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: 956, 957, 958, 960, 961, 964,966,967,968, 969, 970,971, 1087-1094, or an RNA transcribed from the target gene. In 973, 976,978,979,982, 983,985, 987, 988,989, 991, 992, Some embodiments, the polynucleotide comprises one or 994,995,996,997,999, 1006, 1007, 1008, 1009, 1010, 1013, more nucleotide sequences selected from the Trigger 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, Sequences Group. In some embodiments, the polynucleotide 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, is double-stranded RNA. In some embodiments, the agent 1049, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, containing the polynucleotide is formulated for application to 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, fields of crop plants, e.g., in sprayable solutions or emulsions, 1082, 1084, 1085, 1095, 1096, 1097,1098, 1099, 1100, 1101, tank mixes, or powders. In some embodiments, the agent is 1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, 1114, 1118, biologically produced, e.g., in the form of a microbial fer 1119, and 1124. mentation product or expressed in a transgenic plant cell. 0009. In one aspect, a method for controlling a Leptino 0011. In another aspect, a method of causing mortality or tarsa species infestation of a plant comprising contacting the stunting in Leptinotarsa species larvae is provided. In some Leptinotarsa species with a polynucleotide comprising at embodiments, at least one RNA comprising at least one least one segment of 18 or more contiguous nucleotides with silencing element is provided in the diet of a Leptinotarsa a sequence of about 95% to about 100% identity (e.g., a species larvae wherein ingestion of the RNA by the Leptino segment of 21 contiguous nucleotides with a sequence of tarsa species larvae results in mortality or stunting in the 100% identity) with a corresponding fragment of a DNA Leptinotarsa species larvae. In some embodiments, the having a sequence selected from the group consisting of the silencing element is essentially identical or essentially Target Gene Sequences Group, or the DNA complement complementary to a fragment of a target gene sequence of the thereof. In an embodiment, the method for controlling a Lep Leptinotarsa species larvae, wherein the target gene is tinotarsa species infestation of a plant comprises contacting selected from the group consisting of the genes in the Target the Leptinotarsa species with a polynucleotide comprising a Gene Sequences Group In an embodiment, the method of nucleotide sequence that is complementary to at least 21 causing mortality or stunting in larvae of the Leptinotarsa contiguous nucleotides of a target gene having a nucleotide species comprises providing in the diet of the larvae at least sequence selected from the group consisting of SEQ ID one polynucleotide comprising at least one silencing element NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID comprising 21 contiguous nucleotides that are complemen NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, tary to a target gene having a nucleotide sequence selected and SEQIDNOs: 1087-1094, oran RNA transcribed from the from the group consisting of: SEQ ID NO:730, SEQ ID target gene. In some embodiments, the polynucleotide is NO:807, SEQIDNOs: 1-725, SEQID NOs:726-729, SEQID double-stranded RNA. In some embodiments, the polynucle NOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs: otide comprises one or more nucleotide sequences selected 1087-1094, or an RNA transcribed from the target gene. In from the Trigger Sequences Group. In some embodiments, Some embodiments, the silencing element comprises one or the contacting with a polynucleotide is achieved by topical more nucleotide sequences selected from the Trigger application of the polynucleotide, or of a composition or Sequences Group. In some embodiments, the polynucleotide Solution containing the polynucleotide (e.g., by spraying or is double-stranded RNA. Some embodiments relate to a dusting or soaking), directly to the Leptinotarsa species or to method of causing mortality or lower fecundity in Leptino a Surface or matrix (e.g., a plant or soil) contacted by the tarsa species comprising providing in the diet of Leptinotarsa Leptinotarsa species. In some embodiments, the contacting species at least one RNA comprising at least one silencing with a polynucleotide is achieved by providing a polynucle element essentially identical or essentially complementary to otide that is ingested by the Leptinotarsa species. In some a fragment of a target gene sequence of the Leptinotarsa embodiments, the contacting with a polynucleotide is species larvae wherein ingestion of the RNA by the Leptino achieved by providing a transgenic plant that expresses to the tarsa species results in mortality or lower fecundity in the Leptinotarsa species. Leptinotarsa species. In some embodiments, the target gene 0010 Several embodiments relate to a method for control is selected from the group consisting of the genes in the Target ling a Leptinotarsa species infestation of a plant by providing Gene Sequences Group. In some embodiments, the method in the diet of a Leptinotarsa species an agent comprising a causes a decrease in metamorphosis rate or a decrease in polynucleotide having at least one segment of 18 or more feeding activity. In some embodiments, the method is useful contiguous nucleotides with a sequence of about 95% to for providing plants having increased resistance to infestation about 100% identity (e.g., a segment of 21 contiguous nucle by Leptinotarsa species. otides with a sequence of 100% identity) with a correspond 0012 Several embodiments relate to a method of provid ing fragment of a DNA having a sequence selected from the ing a plant having improved resistance to a Leptinotarsa group consisting of the Target Gene Sequences Group, or the species infestation comprising topically applying to the plant DNA complement thereof, and wherein the agent functions a composition comprising at least one polynucleotide having upon ingestion by the Leptinotarsa species to inhibit a bio at least one segment of 18 or more contiguous nucleotides US 2015/O 143580 A1 May 21, 2015 with a sequence of about 95% to about 100% identity (e.g., a nucleotide herbicidal molecule, a non-polynucleotide pesti segment of 21 contiguous nucleotides with a sequence of cide, a safener, and an insect growth regulator. 100% identity) with a corresponding fragment of a DNA 0014 Several embodiments relate to a method of provid having a sequence selected from the group consisting of the ing a plant having improved resistance to a Leptinotarsa Target Gene Sequences Group, or the DNA complement species infestation comprising expressing in the plant at least thereof. In an embodiment, the method of providing a plant one polynucleotide comprising at least one segment of 18 or having improved resistance to a Leptinotarsa species infes more contiguous nucleotides that are essentially identical or tation comprises topically applying to the planta composition complementary to (e.g., a segment of 21 contiguous nucle comprising at least one polynucleotide comprising a nucle otides with a sequence of 100% identity or complementarity otide sequence that is complementary to at least 21 contigu with) the corresponding fragment of a DNA having a ous nucleotides of a target gene having a nucleotide sequence sequence selected from the group consisting of the Target selected from the group consisting of: SEQID NO:730, SEQ Gene Sequences Group, or the DNA complement thereof. In IDNO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ Some embodiments, the polynucleotide comprises one or ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: more nucleotide sequences selected from the Trigger 1087-1094, oran RNA transcribed from the target gene. In an Sequences Group. In some embodiments, the polynucleotide embodiment, the method of providing a plant having is double-stranded RNA. improved resistance to a Leptinotarsa species infestation comprises topically applying to the plant a composition com 0015 Several embodiments relate to a recombinant DNA prising at least one polynucleotide in a manner Such that an construct comprising a heterologous promoter operably effective amount of the polynucleotide is ingested by a Lep linked to a DNA element comprising at least one segment of tinotarsa species feeding on the plant, the polynucleotide 18 or more contiguous nucleotides with a sequence of about comprising at least 21 contiguous nucleotides that are 95% to about 100% identity (e.g., a segment of 21 contiguous complementary to a target gene having a nucleotide sequence nucleotides with a sequence of 100% identity) with the cor selected from the group consisting of: SEQID NO:730, SEQ responding fragment of a DNA having a sequence selected IDNO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ from the group consisting of the Target Gene Sequences ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: Group, or the DNA complement thereof. In some embodi 1087-1094, or an RNA transcribed from the target gene. In ments, the DNA element encodes a double-stranded RNA. In Some embodiments, the polynucleotide comprises one or some embodiments, the double-stranded RNA comprises one more nucleotide sequences selected from the Trigger or more nucleotide sequences selected from the Trigger Sequences Group. In some embodiments, the polynucleotide Sequences Group. Related embodiments include a plant is double-stranded RNA. Several embodiments relate to com chromosome or a plastid or a recombinant plant virus vector positions comprising the polynucleotide, formulated for or a recombinant baculovirus vector comprising the recom application to fields of crop plants, e.g., in sprayable solutions binant DNA construct, or comprising the DNA element with or emulsions, tank mixes, or powders. out the heterologous promoter. 0013 Several embodiments relate to an insecticidal com 0016 Several embodiments relate to a transgenic solana position for controlling a Leptinotarsa species comprising an ceous plant cell having in its genome a recombinant DNA insecticidally effective amount of at least one polynucleotide encoding RNA that Suppresses expression of a target gene in molecule comprising at least one segment of 18 or more a Leptinotarsa species that contacts or ingests the RNA, contiguous nucleotides that are essentially identical or wherein the RNA comprises at least one silencing element complementary (e.g., a segment of 21 contiguous nucleotides having at least one segment of 18 or more contiguous nucle with a sequence of 100% identity or complementarity) with otides complementary to a fragment of a target gene. In some the corresponding fragment of a DNA having a sequence embodiments, the target gene is selected from the the Target selected from the group consisting of the Target Gene Gene Sequences Group. A specific embodiment is a trans Sequences Group, or the DNA complement thereof. In some genic Solanaceous plant cell having in its genome a recombi embodiments, the polynucleotide molecule comprises at least nant DNA encoding RNA for silencing one or more target 21 contiguous nucleotides that are complementary to a target genes selected from the group consisting of exocyst genes, gene having a nucleotide sequence selected from the group ribosomal protein genes, and proteosome genes. In some consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID embodiments, the RNA comprises one or more nucleotide NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, sequences selected from the Trigger Sequences Group. SEQID NOs:808-830, and SEQ ID NOs: 1087-1094, or an 0017 Several embodiments relate to an isolated recombi RNA transcribed from the target gene. In some embodiments, nant RNA molecule that causes mortality or stunting of the polynucleotide comprises one or more nucleotide growth in a Leptinotarsa species when ingested or contacted sequences selected from the Trigger Sequences Group. In by the Leptinotarsa species, wherein the recombinant RNA Some embodiments, the polynucleotide molecule is a recom molecule comprises at least one segment of 18 or more con binant polynucleotide. In some embodiments, the polynucle tiguous nucleotides that are essentially complementary to otide molecule is RNA. In some embodiments, the polynucle (e.g., a segment of 21 contiguous nucleotides with a sequence otide molecule is double-stranded RNA. Related of 100% complementarity with) the corresponding of a DNA embodiments include insecticidal compositions comprising having a sequence selected from the group consisting of the the polynucleotide molecule formulated for application to Target Gene Sequences Group, or the DNA complement fields of crop plants, e.g., in sprayable solutions or emulsions, thereof. In some embodiments, the recombinant RNA mol tank mixes, or powders, and optionally comprising one or ecule is double-stranded RNA. Specific embodiments include more additional components, such as a carrier agent, a Sur an isolated recombinant RNA molecule for Suppressing factant, a cationic lipid, an organosilicone, an organosilicone expression of a ribosomal protein Such as a ribosomal L7 Surfactant, a polynucleotide herbicidal molecule, a non-poly protein or a protein encoded by SEQ ID NO:730, and an US 2015/O 143580 A1 May 21, 2015

isolated recombinant double-stranded RNA molecule having foodstuffs having a detectable amount of a polynucleotide as a sequence selected from the group consisting of SEQ ID described herein) are provided. Several embodiments relate NO:989,988, 1104, or 1105. to polyclonal or monoclonal antibodies that bind a protein 0018 Several embodiments relate to a method of provid encoded by a sequence or a fragment of a sequence selected ing a plant having improved resistance to a Leptinotarsa from the Target Gene Sequences Group. Another aspect species infestation comprising providing to the plant at least relates to polyclonal or monoclonal antibodies that bind a one polynucleotide comprising at least one segment of 18 or protein encoded by a sequence or a fragment of a sequence more contiguous nucleotides that are essentially identical or selected from the Trigger Sequences Group, or the comple complementary to (e.g., a segment of 21 contiguous nucle ment thereof. Suchantibodies are made by routine methods as otides with a sequence of 100% identity or complementarity known to one of ordinary skill in the art. with) the corresponding fragment of a target gene selected 0022. In the various embodiments described herein, the from the Target Gene Sequences Group. In an embodiment, plant can be any plant that is Subject to infestation by a the method of providing a plant having improved resistance to Leptinotarsa species. Of particular interest are embodiments a Leptinotarsa species infestation comprises providing to the wherein the plant is a Solanaceous plant (family Solanaceae). plant at least one polynucleotide comprising at least one Examples include a plant selected from the group consisting segment that is identical or complementary to at least 21 of potato, tomato, and eggplant. Embodiments include those contiguous nucleotides of a target gene or an RNA tran wherein the plant is an ungerminated Solanaceous plant seed, scribed from the target gene, wherein the target gene is a Solanaceous plant in a vegetative stage, or a Solanaceous selected from the group consisting of the genes identified in plant in a reproductive stage. Embodiments include those the Target Gene Sequences Group. In some embodiments, the wherein the plant is a 'seed potato’, meaning a potato tuberor polynucleotide comprises one or more nucleotide sequences piece of potato tuber which can be propagated into new potato selected from the Trigger Sequences Group. In some embodi plants. ments, the polynucleotide is double-stranded RNA. 0023. Other aspects and specific embodiments of this 0019. Several embodiments relate to a method for control invention are disclosed in the following detailed description. ling a Leptinotarsa species infestation of a plant comprising contacting the Leptinotarsa species with a polynucleotide DETAILED DESCRIPTION comprising at least one segment of 18 or more contiguous 0024. Unless defined otherwise, all technical and scien nucleotides that are essentially identical or complementary to tific terms used have the same meaning as commonly under (e.g., a segment of 21 contiguous nucleotides with a sequence stood by one of ordinary skill in the art to which this invention of 100% identity or complementarity with) the corresponding belongs. Where a term is provided in the singular, the inven fragment of equivalent length of a DNA of a target gene tors also contemplate aspects of the invention described by selected from the Target Gene Sequences Group. In some the plural of that term. Where there are discrepancies interms embodiments, the polynucleotide is double-stranded RNA. In and definitions used in references that are incorporated by an embodiment, the method for controlling a Leptinotarsa reference, the terms used in this application shall have the species infestation of a plant comprises contacting the Lepti definitions given herein. Other technical terms used have their notarsa species with an effective amount of a double ordinary meaning in the art in which they are used, as exem stranded RNA, one strand of which is complementary to at plified by various art-specific dictionaries, for example, “The least 21 contiguous nucleotides of a gene that encodes a American Heritage(R) Science Dictionary” (Editors of the ribosomal protein, wherein RNA interference is induced and American Heritage Dictionaries, 2011, Houghton Mifflin mortality occurs. In some embodiments, the double-stranded Harcourt, Boston and New York), the “McGraw-Hill Dictio RNA comprises one or more nucleotide sequences selected nary of Scientific and Technical Terms” (6" edition, 2002, from the Trigger Sequences Group. McGraw-Hill, New York), or the “Oxford Dictionary of Biol 0020 Several embodiments relate to a method of selecting ogy” (6" edition, 2008, Oxford University Press, Oxford and target genes for RNAi-mediated silencing from a plant New York). The inventors do not intend to be limited to a genome or from an animal genome. In various embodiments, mechanism or mode of action. Reference thereto is provided the method provides a Subset of target genes that are present for illustrative purposes only. in single- or low-copy-number (non-repetitive and non-re 0025. Unless otherwise stated, nucleic acid sequences in dundant) in a particular genome, or that have low nucleotide the text of this specification are given, when read from left to diversity, or that have a ratio of synonymous (K) to nonsyn right, in the 5' to 3" direction. One of skill in the art would be onymous (K) nucleotide changes where K.PK. aware that a given DNA sequence is understood to define a 0021 Several embodiments relate to man-made composi corresponding RNA sequence which is identical to the DNA tions comprising at least one polynucleotide as described sequence except for replacement of the thymine (T) nucle herein. In some embodiments, formulations useful for topical otides of the DNA with uracil (U) nucleotides. Thus, provid application to a plant or Substance in need of protection from ing a specific DNA sequence is understood to define the exact a Leptinotarsa species infestation are provided. In some RNA equivalent. A given first polynucleotide sequence, embodiments, recombinant constructs and vectors useful for whether DNA or RNA, further defines the sequence of its making transgenic Solanaceous plant cells and transgenic exact complement (which can be DNA or RNA), a second Solanaceous plants are provided. In some embodiments, for polynucleotide that hybridizes perfectly to the first poly mulations and coatings useful for treating Solanaceous plants, nucleotide by forming Watson-Crick base-pairs. For DNA: Solanaceous plant seeds or propagatable parts such as tubers DNA duplexes (hybridized strands), base-pairs are adenine: are provided. In some embodiments, commodity products thymine or guanine:cytosine; for DNA:RNA duplexes, base and foodstuffs produced from Such Solanaceous plants, seeds, pairs are adenine:uracil or guanine:cytosine. Thus, the or propagatable parts treated with or containing a polynucle nucleotide sequence of a blunt-ended double-stranded poly otide as described herein (especially commodity products and nucleotide that is perfectly hybridized (where there “100% US 2015/O 143580 A1 May 21, 2015 complementarity” between the strands or where the strands otides in length, for example up to the entire length of a target are “complementary') is unambiguously defined by provid gene including coding or non-coding or both coding and ing the nucleotide sequence of one Strand, whether given as non-coding portions of the target gene). Where a polynucle DNA or RNA. By “essentially identical” or “essentially otide is double-stranded, its length can be similarly described complementary' to a target gene or a fragment of a target gene in terms of base pairs. is meant that a polynucleotide Strand (or at least one strand of 0027. The polynucleotides described herein can be single a double-stranded polynucleotide) is designed to hybridize stranded (ss) or double-stranded (ds). “Double-stranded (generally under physiological conditions such as those refers to the base-pairing that occurs between sufficiently found in a living plant or animal cell) to a target gene or to a complementary, anti-parallel nucleic acid strands to form a fragment of a target gene or to the transcript of the target gene double-stranded nucleic acid structure, generally under or the fragment of a target gene; one of skill in the art would physiologically relevant conditions. Embodiments include understand that Such hybridization does not necessarily those wherein the polynucleotide is selected from the group require 100% sequence identity or complementarity. A first consisting of sense single-stranded DNA (ssDNA), sense nucleic acid sequence is “operably” connected or “linked single-stranded RNA (ssRNA), double-stranded RNA with a second nucleic acid sequence when the first nucleic (dsRNA), double-stranded DNA (dsDNA), a double acid sequence is placed in a functional relationship with the stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense second nucleic acid sequence. For example, a promoter ssRNA; a mixture of polynucleotides of any of these types can sequence is “operably linked to a DNA if the promoter be used. In some embodiments, the polynucleotide is double provides for transcription or expression of the DNA. Gener stranded RNA of a length greater than that which is typical of ally, operably linked DNA sequences are contiguous. naturally occurring regulatory Small RNAS (Such as endog 0026. The term “polynucleotide' commonly refers to a enously produced siRNAs and mature miRNAs). In some DNA or RNA molecule containing multiple nucleotides and embodiments, the polynucleotide is double-stranded RNA of generally refers both to "oligonucleotides” (a polynucleotide at least about 30 contiguous base-pairs in length. In some molecule of 18-25 nucleotides in length) and longer poly embodiments, the polynucleotide is double-stranded RNA nucleotides of 26 or more nucleotides. Polynucleotides also with a length of between about 50 to about 500 base-pairs. In include molecules containing multiple nucleotides including Some embodiments, the polynucleotide can include compo non-canonical nucleotides or chemically modified nucle nents other than standard ribonucleotides, e.g., an embodi otides as commonly practiced in the art; see, e.g., chemical ment is an RNA that comprises terminal deoxyribonucle modifications disclosed in the technical manual "RNA Inter otides. ference (RNAi) and DsiRNAs, 2011 (Integrated DNA Tech 0028. In various embodiments, the polynucleotide nologies Coralville, Iowa). Generally, polynucleotides as described herein comprise naturally occurring nucleotides, described herein, whether DNA or RNA or both, and whether such as those which occur in DNA and RNA. In certain single- or double-stranded, include at least one segment of 18 embodiments, the polynucleotide is a combination of ribo or more contiguous nucleotides (or, in the case of double nucleotides and deoxyribonucleotides, for example, Syn Stranded polynucleotides, at least 18 contiguous base-pairs) thetic polynucleotides consisting mainly of ribonucleotides that are essentially identical or complementary to a fragment but with one or more terminal deoxyribonucleotides or one or of equivalent size of the DNA of a target gene or the target more terminal dideoxyribonucleotides or synthetic poly gene's RNA transcript. Throughout this disclosure, “at least nucleotides consisting mainly of deoxyribonucleotides but 18 contiguous” means “from about 18 to about 10,000, with one or more terminal dideoxyribonucleotides. In certain including every whole number point in between”. Thus, embodiments, the polynucleotide comprises non-canonical embodiments of this invention include oligonucleotides hav nucleotides such as inosine, thiouridine, or pseudouridine. In ing a length of 18-25 nucleotides (18-mers, 19-mers, certain embodiments, the polynucleotide comprises chemi 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), cally modified nucleotides. Examples of chemically modified or medium-length polynucleotides having a length of 26 or oligonucleotides or polynucleotides are well known in the art; more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, see, for example, U.S. Patent Publication 2011/0171287, U.S. 32,33,34, 35,36, 37,38,39, 40, 41, 42, 43,44, 45,46, 47, 48, Patent Publication 2011/0171176, U.S. Patent Publication 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 2011/0152353, U.S. Patent Publication 2011/0152346, and 70, about 75, about 80, about 85, about 90, about 95, about U.S. Patent Publication 2011/0160082, which are herein 100, about 110, about 120, about 130, about 140, about 150, incorporated by reference. Illustrative examples include, but about 160, about 170, about 180, about 190, about 200, about are not limited to, the naturally occurring phosphodiester 210, about 220, about 230, about 240, about 250, about 260, backbone of an oligonucleotide or polynucleotide which can about 270, about 280, about 290, or about 300 nucleotides), or be partially or completely modified with phosphorothioate, long polynucleotides having a length greater than about 300 phosphorodithioate, or methylphosphonate internucleotide nucleotides (e.g., polynucleotides of between about 300 to linkage modifications, modified nucleoside bases or modified about 400 nucleotides, between about 400 to about 500 nucle Sugars can be used in oligonucleotide or polynucleotide Syn otides, between about 500 to about 600 nucleotides, between thesis, and oligonucleotides or polynucleotides can be about 600 to about 700 nucleotides, between about 700 to labeled with a fluorescent moiety (e.g., fluorescein or about 800 nucleotides, between about 800 to about 900 nucle rhodamine) or other label (e.g., biotin). otides, between about 900 to about 1000 nucleotides, 0029 Several embodiments relate to a polynucleotide between about 300 to about 500 nucleotides, between about comprising at least one segment of 18 or more contiguous 300 to about 600 nucleotides, between about 300 to about 700 nucleotides with a sequence of about 95% to about 100% nucleotides, between about 300 to about 800 nucleotides, identity with a fragment of equivalent length of a DNA or between about 300 to about 900 nucleotides, or about 1000 target gene having a sequence selected from the Target Gene nucleotides in length, or even greater than about 1000 nucle Sequences Group or the DNA complement thereof. In some US 2015/O 143580 A1 May 21, 2015

embodiments, the contiguous nucleotides number at least 18, from a Leptinotarsa species target gene, which can be coding e.g., between 18-24, or between 18-28, or between 20-30, or sequence or non-coding sequence. These effective polynucle between 20-50, or between 20-100, or between 50-100, or otide molecules that modulate expression may be referred to between 50-500, or between 100-250, or between 100-500, or herein as a “polynucleotide'. “polynucleotide trigger”, “trig between 200-1000, or between 500-2000, or even greater. In ger', or “triggers'. Some embodiments, the contiguous nucleotides number more 0031) Effective polynucleotides of any size can be used, than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or alone or in combination, in the various methods and compo greater than 30, e.g., about 35, about 40, about 45, about 50, sitions described herein. In some embodiments, a single poly about 55, about 60, about 65, about 70, about 75, about 80, nucleotide trigger is used to make a composition (e.g., a about 85, about 90, about 95, about 100, about 110, about 120, composition for topical application, or a recombinant DNA about 130, about 140, about 150, about 160, about 170, about construct useful for making a transgenic plant). In other 180, about 190, about 200, about 210, about 220, about 230, embodiments, a mixture or pool of different polynucleotide about 240, about 250, about 260, about 270, about 280, about triggers is used; in Such cases the polynucleotide triggers can 290, about 300, about 350, about 400, about 450, about 500, be for a single target gene or for multiple target genes. or greater than 500 contiguous nucleotides. In some embodi 0032. As used herein, the term "isolated” refers to sepa ments, the polynucleotide comprises at least one segment of rating a molecule from other molecules normally associated at least 21 contiguous nucleotides with a sequence of 100% with it in its native or natural state. The term "isolated thus identity with a fragment of equivalent length of a DNA or may refer to a DNA molecule that has been separated from target gene having a sequence selected from the Target Gene other DNA molecule(s) which normally are associated with it Sequences Group or the DNA complement thereof. In some in its native or natural state. Such a DNA molecule may be embodiments, the polynucleotide is a double-stranded present in a recombined state. Such as a recombinant DNA nucleic acid (e.g., dsRNA) with one strand comprising at least molecule. Thus, DNA molecules fused to regulatory or cod one segment of at least 21 contiguous nucleotides with 100% ing sequences with which they are not normally associated, identity with a fragment of equivalent length of a DNA or for example as the result of recombinant techniques, are con target gene having a sequence selected from the Target Gene sidered isolated, even when integrated as a transgene into the Sequences Group or the DNA complement thereof; expressed chromosome of a cell or present with other DNA molecules. as base-pairs, such a double-stranded nucleic acid comprises 0033. As used herein, the term “Target Gene Sequences at least one segment of at least 21 contiguous, perfectly Group' refers to the group of sequences consisting of SEQID matched base-pairs which correspond to a fragment of NOs: 1-725 and SEQ ID NOs:726-830 and SEQ ID NOs: equivalent length of a DNA or target gene having a sequence 1087-1094). As used herein, the term “Trigger Sequences selected from the Target Gene Sequences Group or the DNA Group' refers to the group of sequences consisting of SEQID complement thereof. In some embodiments, each segment NOs:831, 842, 849, 898, 910, 925,928,931,932, 937,938, contained in the polynucleotide is of a length greater than that 940, 941, 942, 943, 944, 945, 947, 948,949, 950, 951, 952, which is typical of naturally occurring regulatory Small 955, 956,957, 958, 960, 961, 964, 966,967,968, 969, 970, RNAs, for example, each segment is at least about 30 con 971,973, 976, 978,979,982, 983, 985, 987, 988,989, 991, tiguous nucleotides (or base-pairs) in length. In some 992,994, 995,996, 997,999, 1006, 1007, 1008, 1009, 1010, embodiments, the total length of the polynucleotide, or the 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, length of each segment contained in the polynucleotide, is 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, less than the total length of the DNA or target gene having a 1047, 1049, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, sequence selected from the Target Gene Sequences Group. In 1066,1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, Some embodiments, the total length of the polynucleotide is 1081, 1082, 1084, 1085, 1095, 1096, 1097,1098, 1099, 1100, between about 50 to about 500 nucleotides (for single 1101, 1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, 1114, stranded polynucleotides) or base-pairs (for double-stranded 1118, 1119, and 1124. polynucleotides). In some embodiments, the polynucleotide 0034. In various embodiments, the Leptinotarsa species is is a dsRNA of between about 100 to about 500 base-pairs, at least one selected from the group consisting of Leptino such as a dsRNA of the length of any of the dsRNA triggers tarsa behrensi, Leptinotarsa Collinsi, Leptinotarsa decemlin disclosed in Tables 3, 5, 8, 9, and 10. Embodiments include eata (Colorado potato beetle), Leptinotarsa defecta, Leptino those in which the polynucleotide expressed in the plant is an tarsa haldemani (Haldeman's green potato beetle), RNA comprising a segment having a sequence selected from Leptinotarsa heydeni, Leptinotarsa juncta (false potato the group consisting of: SEQID NOS:831-1085, 1095-1104, beetle), Leptinotarsa lineolata (burrobrush leaf beetle), Lep and 1110-11 14, or the complement thereof, or is an RNA tinotarsa peninsularis, Leptinotarsa rubiginosa, Leptino hairpin encoded by a sequence selected from the group con tarsa texana, Leptinotarsa tilascalana, Leptinotarsa sisting of SEQ ID NOs: 1105-1109. In some embodiments, tumamoca, and Leptinotarsa typographica. In specific the polynucleotide is expressed in a plant. In some embodi embodiments, the Leptinotarsa species is at least one selected ments, the polynucleotide is topically provided to the Surface from the group consisting of Leptinotarsa decemlineata of a plant or Leptinotarsa species. (Colorado potato beetle), Leptinotarsa juncta (false potato 0030 Several embodiments relate to polynucleotides that beetle), Leptinotarsa haldemani (Haldeman's green potato are designed to modulate expression by inducing regulation beetle), and Leptinotarsa lineolata (burrobrush leaf beetle). or Suppression of a Leptinotarsa species target gene. In some Controlling Leptinotarsa Infestations by Contacting with a embodiments, the polynucleotides are designed to have a Polynucleotide nucleotide sequence essentially identical or essentially 0035. Provided herein are methods for controlling a Lep complementary to the nucleotide sequence of a Leptinotarsa tinotarsa species infestation of a plant by contacting the Lep species target gene or cDNA (e.g., The Target Gene tinotarsa species with a polynucleotide comprising at least Sequences Group) or to the sequence of RNA transcribed one segment of 18 or more contiguous nucleotides having US 2015/O 143580 A1 May 21, 2015 about 95% to about 100% identity or complementarity with a embodiment, the polynucleotide comprises at least one seg corresponding fragment of a DNA or target gene selected ment of 21 contiguous nucleotides with 100% identity with from the group consisting of the Target Gene Sequences the corresponding fragment of a target gene having a DNA Group, or the DNA complement thereof. In an embodiment, sequence selected from the group consisting of SEQ ID the method for controlling a Leptinotarsa species infestation NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID of a plant comprises contacting the Leptinotarsa species with NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, a polynucleotide comprising at least 21 contiguous nucle and SEQ ID NOs: 1087-1094, or the DNA complement otides with 100% identity with a corresponding fragment of a thereof. In some embodiments, the polynucleotide comprises target gene having a DNA sequence selected from the group “neutral” sequence (sequence having no sequence identity or consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID complementarity to the target gene) in addition to one or more NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, segments of 21 contiguous nucleotides with 100% identity SEQID NOs:808-830, and SEQID NOs: 1087-1094, or the with the corresponding fragment of the target gene, and there DNA complement thereof. In some embodiments, the poly fore the polynucleotide as a whole is of much lower overall nucleotide is a double-stranded RNA. In some embodiments, sequence identity with a target gene. the polynucleotide (e.g., double-stranded RNA) is chemically 0037. Several embodiments relate to a polynucleotide synthesized or is produced by expression in a microorganism designed to Suppress one or more genes (“target genes'). The or by expression in a plant cell. Embodiments include those in term 'gene' refers to any portion of a nucleic acid that pro which the polynucleotide is a dsRNA comprising a sequence vides for expression of a transcript or encodes a transcript. A selected from the group consisting of SEQID NOS:831-1085, 'gene' can include, but is not limited to, a promoter region, 5' 1095-1104, and 1110-11 14, or the complement thereof, or untranslated regions, transcript encoding regions that can wherein the polynucleotide is encoded by a sequence selected include intronic regions, 3' untranslated regions, or combina from the group consisting of SEQID NOs: 1105-1109. In an tions of these regions. In some embodiments, the target genes embodiment, the method for controlling a Leptinotarsa spe can include coding or non-coding sequence or both. In other cies infestation of a plant comprises contacting the Leptino embodiments, the target gene has a sequence identical to or tarsa species with a polynucleotide comprising a nucleotide complementary to a messenger RNA, e.g., in Some embodi sequence that is complementary to at least 21 contiguous ments the target gene is a cDNA. In specific embodiments, the nucleotides of a target gene encoded by a nucleotide sequence polynucleotide is designed to suppress one or more target selected from the group consisting of: SEQID NO:730, SEQ genes, where each target gene is encoded by a DNA sequence IDNO:807, SEQID NOs: 1-725, SEQID NOs:726-729, SEQ selected from the Target Gene Sequences Group. In various ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: embodiments, the polynucleotide is designed to Suppress one 1087-1094, or an RNA transcribed from the target gene. or more target genes, where each target gene is encoded by a Embodiments include those in which the polynucleotide is a sequence selected from the Target Gene Sequences Group, dsRNA comprising a strand having a sequence selected from and can be designed to Suppress multiple target genes from the Trigger Sequences Group. In some embodiments, the this group, or to target different regions of one or more of method uses a polynucleotide comprising one segment of 127 these target genes. In an embodiment, the polynucleotide contiguous nucleotides (SEQID NO:831) which is the anti comprises multiple segments of 21 contiguous nucleotides sense (reverse complement) sequence of 127 contiguous with 100% identity with a fragment of equivalent length of a nucleotides of the target gene encoded by SEQID NO:825. In DNA or target gene having a sequence selected from the Some embodiments, the method uses a polynucleotide com Target Gene Sequences Group or the DNA complement prising segments of 409 and 403 contiguous nucleotides thereof. In Such cases, each segment can be identical or dif (SEQ ID NO:937 and SEQID NO:938, respectively) which ferent in size or in sequence, and can be sense or anti-sense are the anti-sense (reverse complement) sequences of 409 and relative to the target gene. For example, in one embodiment 403 contiguous nucleotides, respectively, of a target gene the polynucleotide comprises multiple segments in tandem or encoded by SEQ ID NO:732. Polynucleotides of use in the repetitive arrangements, wherein each segment comprises 21 method can be designed for multiple target genes. Related contiguous nucleotides with a sequence of 100% identity aspects of the invention include isolated polynucleotides of with a fragment of equivalent length of a DNA or target gene use in the method and plants having improved Leptinotarsa having a sequence selected from the the Target Gene resistance provided by the method. Sequences Group or the DNA complement thereof. In some 0036. In some embodiments, the contiguous nucleotides embodiments, the segments can be from different regions of have a sequence of about 95%, about 96%, about 97%, about the target gene, e.g., the segments can correspond to different 98%, about 99%, or about 100% identity with a fragment of exon regions of the target gene. In some embodiments, equivalent length of a DNA or target gene having a sequence 'spacer nucleotides which do not correspond to a target gene selected from the group consisting of: SEQ ID NOS:1-725 can optionally be used in between or adjacent to the segments. and SEQID NOs:726-830 and SEQ ID NOs: 1087-1094 or 0038. The total length of the polynucleotide of use in this the DNA complement thereof. In some embodiments, the method can be greater than 18 contiguous nucleotides, and contiguous nucleotides are exactly (100%) identical to a frag can include nucleotides in addition to the contiguous nucle ment of equivalent length of a DNA or target gene having a otides having the sequence of about 95% to about 100% sequence selected from the Target Gene Sequences Group or identity with a fragment of equivalent length of a DNA or the DNA complement thereof. In some embodiments, the target gene having a sequence selected from the group con polynucleotide has an overall sequence of about 95%, about sisting of the Target Gene Sequences Group or the DNA 96%, about 97%, about 98%, about 99%, or about 100% complement thereof. In other words, the total length of the identity with a fragment of equivalent length of a DNA or polynucleotide can be greater than the length of the section or target gene having a sequence selected from the Target Gene segment of the polynucleotide designed to suppress one or Sequences Group or the DNA complement thereof. In an more target genes, where each target gene has a DNA US 2015/O 143580 A1 May 21, 2015

sequence selected from the group consisting of the Target of the length of any of the dsRNA triggers disclosed in Tables Gene Sequences Group. For example, the polynucleotide can 3, 5, 8, 9, and 10. Embodiments include those in which the have nucleotides flanking the “active' segment of at least one polynucleotide is a dsRNA comprising a segment having a segment of 18 or more contiguous nucleotides that Suppresses sequence selected from the group consisting of SEQ ID the target gene, or include “spacer nucleotides between NOs:831-1085, 1095-1104, and 1110-11 14, or the comple active segments, or can have additional nucleotides at the 5' ment thereof, or wherein the polynucleotide is encoded by a end, or at the 3' end, or at both the 5' and 3' ends. In an sequence selected from the group consisting of SEQID NOs: embodiment, the polynucleotide can include additional 1105-1109. Alternatively the polynucleotide can be provided nucleotides that are not specifically related (having a in more complex constructs, e.g., as part of a recombinant sequence not complementary or identical to) to the DNA or expression construct, or included in a recombinant vector, for target gene having a sequence selected from the group con example in a recombinant plant virus vector or in a recombi sisting of the Target Gene Sequences Group or the DNA nant baculovirus vector. In some embodiments such recom complement thereof, e.g., nucleotides that provide stabilizing binant expression constructs or vectors are designed to secondary structure or for convenience in cloning or manu include additional elements, such as expression cassettes for facturing. In an embodiment, the polynucleotide can include expressing a gene of interest (e.g., an insecticidal protein). additional nucleotides located immediately adjacent to one or 0041. In various embodiments of the method, the contact more segment of 18 or more contiguous nucleotides with a ing comprises application to a surface of the Leptinotarsa sequence of about 95% to about 100% identity with a frag species of a suitable composition comprising the polynucle ment of equivalent length of a DNA or target gene having a otide of use in this method; such a composition can be pro sequence selected from the group consisting of the Target vided, e.g., as a solid, liquid (including homogeneous mix Gene Sequences Group or the DNA complement thereof. In tures such as solutions and non-homogeneous mixtures Such an embodiment, the polynucleotide comprises one such seg as Suspensions, colloids, micelles, and emulsions), powder, ment, with an additional 5' G or an additional 3' C or both, Suspension, emulsion, spray, encapsulated or micro-encapsu adjacent to the segment. In another embodiment, the poly lation formulation, in or on microbeads or other carrier par nucleotide is a double-stranded RNA comprising additional ticulates, in a film or coating, or on or within a matrix, or as a nucleotides to forman overhang, for example, a dsRNA com seed treatment. The contacting can be in the form of a seed prising 2 deoxyribonucleotides to form a 3' overhang. Thus in treatment, or in the form of treatment of “seed potato’ tubers various embodiments, the nucleotide sequence of the entire or pieces of tuber (e.g., by soaking, coating, or dusting the polynucleotide is not 100% identical or complementary to a seed potato). Suitable binders, inert carriers, surfactants, and sequence of contiguous nucleotides in the DNA or target gene the like can optionally be included in the composition, as is having a sequence selected from the group consisting of the known to one skilled in formulation of pesticides and seed Target Gene Sequences Group, or the DNA complement treatments. In some embodiments, the contacting comprises thereof. For example, in some embodiments the polynucle providing the polynucleotide in a composition that further otide comprises at least two segments each of 21 contiguous comprises one or more components selected from the group nucleotides with a sequence of 100% identity with a fragment consisting of a carrier agent, a Surfactant, a cationic lipid of a DNA having a sequence selected from the group consist (such as that disclosed in Example 18 of U.S. patent applica ing of the Target Gene Sequences Group, or the DNA tion publication 2011/0296.556, incorporated by reference complement thereof, wherein (1) the at least two segments are herein), an organosilicone, an organosilicone Surfactant, a separated by one or more spacer nucleotides, or (2) the at least polynucleotide herbicidal molecule, a non-polynucleotide two segments are arranged in an order different from that in herbicidal molecule, a non-polynucleotide pesticide, a which the corresponding fragments occur in the DNA having safener, and an insect growth regulator. In some embodi a sequence selected from the group consisting of the Target ments, the contacting comprises providing the polynucle Gene Sequences Group, or the DNA complement thereof. otide in a composition that further comprises at least one 0039. The polynucleotide ofuse in this method is provided pesticidal agent selected from the group consisting of a pata by suitable means known to one in the art. Embodiments tin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis include those wherein the polynucleotide is chemically Syn insecticidal protein, a Xenorhabdus insecticidal protein, a thesized (e.g., by in vitro transcription, Such as transcription Photorhabdus insecticidal protein, a Bacillus laterosporous using a T7 polymerase or other polymerase), produced by insecticidal protein, and a Bacillus sphaericus insecticidal expression in a microorganism or in cell culture (such as plant protein. In one embodiment the contacting comprises provid or insect cells grown in culture), produced by expression in a ing the polynucleotide in a composition that can be ingested plant cell, or produced by microbial fermentation. or otherwise absorbed internally by the Leptinotarsa species. 0040. In some embodiments the polynucleotide of use in 0042. It is anticipated that the combination of certain poly this method is provided as an isolated DNA or RNA fragment. nucleotides of use in this method (e.g., the polynucleotide In some embodiments the polynucleotide of use in this triggers described in the working Examples) with one or more method is not part of an expression construct and is lacking non-polynucleotide pesticidal agents will result in a syner additional elements such as a promoter or terminator getic improvement in prevention or control of Leptinotarsa sequences). Such polynucleotides can be relatively short, species infestations, when compared to the effect obtained Such as single- or double-stranded polynucleotides of with the polynucleotide alone or the non-polynucleotide pes between about 18 to about 300 or between about 50 to about ticidal agent alone. In an embodiment, a composition con 500 nucleotides (for single-stranded polynucleotides) or taining one or more polynucleotides and one or more non between about 18 to about 300 or between about 50 to about polynucleotide pesticidal agent selected from the group 500 base-pairs (for double-stranded polynucleotides). In consisting of a patatin, a plant lectin, a phytoecdysteroid, a some embodiments, the polynucleotide is a dsRNA of Bacillus thuringiensis insecticidal protein, a Xenorhabdus between about 100 to about 500 base-pairs, such as a dsRNA insecticidal protein, a Photorhabdus insecticidal protein, a US 2015/O 143580 A1 May 21, 2015

Bacillus laterosporous insecticidal protein, and a Bacillus emulsion, spray, encapsulated or micro-encapsulation formu sphaericus insecticidal protein, is found to effect synergisti lation, in or on microbeads or other carrier particulates, in a cally improved prevention or control of Leptinotarsa species film or coating, or on or within a matrix, or as a seed treat infestations. ment. The agent comprising a polynucleotide can be provided for dietary uptake by the Leptinotarsa species by applying the Controlling Leptinotarsa Infestations by Providing a Dietary agent to a plant Subject to infestation by the Leptinotarsa Polynucleotide species or by applying the agent to seed of the plant, for 0043. Another aspect of this invention provides a method example by spraying, dusting, or coating the plant, or by for controlling a Leptinotarsa species infestation of a plant application of a soil drench, or by providing in an artificial comprising providing in the diet of a Leptinotarsa species an diet. The agent comprising a polynucleotide can be provided agent comprising a polynucleotide having at least one seg for dietary uptake by the Leptinotarsa species in an artificial ment of 18 or more contiguous nucleotides with a sequence of diet formulated to meet the particular nutritional require about 95% to about 100% identity with a fragment of equiva ments for maintaining the Leptinotarsa species, wherein the lent length of a DNA having a sequence selected from the artificial diet is Supplemented with some amount of the poly group consisting of The Target Gene Sequences Group or the nucleotide obtained from a separate source such as chemical DNA complement thereof, wherein the agent functions upon synthesis or purified from a microbial fermentation; this ingestion by the Leptinotarsa species to inhibit a biological embodiment can be useful, e.g., for determining the timing function within the Leptinotarsa species thereby controlling and amounts of effective polynucleotide treatment regimes. infestation by the Leptinotarsa species. The polynucleotide In some embodiments the agent comprising a polynucleotide can be longer than the segment or segments it contains, but is provided for dietary uptake by the Leptinotarsa species in each polynucleotide segment and corresponding DNA frag the form of a plant cell or in plant cell components, or in a ment are of equivalent length. Polynucleotides of use in the microorganism (such as a bacterium or a yeast) or a microbial method can be designed for multiple target genes. Embodi fermentation product, or in a synthetic or man-made diet. In ments include those in which the agent comprises a dsRNA one embodiment the agent comprising a polynucleotide is comprising a segment having a sequence selected from the provided in the form of bait that is ingested by the Leptino group consisting of: SEQID NOS:831-1085, 1095-1104, and tarsa species. The agent comprising a polynucleotide can be 1110-11 14, or the complement thereof, or wherein the agent provided for dietary uptake by the Leptinotarsa species in the comprises a polynucleotide or RNA encoded by a sequence form of a seed treatment, or in the form of treatment of “seed selected from the group consisting of SEQ ID NOS:1105 potato” tubers or pieces of tuber (e.g., by soaking, coating, or 1109. In an embodiment, a method for controlling a Leptino dusting the seed potato). Suitable binders, inert carriers, Sur tarsa species infestation of a plant comprising providing in factants, and the like can be included in the agent, as is known the diet of the Leptinotarsa species a polynucleotide compris to one skilled in formulation of pesticides and seed treat ing a nucleotide sequence that is complementary to at least 21 ments. In some embodiments, the agent comprising a poly contiguous nucleotides of a target gene having a nucleotide nucleotide further comprises one or more components sequence selected from the group consisting of SEQ ID selected from the group consisting of a carrier agent, a Sur NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID factant, a cationic lipid (Such as that disclosed in Example 18 NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, of U.S. patent application publication 2011/0296.556, incor and SEQIDNOs: 1087-1094, oran RNA transcribed from the porated by reference herein), an organosilicone, an organo target gene is provided. In some embodiments, the polynucle silicone Surfactant, a polynucleotide herbicidal molecule, a otide is a double-stranded RNA. In some embodiments, the non-polynucleotide herbicidal molecule, a non-polynucle polynucleotide (e.g., double-stranded RNA) is chemically otide pesticide, a safener, and an insect growth regulator. In synthesized or is produced by expression in a microorganism Some embodiments, the agent comprising a polynucleotide or by expression in a plant cell. Embodiments include those in further comprises at least one pesticidal agent selected from which the polynucleotide is a dsRNA with a strand having a the group consisting of a patatin, a plant lectin, a phytoecdys sequence selected from the group consisting of the Trigger teroid, a phytoecdysteroid, a Bacillus thuringiensis insecti Sequences Group. Related aspects of the invention include cidal protein, a Xenorhabdus insecticidal protein, a Photo isolated polynucleotides of use in the method and plants rhabdus insecticidal protein, a Bacillus laterosporous having improved Leptinotarsa resistance provided by the insecticidal protein, and a Bacillus sphaericus insecticidal method. protein. In some embodiments, the agent comprising a poly 0044. In various embodiments, the agent comprising a nucleotide comprises at least one implantable formulation polynucleotide comprises a microbial cell or is produced in a selected from the group consisting of a particulate, pellet, or microorganism. For example, the agent can include or can be capsule implanted in the plant; in Such embodiments the produced in bacteria or yeast cells. In other embodiments the method comprises implanting in the plant the implantable agent comprising a polynucleotide comprises a transgenic formulation. In some embodiments, the agent comprising a plant cell or is produced in a plant cell (for example a plant polynucleotide comprises at least one in-furrow formulation cell transiently expressing the polynucleotide); Such plant selected from the group consisting of a powder, granule, cells can be cells in an plant or cells grown in tissue culture or pellet, capsule, spray, or drench, or any otherforms Suited for in cell Suspension. applying to a furrow; in Such embodiments, the method com 0045. In various embodiments, the agent comprising a prises an in-furrow treatment with the in-furrow formulation. polynucleotide is provided for dietary uptake by the Leptino In some embodiments, the method comprises treatment of a tarsa species in a form Suitable for ingestion, for example, as Solanaceous plant seed, potato tuber, or piece of potato tuber a solid, liquid (including homogeneous mixtures such as solu with the agent. tions and non-homogeneous mixtures such as Suspensions, 0046. It is anticipated that the combination of certain poly colloids, micelles, and emulsions), powder, Suspension, nucleotides of use in agents of use in this method (e.g., the US 2015/O 143580 A1 May 21, 2015 polynucleotide triggers described in the working Examples) genes, where each target gene has a DNA sequence selected with one or more non-polynucleotide pesticidal agents will from the group consisting of the Target Gene Sequences result in a synergetic improvement in prevention or control of Group. In various embodiments, the polynucleotide is Leptinotarsa species infestations, when compared to the designed to Suppress one or more target genes, where each effect obtained with the polynucleotide alone or the non target gene has a sequence selected from the group consisting polynucleotide pesticidal agent alone. In an embodiment, a of the Target Gene Sequences Group, and can be designed to composition containing one or more polynucleotides and one Suppress multiple target genes from this group, or to target or more non-polynucleotide pesticidal agent selected from different regions of one or more of these target genes. In an the group consisting of a patatin, a plant lectin, a phytoecdys embodiment, the polynucleotide comprises multiple seg teroid, a Bacillus thuringiensis insecticidal protein, a ments of 21 contiguous nucleotides with a sequence of 100% Xenorhabdus insecticidal protein, a Photorhabdus insecti identity with a fragment of equivalent length of a DNA or cidal protein, a Bacillus laterosporous insecticidal protein, target gene having a sequence selected from The Target Gene and a Bacillus sphaericus insecticidal protein, is found to Sequences Group or the DNA complement thereof. In such effect synergistically improved prevention or control of Lep cases, each segment can be identical or different in size or in tinotarsa species infestations when provided to the Leptino sequence, and can be sense or anti-sense relative to the target tarsa species in a diet. gene. For example, in one embodiment the polynucleotide 0047. In some embodiments, the polynucleotide is a comprises multiple segments in tandem or repetitive arrange dsRNA comprising a segment having a sequence selected ments, wherein each segment comprises 21 contiguous nucle from the group consisting of: SEQID NOs:831-1085, 1095 otides with a sequence of 100% identity with a fragment of 1104, and 1110-11 14, or the complement thereof, or wherein equivalent length of a DNA or target gene having a sequence the polynucleotide is encoded by a sequence selected from selected from The Target Gene Sequences Group or the DNA the group consisting of SEQID NOs: 1105-1109. complement thereof; the segments can be from different 0048. In some embodiments, the contiguous nucleotides regions of the target gene, e.g., the segments can correspond have a sequence of about 95%, about 96%, about 97%, about to different exon regions of the target gene, and 'spacer” 98%, about 99%, or about 100% identity with a fragment of nucleotides which do not correspond to a target gene can equivalent length of a DNA or target gene having a sequence optionally be used in between or adjacent to the segments. selected from The Target Gene Sequences Group or the DNA 0050. The total length of the polynucleotide of use in this complement thereof. In some embodiments the contiguous method can be greater than 18 contiguous nucleotides, and nucleotides are exactly (100%) identical to a fragment of can include nucleotides in addition to the contiguous nucle equivalent length of a DNA or target gene having a sequence otides having the sequence of about 95% to about 100% selected from The Target Gene Sequences Group or the DNA identity with a fragment of equivalent length of a DNA or complement thereof. In some embodiments, the polynucle target gene having a sequence selected from The Target Gene otide has an overall sequence of about 95%, about 96%, about Sequences Group or the DNA complement thereof. In other 97%, about 98%, about 99%, or about 100% identity with a words, the total length of the polynucleotide can be greater fragment of equivalent length of a DNA or target gene having than the length of the section or segment of the polynucleotide a sequence selected from The Target Gene Sequences Group designed to Suppress one or more target genes, where each or the DNA complement thereof. In an embodiment, the target gene has a DNA sequence selected from the group polynucleotide comprises at least one segment of 21 contigu consisting of the Target Gene Sequences Group. For example, ous nucleotides with a sequence of 100% identity with the the polynucleotide can have nucleotides flanking the “active' corresponding fragment of a target gene having a DNA segment of at least one segment of 18 or more contiguous sequence selected from the group consisting of SEQ ID nucleotides that Suppresses the target gene, or include NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID 'spacer nucleotides between active segments, or can have NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, additional nucleotides at the 5' end, or at the 3' end, or at both and SEQ ID NOs: 1087-1094, or the DNA complement the 5' and 3' ends. In an embodiment, the polynucleotide can thereof; in Some embodiments, the polynucleotide comprises include additional nucleotides that are not specifically related “neutral” sequence (having no sequence identity or comple (having a sequence not complementary or identical to) to the mentarity to the target gene) in addition to a segment of 21 DNA or target gene having a sequence selected from The contiguous nucleotides with 100% identity with the corre Target Gene Sequences Group or the DNA complement sponding fragment of the target gene, and therefore the poly thereof, e.g., nucleotides that provide stabilizing secondary nucleotide as a whole is of much lower overall sequence structure or for convenience in cloning or manufacturing. In identity with a target gene. an embodiment, the polynucleotide can include additional 0049. The polynucleotide of use in this method is gener nucleotides located immediately adjacent to one or more ally designed to Suppress one or more genes (“target genes'). segment of 18 or more contiguous nucleotides with a The term “gene' refers to any portion of a nucleic acid that sequence of about 95% to about 100% identity with a frag provides for expression of a transcript or encodes a transcript. ment of equivalent length of a DNA or target gene having a A "gene' can include, but is not limited to, a promoter region, sequence selected from The Target Gene Sequences Group or 5' untranslated regions, transcript encoding regions that can the DNA complement thereof. In an embodiment, the poly include intronic regions, 3' untranslated regions, or combina nucleotide comprises one such segment, with an additional 5' tions of these regions. In some embodiments, the target genes G or an additional 3' C or both, adjacent to the segment. In can include coding or non-coding sequence or both. In other another embodiment, the polynucleotide is a double-stranded embodiments, the target gene has a sequence identical to or RNA comprising additional nucleotides to forman overhang, complementary to a messenger RNA, e.g., in Some embodi for example, a dsRNA comprising 2 deoxyribonucleotides to ments the target gene is a cDNA. In specific embodiments, the form a 3' overhang. Thus in various embodiments, the nucle polynucleotide is designed to suppress one or more target otide sequence of the entire polynucleotide is not 100% iden US 2015/O 143580 A1 May 21, 2015 tical or complementary to a sequence of contiguous nucle comprising at least one silencing element essentially identical otides in the DNA or target gene having a sequence selected or essentially complementary to a fragment of a target gene from The Target Gene Sequences Group, or the DNA comple sequence of the Leptinotarsa species larvae, wherein the tar ment thereof. For example, in some embodiments the poly get gene sequence is selected from the group consisting of the nucleotide comprises at least two segments of 21 contiguous Target Gene Sequences Group, and wherein ingestion of the nucleotides with a sequence of 100% identity with a fragment RNA by the Leptinotarsa species larvae results in mortality or of a DNA having a sequence selected from The Target Gene stunting in the Leptinotarsa species larvae is provided. A Sequences Group, or the DNA complement thereof, wherein related aspect of this invention is an RNA comprising at least (1) the at least two segments are separated by one or more one silencing element, wherein the at least one silencing spacer nucleotides, or (2) the at least two segments are element is essentially identical or essentially complementary arranged in an order different from that in which the corre sponding fragments occur in the DNA having a sequence to a fragment of a target gene of the Leptinotarsa species selected from The Target Gene Sequences Group, or the DNA larvae, wherein the target gene sequence is selected from the complement thereof. group consisting of the Target Gene Sequences Group. The 0051. The polynucleotide ofuse in this method is provided RNA can be longer than the silencing element or silencing by suitable means known to one in the art. Embodiments elements it contains, but each silencing element and corre include those wherein the polynucleotide is chemically Syn sponding fragment of a target gene sequence are of equivalent thesized (e.g., by in vitro transcription, Such as transcription length. RNAs of use in the method can be designed for mul using a T7 polymerase or other polymerase), produced by tiple target genes; embodiments include RNAS comprising at expression in a microorganism or in cell culture (such as plant least one silencing element comprising a sequence selected or insect cells grown in culture), produced by expression in a from the group consisting of: SEQID NOs:831-1085, 1095 plant cell, or produced by microbial fermentation. 1104, and 1110-11 14, or the complement thereof, or wherein 0052. In some embodiments the polynucleotide of use in the silencing element is encoded by a sequence selected from this method is provided as an isolated DNA or RNA fragment. the group consisting of SEQ ID NOs: 1105-1109. Embodi In some embodiments the polynucleotide of use in this ments include those in which the RNA comprises a dsRNA method is not part of an expression construct and is lacking with a strand having a sequence selected from the group additional elements such as a promoter or terminator consisting of the Trigger Sequences Group. In a related sequences. Such polynucleotides can be relatively short. Such aspect, a method of causing mortality or lower fecundity in as single- or double-stranded polynucleotides of between Leptinotarsa species comprising providing in the diet of Lep about 18 to about 300 or between about 50 to about 500 tinotarsa species at least one RNA comprising at least one nucleotides (for single-stranded polynucleotides) or between silencing element essentially identical or essentially comple about 18 to about 300 or between about 50 to about 500 mentary to a fragment of a target gene sequence of the Lep base-pairs (for double-stranded polynucleotides). In some tinotarsa species larvae, wherein the target gene sequence is embodiments, the polynucleotide is a dsRNA of between selected from The Target Gene Sequences Group, or the DNA about 100 to about 500 base-pairs, such as a dsRNA of the complement thereof, and wherein ingestion of the RNA by length of any of the dsRNA triggers disclosed in Tables 3, 5, the Leptinotarsa species results in mortality or lower fecun 8,9, and 10. Alternatively the polynucleotide can be provided dity in the Leptinotarsa species is provided. Related aspects in more complex constructs, e.g., as part of a recombinant of the invention include isolated RNAs of use in the method expression construct, or included in a recombinant vector, for and plants having improved Leptinotarsa resistance provided example in a recombinant plant virus vector or in a recombi by the method. nant baculovirus vector. In some embodiments such recom 0054 Invarious embodiments, the diet providing the RNA binant expression constructs or vectors are designed to comprises a microbial cellor is produced in a microorganism. include additional elements, such as expression cassettes for For example, the diet providing the RNA can include or can expressing a gene of interest (e.g., an insecticidal protein). be produced in bacteria or yeast cells. In similar embodiments the diet providing the RNA comprises a transgenic plant cell Controlling Leptinotarsa Infestations by Providing a Dietary or is produced in a plant cell (for example a plant cell tran RNA siently expressing the polynucleotide); Such plant cells can be 0053 Another aspect of this invention provides a method cells in an plant or cells grown in tissue culture or in cell of causing mortality or stunting in larvae of the Leptinotarsa Suspension. species by providing in the diet of the larvae at least one 0055. In one embodiment the diet providing the RNA is polynucleotide comprising at least one silencing element provided in the form of any plant that is subject to infestation comprising 21 contiguous nucleotides that are complemen by a Leptinotarsa species, wherein the RNA is contained in or tary to a target gene having a nucleotide sequence selected on the plant. Such plants can be stably transgenic plants that from the group consisting of: SEQ ID NO:730, SEQ ID express the RNA, or non-transgenic plants that transiently NO:807, SEQID NOs: 1-725, SEQIDNOs:726-729, SEQID express the RNA or that have been treated with the RNA, e.g., NOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs: by spraying or coating. Stably transgenic plants generally 1087-1094, or an RNA transcribed from the target gene. In contain integrated into their genome a recombinant construct Some embodiments, the polynucleotide is a double-stranded that encodes the RNA. Of particular interest are embodiments RNA. In some embodiments, the polynucleotide (e.g., wherein the plant is a Solanaceous plant (family Solanaceae). double-stranded RNA) is chemically synthesized or is pro Examples include a plant selected from the group consisting duced by expression in a microorganism or by expression in of potato, tomato, and eggplant. Embodiments include those a plant cell. In an embodiment, a method of causing mortality wherein the plant is an ungerminated Solanaceous plant seed, or stunting in Leptinotarsa species larvae comprising provid a Solanaceous plant in a vegetative stage, or a Solanaceous ing in the diet of Leptinotarsa species larvae at least one RNA plant in a reproductive stage. Embodiments include those US 2015/O 143580 A1 May 21, 2015

wherein the plant is a 'seed potato’, meaning a potato tuberor tion. In some embodiments, the method comprises treatment piece of potato tuber which can be propagated into new potato of a Solanaceous plant seed, potato tuber, or piece of potato plants. tuber with the agent. 0057. It is anticipated that the combination of certain 0056 Invarious embodiments, the diet providing the RNA RNAs of use in this method (e.g., the dsRNA triggers is provided in a form Suitable for ingestion by the Leptino described in the working Examples) with one or more non tarsa species, for example, as a Solid, liquid (including homo polynucleotide pesticidal agents will result in a synergetic geneous mixtures such as solutions and non-homogeneous improvement in prevention or control of Leptinotarsa species mixtures such as Suspensions, colloids, micelles, and emul infestations, when compared to the effect obtained with the sions), powder, Suspension, emulsion, spray, encapsulated or RNA alone or the non-polynucleotide pesticidal agent alone. micro-encapsulation formulation, in or on microbeads or In an embodiment, a composition containing one or more other carrier particulates, in a film or coating, or on or within RNAS and one or more non-polynucleotide pesticidal agent a matrix, or as a seed treatment. The diet providing the RNA selected from the group consisting of a patatin, a plant lectin, can be provided by applying the diet to a plant Subject to a phytoecdysteroid, a Bacillus thuringiensis insecticidal pro infestation by the Leptinotarsa species, for example by spray tein, a Xenorhabdus insecticidal protein, a Photorhabdus ing, dusting, or coating the plant, or by application of a soil insecticidal protein, a Bacillus laterosporous insecticidal pro drench, or by providing in an artificial diet. In one embodi tein, and a Bacillus sphaericus insecticidal protein, is found ment the diet providing the recombinant RNA is provided in to effect synergistically improved prevention or control of the form of bait that is ingested by the Leptinotarsa species. Leptinotarsa species infestations. The diet providing the RNA can be an artificial diet formu 0058. The RNA of use in this method can be single lated to meet the particular nutritional requirements for main stranded (ss) or double-stranded (ds). Embodiments of the taining the Leptinotarsa species, wherein the artificial diet is method include those wherein the RNA is at least one selected supplemented with some amount of the RNA obtained from a from the group consisting of sense single-stranded RNA (SS separate source Such as chemical synthesis or purified from a RNA), anti-sense single-stranded (ssRNA), or double microbial fermentation; this embodiment can be useful, e.g., stranded RNA (dsRNA); a mixture of RNAs of any of these for determining the timing and amounts of effective poly types can be used. In one embodiment a double-stranded nucleotide treatment regimes. In some embodiments the diet DNA/RNA hybrid is used. The RNA can include components providing the RNA is provided in the form of a plant cellor in other than standard ribonucleotides, e.g., an embodiment is plant cell components, or in a microorganism (such as a an RNA that comprises terminal deoxyribonucleotides. bacterium or a yeast) or a microbial fermentation product, or 0059. The RNA comprises at least one silencing element, in a synthetic diet. In one embodiment the diet providing the wherein the silencing element is essentially identical (as the RNA is provided in the form of bait that is ingested by the RNA equivalent) or essentially complementary to a fragment Leptinotarsa species. The diet providing the RNA can be of a target gene of the Leptinotarsa species larvae, wherein provided in the form of a seed treatment, or in the form of the target gene sequence is selected from the group consisting treatment of “seed potato” tubers or pieces of tuber (e.g., by of the Target Gene Sequences Group. In some embodiments, soaking, coating, or dusting the seed potato). Suitable bind the silencing element has a sequence of about 95%, about ers, inert carriers, Surfactants, and the like can be included in 96%, about 97%, about 98%, about 99%, or about 100% the diet, as is known to one skilled informulation of pesticides identity with or complementarity to a fragment of equivalent and seed treatments. In some embodiments, the diet provid length of a DNA having a sequence selected from the group ing the RNA further comprises one or more components consisting of the Target Gene Sequences Group. In some selected from the group consisting of a carrier agent, a Sur embodiments the silencing element is exactly (100%) iden factant, a cationic lipid (Such as that disclosed in Example 18 tical or exactly (100%) complementary (as the RNA equiva of U.S. patent application publication 2011/0296.556, incor lent) to a fragment of equivalent length of a DNA having a porated by reference herein), an organosilicone, an organo sequence selected from The Target Gene Sequences Group or silicone Surfactant, a polynucleotide herbicidal molecule, a the DNA complement thereof. In some embodiments, the non-polynucleotide herbicidal molecule, a non-polynucle RNA containing the silencing element(s) has an overall otide pesticide, a safener, and an insect growth regulator. In sequence of about 95%, about 96%, about 97%, about 98%, some embodiments, the diet providing the RNA further com about 99%, or about 100% identity with or complementarity prises at least one pesticidal agent selected from the group to the fragment of a DNA having a sequence selected from the consisting of a patatin, a plant lectin, a phytoecdysteroid, a group consisting of the Target Gene Sequences Group. Bacillus thuringiensis insecticidal protein, a Xenorhabdus 0060. In some embodiments, the silencing element com insecticidal protein, a Photorhabdus insecticidal protein, a prises at least one segment of 18 or more contiguous nucle Bacillus laterosporous insecticidal protein, and a Bacillus otides with a sequence of about 95% to about 100% identity sphaericus insecticidal protein. In some embodiments, the with or complementarity to a fragment of equivalent length of diet providing the RNA includes at least one implantable the target gene. In some embodiments the silencing element formulation selected from the group consisting of a particu comprises at least one segment of 18 or more contiguous late, pellet, or capsule implanted in the plant; in Such embodi nucleotides with a sequence of about 95% to about 100% ments the method comprises implanting in the plant the identity with or complementarity to a fragment of equivalent implantable formulation. In some embodiments, the diet pro length of a DNA having a sequence selected from the group viding the RNA includes at least one in-furrow formulation consisting of the Target Gene Sequences Group. In some selected from the group consisting of a powder, granule, embodiments the silencing element comprises at least one pellet, capsule, spray, or drench, or any other forms Suited for segment of 18 or more contiguous nucleotides, e.g., between applying to a furrow; in Such embodiments, the method 18-24, or between 18-28, or between 20-30, or between includes an in-furrow treatment with the in-furrow formula 20-50, or between 20-100, or between 50-100, or between US 2015/O 143580 A1 May 21, 2015

50-500, or between 100-250, or between 100-500, or between consisting of the Target Gene Sequences Group. In various 200-1000, or between 500-2000, or even greater. In some embodiments, the RNA is designed to Suppress one or more embodiments the silencing element comprises more than 18 genes, where each gene has a sequence selected from the contiguous nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27. group consisting of the Target Gene Sequences Group, and 28, 29, 30, or greater than 30, e.g., about 35, about 40, about can be designed to Suppress multiple genes from this group, 45, about 50, about 55, about 60, about 65, about 70, about 75, or to target different regions of one or more of these genes. In about 80, about 85, about 90, about 95, about 100, about 110, an embodiment, the RNA comprises multiple silencing ele about 120, about 130, about 140, about 150, about 160, about ments each of which comprises at least one segment of 21 170, about 180, about 190, about 200, about 210, about 220, contiguous nucleotides with a sequence of 100% identity about 230, about 240, about 250, about 260, about 270, about with or 100% complementarity to a fragment of equivalent 280, about 290, about 300, about 350, about 400, about 450, length of a DNA having a sequence selected from The Target about 500, or greater than 500 contiguous nucleotides. In Gene Sequences Group or the DNA complement thereof. In particular embodiments, the silencing element comprises at Such cases, each silencing element can be identical or differ least one segment of at least 21 contiguous nucleotides with a ent in size or in sequence, and can be sense or anti-sense sequence of 100% identity with a fragment of equivalent relative to the target gene. For example, in one embodiment length of a DNA or target gene having a sequence selected the RNA can include multiple silencing elements in tandem from The Target Gene Sequences Group or the DNA comple or repetitive arrangements, wherein each silencing element ment thereof. In particular embodiments, the RNA is a comprises at least one segment of 21 contiguous nucleotides double-stranded nucleic acid (e.g., dsRNA) with one strand with a sequence of 100% identity with or 100% complemen comprising at least one segment of at least 21 contiguous tarity to a fragment of equivalent length of a DNA having a nucleotides with a sequence of 100% identity with a fragment sequence selected from the group consisting of the Target of equivalent length of a DNA or target gene having a Gene Sequences Group; the segments can be from different sequence selected from The Target Gene Sequences Group or regions of the target gene, e.g., the segments can correspond the DNA complement thereof; expressed as base-pairs, such to different exon regions of the target gene, and 'spacer” a double-stranded nucleic acid comprises at least one seg nucleotides which do not correspond to a target gene can ment of at least 21 contiguous, perfectly matched base-pairs optionally be used in between or adjacent to the segments. which correspond to a fragment of equivalent length of a 0062. The total length of the RNA can be greater than 18 DNA or target gene having a sequence selected from The contiguous nucleotides, and can include nucleotides in addi Target Gene Sequences Group or the DNA complement tion to the silencing element having a sequence of about 95% thereof. In particular embodiments, each silencing element to about 100% identity with or complementarity to a fragment contained in the RNA is of a length greater than that which is of equivalent length of a DNA or target gene having a typical of naturally occurring regulatory Small RNAS, e.g., sequence selected from the group consisting of the Target each segment is at least about 30 contiguous nucleotides (or Gene Sequences Group. In other words, the total length of the base-pairs) in length. In some embodiments, the total length RNA can be greater than the length of the silencing element of the RNA, or the length of each silencing element contained designed to Suppress one or more target genes, where each in the RNA, is less than the total length of the sequence of target gene has a DNA sequence selected from the group interest (DNA or target gene having a sequence selected from consisting of the Target Gene Sequences Group. For example, the group consisting of the Target Gene Sequences Group). In the RNA can have nucleotides flanking the “active' silencing some embodiments, the total length of the RNA is between element of at least one segment of 18 or more contiguous about 50 to about 500 nucleotides (for single-stranded poly nucleotides that Suppresses the target gene, or include nucleotides) or base-pairs (for double-stranded polynucle 'spacer nucleotides between active silencing elements, or otides). In some embodiments, the RNA is a dsRNA of can have additional nucleotides at the 5' end, or at the 3' end, between about 100 to about 500 base-pairs, such as a dsRNA or at both the 5' and 3' ends. In an embodiment, the RNA of the length of any of the dsRNA triggers disclosed in Tables comprises additional nucleotides that are not specifically 3, 5, 8, 9, and 10. Embodiments include those in which the related (ihaving a sequence not complementary or identical RNA is a dsRNA comprising a segment having a sequence to) to the DNA or target genehaving a sequence selected from selected from the group consisting of: SEQ ID NOs:831 The Target Gene Sequences Group or the DNA complement 1085, 1095-1104, and 1110-11 14, or the complement thereof, thereof, e.g., nucleotides that provide stabilizing secondary or wherein the RNA is encoded by a sequence selected from structure or for convenience in cloning or manufacturing. In the group consisting of SEQID NOs: 1105-1109. an embodiment, the RNA comprises additional nucleotides 0061 The RNA ofuse in this method is generally designed located immediately adjacent to one or more silencing ele to Suppress one or more genes (“target genes'). The term ment of 18 or more contiguous nucleotides with a sequence of “gene' refers to any portion of a nucleic acid that provides for about 95% to about 100% identity with or complementarity to expression of a transcript or encodes a transcript. A "gene’ a fragment of equivalent length of a DNA or target gene can include, but is not limited to, a promoter region, 5' having a sequence selected from the group consisting of the untranslated regions, transcript encoding regions that can Target Gene Sequences Group. In an embodiment, the RNA include intronic regions, 3' untranslated regions, or combina comprises one such silencing element, with an additional 5'G tions of these regions. In some embodiments, the target genes oran additional 3' C or both, adjacent to the silencing element. can include coding or non-coding sequence or both. In other In another embodiment, the RNA is a double-stranded RNA embodiments, the target gene has a sequence identical to or comprising additional nucleotides to form an overhang, for complementary to a messenger RNA, e.g., in Some embodi example, a dsRNA comprising 2 deoxyribonucleotides to ments the target gene is a cDNA. In specific embodiments, the form a 3' overhang. Thus in various embodiments, the nucle RNA is designed to Suppress one or more target genes, where otide sequence of the entire RNA is not 100% identical or each target gene has a DNA sequence selected from the group complementary to a fragment of contiguous nucleotides in US 2015/O 143580 A1 May 21, 2015

the DNA or target gene having a sequence selected from the relative to an untreated plant. In an embodiment, the at least group consisting of the Target Gene Sequences Group. For one polynucleotide comprises at least one segment of 18 or example, in some embodiments the RNA comprises at least more contiguous nucleotides that are essentially identical to a two silencing elements each of 21 contiguous nucleotides fragment of equivalent length of a DNA having a sequence with a sequence of 100% identity with a fragment of a DNA selected from the Target Gene Sequences Group, or the DNA having a sequence selected from the group consisting of the complement thereof. The polynucleotide can be longer than Target Gene Sequences Group4, or the DNA complement the segment or segments it contains, but each segment and thereof, wherein (1) the at least two silencing elements are corresponding fragment of a target gene are of equivalent separated by one or more spacer nucleotides, or (2) the at least length. In an embodiment, this invention provides a method of two silencing elements are arranged in an order different from providing a plant having improved resistance to a Leptino that in which the corresponding fragments occur in the DNA tarsa species infestation comprising topically applying to the having a sequence selected from the group consisting of the plant a composition comprising at least one polynucleotide Target Gene Sequences Group, or the DNA complement comprising a nucleotide sequence that is complementary to at thereof. least 21 contiguous nucleotides of a target gene having a 0063. In some embodiments the RNA consists of naturally nucleotide sequence selected from the group consisting of occurring ribonucleotides. In certain embodiments, the RNA SEQID NO:730, SEQIDNO:807, SEQID NOs: 1-725, SEQ comprises components other than ribonucleotides, for ID NOs:726-729, SEQID NOs:731-806, SEQID NOs:808 example, synthetic RNAS consisting mainly of ribonucle 830, and SEQ ID NOs: 1087-1094, or an RNA transcribed otides but with one or more terminal deoxyribonucleotides or from the target gene. In an embodiment, this invention pro one or more terminal dideoxyribonucleotides. In certain vides a method of providing a plant having improved resis embodiments, the RNA comprises non-canonical nucleotides tance to a Leptinotarsa species infestation comprising topi Such as inosine, thiouridine, or pseudouridine. In certain cally applying to the plant a composition comprising at least embodiments, the RNA comprises chemically modified one polynucleotide in a manner Such that an effective amount nucleotides. of the polynucleotide is ingested by Leptinotarsa species 0064. The RNA of use in this method is provided by suit feeding on the plant, the polynucleotide comprising at least able means known to one in the art. Embodiments include 21 contiguous nucleotides that are complementary to a target those wherein the RNA is chemically synthesized (e.g., by in gene having a nucleotide sequence selected from the group vitro transcription, Such as transcription using a T7 poly consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID merase or other polymerase), produced by expression in a NOs: 1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, microorganism or in cell culture (such as plant or insect cells SEQID NOs:808-830, and SEQ ID NOs: 1087-1094, or an grown in culture), produced by expression in a plant cell, or RNA transcribed from the target gene. In some embodiments, produced by microbial fermentation. this invention provides a method for controlling a Leptino 0065. In some embodiments the RNA is provided as an tarsa species infestation of a plant comprising topically isolated RNA that is not part of an expression construct and is applying to the plant a composition comprising at least one lacking additional elements such as a promoter or terminator polynucleotide in a manner Such that an effective amount of sequences. Such RNAS can be relatively short, such as single the polynucleotide is ingested by Leptinotarsa species feed or double-stranded RNAs of between about 18 to about 300 or ing on the plant, the polynucleotide comprising a nucleotide between about 50 to about 500 nucleotides (for single sequence that is complementary to at least 21 contiguous stranded RNAs) or between about 18 to about 300 or between nucleotides of a target gene having a nucleotide sequence about 50 to about 500 base-pairs (for double-stranded RNAs). selected from the group consisting of: SEQID NO:730, SEQ Alternatively the RNA can be provided in more complex ID NO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ constructs, e.g., as part of a recombinant expression con ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: struct, or included in a recombinant vector, for example in a 1087-1094, or an RNA transcribed from the target gene; recombinant plant virus vector or in a recombinant baculovi wherein the Leptinotarsa species is Leptinotarsa decemlin rus vector. In some embodiments such recombinant expres eata; and wherein the target gene has the sequence of SEQID sion constructs or vectors are designed to include additional NO:730 or wherein the polynucleotide is a double-stranded elements, such as including additional RNA encoding an RNA having a strand with a sequence selected from the group aptamer or ribozyme or an expression cassette for expressing consisting of SEQ ID NO:989, 988, 1104, or 1105. Poly a gene of interest (e.g., an insecticidal protein). nucleotides of use in the method can be designed for multiple target genes. Embodiments include those in which the poly Methods of Providing Plants Having Improved Resistance to nucleotide comprises a segment having a sequence selected Leptinotarsa Infestations, and the Plants, Plant Parts, and from the group consisting of: SEQID NOs:831-1085, 1095 Seeds Thus Provided 1104, and 1110-11 14, or the complement thereof, or wherein 0066. Another aspect of this invention provides a method the polynucleotide is encoded by a sequence selected from of providing a plant having improved resistance to a Leptino the group consisting of SEQ ID NOs: 1105-1109. Embodi tarsa species infestation comprising topically applying to the ments include those in which the composition comprises a plant a composition comprising at least one polynucleotide dsRNA with a strand having a sequence selected from the having at least one segment of 18 or more contiguous nucle group consisting of the Trigger Sequences Group. Related otides with a sequence of about 95% to about 100% identity aspects of the invention include compositions for topical with a fragment of a target gene or DNA having a sequence application and isolated polynucleotides of use in the method, selected from The Target Gene Sequences Group, or the DNA and plants having improved Leptinotarsa resistance provided complement thereof, in a manner Such that the plant treated by the method. with the polynucleotide-containing composition exhibits 0067 By “topical application' is meant application to the improved resistance to a Leptinotarsa species infestation, Surface or exterior of an object, Such as the Surface or exterior US 2015/O 143580 A1 May 21, 2015

of a plant, Such as application to the Surfaces of a plant part tants, e.g., SILWET L-77(R) brand surfactant having CAS Such as a leaf stem, flower, fruit, shoot, root, seed, tuber, Number 27306-78-1 and EPA Number: CALREGNO. flowers, anthers, or pollen, or application to an entire plant, or 5905-50073-AA, currently available from Momentive Per to the above-ground or below-ground portions of a plant. formance Materials, Albany, N.Y. In some embodiments, the Topical application can be carried out on non-living Surfaces, topically applied composition further comprises at least one Such as application to Soil, or to a surface or matrix by which pesticidal agent selected from the group consisting of a pata a Leptinotarsa insect can come in contact with the polynucle tin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis otide. In various embodiments of the method, the composi insecticidal protein, a Xenorhabdus insecticidal protein, a tion comprising at least one polynucleotide is topically Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal applied to the plant in a suitable form, e.g., as a solid, liquid protein. Alternatively such additional components or pesti (including homogeneous mixtures such as Solutions and non cidal agents can be provided separately, e.g., by separate homogeneous mixtures such as Suspensions, colloids, topical application or by transgenic expression in the plant. micelles, and emulsions), powder, Suspension, emulsion, Alternatively the plant is topically treated with the polynucle spray, encapsulated or micro-encapsulation formulation, in or otide-containing composition as well as with a separate (pre on microbeads or other carrier particulates, in a film or coat ceding, following, or concurrent) application of a Substance ing, or on or within a matrix, or as a seed treatment. In some that improves the efficacy of the polynucleotide-containing embodiments of the method, the polynucleotide-containing composition. For example, a plant can be sprayed with a first composition is topically applied to above-ground parts of the topical application of a solution containing a nonionic orga plant, e.g., sprayed or dusted onto leaves, stems, and flower nosilicone surfactant such as SILWETR) brand surfactants, ing parts of the plant. Embodiments of the method include e.g., SILWET L-77(R) brand surfactant, followed by a second topical application of a foliar spray (e.g., spraying a liquid topical application of the polynucleotide-containing compo polynucleotide-containing composition on leaves of a Solana ceous plant) or a foliar dust (e.g., dusting a Solanaceous plant sition, or Vice-versa. with a polynucleotide-containing composition in the form of 0068. It is anticipated that the combination of certain poly a powder or on carrier particulates). In other embodiments, nucleotides useful in the polynucleotide-containing compo the polynucleotide-containing composition is topically sition (e.g., the polynucleotide triggers described in the work applied to below-ground parts of the plant, such as to the ing Examples) with one or more non-polynucleotide roots, e.g., by means of a Soil drench. In other embodiments, pesticidal agents will result in a synergetic improvement in the polynucleotide-containing composition is topically prevention or control of Leptinotarsa species infestations, applied to a seed that is grown into the plant. The topical when compared to the effect obtained with the polynucleotide application can be in the form of topical treatment of fruits of alone or the non-polynucleotide pesticidal agent alone. In an Solanaceous plants or seeds from fruits of Solanaceous plants, embodiment, the polynucleotide-containing composition is or in the form of topical treatment of “seed potato’ tubers or provided as a transgenic plant expressing one or more poly pieces of tuber (e.g., by soaking, coating, or dusting the seed nucleotides and one or more genes encoding a non-poly potato). Suitable binders, inert carriers, Surfactants, and the nucleotide pesticidal agent selected from the group consisting like can optionally be included in the polynucleotide-contain of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus ing composition, as is known to one skilled informulation of thuringiensis insecticidal protein, a Xenorhabdus insecticidal pesticides and seed treatments. In some embodiments, the protein, a Photorhabdus insecticidal protein, a Bacillus lat polynucleotide-containing composition is at least one topi erosporous insecticidal protein, and a Bacillus sphaericus cally implantable formulation selected from the group con insecticidal protein, wherein the transgenic plant is found to sisting of a particulate, pellet, or capsule topically implanted exhibit synergistically improved resistance to Leptinotarsa in the plant; in Such embodiments the method comprises species infestations. topically implanting in the plant the topically implantable 0069. The polynucleotide useful in the polynucleotide formulation. In some embodiments, the polynucleotide-con containing composition is provided by Suitable means known taining composition is at least one in-furrow formulation to one in the art. Embodiments include those wherein the selected from the group consisting of a powder, granule, polynucleotide is chemically synthesized (e.g., by in vitro pellet, capsule, spray, or drench, or any other forms Suited for transcription, such as transcription using a T7 polymerase or topically applying to a furrow; in Such embodiments, the other polymerase), produced by expression in a microorgan method includes an in-furrow treatment with the in-furrow ism or in cell culture (such as plant or insect cells grown in formulation. In one embodiment the polynucleotide-contain culture), produced by expression in a plant cell, or produced ing composition can be ingested or otherwise absorbed inter by microbial fermentation. nally by the Leptinotarsa species. For example, the poly 0070. In many embodiments the polynucleotide useful in nucleotide-containing composition can be in the form of bait. the polynucleotide-containing composition is provided as an In some embodiments, the polynucleotide-containing com isolated DNA or RNA fragment. In some embodiments the position further comprises one or more components selected polynucleotide useful in the polynucleotide-containing com from the group consisting of a carrier agent, a Surfactant, a position is not part of an expression construct and is lacking cationic lipid (such as that disclosed in Example 18 of U.S. additional elements such as a promoter or terminator patent application publication 2011/0296556, incorporated sequences). Such polynucleotides can be relatively short, by reference herein), an organosilicone, an organosilicone Such as single- or double-stranded polynucleotides of Surfactant, a polynucleotide herbicidal molecule, a non-poly between about 18 to about 300 or between about 50 to about nucleotide herbicidal molecule, a non-polynucleotide pesti 500 nucleotides (for single-stranded polynucleotides) or cide, a safener, and an insect growth regulator. In one embodi between about 18 to about 300 or between about 50 to about ment the composition further comprises a nonionic 500 base-pairs (for double-stranded polynucleotides). In organosilicone surfactant such as SILWETR) brand surfac some embodiments, the polynucleotide is a dsRNA of US 2015/O 143580 A1 May 21, 2015

between about 100 to about 500 base-pairs, such as a dsRNA Stranded nucleic acid (e.g., dsRNA) with one strand compris of the length of any of the dsRNA triggers disclosed in Tables ing at least one segment of at least 21 contiguous nucleotides 3, 5, 8, 9, and 10. Alternatively the polynucleotide can be with a sequence of 100% identity with a fragment of equiva provided in more complex constructs, e.g., as part of a recom lent length of a DNA or target gene having a sequence binant expression construct, or included in a recombinant selected from the Target Gene Sequences Group or the DNA vector, for example in a recombinant plant virus vector or in complement thereof expressed as base-pairs, such a double a recombinant baculovirus vector. Such recombinant expres Stranded nucleic acid comprises at least one segment of at sion constructs or vectors can be designed to include addi least 21 contiguous, perfectly matched base-pairs which cor tional elements, such as expression cassettes for expressing a respond to a fragment of equivalent length of a DNA or target gene of interest (e.g., an insecticidal protein). gene having a sequence selected from the Target Gene 0071. The polynucleotide useful in the polynucleotide Sequences Group or the DNA complement thereof. In par containing composition has at least one segment of 18 or ticular embodiments, each segment contained in the poly more contiguous nucleotides with a sequence of about 95% to nucleotide is of a length greater than that which is typical of about 100% identity with a fragment of equivalent length of a naturally occurring regulatory Small RNAS, e.g., each seg DNA having a sequence selected from the Target Gene ment is at least about 30 contiguous nucleotides (or base Sequences Group or the DNA complement thereof. In an pairs) in length. In some embodiments, the total length of the embodiment the polynucleotide comprises at least one seg polynucleotide, or the length of each segment contained in the ment of 18 or more contiguous nucleotides that are essentially polynucleotide, is less than the total length of the sequence of identical or complementary to afragment of equivalent length interest (DNA or target gene having a sequence selected from of a DNA having a sequence selected from the group consist the group consisting of the Target Gene Sequences Group). In ing of the Target Gene Sequences Group. In some embodi Some embodiments, the total length of the polynucleotide is ments, the contiguous nucleotides have a sequence of about between about 50 to about 500 nucleotides (for single 95%, about 96%, about 97%, about 98%, about 99%, or about stranded polynucleotides) or base-pairs (for double-stranded 100% identity with a fragment of a DNA having a sequence polynucleotides). In some embodiments, the polynucleotide selected from the group consisting of: SEQID NOS:1-725 or is a dsRNA of between about 100 to about 500 base-pairs, SEQ ID NOs:726-830 or SEQ ID NOs: 1087-1094 or the such as a dsRNA of the length of any of the dsRNA triggers DNA complement thereof. In some embodiments the con disclosed in Tables 3, 5, 8, 9, and 10. In some embodiments, tiguous nucleotides are exactly (100%) identical to a frag the polynucleotide is a dsRNA comprising a segment having ment of equivalent length of a DNA having a sequence a sequence selected from the group consisting of SEQ ID selected from the Target Gene Sequences Group or the DNA NOs:831-1085, 1095-1104, and 1110-11 14, or the comple complement thereof. In some embodiments, the polynucle ment thereof, or the polynucleotide is encoded by a sequence otide has an overall sequence of about 95%, about 96%, about selected from the group consisting of SEQ ID NOs: 1105 97%, about 98%, about 99%, or about 100% identity with a 1109. fragment of a DNA having a sequence selected from the 0073. The topically applied polynucleotide is generally Target Gene Sequences Group or the DNA complement designed to Suppress one or more genes (“target genes'). thereof. Such target genes can include coding or non-coding sequence 0072 The polynucleotide useful in the polynucleotide or both. In specific embodiments, the polynucleotide is containing composition comprises at least one segment of 18 designed to Suppress one or more target genes, where each or more contiguous nucleotides with a sequence of about 95% target gene has a DNA sequence selected from the group to about 100% identity with a fragment of equivalent length of consisting of the Target Gene Sequences Group. In various a DNA having a sequence selected from the Target Gene embodiments, the topically applied polynucleotide is Sequences Group or the DNA complement thereof. In some designed to suppress one or more genes, where each gene has embodiments the polynucleotide comprises at least one seg a sequence selected from the group consisting of the Target ment of 18 or more contiguous nucleotides, e.g., between Gene Sequences Group, and can be designed to suppress 18-24, or between 18-28, or between 20-30, or between multiple genes from this group, or to target different regions 20-50, or between 20-100, or between 50-100, or between of one or more of these genes. In an embodiment, the topically 50-500, or between 100-250, or between 100-500, or between applied polynucleotide comprises multiple sections or seg 200-1000, or between 500-2000, or even greater. In some ments each of which comprises at least one segment of 21 embodiments the segment comprises more than 18 contigu contiguous nucleotides with a sequence of 100% identity ous nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. with a fragment of equivalent length of a DNA having a 30, or greater than 30, e.g., about 35, about 40, about 45, about sequence selected from the Target Gene Sequences Group or 50, about 55, about 60, about 65, about 70, about 75, about 80, the DNA complement thereof. In such cases, each section can about 85, about 90, about 95, about 100, about 110, about 120, be identical or different in size or in sequence, and can be about 130, about 140, about 150, about 160, about 170, about sense oranti-sense relative to the target gene. For example, in 180, about 190, about 200, about 210, about 220, about 230, one embodiment the topically applied polynucleotide can about 240, about 250, about 260, about 270, about 280, about include multiple sections in tandem or repetitive arrange 290, about 300, about 350, about 400, about 450, about 500, ments, wherein each section comprises at least one segment or greater than 500 contiguous nucleotides. In particular of 21 contiguous nucleotides with a sequence of 100% iden embodiments, the polynucleotide comprises at least one seg tity with a fragment of equivalent length of a DNA having a ment of at least 21 contiguous nucleotides with a sequence of sequence selected from the group consisting of SEQ ID 100% identity with a fragment of equivalent length of a DNA NOs: 1-725 or SEQID NOs:726-830 or SEQID NOs: 1087 or target gene having a sequence selected from the Target 1094 or the DNA complement thereof the segments can be Gene Sequences Group or the DNA complement thereof. In from different regions of the target gene, e.g., the segments particular embodiments, the polynucleotide is a double can correspond to different exon regions of the target gene, US 2015/O 143580 A1 May 21, 2015 and 'spacer nucleotides which do not correspond to a target DNA having a sequence selected from the Target Gene gene can optionally be used in between or adjacent to the Sequences Group or the DNA complement thereof, whereby Segments. the plant treated with the polynucleotide composition exhib 0074 The total length of the topically applied polynucle its improved resistance to a Leptinotarsa species infestation, otide can be greater than 18 contiguous nucleotides, and can relative to an untreated plant. An embodiment is a Solana include nucleotides in addition to the contiguous nucleotides ceous plant having improved resistance to a Leptinotarsa having the sequence of about 95% to about 100% identity species infestation when compared to a control plant, pro with a fragment of equivalent length of a DNA having a vided by topically applying to the plant or to a seed grown into sequence selected from the Target Gene Sequences Group or the plant (or, where the plant is a potato plant, to a seed potato the DNA complement thereof. In other words, the total length grown into the potato plant) a dsRNA trigger having a of the topically applied polynucleotide can be greater than the sequence selected from the group consisting of SEQ ID length of the section or segment of the polynucleotide NOs:831-1085, 1095-1104, and 1110-11 14, or the comple designed to Suppress one or more target genes, where each ment thereof, or a dsRNA trigger encoded by a sequence target gene has a DNA sequence selected from the group selected from the group consisting of SEQ ID NOs: 1105 consisting of the Target Gene Sequences Group. For example, 1109. In yet another aspect, this invention is directed to seed the topically applied polynucleotide can have nucleotides (especially transgenic progeny seed) produced by the plant flanking the “active' segment of at least one segment of 18 or having improved resistance to a Leptinotarsa species infes more contiguous nucleotides that Suppresses the target gene, tation, as provided by this method. Also contemplated is a or include 'spacer nucleotides between active segments, or commodity product produced by the plant having improved can have additional nucleotides at the 5' end, or at the 3' end, resistance to a Leptinotarsa species infestation, as provided or at both the 5' and 3' ends. In an embodiment, the topically by this method, and a commodity product produced from the applied polynucleotide comprises additional nucleotides that transgenic progeny seed of Such a plant. are not specifically related (having a sequence not comple mentary or identical to) to the DNA or target gene having a Insecticidal Compositions for Controlling Leptinotarsa sequence selected from the Target Gene Sequences Group or Species the DNA complement thereof, e.g., nucleotides that provide 0076 Another aspect of this invention provides an insec stabilizing secondary structure or for convenience in cloning ticidal composition for controlling a Leptinotarsa species or manufacturing. In an embodiment, the topically applied comprising an insecticidally effective amount of at least one polynucleotide comprises additional nucleotides located RNA comprising at least one segment of 18 or more contigu immediately adjacent to one or more segment of 18 or more ous nucleotides that is essentially identical or complementary contiguous nucleotides with a sequence of about 95% to to a fragment of a target gene or DNA having a sequence about 100% identity with or complementarity to a fragment selected from the group consisting of the Target Gene of equivalent length of a DNA or target gene having a Sequences Group. In this context “controlling includes sequence selected from the group consisting of the Target inducement of a physiological or behavioural change in a Gene Sequences Group. In an embodiment, the topically Leptinotarsa species (adult or larvae) Such as, but not limited applied polynucleotide comprises one such segment, with an to, growth stunting, increased mortality, decrease in repro additional 5' G or an additional 3' C or both, adjacent to the ductive capacity, decrease in or cessation offeeding behavior segment. In another embodiment, the topically applied poly or movement, or decrease in or cessation of metamorphosis nucleotide is a double-stranded RNA comprising additional stage development. By “insecticidally effective' is meant nucleotides to forman overhang, for example, a dsRNA com effective in inducing a physiological or behavioural change in prising 2 deoxyribonucleotides to form a 3' overhang. Thus in a Leptinotarsa species (adult or larvae) Such as, but not lim various embodiments, the nucleotide sequence of the entire ited to, growth stunting, increased mortality, decrease in topically applied polynucleotide is not 100% identical or reproductive capacity or decreased fecundity, decrease in or complementary to a fragment of contiguous nucleotides in cessation offeeding behavior or movement, or decrease in or the DNA or target gene having a sequence selected from the cessation of metamorphosis stage development; in some group consisting of the Target Gene Sequences Group. For embodiments, application of an insecticidally effective example, in Some embodiments the topically applied poly amount of the RNA to a plant improves the plant's resistance nucleotide comprises at least two segments each of 21 con to infestation by a Leptinotarsa species. The RNA can be tiguous nucleotides with a sequence of 100% identity with a longer than the segment or segments it contains, but each fragment of a DNA having a sequence selected from the segment and corresponding fragment of a target gene are of Target Gene Sequences Group, or the DNA complement equivalent length. RNAs of use in the method can be designed thereof, wherein (1) the at least two segments are separated by for multiple target genes. Embodiments include those in one or more spacer nucleotides, or (2) the at least two seg which the insecticidal composition comprises an insecticid ments are arranged in an order different from that in which the ally effective amount of a polynucleotide comprising at least corresponding fragments occur in the DNA having a 21 contiguous nucleotides that are complementary to a target sequence selected from the Target Gene Sequences Group, or gene having a nucleotide sequence selected from the group the DNA complement thereof. consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID 0075. In a related aspect, this invention is directed to the NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, plant having improved resistance to a Leptinotarsa species SEQID NOs:808-830, and SEQ ID NOs: 1087-1094, or an infestation, provided by this method which comprises topi RNA transcribed from the target gene; or an insecticidally cally applying to the plant a composition comprising at least effective amount of at least one polynucleotide comprising at one polynucleotide having at least one segment of 18 or more least one silencing element that is complementary to at least contiguous nucleotides with a sequence of about 95% to 21 contiguous nucleotides of a target gene or an RNA tran about 100% identity with a fragment of equivalent length of a scribed from the target gene, wherein the target gene has a US 2015/O 143580 A1 May 21, 2015

nucleotide sequence selected from the group consisting of least one strand of the double-stranded RNA is complemen SEQID NO:730, SEQID NO:807, SEQID NOs: 1-725, SEQ tary to at least 21 contiguous nucleotides of a gene that ID NOs:726-729, SEQID NOs:731-806, SEQID NOs:808 encodes a ribosomal protein oran RNA transcribed from the 830, and SEQID NOs: 1087-1094; or an insecticidally effec gene, wherein the Leptinotarsa species is Leptinotarsa tive amount of at least one RNA comprising at least one decemlineata, and wherein RNA interference is induced and segment that is identical or complementary to at least 21 Leptinotarsa decemlineata mortality occurs, and wherein the contiguous nucleotides of a target gene having a nucleotide ribosomal protein is a ribosomal L7 protein or a protein sequence selected from the group consisting of SEQ ID encoded by SEQID NO:730 or wherein the double-stranded NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID RNA comprises a sequence selected from the group consist NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, ing of SEQID NO:989,988, 1104, or 1105. Related aspects and SEQIDNOs: 1087-1094, oran RNA transcribed from the of the invention include isolated RNAs of use in the compo target gene; or an RNA molecule that causes mortality or sition and plants having improved Leptinotarsa resistance stunting of growth in a Leptinotarsa species when ingested or provided by treatment with the composition. contacted by the Leptinotarsa species, wherein the RNA mol 0077. In various embodiments, the insecticidal composi ecule comprises at least 21 contiguous nucleotides that are tion for controlling a Leptinotarsa species is in the form of at complementary to a target gene having a nucleotide sequence least one selected from the group consisting of a Solid, liquid selected from the group consisting of: SEQID NO:730, SEQ (including homogeneous mixtures such as Solutions and non IDNO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ homogeneous mixtures such as Suspensions, colloids, ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: micelles, and emulsions), powder, Suspension, emulsion, 1087-1094, oran RNA transcribed from the target gene; oran spray, encapsulated or micro-encapsulation formulation, in or insecticidal double-stranded RNA molecule that causes mor on microbeads or other carrier particulates, in a film or coat tality or stunting of growth in a Leptinotarsa species when ing, or on or within a matrix, or as a seed treatment. Suitable ingested or contacted by the Leptinotarsa species, wherein at binders, inert carriers, Surfactants, and the like can optionally least one strand of the insecticidal double-stranded RNA mol be included in the polynucleotide-containing composition, as ecule comprises 21 contiguous nucleotides that are comple is knownto one skilled informulation of insecticides and seed mentary to a target gene or an RNA transcribed from the treatments. The Leptinotarsa species to be controlled is gen target gene, wherein the target gene has a sequence selected erally a species that infests a plant. In some embodiments, the from the group consisting of: SEQ ID NO:730, SEQ ID insecticidal composition is at least one implantable formula NO:807, SEQID NOs: 1-725, SEQIDNOs:726-729, SEQID tion selected from the group consisting of aparticulate, pellet, NOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs: or capsule implanted in the plant; in Such embodiments the 1087-1094; or an insecticidally effective amount of at least method comprises implanting in the plant the implantable one double-stranded RNA comprising a sequence selected formulation. In some embodiments, the insecticidal compo from the Trigger Sequences Group. In some embodiments, sition is at least one in-furrow formulation selected from the the polynucleotide is a double-stranded RNA. In some group consisting of a powder, granule, pellet, capsule, spray, embodiments, the polynucleotide (e.g., double-stranded or drench, or any other forms Suited for applying to a furrow; RNA) is chemically synthesized or is produced by expression in Such embodiments, the method comprises an in-furrow in a microorganism or by expression in a plant cell. Embodi treatment with the in-furrow formulation. In one embodiment ments include insecticidal compositions comprising a the insecticidal composition can be ingested or otherwise dsRNA having a sequence selected from the group consisting absorbed internally by the Leptinotarsa species. For example, of SEQID NOs:831-1085, 1095-1104, and 1110-11 14, or the the insecticidal composition can be in the form of bait. In complement thereof, or wherein the insecticidal composition Some embodiments, the insecticidal composition further comprises a polynucleotide or RNA encoded by a sequence comprises one or more components selected from the group selected from the group consisting of SEQ ID NOs: 1105 consisting of a carrier agent, a Surfactant, a cationic lipid 1109. Embodiments include those in which the insecticidal (such as that disclosed in Example 18 of U.S. patent applica composition comprises a dsRNA with a strand having a tion publication 2011/0296.556, incorporated by reference sequence selected from the group consisting of the Trigger herein), an organosilicone, an organosilicone Surfactant, a Sequences Group. In an embodiment this invention provides polynucleotide herbicidal molecule, a non-polynucleotide an insecticidal composition for controlling a Leptinotarsa herbicidal molecule, a non-polynucleotide pesticide, a species comprising an insecticidally effective amount of a safener, and an insect growth regulator. In one embodiment double-stranded RNA molecule that causes mortality or the insecticidal composition further comprises a nonionic stunting of growth in a Leptinotarsa species when ingested or organosilicone surfactant such as SILWETR) brand surfac contacted by the Leptinotarsa species, wherein the insecti tants, e.g., SILWET L-77(R) brand surfactant having CAS cidal double-stranded RNA molecule comprises at least one Number 27306-78-1 and EPA Number: CALREGNO. segment that is complementary to 21 contiguous nucleotides 5905-50073-AA, currently available from Momentive Per of a DNA having a sequence selected from the group consist formance Materials, Albany, N.Y. In some embodiments, the ing of: SEQID NO:730, SEQ ID NO:807, SEQ ID NOs: 1 insecticidal composition further comprises at least one pesti 725, SEQID NOs:726-729, SEQID NOs:731-806, SEQ ID cidal agent selected from the group consisting of a patatin, a NOs:808-830, and SEQID NOs: 1087-1094, oran RNA tran plant lectin, a phytoecdysteroid, a Bacillus thuringiensis scribed from the DNA, and wherein the double-stranded insecticidal protein, a Xenorhabdus insecticidal protein, a RNA molecule is at least 50 base-pairs in length or is between Photorhabdus insecticidal protein, a Bacillus laterosporous about 100 to about 500 base-pairs in length. In an embodi insecticidal protein, and a Bacillus sphaericus insecticidal ment this invention provides an insecticidal composition for protein. Alternatively such additional components or pesti controlling a Leptinotarsa species comprising an insecticid cidal agents can be provided separately, e.g., by separate ally effective amount of a double-stranded RNA, wherein at topical application or by transgenic expression in the plant. US 2015/O 143580 A1 May 21, 2015

Alternatively the plant is topically treated with the insecti coating the plant or a seed of the plant or a seed potato, or by cidal composition as well as with a separate (preceding, fol application of a soil drench or in-furrow treatment, or by lowing, or concurrent) application of a Substance that providing in an artificial diet. The insecticidal composition improves the efficacy of the insecticidal composition. For can be provided for dietary uptake by the Leptinotarsa spe example, a plant can be sprayed with a first topical application cies in an artificial diet formulated to meet the particular of a solution containing a nonionic organosilicone surfactant nutritional requirements for maintaining the Leptinotarsa such as SILWETR) brand surfactants, e.g., SILWET L-77(R) species, wherein the artificial diet is Supplemented with some brand Surfactant, followed by a second topical application of amount of the RNA obtained from a separate source such as the insecticidal composition, or Vice-versa. chemical synthesis or purified from a microbial fermentation; 0078. It is anticipated that the combination of certain this embodiment can be useful, e.g., for determining the tim RNAs of use in this method (e.g., the dsRNA triggers ing and amounts of effective RNA treatment regimes. In some described in the working Examples) with one or more non embodiments the insecticidal composition is provided for polynucleotide pesticidal agents will result in a synergetic dietary uptake by the Leptinotarsa species in the form of a improvement in prevention or control of Leptinotarsa species plant cell or in plant cell components, or in a microorganism infestations, when compared to the effect obtained with the (such as a bacterium or a yeast) or a microbial fermentation RNA alone or the non-polynucleotide pesticidal agent alone. product, or in a synthetic diet. In one embodiment the insec In an embodiment, the insecticidal composition contains one ticidal composition is provided in the form of bait that is or more RNAS and one or more non-polynucleotidepesticidal ingested by the Leptinotarsa species. The insecticidal com agent selected from the group consisting of a patatin, a plant position can be provided for dietary uptake by the Leptino lectin, a phytoecdysteroid, a Bacillus thuringiensis insecti tarsa species in the form of a seed (or seed potato) treatment. cidal protein, a Xenorhabdus insecticidal protein, a Photo I0082 In one embodiment the insecticidal composition is rhabdus insecticidal protein, a Bacillus laterosporous insec provided in the form of any plant that is subject to infestation ticidal protein, and a Bacillus sphaericus insecticidal protein, by a Leptinotarsa species, wherein the RNA is contained in or and is found to effect synergistically improved prevention or on the plant. Such plants can be stably transgenic plants that control of Leptinotarsa species infestations. express the RNA, or non-transgenic plants that transiently 007.9 The Leptinotarsa species to be controlled is gener express the RNA or that have been treated with the RNA, e.g., ally a species that infests a plant. The plant can be any plant by spraying or coating. Stably transgenic plants generally that is subject to infestation by a Leptinotarsa species. Of contain integrated into their genome a recombinant construct particular interest are embodiments wherein the plant is a that encodes the RNA. Of particular interest are embodiments Solanaceous plant (family Solanaceae). Examples include a wherein the plant is a Solanaceous plant (family Solanaceae). plant selected from the group consisting of potato, tomato, Examples include a plant selected from the group consisting and eggplant. Embodiments include those wherein the plant of potato, tomato, and eggplant. Embodiments include those is an ungerminated Solanaceous plant seed, a Solanaceous wherein the plant is an ungerminated Solanaceous plant seed, plant in a vegetative stage, or a Solanaceous plant in a repro a Solanaceous plant in a vegetative stage, or a Solanaceous ductive stage. Embodiments include those wherein the plant plant in a reproductive stage. Embodiments include those is a “seed potato’, meaning, a potato tuber or piece of potato wherein the plant is a “seed potato’, meaning, a potato tuber tuber which can be propagated into new potato plants. In or piece of potato tuber which can be propagated into new Some embodiments, use of the insecticidal composition potato plants. results in control of the Leptinotarsa species, e.g., in growth I0083. The RNA useful in the insecticidal composition can stunting, increased mortality, decrease in reproductive capac be single-stranded (ss) or double-stranded (ds). Embodi ity, decrease in or cessation offeeding behavior or movement, ments include those wherein the RNA is at least one selected or decrease in or cessation of metamorphosis stage develop from the group consisting of sense single-stranded RNA (SS ment. In some embodiments, control of the Leptinotarsa spe RNA), anti-sense single-stranded (ssRNA), or double cies is observed as improved growth or improved yields of stranded RNA (dsRNA); a mixture of RNAs of any of these Solanaceous plants treated with the insecticidal composition, types can be used. In one embodiment a double-stranded in comparison to plants not treated with the insecticidal com DNA/RNA hybrid is used. The RNA can include components position. In some embodiments, control of the Leptinotarsa other than standard ribonucleotides, e.g., an embodiment is species is observed as decreased numbers of eggs, larvae, or an RNA that comprises terminal deoxyribonucleotides. adults of the Leptinotarsa species, decreased defoliation or I0084. The RNA in the insecticidal composition has at least other damage to the plant, or increased yield of harvestable one segment of 18 or more contiguous nucleotides with a fruit (e.g., tomatoes or eggplants) or tubers (e.g., potatoes). sequence of about 95% to about 100% identity with a frag 0080. In various embodiments, the insecticidal composi ment of equivalent length of a target gene or DNA having a tion comprises a microbial cell or is produced in a microor sequence selected from the Target Gene Sequences Group, or ganism. For example, the insecticidal composition can the DNA complement thereof. In an embodiment the RNA include or can be produced in bacteria or yeast cells. In similar comprises at least one segment of 18 or more contiguous embodiments the insecticidal composition comprises a trans nucleotides that are essentially identical or complementary to genic plant cell or is produced in a plant cell (for example a a fragment of equivalent length of a DNA having a sequence plant cell transiently expressing the polynucleotide); Such selected from the group consisting of the Target Gene plant cells can be cells in an plant or cells grown in tissue Sequences Group. In some embodiments, the contiguous culture or in cell Suspension. nucleotides have a sequence of about 95%, about 96%, about 0081. The insecticidal composition can be provided for 97%, about 98%, about 99%, or about 100% identity with a dietary uptake by the Leptinotarsa species by applying the fragment of a DNA having a sequence selected from the composition to a plant or Surface Subject to infestation by the Target Gene Sequences Group, or the DNA complement Leptinotarsa species, for example by spraying, dusting, or thereof. In some embodiments the contiguous nucleotides are US 2015/O 143580 A1 May 21, 2015 20 exactly (100%) identical to a fragment of equivalent length of complement thereof, or wherein the RNA is encoded by a a DNA having a sequence selected from the Target Gene sequence selected from the group consisting of SEQID NOs: Sequences Group, or the DNA complement thereof. In some 1105-1109. embodiments, the RNA has an overall sequence of about I0086. The RNA in the insecticidal composition is gener 95%, about 96%, about 97%, about 98%, about 99%, or about ally designed to suppress one or more genes (“target genes'). 100% identity with a fragment of a DNA having a sequence Such target genes can include coding or non-coding sequence selected from the Target Gene Sequences Group, or the DNA or both. In specific embodiments, the RNA is designed to complement thereof. Suppress one or more target genes, where each target gene has a DNA sequence selected from the group consisting of the 0085. The RNA in the insecticidal composition comprises Target Gene Sequences Group. In various embodiments, the at least one segment of 18 or more contiguous nucleotides RNA is designed to Suppress one or more genes, where each with a sequence of about 95% to about 100% identity with a gene has a sequence selected from the group consisting of the fragment of equivalent length of a DNA having a sequence Target Gene Sequences Group, and can be designed to Sup selected from the Target Gene Sequences Group or the DNA press multiple genes from this group, or to target different complement thereof. In some embodiments the RNA com regions of one or more of these genes. In an embodiment, the prises at least one segment of 18 or more contiguous nucle RNA comprises multiple sections or segments each of which otides, e.g., between 18-24, or between 18-28, or between comprises at least one segment of 21 contiguous nucleotides 20-30, or between 20-50, or between 20-100, or between with a sequence of 100% identity with a fragment of equiva 50-100, or between 50-500, or between 100-250, or between lent length of a DNA having a sequence selected from the 100-500, or between 200-1000, or between 500-2000, or even Target Gene Sequences Group or the DNA complement greater. In some embodiments the segment comprises more thereof. In such cases, each section can be identical or differ than 18 contiguous nucleotides, e.g., 19, 20, 21, 22, 23, 24. ent in size or in sequence, and can be sense or anti-sense 25, 26, 27, 28, 29, 30, or greater than 30, e.g., about 35, about relative to the target gene. For example, in one embodiment 40, about 45, about 50, about 55, about 60, about 65, about 70, the RNA can include multiple sections in tandem or repetitive about 75, about 80, about 85, about 90, about 95, about 100, arrangements, wherein each section comprises at least one about 110, about 120, about 130, about 140, about 150, about segment of 21 contiguous nucleotides with a sequence of 160, about 170, about 180, about 190, about 200, about 210, 100% identity with a fragment of equivalent length of a DNA about 220, about 230, about 240, about 250, about 260, about having a sequence selected from the Target Gene Sequences 270, about 280, about 290, about 300, about 350, about 400, Group or the DNA complement thereof; the segments can be about 450, about 500, or greater than 500 contiguous nucle from different regions of the target gene, e.g., the segments otides. In particular embodiments, the RNA comprises at can correspond to different exon regions of the target gene, least one segment of at least 21 contiguous nucleotides with a and 'spacer nucleotides which do not correspond to a target sequence of 100% identity with a fragment of equivalent gene can optionally be used in between or adjacent to the length of a DNA or target gene having a sequence selected Segments. from the Target Gene Sequences Group or the DNA comple I0087. The total length of the RNA in the insecticidal com ment thereof. In particular embodiments, the RNA is a position can be greater than 18 contiguous nucleotides, and double-stranded nucleic acid (e.g., dsRNA) with one strand can include nucleotides in addition to the contiguous nucle comprising at least one segment of at least 21 contiguous otides having the sequence of about 95% to about 100% nucleotides with a sequence of 100% identity with a fragment identity with a fragment of equivalent length of a DNA having of equivalent length of a DNA or target gene having a a sequence selected from the Target Gene Sequences Group sequence selected from the Target Gene Sequences Group or or the DNA complement thereof. In other words, the total the DNA complement thereof; expressed as base-pairs, such length of the RNA can be greater than the length of the section a double-stranded nucleic acid comprises at least one seg or segment of the RNA designed to Suppress one or more ment of at least 21 contiguous, perfectly matched base-pairs target genes, where each target gene has a DNA sequence which correspond to a fragment of equivalent length of a selected from the group consisting of the Target Gene DNA or target gene having a sequence selected from the Sequences Group. For example, the RNA can have nucle Target Gene Sequences Group or the DNA complement otides flanking the “active' segment of at least one segment of thereof. In particular embodiments, each segment contained 18 or more contiguous nucleotides that Suppresses the target in the RNA is of a length greater than that which is typical of gene, or include 'spacer nucleotides between active seg naturally occurring regulatory Small RNAS, e.g., each seg ments, or can have additional nucleotides at the 5' end, or at ment is at least about 30 contiguous nucleotides (or base the 3' end, or at both the 5' and 3' ends. In an embodiment, the pairs) in length. In some embodiments, the total length of the RNA comprises additional nucleotides that are not specifi RNA, or the length of each segment contained in the RNA, is cally related (having a sequence not complementary or iden less than the total length of the sequence of interest (DNA or tical to) to the DNA or target gene having a sequence selected target gene having a sequence selected from the group con from the Target Gene Sequences Group or the DNA comple sisting of the Target Gene Sequences Group). In some ment thereof, e.g., nucleotides that provide stabilizing sec embodiments, the totallength of the RNA is between about 50 ondary structure or for convenience in cloning or manufac to about 500 nucleotides (for single-stranded RNAs) or base turing. In an embodiment, the RNA comprises additional pairs (for double-stranded RNAs). In some embodiments, the nucleotides located immediately adjacent to one or more RNA comprises at least one RNA strand of between about 50 segment of 18 or more contiguous nucleotides with a to about 500 nucleotides in length. Embodiments include sequence of about 95% to about 100% identity with or those in which the RNA comprises at least one segment complementarity to a fragment of equivalent length of a DNA having a sequence selected from the group consisting of SEQ or target gene having a sequence selected from the group ID NOs:831-1085, 1095-1104, and 1110-11 14, or the consisting of the Target Gene Sequences Group. In an US 2015/O 143580 A1 May 21, 2015 embodiment, the RNA comprises one Such segment, with an plant at least one polynucleotide comprising at least one additional 5' G or an additional 3' C or both, adjacent to the segment of 18 or more contiguous nucleotides that is essen segment. In another embodiment, the RNA is a double tially identical or complementary to a fragment of a target Stranded RNA comprising additional nucleotides to form an gene or DNA having a sequence selected from the group overhang, for example, a dsRNA comprising 2 deoxyribo consisting of the Target Gene Sequences Group, whereby the nucleotides to form a 3' overhang. Thus in various embodi resulting plant has improved resistance to a Leptinotarsa ments, the nucleotide sequence of the entire RNA is not 100% species when compared to a control plant in which the poly identical or complementary to a fragment of contiguous nucleotide is not expressed. In an embodiment, the method nucleotides in the DNA or target gene having a sequence comprises expressing in the plant at least one polynucleotide selected from the group consisting of the Target Gene comprising at least one segment of 18 or more contiguous Sequences Group. For example, in some embodiments the nucleotides with a sequence of about 95% to about 100% RNA comprises at least two segments each of 21 contiguous identity with a fragment of equivalent length of a target gene nucleotides with a sequence of 100% identity with a fragment or DNA having a sequence selected from the Target Gene of a DNA having a sequence selected from the Target Gene Sequences Group or the DNA complement thereof. In an Sequences Group, or the DNA complement thereof, wherein embodiment, the invention provides a method of providing a (1) the at least two segments are separated by one or more plant having improved resistance to a Leptinotarsa species spacer nucleotides, or (2) the at least two segments are infestation comprising expressing in the plant at least one arranged in an order different from that in which the corre polynucleotide comprising at least one segment that is iden sponding fragments occur in the DNA having a sequence tical or complementary to at least 21 contiguous nucleotides selected from the Target Gene Sequences Group, or the DNA of a DNA having a sequence selected from the group consist complement thereof. ing of: SEQID NO:730, SEQ ID NO:807, SEQ ID NOs: 1 0088. In various embodiments the RNA in the insecticidal 725, SEQID NOs:726-729, SEQID NOs:731-806, SEQ ID composition consists of naturally occurring ribonucleotides. NOs:808-830, and SEQID NOs: 1087-1094. By “expressing Embodiments include, for example, synthetic RNAs consist a polynucleotide in the plant' is generally meant “expressing ing wholly of ribonucleotides or mainly of ribonucleotides an RNA transcript in the plant', e.g., expressing in the plant but with one or more terminal deoxyribonucleotides or one or an RNA comprising a ribonucleotide sequence that is anti more terminal dideoxyribonucleotides. In certain embodi sense or essentially complementary to at least a fragment of a ments, the RNA comprises non-canonical nucleotides Such as target gene or DNA having a sequence selected from the inosine, thiouridine, or pseudouridine. In certain embodi group consisting of the Target Gene Sequences Group. ments, the RNA comprises chemically modified nucleotides. Embodiments include those in which the polynucleotide (a) The RNA in the insecticidal composition is provided by expressed in the plant is an RNA comprising at least one Suitable means known to one in the art. Embodiments include segment having a sequence selected from the group consist those wherein the RNA is chemically synthesized (e.g., by in ing of: SEQID NOS:831-1085, 1095-1104, and 1110-1114, vitro transcription, Such as transcription using a T7 poly or the complement thereof, or wherein polynucleotide merase or other polymerase), produced by expression in a expressed in the plant is an RNA hairpin encoded by a microorganism or in cell culture (such as plant or insect cells sequence selected from the group consisting of SEQID NOs: grown in culture), produced by expression in a plant cell, or 1105-1109. Embodiments include those in which the poly produced by microbial fermentation. nucleotide expressed in the plant comprises a dsRNA with a 0089. In some embodiments the RNA is provided as an Strand having a sequence selected from the group consisting isolated RNA that is not part of an expression construct. In of the Trigger Sequences Group. However, the polynucle some embodiments the RNA is provided as an isolated RNA otide expressed in the plant can also be DNA (e.g., a DNA that is lacking additional elements such as a promoter or produced in the plant during genome replication), or the RNA terminator sequences. Such RNAs can be relatively short, encoded by such DNA. Related aspects of the invention such as single- or double-stranded RNAs of between about 18 include isolated polynucleotides of use in the method and to about 300 or between about 50 to about 500 nucleotides plants having improved Leptinotarsa resistance provided by (for single-stranded RNAs) or between about 18 to about 300 the method. or between about 50 to about 500 base-pairs (for double 0091. The method comprises expressing at least one poly stranded RNAs). Alternatively the RNA can be provided in nucleotide inaplant, wherein the polynucleotide comprises at more complex constructs, e.g., as part of a recombinant least one segment of 18 or more contiguous nucleotides that expression construct, or included in a recombinant vector, for is essentially identical or complementary to a fragment of a example in a recombinant plant virus vector or in a recombi target gene or DNA having a sequence selected from the nant baculovirus vector. In some embodiments such recom group consisting of the Target Gene Sequences Group. In binant expression constructs or vectors are designed to Some embodiments, a first polynucleotide is provided to a include additional elements, such as including additional plant in the form of DNA (e.g., in the form of an isolated DNA RNA encoding an aptamer or ribozyme or an expression molecule, or as an expression construct, or as a transforma cassette for expressing a gene of interest (e.g., an insecticidal tion vector), and the polynucleotide expressed in the plant is protein). a second polynucleotide (e.g., the RNA transcript of the first polynucleotide) in the plant. In an embodiment, the poly Methods of Providing Plants Having Improved Resistance to nucleotide is expressed in the plant by transgenic expression, Leptinotarsa Species Infestations, and the Plants and Seeds i.e., by stably integrating the polynucleotide into the plants Thus Provided genome from where it can be expressed in a cell or cells of the 0090 Another aspect of this invention is directed to a plant. In an embodiment, a first polynucleotide (e.g., a recom method of providing a plant having improved resistance to a binant DNA construct comprising a promoteroperably linked Leptinotarsa species infestation comprising expressing in the to DNA comprising at least one segment of 18 or more con US 2015/O 143580 A1 May 21, 2015 22 tiguous nucleotides that is essentially identical or comple factants, e.g., SILWET L-77(R) brand surfactant having CAS mentary to a fragment of a target gene or DNA having a Number 27306-78-1 and EPA Number: CALREGNO. sequence selected from the group consisting of the Target 5905-50073-AA, currently available from Momentive Per Gene Sequences Group) is stably integrated into the plants formance Materials, Albany, N.Y. In some embodiments, the genome from where secondarily produced polynucleotides topically applied composition further comprises at least one (e.g., an RNA transcript comprising the transcript of the seg pesticidal agent selected from the group consisting of a pata ment of 18 or more contiguous nucleotides that is essentially tin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis identical or complementary to a fragment of a target gene or insecticidal protein, a Xenorhabdus insecticidal protein, a DNA having a sequence selected from the group consisting of Photorhabdus insecticidal protein, a Bacillus laterosporous the Target Gene Sequences Group) are expressed in a cell or insecticidal protein, and a Bacillus sphaericus insecticidal cells of the plant. Methods of providing stably transformed protein. Alternatively such additional components or pesti plants are provided in the section headed "Making and Using cidal agents can be provided separately, e.g., by separate Transgenic Plant Cells and Transgenic Plants'. topical application or by transgenic expression in the plant. 0092. In another embodiment the polynucleotide Alternatively the plant is topically treated with the polynucle expressed in the plant is expressed by transient expression otide-containing composition as well as with a separate (pre (i.e., expression not resulting from stable integration of a ceding, following, or concurrent) application of a Substance sequence into the plants genome). In such embodiments the that improves the efficacy of the polynucleotide-containing method can include a step of introducing a polynucleotide composition. For example, a plant can be sprayed with a first (e.g., dsRNA or dsDNA) into the plant by routine techniques topical application of a solution containing a nonionic orga known in the art. For example, transient expression can be nosilicone surfactant such as SILWETR) brand surfactants, accomplished by infiltration of a polynucleotide solution e.g., SILWET L-77(R) brand surfactant, followed by a second using a needle-less Syringe into a leaf of a plant. topical application of the polynucleotide-containing compo 0093. In some embodiments where the polynucleotide sition, or Vice-versa. expressed in the plant is expressed by transient expression, a 0094. It is anticipated that the combination of certain poly first polynucleotide is provided to a plant in the form of RNA nucleotides of use in this method (e.g., the polynucleotide or DNA or both RNA and DNA, and a secondarily produced triggers described in the working Examples) with one or more second polynucleotide is transiently expressed in the plant. In non-polynucleotide pesticidal agents will result in a syner Some embodiments, the first polynucleotide is one or more getic improvement in prevention or control of Leptinotarsa selected from: (a) a single-stranded RNA molecule (ssRNA), species infestations, when compared to the effect obtained (b) a single-stranded RNA molecule that self-hybridizes to with the polynucleotide alone or the non-polynucleotide pes form a double-stranded RNA molecule, (c) a double-stranded ticidal agent alone. In an embodiment, a transgenic plant RNA molecule (dsRNA), (d) a single-stranded DNA mol expressing at least one polynucleotide comprising at least one ecule (ssDNA), (e) a single-stranded DNA molecule that segment of 18 or more contiguous nucleotides that is essen self-hybridizes to form a double-stranded DNA molecule, (f) tially identical or complementary to a fragment of a target a single-stranded DNA molecule comprising a modified Pol gene or DNA having a sequence selected from the group III gene that is transcribed to an RNA molecule, (g) a double consisting of the Target Gene Sequences Group (e.g., the stranded DNA molecule (dsDNA), (h) a double-stranded polynucleotide triggers described in the working Examples) DNA molecule comprising a modified Pol III gene that is and one or more genes encoding a non-polynucleotide pesti transcribed to an RNA molecule, and (i) a double-stranded, cidal agent selected from the group consisting of a patatin, a hybridized RNA/DNA molecule, or combinations thereof. In plant lectin, a phytoecdysteroid, a Bacillus thuringiensis specific embodiments, a first polynucleotide is introduced insecticidal protein, a Xenorhabdus insecticidal protein, a into the plant by topical application to the plant of a poly Photorhabdus insecticidal protein, a Bacillus laterosporous nucleotide-containing composition in a Suitable form, e.g., as insecticidal protein, and a Bacillus sphaericus insecticidal a solid, liquid (including homogeneous mixtures such as solu protein, is found to exhibit synergistically improved resis tions and non-homogeneous mixtures such as Suspensions, tance to Leptinotarsa species infestations. colloids, micelles, and emulsions), powder, Suspension, 0095. In some embodiments where the polynucleotide emulsion, spray, encapsulated or micro-encapsulation formu expressed in the plant is expressed by transient expression, a lation, in or on microbeads or other carrier particulates, in a first polynucleotide is provided to a plant in the form of RNA film or coating, or on or within a matrix, or in the form of a or DNA or both RNA and DNA, and a secondarily produced treatment of a Solanaceous plant seed or treatment of a seed second polynucleotide is transiently expressed in the plant; potato. Suitable binders, inert carriers, Surfactants, and the the site of application of the first polynucleotide need not be like can optionally be included in the composition, as is the same site where the second polynucleotide is transiently known to one skilled in formulation of pesticides and seed expressed. For example, a first polynucleotide can be pro treatments. In Such embodiments, the polynucleotide-con vided to a plant by topical application onto a leaf, or by taining composition can further include one or more compo injection into a stem, and the second polynucleotide can be nents selected from the group consisting of a carrier agent, a transiently expressed elsewhere in the plant, e.g., in the roots Surfactant, a cationic lipid (such as that disclosed in Example or throughout the plant. In some embodiments of the method, 18 of U.S. patent application publication 2011/0296.556, a composition comprising at least one polynucleotide is topi incorporated by reference herein), an organosilicone, an orga cally applied to above-ground parts of the plant, e.g., sprayed nosilicone Surfactant, a polynucleotide herbicidal molecule, a or dusted onto leaves, stems, and flowering parts of the plant. non-polynucleotide herbicidal molecule, a non-polynucle In other embodiments, a composition comprising at least one otide pesticide, a safener, and an insect growth regulator; in polynucleotide is topically applied to below-ground parts of one embodiment the composition further comprises a non the plant, such as to the roots, e.g., by means of a soil drench. ionic organosilicone surfactant such as SILWETR) brand sur In other embodiments, a composition comprising at least one US 2015/O 143580 A1 May 21, 2015 polynucleotide is topically applied to a seed (or, in the case of recombinant expression constructs or vectors are designed to potatoes, topically applied to a seed potato) that is grown into include additional elements, such as expression cassettes for the plant having improved resistance to a Leptinotarsa spe expressing a gene of interest (e.g., an insecticidal protein). cies infestation. In some embodiments the polynucleotide 0098. The polynucleotide expressed in the plant has at expressed in the plant is RNA, which can be single-stranded least one segment of 18 or more contiguous nucleotides with (ss) or double-stranded (ds) RNA or a combination of both. a sequence of about 95% to about 100% identity with a 0096. In some embodiments a first polynucleotide (DNA fragment of equivalent length of a DNA having a sequence or RNA or both) is provided to a plant and a second poly selected from the Target Gene Sequences Group or the DNA nucleotide having a sequence corresponding (identical or complement thereof. In an embodiment the polynucleotide complementary) to the first polynucleotide is Subsequently expressed in the plant comprises at least one segment of 18 or expressed in the plant. In such embodiments the polynucle more contiguous nucleotides that are essentially identical or otide expressed in the plant is an RNA transcript which can be complementary to a fragment of equivalent length of a DNA ssRNA or dsRNA or a combination of both. In some embodi having a sequence selected from the group consisting of the ments where the polynucleotide is expressed by transient Target Gene Sequences Group. In some embodiments, the expression, a first polynucleotide is provided to a plant in the contiguous nucleotides have a sequence of about 95%, about form of RNA or DNA or both RNA and DNA, and a second 96%, about 97%, about 98%, about 99%, or about 100% arily produced second polynucleotide is transiently expressed identity with a fragment of a DNA having a sequence selected in the plant; in Such embodiments, the first polynucleotide from the Target Gene Sequences Group or the DNA comple one or more selected from: (a) a single-stranded RNA mol ment thereof. In some embodiments the contiguous nucle ecule (ssERNA), (b) a single-stranded RNA molecule that self otides are exactly (100%) identical to a fragment of equiva hybridizes to form a double-stranded RNA molecule, (c) a lent length of a DNA having a sequence selected from the double-stranded RNA molecule (dsRNA), (d) a single Target Gene Sequences Group or the DNA complement stranded DNA molecule (ssDNA), (e) a single-stranded DNA thereof. In some embodiments, the polynucleotide expressed molecule that self-hybridizes to form a double-stranded DNA in the plant has an overall sequence of about 95%, about 96%, molecule, (f) a single-stranded DNA molecule comprising a about 97%, about 98%, about 99%, or about 100% identity modified Pol III gene that is transcribed to an RNA molecule, with a fragment of a DNA having a sequence selected from (g) a double-stranded DNA molecule (dsDNA), (h) a double the Target Gene Sequences Group or the DNA complement stranded DNA molecule comprising a modified Pol III gene thereof. that is transcribed to an RNA molecule, and (i) a double I0099. The polynucleotide expressed in the plant is gener stranded, hybridized RNA/DNA molecule, or combinations ally designed to suppress one or more genes (“target genes'). thereof. In such embodiments where the polynucleotide is Such target genes can include coding or non-coding sequence expressed by transient expression the first polynucleotide can or both. In specific embodiments, the polynucleotide consist of naturally occurring nucleotides, such as those expressed in the plant is designed to Suppress one or more which occur in DNA and RNA. In such embodiments where target genes, where each target gene has a DNA sequence the polynucleotide is expressed by transient expression the selected from the group consisting of the Target Gene first polynucleotide can be chemically modified, or comprises Sequences Group. In various embodiments, the polynucle chemically modified nucleotides. The first polynucleotide is otide expressed in the plant is designed to suppress one or provided by suitable means known to one in the art. Embodi more genes, where each gene has a sequence selected from ments include those wherein the first polynucleotide is the group consisting of the Target Gene Sequences Group, chemically synthesized (e.g., by invitro transcription, Such as and can be designed to suppress multiple genes from this transcription using a T7 polymerase or other polymerase), group, or to target different regions of one or more of these produced by expression in a microorganism or in cell culture genes. In an embodiment, the polynucleotide expressed in the (such as plant or insect cells grown in culture), produced by plant comprises multiple sections or segments each of which expression in a plant cell, or produced by microbial fermen comprises at least one segment of 21 contiguous nucleotides tation. The first polynucleotide can be provided as an RNA or with a sequence of 100% identity with a fragment of equiva DNA fragment. Alternatively the first polynucleotide can be lent length of a DNA having a sequence selected from the provided in more complex constructs, e.g., as part of a recom Target Gene Sequences Group or the DNA complement binant expression construct, or included in a recombinant thereof. In such cases, each section can be identical or differ vector, for example in a recombinant plant virus vector or in ent in size or in sequence, and can be sense or anti-sense a recombinant baculovirus vector, Such recombinant expres relative to the target gene. For example, in one embodiment sion constructs or vectors can be designed to include addi the polynucleotide expressed in the plant can include multiple tional elements, such as expression cassettes for expressing a sections in tandem or repetitive arrangements, wherein each gene of interest (e.g., an insecticidal protein). section comprises at least one segment of 21 contiguous 0097. In some embodiments the polynucleotide expressed nucleotides with a sequence of 100% identity with a fragment in the plant is an RNA molecule and can be relatively short, of equivalent length of a DNA having a sequence selected such as single- or double-stranded RNAs of between about 18 from the Target Gene Sequences Group or the DNA comple to about 300 or between about 50 to about 500 nucleotides ment thereofthe segments can be from different regions of the (for single-stranded RNAs) or between about 18 to about 300 target gene, e.g., the segments can correspond to different or between about 50 to about 500 base-pairs (for double exon regions of the target gene, and 'spacer nucleotides stranded RNAs). Alternatively the polynucleotide can be pro which do not correspond to a target gene can optionally be vided in more complex constructs, e.g., as part of a recombi used in between or adjacent to the segments. nant expression construct, or included in a recombinant 0100. The total length of the polynucleotide expressed in vector, for example in a recombinant plant virus vector or in the plant can be greater than 18 contiguous nucleotides, and a recombinant baculovirus vector. In some embodiments such can include nucleotides in addition to the contiguous nucle US 2015/O 143580 A1 May 21, 2015 24 otides having the sequence of about 95% to about 100% species infestation, provided by expressing in the plant at identity with a fragment of equivalent length of a DNA having least one polynucleotide comprising at least one segment of a sequence selected from the Target Gene Sequences Group 18 or more contiguous nucleotides with a sequence of about or the DNA complement thereof. In other words, the total 95% to about 100% identity with a fragment of equivalent length of the polynucleotide expressed in the plant can be length of a DNA having a sequence selected from the Target greater than the length of the section or segment of the poly Gene Sequences Group or the DNA complement thereof, nucleotide designed to suppress one or more target genes, whereby the resulting plant has improved resistance to a where each target gene has a DNA sequence selected from the Leptinotarsa species infestation when compared to a control group consisting of the Target Gene Sequences Group. For plant in which the polynucleotide is not expressed. An example, the polynucleotide expressed in the plant can have embodiment is a Solanaceous plant having improved resis nucleotides flanking the “active' segment of at least one tance to a Leptinotarsa species infestation when compared to segment of 18 or more contiguous nucleotides that Suppresses a control plant, provided by expressing in the plant an RNA the target gene, or include “spacer nucleotides between having a sequence selected from the group consisting of SEQ active segments, or can have additional nucleotides at the 5' ID NOs:831-1085, 1095-1104, and 1110-1114, or the end, or at the 3' end, or at both the 5' and 3' ends. In an complement thereof, or expressing in the plant an RNA hair embodiment, the polynucleotide expressed in the plant com pin encoded by a sequence selected from the group consisting prises additional nucleotides that are not specifically related of SEQID NOs: 1105-1109. In yet another aspect, this inven (i.e., having a sequence not complementary or identical to) to tion is directed to seed (especially transgenic progeny seed) the DNA or target gene having a sequence selected from the produced by the plant having improved resistance to a Lepti Target Gene Sequences Group or the DNA complement notarsa species infestation, as provided by this method. Also thereof, e.g., nucleotides that provide stabilizing secondary contemplated is a commodity product produced by the plant structure or for convenience in cloning or manufacturing. In having improved resistance to a Leptinotarsa species infes an embodiment, the polynucleotide expressed in the plant tation, as provided by this method, and a commodity product comprises additional nucleotides located immediately adja produced from the transgenic progeny seed of Such a plant. cent to one or more segment of 18 or more contiguous nucle otides with a sequence of about 95% to about 100% identity Recombinant DNA Constructs for Controlling a Leptinotarsa with or complementarity to a fragment of equivalent length of Species a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequences Group. In an 0102) Another aspect of this invention provides a recom embodiment, the polynucleotide expressed in the plant com binant DNA construct comprising a heterologous promoter prises one such segment, with an additional 5' G or an addi operably linked to a DNA element comprising at least one tional 3' C or both, adjacent to the segment. In another segment of 18 or more contiguous nucleotides with a embodiment, the polynucleotide expressed in the plant is a sequence of about 95% to about 100% identity with a frag double-stranded RNA comprising additional nucleotides to ment of a DNA having a sequence selected from the Target forman overhang, for example, a dsRNA comprising 2 deox Gene Sequences Group, or the DNA complement thereof. In yribonucleotides to form a 3' overhang. Thus in various some embodiments, the recombinant DNA construct com embodiments, the nucleotide sequence of the entire poly prises a heterologous promoter operably linked to: (a) DNA nucleotide expressed in the plant is not 100% identical or comprising a nucleotide sequence that is complementary to at complementary to a fragment of contiguous nucleotides in least 21 contiguous nucleotides of a target gene having a the DNA or target gene having a sequence selected from the sequence selected from the group consisting of SEQ ID group consisting of the Target Gene Sequences Group. For NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, example, in some embodiments the polynucleotide expressed and SEQIDNOs: 1087-1094, oran RNA transcribed from the in the plant comprises at least two segments each of 21 con target gene; or (b) a DNA comprising 21 or more contiguous tiguous nucleotides with a sequence of 100% identity with a nucleotides having 100% identity to a fragment of equivalent fragment of a DNA having a sequence selected from the length of a DNA having a sequence selected from the group Target Gene Sequences Group, or the DNA complement consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID thereof, wherein (1) the at least two segments are separated by NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, one or more spacer nucleotides, or (2) the at least two seg SEQID NOs:808-830, and SEQID NOs: 1087-1094, or the ments are arranged in an order different from that in which the DNA complement thereof; or (c) DNA encoding at least one corresponding fragments occur in the DNA having a silencing element that is complementary to at least 21 con sequence selected from the Target Gene Sequences Group, or tiguous nucleotides of a target gene or an RNA transcribed the DNA complement thereof. from the target gene, wherein the target gene has a sequence 0101. In a related aspect, this invention is directed to the selected from the group consisting of: SEQID NO:730, SEQ plant having improved resistance to a Leptinotarsa species ID NO:807, SEQIDNOs:1-725, SEQID NOs:726-729, SEQ infestation, provided by expressing in the plant at least one ID NOs:731-806, SEQID NOs:808-830, and SEQID NOs: polynucleotide comprising at least one segment of 18 or more 1087-1094; or (d) DNA encoding at least one silencing ele contiguous nucleotides that are essentially identical or ment comprising at least 21 contiguous nucleotides that are complementary to a fragment of equivalent length of a DNA complementary to a target gene selected from the genes in the having a sequence selected from the group consisting of the Target Gene Sequences Group or an RNA transcribed from Target Gene Sequences Group, whereby the resulting plant the target gene; or (e) DNA encoding a RNA comprising at has improved resistance to a Leptinotarsa species infestation least 21 contiguous nucleotides that are complementary to a when compared to a control plant in which the polynucleotide nucleotide sequence selected from the Trigger Sequences is not expressed. In a related aspect, this invention is directed Group, or the complement thereof, or an orthologous nucle to the plant having improved resistance to a Leptinotarsa otide sequence from a Leptinotarsa species or a Tribolium US 2015/O 143580 A1 May 21, 2015 species, wherein the orthologous nucleotide sequence has at selected from the Target Gene Sequences Group or the DNA least 95% sequence identity with a nucleotide sequence complement thereof. In some embodiments the contiguous selected from the Trigger Sequences Group, wherein the per nucleotides are exactly (100%) identical to a fragment of centage sequence identity is calculated over the same length; equivalent length of a DNA having a sequence selected from or (f) DNA encoding a RNA comprising at least one double the Target Gene Sequences Group or the DNA complement Stranded RNA region, at least one Strand of which comprises thereof. In some embodiments, the DNA has an overall at least 21 contiguous nucleotides that are complementary to sequence of about 95%, about 96%, about 97%, about 98%, a nucleotide sequence selected from the Trigger Sequences about 99%, or about 100% identity with a DNA having a Group, or the complement thereof, or an orthologous nucle sequence selected from the Target Gene Sequences Group or otide sequence from a Leptinotarsa species or a Tribolium the DNA complement thereof. species, wherein the orthologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence 0104. The recombinant DNA construct therefore com selected from the group consisting of the Trigger Sequences prises a heterologous promoter operably linked to DNA com Group, wherein the percentage sequence identity is calcu prising at least one segment of 18 or more contiguous nucle lated over the same length; or (g) DNA encoding RNA com otides designed to Suppress expression of a target gene having prising a nucleotide sequence selected from the Trigger a sequence selected from the Target Gene Sequences Group Sequences Group, or the complement thereof. Embodiments or the DNA complement thereof. In some embodiments the include a recombinant DNA construct comprising a heterolo DNA comprises at least one segment of 18 or more contigu gous promoter operably linked to a DNA element encoding ous nucleotides, e.g., between 18-24, or between 18-28, or an RNA having a sequence selected from the group consisting between 20-30, or between 20-50, or between 20-100, or of: SEQ ID NOs:831-1085, 1095-1104, and 1110-11 14, or between 50-100, or between 50-500, or between 100-250, or the complement thereof, or comprising a heterologous pro between 100-500, or between 200-1000, or between 500 moter operably linked to a DNA element encoding an RNA 2000, or even greater. In some embodiments the segment hairpin encoded by a sequence selected from the group con comprises more than 18 contiguous nucleotides, e.g., 19, 20, sisting of SEQID NOs: 1105-1109. Embodiments include a 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., recombinant DNA construct comprising a heterologous pro about 35, about 40, about 45, about 50, about 55, about 60, moter operably linked to a DNA encoding a dsRNA with a about 65, about 70, about 75, about 80, about 85, about 90, Strand having a sequence selected from the group consisting about 95, about 100, about 110, about 120, about 130, about of the Trigger Sequences Group. The recombinant DNA con 140, about 150, about 160, about 170, about 180, about 190, structs are useful in providing a plant having improved resis about 200, about 210, about 220, about 230, about 240, about tance to a Leptinotarsa species infestation, e.g., by expressing 250, about 260, about 270, about 280, about 290, about 300, in a plant a transcript of Such a recombinant DNA construct. about 350, about 400, about 450, about 500, or greater than The recombinant DNA constructs are also useful in the manu 500 contiguous nucleotides. In particular embodiments, the facture of polynucleotides useful in making compositions DNA encodes an RNA containing at least one segment of at that can be applied to a plant, seed, propagatable plant part, least 21 contiguous nucleotides with a sequence of 100% soil or field, or Surface in need of protection from a Leptino identity with a fragment of equivalent length of a DNA or tarsa species infestation. Related aspects of the invention target gene having a sequence selected from the Target Gene include: compositions comprising the recombinant DNA Sequences Group or the DNA complement thereof. In par construct; a plant chromosome or a plastid or a recombinant ticular embodiments, the DNA encodes a double-stranded plant virus vector or a recombinant baculovirus vector com nucleic acid (e.g., dsRNA) with one strand comprising at least prising the recombinant DNA construct; a transgenic Solana one segment of at least 21 contiguous nucleotides with a ceous plant cell having in its genome the recombinant DNA sequence of 100% identity with a fragment of equivalent construct, optionally comprising in its genome DNA encod length of a DNA or target gene having a sequence selected ing at least one pesticidal agent selected from the group from the Target Gene Sequences Group or the DNA comple consisting of a patatin, a plant lectin, a phytoecdysteroid, a ment thereof; expressed as base-pairs, such a double-stranded Bacillus thuringiensis insecticidal protein, a Xenorhabdus nucleic acid comprises at least one segment of at least 21 insecticidal protein, a Photorhabdus insecticidal protein, a contiguous, perfectly matched base-pairs which correspond Bacillus laterosporous insecticidal protein, and a Bacillus to a fragment of equivalent length of a DNA or target gene sphaericus insecticidal protein, and a transgenic Solanaceous having a sequence selected from the Target Gene Sequences plant including Such a transgenic Solanaceous plant cell, or a Group or the DNA complement thereof. In particularembodi fruit, seed, or propagatable part of the transgenic Solanaceous ments, each segment contained in the DNA is of a length plant; and plants having improved Leptinotarsa resistance greater than that which is typical of naturally occurring regu provided by expression of or treatment with the recombinant latory Small RNAS. In some embodiments, each segment is at DNA construct or the RNA encoded therein. least about 30 contiguous nucleotides (or base-pairs) in 0103) The recombinant DNA construct comprises a heter length. In some embodiments, the total length of the DNA, or ologous promoteroperably linked to DNA comprising at least the length of each segment contained in the polynucleotide, is one segment of 18 or more contiguous nucleotides with a less than the total length of the sequence of interest (DNA or sequence of about 95% to about 100% identity with a frag target gene having a sequence selected from the group con ment of equivalent length of a DNA having a sequence sisting of the Target Gene Sequences Group). In some selected from the Target Gene Sequences Group or the DNA embodiments, the total length of the DNA is between about complement thereof. In some embodiments, the segment of 50 to about 500. In some embodiments, the DNA encodes an 18 or more contiguous nucleotides has a sequence with about RNA having a sequence selected from the group consisting 95%, about 96%, about 97%, about 98%, about 99%, or about of: SEQ ID NOs:831-1085, 1095-1104, and 1110-11 14, or 100% identity with a fragment of a DNA having a sequence the complement thereof. In some embodiments, the recom US 2015/O 143580 A1 May 21, 2015 26 binant DNA construct comprises a sequence selected from located immediately adjacent to one or more segment of 18 or the group consisting of SEQID NOs: 1105-1109. more contiguous nucleotides with a sequence of about 95% to 0105. The recombinant DNA construct comprises a heter about 100% identity with or complementarity to a fragment ologous promoter operably linked to DNA generally of equivalent length of a DNA or target gene having a designed to Suppress one or more genes (“target genes'). sequence selected from the group consisting of the Target Such target genes can include coding or non-coding sequence Gene Sequences Group. In an embodiment, the heterologous or both. In specific embodiments, the recombinant DNA con promoter is operably linked to DNA comprising one Such struct is designed to Suppress one or more target genes, where segment, with an additional 5' Goran additional 3' C or both, each target gene has a DNA sequence selected from the group adjacent to the segment. In another embodiment, the heter consisting of the Target Gene Sequences Group. In various ologous promoter is operably linked to DNA encoding a embodiments, the recombinant DNA construct is designed to double-stranded RNA comprising additional nucleotides to Suppress one or more genes, where each gene has a sequence form an overhang. Thus in various embodiments, the nucle selected from the group consisting of the Target Gene otide sequence of the entire DNA operably linked to the Sequences Group, and can be designed to Suppress multiple heterologous promoter is not 100% identical or complemen genes from this group, or to target different regions of one or tary to a fragment of contiguous nucleotides in the DNA or more of these genes. In an embodiment, the recombinant target gene having a sequence selected from the group con DNA construct comprises a heterologous promoter operably sisting of the Target Gene Sequences Group. For example, in linked to multiple sections or segments each of which com Some embodiments the heterologous promoter is operably prises at least one segment of 21 contiguous nucleotides with linked to DNA comprising at least two segments each of 21 a sequence of 100% identity with a fragment of equivalent contiguous nucleotides with a sequence of 100% identity length of a DNA having a sequence selected from the Target with a fragment of a DNA having a sequence selected from Gene Sequences Group or the DNA complement thereof. In the Target Gene Sequences Group, or the DNA complement Such cases, each section can be identical or different in size or thereof, wherein (1) the at least two segments are separated by in sequence, and can be sense or anti-sense relative to the one or more spacer nucleotides, or (2) the at least two seg target gene. For example, in one embodiment the recombi ments are arranged in an order different from that in which the nant DNA construct can include a heterologous promoter corresponding fragments occur in the DNA having a operably linked to multiple sections in tandem or repetitive sequence selected from the Target Gene Sequences Group, or arrangements, wherein each section comprises at least one the DNA complement thereof. segment of 21 contiguous nucleotides with a sequence of 0107. In recombinant DNA constructs, the heterologous 100% identity with a fragment of equivalent length of a DNA promoter is operably linked to DNA that encodes a transcript having a sequence selected from the Target Gene Sequences that can be single-stranded (SS) or double-stranded (ds) or a Group or the DNA complement thereof the segments can be combination of both. Embodiments of the method include from different regions of the target gene, e.g., the segments those wherein the DNA encodes a transcript comprising sense can correspond to different exon regions of the target gene, single-stranded RNA (ssRNA), anti-sense ssRNA, or double and 'spacer nucleotides which do not correspond to a target stranded RNA (dsRNA), or a combination of any of these. gene can optionally be used in between or adjacent to the 0108. The recombinant DNA construct is provided by Segments. Suitable means known to one in the art. Embodiments include 0106 The recombinant DNA construct comprises a heter those wherein the recombinant DNA construct is synthesized ologous promoter operably linked to DNA which can have a in vitro, produced by expression in a microorganism or in cell total length that is greater than 18 contiguous nucleotides, and culture (such as plant or insect cells grown in culture), pro can include nucleotides in addition to the segment of at least duced by expression in a plant cell, or produced by microbial one segment of 18 or more contiguous nucleotides having the fermentation. sequence of about 95% to about 100% identity with a frag 0109 The heterologous promoter of use in recombinant ment of equivalent length of a DNA having a sequence DNA constructs is selected from the group consisting of a selected from the Target Gene Sequences Group or the DNA promoter functional in a plant, a promoter functional in a complement thereof. In other words, the total length of the prokaryote, a promoter functional in a fungal cell, and a DNA can be greater than the length of the segment of the baculovirus promoter. Non-limiting examples of promoters DNA designed to Suppress one or more target genes, where are described in the section headed “Promoters'. each target gene has a DNA sequence selected from the group 0110. In some embodiments, the recombinant DNA con consisting of the Target Gene Sequences Group. For example, struct comprises a second promoter also operably linked to the DNA can have nucleotides flanking the “active' segment the DNA. For example, the DNA comprising at least one of at least one segment of 18 or more contiguous nucleotides segment of 18 or more contiguous nucleotides can be flanked that Suppresses the target gene, or include 'spacer nucle by two promoters arranged so that the promoters transcribe in otides between active segments, or can have additional nucle opposite directions and in a convergent manner, yielding otides at the 5' end, or at the 3' end, or at both the 5' and 3' ends. opposite-strand transcripts of the DNA that are complemen In an embodiment, the heterologous promoter is operably tary to and capable of hybridizing with each other to form linked to DNA comprising additional nucleotides that are not double-stranded RNA. In one embodiment, the DNA is specifically related (having a sequence not complementary or located between two root-specific promoters, which enable identical to) to the DNA or target gene having a sequence transcription of the DNA in opposite directions, resulting in selected from the Target Gene Sequences Group or the DNA the formation of dsRNA. complement thereof, e.g., nucleotides that provide stabilizing 0111. In some embodiments the recombinant DNA con secondary structure or for convenience in cloning or manu struct comprises other DNA elements in addition to the het facturing. In an embodiment, the heterologous promoter is erologous promoter operably linked to DNA comprising at operably linked to DNA comprising additional nucleotides least one segment of 18 or more contiguous nucleotides with US 2015/O 143580 A1 May 21, 2015 27 a sequence of about 95% to about 100% identity with a cies, or in a composition for ingestion by a Leptinotarsa fragment of equivalent length of a DNA having a sequence species. In various embodiments, such compositions contain selected from the Target Gene Sequences Group or the DNA ing the recombinant DNA construct are provided in the form complement thereof. Such DNA elements are known in the of at least one selected from the group consisting of a solid, art, and include but are not limited to introns, recombinase liquid (including homogeneous mixtures such as solutions recognition sites, aptamers or ribozymes, additional and addi and non-homogeneous mixtures Such as Suspensions, col tional expression cassettes for expressing coding sequences loids, micelles, and emulsions), powder, Suspension, emul (e.g., to express a transgene Such as an insecticidal protein or Sion, spray, encapsulated or micro-encapsulation formula selectable marker) or non-coding sequences (e.g., to express tion, in or on microbeads or other carrierparticulates, in a film additional Suppression elements). Inclusion of one or more or coating, or on or within a matrix, or as a seed treatment. The recognition sites for binding and cleavage by a small RNA topical application can be in the form of topical treatment of (e.g., by a miRNA or an siRNA that is expressed only in a fruits of Solanaceous plants or seeds from fruits of Solana particular cell or tissue) allows for more precise expression ceous plants, or in the form of topical treatment of “seed patterns in a plant, wherein the expression of the recombinant potato’ tubers or pieces of tuber (e.g., by soaking, coating, or DNA construct is suppressed where the small RNA is dusting the seed potato). Suitable binders, inert carriers, Sur expressed. factants, and the like can be included in the composition 0112. In some embodiments, the recombinant DNA con containing the recombinant DNA construct, as is known to struct is provided in a recombinant vector. By “recombinant one skilled in formulation of pesticides and seed treatments. vector” is meant a recombinant polynucleotide molecule that In some embodiments, the composition for topical applica is used to transfer genetic information from one cell to tion containing the recombinant DNA construct is at least one another. Embodiments suitable to this invention include, but topically implantable formulation selected from the group are not limited to, recombinant plasmids, recombinant consisting of a particulate, pellet, or capsule topically cosmids, artificial chromosomes, and recombinant viral vec implanted in the plant; in Such embodiments the method tors such as recombinant plant virus vectors and recombinant comprises topically implanting in the plant the topically baculovirus vectors. Alternative embodiments include implantable formulation. In some embodiments, the compo recombinant plasmids, recombinant cosmids, artificial chro sition for topical application containing the recombinant mosomes, and recombinant viral vectors such as recombinant DNA construct is at least one in-furrow formulation selected plant virus vectors and recombinant baculovirus vectors com from the group consisting of a powder, granule, pellet, cap prising the DNA element without the heterologous promoter. Sule, spray, or drench, or any other forms suited for topically 0113. In some embodiments, the recombinant DNA con applying to a furrow; in Such embodiments, the method struct is provided in a plant chromosome or plastid, e.g., in a includes an in-furrow treatment with the in-furrow formula transgenic plant cell or a transgenic plant. Thus, also encom tion. In one embodiment the composition for topical applica passed by this invention is a transgenic plant cell having in its tion containing the recombinant DNA construct can be genome the recombinant DNA construct, as well as a trans ingested or otherwise absorbed internally by the Leptinotarsa genic plant or partially transgenic plant including Such a species. For example, the composition for topical application transgenic plant cell. Partially transgenic plants include, e.g., containing the recombinant DNA construct can be in the form a non-transgenic Scion grafted onto a transgenic rootstock of bait. In some embodiments, the composition containing the including the transgenic plant cell. Embodiments include a recombinant DNA construct further comprises one or more transgenic tomato rootstock including the transgenic plant components selected from the group consisting of a carrier cell. The plant can be any plant that is subject to infestation by agent, a Surfactant, a cationic lipid (such as that disclosed in a Leptinotarsa species. Of particular interest are embodi Example 18 of U.S. patent application publication 2011/ ments wherein the plant is a Solanaceous plant (family Solan 0296.556, incorporated by reference herein), an organosili aceae). Examples include a plant selected from the group cone, an organosilicone surfactant, a polynucleotide herbi consisting of potato, tomato, and eggplant. Embodiments cidal molecule, a non-polynucleotide herbicidal molecule, a include those wherein the plant is an ungerminated Solana non-polynucleotide pesticide, a safener, and an insect growth ceous plant seed, a Solanaceous plant in a vegetative stage, or regulator. In one embodiment the composition containing the a Solanaceous plant in a reproductive stage. Embodiments recombinant DNA construct further comprises a nonionic include those wherein the plant is a 'seed potato’, meaning a organosilicone surfactant such as SILWETR) brand surfac potato tuber or piece of potato tuber which can be propagated tants, e.g., SILWET L-77(R) brand surfactant having CAS into new potato plants. In yet another aspect, this invention is Number 27306-78-1 and EPA Number: CALREGNO. directed to seed (especially transgenic progeny seed) pro 5905-50073-AA, currently available from Momentive Per duced by the transgenic plant having in its genome a recom formance Materials, Albany, N.Y. In some embodiments, the binant DNA construct as described herein. Embodiments also composition containing the recombinant DNA construct fur encompass a transgenic seed potato having in its genome a ther comprises at least one pesticidal agent selected from the recombinant DNA construct as described herein. Also con group consisting of a patatin, a plant lectin, a phytoecdys templated is a commodity product produced by Such a trans teroid, a phytoecdysteroid, a Bacillus thuringiensis insecti genic plant, and a commodity product produced from the cidal protein, a Xenorhabdus insecticidal protein, a Photo transgenic progeny seed of Such a transgenic plant. rhabdus insecticidal protein, a Bacillus laterosporous 0114. The recombinant DNA construct can be provided in insecticidal protein, and a Bacillus sphaericus insecticidal a composition for topical application to a Surface of a plant or protein. of a plant seed, or for topical application to any substrate 0.115. It is anticipated that the combination of certain needing protection from a Leptinotarsa species infestation. recombinant DNA constructs as described herein (e.g., Likewise, the recombinant DNA construct can be provided in recombinant DNA constructs including the polynucleotide a composition for topical application to a Leptinotarsa spe triggers described in the working Examples), whether trans US 2015/O 143580 A1 May 21, 2015 28 genically expressed or topically applied, with one or more expressing the recombinant DNA construct); Such plant cells non-polynucleotide pesticidal agents, whether transgenically can be cells in an plant or cells grown in tissue culture or in expressed or topically applied, will result in a synergetic cell Suspension. improvement in prevention or control of Leptinotarsa species infestations, when compared to the effect obtained with the Transgenic Solanaceous Plant Cells recombinant DNA constructs alone or the non-polynucle 0118 Several embodiments relate to transgenic solana otide pesticidal agent alone. In an embodiment, a recombi ceous plant cells expressing a polynucleotide useful in the nant DNA construct for expressing one or more polynucle methods described herein for Suppressing expression of a otides as well as one or more genes encoding a non target gene in a Leptinotarsa species or for controlling a polynucleotide pesticidal agent selected from the group Leptinotarsa infestation. In one aspect this invention provides consisting of a patatin, a plant lectin, a phytoecdysteroid, a a transgenic Solanaceous plant cell having in its genome a Bacillus thuringiensis insecticidal protein, a Xenorhabdus recombinant DNA encoding RNA comprising at least one insecticidal protein, a Photorhabdus insecticidal protein, a segment of 18 or more contiguous nucleotides with a Bacillus laterosporous insecticidal protein, and a Bacillus sequence of about 95% to about 100% identity with a frag sphaericus insecticidal protein, is found to provide synergis ment of a DNA having a sequence selected from the Target tically improved resistance to Leptinotarsa species infesta Gene Sequences Group, or the DNA complement thereof. In tions in plants expressing the recombinant DNA construct. one aspect this invention provides a transgenic Solanaceous An embodiment relates to a recombinant DNA construct for plant cell having in its genome a recombinant DNA encoding expressing an RNA comprising a segment having a sequence RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a selected from the group consisting of SEQID NOS:831-1085 target gene sequence of the Leptinotarsa species larvae, and 1095 as well as one or more genes encoding a non wherein the target gene sequence is selected from the Target polynucleotide pesticidal agent selected from the group con Gene Sequences Group, or the DNA complement thereof. In sisting of apatatin, a plant lectin, a phytoecdysteroid, a Bacil one aspect this invention provides a transgenic Solanaceous lus thuringiensis insecticidal protein, a Xenorhabdus plant cell having in its genome a recombinant DNA encoding insecticidal protein, a Photorhabdus insecticidal protein, a RNA that Suppresses expression of a target gene in a Lepti Bacillus laterosporous insecticidal protein, and a Bacillus notarsa species that contacts or ingests the RNA, wherein the sphaericus insecticidal protein. RNA comprises at least one silencing element having at least 0116. The composition containing the recombinant DNA one segment of 18 or more contiguous nucleotides comple construct can be provided for dietary uptake by a Leptino mentary to a fragment of the target gene, and wherein the tarsa species by applying the composition to a plant or Sur target gene is selected from the group consisting of the genes in the Target Gene Sequences Group. A specific embodiment face Subject to infestation by the Leptinotarsa species, for is a transgenic Solanaceous plant cell having in its genome a example by spraying, dusting, or coating the plant, or by recombinant DNA encoding RNA that suppresses expression application of a soil drench, or by providing in an artificial of a target gene in a Leptinotarsa species that contacts or diet. The composition containing the recombinant DNA con ingests the RNA, wherein the RNA comprises at least one struct can be provided for dietary uptake by a Leptinotarsa silencing element having at least one segment of 18 or more species in an artificial diet formulated to meet the particular contiguous nucleotides complementary to a fragment of one nutritional requirements for maintaining the Leptinotarsa or more exocyst target genes; Suitable exocyst target genes species, wherein the artificial diet is Supplemented with some include the Leptinotarsa exocyst genes provided in Table 4 or amount of the recombinant DNA construct obtained from a homologous sequences identified from other insect species. separate source such as in vitro synthesis or purified from a In one aspect this invention provides a transgenic Solanaceous microbial fermentation or other biological source; this plant cell having in its genome a recombinant DNA encoding embodiment can be useful, e.g., for determining the timing an RNA having a sequence selected from the group consisting and amounts of effective treatment regimes. In some embodi of: SEQ ID NOs:831-1085, 1095-1104, and 1110-11 14, or ments the composition containing the recombinant DNA con the complement thereof, or a recombinant DNA selected struct is provided for dietary uptake by the Leptinotarsa spe from the group consisting of SEQ ID NOs: 1105-1109: cies in the form of a plant cell or in plant cell components, or embodiments include a transgenic Solanaceous plant cell hav in a microorganism (Such as a bacterium or a yeast) or a ing in its genome a recombinant DNA encoding a dsRNA microbial fermentation product, or in a synthetic diet. In one with a strand having a sequence selected from the group embodiment the composition containing the recombinant consisting of the Trigger Sequences Group. Such transgenic DNA construct is provided in the form of bait that is ingested Solanaceous plant cells are useful in providing a transgenic by the Leptinotarsa species. The composition containing the Solanaceous plant having improved resistance to a Leptino recombinant DNA construct can be provided for dietary tarsa species infestation when compared to a control plant uptake by the Leptinotarsa species in the form of a seed lacking Such plant cells. The transgenic Solanaceous plant cell treatment. can an isolated transgenic Solanaceous plant cell, or a trans 0117. In various embodiments, the composition contain genic Solanaceous plant cell grown in culture, or a transgenic ing the recombinant DNA construct comprises a microbial cell of any transgenic Solanaceous plant that is subject to cell or is produced in a microorganism. For example, the infestation by a Leptinotarsa species. Examples include a composition for containing the recombinant DNA construct transgenic Solanaceous plant selected from the group consist can include or can be produced in bacteria or yeast cells. In ing of potato, tomato, and eggplant. Embodiments include similar embodiments the composition containing the recom those wherein the transgenic Solanaceous plant is an unger binant DNA construct comprises a transgenic plant cell or is minated transgenic Solanaceous plant seed, a transgenic produced in a plant cell (for example a plant cell transiently Solanaceous plant in a vegetative stage, or a transgenic US 2015/O 143580 A1 May 21, 2015 29

Solanaceous plant in a reproductive stage. Embodiments RNAS. In some embodiments, each segment is at least about include those wherein the transgenic Solanaceous plant is a 30 contiguous nucleotides (or base-pairs) in length. In par potato tuber or piece of potato tuber (“seed potato') which ticular embodiments, the RNA is between about 50 to about can be propagated into new transgenic potato plants. 500 nucleotides in length. In particular embodiments, the 0119. In an embodiment, the recombinant DNA is stably RNA has a sequence selected from the group consisting of integrated into the transgenic Solanaceous plant's genome SEQID NOs:831-1085, 1095-1104, and 1110-11 14, or the from where it can be expressed in a cell or cells of the trans complement thereof, or the RNA is encoded by a sequence genic Solanaceous plant. Methods of providing stably trans selected from the group consisting of SEQ ID NOs: 1105 formed plants are provided in the section headed “Making 1109. and Using Transgenic Plant Cells and Transgenic Plants”. 0121. In some embodiments, the transgenic Solanaceous plant cell is further capable expressing additional heterolo 0120 Several embodiments relate to a transgenic solana gous DNA sequences. In an embodiment, the transgenic ceous plant cell having in its genome a recombinant DNA Solanaceous plant cell has a genome that further comprises encoding RNA that Suppresses expression of a target gene in recombinant DNA encoding at least one pesticidal agent a Leptinotarsa species that contacts or ingests the RNA, selected from the group consisting of a patatin, a plant lectin, wherein the RNA comprises at least one silencing element a phytoecdysteroid, a phytoecdysteroid, a Bacillus thuring complementary to the target gene, and wherein the target gene iensis insecticidal protein, a Xenorhabdus insecticidal pro sequence is selected from the Target Gene Sequences Group tein, a Photorhabdus insecticidal protein, a Bacillus lat or the complement thereof. In some embodiments, the silenc erosporous insecticidal protein, and a Bacillus sphaericus ing element comprises at least one 18 or more contiguous insecticidal protein. In particular embodiments, the trans nucleotides with a sequence of about 95% to about 100% genic Solanaceous plant cell has stably integrated in its complementarity to a fragment of equivalent length of a DNA genome (i) recombinant DNA encoding at least one RNA having a sequence selected from the group consisting of the with a sequence selected from the group consisting of SEQID Target Gene Sequences Group. In some embodiments, the NOs:831-1085 and 1095 and (ii) DNA encoding at least one silencing element comprises at least one 18 or more contigu pesticidal agent selected from the group consisting of a pata ous nucleotides capable of hybridizing in vivo or of hybrid tin, a plant lectin, a phytoecdysteroid, a phytoecdysteroid, a izing under physiological conditions (e.g., such as physi Bacillus thuringiensis insecticidal protein, a Xenorhabdus ological conditions normally found in the cells of a insecticidal protein, a Photorhabdus insecticidal protein, a Leptinotarsa species) to a fragment of equivalent length of a Bacillus laterosporous insecticidal protein, and a Bacillus DNA having a sequence selected from the group consisting of sphaericus insecticidal protein. the Target Gene Sequences Group. The contiguous nucle I0122. In a related aspect, this invention is directed to a otides number at least 18, e.g., between 18-24, or between transgenic Solanaceous plant including the transgenic Solana 18-28, or between 20-30, or between 20-50, or between ceous plant cell, a commodity product produced from the 20-100, or between 50-100, or between 50-500, or between transgenic Solanaceous plant, and transgenic progeny Solana 100-250, or between 100-500, or between 200-1000, or ceous plant seed or transgenic propagatable part of the trans between 500-2000, or even greater. In some embodiments, genic Solanaceous plant. Embodiments include a transgenic the contiguous nucleotides number more than 18, e.g., 19, 20, tomato plant, a transgenic tomato rootstock, a transgenic 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., eggplant, or a transgenic potato plant having improved resis about 35, about 40, about 45, about 50, about 55, about 60, tance to a Leptinotarsa species infestation. Also contem about 65, about 70, about 75, about 80, about 85, about 90, plated is a commodity product produced by the transgenic about 95, about 100, about 110, about 120, about 130, about Solanaceous plant, and a commodity product produced from 140, about 150, about 160, about 170, about 180, about 190, the transgenic progeny Seed of such a transgenic Solanaceous about 200, about 210, about 220, about 230, about 240, about plant. 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, or greater than Insecticidal Compositions for Controlling Leptinotarsa 500 contiguous nucleotides. In particular embodiments, the Species silencing element comprises at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity I0123. Another aspect of this invention provides an insec with a fragment of equivalent length of a DNA or target gene ticidal composition for controlling a Leptinotarsa species, having a sequence selected from the Target Gene Sequences wherein the insecticidal composition consists essentially of Group or the DNA complement thereof. In particular embodi an RNA molecule that causes mortality or stunting of growth ments, the RNA is a double-stranded nucleic acid (e.g., in a Leptinotarsa species when ingested or contacted by the dsRNA) with one strand comprising at least one segment of at Leptinotarsa species, and wherein the RNA molecule com least 21 contiguous nucleotides with a sequence of 100% prises at least one segment of 18 or more contiguous nucle identity with a fragment of equivalent length of a DNA or otides that is essentially complementary to a fragment of a target gene having a sequence selected from the Target Gene DNA having a sequence selected from the Target Gene Sequences Group or the DNA complement thereof; expressed Sequences Group, or the DNA complement thereof. In this as base-pairs, such a double-stranded nucleic acid comprises context "controlling a Leptinotarsa species comprises at least one segment of at least 21 contiguous, perfectly inducement of a physiological or behavioural change in a matched base-pairs which correspond to a fragment of Leptinotarsa species (adult or larvae) Such as, but not limited equivalent length of a DNA or target gene having a sequence to, growth stunting or increased mortality. In some embodi selected from the Target Gene Sequences Group or the DNA ments, “controlling a Leptinotarsa species is achieved by a complement thereof. In particular embodiments, each silenc decrease in reproductive capacity, decrease in or cessation of ing element contained in the RNA is of a length greater than feeding behavior or movement, or decrease in or cessation of that which is typical of naturally occurring regulatory Small metamorphosis stage development in a Leptinotarsa species. US 2015/O 143580 A1 May 21, 2015 30

Generally the RNA molecule has been isolated, that is, sub 140, about 150, about 160, about 170, about 180, about 190, stantially purified from a mixture Such as from a fermentation about 200, about 210, about 220, about 230, about 240, about or from an in vitro synthesis mixture. In one embodiment the 250, about 260, about 270, about 280, about 290, about 300, RNA molecule comprises at least one segment of 18 or more about 350, about 400, about 450, about 500, or greater than contiguous nucleotides with a sequence of about 95% to 500 contiguous nucleotides. In particular embodiments, the about 100% complementarity to a fragment of equivalent RNA molecule comprises at least one segment of at least 21 length of a DNA having a sequence selected from the Target contiguous nucleotides with a sequence of 100% identity Gene Sequences Group or the DNA complement thereof. In with a fragment of equivalent length of a DNA or target gene some embodiments the RNA molecule comprises at least one having a sequence selected from the Target Gene Sequences segment of 18 or more contiguous nucleotides that is essen Group or the DNA complement thereof. In particularembodi tially complementary to a fragment of a DNA having a ments, the RNA is a double-stranded nucleic acid (e.g., sequence selected from the group consisting of SEQ ID dsRNA) with one strand comprising at least one segment of at NOs:831-1085, 1095-1104, and 1110-11 14, or the comple least 21 contiguous nucleotides with a sequence of 100% ment thereof, or wherein the RNA molecule is encoded by a identity with a fragment of equivalent length of a DNA or sequence selected from the group consisting of SEQID NOs: target gene having a sequence selected from the Target Gene 1105-1109. In some embodiments the RNA molecule is Sequences Group or the DNA complement thereof; expressed double-stranded, and the at least one segment is between as base-pairs. Such a double-stranded nucleic acid comprises about 50 to about 500 base-pairs in length. In some embodi at least one segment of at least 21 contiguous, perfectly ments the RNA molecule is a dsRNA with a strand having a matched base-pairs which correspond to a fragment of sequence selected from the group consisting of the Trigger equivalent length of a DNA or target gene having a sequence Sequences Group. In some embodiments, an insecticidal selected from the Target Gene Sequences Group or the DNA composition is provided for controlling a Leptinotarsa spe complement thereof. In particular embodiments, each seg cies, wherein the insecticidal composition comprises a ment contained in the RNA molecule is of a length greater double-stranded RNA, wherein at least one strand of the than that which is typical of naturally occurring regulatory double-stranded RNA is complementary to at least 21 con Small RNAS. In some embodiments, each segment is at least tiguous nucleotides of a gene that encodes a ribosomal pro about 30 contiguous nucleotides (or base-pairs) in length. In tein or an RNA transcribed from the gene, wherein the Lep some embodiments, the total length of the RNA molecule, or tinotarsa species is Leptinotarsa decemlineata, and wherein the length of each segment contained in the RNA molecule, is RNA interference is induced and Leptinotarsa decemlineata less than the total length of the sequence of interest (DNA or mortality occurs, and wherein the ribosomal protein is a ribo target gene having a sequence selected from the group con somal L7 protein or a protein encoded by SEQID NO:730 or sisting of the Target Gene Sequences Group). In some wherein the double-stranded RNA comprises a sequence embodiments, the total length of the RNA molecule is selected from the group consisting of SEQID NO:989,988, between about 50 to about 500 nucleotides (for single 1104, or 1105. stranded polynucleotides) or base-pairs (for double-stranded 0124 Embodiments of the RNA molecule include those polynucleotides). In some embodiments, the RNA molecule wherein the segment of 18 or more contiguous nucleotides is a dsRNA of between about 100 to about 500 base-pairs, has a sequence with about 95%, about 96%, about 97%, about such as a dsRNA of the length of any of the dsRNA triggers 98%, about 99%, or about 100% complementarity to a frag disclosed in Tables 3, 5, 8, 9, and 10. In some embodiments, ment of a DNA having a sequence selected from the Target the insecticidal composition consists essentially of an insec Gene Sequences Group or the DNA complement thereof. In ticidally effective amount of a double-stranded RNA mol Some embodiments the contiguous nucleotides are exactly ecule with one Strand having a sequence selected from the (100%) complementary to a fragment of equivalent length of group consisting of: SEQID NOS:831-1085, 1095-1104, and a DNA having a sequence selected from the Target Gene 1110-11 14, or the complement thereof, or consists essentially Sequences Group or the DNA complement thereof. In some an insecticidally effective amount of an RNA hairpin encoded embodiments, the RNA molecule has an overall sequence of by a sequence selected from the group consisting of SEQID about 95%, about 96%, about 97%, about 98%, about 99%, or NOs: 1105-1109. In some embodiments, the insecticidal about 100% complementarity with a DNA having a sequence composition consists essentially of an insecticidally effective selected from the Target Gene Sequences Group or the DNA amount of a double-stranded RNA molecule with one strand complement thereof. having a sequence selected from the group consisting of the Trigger Sequences Group. 0125 Embodiments of the RNA molecule include at least one segment of 18 or more contiguous nucleotides designed 0.126 The RNA molecule is generally designed to sup to Suppress expression of a target gene having a sequence press one or more genes (“target genes”). Such target genes selected from the Target Gene Sequences Group or the DNA can include coding or non-coding sequence or both. In spe complement thereof. The contiguous nucleotides of the seg cific embodiments, the RNA molecule is designed to suppress ment number at least 18, e.g., between 18-24, or between one or more target genes, where each target gene has a DNA 18-28, or between 20-30, or between 20-50, or between sequence selected from the group consisting of the Target 20-100, or between 50-100, or between 50-500, or between Gene Sequences Group. In various embodiments, the RNA 100-250, or between 100-500, or between 200-1000, or molecule is designed to suppress one or more genes, where between 500-2000, or even greater. In some embodiments, each gene has a sequence selected from the group consisting the contiguous nucleotides number more than 18, e.g., 19, 20, of the Target Gene Sequences Group, and can be designed to 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., Suppress multiple genes from this group, or to target different about 35, about 40, about 45, about 50, about 55, about 60, regions of one or more of these genes. Embodiments of the about 65, about 70, about 75, about 80, about 85, about 90, RNA molecule include at least one segment of 18 or more about 95, about 100, about 110, about 120, about 130, about contiguous nucleotides having a sequence designed to Sup US 2015/O 143580 A1 May 21, 2015 press one or more genes, where each gene has a sequence tiguous nucleotides with a sequence of 100% identity with a selected from the group consisting of the Target Gene fragment of a DNA having a sequence selected from the Sequences Group. In an embodiment, the RNA molecule Target Gene Sequences Group, or the DNA complement comprises multiple sections or segments each of which com thereof, wherein (1) the at least two segments are separated by prises at least one segment of 21 contiguous nucleotides with one or more spacer nucleotides, or (2) the at least two seg a sequence of 100% complementarity to a fragment of ments are arranged in an order different from that in which the equivalent length of a DNA having a sequence selected from corresponding fragments occur in the DNA having a the Target Gene Sequences Group or the DNA complement sequence selected from the Target Gene Sequences Group, or thereof. In such cases, each section can be identical or differ the DNA complement thereof. ent in size or in sequence. For example, in one embodiment I0128. The RNA molecule can be single-stranded (ss) or the RNA molecule comprises multiple sections in tandem or double-stranded (ds) or a combination of both. Embodiments repetitive arrangements, wherein each section comprises at of the RNA molecule include sense single-stranded RNA least one segment of 21 contiguous nucleotides with a (ssRNA), anti-sense ssRNA, or double-stranded RNA sequence of 100% complementarity to a fragment of equiva (dsRNA), or a combination of any of these. The RNA can lent length of a DNA having a sequence selected from the include components other than standard ribonucleotides, e.g., Target Gene Sequences Group or the DNA complement an embodiment is an RNA that comprises terminal deoxyri thereof; the segments can be from different regions of the bonucleotides. In various embodiments the RNA molecule target gene, e.g., the segments can correspond to different consists of naturally occurring ribonucleotides. In certain exon regions of the target gene, and 'spacer nucleotides embodiments, the RNA molecule is a combination of ribo which do not correspond to a target gene can optionally be nucleotides and deoxyribonucleotides, for example, Syn used in between or adjacent to the segments. thetic RNA molecule consisting mainly of ribonucleotides 0127. The RNA molecule can have a total length that is but with one or more terminal deoxyribonucleotides or one or greater than 18 contiguous nucleotides, and can include more terminal. In certain embodiments, the RNA molecule nucleotides in addition to the segment of at least one segment comprises non-canonical nucleotides such as inosine, thiou of 18 or more contiguous nucleotides having the sequence of ridine, or pseudouridine. In certain embodiments, the RNA about 95% to about 100% complementarity to a fragment of molecule comprises chemically modified nucleotides. equivalent length of a DNA having a sequence selected from I0129. The RNA molecule is provided by suitable means the Target Gene Sequences Group or the DNA complement known to one in the art. Embodiments include those wherein thereof. In other words, the total length of the RNA molecule the RNA molecule is synthesized in vitro, produced by can be greater than the length of the segment which is expression in a microorganism or in cell culture (such as plant designed to Suppress one or more target genes, where each or insect cells grown in culture), produced by expression in a target gene has a DNA sequence selected from the group plant cell, or produced by microbial fermentation. consisting of the Target Gene Sequences Group. For example, 0.130. In some embodiments the RNA molecule comprises the RNA molecule can have nucleotides flanking the “active' other RNA elements, such as RNA aptamers or ribozymes, segment of at least one segment of 18 or more contiguous additional non-coding RNA (e.g., additional Suppression ele nucleotides that Suppresses the target gene, or include ments), or one or more recognition sites for binding and 'spacer nucleotides between active segments, or can have cleavage by a small RNA (e.g., by a miRNA oran siRNA that additional nucleotides at the 5' end, or at the 3' end, or at both is expressed only in a particular cell or tissue). the 5' and 3' ends. In an embodiment, the RNA molecule I0131 The insecticidal composition can be provided for comprises additional nucleotides that are not specifically topical application to a Surface of a plant or of a plant seed, or related (having a sequence not complementary oridentical to) for topical application to any Substrate needing protection to the DNA or target gene having a sequence selected from the from a Leptinotarsa species infestation. Likewise, the insec Target Gene Sequences Group or the DNA complement ticidal composition can be provided for topical application to thereof, e.g., nucleotides that provide stabilizing secondary a Leptinotarsa species, or in a composition for ingestion by a structure or for convenience in cloning or manufacturing. In Leptinotarsa species. In various embodiments, the insecti an embodiment, the RNA molecule comprises additional cidal composition is provided in the form of at least one nucleotides located immediately adjacent to one or more selected from the group consisting of a solid, liquid (includ segment of 18 or more contiguous nucleotides with a ing homogeneous mixtures Such as Solutions and non-homo sequence of about 95% to about 100% complementarity to a geneous mixtures such as Suspensions, colloids, micelles, and fragment of equivalent length of a DNA or target gene having emulsions), powder, Suspension, emulsion, spray, encapsu a sequence selected from the group consisting of the Target lated or micro-encapsulation formulation, in or on micro Gene Sequences Group. In an embodiment, the RNA mol beads or other carrier particulates, in a film or coating, or on ecule comprises one Such segment, with an additional 5' G or or within a matrix. Suitable binders, inert carriers, surfac an additional 3' C or both, adjacent to the segment. In another tants, and the like can included in the insecticidal composi embodiment, the RNA molecule is a double-stranded RNA tion, as is known to one skilled in formulation of pesticides comprising additional nucleotides to form an overhang, for and seed treatments. While the insecticidal composition con example, a dsRNA comprising 2 deoxyribonucleotides to sists essentially of an RNA molecule, in some embodiments form a 3' overhang. Thus in various embodiments, the nucle the insecticidal composition further comprises at least one otide sequence of the entire RNA molecule is not 100% non-insecticidal agent selected from the group consisting of a identical or complementary to a fragment of contiguous carrier agent, a salt, a surfactant, a cationic lipid (such as that nucleotides in the DNA or target gene having a sequence disclosed in Example 18 of U.S. patent application publica selected from the group consisting of the Target Gene tion 2011/0296.556, incorporated by reference herein), an Sequences Group. For example, in some embodiments the organosilicone, an organosilicone surfactant, a polynucle RNA molecule comprises at least two segments of 21 con otide herbicidal molecule, a non-polynucleotide herbicidal US 2015/O 143580 A1 May 21, 2015 32 molecule, and a safener. In one embodiment the composition the Tribolium castaneum genes listed in Table 1. In some containing the recombinant RNA molecule further comprises embodiments, the polynucleotide is a double-stranded RNA. a nonionic organosilicone surfactant such as SILWETR In some embodiments, the polynucleotide (e.g., double brand surfactants, e.g., SILWET L-77(R) brand surfactant hav stranded RNA) is chemically synthesized or is produced by ing CAS Number 27306-78-1 and EPA Number: CALREG. expression in a microorganism or by expression in a plant NO. 5905-50073-AA, currently available from Momentive cell. In some embodiments the polynucleotide comprises at Performance Materials, Albany, N.Y. Furthermore, the insec least one segment of 18 or more contiguous nucleotides that ticidal composition can be used in combination with, Subse is essentially identical or complementary to a sequence quently to, or preceding, treatment with a polynucleotide selected from the group consisting of the Target Gene herbicidal molecule, a non-polynucleotide herbicidal mol Sequences Group. In some embodiments the polynucleotide ecule, a non-polynucleotide pesticide (e.g., at least one pes is a dsRNA with a strand having a sequence selected from the ticidal agent selected from the group consisting of a patatin, a group consisting of: SEQID NOS:831-1085, 1095-1104, and plant lectin, a phytoecdysteroid, a Bacillus thuringiensis 1110-11 14, or the complement thereof, or wherein the poly insecticidal protein, a Xenorhabdus insecticidal protein, a nucleotide is encoded by a sequence selected from the group Photorhabdus insecticidal protein, a Bacillus laterosporous consisting of SEQID NOs: 1105-1109. In some embodiments insecticidal protein, and a Bacillus sphaericus insecticidal the polynucleotide comprises a dsRNA with a strand having a protein). Related compositions include combinations of the sequence selected from the Trigger Sequences Group. RNA molecule with a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucle I0134. In one embodiment the method comprises topically otide pesticide. applying to the plant a composition comprising at least one 0132) The insecticidal composition can be provided for polynucleotide comprising at least one segment of 18 or more dietary uptake by a Leptinotarsa species by applying the contiguous nucleotides that are essentially identical or composition to a plant or Surface Subject to infestation by the complementary to a fragment of equivalent length of a DNA Leptinotarsa species, for example by spraying, dusting, or of a target gene selected from the group consisting of the coating the plant, or by application of a Soil drench, or by genes identified in the Target Gene Sequences Group, providing in an artificial diet. The insecticidal composition whereby the plant treated with the polynucleotide composi can be provided for dietary uptake by a Leptinotarsa species tion exhibits improved resistance to a Leptinotarsa species in an artificial diet formulated to meet the particular nutri infestation, relative to an untreated plant. By “topical appli tional requirements for maintaining the Leptinotarsa species, cation' is meant application to the Surface or exterior of an wherein the artificial diet is supplemented with some amount object, such as the surface or exterior of a plant, such as of the recombinant RNA molecule obtained from a separate application to the Surfaces of a plant part Such as a leaf stem, Source such as in vitro synthesis or purified from a microbial flower, fruit, shoot, root, seed, tuber, flowers, anthers, or fermentation or other biological Source; this embodiment can pollen, or application to an entire plant, or to the above be useful, e.g., for determining the timing and amounts of ground or below-ground portions of a plant. Topical applica effective treatment regimes. The insecticidal composition can tion can be carried out on non-living Surfaces, such as appli be provided for dietary uptake by the Leptinotarsa species in cation to Soil, or to a surface or matrix by which a Leptinotarsa insect can come in contact with the polynucle the form of a seed treatment. otide. In various embodiments of the method, the polynucle Methods of Providing Plants Having Improved Resistance to otide-containing composition is topically applied to the plant Leptinotarsa Species Infestations, and the Plants, Plant Parts, in a suitable form, e.g., as a Solid, liquid (including homoge and Seeds Thus Provided neous mixtures Such as solutions and non-homogeneous mix tures such as Suspensions, colloids, micelles, and emulsions), 0133. Several embodiments relate to a method of provid powder, Suspension, emulsion, spray, encapsulated or micro ing a plant having improved resistance to a Leptinotarsa encapsulation formulation, in or on microbeads or other car species infestation comprising providing to the plant at least rier particulates, in a film or coating, or on or within a matrix, one polynucleotide comprising at least one segment of 18 or or as a seed treatment. In some embodiments of the method, more contiguous nucleotides that is essentially identical or the polynucleotide-containing composition is topically complementary to a fragment of a target gene selected from applied to above-ground parts of the plant, e.g., sprayed or the group consisting of the genes identified in the Target Gene dusted onto leaves, stems, and flowering parts of the plant. Sequences Group. In an embodiment, this invention provides Embodiments of the method include topical application of a a method of providing a plant having improved resistance to foliar spray (e.g., spraying a liquid polynucleotide-containing a Leptinotarsa species infestation comprising providing to composition on leaves of a Solanaceous plant) or a foliar dust the plant at least one polynucleotide comprising at least one (e.g., dusting a Solanaceous plant with a polynucleotide-con segment that is identical or complementary to at least 21 taining composition in the form of a powder or on carrier contiguous nucleotides of a target gene or an RNA tran particulates). In other embodiments, the polynucleotide-con scribed from the target gene, wherein the target gene is taining composition is topically applied to below-ground selected from the genes identified in the Target Gene parts of the plant, such as to the roots, e.g., by means of a soil Sequences Group oran RNA transcribed from the target gene. drench. In other embodiments, the polynucleotide-containing Embodiments of these target genes are identified by name in composition is topically applied to a seed that is grown into Tables 1, 2 and 4 and include genes having a sequence the plant. The topical application can be in the form of topical selected from the group consisting of the Target Gene treatment of fruits of Solanaceous plants or seeds from fruits Sequences Group, as well as related genes, including ortho of Solanaceous plants, or in the form of topical treatment of logues from related insect species, for example related genes 'seed potato’ tubers or pieces of tuber (e.g., by soaking, from other Leptinotarsa species, Tribolium species, or other coating, or dusting the seed potato). Suitable binders, inert related genera. Examples of Such related target genes include carriers, Surfactants, and the like can optionally be included in US 2015/O 143580 A1 May 21, 2015 the polynucleotide-containing composition, as is known to ticidal protein, and a Bacillus sphaericus insecticidal protein, one skilled in formulation of pesticides and seed treatments. is found to effect synergistically improved prevention or con In some embodiments, the polynucleotide-containing com trol of Leptinotarsa species infestations when topically position is at least one topically implantable formulation applied to a plant. selected from the group consisting of a particulate, pellet, or 0.136. In some embodiments, the method comprises topi capsule topically implanted in the plant; in Such embodiments cally applying to the plant a composition comprising at least the method comprises topically implanting in the plant the one polynucleotide comprising at least one segment of 18 or topically implantable formulation. In some embodiments, the more contiguous nucleotides that are essentially identical or polynucleotide-containing composition is at least one in-fur complementary to a fragment of equivalent length of a DNA row formulation selected from the group consisting of a pow of a target gene selected from the group consisting of the der, granule, pellet, capsule, spray, or drench, or any other genes identified in the Target Gene Sequences Group. The forms Suited for topically applying to a furrow; in Such polynucleotide topically applied to the plant can be single embodiments, the method includes an in-furrow treatment stranded (ss) or double-stranded (ds). with the in-furrow formulation. In one embodiment the poly 0.137 The polynucleotide topically applied to the plant is nucleotide-containing composition can be ingested or other provided by suitable means known to one in the art. Embodi wise absorbed internally by the Leptinotarsa species. For ments include those wherein the polynucleotide is chemically example, the polynucleotide-containing composition can be synthesized (e.g., by in vitro transcription, Such as transcrip in the form of bait. In some embodiments, the polynucleotide tion using a T7 polymerase or other polymerase), produced containing composition further comprises one or more com by expression in a microorganism or in cell culture (such as ponents selected from the group consisting of a carrier agent, plantorinsect cells grown in culture), produced by expression a surfactant, a cationic lipid (such as that disclosed in in a plant cell, or produced by microbial fermentation. Example 18 of U.S. patent application publication 2011/ 0.138. In many embodiments the polynucleotide topically 0296.556, incorporated by reference herein), an organosili applied to the plant is provided as an isolated DNA or RNA. cone, an organosilicone surfactant, a polynucleotide herbi In some embodiments, the polynucleotide topically applied cidal molecule, a non-polynucleotide herbicidal molecule, a to the plant is not part of an expression construct and is non-polynucleotide pesticide, a safener, and an insect growth lacking additional elements such as a promoter or terminator regulator. In one embodiment the composition further com sequences. Such polynucleotides can be relatively short. Such prises a nonionic organosilicone Surfactant such as SIL as single- or double-stranded polynucleotides of between WETR) brand surfactants, e.g., SILWET L-77R brand surfac about 18 to about 300 or between about 50 to about 500 tant having CAS Number 27306-78-1 and EPA Number: nucleotides (for single-stranded polynucleotides) or between CALREGNO. 5905-50073-AA, currently available from about 18 to about 300 or between about 50 to about 500 Momentive Performance Materials, Albany, N.Y. In some base-pairs (for double-stranded polynucleotides). In some embodiments, the topically applied composition further com embodiments, the polynucleotide is a dsRNA of between prises at least one pesticidal agent selected from the group about 100 to about 500 base-pairs, such as a dsRNA of the consisting of a patatin, a plant lectin, a phytoecdysteroid, a length of any of the dsRNA triggers disclosed in Tables 3, 5, Bacillus thuringiensis insecticidal protein, a Xenorhabdus 8,9, and 10. Alternatively the polynucleotide can be provided insecticidal protein, a Photorhabdus insecticidal protein, a in more complex constructs, e.g., as part of a recombinant Bacillus laterosporous insecticidal protein, and a Bacillus expression construct, or included in a recombinant vector, for sphaericus insecticidal protein. Alternatively such additional example in a recombinant plant virus vector or in a recombi components or pesticidal agents can be provided separately, nant baculovirus vector. Such recombinant expression con e.g., by separate topical application or by transgenic expres structs or vectors can be designed to include additional ele sion in the plant. Alternatively the plant is topically treated ments, such as expression cassettes for expressing a gene of with the polynucleotide-containing composition as well as interest (e.g., an insecticidal protein). with a separate (preceding, following, or concurrent) appli 0.139. The polynucleotide topically applied to the planthas cation of a Substance that improves the efficacy of the poly at least one segment of 18 or more contiguous nucleotides that nucleotide-containing composition. For example, a plant can are essentially identical or complementary to a fragment of be sprayed with a first topical application of a solution con equivalent length of a DNA of a target gene selected from the taining a nonionic organosilicone Surfactant such as SIL group consisting of the genes identified in the Target Gene WETR) brand surfactants, e.g., SILWET L-77R brand surfac Sequences Group, or that have a sequence of about 95% to tant, followed by a second topical application of the about 100% identity with or complementarity to a fragment polynucleotide-containing composition, or Vice-versa. of equivalent length of a DNA of a target gene selected from 0135) It is anticipated that the combination of certain poly the group consisting of the genes identified in the Target Gene nucleotides (e.g., the polynucleotide triggers described in the Sequences Group. In an embodiment the polynucleotide topi working Examples) with one or more non-polynucleotide cally applied to the plant comprises at least one segment of 18 pesticidal agents will result in a synergetic improvement in or more contiguous nucleotides that are essentially identical prevention or control of Leptinotarsa species infestations, or complementary to a fragment of equivalent length of a when compared to the effect obtained with the polynucleotide DNA of a target gene selected from the group consisting of alone or the non-polynucleotide pesticidal agent alone. In an the genes identified in the Target Gene Sequences Group. In embodiment, a composition containing one or more poly Some embodiments, the contiguous nucleotides have a nucleotides and one or more non-polynucleotide pesticidal sequence of about 95%, about 96%, about 97%, about 98%, agent selected from the group consisting of a patatin, a plant about 99%, or about 100% identity with or complementarity lectin, a phytoecdysteroid, a Bacillus thuringiensis insecti to the fragment of equivalent length of a DNA of a target gene cidal protein, a Xenorhabdus insecticidal protein, a Photo selected from the group consisting of the genes identified in rhabdus insecticidal protein, a Bacillus laterosporous insec the Target Gene Sequences Group. In some embodiments the US 2015/O 143580 A1 May 21, 2015 34 contiguous nucleotides are exactly (100%) identical or active segments, or can have additional nucleotides at the 5' complementary to a fragment of equivalent length of a DNA end, or at the 3' end, or at both the 5' and 3' ends. In an of a target gene selected from the group consisting of the embodiment, the polynucleotide topically applied to the plant genesidentified in the Target Gene Sequences Group. In some comprises additional nucleotides that are not specifically embodiments, the polynucleotide has an overall sequence of related (having a sequence not complementary oridentical to) about 95%, about 96%, about 97%, about 98%, about 99%, or to the target gene is selected from the group consisting of the about 100% identity or complementarity with a fragment of a genes identified in the Target Gene Sequences Group, e.g., DNA of a target gene selected from the group consisting of nucleotides that provide stabilizing secondary structure or for the genes identified in the Target Gene Sequences Group. convenience in cloning or manufacturing. In an embodiment, 0140. The polynucleotide topically applied to the plant is the polynucleotide topically applied to the plant comprises generally designed to Suppress one or more genes (“target additional nucleotides located immediately adjacent to one or genes'). In specific embodiments, the polynucleotide is more segment of 18 or more contiguous nucleotides with a designed to Suppress one or more target genes selected from sequence of about 95% to about 100% identity with or the group consisting of the genes identified in the Target Gene complementarity to the target gene selected from the group Sequences Group. Embodiments of the genes identified in the consisting of the genes identified in the Target Gene Target Gene Sequences Group include, but are not limited to, Sequences Group. In an embodiment, the polynucleotide the cDNA sequences selected from the group consisting of topically applied to the plant comprises one such segment, the Target Gene Sequences Group. In various embodiments, with an additional 5' G or an additional 3' C or both, adjacent the polynucleotide topically applied to the plant is designed to to the segment. In another embodiment, the polynucleotide Suppress one or more genes, where each gene is selected from topically applied to the plant is a double-stranded RNA com the group consisting of the genes identified in the Target Gene prising additional nucleotides to form an overhang, for Sequences Group, and can be designed to Suppress multiple example, a dsRNA comprising 2 deoxyribonucleotides to genes from this group, or to target different regions of one or form a 3' overhang. Thus in various embodiments, the nucle more of these genes. In an embodiment, the polynucleotide otide sequence of the entire polynucleotide topically applied topically applied to the plant comprises multiple sections or to the plant is not 100% identical or complementary to a segments each of which comprises at least one segment of 18 fragment of contiguous nucleotides in the target gene selected or more contiguous nucleotides with a sequence of about 95% from the group consisting of the genes identified in the Target to about 100% identity or complementarity with a fragment Gene Sequences Group. For example, in some embodiments of equivalent length of a DNA of a target gene selected from the polynucleotide topically applied to the plant comprises at the group consisting of the genes identified in the Target Gene least two segments each of 21 contiguous nucleotides with a Sequences Group. In Such cases, each section can be identical sequence of 100% identity with a fragment of a target gene or different in size or in sequence, and can be sense or anti selected from the group consisting of the genes identified in sense relative to the target gene. For example, in one embodi the Target Gene Sequences Group, wherein (1) the at least two ment the polynucleotide topically applied to the plant can segments are separated by one or more spacer nucleotides, or include multiple sections in tandem or repetitive arrange (2) the at least two segments are arranged in an order different ments, wherein each section comprises at least one segment from that in which the corresponding fragments occur in the of 21 contiguous nucleotides with a sequence of 100% iden target gene selected from the group consisting of the genes tity or 100% complementarity with a fragment of equivalent identified in the Target Gene Sequences Group. length of a DNA of a target gene selected from the group 0142. In a related aspect, this invention is directed to the consisting of the genes identified in the Target Gene plant having improved resistance to a Leptinotarsa species Sequences Group; the segments can be from different regions infestation, provided by this method which comprises topi of the target gene, e.g., the segments can correspond to dif cally applying to the plant a composition comprising at least ferent exon regions of a cDNA with a sequence selected from one polynucleotide comprising at least one segment of 18 or the group consisting of the Target Gene Sequences Group, more contiguous nucleotides that are essentially identical or and 'spacer nucleotides which do not correspond to a target complementary to a fragment of equivalent length of a DNA gene can optionally be used in between or adjacent to the of a target gene selected from the group consisting of the Segments. genes identified in the Target Gene Sequences Group, 0141. The total length of the polynucleotide topically whereby the plant treated with the polynucleotide composi applied to the plant can be greater than 18 contiguous nucle tion exhibits improved resistance to a Leptinotarsa species otides, and can include nucleotides in addition to the at least infestation, relative to an untreated plant. In yet another one segment of contiguous nucleotides having the sequence aspect, this invention is directed to seed (especially transgenic essentially identical or complementary to a fragment of progeny seed) produced by the plant having improved resis equivalent length of a DNA of a target gene selected from the tance to a Leptinotarsa species infestation, as provided by this group consisting of the genes identified in the Target Gene method. Also contemplated is a commodity product produced Sequences Group. In other words, the total length of the by the plant having improved resistance to a Leptinotarsa polynucleotide topically applied to the plant can be greater species infestation, as provided by this method, and a com than the length of the section or segment of the polynucleotide modity product produced from the transgenic progeny seed of designed to Suppress one or more target genes, where each Such a plant. target gene is selected from the group consisting of the genes 0143. In another embodiment the method comprises identified in the Target Gene Sequences Group. For example, expressing in the plant at least one polynucleotide comprising the polynucleotide topically applied to the plant can have at least one segment of 18 or more contiguous nucleotides that nucleotides flanking the “active' segment of at least one are essentially identical or complementary to a fragment of segment of 18 or more contiguous nucleotides that Suppresses equivalent length of a target gene selected from the group the target gene, or include “spacer nucleotides between consisting of the genes identified in the Target Gene US 2015/O 143580 A1 May 21, 2015

Sequences Group, whereby the plant expressing the poly first polynucleotide is provided to a plant in the form of RNA nucleotide exhibits improved resistance to a Leptinotarsa or DNA or both RNA and DNA, and a secondarily produced species infestation, relative to an plant not expressing the second polynucleotide is transiently expressed in the plant. In polynucleotide. In an embodiment the method comprises Some embodiments, the first polynucleotide is one or more expressing in the plant at least one polynucleotide comprising selected from: (a) a single-stranded RNA molecule (ssRNA), at least one segment of 18 or more contiguous nucleotides (b) a single-stranded RNA molecule that self-hybridizes to with a sequence of about 95% to about 100% identity or complementarity with a fragment of equivalent length of a form a double-stranded RNA molecule, (c) a double-stranded DNA of a target gene selected from the group consisting of RNA molecule (dsRNA), (d) a single-stranded DNA mol the genes identified in the Target Gene Sequences Group. ecule (ssDNA), (e) a single-stranded DNA molecule that Embodiments of these target genes are identified by name in self-hybridizes to form a double-stranded DNA molecule, (f) Tables 1, 2 and 4 and include genes having a sequence a single-stranded DNA molecule comprising a modified Pol selected from the group consisting of the Target Gene III gene that is transcribed to an RNA molecule, (g) a double Sequences Group, as well as related genes, including ortho stranded DNA molecule (dsDNA), (h) a double-stranded logues from related insect species, for example related genes DNA molecule comprising a modified Pol III gene that is from other Leptinotarsa species, Tribolium species, or other transcribed to an RNA molecule, and (i) a double-stranded, related genera. Examples of Such related target genes include hybridized RNA/DNA molecule, or combinations thereof. In the Tribolium castaneum genes listed in Table 1. By “express specific embodiments, a first polynucleotide is introduced ing a polynucleotide in the plant' is generally meant into the plant by topical application to the plant of a poly “expressing an RNA transcript in the plant'. However, the nucleotide-containing composition in a Suitable form, e.g., as polynucleotide expressed in the plant can also be DNA, e.g., a solid, liquid (including homogeneous mixtures such as solu a DNA produced in the plant during genome replication. tions and non-homogeneous mixtures Such as Suspensions, 0144. The method comprises expressing at least one poly colloids, micelles, and emulsions), powder, Suspension, nucleotide in a plant, wherein the polynucleotide comprises at emulsion, spray, encapsulated or micro-encapsulation formu least one segment of 18 or more contiguous nucleotides that lation, in or on microbeads or other carrier particulates, in a is essentially identical or complementary to a fragment of a film or coating, or on or within a matrix, or in the form of a target gene selected from the group consisting of the genes treatment of a Solanaceous plant seed or treatment of a seed identified in the Target Gene Sequences Group. In some potato. Suitable binders, inert carriers, Surfactants, and the embodiments, a first polynucleotide is provided to a plant in like can optionally be included in the composition, as is the form of DNA (e.g., in the form of an isolated DNA known to one skilled in formulation of pesticides and seed molecule, or as an expression construct, or as a transforma treatments. In such embodiments, the polynucleotide-con tion vector), and the polynucleotide expressed in the plant is taining composition can further include one or more compo a second polynucleotide (e.g., the RNA transcript of the first nents selected from the group consisting of a carrier agent, a polynucleotide) in the plant. In an embodiment, the poly Surfactant, a cationic lipid (such as that disclosed in Example nucleotide is expressed in the plant by transgenic expression, 18 of U.S. patent application publication 2011/0296.556, i.e., by stably integrating the polynucleotide into the plants incorporated by reference herein), an organosilicone, an orga genome from where it can be expressed in a cell or cells of the nosilicone Surfactant, a polynucleotide herbicidal molecule, a plant. In an embodiment, a first polynucleotide (e.g., a recom non-polynucleotide herbicidal molecule, a non-polynucle binant DNA construct comprising a promoteroperably linked otide pesticide, a safener, and an insect growth regulator; in to DNA comprising at least one segment of 18 or more con one embodiment the composition further comprises a non tiguous nucleotides that is essentially identical or comple ionic organosilicone surfactant such as SILWETR) brand sur mentary to a fragment of target gene selected from the group factants, e.g., SILWET L-77(R) brand surfactant having CAS consisting of the genes identified in the Target Gene Number 27306-78-1 and EPA Number: CALREGNO. Sequences Group is stably integrated into the plant's genome 5905-50073-AA, currently available from Momentive Per from where secondarily produced polynucleotides (e.g., an formance Materials, Albany, N.Y. In some embodiments, the RNA transcript comprising the transcript of the segment of 18 topically applied composition further comprises at least one or more contiguous nucleotides that is essentially identical or pesticidal agent selected from the group consisting of a pata complementary to a fragment of target gene selected from the tin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis group consisting of the genes identified in the Target Gene insecticidal protein, a Xenorhabdus insecticidal protein, a Sequences Group) are expressed in a cell or cells of the plant. Photorhabdus insecticidal protein, a Bacillus laterosporous Methods of providing stably transformed plants are provided insecticidal protein, and a Bacillus sphaericus insecticidal in the section headed "Making and Using Transgenic Plant protein. Alternatively such additional components or pesti Cells and Transgenic Plants’. cidal agents can be provided separately, e.g., by separate 0145. In another embodiment the polynucleotide topical application or by transgenic expression in the plant. expressed in the plant is expressed by transient expression Alternatively the plant is topically treated with the polynucle (i.e., expression not resulting from stable integration of a otide-containing composition as well as with a separate (pre sequence into the plants genome). In such embodiments the ceding, following, or concurrent) application of a Substance method can include a step of introducing a polynucleotide that improves the efficacy of the polynucleotide-containing (e.g., dsRNA or dsDNA) into the plant by routine techniques composition. For example, a plant can be sprayed with a first known in the art. For example, transient expression can be topical application of a solution containing a nonionic orga accomplished by infiltration of a polynucleotide solution nosilicone surfactant such as SILWETR) brand surfactants, using a needle-less Syringe into a leaf of a plant. e.g., SILWET L-77(R) brand surfactant, followed by a second 0146 In some embodiments where the polynucleotide topical application of the polynucleotide-containing compo expressed in the plant is expressed by transient expression, a sition, or Vice-versa. US 2015/O 143580 A1 May 21, 2015 36

0147. It is anticipated that the combination of certain poly stranded DNA molecule (ssDNA), (e)a single-stranded DNA nucleotides of use in this method (e.g., the polynucleotide molecule that self-hybridizes to form a double-stranded DNA triggers described in the working Examples) with one or more molecule, (f) a single-stranded DNA molecule comprising a non-polynucleotide pesticidal agents will result in a syner modified Pol III gene that is transcribed to an RNA molecule, getic improvement in prevention or control of Leptinotarsa (g) a double-stranded DNA molecule (dsDNA), (h) a double species infestations, when compared to the effect obtained stranded DNA molecule comprising a modified Pol III gene with the polynucleotide alone or the non-polynucleotide pes that is transcribed to an RNA molecule, and (i) a double ticidal agent alone. In an embodiment, a transgenic plant stranded, hybridized RNA/DNA molecule, or combinations expressing at least one polynucleotide comprising at least one thereof. In such embodiments where the polynucleotide is segment of 18 or more contiguous nucleotides that is essen expressed by transient expression the first polynucleotide can tially identical or complementary to a fragment of target gene consist of naturally occurring nucleotides, such as those selected from the group consisting of the genes identified in which occur in DNA and RNA. In such embodiments where Table 1 (e.g., the polynucleotide triggers described in the the polynucleotide is expressed by transient expression the working Examples) and one or more genes encoding a non first polynucleotide can be chemically modified, or comprises polynucleotide pesticidal agent selected from the group con chemically modified nucleotides. The first polynucleotide is sisting of apatatin, a plant lectin, a phytoecdysteroid, a Bacil provided by suitable means known to one in the art. Embodi lus thuringiensis insecticidal protein, a Xenorhabdus ments include those wherein the first polynucleotide is insecticidal protein, a Photorhabdus insecticidal protein, a chemically synthesized (e.g., by in vitro transcription, such as Bacillus laterosporous insecticidal protein, and a Bacillus transcription using a T7 polymerase or other polymerase), sphaericus insecticidal protein, is found to exhibit synergis produced by expression in a microorganism or in cell culture tically improved resistance to Leptinotarsa species infesta (such as plant or insect cells grown in culture), produced by tions. expression in a plant cell, or produced by microbial fermen 0148. In some embodiments where the polynucleotide tation. The first polynucleotide can be provided as an RNA or expressed in the plant is expressed by transient expression, a DNA fragment. Alternatively the first polynucleotide can be first polynucleotide is provided to a plant in the form of RNA provided in more complex constructs, e.g., as part of a recom or DNA or both RNA and DNA, and a secondarily produced binant expression construct, or included in a recombinant second polynucleotide is transiently expressed in the plant; vector, for example in a recombinant plant virus vector or in the site of application of the first polynucleotide need not be a recombinant baculovirus vector, Such recombinant expres the same site where the second polynucleotide is transiently sion constructs or vectors can be designed to include addi expressed. For example, a first polynucleotide can be pro tional elements, such as expression cassettes for expressing a vided to a plant by topical application onto a leaf, or by gene of interest (e.g., an insecticidal protein). injection into a stem, and the second polynucleotide can be 0151. In some embodiments the polynucleotide expressed transiently expressed elsewhere in the plant, e.g., in the roots in the plant is an RNA molecule and can be relatively short, or throughout the plant. In some embodiments of the method, such as single- or double-stranded RNAs of between about 18 a composition comprising at least one polynucleotide is topi to about 300 or between about 50 to about 500 nucleotides cally applied to above-ground parts of the plant, e.g., sprayed (for single-stranded RNAs) or between about 18 to about 300 or dusted onto leaves, stems, and flowering parts of the plant. or between about 50 to about 500 base-pairs (for double In other embodiments, a composition comprising at least one stranded RNAs). Alternatively the polynucleotide can be pro polynucleotide is topically applied to below-ground parts of vided in more complex constructs, e.g., as part of a recombi the plant, such as to the roots, e.g., by means of a soil drench. nant expression construct, or included in a recombinant In other embodiments, a composition comprising at least one vector, for example in a recombinant plant virus vector or in polynucleotide is topically applied to a seed (or, in the case of a recombinant baculovirus vector. In some embodiments such potatoes, topically applied to a seed potato) that is grown into recombinant expression constructs or vectors are designed to the plant having improved resistance to a Leptinotarsa spe include additional elements, such as expression cassettes for cies infestation. expressing a gene of interest (e.g., an insecticidal protein). 0149. In some embodiments the polynucleotide expressed 0152 The polynucleotide expressed in the plant has at in the plant is RNA, which can be single-stranded (ss) or least one segment of 18 or more contiguous nucleotides with double-stranded (ds) RNA or a combination of both. a sequence of about 95% to about 100% identity with or 0150. In some embodiments a first polynucleotide (DNA complementarity to a fragment of equivalent length of a target or RNA or both) is provided to a plant and a second poly gene selected from the group consisting of the genes identi nucleotide having a sequence corresponding (identical or fied in the Target Gene Sequences Group. In an embodiment complementary) to the first polynucleotide is Subsequently the polynucleotide expressed in the plant comprises at least expressed in the plant. In such embodiments the polynucle one segment of 18 or more contiguous nucleotides that are otide expressed in the plant is an RNA transcript which can be essentially identical or complementary to a fragment of ssRNA or dsRNA or a combination of both. In some embodi equivalent length of a target gene selected from the group ments where the polynucleotide is expressed by transient consisting of the genes identified in the Target Gene expression, a first polynucleotide is provided to a plant in the Sequences Group. In some embodiments, the contiguous form of RNA or DNA or both RNA and DNA, and a second nucleotides have a sequence of about 95%, about 96%, about arily produced second polynucleotide is transiently expressed 97%, about 98%, about 99%, or about 100% identity with or in the plant; in Such embodiments, the first polynucleotide complementarity to a fragment of equivalent length of a target one or more selected from: (a) a single-stranded RNA mol gene selected from the group consisting of the genes identi ecule (ssERNA), (b) a single-stranded RNA molecule that self fied in the Target Gene Sequences Group. In some embodi hybridizes to form a double-stranded RNA molecule, (c) a ments the contiguous nucleotides are exactly (100%) identi double-stranded RNA molecule (dsRNA), (d) a single cal or complementary to a fragment of equivalent length of a US 2015/O 143580 A1 May 21, 2015 37 target gene selected from the group consisting of the genes secondary structure or for convenience in cloning or manu identified in the Target Gene Sequences Group. In some facturing. In an embodiment, the polynucleotide expressed in embodiments, the polynucleotide expressed in the plant has the plant comprises additional nucleotides located immedi an overall sequence of about 95%, about 96%, about 97%, ately adjacent to one or more segment of 18 or more contigu about 98%, about 99%, or about 100% identity with or ous nucleotides with a sequence of about 95% to about 100% complementarity to a fragment of a target gene selected from identity with or complementarity to a fragment of equivalent the group consisting of the genes identified in the Target Gene length of a target gene selected from the group consisting of Sequences Group. the genes identified in the Target Gene Sequences Group. In 0153. The polynucleotide expressed in the plant is gener an embodiment, the polynucleotide expressed in the plant ally designed to Suppress one or more genes (“target genes'). comprises one Such segment, with an additional 5' G or an Such target genes can include coding or non-coding sequence additional 3' C or both, adjacent to the segment. In another or both. In specific embodiments, the polynucleotide embodiment, the polynucleotide expressed in the plant is a expressed in the plant is designed to Suppress one or more double-stranded RNA comprising additional nucleotides to target genes selected from the group consisting of the genes forman overhang, for example, a dsRNA comprising 2 deox identified in the Target Gene Sequences Group. In various embodiments, the polynucleotide expressed in the plant is yribonucleotides to form a 3' overhang. Thus in various designed to Suppress one or more target genes selected from embodiments, the nucleotide sequence of the entire poly the group consisting of the genes identified in the Target Gene nucleotide expressed in the plant is not 100% identical or Sequences Group, and can be designed to Suppress multiple complementary to a fragment of contiguous nucleotides in genes from this group, or to target different regions of one or the target gene selected from the group consisting of the genes more of these genes. In an embodiment, the polynucleotide identified in the Target Gene Sequences Group. For example, expressed in the plant comprises multiple sections or seg in some embodiments the polynucleotide expressed in the ments each of which comprises at least one segment of 18 or plant comprises at least two segments of 21 contiguous nucle more contiguous nucleotides with a sequence of about 95% to otides with a sequence of 100% identity with or 100% about 100% identity with or complementarity to a fragment complementarity to a fragment of a target gene selected from of a target gene selected from the group consisting of the the group consisting of the genes identified in the Target Gene genes identified in the Target Gene Sequences Group. In Such Sequences Group, wherein (1) the at least two segments are cases, each section can be identical or different in size or in separated by one or more spacer nucleotides, or (2) the at least sequence, and can be sense or anti-sense relative to the target two segments are arranged in an order different from that in gene. For example, in one embodiment the polynucleotide which the corresponding fragments occur in the target gene expressed in the plant can include multiple sections in tandem selected from the group consisting of the genes identified in or repetitive arrangements, wherein each section comprises at the Target Gene Sequences Group. least one segment of 18 or more contiguous nucleotides with 0.155. In a related aspect, this invention is directed to the a sequence of about 95% to about 100% identity with or plant having improved resistance to a Leptinotarsa species complementarity to a fragment of a target gene selected from infestation, provided by expressing in the plant at least one the group consisting of the genes identified in the Target Gene polynucleotide comprising at least one segment of 18 or more Sequences Group; the segments can be from different regions contiguous nucleotides that are essentially identical or of the target gene, e.g., the segments can correspond to dif complementary to a fragment of equivalent length of a target ferent exon regions of the target gene, and 'spacer nucle gene selected from the group consisting of the genes identi otides which do not correspond to a target gene can optionally fied in the Target Gene Sequences Group, whereby the result be used in between or adjacent to the segments. ing plant has improved resistance to a Leptinotarsa species 0154 The total length of the polynucleotide expressed in infestation when compared to a control plant in which the the plant can be greater than 18 contiguous nucleotides, and polynucleotide is not expressed. In a related aspect, this can include nucleotides in addition to the contiguous nucle invention is directed to the plant having improved resistance otides having the sequence of about 95% to about 100% to a Leptinotarsa species infestation, provided by expressing identity with or complementarity to a fragment of a target in the plant at least one polynucleotide comprising at least one gene selected from the group consisting of the genes identi segment of 18 or more contiguous nucleotides with a fied in the Target Gene Sequences Group. In other words, the sequence of about 95% to about 100% identity with or total length of the polynucleotide expressed in the plant can complementarity to a fragment of a target gene selected from be greater than the length of the section or segment of the the group consisting of the genes identified in the Target Gene polynucleotide designed to Suppress one or more target genes Sequences Group, whereby the resulting plant has improved selected from the group consisting of the genes identified in resistance to a Leptinotarsa species infestation when com the Target Gene Sequences Group. For example, the poly pared to a control plant in which the polynucleotide is not nucleotide expressed in the plant can have nucleotides flank expressed. An embodiment is a Solanaceous plant having ing the “active' segment of at least one segment of 18 or more improved resistance to a Leptinotarsa species infestation contiguous nucleotides that Suppresses the target gene, or when compared to a control plant, provided by expressing in include 'spacer nucleotides between active segments, or can the plant an RNA having a sequence selected from the group have additional nucleotides at the 5' end, or at the 3' end, or at consisting of SEQID NOS:831-1085 and 1095. In yet another both the 5' and 3' ends. In an embodiment, the polynucleotide aspect, this invention is directed to seed or propagatable parts expressed in the plant comprises additional nucleotides that (especially transgenic progeny seed or propagatable parts) are not specifically related (having a sequence not comple produced by the plant having improved resistance to a Lepti mentary or identical to) to the target gene selected from the notarsa species infestation, as provided by this method. Also group consisting of the genes identified in the Target Gene contemplated is a commodity product produced by the plant Sequences Group, e.g., nucleotides that provide stabilizing having improved resistance to a Leptinotarsa species infes US 2015/O 143580 A1 May 21, 2015

tation, as provided by this method, and a commodity product double-stranded RNA is complementary to at least 21 con produced from the transgenic progeny seed or propagatable tiguous nucleotides of a gene that encodes a ribosomal pro parts of Such a plant. tein or an RNA transcribed from the gene, wherein the Lep tinotarsa species is Leptinotarsa decemlineata, and wherein Methods of Controlling Leptinotarsa Species Infestations of RNA interference is induced and Leptinotarsa decemlineata a Plant mortality occurs, and wherein the ribosomal protein is a ribo somal L7 protein or a protein encoded by SEQID NO:730 or 0156 Several embodiments relate to a method for control ling a Leptinotarsa species infestation of a plant comprising wherein the double-stranded RNA comprises a sequence contacting the Leptinotarsa species with a polynucleotide selected from the group consisting of SEQID NO:989,988, comprising at least one segment of 18 or more contiguous 1104, or 1105; in some embodiments, the solution further nucleotides that is essentially identical or complementary to a comprises one or more components selected from the group fragment of equivalent length of a DNA of a target gene consisting of an organosilicone surfactant or a cationic lipid. selected from the group consisting of the genes identified in 0157. In some embodiments, the contiguous nucleotides the Target Gene Sequences Group. In this context “control have a sequence of about 95%, about 96%, about 97%, about ling includes inducement of a physiological or behavioural 98%, about 99%, or about 100% identity with or complemen change in a Leptinotarsa species (adult or larvae) such as, but tarity to a fragment of equivalent length of a target gene not limited to, growth stunting, increased mortality, decrease selected from the group consisting of the genes identified in in reproductive capacity, decrease in or cessation of feeding the Target Gene Sequences Group. In some embodiments the behavior or movement, or decrease in or cessation of meta contiguous nucleotides are exactly (100%) identical or morphosis stage development. In an embodiment, the method complementary to a fragment of equivalent length of a target for controlling a Leptinotarsa species infestation of a plant gene selected from the group consisting of the genes identi comprises contacting the Leptinotarsa species with a poly fied in the Target Gene Sequences Group. In some embodi nucleotide comprising at least one segment that is identical or ments, the polynucleotide has an overall sequence of about complementary to at least 21 contiguous nucleotides of a 95%, about 96%, about 97%, about 98%, about 99%, or about target gene selected from the genes identified in the Target 100% identity with or complementarity to a fragment of Gene Sequences Group oran RNA transcribed from the target equivalent length of a target gene selected from the group gene. In some embodiments, the polynucleotide is a double consisting of the genes identified in the Target Gene stranded RNA. In some embodiments, the polynucleotide Sequences Group. In an embodiment, the polynucleotide (e.g., double-stranded RNA) is chemically synthesized or is comprises at least one segment of 21 contiguous nucleotides produced by expression in a microorganism or by expression with a sequence of 100% identity or complementarity with in a plant cell. In some embodiments, the polynucleotide is a the corresponding fragment of a target gene selected from the double-stranded RNA comprising a strand comprising a group consisting of the genes identified in the Target Gene sequence selected from the Trigger Sequences Group. In an Sequences Group; in Some embodiments, the polynucleotide embodiment, the method for controlling a Leptinotarsa spe comprises “neutral” sequence (having no sequence identity cies infestation of a plant comprises contacting the Leptino or complementarity to the target gene) in addition to a seg tarsa species with an effective amount of a double-stranded ment of 21 contiguous nucleotides with 100% identity with RNA, one strand of which is complementary to at least 21 the corresponding fragment of the target gene, and therefore contiguous nucleotides of a gene that encodes a ribosomal the polynucleotide as a whole is of much lower overall protein, wherein RNA interference is induced and mortality sequence identity with a target gene. occurs. Embodiments of target genes are identified by name 0158. The polynucleotide of use in this method is gener in Tables 1, 2 and 4 and include genes having a sequence ally designed to suppress one or more genes (“target genes'). selected from the group consisting of the Target Gene The term “gene' refers to any portion of a nucleic acid that Sequences Group, as well as related genes including ortho provides for expression of a transcript or encodes a transcript. logues from related insect species, for example, related genes A "gene' can include, but is not limited to, a promoter region, from other Leptinotarsa species, Tribolium species, or other 5' untranslated regions, transcript encoding regions that can related coleopteran genera. Examples of Such related genes include intronic regions, 3' untranslated regions, or combina include the Tribolium castaneum genes listed in Table 1. In tions of these regions. In some embodiments, the target genes Some embodiments the polynucleotide comprises at least one can include coding or non-coding sequence or both. In other segment of 18 or more contiguous nucleotides that is essen embodiments, the target gene has a sequence identical to or tially identical or complementary to a fragment of a target complementary to a messenger RNA, e.g., in Some embodi gene having a sequence selected from the group consisting of ments the target gene is a cDNA. In specific embodiments, the the Target Gene Sequences Group. In some embodiments the polynucleotide is designed to suppress one or more target polynucleotide comprises RNA having a sequence selected genes selected from the group consisting of the genes identi from the group consisting of SEQID NOS:831-1085, 1095 fied in the Target Gene Sequences Group. In various embodi 1104, and 1110-11 14, or the complement thereof, or is an ments, the polynucleotide is designed to suppress one or more RNA hairpin encoded by a sequence selected from the group target genes selected from the group consisting of the genes consisting of SEQID NOs: 1105-1109. In some embodiments identified in the Target Gene Sequences Group, and can be the polynucleotide comprises a dsRNA with a strand having a designed to suppress multiple target genes from this group, or sequence selected from the group consisting of the Trigger to target different regions of one or more of these target genes. Sequences Group. In some embodiments, this invention pro In an embodiment, the polynucleotide comprises multiple vides a method for controlling a Leptinotarsa species infes segments of 21 contiguous nucleotides with a sequence of tation of a plant comprising contacting the Leptinotarsa spe 100% identity with a fragment of equivalent length of a DNA cies with an effective amount of a solution comprising a or target gene having a sequence selected from the Target double-stranded RNA, wherein at least one strand of the Gene Sequences Group or the DNA complement thereof. In US 2015/O 143580 A1 May 21, 2015 39

Such cases, each segment can be identical or different in size 0160 The polynucleotide ofuse in this method is provided or in sequence, and can be sense or anti-sense relative to the by suitable means known to one in the art. Embodiments target gene. For example, in one embodiment the polynucle include those wherein the polynucleotide is chemically Syn otide comprises multiple segments in tandem or repetitive thesized (e.g., by in vitro transcription, Such as transcription arrangements, wherein each segment comprises 18 or more using a T7 polymerase or other polymerase), produced by contiguous nucleotides with a sequence of about 95% to expression in a microorganism or in cell culture (such as plant about 100% identity with or complementarity to a fragment or insect cells grown in culture), produced by expression in a of equivalent length of a target gene selected from the group plant cell, or produced by microbial fermentation. consisting of the genes identified in the Target Gene 0.161. In some embodiments the polynucleotide of use in Sequences Group; the segments can be from different regions this method is provided as an isolated DNA or RNA fragment. of the target gene, e.g., the segments can correspond to dif In some embodiments the polynucleotide of use in this ferent exon regions of the target gene, and 'spacer nucle method is not part of an expression construct and is lacking otides which do not correspond to a target gene can optionally additional elements such as a promoter or terminator be used in between or adjacent to the segments. sequences). Such polynucleotides can be relatively short, 0159. The total length of the polynucleotide of use in this Such as single- or double-stranded polynucleotides of method can be greater than 18 contiguous nucleotides, and between about 18 to about 300 or between about 50 to about can include nucleotides in addition to the contiguous nucle 500 nucleotides (for single-stranded polynucleotides) or otides having the sequence of about 95% to about 100% between about 18 to about 300 or between about 50 to about identity with or complementarity to a fragment of equivalent 500 base-pairs (for double-stranded polynucleotides). In length of a target gene selected from the group consisting of some embodiments, the polynucleotide is a dsRNA of the genes identified in the Target Gene Sequences Group. In between about 100 to about 500 base-pairs, such as a dsRNA other words, the total length of the polynucleotide can be of the length of any of the dsRNA triggers disclosed in Tables greater than the length of the section or segment of the poly 3, 5, 8, 9, and 10. Embodiments include those in which the nucleotide designed to Suppress one or more target genes polynucleotide is a dsRNA comprising a segment having a selected from the group consisting of the genes identified in sequence selected from the group consisting of SEQ ID the Target Gene Sequences Group. For example, the poly NOs:831-1085, 1095-1104, and 1110-11 14, or the comple nucleotide can have nucleotides flanking the “active' seg ment thereof, or wherein the polynucleotide is an RNA hair ment of at least one segment of 18 or more contiguous nucle pin encoded by a sequence selected from the group consisting otides that suppresses the target gene, or include “spacer” of SEQID NOS:1105-1109. Alternatively the polynucleotide nucleotides between active segments, or can have additional can be provided in more complex constructs, e.g., as part of a nucleotides at the 5' end, or at the 3' end, or at both the 5' and recombinant expression construct, or included in a recombi 3' ends. In an embodiment, the polynucleotide can include nant vector, for example in a recombinant plant virus vector or additional nucleotides that are not specifically related (having in a recombinant baculovirus vector. In some embodiments a sequence not complementary or identical to) to the target Such recombinant expression constructs or vectors are gene selected from the group consisting of the genes identi designed to include additional elements, such as expression fied in the Target Gene Sequences Group, e.g., nucleotides cassettes for expressing a gene of interest (e.g., an insecticidal that provide stabilizing secondary structure or for conve protein). nience in cloning or manufacturing. In an embodiment, the (0162. In various embodiments of the method, the contact polynucleotide can include additional nucleotides located ing comprises application to a surface of the Leptinotarsa immediately adjacent to one or more segment of 18 or more species of a suitable composition comprising the polynucle contiguous nucleotides with a sequence of about 95% to otide of use in this method; such a composition can be pro about 100% identity with or complementarity to a fragment vided, e.g., as a solid, liquid (including homogeneous mix of equivalent length of a target gene selected from the group tures such as solutions and non-homogeneous mixtures Such consisting of the genes identified in the Target Gene as Suspensions, colloids, micelles, and emulsions), powder, Sequences Group. In an embodiment, the polynucleotide Suspension, emulsion, spray, encapsulated or micro-encapsu comprises one Such segment, with an additional 5' G or an lation formulation, in or on microbeads or other carrier par additional 3' C or both, adjacent to the segment. In another ticulates, in a film or coating, or on or within a matrix, or as a embodiment, the polynucleotide is a double-stranded RNA seed treatment. The contacting can be in the form of a seed comprising additional nucleotides to form an overhang, for treatment, or in the form of treatment of “seed potato’ tubers example, a dsRNA comprising 2 deoxyribonucleotides to or pieces of tuber (e.g., by soaking, coating, or dusting the form a 3' overhang. Thus in various embodiments, the nucle seed potato). Suitable binders, inert carriers, Surfactants, and otide sequence of the entire polynucleotide is not 100% iden the like can optionally be included in the composition, as is tical or complementary to a sequence of contiguous nucle known to one skilled in formulation of pesticides and seed otides in the target gene selected from the group consisting of treatments. In some embodiments, the contacting comprises the genes identified in the Target Gene Sequences Group. For providing the polynucleotide in a composition that further example, in Some embodiments the polynucleotide com comprises one or more components selected from the group prises at least two segments each of 21 contiguous nucle consisting of a carrier agent, a Surfactant, a cationic lipid otides with a sequence of 100% identity with a fragment of (such as that disclosed in Example 18 of U.S. patent applica equivalent length of the target gene, wherein (1) the at least tion publication 2011/0296.556, incorporated by reference two segments are separated by one or more spacer nucle herein), an organosilicone, an organosilicone Surfactant, a otides, or (2) the at least two segments are arranged in an order polynucleotide herbicidal molecule, a non-polynucleotide different from that in which the corresponding fragments herbicidal molecule, a non-polynucleotide pesticide, a occur in the DNA having a sequence selected from the Target safener, and an insect growth regulator. In embodiments, the Gene Sequences Group, or the DNA complement thereof. contacting comprises providing the polynucleotide in a com US 2015/O 143580 A1 May 21, 2015 40 position that further comprises at least one pesticidal agent of single- or low-copy-number genes from the first species to selected from the group consisting of a patatin, a plant lectin, identify corresponding single- or low-copy-number genes a phytoecdysteroid, a Bacillus thuringiensis insecticidal pro from the second species. The single- or low-copy-number tein, a Xenorhabdus insecticidal protein, a Photorhabdus genes from the second species are useful as target genes for insecticidal protein, a Bacillus laterosporous insecticidal pro RNAi-mediated silencing; the sequences of these target genes tein, and a Bacillus sphaericus insecticidal protein. In one are used for designing polynucleotides (e.g., an SSRNA or embodiment the contacting comprises providing the poly dsRNA trigger, such as the dsRNA triggers described in the nucleotide in a composition that can be ingested or otherwise working Examples, or recombinant DNA constructs for mak absorbed internally by the Leptinotarsa species. ing transgenic plants) and methods of use thereof for prevent 0163. It is anticipated that the combination of certain poly ing or controlling infestations by the second species. nucleotides of use in this method (e.g., the polynucleotide 0166 Embodiments of the method include a further step triggers described in the working Examples) with one or more of estimating nucleotide diversity for low-fsingle-copy genes non-polynucleotide pesticidal agents will result in a syner in a population of the chosen organism and selecting those getic improvement in prevention or control of Leptinotarsa low-single-copy genes that further have the lowest nucle species infestations, when compared to the effect obtained otide diversity. Low-fsingle-copy genes that further have low with the polynucleotide alone or the non-polynucleotide pes nucleotide diversity are selected as targets for RNAi-medi ticidal agent alone. In an embodiment, a composition con ated silencing. taining one or more polynucleotides and one or more non 0.167 Embodiments of the method include a further step polynucleotide pesticidal agent selected from the group of comparing the ratio of synonymous (K) to nonsynony consisting of a patatin, a plant lectin, a phytoecdysteroid, a mous (K) nucleotide changes as an estimate of functional or Bacillus thuringiensis insecticidal protein, a Xenorhabdus evolutionary constraint. In an embodiment, the method com insecticidal protein, a Photorhabdus insecticidal protein, a prises the step of selecting genes where K is at least equal to Bacillus laterosporous insecticidal protein, and a Bacillus or greater than K. In an embodiment, the method comprises sphaericus insecticidal protein, is found to effect synergisti the step of selecting genes where KPK. cally improved prevention or control of Leptinotarsa species 0168 A related aspect of this invention is a set of target infestations. genes for RNAi-mediated silencing identified from a genome by any of the gene selection methods described herein. An Methods of Selecting Target Genes embodiment relates to a set of target genes for RNAi-medi 0164. Another aspect of this invention provides a method ated silencing selected from a genome by identifying single of non-random selection of target genes for RNAi-mediated or low-copy-number target genes from a larger set of genes silencing. In an embodiment, the method provides a Subset of from that genome. One embodiment relates to a set of target target genes that are present in single- or low-copy-number genes for RNAi-mediated silencing selected from an inverte (non-repetitive and non-redundant) in a particular genome. brate genome by identifying single- or low-copy-number tar Such target genes can be genes from a plant genome or genes get genes from a larger set of genes from that invertebrate from an animal genome. In some embodiments, the target genome. A specific embodiment relates to a set of target genes genes are genes of an invertebrate pest, e.g. an invertebrate for RNAi-mediated silencing in a Leptinotarsa species pest of a plant or an invertebrate pest of a vertebrate. In some selected from a Leptinotarsa genome by identifying single embodiments, the target genes are genes of an insect pest of a or low-copy-number target genes from a larger set of genes plant or a nematode pest of a plant. In some embodiments, the from that Leptinotarsa genome. A specific embodiment target genes are genes of a Leptinotarsa species. Further relates to a set of target genes for RNAi-mediated silencing in aspects include manufacturing a polynucleotide (e.g., an a Leptinotarsa species selected from a Leptinotarsa genome ssRNA or dsRNA trigger, such as the dsRNA triggers by identifying single- or low-copy-number target genes from described in the working Examples, or a recombinant DNA a larger set of genes from that Leptinotarsa genome, wherein construct useful for making transgenic plants) based on target the set of sequences is the group consisting of SEQID NOs: genes for RNAi-mediated silencing selected by any of the 1-725, or the DNA complement thereof. methods described herein. 0169. Related aspects of this invention are methods and 0.165. In an embodiment, the method comprises the step of compositions utilising the set of target genes consisting of identifying single- or low-copy-number genes in the chosen SEQID NOS:1-725, or the DNA complement thereof. These genome, or alternatively to identify single- or low-copy-num include: (i) a method for controlling a Leptinotarsa species ber genes in an orthologous database from related organisms infestation of a plant comprising contacting the Leptinotarsa to predict which genes will be single/low copy in the chosen species with a polynucleotide comprising at least one seg organism. Low-copy genes, and in particular single-copy ment of 18 or more contiguous nucleotides with a sequence of genes, are selected as targets for RNAi-mediated silencing. In about 95% to about 100% identity with a segment of equiva one embodiment, the identification of single- or low-copy lent length of a DNA having a sequence selected from the number genes is carried out by sequence comparison between group consisting of: SEQID NOS:1-725, or the DNA comple a set of genes from a first species and a set of genes from a ment thereof; (ii) a method for controlling a Leptinotarsa second species, wherein the set of genes from a second spe species infestation of a plant comprising providing in the diet cies have been identified as single- or low-copy-number in the of a Leptinotarsa species an agent comprising a polynucle second species. In one embodiment, the identification of otide having at least one segment of 18 or more contiguous single- or low-copy-number genes is carried out by applying nucleotides with a sequence of about 95% to about 100% an algorithm performed by a computer to a set of genes from identity with a segment of equivalent length of a DNA having a first species to identify a Subset of single- or low-copy a sequence selected from the group consisting of: SEQ ID number genes in the set of genes from the first species, then NOs:1-725, or the DNA complement thereof, wherein the comparing a set of genes from a second species to the Subset agent functions upon ingestion by the Leptinotarsa species to US 2015/O 143580 A1 May 21, 2015 inhibit a biological function within the Leptinotarsa species (K) nucleotide changes in genes of that genome and select thereby controlling infestation by the Leptinotarsa species; ing genes where K is at least equal to or greater than K. In an (iii) a method of causing mortality or stunting in Leptinotarsa embodiment, the set of target genes for RNAi-mediated species larvae comprising providing in the diet of Leptino silencing are genes where K is at least equal to or greater than tarsa species larvae at least one recombinant RNA compris K. In an embodiment, the set of target genes for RNAi ing at least one silencing element essentially identical or mediated silencing are genes where KCK. An embodiment essentially complementary to a target gene of the Leptino relates to a set of target genes for RNAi-mediated silencing tarsa species larvae, wherein the target gene sequence is selected from an invertebrate genome and where K->K for selected from the group consisting of SEQ ID NOs: 1-725: the selected genes. (iv) a method of providing a plant having improved resistance 0172. In an embodiment, the single- or low-copy-number to a Leptinotarsa species infestation comprising topically target genes are a Subset of target genes of a first invertebrate applying to the plant a composition comprising at least one species selected from a larger set of genes from the first polynucleotide having at least one segment of 18 or more invertebrate species, wherein the selection is by a sequence contiguous nucleotides with a sequence of about 95% to comparison performed by a computer between the larger set about 100% identity with a segment of equivalent length of a of genes from the first invertebrate species and a set of genes DNA having a sequence selected from the group consisting from a second invertebrate species that have been identified as of: SEQID NOS:1-725, or the DNA complement thereof: (v) single- or low-copy-number in the second invertebrate spe a composition for controlling a Leptinotarsa species compris cies. In a specific embodiment, the single- or low-copy-num ing at least one recombinant polynucleotide comprising at ber target genes are a Subset of Leptinotarsa decemlineata least one segment of 18 or more contiguous nucleotides that target genes selected from a larger set of Leptinotarsa decem is essentially identical or complementary to a segment of lineata target genes, wherein the selection is by a sequence equivalent length of a DNA having a sequence selected from comparison performed by a computer between the larger set the group consisting of SEQID NOS:1-725; (vi) a method of of Leptinotarsa decemlineata target genes and a set of genes providing a plant having improved resistance to a Leptino from a second invertebrate species that have been identified as tarsa species infestation comprising expressing in the plant at single- or low-copy-number in the second invertebrate spe least one polynucleotide comprising at least one segment of cies. The Leptinotarsa decemlineata single- or low-copy 18 or more contiguous nucleotides that is essentially identical number target genes selected by the method are particularly or complementary to a segment of equivalent length of a DNA useful in making polynucleotides of this invention, including having a sequence selected from the group consisting of SEQ recombinant DNA constructs useful, e.g., for providing ID NOs: 1-725; (vii) a recombinant DNA construct compris plants having increased resistance to a Leptinotarsa species ing a heterologous promoter operably linked to DNA com infestation, and isolated recombinant RNA molecules useful, prising at least one segment of 18 or more contiguous nucle e.g., in making compositions for topical treatment of a plant otides with a sequence of about 95% to about 100% identity or Leptinotarsa species to provide prevention or control of a with a segment of equivalent length of a DNA having a Leptinotarsa species infestations. In an embodiment, Lepti sequence selected from the group consisting of SEQ ID notarsa decemlineata single- or low-copy-number target NOs: 1-725, or the DNA complement thereof; and (viii) a genes selected by the method are genes having a sequence transgenic Solanaceous plant cell having in its genome a selected from the group consisting of SEQID NOS:1-725. recombinant DNA encoding RNA that suppresses expression 0173 A further aspect of this invention are polyclonal or of a target gene in a Leptinotarsa species that contacts or monoclonal antibodies that bind a protein encoded by a ingests the RNA, wherein the RNA comprises at least one sequence or a fragment of a sequence selected from the group silencing element complementary to the target gene, and consisting of the Target Gene Sequences Group and poly wherein the target gene sequence is a sequence selected from clonal or monoclonal antibodies that bind a protein encoded the group consisting of: SEQID NOS:1-725, or the comple by a sequence or a fragment of a sequence selected from the ment thereof. Trigger Sequences Group, or the complement thereof. Such 0170 Another embodiment relates to a set of target genes antibodies are made by routine methods as known to one of for RNAi-mediated silencing selected from a genome by ordinary skill in the art, for example using routine protocols estimating nucleotide diversity for a given set of genes in a as described in “Antibody Methods and Protocols’ (Proetzel population of individuals of the species having that genome, and Ebersbach, editors, 2012, Humana Press, New York) or and selecting those genes that have the lowest nucleotide “Making and Using Antibodies' (Howard and Kaser, editors, diversity. One embodiment relates to a set of target genes for 2006, CRC Press, Boca Raton). RNAi-mediated silencing selected from an invertebrate genome by estimating nucleotide diversity for a given set of Selection of Effective Polynucleotides by “Tiling genes in a population of individuals of the invertebrate having 0.174 Polynucleotides of use in the embodiments that genome, and selecting those genes that have the lowest described herein need not be of the full length of a target gene, nucleotide diversity. Another embodiment relates to a set of and in many embodiments are much shorter than the target target genes for RNAi-mediated silencing selected from an gene. An example of a technique that is useful for selecting invertebrate genome by estimating nucleotide diversity for effective polynucleotides is “tiling', or evaluation of poly low-single-copy genes in a population of individuals of the nucleotides corresponding to adjacent or partially overlap invertebrate having that genome, and selecting those low-f ping segments of a target gene. single-copy genes that further have the lowest nucleotide 0.175. In some embodiments, effective polynucleotide diversity. triggers can be identified by “tiling’ gene targets in selected 0171 Another embodiment relates to a set of target genes length fragments, e.g., fragments of 200-300 nucleotides in for RNAi-mediated silencing selected from a genome by length, with partially overlapping regions, e.g., of about 25 comparing the ratio of synonymous (K) to nonsynonymous nucleotides, along the length of the target gene. In some US 2015/O 143580 A1 May 21, 2015 42 embodiments, polynucleotide trigger sequences are designed percent sequence identity when compared to the sequence of to correspond to (have a nucleotide identity or complemen 18 or more contiguous nucleotides in either the endogenous tarity with) regions that are unique to the target gene. In some target gene or to an RNA transcribed from the target gene. In embodiments, the selected region of the target gene can certain embodiments, an “essentially complementary' poly include coding sequence or non-coding sequence (e.g., pro nucleotide has 100 percent sequence complementarity or at moter regions, 3' untranslated regions, introns and the like) or least about 83, 84,85, 86, 87,88, 89,90,91, 92,93, 94.95, 96, a combination of both. 97.98, or 99 percent sequence complementarity when com 0176 Where it is of interest to design a target effective in pared to the sequence of 18 or more contiguous nucleotides in Suppressing multiple target genes, the multiple target gene either the target gene or RNA transcribed from the target sequences are aligned and polynucleotide triggers are gene. designed to correspond to regions with high sequence homol 0180 Polynucleotides containing mismatches to the tar ogy in common among the multiple targets. Conversely, get gene or transcript can be used in certain embodiments of where it is of interest to design a target effective in selectively the compositions and methods described herein. In some Suppressing one among multiple target sequences, the mul embodiments, the polynucleotide includes at least 18 or at tiple target gene sequences are aligned and polynucleotide least 19 or at least 21 contiguous nucleotides that are essen triggers designed to correspond to regions with no or low tially identical or essentially complementary to a segment of sequence homology in common among the multiple targets. equivalent length in the target gene or target gene's transcript. In certain embodiments, a polynucleotide of 21 or more con Thermodynamic Considerations in Selection of Effective tiguous nucleotides that is essentially identical or essentially Polynucleotides complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to 0177. In some embodiments, polynucleotide triggers can the target gene or transcript (i.e., 1 or 2 mismatches between be designed or their sequence optimised using thermody the polynucleotide's 21 contiguous nucleotides and the seg namic considerations. For example, polynucleotide triggers ment of equivalent length in the target gene or target gene's can be selected based on the thermodynamics controlling transcript). In certain embodiments, a polynucleotide of hybridization between one nucleic acid strand (e.g., a poly about 50, 100, 150, 200, 250, 300, 350 or more nucleotides nucleotide trigger or an individual siRNA) and another (e.g., that contains a contiguous 21 nucleotide span of identity or a target gene transcript). complementarity to a segment of equivalent length in the 0.178 Methods and algorithms to predict nucleotide target gene or target gene's transcript can have 1 or 2 or more sequences that are likely to be effective at RNAi-mediated mismatches to the target gene or transcript. silencing of a target gene are known in the art. Non-limiting 0181. In designing polynucleotides with mismatches to an examples of Such methods and algorithms include “i-score'. endogenous target gene or to an RNA transcribed from the described by Ichihara et al. (2007) Nucleic Acids Res., 35(18): target gene, mismatches of certain types and at certain posi 123e: “Oligowalk', publicly available at rna.urmc.rochester. tions that are more likely to be tolerated can be used. In certain edu/servers/oligowalk and described by Lu et al. (2008) embodiments, mismatches formed between adenine and Nucleic Acids Res., 36:W 104-108; and “Reynolds score”, cytosine or guanosine and uracil residues are used as described by Khovorova et al. (2004) Nature Biotechnol., described by Du et al. (2005) Nucleic Acids Res., 33:1671 22:326-330. 1677. In some embodiments, mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, Permitted Mismatches 8 or 11 (from the 5' end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17 (from the 5' end of a 0179. By “essentially identical or “essentially comple 19-nucleotide target), and/or at the high tolerance positions at mentary' is meant that a polynucleotide (or at least one strand either end of the region of complementarity, i.e., positions 1, of a double-stranded polynucleotide) has sufficient identity or 2, 18, and 19 (from the 5' end of a 19-nucleotide target) as complementarity to the target gene or to the RNA transcribed described by Du et al. (2005) Nucleic Acids Res., 33:1671 from a target gene (e.g., the transcript) to suppress expression 1677. Tolerated mismatches can be empirically determined in of a target gene (e.g., to effect a reduction in levels or activity routine assays, e.g., in in vitro dietary assays on Leptinotarsa of the target gene transcript and/or encoded protein). Poly species larvae. nucleotides as described herein need not have 100 percent identity or complementarity to a target gene or to the RNA transcribed from a target gene to suppress expression of the Embedding Silencing Elements in Neutral Sequence target gene (e.g., to effect a reduction in levels or activity of 0182. In some embodiments, a silencing element compris the target gene transcript or encoded protein, or to provide ing a sequence corresponding to the target gene and which is control of a Leptinotarsa species). In some embodiments, the responsible for an observed Suppression of the target gene is polynucleotide or a portion thereof is designed to be essen embedded in “neutral sequence, i.e., inserted into additional tially identical to, or essentially complementary to, a nucleotides that have no sequence identity or complementa sequence of at least 18 or 19 contiguous nucleotides in either rity to the target gene. Neutral sequence can be desirable, e.g., the target gene or the RNA transcribed from the target gene. In to increase the overall length of a polynucleotide. For Some embodiments, the polynucleotide or a portion thereof is example, it can be desirable for a polynucleotide to be of a designed to be 100% identical to, or 100% complementary to, particular size for reasons of stability, cost-effectiveness in one or more sequences of 21 contiguous nucleotides in either manufacturing, or biological activity. In some embodiments, the target gene or the RNA transcribed from the target gene. In neutral sequence is also useful informing the loop in a hairpin certain embodiments, an “essentially identical polynucle trigger or as a spacer between trigger regions. otide has 100 percent sequence identity or at least about 83, 0183. It has been reported that in another coleopteran spe 84, 85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, 97,98, or 99 cies, Diabrotica virgifera, dsRNAS greater than or equal to US 2015/O 143580 A1 May 21, 2015 approximately 60 base-pairs (bp) are required for biological can correspond to different exon regions of the target gene, activity in artificial diet bioassays: see Bolognesi et al. (2012) and 'spacer nucleotides which do not correspond to a target PLoS ONE 7(10): e47534. doi:10.1371/journal.pone. gene can optionally be used in between or adjacent to the 0047534. Thus, in one embodiment, a 21-base-pair dsRNA segments), or are from different target genes. In some silencing element corresponding to a target gene in Table 1 embodiments, the insecticidal double-stranded RNA mol and found to provide control of a Leptinotarsa infestation is ecule comprises multiple segments of 18 or more contiguous embedded in neutral sequence of an additional 39 base pairs, nucleotides that are essentially identical or essentially thus forming a polynucleotide of about 60 base pairs. In some complementary to a segment of equivalent length of a target embodiments, the dsRNA trigger includes neutral sequence gene or DNA (cDNA) having a sequence selected from The of between about 60 to about 500, or between 100 to about Target Gene Sequences Group, wherein the segments are 450 base-pairs, in which is embedded at least one segment of from different regions of the target gene and are arranged in 21 contiguous nucleotides with a sequence of 100% identity the insecticidal double-stranded RNA molecule in an order or 100% complementarity with a fragment of equivalent different from the order in which the segments naturally occur length of a target gene having a sequence selected from the in the target gene. In some embodiments, the insecticidal group consisting of SEQ ID NOS:1-725 and SEQ ID NOs: double-stranded RNA molecule comprises multiple seg 726-830 and SEQ ID NOs: 1087-1094. In another embodi ments each of 21 contiguous nucleotides with a sequence of ment, a single 21-base-pair silencing element with a sequence 100% identity or 100% complementary to a segment of of 100% identity or 100% complementarity with a fragment equivalent length of a target gene or DNA (cDNA) having a of equivalent length of a target gene is found to be efficacious sequence selected from The Target Gene Sequences Group, when embedded in larger sections of neutral sequence, e.g., wherein the segments are from different regions of the target where the total polynucleotide length is from about 60 to gene and are arranged in the insecticidal double-stranded about 300 base pairs. In another embodiment, at least one RNA molecule in an order different from the order in which segment of at least 21 contiguous nucleotides of a sequence the segments naturally occur in the target gene. In some selected from the group consisting of: SEQ ID NOs:831 embodiments, the insecticidal double-stranded RNA mol 1085, 1095-1104, and 1110-11 14, or the complement thereof, ecule comprises one Strand comprising a sequence selected is embedded in larger sections of neutral sequence to provide from the group consisting of: SEQID NOs:831-1085, 1095 an efficacious polynucleotide. In another embodiment, seg 1104, and 1110-11 14, or the complement thereof, or com ments from multiple sequences (or multiple copies of a seg prises an RNA hairpin encoded by a sequence selected from ment from one or more sequences) selected from the group the group consisting of SEQ ID NOs: 1105-1109. In some consisting of: SEQID NOS:831-1085, 1095-1104, and 1110 embodiments the insecticidal double-stranded RNA com 1114, or the complement thereof, are embedded in larger prises a dsRNA with a strand having a sequence selected from sections of neutral sequence to provide an efficacious poly the group consisting of the Trigger Sequences Group. The nucleotide. In embodiments where the polynucleotide insecticidal double-stranded RNA molecule can be topically includes regions of neutral sequence, the polynucleotide will applied to a plant, especially a Solanaceous plant such as have relatively low overall sequence identity in comparisonto tomato, eggplant, or potato, to control or prevent infestation the target gene; for example, a dsRNA with an overall length by a Leptinotarsa species. The insecticidal double-stranded of 210 base-pairs, containing a single 21-base-pair trigger (of RNA molecule can be provided in a form suitable for inges 100% identity or complementarity to a 21-nucleotide frag tion or direct contact by a Leptinotarsa species, e.g., in the ment of a target gene) embedded in an additional 189 base form of a spray or powder or bait. Other methods and suitable pairs of neutral sequence, will have an overall sequence iden compositions for providing the insecticidal double-stranded tity with the target gene of about 10%. RNA molecule are similar to those described in the preceding paragraphs for other aspects of this invention. Insecticidal Double-Stranded RNA Molecules 0185. Several embodiments relate to a tank mixture com 0184 Another aspect of this invention provides an insec prising one or more insecticidal polynucleotides and water or ticidal double-stranded RNA molecule that causes mortality other solvent, optionally including a cationic lipid oran orga or stunting of growth in a Leptinotarsa species when ingested nosilicone surfactant or both. Embodiments include tank or contacted by the Leptinotarsa species, wherein the insec mixture formulations of the polynucleotide and optionally at ticidal double-stranded RNA molecule comprises at least one least one pesticidal agent selected from the group consisting segment of 18 or more contiguous nucleotides that is essen of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus tially identical or essentially complementary to a segment of thuringiensis insecticidal protein, a Xenorhabdus insecticidal equivalent length of a target gene or DNA (cDNA) having a protein, a Photorhabdus insecticidal protein, a Bacillus lat sequence selected from The Target Gene Sequences Group. erosporous insecticidal protein, and a Bacillus sphaericus In some embodiments, the insecticidal double-stranded RNA insecticidal protein. Embodiments of Such compositions molecule is between about 50 to about 500 base-pairs in include those where one or more insecticidal polynucleotides length. In some embodiments, the insecticidal double are provided in a living or dead microorganism Such as a Stranded RNA molecule comprises at least one segment of at bacterium or fungal or yeast cell, or provided as a microbial least 30 contiguous nucleotides in length. In some embodi fermentation product, or provided in a living or dead plant ments, the insecticidal double-stranded RNA molecule com cell, or provided as a synthetic recombinant polynucleotide. prises multiple segments of 18 or more contiguous nucle In an embodiment the composition includes a non-pathogenic otides that are essentially identical or essentially strain of a microorganism that contains a polynucleotide as complementary to a segment of equivalent length of a target described herein; ingestion or intake of the microorganism gene or DNA (cDNA) having a sequence selected from The results in stunting or mortality of the Leptinotarsa species; Target Gene Sequences Group, wherein the segments are non-limiting examples of suitable microorganisms include E. from different regions of the target gene (e.g., the segments coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., US 2015/O 143580 A1 May 21, 2015 44

Xenorhabdus sp., Serratia entomophila and related Serratia limiting embodiments of effective amounts of a polynucle sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, otide include a range from about 10 nanograms per milliliter Clostridium bifermentans and other Clostridium species, or to about 100 micrograms per milliliter of a polynucleotide in other spore-forming gram-positive bacteria. In an embodi a liquid form sprayed on a plant, or from about 10 milligrams ment, the composition includes a plant virus vector compris per acre to about 100 grams per acre of polynucleotide ing a polynucleotide as described herein; feeding by a Lepti applied to a field of plants, or from about 0.001 to about 0.1 notarsa species on a plant treated with the plant virus vector microgram per milliliter of polynucleotide in an artificial diet results in stunting or mortality of the Leptinotarsa species. In for feeding the Leptinotarsa species. Where polynucleotides an embodiment, the composition includes abaculovirus vec as described herein are topically applied to a plant, the con tor including a polynucleotide as described herein; ingestion centrations can be adjusted in consideration of the Volume of or intake of the vector results in stunting or mortality of the spray or treatment applied to plant leaves or other plant part Leptinotarsa species. In an embodiment, a polynucleotide as Surfaces, such as flower petals, stems, tubers, fruit, anthers, described herein is encapsulated in a synthetic matrix Such as pollen, leaves, roots, or seeds. In one embodiment, a useful a polymer or attached to particulates and topically applied to treatment for herbaceous plants using 25-mer polynucle the Surface of a plant; feeding by a Leptinotarsa species on the otides as described herein is about 1 nanomole (nmol) of topically treated plant results in stunting or mortality of the polynucleotides per plant, for example, from about 0.05 to 1 Leptinotarsa species. In an embodiment, a polynucleotide as nmol polynucleotides per plant. Other embodiments for her described herein is provided in the form of a plant cell (e.g., a baceous plants include useful ranges of about 0.05 to about transgenic Solanaceous plant cell of this invention) express 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to ing the polynucleotide; ingestion of the plant cell or contents about 10 nmol of polynucleotides per plant. In certain of the plant cell by a Leptinotarsa species results in stunting embodiments, about 40 to about 50 nmol of a ssDNA poly or mortality of the Leptinotarsa species. nucleotide are applied. In certain embodiments, about 0.5 0186. In some embodiments, one or more polynucleotides nmol to about 2 nmol of a dsRNA is applied. In certain as described herein are provided with appropriate Stickers and embodiments, a composition containing about 0.5 to about wetters required for efficient foliar coverage as well as UV 2.0 milligrams per milliliter, or about 0.14 milligrams per protectants to protect polynucleotides such as dsRNAs from milliliter of a dsRNA or an ssDNA (21-mer) is applied. In UV damage. Such additives are commonly used in the bioin certain embodiments, a composition of about 0.5 to about 1.5 secticide industry and are known to those skilled in the art. milligrams per milliliter of a dsRNA polynucleotide of this Compositions for soil application can include granular for invention of about 50 to about 200 or more nucleotides is mulations that serve as bait for Leptinotarsa species larvae. In applied. In certain embodiments, about 1 nmol to about 5 Some embodiments, one or more polynucleotides as nmol of a dsRNA of this invention is applied to a plant. In described herein are further provided with a carrier agent, a certain embodiments, the polynucleotide composition as Surfactant, a cationic lipid (such as that disclosed in Example topically applied to the plant contains at least one polynucle 18 of U.S. patent application publication 2011/0296.556, otide of this invention at a concentration of about 0.01 to incorporated by reference herein), an organosilicone, an orga about 10 milligrams per milliliter, or about 0.05 to about 2 nosilicone Surfactant, a polynucleotide herbicidal molecule, a milligrams per milliliter, or about 0.1 to about 2 milligrams non-polynucleotide herbicidal molecule, a non-polynucle per milliliter. Very large plants, trees, or vines can require otide pesticide, a safener, and an insect growth regulator. In correspondingly larger amounts of polynucleotides. When Some embodiments, the composition further includes at least using long dsRNA molecules of this invention that can be one pesticidal agent selected from the group consisting of a processed into multiple oligonucleotides (e.g., multiple trig patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuring gers encoded by a single recombinant DNA molecule of this iensis insecticidal protein, a Xenorhabdus insecticidal pro invention), lower concentrations can be used. Non-limiting tein, a Photorhabdus insecticidal protein, a Bacillus lat examples of effective polynucleotide treatment regimes erosporous insecticidal protein, and a Bacillus sphaericus include a treatment of between about 0.1 to about 1 nmol of insecticidal protein. polynucleotide molecule per plant, or between about 1 nmol 0187 Such compositions are applied in any convenient to about 10 nmol of polynucleotide molecule per plant, or manner, e.g., by spraying or dusting the Leptinotarsa species between about 10 nmol to about 100 nmol of polynucleotide directly, or spraying or dusting a plant or environment molecule per plant. wherein prevention or control of infestation by that Leptino 0189 In some embodiments, one or more polynucleotides tarsa species is desired, or by applying a coating to a Surface is provided with a “transfer agent', which is an agent that of a plant, or by applying a coating to a seed (or seed potato) enables a topically applied polynucleotide to enter the cells of in preparation for the seeds planting, or by applying a soil an organism. Such transfer agents can be incorporated as part drench around roots of a plant for which prevention or control of a composition comprising a polynucleotide as described of infestation by that Leptinotarsa species is desired. herein, or can be applied prior to, contemporaneously with, or 0188 An effective amount of a polynucleotide as following application of the polynucleotide. In some embodi described herein is an amount sufficient to provide control of ments, a transfer agent is an agent that improves the uptake of the Leptinotarsa species, or to prevent infestation by the a polynucleotide of this invention by a Leptinotarsa species. Leptinotarsa species; determination of effective amounts of a In some embodiments, a transfer agent is an agent that con polynucleotide are made using routine assays such as those ditions the Surface of plant tissue, e.g., seeds, leaves, stems, described in Examples 5 and 6. While there is no upper limit roots, flowers, or fruits, to permeation by a polynucleotide on the concentrations and dosages of an insecticidal poly into plant cells. In some embodiments, the transfer agent nucleotide that can be useful in the methods and compositions enables a pathway for a polynucleotide through cuticle wax provided herein, lower effective concentrations and dosages barriers, stomata, and/or cell wall or membrane barriers into will generally be sought for efficiency and economy. Non plant cells. US 2015/O 143580 A1 May 21, 2015

0190. Suitable transfer agents include agents that increase to, polyethylene glycol or polypropylene glycol. Polyglycol permeability of the exterior of the organism or that increase chains can comprise a mixture that provides an average chain permeability of cells of the organism to polynucleotides. Suit length “n” of about "7.5". In certain embodiments, the aver able transfer agents include a chemical agent, or a physical age chain length “n” can vary from about 5 to about 14. agent, or combinations thereof. Chemical agents for condi Terminal groups can include, but are not limited to, alkyl tioning or transfer include (a) Surfactants, (b) an organic groups such as a methyl group. Organosilicone compounds Solvent oran aqueous Solution oraqueous mixtures of organic useful as transfer agents include, but are not limited to, trisi Solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) loxane ethoxylate surfactants or polyalkylene oxide modified enzymes, or any combination thereof. In some embodiments, heptamethyl trisiloxane. An example of a transfer agent for application of a polynucleotide and a transfer agent option use in this invention is Compound I: ally includes an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Suitable transfer agents can be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or can cause the polynucleotide to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like com position. Embodiments of transfer agents include counter ions or other molecules that are known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines Such as spermine, spermidine, or putrescine, and other cations. Embodiments of transfer agents include organic solvents such as DMSO, 0193 (Compound I: polyalkyleneoxide heptamethyltrisi DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, loxane, average n=7.5). acetonitrile, dioxane, polypropylene glycol, or other solvents 0194 Organosilicone compounds useful as transfer agents miscible with water or that dissolve phosphonucleotides in are used, e.g., as freshly made concentrations in the range of non-aqueous systems (such as is used in synthetic reactions). about 0.015 to about 2 percent by weight (wt percent) (e.g., Embodiments of transfer agents include naturally derived or about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, synthetic oils with or without Surfactants or emulsifiers, e.g., 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, plant-sourced oils, crop oils (such as those listed in the 9" 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, Compendium of Herbicide Adjuvants, publicly available on 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent). line at herbicide.adjuvants.com), paraffinic oils, polyol fatty 0.195 Embodiments of transfer agents include one or more acid esters, or oils with short-chain molecules modified with salts such as ammonium chloride, tetrabutylphosphonium amides or polyamines such as polyethyleneimine or N-pyr bromide, and ammonium sulfate, provided in or used with a rolidine. composition including a polynucleotide. In some embodi 0191 Embodiments of transfer agents include organosili ments, ammonium chloride, tetrabutylphosphonium bro cone preparations. For example, a Suitable transfer agent is an mide, and/or ammonium Sulfate are used at a concentration of organosilicone preparation that is commercially available as about 0.5% to about 5% (w/v), or about 1% to about 3% (w/v), SILWET L-77(R) brand surfactant having CAS Number or about 2% (w/v). In certain embodiments, the composition 27306-78-1 and EPA Number: CALREGNO. 5905-50.073 including a polynucleotide includes an ammonium salt at a AA, and currently available from Momentive Performance concentration greater or equal to 300 millimolar. In certain Materials, Albany, N.Y. One embodiment includes a compo embodiments, the composition including a polynucleotide sition that comprises a polynucleotide and a transfer agent includes an organosilicone transfer agent in a concentration including an organosilicone preparation such as Silwet L-77 of about 0.015 to about 2 percent by weight (wt percent) as in the range of about 0.015 to about 2 percent by weight (wt well as ammonium sulfate at concentrations from about 80 to percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, about 1200 mM or about 150 mM to about 600 mM. 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0196. Embodiments of transfer agents include a phos 0.09, 0.095, 0.1, 0.2,0.3, 0.4,0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, phate salt. Phosphate salts useful in a composition including 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt a polynucleotide include, but are not limited to, calcium, percent). One embodiment includes a composition that com magnesium, potassium, or Sodium phosphate salts. In certain prises a polynucleotide of this invention and a transfer agent embodiments, a composition including a polynucleotide including SILWET L-77(R) brand surfactant in the range of includes a phosphate salt at a concentration of at least about 5 about 0.3 to about 1 percent by weight (wt percent) or about millimolar, at least about 10 millimolar, or at least about 20 0.5 to about 1%, by weight (wt percent). millimolar. In certain embodiments, a composition including 0192 Organosilicone compounds useful as transfer agents a polynucleotide a phosphate salt in a range of about 1 mM to for use in this invention include, but are not limited to, com about 25 mM or in a range of about 5 mM to about 25 mM. In pounds that include: (a) a trisiloxane head group that is certain embodiments, the composition including a polynucle covalently linked to, (b) an alkyl linker including, but not otide Sodium phosphate at a concentration of at least about 5 limited to, an n-propyl linker, that is covalently linked to, (c) millimolar, at least about 10 millimolar, or at least about 20 a polyglycol chain, that is covalently linked to, (d) a terminal millimolar. In certain embodiments, a composition including group. Trisiloxane head groups of Such organosilicone com a polynucleotide includes sodium phosphate at a concentra pounds include, but are not limited to, heptamethyltrisilox tion of about 5 millimolar, about 10 millimolar, or about 20 ane. Alkyl linkers can include, but are not limited to, an millimolar. In certain embodiments, a composition including n-propyl linker. Polyglycol chains include, but are not limited a polynucleotide includes a Sodium phosphate salt in a range US 2015/O 143580 A1 May 21, 2015 46 of about 1 mM to about 25 mM or in a range of about 5 mM gous promoter operably linked to DNA comprising at least to about 25 mM. In certain embodiments, a composition one segment of 18 or more contiguous nucleotides with a including a polynucleotide includes a sodium phosphate salt sequence of about 95% to about 100% identity with a frag in a range of about 10 mM to about 160 mM or in a range of ment of equivalent length of a DNA having a sequence about 20 mM to about 40 mM. In certain embodiments, a selected from the Target Gene Sequences Group or the DNA composition including a polynucleotide includes a sodium complement thereof. Another aspect of the invention provides phosphate buffer at a pH of about 6.8. a recombinant DNA construct comprising a heterologous 0.197 Embodiments of transfer agents include surfactants promoter operably linked to DNA encoding an RNA hairpin and/or effective molecules contained therein. Surfactants having an anti-sense region having a sequence, or a fragment and/or effective molecules contained therein include, but are of a sequence, selected from the group selected from the not limited to, sodium or lithium salts of fatty acids (such as Trigger Sequences Group. In another embodiment, a recom tallow or tallowamines or phospholipids) and organosilicone binant DNA construct comprising a promoteroperably linked Surfactants. In certain embodiments, a composition including to DNA encoding: (a) an RNA silencing element for Sup a polynucleotide is formulated with counter-ions or other pressing a target gene selected from the group consisting of molecules that are known to associate with nucleic acid mol the genes identified in Table 1), and (b) anaptamer, is stably ecules. Non-limiting examples include, tetraalkyl ammonium integrated into the plant's genome from where RNA tran ions, trialkyl ammonium ions, Sulfonium ions, lithium ions, scripts including the RNA aptamer and the RNA silencing and polyamines such as spermine, spermidine, or putrescine. element are expressed in cells of the plant; the aptamer serves In certain embodiments, a composition including a poly to guide the RNA silencing element to a desired location in nucleotide is formulated with a non-polynucleotide herbicide the cell. In another embodiment, inclusion of one or more e.g., glyphosate, auxin-like benzoic acid herbicides including recognition sites for binding and cleavage by a small RNA dicamba, chloramben, and TBA, glufosinate, auxin-like her (e.g., by a miRNA or an siRNA that is expressed only in a bicides including phenoxy carboxylic acid herbicide, pyri particular cell or tissue) allows for more precise expression dine carboxylic acid herbicide, quinoline carboxylic acid her patterns in a plant, wherein the expression of the recombinant bicide, pyrimidine carboxylic acid herbicide, and benazolin DNA construct is suppressed where the small RNA is ethyl herbicide, Sulfonylureas, imidazolinones, bromoxynil. expressed. Such additional elements are described below. delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase Promoters inhibiting herbicides. In certain embodiments, a composition (0199 Promoters of use in the invention are functional in including a polynucleotide is formulated with a non-poly the cell in which the construct is intended to be transcribed. nucleotide pesticide, e.g., a patatin, a plant lectin, a phyto Generally these promoters are heterologous promoters, as ecdysteroid, a Bacillus thuringiensis insecticidal protein, a used in recombinant constructs, i.e., they are not in nature Xenorhabdus insecticidal protein, a Photorhabdus insecti found to be operably linked to the other nucleic elements used cidal protein, a Bacillus laterosporous insecticidal protein, in the constructs described herein. In various embodiments, and a Bacillus sphaericus insecticidal protein. In some the promoter is selected from the group consisting of a con embodiments, a composition including a polynucleotide and stitutive promoter, a spatially specific promoter, a temporally a non-polynucleotide pesticide provides Synergetic improve specific promoter, a developmentally specific promoter, and ment in prevention or control of Leptinotarsa species infes an inducible promoter. In many embodiments the promoter is tations, when compared to the effect obtained with the poly a promoter functional in a plant, for example, a pol II pro nucleotide alone or the non-polynucleotide pesticide alone. moter, a pol III promoter, a pol IV promoter, or a pol V In some embodiments, a composition comprising a double promoter. Stranded RNA with a strand having a sequence selected from the group consisting of the Trigger Sequences Group is com 0200. Non-constitutive promoters suitable for use with the recombinant DNA constructs of this invention include spa bined with a non-polynucleotide pesticide (e.g., a patatin, a tially specific promoters, temporally specific promoters, and plant lectin, a phytoecdysteroid, a Bacillus thuringiensis inducible promoters. Spatially specific promoters can include insecticidal protein, a Xenorhabdus insecticidal protein, a organelle-, cell-, tissue-, or organ-specific promoters (e.g., a Photorhabdus insecticidal protein, a Bacillus laterosporous plastid-specific, a root-specific, a pollen-specific, or a seed insecticidal protein, and a Bacillus sphaericus insecticidal specific promoter for expression in plastids, roots, pollen, or protein), wherein the combination is found to effect synergis seeds, respectively). In many cases a seed-specific, embryo tically improved prevention or control of Leptinotarsa spe specific, aleurone-specific, or endosperm-specific promoter cies infestations, when compared to the effect obtained with is especially useful. Temporally specific promoters can the double-stranded RNA alone or the non-polynucleotide include promoters that tend to promote expression during pesticide alone. certain developmental stages in a plant's growth cycle, or Related Techniques during different times of day or night, or at different seasons in a year. Inducible promoters include promoters induced by 0198 Embodiments of the polynucleotides and nucleic chemicals or by environmental conditions such as, but not acid molecules as described herein can include additional limited to, biotic or abiotic stress (e.g., water deficit or elements, such as promoters, Small RNA recognition sites, drought, heat, cold, high or low nutrient or salt levels, high or aptamers or ribozymes, additional and additional expression low light levels, or pest or pathogen infection). MicroRNA cassettes for expressing coding sequences (e.g., to express a promoters are useful, especially those having a temporally transgene Such as an insecticidal protein or selectable marker) specific, spatially specific, or inducible expression pattern; or non-coding sequences (e.g., to express additional Suppres examples of miRNA promoters, as well as methods for iden sion elements). For example, an aspect of this invention pro tifying miRNA promoters having specific expression pat vides a recombinant DNA construct comprising a heterolo terns, are provided in U.S. Patent Application Publications US 2015/O 143580 A1 May 21, 2015 47

2006/0200878, 2007/0199095, and 2007/0300329, which are some embodiments, Pol III promoters (e.g., U6 or H1 pro specifically incorporated herein by reference. An expression moters) are for adding a short AT-rich transcription termina specific promoter can also include promoters that are gener tion site that results in 2 base-pair overhangs (UU) in the ally constitutively expressed but at differing degrees or transcribed RNA; this is useful, e.g., for expression of siRNA 'strengths of expression, including promoters commonly type constructs. Use of pol III promoters for driving expres regarded as "strong promoters' or as “weak promoters'. sion of siRNA constructs has been reported; see Van de Weter 0201 Promoters of particular interest include the follow ing et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) ing examples: an opaline synthase promoter isolated from Nature Biotechnol., 20: 446-448. Baculovirus promoters T-DNA of Agrobacterium; a cauliflower mosaic virus 35S Such as baculovirus polyhedrin and p10 promoters are known promoter, enhanced promoter elements or chimeric promoter in the art and commercially available; see, e.g., Invitrogen's elements such as an enhanced cauliflower mosaic virus “Guide to Baculovirus Expression Vector Systems (BEVS) (CaMV) 35S promoter linked to an enhancer element (an and Insect Cell Culture Techniques”, 2002 (Life Technolo intron from heat shock protein 70 of Zea mays); root specific gies, Carlsbad, Calif.) and F. J. Haines et al. “Baculovirus promoters such as those disclosed in U.S. Pat. Nos. 5,837. Expression Vectors’, undated (Oxford Expression Technolo 848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter gies, Oxford, UK). disclosed in U.S. Pat. No. 6,433,252; a promoter for a plant 0204 The promoter element can include nucleic acid nuclear gene encoding a plastid-localized aldolase disclosed sequences that are not naturally occurring promoters or pro in U.S. Patent Application Publication 2004/0216189; cold moter elements or homologues thereof but that can regulate inducible promoters disclosed in U.S. Pat. No. 6,084,089: expression of a gene. Examples of Such 'gene independent' salt-inducible promoters disclosed in U.S. Pat. No. 6,140. regulatory sequences include naturally occurring or artifi 078; light-inducible promoters disclosed in U.S. Pat. No. cially designed RNA sequences that include a ligand-binding 6.294,714; pathogen-inducible promoters disclosed in U.S. region or aptamer (see "Aptamers', below) and a regulatory Pat. No. 6,252,138; and water deficit-inducible promoters region (which can be cis-acting). See, for example, Isaacs et disclosed in U.S. Patent Application Publication 2004/ al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke 0.123347 A1. All of the above-described patents and patent (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker publications disclosing promoters and their use, especially in (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and recombinant DNA constructs functional in plants are incor Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et porated herein by reference. al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 0202 Plant vascular- or phloem-specific promoters of 9:644-647, and Mandal and Breaker (2004) Nature Struct. interest include a rolC or rolA promoter of Agrobacterium Mol. Biol., 11:29-35. Such “riboregulators' could be selected rhizogenes, a promoter of a Agrobacterium tumefaciens or designed for specific spatial or temporal specificity, for T-DNA gene 5, the rice sucrose synthase RSS1 gene pro example, to regulate translation of DNA that encodes a silenc moter, a Commelina yellow mottle badnavirus promoter, a ing element for Suppressing a Leptinotarsa target gene only in coconut foliar decay virus promoter, a rice tungro bacilliform the presence (or absence) of a given concentration of the virus promoter, the promoter of a pea glutamine synthase appropriate ligand. One example is a riboregulator that is GS3A gene, a invoD111 and invoD141 promoters of a potato responsive to an endogenous ligand (e.g., jasmonic acid or invertase genes, a promoter isolated from Arabidopsis shown salicylic acid) produced by the plant when under stress (e.g., to have phloem-specific expression in tobacco by Kertbundit abiotic stress such as water, temperature, or nutrient stress, or et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, a biotic stress Such as attach by pests or pathogens); under VAHOX1 promoter region, a pea cell wall invertase gene stress, the level of endogenous ligand increases to a level promoter, an acid invertase gene promoter from carrot, a sufficient for the riboregulator to begin transcription of the promoter of a sulfate transporter gene Sultr1;3, a promoter of DNA that encodes a silencing element for Suppressing a Lep a plant Sucrose synthase gene, and a promoter of a plant tinotarsa target gene. Sucrose transporter gene. 0203 Promoters suitable for use with a recombinant DNA Recombinase Sites construct or polynucleotide of this invention include poly 0205. In some embodiments, the recombinant DNA con merase II (“pol II) promoters and polymerase III (“pol III') struct or polynucleotide of this invention comprises DNA promoters. RNA polymerase II transcribes structural or cata encoding one or more site-specific recombinase recognition lytic RNAs that are usually shorter than 400 nucleotides in sites. In one embodiment, the recombinant DNA construct length, and recognizes a simple run of T residues as a termi comprises at least a pair of loXP sites, wherein site-specific nation signal; it has been used to transcribe siRNA duplexes recombination of DNA between the loxP sites is mediated by (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e 171). Pol II a Cre recombinase. The position and relative orientation of promoters are therefore in certain embodiments where a short the loXP sites is selected to achieve the desired recombina RNA transcript is to be produced from a recombinant DNA tion; for example, when the loxP sites are in the same orien construct of this invention. In one embodiment, the recombi tation, the DNA between the loXP sites is excised in circular nant DNA construct comprises apol II promoter to express an form. In another embodiment, the recombinant DNA con RNA transcript flanked by self-cleaving ribozyme sequences struct comprises DNA encoding one loxP site; in the presence (e.g., self-cleaving hammerhead ribozymes), resulting in a of Cre recombinase and another DNA with aloxP site, the two processed RNA, such as a single-stranded RNA that binds to DNAs are recombined. the transcript of the Leptinotarsa target gene, with defined 5' and 3' ends, free of potentially interfering flanking sequences. Aptamers An alternative approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, i.e., to tran 0206. In some embodiments, the recombinant DNA con scribe an RNA with minimal 5' and 3' flanking sequences. In structor polynucleotide of this invention comprises DNA that US 2015/O 143580 A1 May 21, 2015 48 is processed to an RNA aptamer, that is, an RNA that binds to encoding a spliceable intron. By “intron’ is generally meant a ligand through binding mechanism that is not primarily a segment of DNA (or the RNA transcribed from such a based on Watson-Crick base-pairing (in contrast, for segment) that is located between exons (protein-encoding example, to the base-pairing that occurs between comple segments of the DNA or corresponding transcribed RNA), mentary, anti-parallel nucleic acid strands to form a double wherein, during maturation of the messenger RNA, the intron Stranded nucleic acid structure). See, for example, Ellington present is enzymatically “spliced out” or removed from the and Szostak (1990) Nature, 346:818-822. Examples of RNA strand by a cleavage/ligation process that occurs in the aptamers can be found, for example, in the public Aptamer nucleus in eukaryotes. The term “intron’ is also applied to Database, available on line at aptamericmb.utexas.edu (Lee non-coding DNA sequences that are transcribed to RNA seg et al. (2004) Nucleic Acids Res., 32(1):D95-100). Aptamers ments that can be spliced out of a maturing RNA transcript, useful in the invention can, however, be monovalent (binding but are not introns found between protein-coding exons. One a single ligand) or multivalent (binding more than one indi example of these are spliceable sequences that that have the vidual ligand, e.g., binding one unit of two or more different ability to enhance expression in plants (in some cases, espe ligands). cially in monocots) of a downstream coding sequence; these 0207 Ligands useful in the invention include any mol spliceable sequences are naturally located in the 5' untrans ecule (or part of a molecule) that can be recognized and be lated region of some plant genes, as well as in some viral bound by a nucleic acid secondary structure by a mechanism genes (e.g., the tobacco mosaic virus 5' leader sequence or not primarily based on Watson-Crick base pairing. In this “omega’ leader described as enhancing expression in plant way, the recognition and binding of ligand and aptamer is genes by Gallie and Walbot (1992) Nucleic Acids Res., analogous to that of antigen and antibody, or of biological 20:4631-4638). These spliceable sequences or “expression effector and receptor. Ligands can include single molecules enhancing introns' can be artificially inserted in the 5' (or part of a molecule), or a combination of two or more untranslated region of a plant gene between the promoter but molecules (or parts of a molecule), and can include one or before any protein-coding exons. Examples of Such expres more macromolecular complexes (e.g., polymers, lipid bilay Sion-enhancing introns include, but are not limited to, a maize ers, liposomes, cellular membranes or other cellular struc alcohol dehydrogenase (Zm-Adh1), a maize Bronze-1 tures, or cell Surfaces). Examples of specific ligands include expression-enhancing intron, a rice actin 1 (OS-Act 1) intron, Vitamins such as coenzyme B and thiamine pyrophosphate, a Shrunken-1 (Sh-1) intron, a maize Sucrose synthase intron, flavin mononucleotide, guanine, adenosine, S-adenosylme a heat shock protein 18 (hsp18) intron, and an 82 kilodalton thionine, S-adenosylhomocysteine, coenzyme A, lysine, heat shock protein (hsp82) intron. U.S. Pat. Nos. 5,593.874 tyrosine, dopamine, glucosamine-6-phosphate, caffeine, and 5,859,347, specifically incorporated by reference herein, theophylline, antibiotics such as chloramphenicol and neo describe methods of improving recombinant DNA constructs mycin, herbicides such as glyphosate and dicamba, proteins for use in plants by inclusion of an expression-enhancing including viral or phage coat proteins and invertebrate epi intron derived from the 70 kilodalton maize heat shock pro dermal or digestive tract surface proteins, and RNAs includ tein (hsp70) in the non-translated leader positioned 3' from ing viral RNA, transfer-RNAs (t-RNAs), ribosomal RNA the gene promoter and 5' from the first protein-coding exon. (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNAaptamers useful Ribozymes in the invention are “thermoswitches' that do not bind a ligand but are thermally responsive, that is to say, the aptam 0210. In some embodiments, the recombinant DNA con er's conformation is determined by temperature; see, for struct or polynucleotide of this invention comprises DNA example, Box 3 in Mandal and Breaker (2004) Nature Rev. encoding one or more ribozymes. Ribozymes of particular Mol. Cell Biol. 5:451-463. interest include a self-cleaving ribozyme, a hammerhead Transgene Transcription Units ribozyme, or a hairpin ribozyme. In one embodiment, the 0208. In some embodiments, the recombinant DNA con recombinant DNA construct comprises DNA encoding one or struct or polynucleotide of this invention comprises a trans more ribozymes that serve to cleave the transcribed RNA to gene transcription unit. A transgene transcription unit com provide defined segments of RNA. Such as silencing elements prises DNA sequence encoding a gene of interest, e.g., a for Suppressing a Leptinotarsa target gene. natural protein or a heterologous protein. A gene of interest can be any coding or non-coding sequence from any species Gene Suppression Elements (including, but not limited to, non-eukaryotes Such as bacte ria, and viruses; fungi, protists, plants, invertebrates, and 0211. In some embodiments, the recombinant DNA con Vertebrates. Particular genes of interest are genes encoding at struct or polynucleotide of this invention comprises DNA least one pesticidal agent selected from the group consisting encoding additional gene Suppression element for Suppress of a patatin, a plant lectin, a phytoecdysteroid, a phytoecdys ing a target gene other than a Leptinotarsa target gene. The teroid, a Bacillus thuringiensis insecticidal protein, a target gene to be suppressed can include coding or non Xenorhabdus insecticidal protein, a Photorhabdus insecti coding sequence or both. cidal protein, a Bacillus laterosporous insecticidal protein, 0212 Suitable gene suppression elements are described in and a Bacillus sphaericus insecticidal protein. The transgene detail in U.S. Patent Application Publication 2006/0200878, transcription unit can further include 5' or 3' sequence or both which disclosure is specifically incorporated herein by refer as required for transcription of the transgene. ence, and include one or more of: Introns 0213 (a) DNA that comprises at least one anti-sense 0209. In some embodiments, the recombinant DNA con DNA segment that is anti-sense to at least one segment struct or polynucleotide of this invention comprises DNA of the gene to be Suppressed; US 2015/O 143580 A1 May 21, 2015 49

0214 (b) DNA that comprises multiple copies of at least tion sites, as described in detail in U.S. Patent Application one anti-sense DNA segment that is anti-sense to at least Publication 2006/0200878, specifically incorporated herein one segment of the gene to be suppressed; by reference. 0215 (c) DNA that comprises at least one sense DNA segment that is at least one segment of the gene to be Making and Using Transgenic Plant Cells and Transgenic Suppressed; Plants 0216 (d) DNA that comprises multiple copies of at least 0226 Transformation of a plant can include any of several one sense DNA segment that is at least one segment of well-known methods and compositions. Suitable methods for the gene to be suppressed; plant transformation include virtually any method by which 0217 (e) DNA that transcribes to RNA for suppressing DNA can be introduced into a cell. One method of plant the gene to be suppressed by forming double-stranded transformation is microprojectile bombardment, for RNA and comprises at least one anti-sense DNA seg example, as illustrated in U.S. Pat. No. 5,015,580 (soybean), ment that is anti-sense to at least one segment of the gene U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 5,550,318 to be Suppressed and at least one sense DNA segment (maize), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. that is at least one segment of the gene to be Suppressed; 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. 0218 (f) DNA that transcribes to RNA for suppressing No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. the gene to be suppressed by forming a single double Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 stranded RNA and comprises multiple serial anti-sense (maize), all of which are incorporated by reference for DNA segments that are anti-sense to at least one segment enabling the production of transgenic plants. of the gene to be suppressed and multiple serial sense 0227. Another useful method of plant transformation is DNA segments that are at least one segment of the gene Agrobacterium-mediated transformation by means of Agro to be suppressed; bacterium containing a binary Tiplasmid system, wherein the 0219 (g) DNA that transcribes to RNA for suppressing Agrobacterium carries a first Tiplasmid and a second, chi the gene to be suppressed by forming multiple double meric plasmid containing at least one T-DNA border of a strands of RNA and comprises multiple anti-sense DNA wild-type Ti plasmid, a promoter functional in the trans segments that are anti-sense to at least one segment of formed plant cell and operably linked to a polynucleotide or the gene to be suppressed and multiple sense DNA seg recombinant DNA construct of this invention. See, for ments that are at least one segment of the gene to be example, the binary system described in U.S. Pat. No. 5,159, Suppressed, and wherein the multiple anti-sense DNA 135, incorporated by reference. Also see DeFramond (1983) segments and the multiple sense DNA segments are Biotechnology, 1:262-269; and Hoekema et al., (1983) arranged in a series of inverted repeats; Nature, 303:179. In such a binary system, the smaller plas 0220 (h) DNA that comprises nucleotides derived from mid, containing the T-DNA border or borders, can be conve a plant miRNA; niently constructed and manipulated in a suitable alternative 0221 (i) DNA that comprises nucleotides of a siRNA; host, Such as E. coli, and then transferred into Agrobacterium. 0222 () DNA that transcribes to an RNA aptamer 0228 Detailed procedures for Agrobacterium-mediated capable of binding to a ligand; and transformation of plants, especially crop plants, include pro 0223 (k) DNA that transcribes to an RNA aptamer cedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, and capable of binding to a ligand, and DNA that transcribes 5,518.908 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, to regulatory RNA capable of regulating expression of 5,824,877 and 6,384.301 (soybean): U.S. Pat. Nos. 5,591,616 the gene to be suppressed, wherein the regulation is and 5,981,840 (maize): U.S. Pat. No. 5,463,174 (brassicas dependent on the conformation of the regulatory RNA, including canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. and the conformation of the regulatory RNA is allosteri Pat. No. 6,329,571 (rice), and in U.S. Patent Application cally affected by the binding state of the RNA aptamer. Publications 2004/0244075 (maize) and 2001/0042257 A1 0224. In some embodiments, an intron is used to deliver a (Sugar beet), all of which are specifically incorporated by gene Suppression element in the absence of any protein-cod reference for enabling the production of transgenic plants. ing exons (coding sequence). In one example, an intron, Such U.S. Patent Application Publication 2011/0296555 discloses in Example 5 the transformation vectors (including the vector as an expression-enhancing intron, is interrupted by embed sequences) and detailed protocols for transforming maize, ding within the intron a gene Suppression element, wherein, Soybean, canola, cotton, and Sugarcane) and is specifically upon transcription, the gene Suppression element is excised incorporated by reference for enabling the production of from the intron. Thus, protein-coding exons are not required transgenic plants. Similar methods have been reported for to provide the gene Suppressing function of the recombinant many plant species, both dicots and monocots, including, DNA constructs disclosed herein. among others, peanut (Cheng et al. (1996) Plant Cell Rep., Transcription Regulatory Elements 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant 0225. In some embodiments, the recombinant DNA con Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technol struct or polynucleotide of this invention comprises DNA ogy, 6:10;Zhanget al. (1988) Plant Cell Rep., 7:379; wheat encoding a transcription regulatory element. Transcription (Vasil et al. (1992) Bio/Technology, 10:667: Becker et al. regulatory elements include elements that regulate the (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996) Trans expression level of the recombinant DNA construct of this gen. Res., 5:313); and tomato (Sun et al. (2006) Plant Cell invention (relative to its expression in the absence of Such Physiol., 47:426-431). See also a description of vectors, regulatory elements). Examples of Suitable transcription transformation methods, and production of transformed Ara regulatory elements include riboswitches (cis- or trans-act bidopsis thaliana plants where transcription factors are con ing), transcript stabilizing sequences, and miRNA recogni stitutively expressed by a CaMV35S promoter, in U.S. Patent US 2015/O 143580 A1 May 21, 2015 50

Application Publication 2003/0167537 A1, incorporated by mation methods and materials for making transgenic plants of reference. Transformation methods specifically useful for this invention (e.g., various media and recipient target cells, Solanaceous plants are well known in the art. See, for transformation of immature embryos, and Subsequent regen example, publicly described transformation methods for eration of fertile transgenic plants) are disclosed, for tomato (Sharma et al. (2009), J. Biosci., 34:423-433), egg example, in U.S. Pat. Nos. 6,194,636 and 6.232,526 and U.S. plant (Arpaia et al. (1997) Theor: Appl. Genet., 95:329-334), Patent Application Publication 2004/0216189, which are spe potato (Bannerjee et al. (2006) Plant Sci., 170:732-738; cifically incorporated by reference. Chakravarty et al. (2007) Amer: J. Potato Res., 84:301-311; S. 0230. In general transformation practice, DNA is intro Millam Agrobacterium-mediated transformation of potato.” duced into only a small percentage of target cells in any one Chapter 19 (pp. 257-270), “Transgenic Crops of the World: transformation experiment. Marker genes are generally used Essential Protocols, Ian S. Curtis (editor), Springer, 2004), to provide an efficient system for identification of those cells and peppers (Li et al. (2003) Plant Cell Reports, 21: 785 that are stably transformed by receiving and integrating a 788). Stably transgenic potato, tomato, and eggplant have transgenic DNA construct into their genomes. Preferred been commercially introduced in various regions; see, e.g., K. marker genes provide selective markers which confer resis Redenbaugh et al. “Safety Assessment of Genetically Engi tance to a selective agent, such as an antibiotic or herbicide. neered Fruits and Vegetables: A Case Study of the FLAVR Any of the antibiotics or herbicides to which a plant cell is SAVR Tomato', CRC Press, Boca Raton, 1992, and the resistant can be a useful agent for selection. Potentially trans extensive publicly available documentation of commercial formed cells are exposed to the selective agent. In the popu genetically modified crops in the GM Crop Database; see: lation of surviving cells will be those cells where, generally, CERA. (2012). GM Crop Database. Center for Environmen the resistance-conferring gene is integrated and expressed at tal Risk Assessment (CERA), ILSI Research Foundation, sufficient levels to permit cell survival. Cells can be tested Washington D.C., available electronically at www.cera-gmc. further to confirm stable integration of the recombinant DNA. org/?action gm crop database. Various methods of trans Commonly used selective marker genes include those con formation of other plant species are well known in the art, see, ferring resistance to antibiotics such as kanamycin or paro for example, the encyclopedic reference, “Compendium of momycin (nptll), hygromycin B (aph IV) and gentamycin Transgenic Crop Plants', edited by Chittaranjan Kole and (aac3 and aacC4) or resistance to herbicides such as glufosi Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN nate (bar or pat) and glyphosate (EPSPS). Examples of useful 978-1-405-16924-0 (available electronically at mrw.inter selective marker genes and selection agents are illustrated in science.wiley.com/emrw/9781405181099/hpt/toc), which U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118, describes transformation procedures for cereals and forage 047, all of which are specifically incorporated by reference. grasses (rice, maize, wheat, barley, oat, Sorghum, pearl millet, Screenable markers or reporters, such as markers that provide finger millet, cool-season forage grasses, and bahiagrass), an ability to visually identify transformants can also be oilseed crops (soybean, oilseed brassicas, Sunflower, peanut, employed. Examples of useful screenable markers include, flax, sesame, and safflower), legume grains and forages (com for example, a gene expressing a protein that produces a mon bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic detectable color by acting on a chromogenic Substrate (e.g., beans, pigeonpea, Vetch, chickpea, lupin, alfalfa, and clo beta glucuronidase (GUS) (uidA) or luciferase (luc)) or that Vers), temperate fruits and nuts (apple, pear, peach, plums, itself is detectable, such as green fluorescent protein (GFP) berry crops, cherries, grapes, olive, almond, and Persian wal (gfp) or an immunogenic molecule. Those of skill in the art nut), tropical and Subtropical fruits and nuts (citrus, grape will recognize that many other useful markers or reporters are fruit, banana and plantain, pineapple, papaya, mango, avo available for use. cado, kiwifruit, passionfruit, and persimmon), vegetable 0231. Detecting or measuring transcription of a recombi crops (tomato, eggplant, peppers, vegetable brassicas, radish, nant DNA construct in a transgenic plant cell can be achieved carrot, cucurbits, alliums, asparagus, and leafy vegetables), by any suitable method, including protein detection methods Sugar, tuber, and fiber crops (Sugarcane, Sugar beet, Stevia, (e.g., western blots, ELISAS, and other immunochemical potato, Sweet potato, cassava, and cotton), plantation crops, methods), measurements of enzymatic activity, or nucleic ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, acid detection methods (e.g., Southern blots, northern blots, rubber tree, medicinal plants, ornamentals, and turfgrasses), PCR, RT-PCR, fluorescent in situ hybridization). and forest tree species. 0232 Other suitable methods for detecting or measuring 0229 Transformation methods to provide transgenic plant transcription in a plant cell of a recombinant polynucleotide cells and transgenic plants containing stably integrated of this invention targetting a Leptinotarsa species target gene recombinant DNA are preferably practiced in tissue culture include measurement of any other trait that is a director proxy on media and in a controlled environment. “Media' refers to indication of the level of expression of the target gene in the the numerous nutrient mixtures that are used to grow cells in Leptinotarsa species, relative to the level of expression vitro, that is, outside of the intact living organism. Recipient observed in the absence of the recombinant polynucleotide, cell targets include, but are not limited to, meristem cells, e.g., growth rates, mortality rates, or reproductive or recruit callus, immature embryos or parts of embryos, and gametic ment rates of the Leptinotarsa species, or measurements of cells such as microspores, pollen, sperm, and egg cells. Any injury (e.g., root injury) or yield loss in a plant or field of cell from which a fertile plant can be regenerated is contem plants infested by the Leptinotarsa species. In general, Suit plated as a useful recipient cell for practice of this invention. able methods for detecting or measuring transcription in a Callus can be initiated from various tissue sources, including, plant cell of a recombinant polynucleotide of interest include, but not limited to, immature embryos or parts of embryos, e.g., gross or microscopic morphological traits, growth rates, seedling apical meristems, microspores, and the like. Those yield, reproductive or recruitment rates, resistance to pests or cells which are capable of proliferating as callus can serve as pathogens, or resistance to biotic or abiotic stress (e.g., water recipient cells for genetic transformation. Practical transfor deficit stress, salt stress, nutrient stress, heat or cold stress). US 2015/O 143580 A1 May 21, 2015

Such methods can use direct measurements of a phenotypic progeny generations of plants, including inbred or hybrid trait or proxy assays (e.g., in plants, these assays include plant plant lines, made by crossing a transgenic plant grown part assays such as leaf or root assays to determine tolerance directly from transgenic seed to a second plant not grown ofabiotic stress). Such methods include direct measurements from the same transgenic Seed. Crossing can include, for of resistance to an invertebrate pest or pathogen (e.g., damage example, the following steps: to plant tissues) or proxy assays (e.g., plant yield assays, or 0237 (a) plant seeds of the first parent plant (e.g., non bioassays such as the Western corn rootworm (Diabrotica transgenic or a transgenic) and a second parent plant that virgifera virgifera LeConte) larval bioassay described in is transgenic according to the invention; International Patent Application Publication WO2005/ 0238 (b) grow the seeds of the first and second parent 110068A2 and U.S. Patent Application Publication US 2006/ plants into plants that bear flowers; 0021087 A1, specifically incorporated by reference, or the 0239 (c) pollinate a flower from the first parent with soybean cyst nematode bioassay described by Steeves et al. pollen from the second parent; and (2006) Funct. Plant Biol., 33:991-999, wherein cysts per 0240 (d) harvest seeds produced on the parent plant plant, cysts per gram root, eggs perplant, eggs per gram root, bearing the fertilized flower. and eggs per cyst are measured, or the Colorado potato beetle 0241. It is often desirable to introgress recombinant DNA (Leptinotarsa decemlineata) bioassay described herein in the into elite varieties, e.g., by backcrossing, to transfer a specific working Examples. desirable trait from one source to an inbred or other plant that 0233. The recombinant DNA constructs of this invention lacks that trait. This can be accomplished, for example, by can be stacked with other recombinant DNA for imparting first crossing a superior inbred (A') (recurrent parent) to a additional traits (e.g., in the case of transformed plants, traits donor inbred (“B”) (non-recurrent parent), which carries the including herbicide resistance, pest resistance, cold germina appropriate gene(s) for the trait in question, for example, a tion tolerance, water deficit tolerance, and the like) for construct prepared in accordance with the current invention. example, by expressing or Suppressing other genes. Con The progeny of this cross first are selected in the resultant structs for coordinated decrease and increase of gene expres progeny for the desired trait to be transferred from the non sion are disclosed in U.S. Patent Application Publication recurrent parent “B”, and then the selected progeny are mated 2004/0126845A1, specifically incorporated by reference. back to the superior recurrent parent 'A'. After five or more 0234 Seeds of fertile transgenic plants can be harvested backcross generations with selection for the desired trait, the and used to grow progeny generations, including hybridgen progeny can be essentially hemizygous for loci controlling erations, of transgenic plants of this invention that include the the characteristic being transferred, but are like the Superior recombinant DNA construct in their genome. Thus, in addi parent for most or almost all other genes. The last backcross tion to direct transformation of a plant with a recombinant generation would be selfed to give progeny which are pure DNA construct of this invention, transgenic plants of this breeding for the gene(s) being transferred, e.g., one or more invention can be prepared by crossing a first plant having the transformation events. recombinant DNA with a second plant lacking the construct. 0242 Through a series of breeding manipulations, a For example, the recombinant DNA can be introduced into a selected DNA construct can be moved from one line into an plant line that is amenable to transformation to produce a entirely different line without the need for further recombi transgenic plant, which can be crossed with a second plant nant manipulation. One canthus produce inbred plants which line to introgress the recombinant DNA into the resulting are true breeding for one or more DNA constructs. By cross progeny. A transgenic plant of this invention can be crossed ing different inbred plants, one can produce a large number of with a plant line having other recombinant DNA that confers different hybrids with different combinations of DNA con one or more additional trait(s) (such as, but not limited to, structs. In this way, plants can be produced which have the herbicide resistance, pest or disease resistance, environmen desirable agronomic properties frequently associated with tal stress resistance, modified nutrient content, and yield hybrids (“hybrid vigor”), as well as the desirable character improvement) to produce progeny plants having recombinant istics imparted by one or more DNA constructs. DNA that confers both the desired target sequence expression 0243 In certain transgenic plant cells and transgenic behavior and the additional trait(s). plants of this invention, it is sometimes desirable to concur 0235. In such breeding for combining traits the transgenic rently express a gene of interest while also modulating plant donating the additional trait can be a male line (polli expression of a Leptinotarsa target gene. Thus, in some nator) and the transgenic plant carrying the base traits can be embodiments, the transgenic plant contains recombinant the female line. The progeny of this cross segregate Such that DNA further comprising a gene expression element for some of the plant will carry the DNA for both parental traits expressing at least one gene of interest, and transcription of and some will carry DNA for one parental trait; such plants the recombinant DNA construct of this invention is effected can be identified by markers associated with parental recom with concurrent transcription of the gene expression element. binant DNA Progeny plants carrying DNA for both parental 0244. In some embodiments, the recombinant DNA con traits can be crossed back into the female parent line multiple structs of this invention can be transcribed in any plant cell or times, e.g., usually 6 to 8 generations, to produce a homozy tissue or in a whole plant of any developmental stage. Trans gous progeny plant with Substantially the same genotype as genic plants can be derived from any monocot or dicot plant, one original transgenic parental line as well as the recombi Such as, but not limited to, plants of commercial or agricul nant DNA of the other transgenic parental line. tural interest, such as crop plants (especially crop plants used 0236. Yet another aspect of this invention is a transgenic for human food or animal feed), wood- or pulp-producing plant grown from the transgenic seed (or in the case of pota trees, vegetable plants, fruit plants, and ornamental plants. toes, a transgenic seed potato) of this invention. This inven Examples of plants of interest include grain crop plants (such tion contemplates transgenic plants grown directly from as wheat, oat, barley, maize, rye, triticale, rice, millet, Sor transgenic seed containing the recombinant DNA as well as ghum, quinoa, amaranth, and buckwheat); forage crop plants US 2015/O 143580 A1 May 21, 2015 52

(such as forage grasses and forage dicots including alfalfa, 0256 () improved harvest, storage, or processing qual Vetch, clover, and the like); oilseed crop plants (such as cot ity. ton, safflower, Sunflower, soybean, canola, rapeseed, flax, 0257. In some embodiments, the transgenic plant is char peanuts, and oil palm); tree nuts (such as walnut, cashew, acterized by: improved tolerance of abiotic stress (e.g., toler hazelnut, pecan, almond, and the like); Sugarcane, coconut, ance of water deficit or drought, heat, cold, non-optimal nutri date palm, olive, Sugarbeet, tea, and coffee; wood- or pulp ent or salt levels, non-optimal light levels) or of biotic stress producing trees; vegetable crop plants such as legumes (for (e.g., crowding, allelopathy, or wounding); by a modified example, beans, peas, lentils, alfalfa, peanut), lettuce, aspara primary metabolite (e.g., fatty acid, oil, amino acid, protein, gus, artichoke, celery, carrot, radish, the brassicas (for Sugar, or carbohydrate) composition; a modified secondary example, cabbages, kales, mustards, and other leafy brassi metabolite (e.g., alkaloids, terpenoids, polyketides, non-ribo cas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi). Somal peptides, and secondary metabolites of mixed biosyn edible cucurbits (for example, cucumbers, melons, Summer thetic origin) composition; a modified trace element (e.g., squashes, winter squashes), edible alliums (for example, iron, Zinc), carotenoid (e.g., beta-carotene, lycopene, lutein, onions, garlic, leeks, shallots, chives), edible members of the Zeaxanthin, or other carotenoids and Xanthophylls), or Vita Solanaceae (for example, tomatoes, eggplants, potatoes, pep min (e.g., tocopherols) composition; improved yield (e.g., pers, groundcherries), and edible members of the Chenopo improved yield under non-stress conditions or improved yield diaceae (for example, beet, chard, spinach, quinoa, ama under biotic or abiotic stress); improved ability to use nitro ranth); fruit crop plants such as apple, pear, citrus fruits (for gen, phosphate, or other nutrients; modified agronomic char example, orange, lime, lemon, grapefruit, and others), Stone acteristics (e.g., delayed ripening; delayed senescence; ear fruits (for example, apricot, peach, plum, nectarine), banana, lier or later maturity; improved shade tolerance; improved pineapple, grape, kiwifruit, papaya, avocado, and berries; resistance to root or stalk lodging; improved resistance to plants grown for biomass or biofuel (for example, Miscanthus 'green Snap' of stems; modified photoperiod response); grasses, Switchgrass, jatropha, oil palm, eukaryotic microal modified growth or reproductive characteristics (e.g., inten gae Such as Botryococcus braunii, Chlorella spp., and tional dwarfing; intentional male sterility, useful, e.g., in Dunaliella spp., and eukaryotic macroalgae Such as improved hybridization procedures; improved vegetative Graciliaria spp., and Sargassum spp.); and ornamental plants growth rate; improved germination; improved male or female including ornamental flowering plants, ornamental trees and fertility); improved harvest, storage, or processing quality shrubs, ornamental groundcovers, and ornamental grasses. (e.g., improved resistance to pests during storage, improved 0245. This invention also provides commodity products resistance to breakage, improved appeal to consumers); or produced from a transgenic plant cell, plant, or seed of this any combination of these traits. invention, including, but not limited to, harvested leaves, 0258. In another embodiment, transgenic seed, or seed roots, shoots, tubers, stems, fruits, seeds, or other parts of a produced by the transgenic plant, has modified primary plant, meals, oils, extracts, fermentation or digestion prod metabolite (e.g., fatty acid, oil, amino acid, protein, Sugar, or ucts, crushed or whole grains or seeds of a plant, or any food carbohydrate) composition, a modified secondary metabolite or non-food product including Such commodity products pro composition, a modified trace element, carotenoid, or vitamin duced from a transgenic plant cell, plant, or seed of this composition, an improved harvest, storage, or processing invention. The detection of one or more of nucleic acid quality, or a combination of these. In another embodiment, it sequences of the recombinant DNA constructs of this inven can be desirable to change levels of native components of the tion in one or more commodity or commodity products con transgenic plant or seed of a transgenic plant, for example, to templated herein is de facto evidence that the commodity or decrease levels of an allergenic protein or glycoprotein or of commodity product contains or is derived from a transgenic a toxic metabolite. plant cell, plant, or seed of this invention. 0259 Generally, screening a population of transgenic 0246 Generally a transgenic plant having in its genome a plants each regenerated from a transgenic plant cell is per recombinant DNA construct of this invention exhibits formed to identify transgenic plant cells that develop into increased resistance to a Leptinotarsa species infestation. In transgenic plants having the desired trait. The transgenic various embodiments, for example, where the transgenic plants are assayed to detect an enhanced trait, e.g., enhanced plant expresses a recombinant DNA construct of this inven water use efficiency, enhanced cold tolerance, increased tion that is stacked with other recombinant DNA for impart yield, enhanced nitrogen use efficiency, enhanced seed pro ing additional traits, the transgenic plant has at least one tein, and enhanced seed oil. Screening methods include direct additional altered trait, relative to a plant lacking the recom screening for the trait in a greenhouse or field trial or screen binant DNA construct, selected from the group of traits con ing for a Surrogate trait. Such analyses are directed to detect sisting of: ing changes in the chemical composition, biomass, physi 0247 (a) improved abiotic stress tolerance: ological properties, or morphology of the plant. Changes in 0248 (b) improved biotic stress tolerance; chemical compositions such as nutritional composition of 0249 (c) modified primary metabolite composition; grain are detected by analysis of the seed composition and 0250 (d) modified secondary metabolite composition; content of protein, free amino acids, oil, free fatty acids, 0251 (e) modified trace element, carotenoid, or vitamin starch, tocopherols, or other nutrients. Changes in growth or composition; biomass characteristics are detected by measuring plant (0252 (f) improved yield; height, stem diameter, internode length, root and shoot dry 0253) (g) improved ability to use nitrogen, phosphate, weights, and (for grain-producing plants such as maize, rice, or other nutrients; or wheat) ear or seed head length and diameter. Changes in 0254 (h) modified agronomic characteristics; physiological properties are identified by evaluating 0255 (i) modified growth or reproductive characteris responses to stress conditions, e.g., assays under imposed tics; and stress conditions such as water deficit, nitrogen orphosphate US 2015/O 143580 A1 May 21, 2015

deficiency, cold or hot growing conditions, pathogen or insect surfactant, e.g., 0.2% Silwet L77) to the desired concentra attack, light deficiency, or increased plant density. Other tion, and applied to the plants using a track sprayer at a rate of selection properties include days to flowering, days to pollen 15 gallons per acre. A higher concentration (e.g., 100 micro shed, days to fruit maturation, fruit or tuber quality or amount grams/milliliter) can be used for initially assaying a poly produced, days to silking in maize, leaf extension rate, chlo nucleotide for activity, and lower concentrations (e.g., rophyll content, leaf temperature, stand, seedling vigor, inter between about 0.1 to about 1 microgram per milliliter) can be node length, plant height, leaf number, leaf area, tillering, used in Subsequent assays such as those for determining rela brace roots, staying green, Stalk lodging, root lodging, plant tive efficacy of various polynucleotides. Plants are caged health, fertility, green Snap, and pest resistance. In addition, individually with mesh sleeves, and infested with 6 neonatal phenotypic characteristics of harvested fruit, seeds, or tubers Leptinotarsa decemlineata (Colorado potato beetle) larvae. can be evaluated; for example, intomato and eggplant this can Infested plants are incubated in the growth chamber (27 include the total number or weight of fruit harvested or the degree Celsius, 50% relative humidity) for 12-14 days. At the color, acidity, Sugar content, or flavor of Such fruit, and in end of this period, plants are evaluated for level of defoliation, potato this can include the number or total weight of tubers rated as “percent control, and insects are collected from harvested and the quality of such tubers. plants and soil to evaluate “percent viable insects recovered 0260 Specific assays with the compositions and methods and “average weight of viable insects recovered”. of this invention can be carried out in Solanaceous plants 0263. The following Examples are presented for the pur including potato, tomato, eggplant, and peppers, either as poses of illustration and should not be construed as limita hybrids or inbreds; such assays are useful, e.g., for identifying tions. or selecting plants with improved resistance to Colorado potato beetle (larvae or adults), for determining insecticidally EXAMPLES effective amounts of a given composition, or for determining effective treatment regimes. Non-limiting examples of Such Example 1 assays include the following. Generation of Leptinotarsa cDNA Library 0261) An in planta Colorado potato beetle (larvae or adults) assay is carried out in tomato plants with 6 replicates 0264. A cDNA library was generated from Leptinotarsa per treatment. Big Cherry tomato plants are seeded in Readi decemlineata (Colorado potato beetle, “CPB) neonate lar Earth soil containing 6 pounds/cubic yard 14-14-14 fertilizer vae, as follows. Total RNA was isolated from 800 third instar and maintained in a 27 degree Celsius, 50% relative humidity Leptinotarsa decemlineata larvae (whole body) using an growth chamber for three weeks. On the day of the assay, Ambion Totally RNA isolation kit (catalogue number double-stranded RNA is diluted into 25 milliliters of spray AM1910, Life Technologies, Carlsbad, Calif.) with the solution (20 millimolar sodium phosphate buffer (pH 6.8), optional LiCL precipitation procedure. PolyA RNA was iso optionally containing a surfactant, e.g., 0.2% Silwet L77) to lated using Ambion MicroPoly(A) Purist (catalogue number the desired concentration, and applied to the plants using a AM1919, Life Technologies, Carlsbad, Calif.). Random track sprayer at a rate of 15 gallons per acre. A higher con primed cDNA synthesis was performed using a SuperScript centration (e.g., 100 micrograms/milliliter) can be used for Double-Stranded cDNA synthesis kit (catalogue number initially assaying a polynucleotide for activity, and lower 11917-010, Life Technologies, Carlsbad, Calif.) with a ran concentrations (e.g., between about 0.1 to about 1 microgram dom hexamer kit (catalogue number 12328-032, Life Tech per milliliter) can be used in Subsequent assays such as those nologies, Carlsbad, Calif.). The cDNA library was obtained for determining relative efficacy of various polynucleotides. by high-throughput sequencing using commercially available Plants are caged individually with mesh sleeves, and infested 454 technology (454 Life Sciences, 15 Commercial St., Bran with 12 neonatal Leptinotarsa decemlineata (Colorado ford, Conn. 06405, USA), as described in Margulies et al. potato beetle) larvae. Infested plants are incubated in the (2005) Nature, 437:376-380. This provided 1,446,014 reads growth chamber (27 degrees Celsius, 50% relative humidity) (averaging ~350 base-pairs in length), which were Supple for 12-14 days. At the end of this period, plants are evaluated mented with publicly available Leptinotarsa decemlineata for level of defoliation, rated as “percent control, and insects sequence data from NCBI (including 8,835 expressed are collected from plants and soil to evaluate “percent viable sequence tag sequences, 150 full-length cDNAs, 839,061 insects recovered and “average weight of viable insects high-throughput DNA and RNA archived sequence reads) to recovered'. provide a total of 2294,087 combined reads. The combined 0262 An in planta Colorado potato beetle (larvae or sequence data were assembled into contigs de novo using the adults) assay is carried out in potato plants with 9 replicates Newbler (version 2.3) software package (454 Life Sciences, per treatment. Cuttings are prepared from mature Atlantic 15 Commercial St., Branford, Conn. 06405, USA). Approxi potato plants by cutting the stem at an angle below the second mately 38,164 assembled contigs were identified from the node from the youngest growth. The cutting is dipped into sequence data. rooting hormone (Rhizopon #1, 0.1% IBA) and immediately inserted into pre-wet Readi-Earth soil containing 6 pounds/ Example 2 cubic yard 14-14-14 fertilizer. Flats of cuttings are covered to decrease light exposure and placed in a sealed plastic bag to Selection of Low-Copy Target Genes increase humidity. Over the next week, the cover is removed 0265 Leptinotarsa target gene sequences predicted to be and flats are removed from the plastic bags. Plants that are 6-9 effective targets for RNAi-mediated silencing were identified inches tall (usually 3 weeks from cutting date) are used in the as follows. Low-copy genes, and in particular single-copy assay. On the day of the assay, double-stranded RNA is genes, were selected as targets for RNAi-mediated silencing diluted into 25 milliliters of spray solution (20 millimolar as these genea are unlikely to have their function recapitu Sodium phosphate buffer (pH 6.8), optionally containing a lated by a paralogue. A public database of orthologous genes, US 2015/0 143580 A1 May 21, 2015 54

OrthoDB6 (available at cegg.unige.ch/orthodb6 and ID NOs:726-830. It is recognized that analogous sequences described in Waterhouse et al. (2012) Nucleic Acids Res., can be obtained from any other Leptinotarsa species referred PMID:23180791; doi:10.1093/margks 1116) was filtered to to herein. selecta subset of 766 genes that were single-copy or low-copy in Tribolium castaneum (red flour beetle, a coleopteran spe Example 4 cies) as well as single-copy or low-copy in all available arthropod genomes in the database (at the time this applica Selection of Polynucleotide Triggers by "Tiling” tion is filed 33 other arthropod genomes were available). Tribolium castaneum is a coleopteran species and is therefore 0269. One non-limiting example of a method for selecting closely related to Leptinotarsa, which makes it likely that a a polynucleotide triggerfor expression in a transgenic plant or single-copy or low-copy gene present in the Tribolium cas use in a composition for topical application to the surface of taneum genome database will also be a single-copy or low a transgenic or non-transgenic plant involves the mapping of copy gene in the Leptinotarsa decemlineata genome, at least efficacious polynucleotide sequences (or segments of for genes that have high sequence similarity between the two sequences) using a whole-gene (or full-length reference organisms. From the 38,164 unigenes obtained from the Lep sequence) tiling array approach. Sequences selected from tinotarsa decemlineata (Colorado potato beetle, CPB) SEQID NOs: 1-725 and SEQID NOs:726-830 and SEQID sequencing and assembly described in Example 1, a subset of NOs: 1087-1094 are divided into “tiling sequences” or seg 725 genes were identified using a translated nucleotide ments of 200-300 contiguous nucleotides along the entire BLAST search (tblastx) as genes having high sequence simi length of the selected target sequence. The tiling sequences larity (significance ore-value of less than or equal to 1x10') can be designed to be contiguous segments of the selected to the 766 single-copy or low-copy Tribolium castaneum sequence with no overlap or to overlap about 18, 19, 20, 21. genes in the OrthoDB database. 22, 23, 24 or 25 nucleotides in adjacent segments of the 0266 For sequence annotation, SmartBlast annotation selected sequence. Polynucleotide triggers corresponding to was performed by using NCBI's Blastall 2.2.21 software to each 200-300 nucleotide tiling sequence (in sense, anti-sense. search Leptinotarsa decemlineata contigs against the pub or both sense and anti-sense orientation) are synthesized for licly available uniref)0.fasta database (ftp.uniprot.org/pub/ efficacy screening. databases/uniprot/current release/unirefuniref)0/). The (0270. The polynucleotide triggers are tested by any con blast search was performed in blastX mode (translated Lepti venient means for efficacy in silencing the Leptinotarsa spe notarsa decemlineata nucleotide queries searched against the cies target gene. An example of a suitable test is a diet bioas uniref)0 protein database). Only blast hits with an e-value say such as that described in Examples 5 and 6. Another less than or equal to 9e-9 were retained. For each Leptinotarsa suitable test involves the topical application of the polynucle decemlineata contig the description line from the uniref)0 otide triggers either directly to Leptinotarsa individuals or to best hit was used as an annotation. When no SmartBlast hits the surface of a plant to be protected from a Leptinotarsa were found, the sequence was subjected to a supplementary species infestation. One desired result of treatment with a Pfam search. To accomplish this, the longest open reading polynucleotide trigger is prevention or control of a Leptino frame (ORF) was identified for each Leptinotarsa decemlin tarsa species infestation, e.g., by inducing in a Leptinotarsa eata contig and used to query the publicly available Pfam-A insect a physiological or behavioural change such as, but not database (ftp.sanger.ac.uk/pub/databases/Pfam/current re limited to, growth stunting, increased mortality, decrease in lease) using the publicly available HMMER 3.0 software reproductive capacity, decrease in or cessation of feeding package (hmmer janelia.org/). Leptinotarsa decemlineata behavior or movement, or decrease in or cessation of meta contigs with a Pfam hit with an e-value less than or equal to morphosis stage development. Another desired result of treat 1e–5 were annotated with the protein family name and the ment with a polynucleotide trigger is provision of a solana Pfam identifier. Leptinotarsa decemlineata contigs with no ceous plant that exhibits improved resistance to a SmartBlast or Pfam hit were annotated as “novel protein'. Leptinotarsa species infestation, such as a potato, tomato, 0267. The 725 Leptinotarsa decemlineata genes identified eggplant, or pepper plant that exhibits improved resistance to as having high sequence similarity to single-copy or low an infestation by Leptinotarsa decemlineata (Colorado copy Tribolium castaneum genes as described above are pro potato beetle, CPB) or other Leptinotarsa species. Polynucle vided as SEQID NOS:1-725, with each gene annotated based otide tiggers may be screened in sets. For example, sets offive on sequence similarity to Tribolium castaneum and/or individual polynucleotide tiggers are pooled into a single OrthoDB sequences, or by conserved Pfam domains. For polynucleotide composition and topically applied to plants. each Leptinotarsa decemlineata gene, the homologous Tri Those sets showing better efficacy are then re-screened by bolium castaneum gene is also identified in the annotation, testing the individual component polynucleotide tiggers for together with the similarity e-value for each pair. efficacy. (0271 The tiling procedure can be repeated, if desired. A Example 3 polynucleotide trigger found to provide desired activity can itself be tiled. The parent polynucleotide trigger is divided Selection of Leptinotarsa Target Genes into smaller overlapping or non-overlapping segments along the length of the parent polynucleotide trigger. For example, 0268 cDNA sequences corresponding to useful target the parent polynucleotide trigger is divided into segments of genes for controlling Leptinotarsa species by RNAi-medi 50-60 nucleotides in length along the entire length of the ated silencing were selected from the sequences obtained parent polynucleotide trigger. Polynucleotide triggers corre from the Leptinotarsa decemlineata (Colorado potato beetle, sponding to each 50-60 nucleotide tiling sequence (in sense, CPB) sequencing and assembly described in Example 1. This anti-sense, or both sense and anti-sense orientation) are Syn subset of cDNA sequences or target genes is provided in SEQ thesized for efficacy screening. Additional rounds of tiling US 2015/O 143580 A1 May 21, 2015

analysis can be carried out, where triggers as short as 18, 19. 0275. The dsRNA triggers (Table 1) for suppressing the 20, 21, 22, 23, 24, or 25 nucleotides are tested. Leptinotarsa target genes were tested using the following 0272 Effective polynucleotide triggers of any size are methodology to assay mortality or stunting of Leptinotarsa used to make a composition for topical application or a decemlineata larvae due to contact with or ingestion of the recombinant DNA construct useful for making a transgenic polynucleotide triggers. Bioassays with the Colorado potato plant. beetle (CPB), Leptinotarsa decemlineata, were conducted Example 5 using an artificial diet consisting of 13.2 grams/liter agar 0273. This example illustrates a non-limiting assay useful (Serva 11393), 140.3 grams/liter Bio-Serve pre-mix for evaluating the Leptinotarsa-controlling efficacy of poly (F9380B), 5 milliliters/liter KOH (18.3% w/w), and 1.25 nucleotide triggers. More specifically, this example illustrates milliliters/liter formalin (37%). The diet was dispensed in 200 double-stranded RNA triggers comprising a nucleotide microliter aliquots into 96-well plates and dried briefly prior sequence that is complementary to at least 21 contiguous to sample application. Twenty microliters of test sample were nucleotides of a Leptinotarsa target gene (e.g., a target gene applied per well, with sterile water serving as the untreated selected from the Target Gene Sequences Group, or having a control (UTC). Plates were allowed to dry before adding DNA sequence selected from the group consisting of: SEQID insect larvae. One neonate CPB larva was added per well with NOs: 1-725 and SEQ ID NOs:726-830 and SEQ ID NOs: a fine paintbrush. Plates were sealed with Mylar and venti 1087-1094, or the DNA complement thereof), and a bioassay lated using an insect pin. Thirty-two larvae were tested per useful for evaluating the Leptinotarsa-controlling efficacy of treatment. The bioassay plates were incubated at 27 degrees these dsRNA triggers. Celsius, 60% relative humidity, in complete darkness for (0274 Triggers of between about 50 to about 500 base 10-12 days. The plates were scored for larval stunting and pairs (more specifically, of between about 100 to about 450 mortality. Data was analyzed using JMPC4 statistical soft base-pairs) in length were designed for Leptinotarsa target ware (SAS Institute, 1995) and a full factorial ANOVA was genes (see Examples 2 and 3). Blunt-ended double-stranded conducted with a Dunnet's test to look for treatment effects RNAs (dsRNAs) with the anti-sense strand sequences pro compared to the untreated control (P<0.05). ATukey-Kramer vided in SEQID NOs: 831-1085 were manufactured for the post hoc test was performed to compare all pairs of the treat target genes listed in Table 1. ments (P<0.05). Results are provided in Table 1. TABLE 1 SEQ ID dsRNA SEQ NO. OF CPB Diet concen ID TARGET Bioassay tration Exon NO.* Target Gene GENE Results (ppm) No. 831 26S proteasome non-ATPase regulatory subunit 1 825 (+) O. 832 26S proteasome non-ATPase regulatory subunit 1 825 (-) O. 833 26S proteasome non-ATPase regulatory subunit 1 825 (-) O. 834 26S proteasome non-ATPase regulatory subunit 1 825 (-) O. 835 26S proteasome non-ATPase regulatory subunit 1 825 (-) O. 2 836 Actin 821 (-) O. 837 Actin 821 (-) O. 838 Actin 821 (-) O. 839 Actin 821 (-) O. 840 Actin 821 (-) O. 841 Coatomer subunit beta 822 (-) O. 842 Coatomer subunit beta 822 (+) O. 843 Coatomer subunit beta 822 NT O. 844 Coatomer subunit beta 822 NT O. 845 Coatomer subunit beta 822 (-) O. 846 26S proteasome non-ATPase regulatory subunit 2 805 (-) O. 847 26S proteasome non-ATPase regulatory subunit 2 805 (-) O. 848 26S proteasome non-ATPase regulatory subunit 2 805 (-) O. 849 26S proteasome non-ATPase regulatory subunit 2 805 (+) O. 850 26S proteasome non-ATPase regulatory subunit 2 805 (-) O. 2. 851 26S proteasome non-ATPase regulatory subunit 12 806 (-) O. 852 26S proteasome non-ATPase regulatory subunit 12 806 (-) O. 853 26S proteasome non-ATPase regulatory subunit 12 806 NT O. 2. 854 26S proteasome non-ATPase regulatory subunit 12 806 NT O. 855 26S proteasome non-ATPase regulatory subunit 12 806 NT O. 856 Probable 26S proteasome non-ATPase regulatory subunit 3 807 (-) O. 857 Probable 26S proteasome non-ATPase regulatory subunit 3 807 (-) O. 858 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT O. 859 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT O. 860 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT O. 861 26S proteasome non-ATPase regulatory subunit 7 808 (-) O. 862 26S proteasome non-ATPase regulatory subunit 7 808 (-) O. 863 26S proteasome non-ATPase regulatory subunit 7 808 NT O. 864. 26S proteasome non-ATPase regulatory subunit 7 808 NT O. 865 26S proteasome non-ATPase regulatory subunit 7 808 NT O. 866 26S proteasome non-ATPase regulatory subunit 2 809 (-) O. 867 26S proteasome non-ATPase regulatory subunit 2 809 (-) O. 2. US 2015/O 143580 A1 May 21, 2015 56

TABLE 1-continued SEQID dsRNA SEQ NO. OF CPB Diet concen ID TARGET Bioassay tration Exon NO.* Target Gene GENE Results * (ppm) No. 868 26S proteasome non-ATPase regulatory subunit 2 809 (-) O. 2 869 26S proteasome non-ATPase regulatory subunit 2 809 (-) O. 870 26S proteasome non-ATPase regulatory subunit 2 809 (-) O. 871 26S proteasome non-ATPase regulatory subunit 4 810 (-) O. 872 26S proteasome non-ATPase regulatory subunit 4 810 NT O. 2 873 26S proteasome non-ATPase regulatory subunit 4 810 NT O. 874 26S proteasome non-ATPase regulatory subunit 4 810 NT O. 875 26S proteasome non-ATPase regulatory subunit 4 810 NT O. 876 26S protease regulatory subunit 8 811 NT O. 877 26S protease regulatory subunit 8 811 NT O. 878 26S protease regulatory subunit 8 811 NT O. 2 879 26S protease regulatory subunit 8 811 (-) O. 88.0 26S protease regulatory subunit 8 811 (-) O. 881 26S proteasome non-ATPase regulatory subunit 13 812 NT O. 2 882 26S proteasome non-ATPase regulatory subunit 13 812 NT O. 883 26S proteasome non-ATPase regulatory subunit 13 812 (-) O. 884 26S proteasome non-ATPase regulatory subunit 13 812 (-) O. 885 26S proteasome non-ATPase regulatory subunit 13 812 (-) O. 886 Putative uncharacterized protein 813 NT O. 887 Putative uncharacterized protein 813 NT O. 888 Putative uncharacterized protein 813 (-) O. 889 ADP-ribosylation factor GTPase-activating protein, putative 814 NT O. 890 ADP-ribosylation factor GTPase-activating protein, putative 814 NT O. 891 ADP-ribosylation factor GTPase-activating protein, putative 814 (-) O. 892 ADP-ribosylation factor GTPase-activating protein, putative 814 (-) O. 893 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 NT O. 894 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 NT O. 895 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 NT O. 896 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 NT O. 897 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 NT O. 2 898 Sec24 protein, putative 816 (+) O. 2 899 Sec24 protein, putative 816 (-) O. 2 900 Sec24 protein, putative 816 (-) O. 2 901 Sec24 protein, putative 816 (-) O. 2 902 Sec24 protein, putative 816 (-) O. 903 Protein transport protein Sec24B 817 (-) O. 904 Protein transport protein Sec24B 817 (-) O. 905 Protein transport protein Sec24B 817 (-) O. 906 Protein transport protein Sec24B 817 (-) O. 907 Protein transport protein Sec24B 817 (-) O. 908 Protein transport protein sec31A 818 (-) O. 909 Protein transport protein sec31A 818 (-) O. 910 Protein transport protein sec31A 818 (+) O. 911 Protein transport protein sec31A 818 (-) O. 2 912 Protein transport protein sec31A 818 (-) O. 913 GTP-binding protein SAR1B 819 (-) O. 914 GTP-binding protein SAR1B 819 (-) O. 915 GTP-binding protein SAR1B 819 NT O. 2 916 GTP-binding protein SAR1B 819 NT O. 917 GTP-binding protein SAR1B 819 NT O. 918 Protein transport protein sec13 82O (-) O. 2 919 Protein transport protein sec13 82O (-) O. 920 Protein transport protein sec13 82O (-) O. 921 Protein transport protein sec13 82O (-) O. 922 Ribosomal protein L13A 741 NT O 2 923 Ribosomal protein L13A 741 NT O 2 924 60S ribosomal protein L5 728 NT O 2 925 60S ribosomal protein L5 728 (+) O 2? 926 Ribosomal protein S7 776 NT O 1 927 Ribosomal protein S7 776 (-) O 1 928 Ribosomal protein L9 735 (+) O 2 929 Ribosomal protein L9 735 NT O 1 930 Ribosomal protein L3 726 NT O 2 931 Ribosomal protein L3 726 (+) O 2 932 60S ribosomal protein L32 755 (+) O 3 933 Ribosomal protein L8 734 NT O 2 934 Ribosomal protein L8 734 NT O 2 935 Ribosomal protein S15 785 NT O 2 936 Ribosomal protein S15 785 NT O 2 937 Ribosomal protein L7A 732 (+) O 3 938 Ribosomal protein L7A 732 (+) O 3 939 40S ribosomal protein S14 784 NT O 2 US 2015/O 143580 A1 May 21, 2015 57

TABLE 1-continued SEQID dsRNA SEQ NO. OF CPB Diet concen ID TARGET Bioassay tration Exon NO.* Target Gene GENE Results * (ppm) No. 940 40S ribosomal protein S14 784 (+) O 2 941 40S ribosomal protein S24 796 (+) O 2? 942 60S ribosomal protein L10A 737 (+) O 1 943 Ribosomal protein L13 740 (+) O 1 944 Ribosomal protein L13 740 (+) O 1 945 Ribosomal protein S13 783 (+) O 3 946 Ribosomal protein S13 783 NT O 2 947 Ribosomal protein L4e 727 (+) O 3 948 Ribosomal protein L4e 727 (+) O 2 949 Ribosomal protein S30 803 (+) O 2 950 Ribosomal protein S30 803 (+) O 2 951 Ribosomal protein L26 749 (+) O 2? 952 Ribosomal protein L26 749 (+) O 2? 953 Ribosomal protein L31 754 NT O 3 954 60S Ribosomal protein L10 736 NT O 2 955 60S Ribosomal protein L10 736 (+) O 2 956 Ribosomal protein S4 772 (+) O 3 957 Ribosomal protein S4 772 (+) O 2 958 Ribosomal protein L11e 738 (+) O 2 959 Ribosomal protein S6 774 (-) O 1 960 Ribosomal protein S11 782 (+) O 3 961 Ribosomal protein S11 782 (+) O 3 962 Ribosomal protein S11 781 NT O 3 963 Ribosomal protein S11 781 NT O 3 964 Ribosomal protein L12e 739 (+) O 2 965 Ribosomal protein L12e 739 NT O 2 966 Ribosomal protein S5 773 (+) O 2 967 Ribosomal protein SS 773 (+) O 3 968 Ribosomal protein S18 790 (+) O 2 969 Ribosomal protein S18 790 (+) O 2 970 Ribosomal protein L23A 747 (+) O 2 971 Ribosomal protein L23A 747 (+) O 2 972 Ribosomal protein L35A 759 NT O 1 973 Ribosomal protein L35A 759 (+) O 2 974 Ribosomal protein L21 746 NT O 2? 975 Ribosomal protein L21 746 NT O 2? 976 Ribosomal protein L21 745 (+) O 1 977 Ribosomal protein L21 745 (-) O 2? 978 Ribosomal protein S8 777 (+) O 2 979 Ribosomal protein S8 777 (+) O 3 980 Ribosomal protein S16 788 NT O 1 981 Ribosomal protein S16 799 NT O 2 982 Ribosomal protein L18Ae 744 (+) O 2 983 Ribosomal protein S6 775 (+) O 1 984 Ribosomal protein S3 768 NT O 2 985 Ribosomal protein S3 768 (+) O 2 986 Ribosomal protein S17 789 NT O 2 987 Ribosomal protein S15A 786 (+) O 2 988 Ribosomal protein L7 730 (+) O 2? 989 Ribosomal protein L7 730 (+) O 2 990 Ribosomal protein S4 771 NT O 2 991 Ribosomal protein S4 771 (+) O 2 992 40S ribosomal protein S3A 769 (+) O 993 40S ribosomal protein S3A 769 NT O 994 Ribosomal protein L36 760 (+) O 995 Ribosomal protein L37 762 (+) O 2 996 Ribosomal protein L37 763 (+) O 2 997 Ribosomal protein S19 792 (+) O 998 Ribosomal protein S19 792 NT O 999 Ribosomal protein S19 792 (+) O 1000 Ribosomal protein S20 794 NT O 1001 Ribosomal protein L15 743 NT O 2 1002 Ribosomal protein L35A 758 NT O 1003 Ribosomal protein L35A 758 NT O 1004 40S ribosomal protein S21 795 NT O 3 1005 Ribosomal protein S29 802 NT O 1006 Ribosomal protein S8 778 (+) O 1007 40S ribosomal protein S3A 770 (+) O 1008 Ribosomal protein L24 748 (+) O 2 1009 Ribosomal protein S16 787 (+) O 2 1010 Ribosomal protein L7A 733 (+) O 1011 40S ribosomal protein S9 78O NT O 2 US 2015/O 143580 A1 May 21, 2015 58

TABLE 1-continued SEQID dsRNA SEQ NO. OF CPB Diet concen ID TARGET Bioassay tration Exon NO.* Target Gene GENE Results * (ppm) No. 012 40S ribosomal protein SA 804 NT O 1 013 40S ribosomal protein SA 804 (+) O 1 014 Ribosomal protein L37Ae 764 (-) O 2? 015 60S Ribosomal protein L23 797 NT O 1 O16 Ribosomal protein L7 731 NT O 2 O17 Ribosomal protein L36 761 NT O 1 018 40S ribosomal protein S9 779 (+) O 2? O19 Ribosomal protein S26 798 (+) O 3 020 Ribosomal protein L34A 756 (+) O 2 O21 Ribosomal protein L27Ae 751 NT O 1 022 Ribosomal protein L27Ae 751 (+) O 1 O23 40S ribosomal protein S28 8O1 (-) O 2? O24 Ribosomal protein L29 753 (-) O 3 O25 Ribosomal protein L28 752 (+) .0 4 026 Ribosomal protein L28 752 NT .0 4 027 Ribosomal biogenesis protein RLP24 765 NT O 2 028 Ribosomal biogenesis protein RLP24 765 (-) O 1 O29 Ribosomal protein L27 750 (+) O 2 O30 Ribosomal protein L27 750 (+) O 2 O31 39S ribosomal protein L13 766 (-) O 3 O32 39S ribosomal protein L13 766 (-) O 3 033 Ribosomal protein S2 767 (+) O 1 O34 40S ribosomal protein S28 800 (-) O 2? O35 Ribosomal protein L14 742 (+) O 2 O36 Ribosomal protein L6 729 (+) O 2 O37 Coatomer subunit beta 822 (+) O 2 038 Coatomer subunit gamma 828 (+) O 2 O39 Myosin Vila 824 (+) O 2 040 Myosin Vila 823 (+) O 1 041 Actin 821 (+) O 1 042 26S proteasome non-ATPase regulatory subunit 1 826 (+) O 2 043 26S proteasome non-ATPase regulatory subunit 1 825 (+) O 2 O44 crooked neck 830 NT O 1 045 crooked neck 829 (+) O 2 O46 Predicted putative protein 827 (+) O 2 O47 26S proteasome non-ATPase regulatory subunit 2 805 (+) O 2 048 26S proteasome non-ATPase regulatory subunit, putative 806 (-) O 2 049 Probable 26S proteasome non-ATPase regulatory subunit 3 807 (+) O 1 050 26S proteasome non-ATPase regulatory subunit 7 808 (+) O 2 051 26S proteasome non-ATPase regulatory subunit 2 809 NT O 2 052 26S proteasome non-ATPase regulatory subunit 4 810 (-) O 3 053 26S protease regulatory subunit 8 811 (+) O 3 054 26S proteasome non-ATPase regulatory subunit 13 812 (+) O 3 055 Putative uncharacterized protein 813 (-) O 2 056 ADP-ribosylation factor GTPase-activating protein, putative 814 (-) O 2 057 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 (-) O 2? 058 Sec24 protein, putative 816 (+) O 2 059 Protein transport protein Sec24B 817 (-) O 1 060 Protein transport protein sec31A 818 (+) O 2 O61 GTP-binding protein SAR1B 819 (+) O 2 062 Protein transport protein sec13 82O (-) O 2? O63 Sec24B protein 817 (-) O 1 064 Coatomer subunit beta 822 (+) O 2 O65 Coatomer subunit gamma 828 (+) O 2 066 Myosin Vila 824 (+) O 2 067 Myosin Vila 823 (+) O 2 068 Actin 821 (+) O 1 O69 26S proteasome non-ATPase regulatory subunit 1 825 NT O 2 O70 Crooked neck 829 (+) O 2 071 26S proteasome non-ATPase regulatory subunit 2 805 (-) O 2 072 26S proteasome non-ATPase regulatory subunit 12 806 (-) O 2 073 Probable 26S proteasome non-ATPase regulatory subunit 3 807 (+) O 1 074 26S proteasome non-ATPase regulatory subunit 7 808 (+) O 2 075 26S proteasome non-ATPase regulatory subunit 2 809 (+) O 2 076 26S proteasome non-ATPase regulatory subunit 4 810 (-) O 2 077 26S protease regulatory subunit 8 811 (+) O 3 078 26S proteasome non-ATPase regulatory subunit 13 812 (+) O 3 079 ADP-ribosylation factor GTPase-activating protein, putative 814 (-) O 2 080 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor, putative 815 (+) O 1 081 Sec24 protein, putative 816 (+) O 1 082 Protein transport protein Sec24B 817 (+) O 1 083 Protein transport protein sec31A 818 (-) O 1 US 2015/O 143580 A1 May 21, 2015 59

TABLE 1-continued SEQID dsRNA SEQ NO. OF CPB Diet concen ID TARGET Bioassay tration Exon NO.* Target Gene GENE Results * (ppm) No. 1084 GTP-binding protein SAR1B 819 (+) 1.O 1 1085 Protein transport protein sec13 82O (+) 1.O 1 *sequence of anti-sense strand of the dsRNA trigger **(+) significant stunting or mortality compared with water-treated control; (-) no significant stunting or mortality compared with water-treated control; NT = either (1) trigger was not tested, or (2) both of the following occurred: the sample did not provide significant stunting mortality and the positive control did not provide significant stunting mortality in that test, Positive control used in this assay was the dsRNA trigger targetting beta coatomer and having the sense strand sequence of SEQID NO:1086, previously disclosed as SEQD NO:880 in U.S. Pat. No. 7,943,819. 0276. Where available genomic sequence data permitted, Example 6 the number of exons spanned by a given trigger sequence was determined and is provided in Table 1: “1” indicates the trigger sequence appears to be contained in a single contigu 0280. This example illustrates non-limiting embodiments ous genomic locus; “2?” indicates that the full length of the of polynucleotides of this invention, insecticidal composi trigger did not align to the genome, with at least 40 base-pairs tions for controlling a Leptinotarsa species, and a represen missing, which may indicate incompleteness of the available tative assay useful for evaluating the Leptinotarsa-control genomic sequence data. ling efficacy of Such polynucleotides. 0277 Additional cDNA sequences encoding subunits of a Leptinotarsa decemlineata (Colorado potato beetle, CPB) 0281 Five dsRNA triggers (having anti-sense strand exocyst complex were identified from a separate sequencing sequences of SEQID NOs:989, 1049, 1050, 1078, and 1084; and assembly project as Leptinotarsa target genes. These see Table 1) for Suppressing Leptinotarsa target genes were Leptinotarsa exocyst target genes, SEQID NOs: 1087-1094, tested using the following leaf disc methodologies to assay are useful in designing polynucleotide triggers comprising at mortality or stunting of Leptinotarsa decemlineata larvae due least 21 contiguous nucleotides complementary to an exocyst to contact with or ingestion of the polynucleotide triggers. target gene and useful for controlling Leptinotarsa species infestations, and in making transgenic plants expressing Such 0282 For the leaf disc bioassay with adult insects, newly polynucleotide triggers for resistance to Leptinotarsa species emerged Colorado potato beetle (CPB, Leptinotarsa decem infestations. lineata) adults were collected and maintained on potato foli age for up to 7 days, and then fasted for 6-8 hours prior to (0278 Triggers of between about 50 to about 500 base pairs (more specifically, of between about 100 to about 450 beginning the bioassay. Fifteen adults per treatment (trigger/ base-pairs) in length are designed for each of the Leptinotarsa dose) were used. Ten microliters containing 250, 83.3, 27.8, exocyst target genes (SEQID NOs: 1087-1094) as described or 9.3 nanograms of dsRNA trigger in a 0.1% Silwet L77 in Example 4. These triggers are tested using the same meth solution in UltraPure water (Invitrogen) was applied to odology as that described above for the polynucleotides in 15-millimeter-diameter potato (Atlantic variety) leaf discs; Table 1. control leaf discs were treated with either the formulation 0279. In a non-limiting example, a polynucleotide trigger, 0.1% Silwet L77 solution or with a negative control trigger designed to target the Leptinotarsa decemlineata Exo70 gene designed to silence green fluorescent protein (GFP). Treated (SEQ ID NO:1093), was produced as a blunt-ended double leaf discs were placed individually into wells of 6-well cluster stranded RNA having the anti-sense strand sequence of SEQ plates containing 2 milliliters/well of a solidified 2% agar ID NO:1095. This trigger gave significant stunting and sig nificant mortality at both concentrations tested, using the agar/distilled water matrix. A single CPB adult was placed in methodology described above. Results are provided in Table each well and incubated overnight to allow it to consume the 2. leaf disc; in cases where the leaf disc was not totally con Sumed, the insect was likely dead or damaged from handling TABLE 2 and was excluded from the assay. The next day, the CPB adults from a given trigger/dose treatment were collectively SEQID SEQ Trigger NO. OF CPB Diet dsRNA transferred to a feeding arena made from a covered, aerated ID Length Target TARGET Bioassay concentration 16-ounce translucent plastic container lined at its base with NO.: (bp) Gene GENE Results: (ppm) filter paper and containing potato (Atlantic variety) foliage 109S 277 Exof O 1093 (+) O.1 with stems inserted in a water-filled tube for freshness. The 109S 277 Exof O 1093 (+) O.O33 insects were incubated in the feeding arena in an environmen *sequence of anti-sense strand of the dsRNA trigger tal chamber (27 degrees Celsius; 60% relative humidity; 16 (+) significant stunting or mortality compared with water-treated control; (-) no signifi hours light/8 hours dark) with potato foliage replenished as cant stunting or mortality compared with water-treated control; NT = either (1) trigger was not tested, or (2) both of the following occurred: the sample did not provide significant stunting mortality and the positive control did not provide significant stunting mortality in needed. Insect viability was monitored daily. Insects were that test. Positive control used in this assay was the dsRNA trigger targetting beta coatomer recorded as active (viable), moribund (does not return to feet and having the sense strand sequence of SEQDNO:1086, previously disclosed as SEQD NO:880 in U.S. Pat No. 7,943,819. after 10 seconds after being placed on its back), or dead. Viability results are provided in Table 3. US 2015/O 143580 A1 May 21, 2015 60

TABLE 3 CPB Target gene SEQ Days since treatment

Treatment ID NO. 5 6 7 8 9 10 12 14 16

Formulation-1 na 1OO 100 100 1OO 100 100 1OO 1OO 100 Formulation-2 na 93 93 93 93 93 93 86 86 86 SEQID NO. 1115, GFP-1 na 1OO 100 100 1OO 100 100 8O 8O 60 SEQID NO. 1115, GFP-2 na 93 93 87 87 8O 8O 8O 8O 8O SEQID NO.989*, 250 ng 730 87 87 8O 33 O O O O O SEQID NO.989*, 83 ng 730 1OO 100 79 43 29 7 O O O SEQID NO. 989*, 28 ng 730 1OO 100 8O 47 27 O O O O SEQID NO. 989*, 9 ng 730 93 93 73 60 33 O O O O SEQID NO. 1049*, 250 ng 807 40 13 O O O O O O O SEQID NO. 1049*, 83 ng 807 8O 7 7 7 O O O O O SEQID NO. 1049*, 28 ng 807 8O 13 13 13 13 7 13 7 7 SEQID NO. 1049*,9ng 807 87 73 60 60 60 60 53 53 53 SEQID NO. 1050*,250 ng 808 60 13 O O O O O O O SEQID NO. 1050*, 83 ng 808 60 2O O O O O O O O SEQID NO. 1050*, 28 ng 808 86 29 29 14 14 14 14 14 14 SEQID NO. 1050*,9ng 808 8O 60 60 53 53 53 47 40 40 SEQID NO. 1078*, 250 ng 812 67 27 2O O O O O O O SEQID NO. 1078*, 83 ng 812 60 13 7 7 7 7 7 7 7 SEQID NO. 1078*, 28 ng 812 73 33 2O 13 13 13 13 13 13 SEQID NO. 1078*,9ng 812 1OO 8O 8O 67 60 60 53 47 47 SEQID NO. 1084*, 250 ng 819 33 O O O O O O O O SEQID NO. 1084*, 83 ng 819 73 33 7 O O O O O O SEQID NO. 1084*, 28 ng 819 73 40 33 33 33 33 2O 2O 2O SEQID NO. 1084*,9ng 819 8O 60 53 53 53 53 47 47 40 sequence of anti-sense strand of the dsRNA trigger, unless otherwise noted, “Formulation-1” and “Formulation-2” are duplicates of a null control (0.1% Silwet in water), “GFP-1” and “GFP-2 are duplicates of Egil's rising a 377 bp dsRNA trigger targetting green fluorescent protein (GFP) and having the sense strand sequence of “na’ = not applicable.

0283 For the leaf disc bioassay with larvae, neonate Colo overnight to allow it to consume the leaf disc; in cases where rado potato beetle (CPB, Leptinotarsa decemlineata) larvae the leaf disc was not totally consumed, the insect was likely hatched within 24 hours of the bioassay were used. Sixteen dead or damaged from handling and was excluded from the larvae per treatment (trigger/dose) were used. Two microli assay. The next day, the CPB larvae from a given trigger/dose ters containing 250, 83.3, 27.8, or 9.3 nanograms of dsRNA treatment were collectively transferred to a feeding arena trigger in a 0.1% Silwet L77 solution in UltraPure water made from a covered, aerated 16-ounce translucent plastic (Invitrogen) was applied to 7-millimeter-diameter potato (At container lined at its base with filter paper and containing lantic variety) leaf discs; control leaf discs were treated with potato (Atlantic variety) foliage with stems inserted in a either the formulation 0.1% Silwet L77 solution or with a water-filled tube for freshness. The insects were incubated in negative control trigger designed to silence green fluorescent the feeding arena in an environmental chamber (27 degrees protein (GFP). Treated leaf discs were placed individually Celsius; 60% relative humidity; 16 hours light/8 hours dark) into wells of 128-well clusterplates containing 0.5 milliliters/ with potato foliage replenished as needed. Larval viability well of a solidified 2% agar agar/distilled water matrix. A was monitored daily. Larvae were recorded as alive or dead. single CPB neonate was placed in each well and incubated Viability results are provided in Table 4. TABLE 4 CPB Target gene SEQ Days since treatment

Treatment ID NO. 5 6 7 8 9 10 12 14 16

Formulation-1 na 1OO 100 100 100 100 100 92 S4 15 Formulation-2 na 87 87 87 87 73 73 73 27 20 SEQID NO . 1115, GFP-1 na 69 69 69 69 69 69 69 SO 38 SEQID NO . 1115, GFP-2 na 1OO 100 94 94 75 75 56 19 19 SEQID NO. 989*, 250 ng 730 44 38 31 13 13 O O O O SEQID NO. 989*, 83 ng 730 19 19 13 O O O O O O SEQID NO. 989* 28 ng 730 69 50 38 13 13 6 6 6 6 SEQID NO. 989*,9ng 730 38 13 13 13 6 6 6 6 6 SEQID NO. 1049*, 250 ng 807 2O 7 7 7 7 O O O O SEQID NO. 1049*, 83 ng 807 38 13 13 13 13 13 13 13 13 SEQID NO. 1049*, 28 ng 807 38 13 13 6 6 6 6 6 6 SEQID NO. 1049*, 9 ng 807 57 21 21 21 21 21 21 21 14 SEQID NO. 1050*, 250 ng 808 44 31 31 25 19 19 19 O O SEQID NO. 1050*, 83 ng 808 38 19 19 6 O O O O O SEQID NO. 1050*, 28 ng 808 13 13 13 13 13 13 O O O US 2015/O 143580 A1 May 21, 2015 61

TABLE 4-continued CPB Target gene SEQ Days since treatment

Treatment ID NO. 5 6 7 8 9 10 12 14 16 SEQID NO. 1050*,9ng 808 O O O O O O O O O SEQID NO. 1078*, 250 ng 812 19 13 O O O O O O O SEQID NO. 1078*, 83 ng 812 29 14 14 7 7 O O O O SEQID NO. 1078*, 28 ng 812 50 31 19 13 13 6 6 O O SEQID NO. 1078*,9ng 812 60 47 40 27 27 27 27 27 13 SEQID NO. 1084*, 250 ng 819 79 43 43 43 29 21 21 14 14 SEQID NO. 1084*, 83 ng 819 56 38 19 19 19 13 13 13 13 SEQID NO. 1084*, 28 ng 819 50 38 25 19 19 19 19 19 19 SEQID NO. 1084*,9ng 819 75 50 44 44 38 38 38 31 31 sequence of anti-sense strand of the dsRNA trigger, unless otherwise noted, “Formulation-1” and “Formulation-2 are duplicates of a null control (0.1% Silwet in water), “GFP-1” and “GFP-2 are duplicates of a negative control using a 377 bp dsRNA trigger targetting green fluorescent protein (GFP) and having the sense strand sequence of SEQID NO: 1115. “na' = not applicable.

Example 7 decemlineata (see Tables 1, 3, and 4). Hairpin triggers are 0284. This example illustrates non-limiting embodiments suitable for in vitro expression or in vivo expression when of polynucleotide triggers for Suppressing Leptinotarsa target provided in an expression construct with appropriate promot genes. More specifically, this example illustrates embodi ers or other elements to permit their expression, e.g., in a ments of blunt-ended dsRNA triggers consisting of a sense bacterial cellor in a plant cell. The non-limiting embodiments and a separate anti-sense Strand, as well as embodiments of disclosed in Table 6 each contain a T7 promoter (located at dsRNA triggers in the form of a hairpin (a single RNA tran nucleotide positions 1-17 in each hairpin sequence) and a Script containing both a sense region and an anti-sense “loop' or spacer located between the sense and the anti-sense region). regions; the loop contains non-specific (not complementary 0285 Table 5 provides blunt-ended dsRNA triggers with or identical to any part of the target gene) nucleotides. One of sequences related to a "parent trigger” (see Table 1), where skill would immediately understand that the sense and anti the parent trigger had been determined to have insecticidal sense regions of the hairpin are useful in combination with activity against Leptinotarsa decemlineata (see Tables 1, 3, different suitable promoters for expression in a given cell and 4) and the derivative triggers are blunt-ended dsRNAs type, and with different spacer or loop sequences (or none at corresponding to Sub-regions of the parent trigger. all, where nucleotides at the junction of the sense and anti TABLE 5 Trigger Target Parent Diet Activity Diet Activity SEQID gene SEQ trigger SEQ vs. CPB vs. CPB NO:* Target gene name ID NO: ID NO: (0.1 ppm) (0.025 ppm) 096 GTP-binding protein 819 1084 (-) (-) SAR1B 097 GTP-binding protein 819 1084 (-) (-) SARI1B 098 GTP-binding protein 819 1084 (+) (-) SAR1B 099 GTP-binding protein 819 1084 (+) (-) SAR1B 100 Probable 26S 807 1049 (-) (-) proteasome non-ATPase regulatory Subunit 3 101 26S proteasome non- 808 1 OSO (-) (-) ATPase regulatory subunit 7 102 26S proteasome non- 812 1078 (-) ATPase regulatory subunit 13 103 Ribosomal protein L7 730 989 (-) 104 Ribosomal protein L7 730 989 (+) *sequence of anti-sense strand of the dsRNA trigger

0286 Table 6 provides dsRNA triggers in the form of a sense regions form the necessary “turn” or minimal loop in hairpin (a single RNA transcript containing both a sense the hairpin). One of skill would also recognize that similar region and an anti-sense region that hybridize to form recombinant DNA constructs are easily designed to encode dsRNA), with sequences derived from or related to a “parent hairpin dsRNA triggers corresponding to the blunt-ended trigger” (see Table 1), where the parent trigger had been dsRNA triggers provided in Tables 1-5 or targeting the target determined to have insecticidal activity against Leptinotarsa genes provided in the Target Gene Sequences Group. US 2015/O 143580 A1 May 21, 2015

TABLE 6

nucleotide position of trigger nucleotide nucleotide Blunt-ended Hairpin trigger anti-sense position position dsRNA Trigger anti-sense region in of loop or of trigger Trigger CPB Target SEQID region in hairpin, SEQ spacer in sense region SEQID Gene SEQ NO:s hairpin ID NO: hairpin in hairpin NO: ID NO: 1105 21-417 1110 418-566 567-963 989** 730 1106 21-300 1111 301-4SO 451-730 1086 1107 21-453 1112 454-603 604-1036 1084** 819 1108 21-458 1113 459-608 609-1046 1050** 808 1109 21-448 1114 449-598 599-1026 1038.** 828 *sequence of DNA construct encoding the hairpin dsRNA trigger **sequence of anti-sense strand of the dsRNA trigger SEQ DNO: 1086 corresponds to the sense strand sequence of a blunt-ended dsRNA targetting beta coatomer, previously disclosed as SEQID NO: 880 in U.S. Pat. No. 7,943,819.

0287. It is anticipated that the combination of certain mercial growing practices. Foliar spray treatments were per recombinant RNAs as described herein (e.g., the dsRNA trig formed twice: a first treatment 36 days after planting and a gers described in Tables 1-6 or their hairpin equivalents, or second treatment 43 days after planting. All foliar treatments active fragments of these triggers) with one or more non were applied with a 4-nozzle boom equipped with 110003VS polynucleotide pesticidal agents will result in a synergetic spray tips spaced 20 inches apart, spraying 2 rows at a time, improvement in prevention or control of Leptinotarsa species and powered by a carbon dioxide-powered backpack sprayer infestations, when compared to the effect obtained with the at 40 pounds per square inch, delivering 38 gallons per acre. recombinant RNA alone or the non-polynucleotide pesticidal All life stages of Colorado potato beetle were recorded for ten agent alone. Routine insect bioassays such as the bioassay randomly selected stems perplot at 3 time points: 3 days after employing an artificial diet described here are useful for defining dose-responses for larval mortality or growth inhi the first foliar spray treatment (39 days after planting), 7 days bition using combinations of the polynucleotide triggers and after the first foliar spray treatment (43 days after planting), one or more non-polynucleotide pesticidal agents (e.g., a and 3 days after the second foliar spray treatment (46 days patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuring after planting). Defoliation, which is caused primarily by iensis insecticidal protein, a Xenorhabdus insecticidal pro small larvae, was measured at 9 days after the first foliar spray tein, a Photorhabdus insecticidal protein, a Bacillus lat treatment (45 days after planting). Two commercial synthetic erosporous insecticidal protein, and a Bacillus sphaericus (Small molecule) insecticides were used as positive controls: insecticidal protein). One of skill in the art can test combina CoragenR (chlorantraniliprole, DuPont) and Radiant(R) (spin tions of polynucleotides and non-polynucleotide pesticidal etoram, Dow AgroSciences). Results are presented in Table 7: agents in routine bioassays to identify combinations of bio statistically different values are indicated by different letters actives that are synergistic and desirable for use in protecting (a, b, c, d, e). Those treatments that share a letter, for example plants from Leptinotarsa species infestations. the Untreated Control and 5 grams per acre SEQID NO:989 Treatment at 3 days after first spray which share the letter “a”, Example 8 are not statistically different; while those treatments that do not share a letter, for example the Untreated Control and Field Efficacy of RNAi-Mediated Control of Coragen R. Treatment at 3 days after first spray, are statisti Leptinotarsa decemlineata cally different. The effects of the dsRNA triggers increased over time and showed a dose-dependent response; at 3 days 0288 A field trial was performed to test efficacy of topi after the second foliar spray, all of the dsRNA trigger treat cally applied dsRNA triggers on controlling Leptinotarsa ments except for the lowest dose of the dsRNA trigger having decemlineata (Colorado potato beetle, CPB) infestations of potato plants under field conditions. Three dsRNA triggers an anti-sense strand sequence of SEQID NO: 1049 resulted in were tested using topical (foliar spray) application: a blunt a decrease in large larvae that was not significantly different ended dsRNA having an anti-sense strand sequence of SEQ from the synthetic insecticide positive controls (CoragenR) IDNO:989, which targets Ribosomal Protein L7 (encoded by and Radiant Treatments) and that was significantly different SEQID NO: 730); a blunt-ended dsRNA having an anti-sense from the Untreated Control. Defoliation also showed a dose strand sequence of SEQID NO: 1049, which targets Probable dependent response to the dsRNA treatments; several of the 26S proteosome non-ATPase regulatory subunit 3 (encoded dsRNA treatments were significantly different from the by SEQ ID NO: 807); and a hairpin dsRNA encoded by the Untreated Control and all of the dsRNA triggers at the highest DNA construct of SEQID NO:1105, which targets Riboso dose tested provided defoliation protection that was not sig mal Protein L7 (encoded by SEQ ID NO: 730). SEQ ID nificantly different from that provided by the synthetic insec NO:1105 encodes a hairpin dsRNA having an anti-sense ticide positive controls (CoragenR) and Radiant Treatments). strand corresponding to SEQ ID NO:989 (see Example 7). The decreased number of larvae and decreased defoliation or The experiment was designed with 11 treatments arranged in plant damage indicated improved resistance of the dsRNA a random complete block design with four replicates. Test treated potato plants to Leptinotarsa decemlineata; these plots consisted of potato plants (variety “Superior) planted plants with improved resistance to Leptinotarsa decemlin in the spring in two 20-foot rows with 6-foot row center eata are expected to exhibit improved yield (increased har spacing; plots were maintained according to standard com vestable tubers). US 2015/O 143580 A1 May 21, 2015

TABLE 7 Mean number of Colorado potato beetles/10 stems Small larvae Large larvae Rate (grams 3 days after 7 days after 3 days after 3 days after 7 days after 3 days after % Treatment per acre) first spray first spray second spray first spray first spray second spray Defoliation Untreated Control Ila. 115.8 a 2013 a 72O ab O 45.3 ab 108.0 a 72.S a SEQID NO:989* 5 63.5 ab 146.5 ab 98.0 alb 3 8.5 bcd 8.3 b 9.8 de SEQID NO:989* 1 93.3 ab 1595 a 144.5 a. 1.3 33.0 abcd 21.8 b 28.8 cd SEQID NO:989* O.2 87.8 ab 116.0 abc 118.0 a O 25.3 abcd 33.8 b 45.0 abc SEQID NO: 1049* 5 66.5 ab 135.5 abc 126.0 a O 2.0 cc 12.8 b 15.0 code SEQID NO: 1049* 1 91.0 ab 17SO a 102.5 ab O 41.3 abc 33.8 b 32.5 bcd Untreated Control Ila. 115.8 a 2013 a 72O ab O 45.3 ab 108.0 a 72.S a SEQID NO: 1049* O.2 93.5 ab 113.8 abc 99.3 ab O.8 59.0 a 8O.O a 68.8 ab SEQID NO: 1105* 5 61.0 ab 91.3 abc 117.8 a O 9.0 bcd 14.0 b 12.5 cde SEQID NO: 1105* 1 72.3 ab 104.8 abc 87.3 ab O 17.8 bcd 8.8 b 18.8 cd Coragen (R) 5:8: 9.8 b 6.0 c O.3 b O 0.0 d O.O. b O.O e Radiant 8 * * 1.3 b 16.8 bc O.O. b O OS d O.O. b O.O e P-Value from Anova O.OOS3 O.OOO4 O.OOO9 S O.OOO1

0289 All of the materials and methods disclosed and applied to the materials and methods described herein with claimed herein can be made and used without undue experi out departing from the concept, spirit and scope of this inven mentation as instructed by the above disclosure. Although the tion. All Such similar Substitutes and modifications apparent materials and methods of this invention have been described to those skilled in the art are deemed to be within the spirit, in terms of embodiments and illustrative examples, it will be Scope and concept of this invention as defined by the apparent to those of skill in the art that variations can be appended claims.

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US2015.0143580A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A method for controlling a Leptinotarsa species infes (c) causing mortality or stunting in larvae of said Leptino tation of a plant comprising: tarsa species by providing in the diet of said larvae at least one polynucleotide comprising at least one silenc (a) contacting said Leptinotarsa species with a polynucle ing element comprising 21 contiguous nucleotides that otide comprising a nucleotide sequence that is comple are complementary to a target gene having a nucleotide mentary to at least 21 contiguous nucleotides of a target sequence selected from the group consisting of SEQID gene having a nucleotide sequence selected from the NO:730, SEQID NO:807, SEQID NOs: 1-725, SEQID group consisting of: SEQID NO:730, SEQID NO:807, NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs: SEQ ID NOs: 1-725, SEQ ID NOs:726-729, SEQ ID 808-830, and SEQID NOs: 1087-1094, oran RNA tran NOs:731-806, SEQID NOs:808-830, and SEQID NOs: scribed from said target gene; or 1087-1094, or an RNA transcribed from said target (d) topically applying to said planta composition compris gene; or ing at least one polynucleotide comprising a nucleotide (b) providing in the diet of said Leptinotarsa species a sequence that is complementary to at least 21 contiguous polynucleotide comprising a nucleotide sequence that is nucleotides of a target gene having a nucleotide complementary to at least 21 contiguous nucleotides of sequence selected from the group consisting of SEQID a target gene having a nucleotide sequence selected from NO:730, SEQID NO:807, SEQID NOs: 1-725, SEQID the group consisting of: SEQ ID NO:730, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs: NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, 808-830, and SEQID NOs: 1087-1094, oran RNA tran SEQID NOs:731-806, SEQID NOs:808-830, and SEQ scribed from said target gene; or ID NOS: 1087-1094, or an RNA transcribed from said (e) topically applying to said plant a composition compris target gene, or ing at least one polynucleotide in a manner Such that an US 2015/O 143580 A1 May 21, 2015 64

effective amount of said polynucleotide is ingested by NOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs: Leptinotarsa species feeding on said plant, said poly 1087-1094; and wherein said composition further comprises nucleotide comprising at least 21 contiguous nucle one or more components selected from the group consisting otides that are complementary to a target gene having a of a carrier agent, a surfactant, a cationic lipid, an organosili nucleotide sequence selected from the group consisting cone, an organosilicone surfactant, a polynucleotide herbi of: SEQID NO:730, SEQID NO:807, SEQID NOs: 1 cidal molecule, a nonpolynucleotide herbicidal molecule, a 725, SEQIDNOs:726-729, SEQID NOs:731-806, SEQ non-polynucleotide pesticide, a safener, and an insect growth ID NOs:808-830, and SEQ ID NOs: 1087-1094, or an regulator. RNA transcribed from said target gene; or 6. The method of claim 1, wherein said method comprises (f) expressing in said plant at least one polynucleotide contacting said Leptinotarsa species with an effective amount comprising at least one segment that is identical or of a solution comprising a double-stranded RNA, wherein at complementary to at least 21 contiguous nucleotides of least one strand of the double-stranded RNA is complemen a DNA having a sequence selected from the group con tary to at least 21 contiguous nucleotides of a gene that sisting of: SEQID NO:730, SEQID NO:807, SEQ ID encodes a ribosomal protein oran RNA transcribed from said NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731 gene, wherein said Leptinotarsa species is Leptinotarsa 806, SEQ ID NOs:808-830, and SEQ ID NOs: 1087 decemlineata, and wherein RNA interference is induced and 1094; or Leptinotarsa decemlineata mortality occurs, and wherein (g) providing to said plant at least one polynucleotide com said ribosomal protein is a ribosomal L7 protein or a protein prising at least one segment that is identical or comple encoded by SEQID NO:730 or wherein said double-stranded mentary to at least 21 contiguous nucleotides of a target RNA comprises a sequence selected from the group consist gene or an RNA transcribed from said target gene, ing of SEQID NO:989,988, 1104, or 1105. wherein said target gene is selected from the group con 7. The method of claim 6, wherein said solution further sisting of the genes identified in the Target Gene comprises one or more components selected from the group Sequences Group or an RNA transcribed from said tar consisting of an organosilicone surfactant or a cationic lipid. get gene; or 8. The method of claim 1, wherein said method comprises (h) contacting said Leptinotarsa species with an effective topically applying to said plant a composition comprising at amount of a double-stranded RNA, one strand of which least one polynucleotide in a manner Such that an effective is complementary to at least 21 contiguous nucleotides amount of said polynucleotide is ingested by Leptinotarsa of a gene that encodes a ribosomal protein, wherein species feeding on said plant, said polynucleotide comprising RNA interference is induced and mortality occurs; or a nucleotide sequence that is complementary to at least 21 (i) contacting said Leptinotarsa species with a polynucle contiguous nucleotides of a target gene having a nucleotide otide comprising at least one segment that is identical or sequence selected from the group consisting of SEQ ID complementary to at least 21 contiguous nucleotides of NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID a target gene selected from the group consisting of the NOs:726-729, SEQID NOs:731-806, SEQID NOs:808-830, genes identified in the Target Gene Sequences Group or and SEQ ID NOs: 1087-1094, or an RNA transcribed from an RNA transcribed from said target gene. said target gene; wherein said Leptinotarsa species is Lepti 2. The method of claim 1, wherein said polynucleotide is a notarsa decemlineata; and wherein said target gene has the double-stranded RNA. sequence of SEQID NO:730 or wherein said polynucleotide 3. The method of claim 2, wherein said double-stranded is a double-stranded RNA having a strand with a sequence RNA is chemically synthesized or is produced by expression selected from the group consisting of SEQID NO:989,988, in a microorganism or by expression in a plant cell. 1104, or 1105. 4. The method of claim 2, wherein said double-stranded 9. The method of claim 1, wherein said Leptinotarsa spe RNA comprises a strand comprising a sequence selected from cies is selected from the group consisting of Leptinotarsa the group consisting of: SEQ ID NOs:989, 1049, 831, 842, behrensi, Leptinotarsa collinsi, Leptinotarsa decemlineata 849, 898, 910, 925,928,931, 932, 937,938,940, 941, 942, (Colorado potato beetle), Leptinotarsa defecta, Leptinotarsa 943, 944, 945, 947, 948,949, 950, 951, 952,955, 956, 957, haldemani (Haldeman's green potato beetle), Leptinotarsa 958, 960, 961,964,966,967,968, 969, 970,971,973, 976, heydeni, Leptinotarsa juncta (false potato beetle), Leptino 978,979,982,983,985, 987, 988, 991, 992,994, 995, 996, tarsa lineolata (burrobrush leaf beetle), Leptinotarsa penin 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, sularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Lep 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, tinotarsa tilascalana, Leptinotarsa tumamoca, and 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, Leptinotarsa typographica. 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 10. A plant having improved resistance to a Leptinotarsa 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, species infestation, provided by the method of claim 1, or a 1095, 1096, 1097,1098, 1099, 1100, 1101, 1102, 1103, 1104, fruit, seed, or propagatable part of said plant. 1105, 1110, 1111, 1112, 1113, and 1114, 1118, 1119, and 11. The plant of claim 10, wherein said plant is selected 1124. from the group consisting of potato, tomato, and eggplant. 5. The method of claim 1, wherein said method comprises 12. An insecticidal composition for controlling a Leptino topically applying to said plant a composition comprising at tarsa species, comprising: least one polynucleotide comprising a nucleotide sequence (a) an insecticidally effective amount of a polynucleotide that is complementary to at least 21 contiguous nucleotides of comprising at least 21 contiguous nucleotides that are a target gene or an RNA transcribed from said target gene, complementary to a target gene having a nucleotide wherein said target gene has a nucleotide sequence selected sequence selected from the group consisting of SEQID from the group consisting of: SEQ ID NO:730, SEQ ID NO:730, SEQID NO:807, SEQID NOs: 1-725, SEQID NO:807, SEQID NOs: 1-725, SEQIDNOs:726-729, SEQID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs: US 2015/O 143580 A1 May 21, 2015 65

808-830, and SEQID NOs: 1087-1094, oran RNA tran- 14. The insecticidal composition of claim 12, further com scribed from said target gene; or prising at least one component selected from the group con (b) an insecticidally effective amount of at least one poly- sisting of a carrier agent, a surfactant, a cationic lipid, an nucleotide comprising at least one silencing element that organosilicone, an organosilicone surfactant, a polynucle is complementary to at least 21 contiguous nucleotides otide herbicidal molecule, a non-polynucleotide herbicidal of a target gene oran RNA transcribed from said target molecule, a nonpolynucleotide pesticide, a safener, and an gene, wherein said target gene has a nucleotide sequence insect growth regulator. selected from the group consisting of: SEQID NO:730, 15. The insecticidal composition of claim 12, wherein said SEQ ID NO:807, SEQ ID NOs: 1-725, SEQ ID NOs: insecticidal composition comprises an insecticidal double 726-729, SEQID NOs:731-806, SEQID NOs:808-830, stranded RNA molecule that causes mortality or stunting of and SEQID NOs: 1087-1094; or growth in a Leptinotarsa species when ingested or contacted (c) an insecticidally effective amount of at least one RNA by said Leptinotarsa species, wherein said insecticidal comprising at least one segment that is identical or double-stranded RNA molecule comprises at least one seg complementary to at least 21 contiguous nucleotides of ment that is complementary to 21 contiguous nucleotides of a a target gene having a nucleotide sequence selected from DNA having a sequence selected from the group consisting the group consisting of: SEQ ID NO:730, SEQ ID of: SEQID NO:730, SEQID NO:807, SEQ ID NOs: 1-725, SEQID NOs:726-729, SEQID NOs:731-806, SEQID NOs: NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, 808-830, and SEQ ID NOs: 1087-1094, or an RNA tran SEQID NOs:731-806, SEQID NOs:808-830, and SEQ scribed from said DNA, and wherein said double-stranded ID NOS: 1087-1094, or an RNA transcribed from said RNA molecule is at least 50 base-pairs in length or is between target gene, or about 100 to about 500 base-pairs in length. (d) an RNA molecule that causes mortality or stunting of 16. A recombinant DNA construct comprising a heterolo growth in a Leptinotarsa species when ingested or con gous promoter operably linked to: tacted by said Leptinotarsa species, wherein said RNA (a) DNA comprising a nucleotide sequence that is comple molecule comprises at least 21 contiguous nucleotides mentary to at least 21 contiguous nucleotides of a target that are complementary to a target gene having a nucle gene having a sequence selected from the group consist otide sequence selected from the group consisting of: ing of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID SEQID NO:730, SEQID NO:807, SEQID NOs:1-725, NOs: 1-725, SEQ ID NOs:726-729, SEQ ID NOs:731 SEQID NOs:726-729, SEQID NOs:731-806, SEQ ID 806, SEQ ID NOs:808-830, and SEQID NOs: 1087 NOs:808-830, and SEQID NOs: 1087-1094, oran RNA 1094, or an RNA transcribed from said target gene; or transcribed from said target gene; or (b) a DNA comprising 21 or more contiguous nucleotides (e) an insecticidal double-stranded RNA molecule that having 100% identity to a fragment of equivalent length causes mortality or stunting of growth in a Leptinotarsa of a DNA having a sequence selected from the group species when ingested or contacted by said Leptinotarsa consisting of: SEQID NO:730, SEQ ID NO:807, SEQ species, wherein at least one Strand of said insecticidal ID NOs: 1-725, SEQ ID NOs:726-729, SEQ ID NOs: double-stranded RNA molecule comprises 21 contigu- 731-806, SEQ ID NOs:808-830, and SEQ ID NOS: ous nucleotides that are complementary to a target gene 1087-1094, or the DNA complement thereof; or or an RNA transcribed from said target gene, wherein (c) DNA encoding at least one silencing element that is said target gene has a sequence selected from the group complementary to at least 21 contiguous nucleotides of consisting of: SEQID NO:730, SEQ ID NO:807, SEQ a target gene or an RNA transcribed from said target ID NOs: 1-725, SEQ ID NOs:726-729, SEQ ID NOs: gene, wherein said target gene has a sequence selected 731-806, SEQ ID NOs:808-830, and SEQ ID NOs: from the group consisting of: SEQID NO:730, SEQID 1087-1094; or NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, (f) an insecticidally effective amount of at least one double- SEQID NOs:731-806, SEQID NOs:808-830, and SEQ stranded RNA comprising a sequence selected from the ID NOs: 1087-1094; or group consisting of: SEQID NOs:989, 1049, 831, 842, (d) DNA encoding at least one silencing element compris 849, 898, 910, 925,928,931, 932, 937,938, 940, 941, ing at least 21 contiguous nucleotides that are comple 942, 943,944, 945, 947, 948,949, 950, 951, 952,955, mentary to a target gene selected from the group con 956, 957, 958, 960, 961, 964, 966,967,968, 969, 970, sisting of the genes in the Target Gene Sequences Group 971,973, 976,978,979,982, 983,985, 987, 988, 991, or an RNA transcribed from said target gene; or 992,994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, (e) DNA encoding a RNA comprising at least 21 contigu 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, ous nucleotides that are complementary to a nucleotide 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, sequence selected from the group consisting of SEQID 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, NOs:989, 1049, 831, 842, 849, 898,910, 925,928,931, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 932, 937,938, 940, 941, 942, 943,944, 945, 947, 948, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 949, 950, 951, 952,955, 956,957, 958, 960, 961,964, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 966,967,968, 969, 970,971,973, 976,978,979,982, 1105, 1110, 1111, 1112, 1113, and 1114. 983, 985, 987, 988,991, 992,994, 995, 996, 997, 999, 13. The insecticidal composition of claim 12, wherein said 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, insecticidal composition is in the form of at least one selected 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, from the group consisting of a solid, liquid, powder, Suspen- 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, Sion, emulsion, spray, encapsulation, microbeads, carrier par- 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, ticulates, film, matrix, seed treatment, soil drench, implant- 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, able formulation, and in-furrow formulation. 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, US 2015/O 143580 A1 May 21, 2015 66

1101, 1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, 949, 950, 951, 952,955, 956,957, 958, 960, 961,964, and 1114, or the complement thereof, or an orthologous 966,967,968, 969, 970,971,973, 976,978,979,982, nucleotide sequence from a Leptinotarsa species or a 983, 985, 987, 988,991, 992,994, 995, 996, 997, 999, Tribolium species, wherein the orthologous nucleotide 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, sequence has at least 95% sequence identity with a 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, nucleotide sequence selected from the group consisting 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, of: SEQ ID NOs:989, 1049, 831, 842, 849, 898, 910, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 925,928,931,932, 937,938, 940, 941, 942, 943, 944, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 945, 947, 948,949, 950, 951, 952,955, 956, 957, 958, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 960, 961,964,966,967,968, 969, 970,971,973, 976, 1101, 1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, 978,979,982,983,985, 987, 988,991, 992,994, 995, and 1114, wherein the percentage sequence identity is 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, calculated over the same length; or 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, (g) DNA encoding RNA comprising a nucleotide sequence 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, selected from the group consisting of: SEQID NOs:989, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1049, 831, 842, 849, 898,910, 925,928,931,932, 937, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 938, 940, 941, 942, 943, 944, 945, 947, 948,949, 950, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 951, 952,955, 956,957, 958, 960, 961,964, 966,967, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1110, 968, 969, 970,971,973, 976,978,979,982, 983,985, 1111, 1112, 1113, and 1114, wherein the percentage 987,988,991,992,994,995,996,997,999, 1006, 1007, sequence identity is calculated over the same length; or 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, (f) DNA encoding a RNA comprising at least one double 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, stranded RNA region, at least one strand of which com 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, prises at least 21 contiguous nucleotides that are 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, complementary to a nucleotide sequence selected from 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, the group consisting of: SEQ ID NOs:989, 1049, 831, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 842, 849, 898, 910, 925,928,931,932, 937,938, 940, 1103, 1104, 1105, 1110, 1111, 1112, 1113, and 1114, or 941, 942, 943,944, 945, 947, 948,949, 950, 951, 952, the complement thereof. 955, 956,957, 958, 960, 961, 964,966,967,968, 969, 17. A plant chromosome or a plastid or a recombinant plant 970,971,973, 976,978,979,982,983,985, 987, 988, virus vector or a recombinant baculovirus vector comprising 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, the recombinant DNA construct of claim 16. 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 18. A transgenic Solanaceous plant cell having in its 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, genome the recombinant DNA construct of claim 16. 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 19. The transgenic solanaceous plant cell of claim 18, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, wherein said transgenic Solanaceous plant cell further has in 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, its genome DNA encoding at least one pesticidal agent 1095, 1096, 1097,1098, 1099, 1100, 1101, 1102, 1103, selected from the group consisting of a patatin, a plant lectin, 1104, 1105, 1110, 1111, 1112, 1113, and 1114, or the a phytoecdysteroid, a Bacillus thuringiensis insecticidal pro complement thereof, or an orthologous nucleotide tein, a Xenorhabdus insecticidal protein, a Photorhabdus sequence from a Leptinotarsa species or a Tribolium insecticidal protein, a Bacillus laterosporous insecticidal pro species, wherein the orthologous nucleotide sequence tein, and a Bacillus sphaericus insecticidal protein. has at least 95% sequence identity with a nucleotide 20. A transgenic Solanaceous plant comprising the trans sequence selected from the group consisting of SEQID genic Solanaceous plant cell of claim 18, or a fruit, seed, or NOs:989, 1049, 831, 842, 849, 898,910, 925,928,931, propagatable part of said transgenic Solanaceous plant. 932, 937,938,940, 941, 942, 943,944, 945, 947, 948, k k k k k