Compositions and methods for controlling leptinotarsa

Abstract

Disclosed herein are methods of controlling insect pests, in particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with improved resistance to infestation by Leptinotarsa spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of Leptinotarsa spp.

1 . A method for controlling a  Leptinotarsa  species infestation of a plant comprising: 
 (a) contacting said  Leptinotarsa  species with a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs: 1087 – 1094, or an RNA transcribed from said target gene; or 
 (b) providing in the diet of said  Leptinotarsa  species a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (c) causing mortality or stunting in larvae of said  Leptinotarsa  species by providing in the diet of said larvae at least one polynucleotide comprising at least one silencing element comprising 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of: 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (d) topically applying to said plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (e) topically applying to said plant a composition comprising at least one polynucleotide in a manner such that an effective amount of said polynucleotide is ingested by  Leptinotarsa  species feeding on said plant, said polynucleotide comprising at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting NOs:1 – 725, SEQ ID NOs:726 - 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (f) expressing in said plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a DNA having a sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 - 729, SEQ ID NOs: 732 - 806, SEQ ID NOs:808 - 830, and SEQ ID NOs:1087 - 1094; or 
 (g) providing to said plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene is selected from the group consisting the genes identified in the Target Gene Sequences Group or an RNA transcribed from said target gene; or 
 (h) contacting said  Leptinotarsa  species with an effective amount of a double-stranded RNA, one strand of which is complementary to at least 21 contiguous nucleotides of a gene that encodes a ribosomal protein, wherein RNA interference is induced and mortality occurs; or 
 (i) contacting said  Leptinotarsa  species with a polynucleotide comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a target gene selected from the group consisting the genes identified in the Target Gene Sequences Group or an RNA transcribed from said target gene. 
 
     
     
         2 . The method of  claim 1 , wherein said polynucleotide is a double-stranded RNA. 
     
     
         3 . The method of  claim 2 , wherein said double-stranded RNA is chemically synthesized or is produced by expression in a microorganism or by expression in a plant cell. 
     
     
         4 . The method of  claim 2 , wherein said double-stranded RNA comprises a strand comprising a sequence selected from the group consisting SEQ ID NOs: 831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1115,1120, 1121, 1122, 1123, 1124,1125, and 1126. 
     
     
         5 . The method of  claim 1 , wherein said method comprises topically applying to said plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094; and wherein said composition further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, and an insect growth regulator. 
     
     
         6 . The method of  claim 1 , wherein said method comprises contacting said  Leptinotarsa  species with an effective amount of a solution comprising a double-stranded RNA, wherein at least one strand of the double-stranded RNA is complementary to at least 21 contiguous nucleotides of a gene that encodes a ribosomal protein or an RNA transcribed from said gene, wherein said  Leptinotarsa  species is  Leptinotarsa decemlineata , and wherein RNA interference is induced and  Leptinotarsa decemlineata  mortality occurs, and wherein said ribosomal protein is a ribosomal L7 protein. 
     
     
         7 . The method of  claim 6 , wherein said solution further comprises one or more components selected from the group consisting of an organosilicone surfactant or a cationic lipid. 
     
     
         8 . The method of  claim 1 , wherein said method comprises topically applying to said plant a composition comprising at least one polynucleotide in a manner such that an effective amount of said polynucleotide is ingested by  Leptinotarsa  species feeding on said plant, said polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; wherein said  Leptinotarsa  species is  Leptinotarsa   decemlineata ; and wherein said polynucleotide is a double-stranded RNA having a strand with a sequence selected from the group consisting of SEQ ID NOs: 831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126. 
     
     
         9 . The method of  claim 1 , wherein said  Leptinotarsa  species is selected from the group consisting of:  Leptinotarsa behrensi ,  Leptinotarsa collinsi ,  Leptinotarsa decemlineata  (Colorado potato beetle),  Leptinotarsa defecta ,  Leptinotarsa haldemani  (Haldeman’s green potato beetle),  Leptinotarsa heydeni ,  Leptinotarsa juncta  (false potato beetle),  Leptinotarsa lineolata  (burrobrush leaf beetle),  Leptinotarsa peninsularis ,  Leptinotarsa rubiginosa ,  Leptinotarsa texana ,  Leptinotarsa tlascalana ,  Leptinotarsa tumamoca , and  Leptinotarsa typographica . 
     
     
         10 . A plant having improved resistance to a  Leptinotarsa  species infestation, provided by the method of  claim 1 , or a fruit, seed, or propagatable part of said plant. 
     
     
         11 . The plant of  claim 10 , wherein said plant is selected from the group consisting of potato, tomato, and eggplant. 
     
     
         12 . An insecticidal composition for controlling a  Leptinotarsa  species, comprising: 
 (a) an insecticidally effective amount of a polynucleotide comprising at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (b) an insecticidally effective amount of at least one polynucleotide comprising at least one silencing element that is complementary to at least 21 contiguous nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094; or 
 (c) an insecticidally effective amount of at least one RNA comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (d) an RNA molecule that causes mortality or stunting of growth in a  Leptinotarsa  species when ingested or contacted by said  Leptinotarsa  species, wherein said RNA molecule comprises at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or 
 (e) an insecticidal double-stranded RNA molecule that causes mortality or stunting of growth in a  Leptinotarsa  species when ingested or contacted by said  Leptinotarsa  species, wherein at least one strand of said insecticidal double-stranded RNA molecule comprises 21 contiguous nucleotides that are complementary to a target gene or an RNA transcribed from said target gene, wherein said target gene has a sequence selected from the group consisting SEQ ID NOs: 1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs: 1087 – 1094; or 
 (f) an insecticidally effective amount of at least one double-stranded RNA comprising a sequence selected from the group consisting SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1120, 1121, 1122, 1123, 1124, 1125, and 1126. 
 
     
     
         13 . The insecticidal composition of  claim 12 , wherein said insecticidal composition is in the form of at least one selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, seed treatment, soil drench, implantable formulation, and in-furrow formulation. 
     
     
         14 . The insecticidal composition of  claim 12 , further comprising at least one component selected from the group consisting of a carrier agent, a surfactant, a cationic lipid, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, and an insect growth regulator. 
     
     
         15 . The insecticidal composition of  claim 12 , wherein said insecticidal composition comprises an insecticidal double-stranded RNA molecule that causes mortality or stunting of growth in a  Leptinotarsa  species when ingested or contacted by said  Leptinotarsa  species, wherein said insecticidal double-stranded RNA molecule comprises at least one segment that is complementary to 21 contiguous nucleotides of a DNA having a sequence selected from the group consisting SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said DNA, and wherein said double-stranded RNA molecule is at least 50 base-pairs in length or is between about 100 to about 500 base-pairs in length. 
     
     
         16 . A recombinant DNA construct comprising a heterologous promoter operably linked to: 
 (a) DNA comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or an RNA transcribed from said target gene; or   (b) a DNA comprising 21 or more contiguous nucleotides having 100% identity to a fragment of equivalent length of a DNA having a sequence selected from the group consisting SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs:1087 – 1094, or the DNA complement thereof; or   (c) DNA encoding at least one silencing element that is complementary to at least 21 contiguous nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a sequence selected from the group consisting of: SEQ ID NOs:1 – 725, SEQ ID NOs:726 – 729, SEQ ID NOs: 732 – 806, SEQ ID NOs:808 – 830, and SEQ ID NOs: 1087 – 1094; or   (d) DNA encoding at least one silencing element comprising at least 21 contiguous nucleotides that are complementary to a target gene selected from the group consisting of: the genes in the Target Gene Sequences Group or an RNA transcribed from said target gene; or   (e) DNA encoding a RNA comprising at least 21 contiguous nucleotides that are complementary to a nucleotide sequence selected from the group consisting of: SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098,1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126, or the complement thereof, or an orthologous nucleotide sequence from a  Leptinotarsa  species or a  Tribolium  species, wherein the orthologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence selected from the group consisting of: SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126, wherein the percentage sequence identity is calculated over the same length; or   (f) DNA encoding a RNA comprising at least one double-stranded RNA region, at least one strand of which comprises at least 21 contiguous nucleotides that are complementary to a nucleotide sequence selected from the group consisting of: SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126, or the complement thereof, or an orthologous nucleotide sequence from a  Leptinotarsa  species or a  Tribolium  species, wherein the orthologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126, wherein the percentage sequence identity is calculated over the same length; or   (g) DNA encoding RNA comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs:831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1111, 1112, 1113, 1114, 1115, 1120, 1121, 1122, 1123, 1124, 1125, and 1126, or the complement thereof.   
     
     
         17 . A plant chromosome or a plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the recombinant DNA construct of  claim 16 . 
     
     
         18 . A transgenic solanaceous plant cell having in its genome the recombinant DNA construct of  claim 16 . 
     
     
         19 . The transgenic solanaceous plant cell of  claim 18 , wherein said transgenic solanaceous plant cell further has in its genome DNA encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein. 
     
     
         20 . A transgenic solanaceous plant comprising the transgenic solanaceous plant cell of  claim 18 , or a fruit, seed, or propagatable part of said transgenic solanaceous plant.