pest resistance management 0008. Several embodiments relate to suppression of a tar schemes. get geneina Leptinotarsa species by a polynucleotide trigger. 0004 RNA interference (RNAi, RNA-mediated gene Sup Some embodiments relate to methods for selecting Leptino pression) is another approach used for pest control. In inver tarsa target genes that are likely to be effective targets for tebrates, RNAi-based gene Suppression was first demon RNAi-mediated control of a Leptinotarsa species. In some strated innematodes (Fire et al., (1998) Nature, 391:806-811; embodiments, target genes selected for RNAi-mediated Sup Timmons & Fire (1998) Nature, 395:854). Subsequently, pression are genes that are non-repetitive and non-redundant RNAi-based suppression of invertebrate genes using recom in a Leptinotarsa species genome, or that have low nucleotide US 2015/O 143580 A1 May 21, 2015 diversity, or that are evolutionarily or functionally con logical function within the Leptinotarsa species thereby con strained to have a more synonymous (K) than nonsynony trolling infestation by the Leptinotarsa species. In an embodi mous (K) nucleotide changes. Provided herein are nucle ment, the method for controlling a Leptinotarsa species otide sequences referred to herein as the “Target Gene infestation of a plant comprises providing in the diet of the Sequences Group', which consists of SEQID NOS:1-725 and Leptinotarsa species a polynucleotide comprising a nucle SEQ ID NOs:726-830 and SEQ ID NOs: 1087-1094. Also otide sequence that is complementary to at least 21 contigu provided are nucleotide sequences referred to herein as the ous nucleotides of a target gene having a nucleotide sequence “Trigger Sequences Group', which consists of SEQID NOs: selected from the group consisting of: SEQID NO:730, SEQ 831, 842, 849, 898, 910, 925,928,931, 932, 937,938, 940, ID NO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ 941, 942, 943,944, 945, 947, 948,949, 950, 951, 952,955, ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: