RNA-based control of lepidopteran pests

Abstract

To provide a composition and method for controlling pests, which includes administering an RNA interferant. [Solution] A method for preventing the invasion of lepidopteran pests into plants, comprising: contacting lepidopteran pests with at least one polynucleotide having a target gene having a nucleotide sequence selected from a specific group, or a nucleotide sequence that is complementary to a sequence of nucleotides of transcribed RNA, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with a sequence of nucleotides; providing the polynucleotide to the diet of lepidopteran pests; causing death or inhibition of growth in the larvae of lepidopteran pests; applying the polynucleotide topically to plants; or applying at least one polypeptide topically to plants in such a manner that an effective amount of polynucleotide is ingested by lepidopteran pests that feed on polynucleotides; or expressing at least one polynucleotide in plant material.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 63 / 024,133 and U.S. Provisional Application No. 63 / 106,614, which are hereby incorporated by reference in their entirety.

Background Art

[0002] Crops are often targets of insect attacks. According to the United Nations Food and Agriculture Organization, globally, farmers lose 30 - 40% of their crops to pests and diseases. Maintaining crops and crop health is essential for crop yields and quality, and ultimately they require long - term strategies to minimize the occurrence of pests and diseases. The annual cost for controlling crop pests (e.g., Lepidoptera, Diptera, Coleoptera, Hemiptera, etc.) is estimated to be in the tens of millions of dollars, and if left uncontrolled, the annual cost of crop losses is predicted to reach billions of dollars.

[0003] Chemical insecticides have been one solution for eradicating pest infestations, but there is a need for more environmentally safe alternative solutions. Chemical insecticides are harmful to the environment, may lack specificity or selectivity, and ultimately result in non - target effects. Also, chemical insecticides have slow metabolism, are prone to accumulation, and resistance is likely to occur. Therefore, a more environmentally friendly method for controlling or eradicating insect infestations that is more selective, environmentally safe, and biodegradable has long been needed.

Summary of the Invention

[0004] This embodiment relates to the control of lepidopteran pests, particularly those of economic or agricultural importance. In various embodiments, the lepidopteran pest is at least one selected from the group consisting of the genera Spodoptera (e.g., S. frugiperda, also known as the fall armyworm) and Plutella (e.g., P. xylostella, also known as the diamondback moth).

[0005] The compositions and methods described herein include recombinant polynucleotide molecules, for example, recombinant DNA components for making transgenic plants resistant to invasion by lepidopteran pests, and single-stranded or double-stranded DNA or RNA molecules (referred to herein as “triggers”) useful for controlling or preventing plant invasion by lepidopteran pests. In some embodiments, the polynucleotide trigger is provided as a topically applied agent for controlling or preventing plant invasion by lepidopteran pests. In some embodiments, crop plants with improved resistance to lepidopteran pest invasion are provided, such as transgenic crop plants expressing polynucleotide triggers (including reproductive parts such as seeds or tubers). In some embodiments, crop plants (including reproductive parts such as seeds or tubers) are provided that have been topically treated with a composition containing a polynucleotide trigger (for example, crop plants sprayed with a solution of a dsRNA molecule). Also provided are polynucleotide-containing compositions that are topically applied to plants, plant parts, or seeds to be protected from lepidopteran pests or invasion by lepidopteran pests.

[0006] Some embodiments relate to the repression of target genes in lepidopteran pests by polynucleotide triggers. Some embodiments relate to a method for selecting lepidopteran target genes that may be effective targets for RNAi-mediated control of lepidopteran pests. In some embodiments, the target genes selected for RNAi-mediated repression are non-repetitive and non-duplication genes in the lepidopteran pest genome, i.e., genes that have low nucleotide diversity or are evolutionarily or functionally constrained to have more synonymous (Ks) nucleotide changes than non-synonymous (Ka) nucleotide changes. Provided herein are nucleotide sequences referred to herein as “target gene sequences,” the sequences being sequence numbers 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623. Furthermore, nucleotide sequences referred to herein as "trigger sequences" are also provided, consisting of sequence numbers 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683. The sequence numbers relate to the sequences provided in the sequence list provided herein.

[0007] In one embodiment, a method for controlling the invasion of lepidopteran plant pests includes contacting a lepidopteran pest with a polynucleotide containing at least one segment of 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity of the sequence (for example, containing a segment of 21 consecutive nucleotides having 100% identity of the sequence) or complementary DNA, to a corresponding fragment of DNA having a sequence selected from the target gene sequence group or trigger sequence group. In one embodiment, the method for preventing the invasion of lepidopteran plant pests is described by Sequence IDs 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 115 7, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623 The invention involves contacting a lepidopteran pest with a polynucleotide comprising a nucleotide sequence complementary to at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of, or RNA transcribed from, the target gene. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the contact with the polynucleotide is achieved by direct application of the polynucleotide, or a composition or solution containing the polynucleotide (e.g., by spraying, dispersing, or immersion), to the lepidopteran pest or topically to a surface or matrix (e.g., a plant or soil) in contact with the lepidopteran pest. In some embodiments, the contact with the polynucleotide is achieved by providing a polynucleotide to be ingested by the lepidopteran pest. In some embodiments, the contact with the polynucleotide is achieved by providing a transgenic plant that expresses against the lepidopteran pest.

[0008] Some embodiments relate to a method for controlling the invasion of lepidopteran pests into plants by providing a polynucleotide in the diet of lepidopteran pests, which comprises a polynucleotide having 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity with a corresponding DNA fragment having a sequence selected from a group of target gene sequences and a group of trigger sequences, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having a sequence with 100% identity), wherein the polynucleotide functions to inhibit biological functions within the lepidopteran pest in response to its invasion, thereby controlling the invasion of the lepidopteran pest. In one embodiment, a method for preventing the invasion of lepidopteran plant pests is described by Sequence IDs 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 5 04, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 7 37, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157 , 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623 The invention involves providing a polynucleotide in the diet of a lepidopteran pest, comprising at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of, or a nucleotide sequence complementary to the RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the agent comprising the polynucleotide is formulated, for example, into a sprayable solution or emulsion, a tank mixture, or a powder for application to a field of crop plants. In some embodiments, the agent is biologically produced, for example, in the form of a microbial fermentation product or expressed in transgenic plant cells.

[0009] In another embodiment, a method is provided for inducing death or inhibiting growth in lepidopteran pest larvae. In some embodiments, at least one RNA containing at least one silencing element is provided in the diet of a lepidopteran pest larva, where ingestion of the RNA by the lepidopteran pest larva results in the death or inhibiting growth of the lepidopteran pest larva. In some embodiments, the silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence of the lepidopteran pest larva, where the target gene is selected from a group consisting of genes in the target gene sequence group. In one embodiment, a method for inducing death or inhibiting growth in a lepidopteran pest larva includes providing the larva's diet with at least one polynucleotide containing at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a target gene having a nucleotide sequence selected from the target gene sequence group.In a specific embodiment, the target genes are SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510 ,530,532,533,542,545,546,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,596,602,611,612,620,665,666,672,717,730,731,736,737,738,740,741,742,743,7 49, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195 , selected from the group consisting of 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or RNA transcribed from the target gene. In some embodiments, the silencing element comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments describe a method for inducing death or reduced reproductive capacity in lepidopteran pests, comprising providing the lepidopteran pests' diet with at least one RNA containing at least one silencing element essentially identical or essentially complementary to a target gene sequence fragment of the lepidopteran pest larva, wherein inoculation of the RNA by the lepidopteran pest results in death and reduced reproductive capacity in the lepidopteran pest. In some embodiments, the target gene is selected from a group consisting of genes in the target gene sequence group. In some embodiments, the method induces a decrease in the rate of metamorphosis or a decrease in feeding activity.In some embodiments, the method is useful in providing plants with increased resistance to invasion by lepidopteran pests.

[0010] Some embodiments relate to a method for providing improved resistance to lepidopteran pest invasion, comprising topically applying to a plant a composition comprising 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity with a corresponding DNA fragment having a sequence selected from the target gene sequence group and the trigger sequence group, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having a sequence with 100% identity). In one embodiment, a method for providing plants with improved resistance to the invasion of lepidopteran pests is described in SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 53 2, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 8 14, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283 The method involves topically applying to a plant a composition comprising at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or at least one polynucleotide comprising a nucleotide sequence complementary to the RNA transcribed from the target gene.In one embodiment, a method for providing a plant having improved resistance to lepidopteran insect infestation includes topically applying a composition comprising at least one polynucleotide to a plant in such a manner that an effective amount of the polynucleotide is ingested by a lepidopteran insect that feeds on the plant, wherein the polynucleotide is one of the SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309 ,318,319,320,321,323,324,328,341,343,346,356,358,359,503,504,505,506,509,510,530,532,533,542,545,546,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,596,602,611,612,620,665,666,672,717,73 0, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196 The polynucleotide comprises at least 18 consecutive nucleotides complementary to a target gene having a nucleotide sequence selected from the group consisting of 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments relate to compositions comprising a polynucleotide that are formulated, for example, as a sprayable solution or emulsion, tank mixture, or powder for application to a field of crop plants.

[0011] Some embodiments relate to an insecticidal composition for controlling lepidopteran pests, comprising an insecticidal amount of at least one polynucleotide molecule containing 18 or more consecutive nucleotides that are essentially identical or complementary to a corresponding fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having a 100% identical or complementary sequence). In several embodiments, the polynucleotide molecules are SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 5 32, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763 ,766,808,809,810,814,815,817,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251 The polynucleotide comprises at least 18 consecutive nucleotides complementary to a target gene having a nucleotide sequence selected from the group consisting of 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the polynucleotide molecule is a recombinant polynucleotide.In some embodiments, the polynucleotide molecule is RNA. In some embodiments, the polynucleotide molecule is double-stranded RNA. Relevant embodiments include, for example, an insecticidal composition comprising a polynucleotide molecule formulated for application to a field of crop plants in a sprayable solution or emulsion, tank mixture, or powder, and optionally comprising one or more additional components such as a carrier, surfactant, cationic lipid, organosilicone, organosilicone surfactant, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators.

[0012] Some embodiments relate to a method for providing plants with improved resistance to lepidopteran pest invasion, comprising expressing in a plant at least one polynucleotide comprising 18 or more consecutive nucleotides that are essentially identical or complementary to a corresponding fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having a 100% identical or complementary sequence). In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the trigger sequence group. In some embodiments, the polynucleotide is double-stranded RNA.

[0013] Some embodiments relate to recombinant DNA constructs comprising a heterologous promoter operably ligated to a DNA element containing 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity to a corresponding fragment of DNA having a sequence selected from the target gene sequence group and the trigger sequence group, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having a sequence with 100% identity). In some embodiments, the DNA element encodes double-stranded RNA. In some embodiments, the double-stranded RNA contains one or more nucleotide sequences selected from the trigger sequence group. Relevant embodiments include plant chromosomes or plastids or recombinant plant virus vectors, recombinant baculovirus vectors containing recombinant DNA constructs, or DNA elements without heterologous promoters.

[0014] Some embodiments relate to transgenic crop plant cells having recombinant DNA in its genome that encodes RNA that suppresses the expression of a target gene in a lepidopteran pest that comes into contact with or ingests the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides complementary to a fragment of the target gene. In some embodiments, the target gene is selected from the group of target gene sequences. A specific embodiment is a transgenic crop plant cell having recombinant DNA in its genome that encodes RNA for silencing one or more target genes selected from the group of target gene sequences. In some embodiments, the RNA comprises one or more nucleotide sequences selected from the group of trigger sequences.

[0015] Some embodiments relate to isolated recombinant RNA molecules that, upon ingestion or contact with lepidopteran pests, cause death or inhibit growth in lepidopteran pests, wherein the recombinant RNA molecule comprises 18 or more consecutive nucleotides that are essentially complementary to a corresponding fragment of DNA having sequences selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these (e.g., a segment of 21 consecutive nucleotides having 100% complementarity to that fragment). In some embodiments, the recombinant RNA molecule is double-stranded RNA. Specific embodiments are shown in Sequence IDs 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 52 8, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1 038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 142 This includes isolated recombinant double-stranded RNA molecules having a strand containing a sequence selected from the group consisting of 6, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or combinations thereof.Another embodiment relates to isolated recombinant double-stranded RNA molecules having a strand containing a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539, or combinations thereof.

[0016] Some embodiments relate to a method for providing plants with improved resistance to lepidopteran pest invasion, comprising providing the plants with at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that is essentially identical or complementary to a corresponding fragment of a target gene selected from the group of target gene sequences (for example, a segment of 21 consecutive nucleotides having a sequence that is 100% identical or complementary to that fragment). In one embodiment, the method for providing plants with improved resistance to lepidopteran pest invasion comprises providing the plants with at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 consecutive nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene is selected from the group of genes identified in the group of target gene sequences. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group of trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA.

[0017] Some embodiments relate to a method for controlling the invasion of lepidopteran pests into plants, comprising exposing lepidopteran pests to a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides (e.g., a segment of 21 consecutive nucleotides having a 100% identical or complementary sequence) that is essentially identical or complementary to a corresponding fragment of equivalent length of DNA of a target gene selected from the group of target gene sequences. In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments relate to a method for selecting target genes for RNAi-mediated silencing from plant genomes or animal genomes (e.g., insects and arthropods). In various embodiments, the method provides a subset of target genes that exist as single or low copy number (non-repeating and non-duplication) in a specific genome, or have low nucleotide diversity, or have a synonymous (Ks) ratio to non-synonymous (Ka) nucleotide changes where Ks >> Ka.

[0018] Some embodiments relate to artificial compositions comprising at least one polynucleotide described herein. Some embodiments provide formulations useful for topical application to plants or substances requiring protection from lepidopteran pest infestation. Some embodiments provide recombinant constructs and vectors useful for creating transgenic crop plant cells and transgenic crop plants. Some embodiments provide formulations and coatings useful for treating crop plants, seeds or tubers or other fertile parts of crop plants. Some embodiments provide commodities and food products (in particular commodities and food products having a detectable amount of polynucleotide described herein) produced from such crop plants, seeds or fertile parts that have been treated with or contain the polynucleotide described herein. Some embodiments relate to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the target gene sequence group. Another embodiment relates to proteins encoded by sequences or fragments of sequences selected from the trigger sequence group, or polyclonal or monoclonal antibodies that bind complementaryly thereto. Such antibodies are prepared by routine methods known to those skilled in the art.

[0019] In the various embodiments described herein, the plant may be any plant that is susceptible to invasion by lepidopteran pests. The particular area of ​​interest is the embodiment in which the plant is [plant genus name]. The examples include plants selected from the group consisting of [named crop plants]. Embodiments include cases where the plant is an ungerminated seed of a crop plant, a crop plant in the vegetative growth stage, or a crop plant in the reproductive stage.

[0020] Other aspects and specific embodiments of the present invention are disclosed in the following detailed description.

[0021] Detailed explanation I. Definition Unless otherwise defined, all technical and scientific terms used have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Where a term is provided in the singular form, the inventors also intend aspects of the invention described in the plural form of that term. In the event of any inconsistency between terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions set forth herein. Other technical terms used have the common meanings in the art to which they pertain, as exemplified in dictionaries specific to various art fields, such as “The American Heritage(R) Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to mechanisms of action or modes of action. References are provided for illustrative purposes only.

[0022] Unless otherwise specified, nucleic acid sequences in this specification are given in the 5' to 3' direction when read from left to right. Those skilled in the art will recognize that a given DNA sequence is understood to define a corresponding RNA sequence that is identical to the DNA sequence except that the thymine (T) nucleotides of the DNA are replaced with uracil (U) nucleotides. Thus, providing a particular DNA sequence is understood to define an exact RNA equivalent, and the terms “identity” or “essentially identical” with respect to a DNA sequence include RNA sequences that satisfy these criteria except that the thymine nucleotides are replaced with uracil nucleotides. A given first polynucleotide sequence further defines a second polynucleotide that completely hybridizes to the first polynucleotide by forming a Watson-Crick base pair, which is its exact complementary sequence (which may be DNA or RNA). If DNA is a double-stranded DNA (hybridized strand), the base pairs are adenine:thymine or guanine:cytosine; if DNA is a double-stranded RNA, the base pairs are adenine:uracil or guanine:cytosine. Thus, the nucleotide sequence of a blunt-ended double-stranded polynucleotide that is fully hybridized (there is "100% complementarity" between the strands, or the strands are "complementary") is clearly defined by providing the nucleotide sequence of one strand, whether given as DNA or RNA. "Essentially identical" or "essentially complementary" to a target gene or fragment of a target gene means that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to a target gene or fragment of a target gene, or a transcript of a target gene or fragment of a target gene (generally under physiological conditions such as those found in living plant or animal cells); those skilled in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity. The first nucleic acid sequence is "operably" connected to or "linked" to the second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.For example, when a promoter provides DNA transcription or expression, the promoter sequence is "manipulatively ligated" to the DNA. Generally, manipulatively ligated DNA sequences are contiguous.

[0023] The term “polynucleotide” generally refers to a DNA or RNA molecule containing multiple nucleotides, and generally refers to both “oligonucleotides” (polynucleotide molecules with a length of 18 to 25 nucleotides) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides, including non-standard nucleotides or chemically modified nucleotides, as is commonly practiced in the art; see, for example, the chemical modifications disclosed in the technical manual “RNA Interference (RNAi) and DsiRNAs,” 2011 (Integrated DNA Technologies, Coralville, Iowa). Generally, the polynucleotides described herein, whether DNA or RNA or both, and whether single-stranded or double-stranded, include at least one segment of 18 or more consecutive nucleotides (or, in the case of double-stranded polynucleotides, at least 18 consecutive base pairs), which are essentially identical or complementary to a DNA-equivalent fragment of the target gene or the RNA transcript of the target gene. Throughout this disclosure, “at least 18 consecutive” means “including all integer points between about 18 and about 10,000.”Therefore, embodiments of the present invention include oligonucleotides having a length of 18 to 25 nucleotides (18mers, 19mers, 20mers, 21mers, 22mers, 23mers, 24mers, or 25mers), or medium-length polynucleotides having a length of 26 or more nucleotides (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, about 65 pieces, about 70 pieces, about 75 pieces, about 80 pieces, about 85 pieces, about 90 pieces, about 95 pieces, about 100 pieces, about 11 0 pieces, about 120 pieces, about 130 pieces, about 140 pieces, about 150 pieces, about 160 pieces, about 170 pieces, about 180 pieces, about 190 pieces, about 200 pieces, about 210 pieces, about 220 pieces, about 230 pieces, about 240 pieces, about 250 pieces, about 260 pieces, about 270 pieces, about 280 pieces, about 29 0 or approximately 300 nucleotides), or long polynucleotides having a length longer than approximately 300 nucleotides (for example, between approximately 300 and approximately 400 nucleotides, between approximately 400 and approximately 500 nucleotides, between approximately 500 and approximately 600 nucleotides, between approximately 600 and approximately 700 nucleotides, between approximately 700 and approximately 800 nucleotides, between approximately 800 and approximately 900 nucleotides, between approximately 900 and approximately 1000 nucleotides, between approximately 300 and approximately 500 nucleotides, between approximately 300 and approximately 600 nucleotides, between approximately 300 and approximately 700 nucleotides, between approximately 300 and approximately 800 nucleotides, between approximately 300 and approximately 900 nucleotides, a length of approximately 1000 nucleotides, or a polynucleotide with a length of approximately 1000 or more nucleotides, for example, up to the coding portion or non-coding portion of the target gene, or the full length of the target gene including both the coding and non-coding portions). If a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.

[0024] The polynucleotides described herein may be single-stranded (ss) or double-stranded (ds). “Double-stranded” refers to base pairing that occurs between complementary, antiparallel-stranded nucleic acids, generally sufficient to form a double-stranded nucleic acid structure under physiologically reasonable conditions. Embodiments include those in which the polynucleotide is selected from sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrids, antisense ssDNA, or antisense ssRNA; and mixtures of any of these types of polynucleotides may be used. In some embodiments, the polynucleotide is a double-stranded RNA longer than the typical length of naturally occurring regulatory small RNAs (such as endogenously produced siRNA and mature miRNA). In some embodiments, the polynucleotide is a double-stranded RNA with a length of at least about 30 consecutive base pairs. In some embodiments, the polynucleotide is a double-stranded RNA having a length of about 50 to about 500 base pairs. In some embodiments, the polynucleotide may contain components other than standard ribonucleotides; for example, one embodiment is RNA containing terminal deoxyribonucleotides.

[0025] Embodiments provide protection for crop plants against Lepidoptera pests. The crop plants include cereal crop plants (e.g., wheat, barley, oats, corn, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (e.g., forage grasses and forage dicotyledons including alfalfa, clover, etc.); oilseed crop plants (e.g., cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanut, and oil palm); nuts (e.g., walnut, cashew, hazelnut, pecan, almond, etc.); sugarcane, coconut, date palm, olive, sugarcane, tea, coffee; trees that produce wood and pulp; vegetable crop plants such as legumes (e.g., beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, Brassicaceae (e.g., cabbage, kale, mustard, other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (e.g., cucumber, melon, summer squash, winter squash), edible alliums (e.g., onion, garlic, leek, shallot, chive), edible parts of Solanaceae (e.g., tomato, eggplant, potato, pepper, ground cherry), and edible parts of Chenopodiaceae (e.g., beet, chard, spinach, quinoa, amaranth); fruit crop plants including, but not limited to, apples, pears, citrus fruits (e.g., orange, lime, lemon, grapefruit, etc.), stone fruits (e.g., apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries.

[0026] In various embodiments, the polynucleotides described herein include naturally occurring nucleotides such as those found in DNA and RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides (for example, a synthetic polynucleotide consisting mainly of ribonucleotides but having one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides, or a synthetic polynucleotide consisting mainly of deoxyribonucleotides but having one or more terminal dideoxyribonucleotides). In certain embodiments, the polynucleotide includes non-standard nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publications 2011 / 0171287, 2011 / 0171176, 2011 / 0152353, 2011 / 0152346, and 2011 / 0160082, which are incorporated herein by reference. Exemplary examples include, but are not limited to, phosphodiester backbones of naturally occurring oligonucleotides or polynucleotides that can be partially or completely modified using nucleotide-nucleotide bond modifications of sphorothioates, phosphorodithioates, or methylphosphonates, modified nucleoside bases, or modified sugars, which can be used in oligonucleotide or polynucleotide synthesis, and which can be labeled with fluorescent moieties (e.g., fluorescein or rhodamine) or other labels (e.g., biotin).

[0027] Some embodiments relate to polynucleotides comprising 18 or more consecutive nucleotides having sequences that are approximately 95% to approximately 100% identical to DNA or a fragment of equivalent length of a target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or at least one segment of DNA or RNA complementary to either of the above. In some embodiments, the number of consecutive nucleotides is at least 18, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or more. In some embodiments, the number of consecutive nucleotides is greater than 18, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, A sequence of nucleotides consisting of at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 280, at least approximately 270, at least approximately 280, at least approximately 290, at least approximately 300, at least approximately 350, at least approximately 400, at least approximately 450, at least approximately 500, or more than 500.In some embodiments, the polynucleotide comprises at least 18, 19, 20, or 21 consecutive nucleotides (referring to at least 18, 19, 20, or 21 as used throughout means that any of these lower limits of the group can be individualized) having 100% identity with a DNA or equivalent-length fragment of the target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or at least one segment of DNA or RNA complementary to any of the above. In some embodiments, the polynucleotide is a single-stranded double-stranded nucleic acid (e.g., dsRNA) having 100% identity to DNA or a fragment of equivalent length to the target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or to an RNA transcript of either thereof, or to at least one segment of DNA or RNA complementary to either of the above; expressing as base pairs, such double-stranded nucleic acid comprises at least 18 consecutive, perfectly matching base pairs, or an RNA transcript of either thereof, or to at least one segment of DNA or RNA complementary to either of the above, corresponding to DNA or a fragment of equivalent length to the target gene having a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the length of each segment contained in the polynucleotide is longer than the length typical of naturally occurring regulatory small RNA molecules, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the polynucleotide, or the length of each segment contained within the polynucleotide, is shorter than the total length of the DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the total length of the polynucleotide is between about 50 and about 500 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides).In some embodiments, the polynucleotide is a dsRNA between about 100 and about 500 base pairs, such as a dsRNA of any of the dsRNA trigger lengths disclosed in Tables 1A, 1B, and 1C. Embodiments include polynucleotides expressed in the plant such as SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 50 9, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1 The present invention comprises RNA or its complement, which includes a segment having a sequence selected from the group consisting of 196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623. In some embodiments, the polynucleotide is expressed in a plant. In some embodiments, the polynucleotide is supplied locally to the surface of a plant or a lepidopteran pest.

[0028] Some embodiments relate to polynucleotides designed to regulate expression by inducing the regulation or suppression of a target gene of a Lepidoptera pest. In some embodiments, the polynucleotide may have a nucleotide sequence that is essentially identical or essentially complementary to the nucleotide sequence of a Lepidoptera pest target gene or cDNA (e.g., the target gene sequence group), or the sequence of RNA transcribed from a Lepidoptera pest target gene that may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules that regulate expression may be referred to herein as "polynucleotides", "polynucleotide triggers", "triggers", or "trigger species".

[0029] Effective polynucleotides of any size can be used alone or in combination in the various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to create a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for creating transgenic plants). In other embodiments, a mixture or pool of various polynucleotide triggers is used; in such cases, the triggers of the polynucleotides may be for a single target gene or multiple target genes.

[0030] As used herein, the term "isolated" refers to separating a molecule from other molecules to which it is bound in its original or natural state. Thus, the term "isolated" may refer to a DNA molecule that has been separated from other DNA molecules to which it is normally bound in its original or natural state. Such a DNA molecule may exist in a recombinant state, such as a recombinant DNA molecule. Thus, for example, as a result of recombinant techniques, a DNA molecule fused to a regulatory sequence or coding sequence that is not normally bound is considered isolated, whether it is integrated into the chromosome of a cell as a transgene or present together with other DNA molecules.

[0031] As used herein, the term "target gene sequence group" refers to the group of sequences consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623. As used herein, the term "trigger sequence group" refers to the group of sequences consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683.

[0032] In various embodiments, the lepidopteran pest is at least one insect species selected from the group consisting of the genus Spodoptera and the tribe Diamondback moths. An example of a Spodoptera species is S. frugiperda (fall armyworm). An example of a Plutella species is P. xylostella (diamondback moth). The lepidopteran pest may be at any of the developmental stages.

[0033] Some embodiments relate to polynucleotides designed to repress one or more genes ("target genes"). The term "gene" refers to any portion of nucleic acid that provides expression for a transcript or codes for a transcript. A "gene" may include, but is not limited to, a transcript coding region that may include a promoter region, a 5' untranslated region, an intron region, a 3' untranslated region, or a combination of these regions. In some embodiments, the target gene may include a coding sequence, a non-coding sequence, or both. In other embodiments, the target gene may have a sequence identical or complementary to messenger RNA; for example, in some embodiments, the target gene is a cDNA. In specific embodiments, the polynucleotide is designed to repress one or more target genes, each of which is coded by a DNA sequence selected from the target gene sequence set. In various embodiments, the polynucleotide is designed to repress one or more target genes, each of which is coded by a sequence selected from the target gene sequence set, and may be designed to repress multiple target genes from this set, or to target one or more different regions of these target genes. In one embodiment, the polynucleotide comprises 21 consecutive nucleotides having 100% identity with a DNA or equivalent-length fragment of the target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or multiple segments of DNA or RNA complementary to either of the above. In such a case, each segment may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide comprises multiple segments in a tandem or repeat sequence, where each segment comprises 21 consecutive nucleotides having 100% identity with a DNA or equivalent-length fragment of the target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or DNA or RNA complementary to either of the above.In some embodiments, the segments can originate from various regions of the target gene; for example, the segments can correspond to various exon regions of the target gene. In some embodiments, “spacer” nucleotides that do not correspond to the target gene can be optionally used between or adjacent to segments.

[0034] Further definitions are provided in the following sections.

[0035] II. To prevent the invasion of lepidopterans by contact with polynucleotides. Provided herein is a method for preventing lepidopteran pests from invading plants by exposing lepidopteran pests to polynucleotides comprising 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity or complementarity with DNA or a fragment of equivalent length of a target gene having a sequence selected from the group consisting of the target gene sequence group or the group consisting of the trigger sequence group, or an RNA transcript thereof, or at least one segment of DNA or RNA complementary to either of the above. In one embodiment, a method for preventing the invasion of lepidopteran pests into plants is described using sequence numbers: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 54 2, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 8 17, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 13 The method involves contacting a lepidopteran pest with a polynucleotide containing at least 18 consecutive nucleotides having 100% identity with a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of 08, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or an RNA transcript thereof, or DNA or RNA complementary to any of the above.In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is synthesized chemically or enzymatically, or produced by expression in microorganisms or plant cells. Embodiments include those in which the polynucleotide is dsRNA comprising a strand having a sequence selected from the trigger sequence group. The polynucleotide used in the method can be designed for multiple target genes. Related aspects of the present invention include isolated polynucleotides used in the method and modified lepidopteran-resistant plants provided by the method. In a specific embodiment, the polynucleotide therein is sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 102 3, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 14 This includes embodiments of a dsRNA consisting of a sequence selected from the group consisting of 22, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a complementary thereof.Other specific embodiments include embodiments in which the polynucleotide is a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539, or a combination thereof.

[0036] In some embodiments, the consecutive nucleotides have sequences that are approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to DNA or a target gene having sequences selected from the target gene sequence group, or an RNA transcript thereof, or a fragment of DNA or RNA of equivalent length complementary to either of the above. In some embodiments, the consecutive nucleotides are exactly (100%) identical to DNA or a target gene having sequences selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or a fragment of DNA or RNA of equivalent length complementary to either of the above. In some embodiments, the polynucleotides have approximately 95%, 96%, 97%, 98%, 99%, or 100% of all sequences to DNA or a target gene having sequences selected from the target gene sequence group or the trigger sequence group, or a fragment of DNA or RNA of equivalent length complementary to either of the above.

[0037] In one embodiment, the polynucleotides are sequence numbers 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815 ,817,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251,1263,1264,1265,1270,1275,1279,12 The polynucleotide comprises 21 consecutive nucleotides having 100% identity with a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of 83, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or an RNA transcript thereof, or at least one segment of DNA or RNA complementary to either of the above. In some embodiments, the polynucleotide includes one or more segments of 21 consecutive nucleotides having 100% identity with a corresponding fragment of the target gene, in addition to a “neutral” sequence (a sequence that does not have sequence identity or complementarity to the target gene), and therefore the polynucleotide as a whole has much lower overall sequence identity with the target gene.

[0038] The total length of the polynucleotide used in this method may be longer than 18 consecutive nucleotides and may include nucleotides added to DNA having a sequence selected from the group consisting of target gene sequences or trigger sequences, or to a sequence of about 95% to about 100% identity with a fragment of the target gene of equivalent length, or to an RNA transcript of either thereof, or to DNA or RNA complementary to either of the above. In other words, the total length of the polynucleotide may be longer than the length of a section or segment of the polynucleotide designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of target gene sequences. For example, the polynucleotide may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the polynucleotide may include additional nucleotides that are not specifically related to (have sequences that are not complementary or identical to) the target gene sequence group, the target gene having a sequence selected from the trigger sequence group, or an RNA transcript thereof, or the complementary DNA or RNA, for example, nucleotides that provide a stabilized second structure or convenience in cloning or manufacturing. In one embodiment, the polynucleotide may include 18 or more consecutive nucleotides having about 95% to about 100% identity with equivalent length fragments of the sequence selected from the group consisting of the target gene sequence group, the trigger sequence group, or an RNA transcript thereof, or additional nucleotides located immediately adjacent to one or more segments of the complementary DNA or RNA. In one embodiment, the polynucleotide may include one such segment having an additional 5'G or an additional 3'C or both adjacent to the segment.In another embodiment, the polynucleotide is a double-stranded RNA containing additional nucleotides to form an overhang, for example, a dsRNA containing two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to the sequence of consecutive nucleotides in DNA or a target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or in DNA complementary to either of the above. For example, in some embodiments, the polynucleotide comprises 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least two segments of DNA complementary to either of the above, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in DNA having a sequence selected from the group consisting of the target gene sequence group or the trigger sequence group, or in DNA complementary to either of the above.

[0039] The polynucleotides used in this method are provided by appropriate means known to those skilled in the art, and embodiments include those in which the polynucleotides are synthesized chemically or enzymatically (for example, by in vitro transcription such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or cell cultures (such as plant or insect cells grown in culture), produced by expression in plant cells, or produced by microbial fermentation.

[0040] In some embodiments, the polynucleotide used in this method is provided as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide used in this method is not part of an expression construct and lacks additional elements such as a promoter or terminator sequence. Such polynucleotides may be relatively short, e.g., between about 18 and about 300 nucleotides or between about 50 and about 500 nucleotides (in the case of a single-stranded polynucleotide), or single-stranded or double-stranded polynucleotides with about 18 to about 300 or about 50 to about 500 base pairs (in the case of a double-stranded polynucleotide). In some embodiments, the polynucleotide is a dsRNA with between about 100 and about 500 base pairs, such as a dsRNA of the length of one of the dsRNA triggers disclosed in Tables 1A, 1B, and 1C. Alternatively, the polynucleotide may be provided in a more complex construct, for example, as part of a recombinant expression construct, or may be included, for example, in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette, for expressing the gene of interest (e.g., an insecticidal protein).

[0041] Some embodiments relate to a method for preventing the invasion of lepidopteran pests into plants, comprising contacting lepidopteran pests with a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that is essentially identical or complementary to a comparable length of DNA fragment of a target gene selected from the group consisting of genes identified in the target gene sequence group. In some embodiments, the polynucleotide comprises dsRNA having a strand having a sequence selected from the group consisting of trigger sequences. In some embodiments, the present invention provides a method for preventing the invasion of lepidopteran pests into plants, comprising contacting lepidopteran pests with an effective amount of a solution comprising double-stranded RNA from the trigger sequence group, the solution further comprising one or more components selected from the group consisting of organosilicone surfactants or cationic lipids.

[0042] In various embodiments of the method described above, the contact comprises the application of a suitable composition containing the polynucleotide used in this method to the surface of a lepidopteran pest; such compositions can be provided, for example, as solids, liquids (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, in or on microbeads or other carrier particles, in films or coatings, or on or within a matrix, or as a seed treatment. The contact can exist in the form of a seed treatment or as a treatment of tubers or fragments of "seed potatoes" (for example, by dipping seed potatoes, coating seed potatoes, or spraying seed potatoes). Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art in the formulation of insecticides and seed treatments, can be optionally included in the composition. In some embodiments, the contact provides the polynucleotide in a composition further comprising one or more components selected from the group consisting of carriers, surfactants, cationic lipids (e.g., those disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In some embodiments, the contact provides the polynucleotide in a composition further comprising at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. In one embodiment, the contact provides the polynucleotide in a composition that can be ingested or otherwise absorbed internally by lepidopteran pests.

[0043] The combination of a specific polynucleotide used in this method (e.g., the polynucleotide trigger described in the examples) with one or more non-polynucleotide insecticides is expected to result in an accelerated improvement in the prevention or control of lepidopteran pest invasion compared to the effects obtained by polynucleotides alone or non-polynucleotide insecticides alone. In one embodiment, a composition containing one or more polynucleotides selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein, and one or more non-polynucleotide insecticides is found to result in an improved prevention or control effect of lepidopteran pest invasion.

[0044] III. Control of Lepidopteran invasion by providing dietary polynucleotides Another aspect of the present invention is a method for controlling the invasion of lepidopteran pests of plants, comprising providing in the diet of lepidopteran pests an agent comprising a polynucleotide having 18 or more consecutive nucleotides having about 95% to about 100% identity with respect to equivalent length fragments of DNA having sequences selected from the group consisting of target gene sequences and the group consisting of trigger sequences, wherein the agent functions to inhibit biological functions within the lepidopteran pest upon ingestion, thereby controlling the invasion by the lepidopteran pest. The polynucleotides are of equivalent length in each polynucleotide segment and corresponding DNA fragment, but can be longer than one or more segments contained in the polynucleotide. The polynucleotides used in the method can be designed for multiple target genes.The embodiment includes a dsRNA comprising a strand having a sequence selected from the trigger sequence group or a complementary strand thereof, or SEQ ID NOs: 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476 ,479,489,491,492,521,522,523,524,527,528,536,538,539,823,826,827,828,830,832,833,834,840,841,842,844,845,851,853,854,862,874,877,883,892,893,901,946,947,953,998,1011,1012,1017,1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, It contains a polynucleotide or RNA encoded by a sequence selected from the group consisting of 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683. In another embodiment, the agent comprises a polynucleotide or RNA, or a combination thereof, encoded by a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539.In one embodiment, a method for preventing the invasion of lepidopteran plant pests is described in SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533 ,542,545,546,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,596,602,611,612,620,665,666,672,717,730,731,736,737,738,740,741,742,743,749,757,760,761,763,766,808,809,810,8 14, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279 The invention includes providing a polynucleotide in the diet of a lepidopteran pest, which comprises a target gene having a nucleotide sequence selected from the group consisting of 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or a polynucleotide having a nucleotide sequence complementary to at least 18 consecutive nucleotides of RNA transcribed from the target gene. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is synthesized chemically or enzymatically, or produced by expression in microorganisms or by expression in plant cells. Related aspects of the invention include isolated polynucleotides used in the present method and modified lepidopteran-resistant plants provided by the method.

[0045] In various embodiments, the agent comprising polynucleotides comprises or is produced within microbial cells. For example, the agent may comprise or be produced within bacterial or yeast cells. In other embodiments, the agent comprising polynucleotides comprises or is produced within transgenic plant cells (e.g., plant cells that transiently express polynucleotides); such plant cells may be cells in a plant or cells grown in tissue culture medium or cell suspension.

[0046] In various embodiments, the polynucleotide-containing agent can be provided for feeding by lepidopteran pests in a form suitable for ingestion, such as a solid, liquid (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, on or in a matrix, or as a seed treatment. The polynucleotide-containing agent can be provided for feeding by lepidopteran pests by applying the agent to plants that are susceptible to invasion by lepidopteran pests, or by applying the agent to the seeds of such plants, for example by spraying, scattering, or coating the plants, or by applying it to soil trenches, or by applying it to artificial feeds. The polynucleotide-containing agent can be provided for dietary intake by lepidopteran pests in an artificial diet formulated to meet specific nutritional requirements for maintaining lepidopteran pests, wherein the artificial diet is supplemented with a certain amount of polynucleotides obtained from another source, such as chemical synthesis, or purified from microbial fermentation; this embodiment may be useful, for example, for determining the timing and amount of an effective polynucleotide treatment regimen. In some embodiments, the polynucleotide-containing agent is provided for dietary intake by lepidopteran pests in the form of plant cells or plant cell components, or microorganisms (e.g., bacteria or yeast) or microbial fermentation products, or in a synthetic or artificial diet. In one embodiment, the polynucleotide-containing agent is provided in the form of a feed ingested by lepidopteran pests. The agent containing polynucleotides can be provided in the form of a seed treatment or a treatment of tubers or tuber fragments of seed potatoes (for example, by dipping the seed potatoes, coating the seed potatoes, or spraying the seed potatoes) for feeding by lepidopteran pests. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art in the formulation of insecticides and seed treatments, can be included in the agent.In some embodiments, the polynucleotide-containing agent further comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids (e.g., those disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In some embodiments, the polynucleotide-containing agent further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids (pecdysteroidhyto), Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. Other proteins include plant-derived proteins described in Toxins (Basel). 2019 Jul 1;11(7). In some embodiments, the agent comprising a polynucleotide comprises at least one transplantable formulation selected from the group consisting of particles, pellets, or capsules transplanted into a plant; in such embodiments, the method comprises transplanting the transplantable formulation into a plant. In some embodiments, the agent comprising a polynucleotide comprises at least one in-furrow formulation selected from the group consisting of powder, granules, pellets, capsules, spray, or any other form suitable for application to a trench or furrow; in such embodiments, the method comprises in-furrow treatment using the in-furrow formulation. In some embodiments, the method comprises treatment of seeds of a Solanaceae plant, potato tubers, or fragments of potato tubers with the agent.

[0047] The combination of a specific polynucleotide used in the agent used in this method (e.g., the polynucleotide trigger described in the examples) and one or more non-polynucleotide insecticides is expected to result in an enhanced improvement in preventing or controlling lepidopteran pest invasion compared to the effects obtained by polynucleotides alone or non-polynucleotide insecticides alone. In one embodiment, a composition containing one or more polynucleotides selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein, and one or more non-polynucleotide insecticides, when provided to lepidopteran pests in their diet, is found to have an improved effect in preventing or controlling lepidopteran pest invasion.

[0048] In some embodiments, the polynucleotide used in this method is a dsRNA containing a segment having a sequence selected from the trigger sequence group, or a complementary segment thereof, where the polynucleotide is sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 44 2, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 94 7, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 13 It is coded by an array selected from the group consisting of 77, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683. In another embodiment, the agent comprises a polynucleotide or RNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539, or a combination thereof.

[0049] In some embodiments, the consecutive nucleotides have sequences that are at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to DNA or a target gene or an RNA transcript thereof having sequences selected from the target gene sequence group or the trigger sequence group, or to a DNA or RNA fragment of equivalent length complementary to either of the above. In some embodiments, the consecutive nucleotides are exactly (100%) identical to DNA or a target gene or an RNA transcript thereof having sequences selected from the target gene sequence group or the trigger sequence group, or to a DNA or RNA fragment of equivalent length complementary to either of the above. In some embodiments, the polynucleotides have at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% all of the sequences to DNA or a target gene or an RNA transcript thereof having sequences selected from the target gene sequence group or the trigger sequence group, or to a DNA or RNA fragment of equivalent length complementary to either of the above.In one embodiment, the polynucleotides are SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 11 For the corresponding fragment of a target gene having a DNA sequence selected from the group consisting of 32, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623 The polynucleotide comprises 21 consecutive nucleotides having 100% identity or at least one segment of complementary DNA; in some embodiments, the polynucleotide includes a "neutral" sequence (a sequence that does not have sequence identity or complementarity to the target gene) in addition to the segment of 21 consecutive nucleotides having 100% identity to the corresponding fragment of the target gene, and therefore the polynucleotide as a whole has much lower overall sequence identity to the target gene.

[0050] The polynucleotides used in this method are generally designed to repress one or more genes ("target genes"). In other embodiments, the target genes have sequences identical or complementary to messenger RNA, and in some embodiments, for example, the target genes are cDNA. In a particular embodiment, the polynucleotide is designed so that each target gene represses one or more target genes having DNA sequences selected from the group consisting of the target gene sequences. In various embodiments, the polynucleotide is designed so that each target gene represses one or more target genes having DNA sequences selected from the group consisting of the target gene sequences, and can be designed to repress multiple target genes from this group, or to target one or more different regions of these target genes. In one embodiment, the polynucleotide comprises multiple segments of 21 consecutive nucleotides having sequences that are 100% identical to DNA or a target gene, or an RNA transcript thereof, or an equivalent length fragment of DNA or RNA complementary to either of the above. In such cases, each segment may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide comprises a plurality of segments in a tandem or repeat sequence, where each segment comprises 21 consecutive nucleotides having a sequence of 100% identity to DNA or a fragment of the target gene of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript thereof, or DNA or RNA complementary to either of the above; the segments may originate from various regions of the target gene, for example, they may correspond to various exon regions of the target gene, and “spacer” nucleotides that do not correspond to the target gene may be optionally used between or adjacent to segments.

[0051] The total length of the polynucleotide used in this method may be longer than 18 consecutive nucleotides and may include nucleotides added to DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or to a sequence of nucleotides having about 95% to about 100% identity with a fragment of the target gene of equivalent length, or to an RNA transcript of either thereof, or to DNA or RNA complementary to either of the above. In other words, the total length of the polynucleotide may be longer than the length of a section or segment of the polynucleotide designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequence group or the trigger sequence group. For example, the polynucleotide may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the polynucleotide may include additional nucleotides that are not specifically associated with (have sequences that are not complementary to or identical to) DNA having sequences selected from the target gene sequence group, the trigger sequence group, or the target gene, or DNA complementary to either of them, such as nucleotides that provide a stabilized second structure or provide convenience in cloning or manufacturing. In one embodiment, the polynucleotide may include 18 or more consecutive nucleotides having sequences of about 95% to about 100% identity with equivalent length fragments of DNA having sequences selected from the target gene sequence group, the trigger sequence group, or the target gene, or additional nucleotides located immediately adjacent to one or more segments of DNA complementary to either of them. In one embodiment, the polynucleotide may include one such segment having an additional 5'G or an additional 3'C or both adjacent to the segment.In another embodiment, the polynucleotide is a double-stranded RNA containing additional nucleotides to form an overhang, for example, a dsRNA containing two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to the sequence of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a sequence of consecutive nucleotides in the target gene, or DNA complementary to either of these. For example, in some embodiments, the polynucleotide comprises 21 consecutive nucleotides having a sequence 100% identical to a fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or at least two segments of DNA complementary to either of these, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in the DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of these.

[0052] The polynucleotides used in this method are provided by appropriate means known to those skilled in the art. Embodiments include those in which the polynucleotides are synthesized chemically or enzymatically (for example, by in vitro transcription such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or cell cultures (such as cells of plants or insects grown in culture), produced by expression in plant cells, or produced by microbial fermentation.

[0053] In some embodiments, the polynucleotide used in this method is provided as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide used in this method is not part of an expression construct and lacks additional elements such as a promoter or terminator sequence. Such polynucleotides may be relatively short, e.g., between about 18 and about 300 nucleotides or between about 50 and about 500 nucleotides (in the case of a single-stranded polynucleotide), or single-stranded or double-stranded polynucleotides with about 18 to about 300 or about 50 to about 500 base pairs (in the case of a double-stranded polynucleotide). In some embodiments, the polynucleotide is a dsRNA with between about 100 and about 500 base pairs, such as a dsRNA of the length of one of the dsRNA triggers disclosed in Tables 1A, 1B, and 1C. Alternatively, the polynucleotide may be provided in a more complex construct, for example, as part of a recombinant expression construct, or may be included, for example, in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette, for expressing the gene of interest (e.g., an insecticidal protein).

[0054] IV. Control of Lepidopteran invasion by providing dietary RNA Another aspect of the present invention provides a method for causing death or inhibiting the growth of lepidopteran pest larvae by providing the larvae with at least one polynucleotide comprising at least 18, 19, 20, or 21 consecutive nucleotides, or at least one silencing element comprising RNA transcribed from the target gene, which is complementary to a target gene having a nucleotide sequence selected from the group of target gene sequences. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is synthesized chemically or enzymatically, or produced by expression in microorganisms or plant cells. In one embodiment, a method is provided to cause death or inhibit the growth of a lepidopteran pest larva, comprising providing the lepidopteran pest larva with the diet of the lepidopteran pest larva, wherein the target gene sequence is selected from the group of target gene sequences, and the ingestion of the RNA by the lepidopteran pest larva results in death or inhibiting the growth of the lepidopteran pest larva. Related aspects of the present invention provide an RNA comprising at least one silencing element, wherein the at least one silencing element is at least identical to or complementary to a fragment of a target gene of a lepidopteran pest larva, and wherein the target gene sequence is an RNA selected from the group of target gene sequences. The RNA may be longer than the silencing element or the silencing element it contains, although the lengths of each silencing element and the corresponding fragment of the target gene sequence are equivalent. The RNA used in the method can be designed for multiple target genes. Embodiments include dsRNA having a strand having a sequence selected from the trigger sequence group. In a related embodiment, a method is provided for causing death or reduced reproductive capacity in a lepidopteran pest, comprising providing the lepidopteran pest's diet at least one RNA containing at least one silencing element that is essentially identical or essentially complementary to a fragment of a target gene sequence of the lepidopteran pest larva, wherein the target gene sequence is selected from the target gene sequence group, the trigger sequence group, or DNA complementary to any of them, and the ingestion of the RNA by the lepidopteran pest results in death or reduced reproductive capacity in the lepidopteran pest. Related embodiments of the present invention include isolated RNA used in the method and modified lepidopteran-resistant plants provided by the method.

[0055] In various embodiments, the RNA-providing diet may include or be produced within microbial cells. For example, the RNA-providing diet may include or be produced within bacterial or yeast cells. In similar embodiments, the RNA-providing diet may include or be produced within transgenic plant cells (e.g., plant cells that transiently express polynucleotides); such plant cells may be plant cells, or cells grown in tissue culture medium or cell suspension.

[0056] In one embodiment, the RNA-providing diet is provided in the form of any plant that is susceptible to invasion by lepidopteran pests, wherein the RNA is contained in or on the plant. Such plants are stable transgenic plants that express RNA, non-transgenic plants that temporarily express RNA, or non-transgenic plants that have been treated with RNA, for example, by spraying or coating. Generally, stable transgenic plants generally contain recombinant structures that encode RNA integrated into their genome. The specific area of ​​interest is an embodiment in which the plant is a crop plant.

[0057] In various embodiments, the RNA-providing diet is provided in a form suitable for ingestion by lepidopteran pests, for example, as a solid, liquid (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in or on a film or coating, in or in a matrix, or as a seed treatment. The RNA-providing diet may be provided by applying the diet to plants that are susceptible to invasion by lepidopteran pests, for example, by spraying, scattering, coating, by applying a soil trench, or by applying it in an artificial diet. In one embodiment, the recombinant RNA-providing diet is provided in the form of a bait consumed by lepidopteran pests. The RNA-providing diet is an artificial diet formulated to meet specific nutritional requirements for maintaining lepidopteran pests, wherein the artificial diet may be supplemented with a certain amount of RNA obtained from another source such as chemical synthesis or purified from microbial fermentation; this embodiment may be useful, for example, for determining the timing and amount of an effective polynucleotide treatment regimen. In some embodiments, the polynucleotide-containing agent is provided for feeding by lepidopteran pests in the form of plant cells or in plant cell components or in microorganisms (such as bacteria or yeast) or microbial fermentation products or in synthetic or artificial diets. In one embodiment, the polynucleotide-containing agent is provided for feeding by lepidopteran pests in the form of a feed consumed by lepidopteran pests. The polynucleotide-containing agent can be provided for feeding by lepidopteran pests in the form of a seed treatment or in the form of a treatment of seed tubers or tuber fragments (e.g., by dipping seed tubers, coating seed tubers, or spraying seed tubers). Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art regarding the formulation of insecticides and seed treatment, can be included in the aforementioned feed.In some embodiments, the RNA-providing diet further comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids (e.g., those disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In some embodiments, the RNA-providing diet further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins. In some embodiments, the RNA-providing diet comprises at least one transplantable formulation selected from the group consisting of particles, pellets, or capsules transplanted into a plant; in such embodiments, the method comprises transplanting the transplantable formulation into a plant. In some embodiments, the RNA-providing diet comprises at least one in-furrow formulation selected from the group consisting of powder, granules, pellets, capsules, spray, or any other form suitable for application to a trench or furrow; in such embodiments, the method comprises in-furrow treatment using the in-furrow formulation. In some embodiments, the method comprises treatment of seeds of a Solanaceae plant, potato tubers, or fragments of potato tubers with the formulation.

[0058] The combination of a specific RNA used in this method (e.g., the dsRNA trigger described in the examples) with one or more non-polynucleotide insecticides is expected to result in a more advanced improvement in preventing or controlling lepidopteran pest invasion compared to the effects obtained with RNA alone or with non-polynucleotide insecticides alone. In one embodiment, a composition containing one or more RNAs and one or more non-polynucleotide insecticides selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein shows an improved effect in preventing or controlling lepidopteran pest invasion.

[0059] The RNA used in this method may be single-stranded (ss) or double-stranded (ds). Embodiments of the method may be at least one selected from the group consisting of sense single-stranded RNA (ssRNA), antisense single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA), or mixtures of these types; any mixture of these types may also be used. In one embodiment, a double-stranded DNA / RNA hybrid is used as the RNA. The RNA may contain components other than standard ribonucleotides; for example, in one embodiment, the RNA contains terminal deoxyribonucleotides.

[0060] The RNA comprises at least one silencing element, wherein the silencing element is essentially identical (as an RNA equivalent) or essentially complementary to a fragment of a target gene of a lepidopteran insect larva, wherein the target gene sequence is selected from the group of target gene sequences. In some embodiments, the silencing element has a sequence that is at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or complementary to a fragment of DNA of equivalent length having a sequence selected from the group of target gene sequences. In some embodiments, the silencing element is exactly (100%) identical or exactly (100%) complementary (as an RNA equivalent) to a fragment of DNA of equivalent length having a sequence selected from the group of target gene sequences, a sequence selected from the group of trigger sequences, or a fragment of DNA complementary to any of them. In some embodiments, the RNA containing the silencing element has all sequences that are approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or complementary to a DNA fragment having sequences selected from the group of target gene sequences.

[0061] In some embodiments, the silencing element comprises at least one segment of 18 or more consecutive nucleotides having a sequence that is about 95% to about 100% identical or complementary to a comparable fragment of the target gene. In some embodiments, the silencing element comprises at least one segment of DNA complementary to either of these sequences, comprising 18 or more consecutive nucleotides having a sequence that is about 95% to about 100% identical or complementary to a comparable fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the silencing element includes 18 or more segments of consecutive nucleotides, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or even more.In some embodiments, the silencing elements are more than 18, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, and fewer It contains at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 270, at least 280, at least 290, at least 300, at least 350, at least 400, at least 450, at least 500, or more than 500 consecutive nucleotides. In certain embodiments, the silencing element includes at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of DNA complementary to any of the above, having sequences that are 100% identical to DNA or equivalent-length fragments of the target gene, which have sequences selected from the target gene sequence group or the trigger sequence group.In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having a single strand containing at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of DNA complementary to any of them, having a sequence that is 100% identical to DNA or a fragment of the target gene of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group; in certain embodiments, the length of each silencing element contained in the RNA is longer than the length typical of naturally occurring regulatory small RNAs, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the RNA, or the length of each silencing element contained in the RNA, is shorter than the total length of the sequence of interest (DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group). In some embodiments, the total length of the RNA is between about 50 and about 500 nucleotides (in the case of a single-stranded polynucleotide) or base pairs (in the case of a double-stranded polynucleotide). In some embodiments, the RNA is a dsRNA of about 100 to about 500 base pairs, for example, a dsRNA of any length of the dsRNA triggers disclosed in Tables 1A, 1B, and 1C.In this embodiment, the RNA is sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524 ,527,528,536,538,539,823,826,827,828,830,832,833,834,840,841,842,844,845,851,853,854,862,874,877,883,892,893,901,946,947,953,998,1011,1012,1017,1018,1019,1021,1022,1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 142 The segment or combination thereof includes sequences selected from the group consisting of 0, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683. In another embodiment, the agent comprises a polynucleotide or RNA, or a combination thereof, encoded by a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539.

[0062] The RNA used in this method is generally designed to repress one or more genes ("target genes"). In some embodiments, the target genes may include coding sequences, non-coding sequences, or both. In other embodiments, the target genes have sequences identical or complementary to the messenger RNA; for example, in some embodiments, the target genes are cDNA. In certain embodiments, the RNA is designed to repress one or more target genes, each having a DNA sequence selected from the target gene sequence group or the trigger sequence group. In various embodiments, the RNA is designed to repress one or more genes, each having a sequence selected from the target gene sequence group or the trigger sequence group, and can be designed to repress multiple genes from this group, or to target one or more different regions of these genes. In one embodiment, the RNA comprises a plurality of silencing elements, each comprising 21 consecutive nucleotides having a sequence that is 100% identical or 100% complementary to a comparable length of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In such a case, each silencing element may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the RNA may include a plurality of silencing elements in tandem or repeat sequences, where each silencing element includes 21 consecutive nucleotides having a sequence that is 100% identical or 100% complementary to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or DNA complementary to either of these; the segments may originate from various regions of the target gene, for example, the segments may correspond to various exon regions of the target gene, and “spacer” nucleotides that do not correspond to the target gene may be optionally used between or adjacent to the segments.

[0063] The total length of the RNA may be more than 18 consecutive nucleotides and may include nucleotides, or DNA complementary to either, in addition to a silencing element having a sequence of approximately 95% to approximately 100% identity or complementarity to DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a fragment of the target gene of equivalent length. In other words, the total length of the RNA may be longer than the length of a silencing element designed to repress one or more target genes having a DNA sequence selected from the group consisting of the target gene sequence group. For example, the RNA may have nucleotides adjacent to the “active” silencing element of at least one segment of 18 or more consecutive nucleotides that represses the target gene, or may include “spacer” nucleotides between the active segments, or may have additional nucleotides at the 5' end, or at the 3' end, or at both the 5' and 3' ends. In one embodiment, the RNA includes additional nucleotides that are not specifically associated with (have sequences that are not complementary or identical to) the DNA or target gene having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of them, for example, nucleotides that provide a stabilized second structure or provide convenience in cloning or manufacturing. In one embodiment, the RNA includes 18 or more consecutive nucleotides having a sequence that is about 95% to about 100% identical or complementary to a comparable length fragment of the DNA or target gene having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of them, or additional nucleotides located immediately adjacent to one or more silencing elements. In one embodiment, the RNA includes one such silencing element having an additional 5'G or an additional 3'C or both adjacent to the silencing element. In another embodiment, the RNA is a double-stranded RNA including additional nucleotides to form an overhang, for example, a dsRNA including two deoxyribonucleotides to form a 3' overhang.Therefore, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a contiguous nucleotide fragment in DNA or a target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or in DNA complementary to either of the above. For example, in some embodiments, the RNA comprises 21 contiguous nucleotides having a sequence that is 100% identical to a fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least two silencing elements of DNA complementary to either of the above, wherein (1) the at least two silencing elements are separated by one or more spacer nucleotides, or (2) the at least two silencing elements are arranged in an order different from the order in which the corresponding fragments exist in DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or in DNA complementary to either of the above.

[0064] In some embodiments, the RNA consists of naturally occurring ribonucleotides. In certain embodiments, the RNA includes components other than ribonucleotides, such as synthetic RNA that is mainly composed of ribonucleotides but has one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain embodiments, the RNA includes non-standard nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the RNA includes chemically modified nucleotides.

[0065] The RNA used in this method is provided by appropriate means known to those skilled in the art. Embodiments include the RNA being synthesized chemically or enzymatically (e.g., by in vitro transcription such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or cell cultures (such as plant or insect cells grown in a culture medium), produced by expression in plant cells, or produced by microbial fermentation.

[0066] In some embodiments, the RNA is provided as isolated RNA that is not part of an expression construct and lacks additional elements such as a promoter or terminator sequence. Such RNA is relatively short, e.g., about 18 to about 300, or about 50 to about 500 (for single-stranded RNA), or about 18 to about 300, or about 50 to about 500 (for double-stranded RNA) nucleotides of single-stranded or double-stranded RNA. Alternatively, the RNA may be provided in a more complex structure, for example, as part of a recombinant expression component, or it may be included in a recombinant vector, for example, in a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression components or vectors are designed to include additional elements, such as an expression cassette for expressing additional RNA encoding an aptamer or ribozyme or a gene of interest (e.g., an insecticidal protein).

[0067] V. A method for providing a plant having improved resistance to lepidopteran invasion, and the plant, plant parts, and seeds provided in this manner. Some embodiments relate to a method for providing plants with improved resistance to lepidopteran pest invasion, comprising providing a plant with at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that is essentially identical or complementary to a fragment of a target gene selected from the group consisting of genes identified in the target gene sequence set. In one embodiment, the present invention provides a method for providing a plant with improved resistance to lepidopteran pest invasion, comprising providing a plant with at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 consecutive nucleotides of a target gene or RNA transcribed from a target gene, wherein the target gene is selected from the gene identified in the target gene sequence set or RNA transcribed from a target gene. Embodiments of gene-targeting sequences are identified by the sequence numbers in Tables 1A, 1B and 1C and include sequences that target genes having sequences selected from the group consisting of the target gene sequence set, as well as sequences that target related genes including related genes, such as orthologues from related pests, for example, from other lepidopteran pests. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is synthesized chemically or enzymatically, or produced by expression in microorganisms or plant cells. In some embodiments, the polynucleotide comprises 18 or more consecutive nucleotides that are essentially identical or complementary to the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the polynucleotide is a dsRNA having a chain with a sequence selected from the trigger sequence group, or a complement thereof. In some embodiments, the polynucleotide comprises a dsRNA having a chain with a sequence selected from the trigger sequence group.

[0068] In a related embodiment, the present invention relates to a plant having improved resistance to lepidopteran pest invasion, provided by expressing in a plant at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that is essentially identical or complementary to a fragment of a target gene of comparable length selected from the group of genes identified in the target gene sequence group, the resulting plant having improved resistance to lepidopteran pest invasion compared to a control plant in which the polynucleotide is not expressed. In a related embodiment, the present invention relates to a plant having improved resistance to lepidopteran pest invasion, provided by expressing in a plant at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence of approximately 95% to approximately 100% identity or complementarity to a fragment of a target gene selected from the group of genes identified in the target gene sequence group, the resulting plant having improved resistance to lepidopteran pest invasion compared to a control plant in which the polynucleotide is not expressed.

[0069] In yet another aspect of the present invention, the present invention relates to seeds or reproductive parts (in particular, transgenic offspring seeds or reproductive parts) produced by plants having improved resistance to lepidopteran pest invasion, provided by this method. Also intended are commodity products produced by plants having improved resistance to lepidopteran pest invasion, provided by this method, and commodity products produced from transgenic offspring seeds or reproductive parts of such plants.

[0070] Another aspect of the present invention provides a method for providing plants with improved resistance to lepidopteran pest invasion, comprising topically applying to plants a composition comprising at least one polynucleotide having 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity with a target gene or DNA fragment having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these, such that plants treated with the polynucleotide-containing composition exhibit improved resistance to lepidopteran pest invasion compared to untreated plants. In one embodiment, the at least one polynucleotide comprises 18 or more consecutive nucleotides that are essentially identical to a DNA fragment of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. The length of each segment of the target gene and the corresponding fragment of the polynucleotide is equivalent, but the length may be longer than the segment or the multiple segments it comprises.In one embodiment, the present invention relates to Sequence IDs 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1 104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1 The present invention provides a method for providing plants with improved resistance to lepidopteran pest invasion, comprising topically applying to a plant a composition comprising at least 18 consecutive nucleotide sequences of a target gene having a nucleotide sequence selected from the group consisting of 318, 1320, 1564, 1565, 1569, 1622, and 1623, or at least one polynucleotide comprising RNA transcribed from the target gene.In one embodiment, the present invention provides a method for providing a plant with improved resistance to lepidopteran pest invasion, comprising topically applying a composition containing at least one polynucleotide to a plant in such a manner that an effective amount of polynucleotide is ingested by lepidopteran pests that feed on the plant, wherein the polynucleotide is SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 30 1, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 71 7, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1 It contains at least 18 consecutive nucleotides complementary to a target gene having a nucleotide sequence selected from the group consisting of 196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or RNA transcribed from the target gene.

[0071] The polynucleotides used in this method can be designed for multiple target genes. Embodiments include the composition comprising a dsRNA having a chain containing a sequence selected from the group comprising the trigger sequence group. Related aspects of the present invention include compositions for topical application, isolated polynucleotides used in the method, and plants having improved lepidopteran resistance provided by this method.

[0072] As used throughout this Specification, “topical application” means application to the surface or external of a target, such as application to the surface or external of a plant, such as application to the surface of a part of a plant, such as leaves, stems, flowers, fruits, shoots, roots, seeds, tubers, flowers, anthers, pollen, etc., or application to the whole plant, or application to the surface or external of a plant, such as application to the above-ground or underground parts of a plant. Topical applications can be carried out on non-living surfaces, such as application to soil, or application to a surface or matrix on which lepidopteran pests can thereby come into contact with the polynucleotide. In various embodiments of the method, a composition comprising at least one polynucleotide is applied topically to a plant in a suitable form, such as, for example, as a solid, liquid (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, or on or in a matrix, or as a seed treatment. In some embodiments of the above method, the polynucleotide-containing composition is applied locally to the above-ground parts of a plant, for example, by spraying or smearing it on the leaves, stems, and flowering parts of the plant.

[0073] Embodiments of the method include local application by foliar spraying (e.g., spraying a liquid polynucleotide-containing composition onto the leaves of a Solanaceae plant) or foliar application (e.g., applying a polynucleotide-containing composition in the form of a powder or carrier particles to a crop). In other embodiments, the polynucleotide-containing composition is applied locally to the underground parts of a plant, such as roots, by means of soil drenching, for example. In other embodiments, the polynucleotide-containing composition is applied locally to seeds grown in a plant. The local application may take the form of local treatment of the fruit of a crop plant or seeds obtained from the fruit of a crop plant, or the form of local treatment of tubers or tuber fragments (e.g., by dipping, coating, or spraying the tubers). The polynucleotide-containing composition may optionally contain suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of insecticide formulation and seed treatment.

[0074] In some embodiments, the polynucleotide-containing composition is at least one locally transplantable formulation selected from the group consisting of particles, pellets, or capsules locally transplanted to a plant; in such embodiments, the method includes locally transplanting the locally transplantable formulation to a plant. In some embodiments, the polynucleotide-containing composition is at least one in-furrow formulation selected from the group consisting of powder, granules, pellets, capsules, spray, or drench, or any other form suitable for local application to a furrow; in such embodiments, the method includes in-furrow treatment with the in-furrow formulation. In one embodiment, the polynucleotide-containing composition can be ingested by lepidopteran pests or otherwise absorbed internally. For example, the polynucleotide-containing composition can be in the form of bait. In some embodiments, the polynucleotide-containing composition further comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids (for example, disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In one embodiment, the composition further comprises a nonionic organosilicon surfactant, such as a SILWET® brand surfactant, currently available from Momentive Performance Materials, Albany, NY, for example, a SILWET L-77® brand surfactant having CAS number 27306-78-1 and EPA number CAL.REG.No.5905-50073-AA. In some embodiments, the topically applied composition further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein.Alternatively, such additional components or insecticides may be provided separately, for example, by separate topical applications or transgenic expression in plants. Alternatively, the plants may be treated topically with separate (before, after, or in between) applications of the polynucleotide-containing composition in addition to a substance that improves the effectiveness of the polynucleotide-containing composition. For example, plants may be sprayed with a second topical application of the polynucleotide-containing composition (and vice versa), following a first topical application of a solution containing a nonionic organosilicone surfactant, such as a surfactant of the SILWET® brand, for example, a surfactant of the SILWET L-77® brand.

[0075] It is predicted that a combination of a specific polynucleotide useful in the polynucleotide-containing composition (e.g., the polynucleotide trigger described in the above examples) and one or more non-polynucleotide insecticides will result in an accelerated improvement in preventing or controlling the invasion of lepidopteran pests compared to the effects obtained by the polynucleotide alone or the non-polynucleotide insecticide alone. In one embodiment, a composition containing one or more non-polynucleotide insecticides, when provided to lepidopteran pests in their diet, is found to have an improved effect in preventing or controlling the invasion of lepidopteran pests. In one embodiment, the polynucleotide-containing composition is provided as a transgenic plant expressing one or more polynucleotides and one or more genes, which encode a non-polynucleotide insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein, wherein the transgenic plant is found to exhibit improved resistance to invasion by lepidopteran pests.

[0076] The polynucleotides useful in the polynucleotide-containing composition are provided by appropriate means known to those skilled in the art. Embodiments include those in which the polynucleotides are synthesized chemically or enzymatically (e.g., by in vitro transcription such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or cell culture media (e.g., plant or insect cells grown in culture media), produced by expression in plant cells, or produced by microbial fermentation.

[0077] In many embodiments, the polynucleotides useful in the polynucleotide-containing composition are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides useful in the polynucleotide-containing composition are not part of an expression construct and lack additional elements such as promoter or terminator sequences. Such polynucleotides may be relatively short, e.g., single-stranded or double-stranded polynucleotides of about 18 to about 300 or about 50 to about 500 nucleotides (in the case of single-stranded polynucleotides), or about 18 to about 300 or about 50 to about 500 base pairs (in the case of double-stranded polynucleotides). In some embodiments, the polynucleotides are dsRNAs of between about 100 and about 500 base pairs, such as dsRNAs of the length of any of the dsRNA triggers disclosed in Tables 1A, 1B, and 1C. Alternatively, the polynucleotides may be provided in a more complex construct, for example, as part of a recombinant expression construct, or may be included, for example, in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette, for expressing the gene of interest (e.g., an insecticidal protein).

[0078] In the polynucleotide-containing composition, the useful polynucleotides consist of 18 or more consecutive nucleotides having about 95% to about 100% identity with a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In one embodiment, the polynucleotide comprises 18 or more consecutive nucleotides that are essentially identical or complementary to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the consecutive nucleotides have sequences of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% with respect to a fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the polynucleotide has sequences that are approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to DNA having sequences selected from the target gene sequence group, the trigger sequence group, or a DNA fragment complementary to either of them.

[0079] The polynucleotide useful in the polynucleotide-containing composition includes at least one segment of a continuous nucleotide sequence of 18 or more nucleotides, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or even more. In some embodiments, the segments are more than 18 consecutive segments, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130 , containing at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 280, at least approximately 270, at least approximately 280, at least approximately 290, at least approximately 300, at least approximately 350, at least approximately 400, at least approximately 450, at least approximately 500, or more than 500 consecutive nucleotides. In certain embodiments, the polynucleotide comprises at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of DNA complementary to any of them, having 100% identity with DNA or a fragment of the target gene of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group.In certain embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA) having a single strand containing at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of DNA complementary to either of these, which are expressed as base pairs, each containing at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs corresponding to a DNA or target gene having a sequence selected from the target gene sequence group, the trigger sequence group, or a fragment of DNA of equivalent length complementary to either of the above. In certain embodiments, the length of each segment contained in the polynucleotide is longer than the length typical of naturally occurring regulatory small RNA molecules, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is shorter than the total length of the sequence of interest (DNA or target gene having a sequence selected from a group of target gene sequences). In some embodiments, the total length of the polynucleotide is between about 50 and about 500 nucleotides (in the case of a single-stranded polynucleotide) or base pairs (in the case of a double-stranded polynucleotide). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 500 base pairs, such as a dsRNA of any length disclosed in Tables 1A, 1B, and 1C for the dsRNA triggers.In the several embodiments, the polynucleotides are SEQ ID NOs: 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 5 23, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 102 3, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1 dsRNA, or a combination thereof, encoded by a sequence selected from the group consisting of 422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683. In another embodiment, the agent comprises a polynucleotide or RNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539, or a combination thereof.

[0080] Locally applied polynucleotides are generally designed to repress one or more genes ("target genes"). Such target genes include coding sequences, non-coding sequences, or both. In certain embodiments, the polynucleotide is designed to repress one or more target genes, each of which has a DNA sequence selected from the group comprising the target gene sequences. In various embodiments, the locally applied polynucleotide is designed to repress one or more genes, each of which has a sequence selected from the group comprising the target gene sequences, and can be designed to repress multiple genes from this group, or to target one or more different regions of these genes. In one embodiment, the locally applied polynucleotide comprises multiple sections or segments, each of which comprises 21 consecutive nucleotides having a sequence that is 100% identical to a comparable length fragment of DNA having a sequence selected from the target gene sequences, the trigger sequence group, or at least one segment of DNA complementary to either of these. In such a case, each section may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the locally applied polynucleotide may include multiple sections in a tandem or repeat sequence, where each section includes at least one segment of 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of DNA of equivalent length having a sequence selected from a group of target gene sequences.

[0081] The total length of the locally applied polynucleotide can be longer than 18 or more consecutive nucleotides and may include consecutive nucleotides having approximately 95% to approximately 100% identity with a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or nucleotides added to DNA complementary to either of these. In other words, the total length of the locally applied polynucleotide can be longer than the length of a section or segment of polynucleotide designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequence group. For example, the locally applied polynucleotide may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the locally applied polynucleotide includes additional nucleotides that are not specifically related to (have non-complementary or non-identical) the DNA or target gene having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of them, such as nucleotides that provide a stabilized second structure or provide convenience in cloning or manufacturing.

[0082] In one embodiment, the locally applied polynucleotide includes additional nucleotides located adjacent to one or more segments of 18 or more consecutive nucleotides having a sequence that is approximately 95% to approximately 100% identical or complementary to a DNA or equivalent length fragment of the target gene having a sequence selected from the group consisting of the target gene sequences. In one embodiment, the locally applied polynucleotide includes one such segment having an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the locally applied polynucleotide is a double-stranded RNA containing additional nucleotides to form an overhang, for example, a dsRNA containing two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire locally applied polynucleotide is not 100% identical or complementary to a consecutive nucleotide in the target gene having a sequence selected from the group consisting of the target gene sequences, the group consisting of the trigger sequences, or a fragment of DNA complementary to either of these. For example, in some embodiments, the locally applied polynucleotide comprises at least two segments of DNA complementary to either of the following 21 consecutive nucleotides having sequences of 100% identity to the fragments of the target gene sequence group, the DNA having sequences selected from the trigger sequence group, and where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in the DNA complementary to the target gene sequence group, the DNA having sequences selected from the trigger sequence group, or either of the following.

[0083] In a related embodiment, the present invention relates to plants having improved resistance to lepidopteran pest invasion, provided by a method comprising topically applying to plants a composition comprising a polynucleotide having 18 or more consecutive nucleotides having sequences of about 95% to about 100% identity with respect to equivalent length fragments of DNA having sequences selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these, thereby exhibiting improved resistance to lepidopteran pest invasion compared to untreated plants.

[0084] One embodiment involves a dsRNA trigger having a sequence selected from the trigger sequence group, or its complement, applied to the plant, or to seeds grown in the plant (or, if the plant is a potato plant, to seed tubers grown in the potato plant), or sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 4 52, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1 019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, 144 A crop plant having improved resistance to lepidopteran pest invasion compared to a control plant, provided by locally applying a dsRNA trigger encoded by a sequence selected from the group consisting of 7, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a combination thereof.In another embodiment, the agent comprises a polynucleotide or RNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, and 539, or a combination thereof. In yet another embodiment, the present invention relates to seeds or fertile parts (in particular, transgenic offspring seeds or fertile parts) produced by a plant having improved resistance to lepidopteran pest invasion, provided by this method. Also intended are commodity products produced by a plant having improved resistance to lepidopteran pest invasion, provided by this method, and commodity products produced from transgenic offspring seeds or fertile parts of such plants.

[0085] VI. Insecticidal compositions for controlling lepidopteran pests Another aspect of the present invention provides an insecticidal composition for controlling lepidopteran pests comprising an insecticidal effect amount of at least one RNA comprising 18 or more consecutive nucleotides that are essentially identical or complementary to a target gene or DNA fragment having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In this context, “controlling” includes, but is not limited to, the induction of physiological or behavioral changes in lepidopteran pests (adults or larvae), such as inhibition of growth, increased mortality rates, decreased reproductive capacity, decreased or cessation of feeding behavior or movement, or decreased or cessation of development at the metamorphosis stage. “Insecticidal effective” means, for example, effective in inducing physiological or behavioral changes in lepidopteran pests (adults or larvae), such as inhibition of growth, increased mortality rates, decreased or reduced reproductive capacity, decreased or cessation of feeding behavior or movement, or decreased or cessation of development at the metamorphosis stage. In some embodiments, the application of an insecticidal amount of RNA to plants improves their resistance to invasion by lepidopteran pests. The RNA is of similar length to each segment and corresponding fragment of the target gene, but can be longer than a segment or the multiple segments it contains. The RNA used in the method can be designed for multiple target genes. Embodiments include an insecticidal amount of polynucleotides comprising at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group of target gene sequences or RNA transcribed from the target gene; Alternatively, at least one polynucleotide in an insecticidal amount comprising at least one silencing element complementary to at least 21 consecutive nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene has a nucleotide sequence selected from the group of target gene sequences; Alternatively, Sequence IDs 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822 ,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251,1263,1264,1265,1270,1275,1279,1283,1290,1308,13 At least one insecticidal amount of RNA having a target gene having a nucleotide sequence selected from the group consisting of 10, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or at least one segment of RNA transcribed from the target gene that is identical to or complementary to at least 18, 19, 20, or 21 consecutive nucleotides; Alternatively, RNA molecules that cause death or inhibit growth in lepidopteran pests when ingested or come into contact with them, where the RNA molecules are SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 5 05, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, A target gene having a nucleotide sequence selected from the group consisting of 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or containing at least 18, 19, 20, or 21 consecutive nucleotides complementary to the RNA transcribed from the target gene; Alternatively, an insecticidal double-stranded RNA molecule that causes death or inhibits growth in lepidopteran pests when ingested or come into contact with them, wherein at least one strand of the insecticidal double-stranded RNA molecule contains 21 consecutive nucleotides complementary to a target gene or RNA transcribed from the target gene, wherein the target gene is sequence numbers 22, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44 ,47,87,95,99,103,301,309,318,319,320,321,323,324,328,341,343,346,356,358,359,503,504,505,506,509,510,530,532,533,542,545,546,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,5 96, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, Does it have a sequence selected from the group consisting of 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623? Alternatively, it comprises at least one insecticidal amount of double-stranded RNA, including a sequence selected from the trigger sequence group. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is synthesized chemically or enzymatically, produced by expression in microorganisms, or produced by expression in plant cells. Embodiments include sequences selected from the trigger sequence group (target sequence group), or, in more specific embodiments, sequences 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1 023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, The present invention comprises an insecticidal composition containing dsRNA having a sequence selected from the group consisting of 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a combination thereof, or a complement thereof.

[0086] In various embodiments, the insecticidal composition for controlling lepidopteran pests exists in at least one form selected from the group consisting of solids, liquids (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated, or microencapsulated formulations, and exists in or on microbeads or other carrier particles, in a film or coating, on or in a matrix, or as a seed treatment. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of insecticide formulation and seed treatment, may be optionally included in the polynucleotide-containing composition. The lepidopteran pests to be controlled are generally invading plant pests. In some embodiments, the insecticidal composition comprises at least one transplantable formulation selected from the group consisting of particles, pellets, or capsules transplanted into a plant; in such embodiments, the method comprises transplanting the transplantable formulation into a plant. In some embodiments, the insecticidal composition is at least one in-furrow formulation selected from the group consisting of powder, granules, pellets, capsules, sprays, or any other form suitable for application to a trench or furrow; in such embodiments, the method includes treatment of a furrow with the in-furrow formulation. In one embodiment, the insecticidal composition may be ingested by lepidopteran pests or otherwise absorbed internally. For example, the insecticidal composition may exist in the form of a bait. In some embodiments, the insecticidal composition further comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids (for example, disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators.In one embodiment, the insecticidal composition further comprises a nonionic organosilicone surfactant, such as a surfactant of the SILWET® brand, currently available from Momentive Performance Materials, Albany, NY, for example, a surfactant of the SILWET L-77® brand, having CAS number 27306-78-1 and EPA number CAL.REG.No.5905-50073-AA. In some embodiments, the insecticidal composition further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. Alternatively, such additional components or insecticides can be provided separately, for example, by separate topical application or transgenic expression in plants. Alternatively, the plants may be treated topically with the insecticidal composition and with separate applications (before, after, or in between) of substances that improve the effectiveness of the insecticidal composition. For example, the plants may be sprayed by topical application of a first solution containing a nonionic organosilicone surfactant, such as a surfactant of the SILWET® brand, for example, a surfactant of the SILWET L-77® brand, and then sprayed by topical application of the second insecticidal composition (and vice versa).

[0087] The combination of a specific RNA used in this method (e.g., the dsRNA trigger described in the examples) with one or more non-polynucleotide insecticides is expected to result in an enhanced improvement in preventing or controlling lepidopteran pest invasion compared to the effects obtained with RNA alone or with non-polynucleotide insecticides alone. In one embodiment, the insecticidal composition containing one or more RNAs and one or more non-polynucleotide insecticides selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein shows an improved effect in preventing or controlling lepidopteran pest invasion.

[0088] In various embodiments, the insecticidal composition comprises or is produced within microbial cells. For example, the insecticidal composition may comprise or be produced within bacterial or yeast cells. In similar embodiments, the insecticidal composition comprises or is produced within transgenic plant cells (e.g., plant cells that transiently express polynucleotides), and such plant cells may be cells in a plant or cells grown in tissue culture medium or cell suspension.

[0089] The insecticidal composition may be provided for feeding by lepidopteran pests by applying the composition to plants or surfaces that are susceptible to invasion by lepidopteran pests, for example by spraying, scattering, or coating plants, plant seeds, or seed potatoes, or by applying soil drenches, or by drenching, or by providing it in artificial feed. The insecticidal composition may be provided for feeding by lepidopteran pests in artificial feed formulated to meet specific nutritional requirements for maintaining lepidopteran pests, wherein the artificial feed is supplemented with a certain amount of RNA obtained from another source such as chemical synthesis or purified from microbial fermentation; this embodiment may be useful, for example, for determining the timing and amount of an effective RNA treatment regimen. In some embodiments, the insecticidal composition may be provided for feeding by lepidopteran pests in the form of plant cells, or in plant cell components, or in microorganisms (e.g., bacteria or yeast) or microbial fermentation products, or in synthetic feed. In one embodiment, the insecticidal composition is provided in the form of bait ingested by lepidopteran pests. The insecticidal composition can also be provided in the form of a treatment for seeds (or seed potatoes) for feeding by lepidopteran pests.

[0090] In one embodiment, the insecticidal composition is provided in the form of any plant that is susceptible to invasion by lepidopteran pests, wherein the RNA is contained in or on the plant. Such a plant is a stable transgenic plant that expresses the RNA, or a non-transgenic plant that temporarily expresses the RNA or has been treated with the RNA, for example, by spraying or coating. Stable transgenic plants generally contain recombinant structures that encode RNA integrated into their genome.

[0091] The RNA useful in the insecticidal composition may be single-stranded (ss) or double-stranded (ds). Embodiments include a configuration in which the RNA is at least one selected from the group consisting of sense single-stranded RNA (ssRNA), antisense single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA); mixtures of any of these types of RNA may be used. In one embodiment, a double-stranded DNA / RNA hybrid is used. The RNA may contain components other than standard ribonucleotides (for example, RNA containing terminal deoxyribonucleotides in one embodiment).

[0092] The RNA in the insecticidal composition has 18 or more consecutive nucleotides having a sequence of approximately 95% to approximately 100% identity with respect to a target gene or equivalent length fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In one embodiment, the RNA includes 18 or more consecutive nucleotides that are essentially identical or complementary to a sequence of equivalent length fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the consecutive nucleotides have a sequence of approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with respect to a fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group, or a DNA complementary to either of them. In some embodiments, the RNA has all sequences of approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with a fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a DNA complementary to either of them.

[0093] The RNA in the insecticidal composition comprises 18 or more consecutive nucleotides having approximately 95% to approximately 100% identity with a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the RNA comprises at least one of 18 or more consecutive nucleotides, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or more. In some embodiments, the segments are 18 or more, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about It contains 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 280, at least approximately 270, at least approximately 280, at least approximately 290, at least approximately 300, at least approximately 350, at least approximately 400, at least approximately 450, at least approximately 500, or more than 500 consecutive nucleotides.In certain embodiments, the RNA comprises at least 18, 19, 20, or 21 consecutive nucleotides having sequences of 100% identity with DNA or a fragment of the target gene of equivalent length having sequences selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to any of these. In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of DNA complementary to any of them, having a sequence of 100% identity with respect to DNA or a fragment of the target gene of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group; expressible as base pairs, such a double-stranded nucleic acid contains at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs, or at least one segment of DNA complementary to any of them, corresponding to DNA or a fragment of the target gene of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group. In certain embodiments, the length of each segment contained in the RNA is longer than the length typical of naturally occurring regulatory small RNA molecules, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the RNA, or the length of each segment contained in the RNA, is shorter than the total length of the sequence of interest (DNA or target gene having a sequence selected from the group consisting of the target gene sequences). In some embodiments, the total length of the RNA is between about 50 and about 500 nucleotides (for single-stranded RNA) or base pairs (for double-stranded RNA). In some embodiments, the RNA comprises at least one RNA strand of about 50 to about 500 nucleotides in length.

[0094] The RNA in the insecticidal composition is generally designed to repress one or more genes ("target genes"). Such target genes may include coding sequences, non-coding sequences, or both. In certain embodiments, the RNA is designed to repress one or more target genes, where each target gene has a DNA sequence selected from the group consisting of the target gene sequences. In various embodiments, the RNA is designed to repress one or more genes, where each gene has a sequence selected from the group consisting of the target gene sequences, and can be designed to repress multiple genes from this group, or to target one or more different regions of these genes. In one embodiment, the RNA comprises multiple sections or segments, each containing 21 consecutive nucleotides having 100% identity to equivalent-length fragments of DNA having sequences selected from the target gene sequences, the trigger sequence group, or at least one segment of DNA complementary to either of these. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the RNA may include multiple sections in a tandem or repeat sequence, where each section includes 21 consecutive nucleotides having a sequence of 100% identity to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these; the segments may originate from various regions of the target gene, for example, the segments may correspond to various exon regions of the target gene, and “spacer” nucleotides not corresponding to the target gene may be optionally used between or adjacent to the segments.

[0095] The total length of the RNA in the insecticidal composition may be longer than 18 consecutive nucleotides and may include consecutive nucleotides having approximately 95% to approximately 100% identity with a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group and the trigger sequence group, or nucleotides added to DNA complementary to either of these. In other words, the total length of the RNA may be longer than the length of a section or segment of RNA designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequence group and the trigger sequence. For example, the RNA may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the RNA includes additional nucleotides that are not specifically related to (have sequences that are not complementary or identical to) the DNA or target gene having sequences selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of them, for example, nucleotides that provide a stabilized second structure or provide convenience in cloning or manufacturing. In one embodiment, the RNA includes 18 or more consecutive nucleotides having sequences of about 95% to about 100% identity to equivalent length fragments of the DNA or target gene having sequences selected from the target gene sequence group, the trigger sequence group, or additional nucleotides located immediately adjacent to one or more segments of DNA complementary to either of them. In one embodiment, the RNA includes one such segment having an additional 5'G or an additional 3'C or both adjacent to the segment. In another embodiment, the RNA is a double-stranded RNA including additional nucleotides to form an overhang, for example, a dsRNA including two deoxyribonucleotides to form a 3' overhang.Therefore, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical to or complementary to the DNA or a contiguous nucleotide fragment in the target gene having a sequence selected from the target gene sequence group or the trigger sequence group. For example, in some embodiments, the RNA comprises at least two segments of DNA each having a sequence that is 100% identical to a fragment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in the DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or the DNA complementary to either of them.

[0096] In various embodiments, the RNA in the insecticidal composition consists of naturally occurring ribonucleotides. Embodiments include, for example, synthetic RNA consisting entirely of ribonucleotides, or synthetic RNA consisting mainly of ribonucleotides but having one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain embodiments, the RNA includes non-standard nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the RNA includes chemically modified nucleotides. (a) The RNA in the insecticidal composition is provided by suitable means known to those skilled in the art. Embodiments include the RNA being synthesized chemically or enzymatically (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or in cell culture media (such as plant or insect cells grown in culture media), produced by expression in plant cells, or produced by microbial fermentation.

[0097] In some embodiments, the RNA is provided as isolated RNA that is not part of an expression construct. In some embodiments, the RNA is provided as isolated RNA lacking additional elements such as a promoter or terminator sequence. Such RNA can be relatively short, for example, single-stranded or double-stranded RNA having between about 18 and about 300 nucleotides, or between about 50 and about 500 nucleotides (for single-stranded RNA), or between about 18 and about 300 base pairs, or between about 50 and about 500 base pairs (for double-stranded RNA). Alternatively, the RNA can be provided in a more complex construct, for example, as part of a recombinant expression construct, and may be contained in a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such a recombinant expression construct or vector is designed to include additional elements, such as additional RNA encoding an aptamer or ribozyme, or an expression cassette for expressing the gene of interest (e.g., an insecticidal protein). VII. Methods for providing plants with improved resistance to the invasion of lepidopteran pests, and plants and seeds to which such methods are applied.

[0098] Another aspect of the present invention relates to a method for providing a plant with improved resistance to lepidopteran pest invasion, comprising expressing in a plant at least one polynucleotide comprising 18 or more consecutive nucleotides having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these, the resulting plant having improved resistance to lepidopteran pests compared to a control plant in which the polynucleotide is not expressed. In one embodiment, the method comprises expressing in a plant at least one polynucleotide comprising 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity with a target gene or DNA fragment of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these.In one embodiment, the present invention relates to Sequence IDs 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 55 1, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104 ,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251,1263,1264,1265,1270,1275,1279,1283,1290,1308,1310,1316,1318, The present invention provides a plant having improved resistance to lepidopteran pest invasion, comprising expressing in the plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18, 19, 20, or 21 consecutive nucleotides of DNA having a sequence selected from the group consisting of 1320, 1564, 1565, 1569, 1622, and 1623. "Expressing a polynucleotide in a plant" generally means "expressing an RNA transcript in a plant," and for example, means expressing in the plant RNA comprising a ribonucleotide sequence that is antisense or essentially complementary to at least one fragment of DNA having a target gene sequence or DNA having a sequence selected from the group of target gene sequences or the group of trigger sequences, or to any of the above.Embodiments include a method in which the polynucleotide expressed in the plant is an RNA or its complement comprising at least one segment having a sequence selected from the trigger sequence group. However, the polynucleotide expressed in the plant may also be DNA (e.g., DNA produced in the plant during genome replication) or RNA encoded by such DNA. Related aspects of the present invention include the isolated polynucleotide used in the method and the modified lepidopteran-resistant plant provided by the method.

[0099] The method comprises expressing at least one polynucleotide in a plant, wherein the polynucleotide comprises 18 or more consecutive nucleotides that are essentially identical or complementary to a target gene or DNA fragment having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the first polynucleotide is provided to the plant in the form of DNA (e.g., in the form of an isolated DNA molecule, or as an expression constructor, or as a transformation vector), and the polynucleotide expressed in the plant is a second polynucleotide in the plant (e.g., an RNA transcript of the first polynucleotide). In one embodiment, the polynucleotide is expressed in the plant by transgenic expression, i.e., by stably incorporating the polynucleotide into the plant genome from a site in one or more cells of the plant where it can be expressed. In one embodiment, a first polynucleotide (for example, a recombinant DNA construct including a promoter operably linked to DNA containing 18 or more consecutive nucleotides that are essentially identical or complementary to a target gene or DNA fragment having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these) is stably incorporated into the plant genome, and a second produced polynucleotide (for example, an RNA transcript containing a transformant of 18 or more consecutive nucleotides that are essentially identical or complementary to a target gene or DNA fragment having a sequence selected from the target gene sequence group, the trigger sequence group, or at least one segment of DNA complementary to either of these) is expressed in one or more cells of the plant. A method for stably providing a transgenic plant is provided in the section titled "Production and Use of Transgenic Plant Cells and Transgenic Plants".

[0100] In another embodiment, the polynucleotide expressed in the plant is expressed by transient expression (i.e., expression not resulting from the stable incorporation of the sequence into the plant genome). In such an embodiment, the method may include the step of introducing the polynucleotide (e.g., dsRNA or dsDNA) into the plant by routine techniques known in the art. For example, transient expression can be achieved by permeating the plant leaves with a polynucleotide solution using a needleless syringe.

[0101] In some embodiments in which the polynucleotide expressed in a plant is expressed by transient expression, the first polynucleotide is supplied to the plant in the form of RNA or DNA, or both RNA and DNA, and the second polynucleotide produced secondarily is transiently expressed in the plant. In some embodiments, the first polynucleotide is selected from one or more of the following: (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule containing a modified PolIII gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule containing a modified PolIII gene that is transcribed into an RNA molecule, and (i) a double-stranded, hybridized RNA / DNA molecule, or a combination thereof. In certain embodiments, the first polynucleotide is introduced into plants by topical application of the polynucleotide-containing composition to plants, in or on microbeads or other carrier particles, in a film or coating, or on or within a matrix, or in the form of treatment of crop plant seeds or seed tubers, in the form of treatment of crop plant seeds or seed tubers, in a suitable form such as solid, liquid (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulations. The composition may optionally include suitable binders, inert carriers, surfactants, etc., known to those skilled in the art in the formulation of insecticides and seed treatments.In such embodiments, the polynucleotide-containing composition may further comprise one or more components selected from the group consisting of carriers, surfactants, cationic lipids (e.g., those disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators; in one embodiment, the composition further comprises a nonionic organosilicon surfactant, such as a SILWET® brand surfactant, currently available from Momentive Performance Materials, Albany, NY, for example, a SILWET L-77® brand surfactant having CAS number 27306-78-1 and EPA number CAL.REG.No.5905-50073-AA. In some embodiments, the topically applied composition further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. Alternatively, such additional components or insecticides may be provided separately, for example, by separate topical application or transgenic expression in plants. Alternatively, the plants may be treated topically with separate (before, after, or in between) applications of the polynucleotide-containing composition in addition to the polynucleotide-containing composition. For example, the plants may be sprayed with a second topical application of the polynucleotide-containing composition (and vice versa), following a first topical application of a solution containing a nonionic organosilicon surfactant, such as a SILWET® brand surfactant, e.g., a SILWET L-77® brand surfactant.

[0102] The combination of specific polypeptides used in this method (e.g., the polynucleotide triggers described in the examples) with one or more non-polynucleotide insecticides is expected to result in a more accelerated improvement in preventing or controlling lepidopteran pest invasion compared to the effects obtained with polynucleotides alone or non-polynucleotide insecticides alone. In one embodiment, a transgenic plant expressing at least one polynucleotide comprising 18 or more consecutive nucleotides that are essentially identical or complementary to a target gene or DNA fragment having a sequence selected from the target gene sequence group, the trigger sequence group (e.g., the polynucleotide trigger described in the examples), or at least one DNA segment complementary to either of these, and one or more genes encoding a non-polynucleotide insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein, are found to exhibit improved resistance to lepidopteran pest invasion.

[0103] In some embodiments in which the polynucleotide expressed in the plant is expressed by transient expression, a first polynucleotide is provided to the plant in the form of RNA or DNA, or both RNA and DNA, and a second polynucleotide produced secondarily is transiently expressed in the plant; the site of application of the first polynucleotide does not have to be the same as the site where the second polynucleotide is transiently expressed. For example, the first polynucleotide can be provided to the plant by topical application to the leaves or by injection into the stem, and the second polynucleotide can be transiently expressed at other locations in the plant, e.g., in the roots or throughout the entire plant. In some embodiments of the method, a composition containing at least one polynucleotide is applied topically to the above-ground parts of the plant, e.g., by spraying or spraying on the leaves, stems, and flowering parts of the plant. In other embodiments, a composition containing at least one polynucleotide is applied topically to the underground parts of the plant, e.g., in the roots, e.g., using a soil trench. In other embodiments, a composition comprising at least one polynucleotide is applied topically to seeds (or, in the case of potatoes, to seed tubers) of plants that have grown to have improved resistance to lepidopteran pest invasion. In some embodiments, the polynucleotide expressed in the plant is RNA, which may be single-stranded (ss) or double-stranded (ds) RNA, or a combination of both.

[0104] In some embodiments, a first polynucleotide (DNA or RNA or both) is provided to a plant, and then a second polynucleotide having a sequence corresponding to (identical or complementary to) the first polynucleotide is expressed in the plant. In such embodiments, the polynucleotide expressed in the plant is an RNA transcript which may be ssRNA or dsRNA, or a combination of both. In some embodiments in which the polynucleotide is expressed by transient expression, a first polynucleotide is provided to the plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide produced secondarily is transiently expressed in the plant; in such embodiments, the first polynucleotide is selected from one or more of the following: (a) single-stranded RNA molecules (ssRNA), (b) single-stranded RNA molecules that self-hybridize to form double-stranded RNA molecules, (c) double-stranded RNA molecules (dsRNA), (d) single-stranded DNA molecules (ssDNA), (e) single-stranded DNA molecules that self-hybridize to form double-stranded DNA molecules, (f) single-stranded DNA molecules containing a modified PolIII gene that is transcribed into an RNA molecule, (g) double-stranded DNA molecules (dsDNA), (h) double-stranded DNA molecules containing a modified PolIII gene that is transcribed into an RNA molecule, and (i) double-stranded, hybridized RNA / DNA molecules, or combinations thereof. In such embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide may consist of nucleotides that are naturally occurring, such as those found in DNA and RNA. In such embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide may be chemically modified, i.e., include chemically modified nucleotides. The first polynucleotide is provided by suitable means known to those skilled in the art. Embodiments include the first polynucleotide being synthesized chemically or enzymatically (e.g., by in vitro transcription such as transcription using T7 polymerase or other polymerases), produced by expression in microorganisms or cell culture media (plant or insect cells grown in culture media), produced by expression in plant cells, or produced by microbial fermentation.The first polynucleotide may be provided as a fragment of RNA or DNA. Alternatively, the first polynucleotide may be provided in a more complex component, for example, as part of a recombinant expression component, or contained in a recombinant vector, for example, in a recombinant plant virus vector or a recombinant baculovirus vector; such recombinant expression components or vectors may be designed to include additional elements, such as an expression cassette for expressing the gene of interest (e.g., an insecticidal protein).

[0105] In some embodiments, the polynucleotide expressed in the plant is an RNA molecule and may be relatively short, for example, a single-stranded or double-stranded polynucleotide of about 18 to about 300 or about 50 to about 500 nucleotides (in the case of a single-stranded polynucleotide), or about 18 to about 300 or about 50 to about 500 base pairs (in the case of a double-stranded polynucleotide). Alternatively, the polynucleotide may be provided in a more complex structure, for example, as part of a recombinant expression structure, or contained in a recombinant vector, for example, in a recombinant plant virus vector or a recombinant baculovirus vector. Some such recombinant expression structures or vectors may be designed to include additional elements, such as an expression cassette for expressing a gene of interest (e.g., an insecticidal protein).

[0106] The polynucleotide expressed in the plant has 18 or more consecutive nucleotides having a sequence that is approximately 95% to approximately 100% identical to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of complementary DNA as described above. In one embodiment, the polynucleotide expressed in the plant includes 18 or more consecutive nucleotides that are essentially identical or complementary to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the consecutive nucleotides have a sequence that is approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to a fragment of DNA complementary to a sequence selected from the target gene sequence group or the trigger sequence group. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group, the trigger sequence group, or a DNA complementary to either of them. In some embodiments, the polynucleotides expressed in the plant have all sequences with approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with a fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a DNA complementary to either of them.

[0107] Generally, the polynucleotide expressed in the plant is designed to repress one or more genes ("target genes"). Such target genes include coding sequences, non-coding sequences, or both. In certain embodiments, the polynucleotide expressed in the plant is designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequences. In various embodiments, the polynucleotide expressed in the plant is designed to repress one or more genes, each of which has a sequence selected from the group consisting of the target gene sequences, and can be designed to repress multiple genes from this group, or to target one or more different regions of these genes. In one embodiment, the polynucleotide expressed in the plant comprises multiple sections or segments, each segment comprising 21 consecutive nucleotides having a sequence of 100% identity with an equivalent length of DNA having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of DNA complementary to either of the above. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide expressed in the plant may include multiple sections in a tandem or repeat sequence, where each section includes at least one segment of 21 consecutive nucleotides having a sequence that is 100% identical to a comparable length of DNA having a sequence selected from the target gene sequence group, the segment may originate from various regions of the target gene, for example, the segment may correspond to various exon regions of the target gene, and “spacer” nucleotides that do not correspond to the target gene may be optionally used between or adjacent to the segments.

[0108] The total length of the polynucleotide expressed in the plant may be longer than 18 or more consecutive nucleotides and may include consecutive nucleotides having approximately 95% to approximately 100% identity with a comparable length of DNA fragment having a sequence selected from the target gene sequence group, or nucleotides added to DNA complementary to either of these. In other words, the total length of the polynucleotide expressed in the plant may be longer than the length of a section or segment of polynucleotide designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequence group. For example, the polynucleotide expressed in the plant may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the polynucleotide expressed in the plant includes additional nucleotides that are not specifically related to (i.e., have sequences that are not complementary or identical to) the DNA or target gene having a sequence selected from the group of target gene sequences, or to its complementary DNA, for example, nucleotides that provide a stabilized second structure or provide convenience in cloning or production. In one embodiment, the polynucleotide expressed in the plant includes additional nucleotides located immediately adjacent to one or more segments of 18 or more consecutive nucleotides having sequences that are about 95% to about 100% identical or complementary to equivalent-length fragments of the DNA or target gene having a sequence selected from the group consisting of the group of target gene sequences. In one embodiment, the polynucleotide expressed in the plant includes one such segment adjacent to the segment having an additional 5'G or an additional 3'C or both. In another embodiment, the polynucleotide expressed in the plant is a double-stranded RNA containing additional nucleotides to form an overhang, for example, a dsRNA containing two deoxyribonucleotides to form a 3' overhang.Therefore, in various embodiments, the nucleotide sequence of the entire polynucleotide expressed in the plant is not 100% identical or complementary to the DNA having sequences selected from the target gene sequence group, the trigger sequence group, or a sequence of nucleotides in the target gene, or a DNA fragment complementary to either of these. For example, in some embodiments, the polynucleotide expressed in the plant comprises at least two segments of DNA each of 21 sequences having 100% identity to the DNA having sequences selected from the target gene sequence group, the trigger sequence group, or a DNA fragment complementary to either of these, wherein (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in the DNA having sequences selected from the target gene sequence group, the trigger sequence group, or a DNA fragment complementary to either of these.

[0109] In a related aspect, the present invention relates to a plant having improved resistance to lepidopteran pest invasion, provided by expressing in the plant at least one polynucleotide comprising 18 or more consecutive nucleotides having sequences selected from the target gene sequence group and the trigger sequence group of equivalent lengths, or at least one segment of DNA complementary to either of these, the resulting plant having improved resistance to lepidopteran pest invasion compared to a control plant in which the polynucleotide is not expressed. In a related aspect, the present invention relates to a plant having improved resistance to lepidopteran pest invasion, provided by expressing in the plant at least one polynucleotide comprising 18 or more consecutive nucleotides having sequences of about 95% to about 100% identity with sequences selected from the target gene sequence group and the trigger sequence group, or at least one segment of DNA complementary to either of these, the resulting plant having improved resistance to lepidopteran pest invasion compared to a control plant in which the polynucleotide is not expressed. One embodiment is a crop plant having improved resistance to lepidopteran pest invasion compared to a control plant, provided by expressing an RNA having a sequence selected from the trigger sequence group, or a complement thereof, in the plant. In yet another embodiment, the present invention relates to seeds (particularly transgenic offspring seeds) produced by the plant having improved resistance to lepidopteran pest invasion provided by this method. Also intended are commodity products produced by the plant having improved resistance to lepidopteran pests provided by this method, and commodity products produced from transgenic offspring seeds of such plants.

[0110] VIII. Recombinant DNA components for controlling lepidopteran insect pests Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably ligated to a DNA element comprising 18 or more consecutive nucleotides having sequences of approximately 95% to approximately 100% identity with respect to a DNA fragment having sequences selected from the target gene sequence group and the trigger sequence group, or at least one segment of DNA complementary to either of these. In some embodiments, the recombinant DNA construct comprises (a) SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265 A target gene having a sequence selected from the group consisting of 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or DNA containing a nucleotide sequence complementary to at least 18, 19, 20, or 21 consecutive nucleotides of RNA transcribed from the target gene;or (b) SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546 ,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,596,602,611,612,620,665,666,672,717,730,731,736,737,738,740,741,742,743,749,757,760,761,763,766,808,809,81 0, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1 DNA containing 18, 19, or 21 or more consecutive nucleotides, or complementary DNA thereof, that have 100% identity with a fragment of equivalent length of DNA having a sequence selected from the group consisting of 265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623;or (c) DNA encoding at least one silencing element complementary to at least 18, 19, 20, or 21 consecutive nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene is sequence numbers 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 76 0, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, (d) DNA having a sequence selected from the group consisting of 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623; or (d) DNA encoding at least one silencing element containing at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to the target gene selected from the trigger gene sequence group or the RNA transcribed from the target gene;or (e) DNA encoding RNA containing at least 18, 19, 20, or 21 consecutive nucleotides complementary to a nucleotide sequence selected from the trigger sequence group or its complement, or an orthologous nucleotide sequence derived from a lepidopteran insect, wherein the orthologous nucleotide sequence has at least 95% sequence identity with respect to the nucleotide sequence selected from the trigger sequence group, where the percentage sequence identity is calculated over the same length; or (f) DNA encoding RNA containing at least one strand containing at least 18, 19, 20, or 21 consecutive nucleotides complementary to at least one double-stranded RNA region, the trigger sequence group, its complement, or a nucleotide sequence selected from an orthologous nucleotide sequence derived from a lepidopteran insect, wherein the orthologous nucleotide sequence has at least 95% sequence identity with respect to a nucleotide sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to any of them, where the percentage sequence identity is calculated over the same length;or (g) a heterogeneous promoter operably ligated to DNA encoding RNA containing a nucleotide sequence selected from the trigger sequence group or its complement. Embodiments include SEQ ID NOs: 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 53 8, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 104 2, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470 A recombinant DNA construct or its complement comprises a heterologous promoter operably bound to a DNA element encoding an RNA having a sequence selected from the group consisting of 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683.

[0111] The embodiment includes a recombinant DNA construct comprising a heterologous promoter operably ligated to DNA encoding a dsRNA having a strand or fragment thereof having a sequence selected from a group of trigger sequences, or a strand having a sequence selected from any complement thereof. The recombinant DNA construct is useful, for example, in providing plants with improved resistance to lepidopteran pest invasion by expressing a transcript of such recombinant DNA construct in plants. The recombinant DNA construct is also useful in producing polynucleotides useful for creating compositions that can be applied to plants, seeds, parts of fertile plants, soil or fields, or surfaces that require protection from lepidopteran pest invasion. Related aspects of the present invention include compositions comprising recombinant DNA components; plant chromosomes or plastids or recombinant plant virus vectors or recombinant baculovirus vectors comprising the recombinant DNA components; transgenic Solanaceae plant cells having the recombinant DNA components in their genome and optionally containing DNA encoding at least one insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein; transgenic Solanaceae plants comprising such transgenic Solanaceae plant cells; or fruits, seeds, or fertile parts of such transgenic Solanaceae plants; and plants having improved lepidopteran resistance provided by expression of or treatment with recombinant DNA components or RNA encoded therein.

[0112] The recombinant DNA component includes a heterologous promoter operably ligated to DNA containing 18 or more consecutive nucleotides having sequences of approximately 95% to approximately 100% identity with respect to a comparable fragment of DNA having sequences selected from the target gene sequence group, or at least one segment of complementary DNA. In some embodiments, the segment of 18 or more consecutive nucleotides has approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with respect to a comparable fragment of DNA having sequences selected from the target gene sequence group, or complementary DNA. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a comparable fragment of DNA having sequences selected from the target gene sequence group, or complementary DNA. In some embodiments, the DNA has all sequences of approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with respect to a comparable fragment of DNA having sequences selected from the target gene sequence group, or complementary DNA.

[0113] Accordingly, the recombinant DNA component includes a heterologous promoter operably ligated to DNA containing 18 or more consecutive nucleotides or at least one segment of DNA complementary thereto, designed to suppress the expression of a target gene having a sequence selected from a group of target gene sequences. In some embodiments, the DNA contains at least 18 consecutive nucleotides, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or more. In some embodiments, the number of segments may be greater than 18, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, or less Each contains approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 280, at least approximately 270, at least approximately 280, at least approximately 290, at least approximately 300, at least approximately 350, at least approximately 400, at least approximately 450, at least approximately 500, or more than 500 consecutive nucleotides.In certain embodiments, the DNA encodes RNA comprising at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides or their complementary DNA having a sequence of 100% identity with respect to DNA or a fragment of equivalent length of the target gene having a sequence selected from the target gene sequence group. In certain embodiments, the DNA encodes a single-stranded double-stranded nucleic acid (e.g., dsRNA) comprising one strand containing at least 18, 19, 20, or 21 consecutive nucleotides or their complementary DNA having a sequence of 100% identity with respect to DNA or a fragment of equivalent length of the target gene having a sequence selected from the target gene sequence group; comprising at least 18, 19, 20, or 21 consecutive perfectly matching base pairs or their complementary DNA at least one segment corresponding to DNA or a fragment of equivalent length of the target gene having a sequence selected from the target gene sequence group. In certain embodiments, the length of each segment contained in the DNA is longer than the typical length of naturally occurring regulatory small RNA molecules. In some embodiments, the length of each segment is the length of at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the DNA, or the length of each segment contained in the polynucleotide, is shorter than the total length of the sequence of interest (DNA or target gene having a sequence selected from the group consisting of the target gene sequences). In some embodiments, the total length of the DNA is between about 50 and about 500 nucleotides.In some embodiments, the DNA is sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524 ,527,528,536,538,539,823,826,827,828,830,832,833,834,840,841,842,844,845,851,853,854,862,874,877,883,892,893,901,946,947,953,998,1011,1012,1017,1018,1019,1021,1022,1023,102 4, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, It encodes RNA having a sequence selected from the group consisting of 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a combination thereof, or its complement.

[0114] The recombinant DNA construct generally includes a heterologous promoter operably ligated to DNA designed to repress one or more genes ("target genes"). Such target genes may include coding sequences, non-coding sequences, or both. In certain embodiments, the recombinant DNA construct is designed to repress one or more target genes, each having a DNA sequence selected from the group consisting of the target gene sequences. In various embodiments, the recombinant DNA construct may be designed to repress one or more genes, each having a sequence selected from the group consisting of the target gene sequences, and to repress multiple genes from this group, or to target one or more different regions of these genes. In one embodiment, the recombinant DNA construct includes a heterologous promoter operably ligated to a plurality of sections or segments, each containing at least one segment of DNA or a complementary sequence of 21 consecutive nucleotides having a sequence of 100% identity with respect to equivalent-length fragments of DNA having a sequence selected from the group of target gene sequences. In such a case, each section may be identical or different in size or sequence, and may be sense or antisense with respect to the target gene. For example, in one embodiment, the recombinant DNA component may include a heterologous promoter operably linked to a plurality of sections in a tandem or repeat sequence, where each section includes 21 consecutive nucleotides or at least one segment of complementary DNA having a sequence of 100% identity to a comparable length fragment of DNA having a sequence selected from the target gene sequence group, the segment may originate from various regions of the target gene, for example, the segment may correspond to various exon regions of the target gene, and “spacer” nucleotides not corresponding to the target gene may be optionally used between or adjacent to the segments.

[0115] The recombinant DNA construct comprises a heterologous promoter operably ligated to DNA that can have a total length longer than 18 consecutive nucleotides, and may include nucleotides added to at least one segment of DNA having 18 or more consecutive nucleotides or complementary DNA having sequences of approximately 95% to approximately 100% identity with a comparable length fragment of DNA having sequences selected from the target gene sequence group. In other words, the total length of the DNA can be longer than the length of a segment of DNA designed to repress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequence group. For example, the DNA may have nucleotides adjacent to the “active” segment of at least one segment of 18 or more consecutive nucleotides that repress the target gene, or may include “spacer” nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' and 3' ends. In one embodiment, the heterologous promoter is operably ligated to DNA containing a sequence selected from the target gene sequence group or complementary DNA, or to DNA containing additional nucleotides that are not specifically related to (i.e., have sequences that are not complementary or identical to) the target gene, for example, nucleotides that provide a stabilized second structure or provide convenience in cloning or manufacturing. In one embodiment, the heterologous promoter is operably ligated to DNA containing 18 or more consecutive nucleotides having sequences that are approximately 95% to approximately 100% identical or complementary to equivalent-length fragments of the target gene, or to additional nucleotides located immediately adjacent to one or more segments of DNA complementary to either of these. In one embodiment, the heterologous promoter is operably ligated to DNA containing one such segment having an additional 5'G or an additional 3'C or both adjacent to the segment.In another embodiment, the heterologous promoter is operably ligated to DNA encoding double-stranded RNA containing additional nucleotides to form an overhang. Therefore, in various embodiments, the heterologous promoter is neither 100% identical nor complementary to DNA having sequences selected from the target gene sequence group, DNA having sequences selected from the trigger sequence group, or a sequence of nucleotides in the target gene, or a fragment of DNA complementary to either of these. For example, in some embodiments, the heterologous promoter comprises at least two segments of DNA each of 21 sequences having 100% identity to DNA having sequences selected from the target gene sequence group, DNA having sequences selected from the trigger sequence group, or a fragment of DNA complementary to either of these, wherein (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments exist in DNA having sequences selected from the target gene sequence group, DNA having sequences selected from the trigger sequence group, or a fragment of DNA complementary to either of these.

[0116] In a recombinant DNA component, a heterologous promoter is operably ligated to DNA encoding a transcript that may be single-stranded (ss), double-stranded (ds), or a combination of both. Embodiments of the method include DNA encoding a transcript containing sense single-stranded RNA (ssRNA), antisense ssRNA, or double-stranded RNA (dsRNA), or any combination thereof.

[0117] The recombinant DNA components are provided by appropriate means known to those skilled in the art. Embodiments include the recombinant DNA components being synthesized in vitro, produced by expression in microorganisms or cell culture media (such as plant or insect cells grown in culture), produced by expression in plant cells, or produced by microbial fermentation.

[0118] The heterologous promoters used in recombinant DNA components are selected from a group consisting of promoters that function in plants, promoters that function in prokaryotes, promoters that function in fungal cells, and baculovirus promoters. Non-exclusive examples of promoters are described in the "Promoters" section.

[0119] In some embodiments, the recombinant DNA component also includes a second promoter that is operablely ligated to the DNA. For example, DNA containing at least one segment of 18 or more consecutive nucleotides can be positioned adjacent to two promoters arranged so that the promoters transcribe in a convergent manner in opposite directions, thereby producing a reverse-strand transcript of DNA that can complement and hybridize with each other to form double-stranded RNA. In one embodiment, the DNA is positioned between two root-specific promoters, thereby enabling transcription of the DNA in opposite directions and resulting in the formation of dsRNA.

[0120] In some embodiments, the recombinant DNA component includes other DNA elements in addition to a heterologous promoter operably ligated to DNA containing 18 or more consecutive nucleotides or at least one complementary DNA segment having about 95% to about 100% identity with a comparable length of DNA fragment having a sequence selected from the target gene sequence group. Such DNA elements are known in the art and include, but are not limited to, introns, recombinase recognition sites, aptamers or ribozymes, as well as additional expression cassettes for expressing coding sequences (e.g., for expressing transgenes such as insecticidal proteins or selectable markers) or non-coding sequences (e.g., for expressing additional repressors). By including one or more recognition sites for binding and cleavage by small RNAs (e.g., miRNAs or siRNAs expressed only in specific cells or tissues), a more precise expression pattern in plants is possible, where the expression of the recombinant DNA component is repressed at the site where the small RNA is expressed.

[0121] In some embodiments, the recombinant DNA components are provided in a recombinant vector. “Recombinant vector” means a recombinant polynucleotide molecule used to transfer genetic information from one cell to another. Suitable embodiments of the present invention include, but are not limited to, recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors such as recombinant plant virus vectors and recombinant baculovirus vectors. Alternative embodiments include recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors such as recombinant plant virus vectors and recombinant baculovirus vectors, which include DNA elements that do not contain heterologous promoters.

[0122] In some embodiments, the recombinant DNA component is provided in the chromosomes or plastids of a plant, for example, in a transgenic plant cell or a transgenic plant. Thus, transgenic plant cells having the recombinant DNA component in their genome, as well as transgenic plants or partially transgenic plants containing such transgenic plant cells, are also included in the present invention. A partially transgenic plant includes, for example, a non-transgenic scion grafted onto a transgenic rhizome containing the transgenic plant cells. Embodiments include a transgenic tomato rhizome rootstock containing transgenic plant cells. The plant may be any plant that is susceptible to invasion by lepidopteran pests. A specific area of ​​interest is an embodiment in which the plant is a crop plant. Embodiments include where the plant is an ungerminated crop plant seed, a crop plant in the vegetative growth stage, or a crop plant in the reproductive stage. Embodiments include where the plant is a “seed potato,” meaning a potato tuber or a portion of a potato tuber that can be propagated into a new potato. In yet another embodiment, the present invention relates to seeds (particularly transgenic offspring seeds) produced by transgenic plants having the recombinant DNA components described herein in their genome. Embodiments also include transgenic seed tubers having the recombinant DNA components described herein in their genome. Furthermore, commodity products produced by such transgenic plants, and commodity products produced from the transgenic offspring seeds of such transgenic plants are also intended.

[0123] The recombinant DNA components may be provided in compositions for topical application to the surface of plants or plant seeds, or for topical application to any substrate requiring protection from invasion by lepidopteran pests. Similarly, the recombinant DNA components may be provided in compositions for topical application to lepidopteran pests, or for ingestion by lepidopteran pests. In various embodiments, such compositions containing the recombinant DNA components may be provided in at least one form selected from the group consisting of solids, liquids (including homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, and may be provided in or on microbeads or other carrier particles, in films or coatings, on or in a matrix, or as a seed treatment. The aforementioned topical application may exist in the form of a topical treatment of the fruit of a Solanaceae plant or seeds obtained from the fruit of a Solanaceae plant, or in the form of a treatment of the tuber or fragments of a “seed potato” (for example, by dipping the seed potato, coating the seed potato, or spraying the seed potato). Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art in the formulation of insecticides and seed treatments, may be optionally included in the composition. In some embodiments, the composition for topical application containing the recombinant DNA component is at least one locally transplantable formulation selected from the group consisting of particles, pellets, or capsules locally transplanted into the plant; in such embodiments, the method includes locally transplanting the locally transplantable formulation into the plant. In some embodiments, the composition for topical application containing the recombinant DNA component is at least one in-furrow formulation selected from the group consisting of powder, granules, pellets, capsules, spray, or drench, or any other form suitable for topical application to a furrow; in such embodiments, the method includes in-furrow treatment using the in-furrow formulation. In one embodiment, the topical application composition containing the recombinant DNA component can be ingested by lepidopteran insects or otherwise absorbed internally. For example, the topical application composition containing the recombinant DNA component can exist in the form of bait.In some embodiments, the composition containing the recombinant DNA component further comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids (e.g., those disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicone surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In one embodiment, the composition containing the recombinant DNA component further comprises a nonionic organosilicone surfactant, such as a SILWET® brand surfactant, currently available from Momentive Performance Materials, Albany, NY, for example, a SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number CAL.REG.No.5905-50073-AA. In some embodiments, the composition comprising the recombinant DNA component further comprises at least one insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein.

[0124] The combination of certain recombinant DNA components described herein (e.g., recombinant DNA components containing polynucleotide triggers as described in the Examples), whether transgenically expressed or topically applied, with one or more non-polynucleotide insecticides is expected to result in an enhanced improvement in preventing or controlling lepidopteran pest invasion compared to the effects obtained by the transgenic DNA component alone or the non-polynucleotide insecticide alone, whether transgenically expressed or topically applied. In one embodiment, a recombinant DNA component for expressing one or more polypeptides and one or more polynucleotides, encoding a non-polynucleotide insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein, is found to provide improved resistance to lepidopteran pest invasion in plants expressing the recombinant DNA component. One embodiment relates to a recombinant DNA for expressing RNA containing a segment containing a sequence selected from the trigger sequence group, and one or more genes encoding non-polynucleotides selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein.

[0125] The composition containing the recombinant DNA component can be provided for dietary intake by lepidopteran pests by applying the composition to plants or surfaces that are susceptible to invasion by lepidopteran pests, for example by spraying, scattering, or coating the plants, or by applying a soil drench, or by providing it in an artificial diet. The composition containing the recombinant DNA component can also be provided for dietary intake by lepidopteran pests in an artificial diet formulated to meet specific nutritional requirements for maintaining lepidopteran pests, where the artificial diet is supplemented with some amount of the recombinant DNA component obtained from another source such as in vitro synthesis, or purified from microbial fermentation or other biological sources; this embodiment may be useful, for example, for determining the timing and amount of an effective treatment regimen. In some embodiments, the composition containing the recombinant DNA component can be provided for dietary intake by lepidopteran pests in the form of plant cells or in plant cell components, or in microorganisms (e.g., bacteria or yeast) or in microbial fermentation products, or in a synthetic diet. In one embodiment, the composition containing the recombinant DNA component is provided in the form of bait ingested by lepidopteran insects. The composition containing the recombinant DNA component can also be provided in the form of seed treatment for feeding by lepidopteran insects.

[0126] In various embodiments, the composition containing the recombinant DNA component includes or is produced in microbial cells. For example, the composition containing the recombinant DNA component may include or be produced in bacterial or yeast cells. In similar embodiments, the composition containing the recombinant DNA component includes or is produced in transgenic plant cells (e.g., in plant cells that transiently express the recombinant DNA component); such plant cells may be cells in a plant or cells grown in tissue culture medium or cell suspension.

[0127] IX. Transgenic plant cells Some embodiments relate to transgenic crop plant cells expressing polynucleotides useful in the methods described herein for suppressing the expression of target genes in lepidopteran pests or for controlling lepidopteran invasion. In one embodiment, the present invention provides transgenic Solanaceae plant cells having recombinant DNA in its genome that encodes RNA containing 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity with a DNA fragment having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of DNA complementary to either of these. In one embodiment, the present invention provides transgenic Solanaceae plant cells having recombinant DNA in its genome that encodes RNA containing at least one silencing element essentially identical or essentially complementary to a target gene sequence fragment of a lepidopteran pest larva, wherein the target gene sequence is selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of these. In one embodiment, the present invention provides a transgenic Solanaceae plant cell having recombinant DNA in its genome that encodes RNA that suppresses the expression of a target gene in lepidopteran pests that come into contact with or ingest the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides complementary to a fragment of the target gene, and wherein the target gene is selected from the group consisting of genes in the target gene sequence group. A specific embodiment is a transgenic crop plant cell having recombinant DNA in its genome that encodes RNA that suppresses the expression of a target gene in lepidopteran pests that come into contact with or ingest the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides complementary to a fragment of one or more target gene sequence groups. In one embodiment, the present invention provides a transgenic crop plant cell having recombinant DNA in its genome that encodes RNA having a sequence selected from the trigger sequence group.Such transgenic crop plant cells are useful in providing transgenic crop plants that have improved resistance to lepidopteran pest invasion compared to control plants lacking such plant cells. The transgenic crop plant cells may be isolated transgenic Solanaceae plant cells, or transgenic Solanaceae plant cells grown in a culture medium, or transgenic cells of any transgenic crop plant that is susceptible to lepidopteran pest invasion.

[0128] In one embodiment, the recombinant DNA is stably incorporated into the genome of the transgenic crop plant and can be expressed therein in one or more cells of the transgenic Solanaceae plant. A method for providing a stably transformed plant is provided in the section titled "Production and Use of Transgenic Plant Cells and Transgenic Plants".

[0129] Some embodiments relate to transgenic Solanaceae plant cells having recombinant DNA in their genome that encodes RNA that suppresses the expression of a target gene in lepidopteran pests that come into contact with or ingest the RNA, wherein the RNA comprises at least one silencing element complementary to the target gene, and the target gene sequence is selected from a group of target gene sequences or their complements. In some embodiments, the silencing element comprises at least one sequence of 18 or more nucleotides having a sequence that is approximately 95% to approximately 100% complementary to a comparable length of DNA having a sequence selected from the group of target gene sequences or the group of trigger sequences, or DNA complementary to either of these. In some embodiments, the silencing element comprises at least one sequence of 18 or more nucleotides, or DNA complementary to either of these, which can hybridize in vivo or under physiological conditions (e.g., physiological conditions commonly found in the cells of lepidopteran insects) to a fragment of DNA of equivalent length having a sequence selected from the target gene sequence group or the trigger sequence group. The number of the sequence of nucleotides is at least 18, for example, between 18 and 24, or between 18 and 28, or between 20 and 30, or between 20 and 50, or between 20 and 100, or between 50 and 100, or between 50 and 500, or between 100 and 250, or between 100 and 500, or between 200 and 1000, or between 500 and 2000, or more.In some embodiments, the number of consecutive nucleotides is greater than 18, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, for example, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, A sequence of at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 280, at least approximately 270, at least approximately 280, at least approximately 290, at least approximately 300, at least approximately 350, at least approximately 400, at least approximately 450, at least approximately 500, or more than 500 consecutive nucleotides. In certain embodiments, the silencing element includes at least 18, 19, 20, or 21 consecutive nucleotides having sequences that are 100% identical to DNA or equivalent-length fragments of the target gene, or at least one complementary segment of DNA having sequences selected from the target gene sequence group or the trigger sequence group.In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least 18, 19, 20, or 21 consecutive nucleotides, or at least one segment of its complementary DNA, having a sequence that is 100% identical to a DNA or equivalent-length fragment of the target gene having a sequence selected from the target gene sequence group or the trigger sequence group; expressing as base pairs, such a double-stranded nucleic acid contains at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs, or at least one segment of its complementary DNA, corresponding to a DNA or equivalent-length fragment of the target gene having a sequence selected from the target gene sequence group or the trigger sequence group. In certain embodiments, the length of each silencing element contained in the RNA is longer than the typical length of naturally occurring regulatory small RNAs. In some embodiments, the length of each segment is the length of at least about 30 consecutive nucleotides (or base pairs). In certain embodiments, the length of the RNA is between approximately 50 and 500 nucleotides. In certain embodiments, the RNA has a sequence selected from the group of trigger sequences.

[0130] In some embodiments, transgenic crop plant cells can further express additional heterologous DNA sequences. In one embodiment, the transgenic Solanaceae plant cell has a genome further comprising recombinant DNA encoding at least one insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein. In a particular embodiment, the transgenic crop plant cell stably incorporates into its genome (i) recombinant DNA encoding at least one RNA having a sequence selected from the trigger sequence group, and (ii) DNA encoding at least one insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein.

[0131] In related embodiments, the present invention relates to transgenic crop plants comprising transgenic crop plant cells, commodity products produced from the transgenic crop plants, and transgenic Solanaceae plant seeds or transgenic reproductive parts of transgenic Solanaceae plants. Also intended are commodity products produced by transgenic crop plants and commodity products produced from transgenic offspring seeds of such transgenic crop plants.

[0132] X. How to select target genes Another aspect of the present invention provides a method for non-random selection of target genes for RNAi-mediated silencing. In one embodiment, the method provides a subset of target genes that exist in a single or low copy number (non-repeating and non-duplication) in a particular genome. Such target genes may be genes derived from a plant genome or genes from an animal genome. In some embodiments, the target genes are genes of invertebrate pests, e.g., plant invertebrate pests or vertebrate invertebrate pests. In some embodiments, the target genes are genes of plant insect pests or plant nematode pests. In some embodiments, the target genes are genes of lepidopteran pests. Further embodiments include producing polynucleotides based on target genes for RNAi-mediated silencing selected by any of the methods described herein (e.g., ssRNA or dsRNA triggers such as the dsRNA trigger described in the examples, or recombinant DNA components useful for creating transgenic plants).

[0133] In one embodiment, the method includes the steps of identifying single-copy or low-copy genes in a selected genome, or alternatively, identifying single-copy or low-copy genes in an ortholog database of relevant organisms to predict which genes will be single / low-copy in the selected organism. Low-copy genes, and in particular single-copy genes, are selected as targets for RNAi-mediated silencing. In one embodiment, the identification of single-copy or low-copy genes is performed by sequence comparison between a set of genes from a first species and a set of genes from a second species, where the set of genes from the second species is identified as single-copy or low-copy in the second species. In another embodiment, the identification of single-copy or low-copy genes is performed by applying a computer-executed algorithm to the set of genes from the first species to identify a subset of single-copy or low-copy genes in the set of genes from the first species, and then comparing the set of genes from the second species with the subset of single-copy or low-copy genes from the first species to identify the corresponding single-copy or low-copy genes from the second species. Single or low-copy-number genes derived from a second species are useful as target genes for RNAi-mediated silencing; the sequences of these target genes are useful for designing polynucleotides (e.g., ssRNA or dsRNA triggers such as the dsRNA trigger described in the examples, or recombinant DNA components for creating transgenic plants) and methods for their use to prevent or control invasion by the second species. Another embodiment relates to identifying genes that are lethal to the survival of an organism by searching a database containing such information for an orthologous model species for a target organism.

[0134] Embodiments of the method include the further step of estimating the nucleotide diversity of low / single copy genes in a selected population of organisms, and selecting those low / single copy genes that have the lowest nucleotide diversity. Low / single copy genes with further low nucleotide diversity are selected as targets for RNAi-mediated silencing. Another embodiment relates to identifying genes that are lethal to the survival of the target organism by searching a database containing such information for an orthologous model species for the target organism.

[0135] Embodiments of the method include a further step of comparing the ratio of synonymous (Ks) nucleotide changes to non-synonymous (Ka) changes as an estimate of a functional or evolutionary constraint. In one embodiment, the method includes a step of selecting genes in which Ks is equal to or greater than Ka. In one embodiment, the method includes a step of selecting genes in which Ka >> Ka.

[0136] Related aspects of the present invention are sets of target genes for RNAi-mediated silencing identified from a genome by any of the gene selection methods described herein. One embodiment relates to a set of target genes for RNAi-mediated silencing selected from a genome by identifying a single or low copy number target gene from a larger set of genes derived from the genome. One embodiment relates to a set of target genes for RNAi-mediated silencing selected from an invertebrate genome by identifying a single or low copy number target gene from a larger set of genes derived from the invertebrate genome. A particular embodiment relates to a set of target genes for RNAi-mediated silencing in lepidopteran pests selected from a lepidopteran genome by identifying a single or low copy number target gene from a larger set of genes derived from the lepidopteran genome. A particular embodiment relates to a set of target genes for RNAi-mediated silencing in lepidopteran pests selected from a lepidopteran genome by identifying a single or low copy number target gene from a larger set of genes derived from the lepidopteran genome.

[0137] Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from genomes by estimating nucleotide diversity for a given set of genes in a population of individuals of the pest having the genome, and selecting these genes having the lowest nucleotide diversity. Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from invertebrate genomes by estimating nucleotide diversity for a given set of genes in individual populations of the invertebrate having the genome, and selecting those genomes having the lowest nucleotide diversity. Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from invertebrate genomes by estimating nucleotide diversity for low / single copy genes in individual populations of invertebrate having the genome, and selecting these low / single copy genes having the lowest nucleotide diversity.

[0138] Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from a genome, by comparing the ratio of synonymous (Ks) to non-synonymous (Ka) nucleotide changes in the genes of the genome, and by selecting genes in which Ks is equal to or greater than Ka. In one embodiment, the set of target genes for RNAi-mediated silencing is a set of genes in which Ks is equal to or greater than Ka. In one embodiment, the set of target genes for RNAi-mediated silencing is a set of genes in which Ks >> Ka. One embodiment relates to a set of target genes for RNAi-mediated silencing selected from an invertebrate genome, where the selected genes are Ks >> Ka.

[0139] Further aspects of the present invention include polyclonal or monoclonal antibodies that conjugate to proteins encoded by sequences or sequence fragments selected from the trigger gene sequence group or their complements, and polyclonal or monoclonal antibodies or their complements that conjugate to proteins encoded by sequences or sequence fragments selected from the trigger sequence group and their complements; such antibodies are prepared by routine methods known to those skilled in the art, for example, using routine protocols described in “Antibody Methods and Protocols” (Proetzel and Ebersbach, editors, 2012, Humana Press, New York) or “Making and Using Antibodies” (Howard and Kaser, editors, 2006, CRC Press, Boca Raton).

[0140] XI. Selection of effective polynucleotides by "tiling" The polynucleotides used in the embodiments described herein do not need to be the full length of the target gene, and in many embodiments, they are much shorter than the target gene. An example of a useful technique for selecting effective polynucleotides is "tiling," i.e., evaluating polynucleotides corresponding to adjacent or partially overlapping segments of the target gene.

[0141] In some embodiments, an effective polynucleotide trigger can be identified by "tiling" the gene along the length of the target gene from selected fragments of length, e.g., partially overlapping regions, fragments of 200 to 300 nucleotides (e.g., 25 nucleotides). In some embodiments, the polynucleotide trigger sequence is designed to have corresponding (nucleotide identity or complementarity) nucleotides that are unique to the target gene (or identical to or complementary to it). In some embodiments, the selected region of the target gene may include coding sequences or non-coding sequences (e.g., promoter regions, 3' untranslated regions, introns, etc.), or a combination of both.

[0142] If the design of a target effective in repressing multiple target genes is of interest, the multiple target gene sequences are aligned, and the polynucleotide trigger is designed to correspond to a region having high sequence homology common to the multiple targets. Conversely, if the design of a target effective in selectively repressing one of the multiple target sequences is of interest, the multiple target gene sequences are aligned, and the polynucleotide trigger is designed to correspond to a region having little to no sequence homology common to the multiple targets.

[0143] XII. Thermodynamic considerations in the selection of effective polynucleotides In some embodiments, polynucleotide triggers can be designed or their sequence optimized using thermodynamic considerations. For example, polynucleotides can be selected based on thermodynamically controlled hybridization between one nucleic acid strand (e.g., a polynucleotide trigger or individual siRNA) and another nucleic acid strand (e.g., a target gene transcript).

[0144] Methods and algorithms for predicting nucleotide sequences that may be effective for RNAi-mediated silencing of target genes are publicly known in the art. Non-limiting examples of such methods and algorithms include: "i-score" Ichihara et al. (2007) Nucleic Acids Res., 35(18): 123e; "Oligowalk" published at rna.urmc.rochester.edu / servers / oligowalk and described in Lu et al. (2008) Nucleic Acids Res., 36:W104-108; "Reynolds score" Khovorova et al. (2004) Nature Biotechnol., 22:326-330; and "si-Fi" Luck et al. (2019) Front. Plant Sci and published at http: / / www.snowformatics.com / si-fi.html; and "SnapDragon" Hu et al. (2016) Nucleic Acids Res., 45: This includes those described in D672-D678 and published at https: / / www.flyrnai.org / snapdragon; and "E-RNAi" Horn et al. (2010) Nucleic Acids Res., 38: W332-W339 and published at https: / / www.dkfz.de / signaling / e-rnai3 / .

[0145] XIII. Acceptable Mismatches As used herein, “essentially identical” or “essentially complementary” means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to a target gene or RNA (e.g., transcript) transcribed from the target gene to suppress the expression of the target gene (e.g., resulting in a decrease in the level or activity of the target gene transcript and / or encoded protein). The polynucleotides described herein do not need to have 100% identity or complementarity to the target gene or sequence or RNA transcribed from the target gene in order to suppress the expression of the target gene (e.g., to result in a decrease in the level or activity of the target gene transcript or encoded protein, or to provide control of lepidopteran pests). In some embodiments, the polynucleotide or a portion thereof is designed to be essentially identical or essentially complementary to a sequence of at least 18 or 19 consecutive nucleotides in either the target gene or RNA transcribed from the target gene. In some embodiments, the polynucleotide or a portion thereof is designed to be 100% identical or 100% complementary to one or more sequences of 21 consecutive nucleotides in either the target gene or RNA transcribed from the target gene. In certain embodiments, the “essentially identical” polynucleotide has 100% sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity compared to a sequence of 18 or more consecutive nucleotides in either the endogenous target gene or RNA transcribed from the target gene. In certain embodiments, the “essentially complementary” polynucleotides have 100% sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence complementarity compared to a sequence of 18 or more consecutive nucleotides in either the target gene or RNA transcribed from the target gene.

[0146] As used herein in the context of two polynucleotides or polypeptides, the term “sequence identity” or “identity” refers to residues in the sequences of two molecules that are identical when aligned in the greatest correspondence beyond a specified comparison window.

[0147] As used herein, the term “percentage of sequence identity” may refer to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or polypeptide sequences) of molecules across a comparison window, where the portion of the sequence within the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleotide or amino acid residues exist in both sequences, giving the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to give the percentage of sequence identity. A sequence that is identical in all positions compared to a reference sequence is said to be 100% identical to the reference sequence, and vice versa. The term “approximately” in relation to numerical values ​​of sequence length means a value with a + / - variance of 1 to 5%. For example, approximately 30 consecutive nucleotides means a range of 27 to 33 consecutive nucleotides, or any range in between. The term "approximately" in numerical values ​​for sequence identity percentages refers to a value with a + / - variance of 1 to 3 percent, rounded to the nearest integer. For example, approximately 90% sequence identity means a range of 87 to 93%. However, the aforementioned sequence identity percentage cannot exceed 100%. Therefore, approximately 98% sequence identity means a range of 95 to 100%.

[0148] Polynucleotides containing mismatches to a target gene or transcript can be used in specific embodiments of the compositions and methods described herein. In some embodiments, the polynucleotide comprises at least 18, at least 19, or at least 21 consecutive nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcript of the target gene. In certain embodiments, a polynucleotide of 18, 19, 20, or 21 or more consecutive nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcript of the target gene may have one or two mismatches to the target gene or transcript (i.e., one or two mismatches between the 21 consecutive nucleotides of the polynucleotide and a segment of equivalent length in the target gene or transcript of the target gene). In certain embodiments, a polynucleotide of about 50, 100, 150, 200, 250, 300, or 350 nucleotides containing 18, 19, 20, or 21 or more consecutive nucleotide spans of identical or complementary length to a sequence of equivalent length in the target gene or a transcript of the target gene may have one or more mismatches with respect to the target gene or transcript.

[0149] In designing polynucleotides having mismatches to an endogenous target gene or to RNA transcribed from said target gene, specific types of mismatches and mismatches at specific, highly tolerable locations can be used. In certain embodiments, mismatches formed between adenine and cytosine or between guanosine and uracil residues are used as described in Du et al. (2005) Nucleic Acids Res., 33:1671-1677. In some embodiments, mismatches in the 19-base pair overlap region are located at low-tolerance positions 5, 7, 8, or 11 (from the 5' end of the 19-nucleotide target), at medium-tolerance positions 3, 4, and 12-17 (from the 5' end of the 19-nucleotide target), and / or at high-tolerance positions at either end of the complementary region, i.e., positions 1, 2, 18, and 19 (from the 5' end of the 19-nucleotide target), as described in Du et al. (2005) Nucleic Acids Res., 33:1671-1677. Acceptable mismatches can be empirically determined in routine assays, such as in vitro diet assays for lepidopteran insect larvae.

[0150] XIV. Embedding of silencing elements in neutral sequences In some embodiments, the silence element having a sequence corresponding to the target gene and involved in the observed suppression of the target gene is embedded in a "neutral" sequence, i.e., inserted into an additional nucleotide that is not identical in sequence to or complementary to the target gene. The neutral sequence may be desirable, for example, to increase the total length of the polynucleotide. For example, the polynucleotide may be desirable to be of a certain size for reasons such as manufacturing stability, cost-effectiveness, or biological activity. In some embodiments, the neutral sequence is also useful for forming loops in the hairpin trigger or as a spacer between trigger regions.

[0151] In another Coleopteran species, Diabrotica virgifera, dsRNAs longer than or equivalent to approximately 60 base pairs (bp) have been reported to be required for biological activity in artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi:10.1371 / journal.pone.0047534. Therefore, in one embodiment, a 21-base-pair dsRNA silencing element, corresponding to the target gene in the aforementioned target gene sequence group and found to provide control of lepidopteran invasion, is embedded in an additional 39-base-pair neutral sequence, thereby forming a polynucleotide of approximately 60 base pairs. In some embodiments, the dsRNA trigger comprises a neutral sequence of base pairs between approximately 60 and 500, or between approximately 100 and 450, in which at least one segment of 21 consecutive nucleotides having a sequence that is 100% identical or 100% complementary to a comparable-length fragment of the target gene having a sequence selected from the target gene sequence set. In another embodiment, it is found that a single 21-base-pair silencing element having a sequence that is 100% identical or 100% complementary to a comparable-length fragment of the target gene is effective when embedded in a neutral sequence of a larger section (e.g., with a total polynucleotide length of approximately 60 to 300 base pairs). In embodiments in which the polynucleotide includes a region of neutral sequence, the polynucleotide has relatively low overall sequence identity with respect to the target gene; for example, a dsRNA having a full length of 210 base pairs, containing a single 21-base-pair trigger (which has 100% identity or complementarity with the 21-nucleotide fragment of the target gene) embedded in an additional 189-base-pair neutral sequence, has about 10% overall sequence identity with respect to the target gene.

[0152] XV. Insecticidal double-stranded RNA molecules Another aspect of the present invention provides an insecticidal double-stranded RNA molecule that causes death or growth inhibition in lepidopteran pests when ingested by or in contact with them, wherein the insecticidal double-stranded RNA molecule comprises at least one segment of 18 or more consecutive nucleotides that is essentially identical or essentially complementary to a target gene or a segment of DNA (cDNA) of equivalent length having a sequence selected from the target gene. In some embodiments, the insecticidal double-stranded RNA molecule is about 50 to about 500 base pairs long. In some embodiments, the insecticidal double-stranded RNA molecule comprises at least one segment of consecutive nucleotides with a length of at least 30. In some embodiments, the insecticidal double-stranded RNA molecule comprises a plurality of segments of 18 or more consecutive nucleotides that are essentially identical or essentially complementary to a target gene or a segment of DNA (cDNA) of equivalent length having a sequence selected from the target gene sequence group, wherein the segments originate from various regions of the target gene (for example, the segments may correspond to various exon regions of the target gene, and “spacer” nucleotides that do not correspond to the target gene may be optionally used between or adjacent to the segments) or from different target genes. In some embodiments, the insecticidal double-stranded RNA molecule comprises a plurality of segments of 18 or more consecutive nucleotides that are essentially identical or essentially complementary to a segment of DNA (cDNA) of equivalent length having a sequence selected from the target gene sequence group, wherein the segments originate from various regions of the target gene and are arranged in the insecticidal double-stranded RNA molecule in an order different from the order in which the segments naturally exist in the target gene.In some embodiments, the insecticidal double-stranded RNA molecule comprises multiple segments of 21 consecutive nucleotides having sequences that are 100% identical or 100% complementary to a target gene or a segment of DNA (cDNA) of equivalent length having a sequence selected from the target gene sequence group, wherein the segments originate from various regions of the target gene and are arranged in the insecticidal double-stranded RNA molecule in an order different from the order in which the segments naturally exist in the target gene. In some embodiments, the insecticidal double-stranded RNA molecule comprises a single strand containing a sequence selected from the trigger sequence group, or a complement thereof. The insecticidal double-stranded RNA molecule can be applied topically to plants to control or prevent the invasion of lepidopteran pests. The insecticidal double-stranded RNA molecule can be provided in a form suitable for ingestion or direct contact by lepidopteran pests, for example, in the form of a spray, powder, or bait. Other methods and suitable compositions for providing the insecticidal double-stranded RNA molecule are similar to those described in the preceding paragraphs with respect to other aspects of the present invention.

[0153] Some embodiments relate to tank mixtures comprising one or more insecticidal polynucleotides and water or other solvents, optionally comprising cationic lipids or organosilicone surfactants, or both. Embodiments include a tank mixture formulation of the polynucleotides and at least one insecticide optionally selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. Embodiments of such compositions include those in which one or more insecticidal polynucleotides are provided in living or dead microorganisms such as bacteria, fungi, or yeast cells, or as microbial fermentation products, or in living or dead plant cells, or as synthetic recombinant polynucleotides. In one embodiment, the composition comprises a non-pathogenic strain of a microorganism containing the polynucleotide described herein; ingestion or uptake of the microorganism results in inhibition of growth or death of the lepidopteran pest; non-limiting examples of suitable microorganisms include E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and related Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium bifermentans and other Clostridium species, or other spore-forming Gram-positive bacteria. In one embodiment, the composition comprises a plant virus vector containing the polynucleotide described herein; ingestion by a lepidopteran pest of a plant treated with the plant virus vector results in inhibition of growth or death of the lepidopteran pest. In one embodiment, the composition comprises a baculovirus vector containing a polynucleotide as described herein; ingestion or uptake by a vector results in inhibition of growth or death of a lepidopteran pest.In one embodiment, the polynucleotide described herein is encapsulated in a synthetic matrix such as a polymer or attached to microparticles and applied topically to the surface of a plant; feeding of the topically treated plant by lepidopteran pests results in inhibition of the growth of the lepidopteran pests or death of the lepidopteran pests. In another embodiment, the polynucleotide described herein is provided in the form of plant cells expressing the polynucleotide (e.g., transgenic Solanaceae plant cells of the present invention); feeding of the plant cells or the contents of the plant cells by lepidopteran pests results in inhibition of the growth of the lepidopteran pests or death of the lepidopteran pests.

[0154] In some embodiments, one or more polynucleotides described herein are provided together with suitable adhesives and wetting agents necessary for efficient leaf covering, as well as UV protectants to protect polynucleotides such as dsRNA from UV damage. Such additives are commonly used in the biopesticide industry and are known to those skilled in the art. Compositions for soil application may include granular formulations that serve as food for lepidopteran insect larvae. In some embodiments, one or more polynucleotides described herein are further provided together with carriers, surfactants, cationic lipids (e.g., disclosed in Example 18 of U.S. Patent Application Publication 2011 / 0296556, incorporated herein by reference), organosilicones, organosilicon surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators. In some embodiments, the composition further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein.

[0155] Such compositions may be applied in any convenient manner, such as by spraying or scattering them directly onto lepidopteran pests, or by spraying or scattering them onto plants or environments where prevention or control of lepidopteran pest infestation is desired, or by applying a coating to the surface of plants, or by applying a coating to seeds (or seed potatoes) in preparation for planting, or by applying soil trenches around the roots of plants where prevention or control of lepidopteran pest infestation is desired.

[0156] The effective amounts of polynucleotides described herein are sufficient to provide control of lepidopteran pests or to prevent invasion by lepidopteran pests; the determination of the effective amount of polynucleotide is performed using routine assays. There is no upper limit to the concentration and dose of insecticidal polynucleotides that may be useful in the methods and compositions provided herein, but lower effective concentrations and doses are generally desired for efficiency and economic reasons. Non-limiting embodiments of an effective amount of polynucleotide include polynucleotides in the form of a liquid sprayed on one plant, ranging from about 10 nanograms to about 100 micrograms per milliliter, or polynucleotides in the range of about 10 milligrams to about 100 grams per acre when applied to a field of plants, or polypeptides in the range of about 0.001 micrograms to about 0.1 micrograms per milliliter in an artificial feed for feeding lepidopteran pests. When the polynucleotides described herein are applied topically to plants, the concentration can be adjusted considering the amount of spray or treatment applied to the surface of the plant's leaves or other plant parts, e.g., petals, stems, tubers, fruits, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using the 25-amino acid length polynucleotides described herein is about 1 nanomol (nmol) of polynucleotides per plant, e.g., about 0.05 to 1 nmol of polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of ssDNA polynucleotides are applied. In certain embodiments, about 0.5 nmol to about 2 nmol of dsRNA are applied. In certain embodiments, compositions containing about 0.5 to about 2.0 milligrams of dsRNA or ssDNA (21mer) per milliliter are applied. In certain embodiments, compositions of the dsRNA polynucleotide of the present invention containing about 50 to about 200 or more nucleotides, with a content of about 0.5 to about 1.5 milligrams per milliliter, are applied.In certain embodiments, about 1 to about 5 nanomoles of the dsRNA of the present invention is applied to a single plant. In certain embodiments, the polynucleotide composition applied topically to the plant contains at least one polynucleotide of the present invention at a concentration of about 0.01 to about 10 milligrams, or about 0.05 to about 2 milligrams, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines may require correspondingly larger amounts of polynucleotides. Lower concentrations can be used when using the long dsRNA molecule of the present invention, which can be processed into multiple oligonucleotides (e.g., multiple triggers encoded by a single recombinant DNA molecule of the present invention). Non-limiting examples of effective polynucleotide treatment regimens include treatment of about 0.1 to about 1 nmol of polynucleotide molecules per plant, or about 1 nmol to about 10 nmol of polynucleotide molecules per plant, or about 10 nmol to about 100 nmol of polynucleotide molecules per plant.

[0157] In some embodiments, one or more polynucleotides are provided together with a “mobilization agent,” which is an agent that enables locally applied polynucleotides to enter the cells of an organism. Such a mobile agent can be incorporated as part of a composition comprising the polynucleotides described herein, or can be applied prior to, simultaneously with, or after the application of the polynucleotides. In some embodiments, the mobile agent is an agent that improves the uptake of the polynucleotides of the present invention by lepidopteran pests. In some embodiments, the mobile agent is an agent that conditions the surface of plant tissue, e.g., seeds, leaves, stems, roots, flowers, or fruits, for the permeation of plant cells by polynucleotides. In some embodiments, the mobile agent enables a pathway for the polynucleotides to enter plant cells through the epidermal wax barrier, stomata, and / or cell wall or membrane barrier.

[0158] Suitable transfer agents include agents that increase the permeability of the organism to the outside world or to the permeability of the organism's cells to the polynucleotide. Suitable transport agents include chemical agents, physical agents, or combinations thereof. Chemicals for conditioning or transport include (a) surfactants, (b) organic solvents or aqueous solvents or mixtures of aqueous organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or any combination thereof. In some embodiments, the application of the polynucleotide and transport agent optionally includes an incubation step, a neutralization step (e.g., for neutralizing an acid, base, or oxidizing agent, or for inactivating an enzyme), a washing step, or a combination thereof. Suitable transport agents may be in the form of emulsions, reversed-phase emulsions, liposomes, or other micelle-like compositions, or the polynucleotide can be made to take the form of emulsions, reversed-phase emulsions, liposomes, or other micelle-like compositions. Embodiments of the transfer agent include counterions or other molecules known to associate with nucleic acid molecules, such as inorganic ammonium ions, alkylammonium ions, lithium ions, spermine, spermidine, or putrescine, and other polyamines, and other cations. Embodiments of the transfer agent include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoroamide, acetonitrile, dioxane, polypropylene glycol, or solvents miscible with water or that dissolve phosphonucleotides in a non-aqueous system (e.g., those used in synthesis reactions). Embodiments of the transfer agent include naturally derived or synthetic oils, with or without surfactants or emulsifiers, such as plant-derived oils, crop oils (e.g., those listed in the 9th Compendium of Herbicide Adjuvants and published online in the 9th Compendium of Herbicide Adjuvants), paraffinic oils, polyol fatty acid esters, or oils having short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0159] Embodiments of the transport agent include organosilicone formulations. For example, a suitable transport agent is an organosilicone formulation, commercially available as a SILWET L-77 (trademark registered) brand surfactant with CAS number 27306-78-1 and EPA number CAL.REG.No.5905-50073-AA, and is currently available from Momentive Performance Materials, Albany, NY. One embodiment comprises a composition containing a polynucleotide and a transfer agent containing an organosilicone formulation such as SILWET L-77 in an amount ranging from about 0.015 to about 2% by weight (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 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, 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% by weight). One embodiment comprises a composition containing the polynucleotide of the present invention and a transfer agent containing a surfactant of the SILWET L-77 (trademark registered) brand in an amount ranging from about 0.3 to about 1% (weight percent) or from about 0.5 to about 1% (weight percent).

[0160] Organosilicone compounds useful as transfer agents for use in the present invention include, but are not limited to, compounds comprising (a) a covalently linked trisiloxane tip group, (b) a covalently linked alkyl linker including, but not limited to, an n-propyl linker, (c) a covalently linked polyglycol chain, and (d) a terminal group. The trisiloxane tip group of such organosilicone compounds includes, but is not limited to, heptamethyltrisiloxane. The alkyl linker may include, but is not limited to, an n-propyl linker. The polyglycol chain includes, but is not limited to, polyethylene glycol or polypropylene glycol. The polyglycol chain may include a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14. The terminal group may include, but is not limited to, an alkyl group such as a methyl group. Organosilicone compounds useful as transfer agents include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide-modified heptamethyltrisiloxane. An example of a mobile agent for use in the present invention is compound I: [ka] (Compound I: Polyalkylene oxide heptamethyltrisiloxane, average n=7.5) That is the case. Organosilicone compounds useful as transport agents are used in newly created concentrations ranging from approximately 0.015% to approximately 2% by weight (weight percent) (e.g., approximately 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 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, 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 weight percent).

[0161] Embodiments of the transfer agent include one or more salts of ammonium chloride, tetrabutylphosphonium bromide, ammonium sulfate, etc., provided in or used in a composition containing a polynucleotide. In some embodiments, ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate are used at concentrations of about 0.5% to about 5% (w / v), or about 1% to about 3% (w / v), or about 2% (w / v). In certain embodiments, the composition containing a polynucleotide contains the ammonium salt at a concentration of 300 millimolar or more or equivalent. In certain embodiments, the composition containing a polynucleotide contains an organosilicon transfer agent at a concentration of about 0.015% to about 2% (weight percent) by weight and ammonium sulfate at a concentration of about 80 to about 1200 mM or about 150 mM to about 600 mM.

[0162] Embodiments of the transfer agent include phosphates. Useful phosphates in compositions containing polynucleotides include, but are not limited to, calcium, magnesium, potassium, or sodium phosphates. In certain embodiments, the composition containing polynucleotides contains phosphates at concentrations of at least about 5 mM, at least about 10 mM, or at least about 20 mM. In certain embodiments, the composition contains polynucleotide phosphates in the range of about 1 mM to about 25 mM or about 5 mM to about 25 mM. In certain embodiments, the composition contains polynucleotide sodium phosphate at concentrations of at least about 5 mM, at least about 10 mM, or at least about 20 mM. In certain embodiments, the composition containing polynucleotides contains sodium phosphate at concentrations of about 5 mM, about 10 mM, or about 20 mM. In certain embodiments, the composition containing polynucleotides contains sodium phosphate salts in the range of about 1 mM to about 25 mM or about 5 mM to about 25 mM. In certain embodiments, the polynucleotide-containing composition contains sodium phosphate in a concentration ranging from about 10 mM to about 160 mM or from about 20 mM to about 40 mM. In certain embodiments, the polynucleotide-containing composition contains a sodium phosphate buffer at a pH of about 6.8.

[0163] Embodiments of the transfer agent include surfactants and / or effective molecules contained herein. These surfactants and / or effective molecules include, but are not limited to, sodium or lithium salts of fatty acids (e.g., animal fats, beef tallow fatty acids, or phospholipids) and organosilicon surfactants. In certain embodiments, compositions containing polynucleotides are formulated with counterions or other molecules known to associate with nucleic acid molecules. Non-limiting examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, compositions containing polynucleotides are formulated together with non-polynucleotide herbicides, such as auxin-like benzoic acid herbicides containing glyphosate, dicamba, chloramben, and TBA; auxin-like herbicides containing glufosinate and phenoxycarboxylic acid herbicides; pyridinecarboxylic acid herbicides; quinoline carboxylic acid herbicides; pyrimidinecarboxylic acid herbicides; and benazoline ethyl herbicides; sulfonylurea, imidazolinones, bromoxynil, derapon, cyclohezandione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenylpyruvate-dioxygenase inhibitory herbicides. In certain embodiments, compositions containing polynucleotides are formulated together with non-polynucleotide insecticides, such as patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. In some embodiments, compositions containing polynucleotides and non-polynucleotide insecticides provide enhanced improvements in preventing or controlling lepidopteran pest infestations compared to the effects obtained by polynucleotides alone or non-polynucleotide insecticides alone.In some embodiments, compositions comprising double-stranded RNA having strands containing sequences selected from the trigger sequence group are combined with non-polynucleotide insecticides (patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein), where the combination is found to be effective in improved prevention or control of lepidopteran pest invasion compared to the effects obtained by double-stranded RNA alone or by non-polynucleotide insecticide alone.

[0164] XVI. Related Technologies Implementations of polynucleotides and nucleic acid molecules described herein may include additional elements, such as additional expression cassettes for expressing coding sequences (e.g., for expressing transgenes such as insecticidal proteins or selection markers) or non-coding sequences (e.g., for expressing additional repressors), in addition to promoters, small RNA recognition sites, aptamers, or ribozymes. For example, one aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably ligated to DNA comprising 18 or more consecutive nucleotides having about 95% to about 100% identity to equivalent length fragments of DNA having sequences selected from the target gene sequence group or the trigger sequence group, or at least one complementary segment of DNA. Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably ligated to DNA encoding an RNA hairpin having an antisense region having a sequence or a fragment of a sequence selected from the group selected from the trigger sequence group. In another embodiment, a recombinant DNA construct comprising (a) an RNA silencing element for repressing a target gene selected from a group of target gene sequences, and (b) a promoter operably ligated to the DNA encoding the aptamer, is stably integrated into the plant genome from the site where the RNA transcript containing the RNA aptamer and the RNA silencing element are expressed in the plant cell; the aptamer plays a role in guiding the RNA silencing element to a desired location in the cell. In another embodiment, the inclusion of one or more recognition sites for binding and cleavage by small RNA molecules (e.g., by miRNA or siRNA expressed only in specific cells or tissues) enables a more precise expression pattern in the plant, where the expression of the recombinant DNA construct is repressed at the site where the small RNA molecules are expressed. Such additional elements are described below.

[0165] XVII. Promoter The promoters used in the present invention are functional in cells in which the constituents are intended to be transcribed. Generally, these promoters are heterologous promoters used in recombinant constituents, i.e., they are not naturally found to be operablely linked to other nucleic acid elements used in the constituents described herein. In various embodiments, the promoter is selected from the group consisting of structural promoters, spatially specific promoters, temporally specific promoters, developmentally specific promoters, and inducible promoters. In many embodiments, the promoter is a promoter that functions in plants, such as the polII promoter, polIII promoter, polV promoter, or polV promoter.

[0166] Non-structural promoters suitable for use with the recombinant DNA components of the present invention include spatially specific promoters, time-specific promoters, and inducible promoters. Spatially specific promoters include organelle, cell, tissue, or organ-specific promoters (e.g., plastid-specific, root-specific, pollen-specific, or seed-specific promoters for expression in plastids, roots, pollen, or seeds, respectively). Seed-specific, embryo-specific, starch-specific, or endosperm-specific promoters are often particularly useful. Time-specific promoters may include promoters that tend to promote expression at specific developmental stages in the plant growth cycle, at different times of day or night, or at different seasons of the year. Inducible promoters include promoters that are induced by chemicals or by environmental conditions (not limited to) such as biological or abiotic stress (e.g., water shortage or drought, heat, cold, high or low levels of nutrients or salinity, high or low levels of light, pest or pathogen infection). MicroRNA promoters are particularly useful for those having temporally specific, spatially specific, or induceable expression patterns; examples of miRNA promoters, and methods for identifying miRNA promoters having specific expression patterns, are provided in U.S. Patent Publications 2006 / 0200878, 2007 / 0199095, and 2007 / 0300329, which are incorporated herein by special reference. Expression-specific promoters may also include promoters that are generally structurally expressed but differ in degree or "strength" of expression, and may include promoters generally considered to be "strong promoters" or "weak promoters."

[0167] Specific promoters of interest include: the opaline synthase promoter isolated from Agrobacterium T-DNA; the cauliflower mosaic virus 35S promoter; for example, the enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer element; root-specific promoters such as those disclosed in U.S. Patents 5,837,848, 6,437,217 and 6,426,446; the maize L3 oleosin promoter disclosed in U.S. Patent 6,433,252; and rice. Examples include the promoter for plant nuclear genes encoding plastid-localized aldolases disclosed in U.S. Patent Application Publication No. 2004 / 0216189; the cold-inducible promoter disclosed in U.S. Patent No. 6,084,089; the salt-inducible promoter disclosed in U.S. Patent No. 6,140,078; the light-inducible promoter disclosed in U.S. Patent No. 6,294,714; the pathogen-inducible promoter disclosed in U.S. Patent No. 6,252,138; and the water-deficiency-inducible promoter disclosed in U.S. Patent Application Publication No. 2004 / 0123347A1. All of the above patents and patent publications disclosing promoters and their use, particularly their use in recombinant DNA components functioning in plants, are incorporated herein by reference.

[0168] Promoters specific to the blood vessels or phloem of the plant of interest include the rolC or rolA promoter of Agrobacterium rhizogenes, the promoter of the Agrobacterium tumefaciens T-DNA gene 5, the inescrose synthase RS1 gene promoter, the Commelina yellow spotted budna virus promoter, the coconut leaf blight virus promoter, the rice tungrobacillus-like virus promoter, the pea glutamine synthetase GS3A gene promoter, and the invCD111 and invCD141 promoters of the potato invertase gene, according to Kertbundit et al. (1991) Proc. Natl. Acad. Sci. USA. This includes promoters isolated from Arabidopsis thaliana, which were shown to have phloem-specific expression in tobacco by 88:5212-5216; the VAHOX1 promoter region; the pea cell wall invertase gene promoter; the carrot-derived acid invertase gene promoter; the sulfate transporter gene Sultr1;3 promoter; the plant sucrose synthetase gene promoter; and the plant sucrose transporter gene promoter.

[0169] Promoter suitable for use with recombinant DNA components or polynucleotides of the present invention include polymerase II ("polII") promoters and polymerase III ("polIII") promoters. RNA polymerase II transcribes structural or catalytic RNA typically shorter than 400 nucleotides in length and recognizes a simple run of T residues as a termination signal; it is used for transcription of siRNA double-stranded RNA (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Therefore, the PolII promoter is included in certain embodiments in which short RNA transcripts are produced from recombinant DNA components of the present invention. In one embodiment, the recombinant DNA component comprises a polII promoter and expresses an RNA transcript adjacent to a self-cleaving ribozyme sequence (e.g., a self-cleaving hammerhead ribozyme) to result in a modified RNA, such as a single-stranded RNA that binds to a lepidopteran target gene transcript, having distinct 5' and 3' ends without potentially interfering adjacent sequences. Another approach involves using the polIII promoter to produce transcripts with relatively distinct 5' and 3' ends, i.e., to transcribe RNA with minimal 5' and 3' contiguous sequences. In some embodiments, the PolIII promoter (e.g., U6 or H1 promoter) is intended to add a short AT-rich transcription termination site to the transcribed RNA, resulting in a two-base-pair overhang (UU); this is useful, for example, for the expression of siRNA-type components. The use of the polIII promoter to drive the expression of siRNA components has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448.Baculovirus polyhedrins and baculovirus promoters such as the p10 promoter are publicly known and commercially available in the art; see, for example, Invitrogen's “Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques”, 2002 (Life Technologies, Carlsbad, Calif.) and FJ Haines et al. “Baculovirus Expression Vectors”, undated (Oxford Expression Technologies, Oxford, UK).

[0170] The promoter element may include nucleic acid sequences that are not naturally occurring promoters or promoter elements or their analogues, but are capable of regulating gene expression. Examples of such “gene-independent” regulatory sequences include naturally occurring or artificially designed RNA sequences that include ligand-binding regions or aptamers (see “Aptamers” below) and regulatory regions (which can act cis-acting). For example, see Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such “riboregulators” can be selected or designed for specific spatial or temporal specificity, for example, to regulate the translation of DNA encoding silencing elements for repressing lepidopteran target genes only in the presence (or absence) of a suitable ligand at a given concentration. One example is a riboregulator that responds to endogenous ligands (e.g., jasmonic acid or salicylic acid) produced by plants when subjected to stress (e.g., abiotic stress such as water or temperature, or biological stress such as nutrient stress or attachment by pests or pathogens); when subjected to stress, the level of the endogenous ligand increases to a level sufficient for the riboregulator to initiate transcription of DNA encoding silencing elements for repressing lepidopteran target genes. XVIII. Recombinase site In some embodiments, the recombinant DNA component or polynucleotide of the present invention comprises DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA component comprises at least one pair of loxP sites, where site-specific recombination of DNA between loxP sites is mediated by Cre recombinase. The position and relative orientation of the loxP sites are selected to achieve the desired recombination; for example, if the loxP sites are oriented in the same direction, the DNA between the loxP sites is cut in a ring. In another embodiment, the recombinant DNA component comprises DNA encoding one loxP site; the two DNAs are recombined in the presence of Cre recombinase and another DNA having a loxP site.

[0171] XIX. Aptamer In some embodiments, the recombinant DNA components or polynucleotides of the present invention include DNA that is processed into RNA aptamers, i.e., RNA that binds to a ligand via a binding mechanism not primarily based on Watson-Crick base pairing (in contrast, for example, base pairing occurring between complementary antiparallel nucleic acid strands results in the formation of a double-stranded nucleic acid structure). See, for example, Ellington and Szostak (1990) Nature, 346:818-822. Examples of aptamers are available online, for example, in the publicly available aptamer database aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100). However, aptamers useful in the present invention may be monovalent (binding to a single ligand) or polyvalent (binding to two or more individual ligands, e.g., binding to one unit of two or more different ligands).

[0172] Ligands useful in the present invention include any molecule (or part of a molecule) that can be recognized and bound to nucleic acid secondary structures by a mechanism not based primarily on Watson-Crick base pairing. Thus, the recognition and binding of ligands and aptamers is analogous to the recognition and binding of antigens and antibodies, or biological effectors and receptors. Ligands may include a single molecule (or part of a molecule) or a combination of two or more molecules (or parts of molecules), and may include a complex of one or more macromolecules (e.g., polymers, lipid bilayers, liposomes, cell membranes or other cellular structures, or cell surfaces). Examples of specific ligands include vitamins such as coenzyme B12 and thiamine pyrophosphate; flavin mononucleotides, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, chloramphenicol and neomycin; herbicides such as glyphosate and dicamba; proteins such as virus or phage coat proteins and invertebrate epidermal or gastrointestinal surface proteins; and RNAs such as viral RNA, transfer RNA (t-RNA), ribosomal RNA (rRNA), and RNA polymerase (RdRP). One class of RNA aptamers useful in the present invention is one that does not bind to a ligand but is thermally responsive, i.e., a "thermal switch" whose conformation is determined by temperature; see, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

[0173] XX. Transgene transcription unit In some embodiments, the recombinant DNA components or polynucleotides of the present invention include a transgene transcription unit. The transgene transcription unit includes a DNA sequence encoding a gene of interest, such as a native protein or a heterologous protein. The gene of interest may be any coding or non-coding sequence derived from any species (non-eukaryotes such as bacteria and viruses; including, but not limited to, fungi, protists, plants, invertebrates, and vertebrates). A specific gene of interest is a gene encoding at least one insecticide selected from the group consisting of patatin, plant lectins, plant ecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein. The transgene transcription unit may further include a 5' or 3' sequence, or both, necessary for the transcription of the transgene.

[0174] XXI.Intron In some embodiments, the recombinant DNA components or polynucleotides of the present invention include DNA encoding splicable introns. “Intron” generally refers to a segment of DNA (or RNA transcribed from such a segment) located between exons (protein-coding segments of DNA or corresponding transcribed RNA), where, during messenger RNA maturation, any present introns are enzymatically “spliced” or removed from the RNA strand by a cleavage / ligation process that occurs in the nucleus in eukaryotes. The term “intron” also applies to non-coding DNA sequences transcribed from a mature RNA transcript into a splicable RNA segment, but not to introns found between protein-coding exons. One example of these is a splicable sequence that has the ability to promote the expression of a downstream coding sequence in plants (in some cases, especially monocots); these splicable sequences are naturally present in the 5' untranslated regions of some plant genes, as well as in the tobacco mosaic virus leader sequence or "omega" leader, which is described as promoted expression in plant genes by some viral genes (e.g., Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These splicable sequences or "expression-promoting introns" can be artificially inserted into the 5' untranslated region between promoters of plant genes (not before any protein-coding exons). Examples of introns that promote such expression include, but are not limited to, the maize alcohol dehydrogenase (Zm-Adh1), the maize bronze-1 expression-promoting intron, the rice actin-1 (Os-Act1) intron, the Schrunken-1 (Sh-1) intron, the maize sucrose synthetase intron, the heat shock protein 18 (hsp18) intron, and the 82 kilodalton heat shock protein (hsp82) intron.U.S. Patents 5,593,874 and 5,859,347, incorporated herein by reference, describe a method for improving recombinant DNA constructs for use in plants by including an intron that promotes expression derived from 70 kilodaltons of maize heat shock protein (hsp70) in an untranslated reader located at the 3' position from the gene promoter and the 5' position from the exon encoding the first protein.

[0175] XXII. Ribozyme In some embodiments, the recombinant DNA component or polynucleotide of the present invention comprises DNA encoding one or more ribozymes. Specific ribozymes of interest include self-cleaving ribozymes, hammerhead ribozymes, or hairpin ribozymes. In one embodiment, the recombinant DNA component comprises DNA encoding one or more ribozymes that help to cleave the transcribed RNA to provide a specific segment of RNA, such as a silencing element for suppressing a target gene in lepidopterans.

[0176] XXIII. Gene suppression factors In some embodiments, the recombinant DNA components or polynucleotides of the present invention include DNA encoding additional gene repressor elements for repressing target genes other than lepidopteran target genes. The repressed target genes may include coding sequences, non-coding sequences, or both.

[0177] Appropriate gene suppressors are described in detail in U.S. Patent Application Publication No. 2006 / 0200878, the disclosure of which is specifically incorporated herein by reference, below: (a) DNA comprising at least one antisense DNA segment that is antisense with respect to at least one segment of the gene to be repressed; (b) DNA comprising multiple copies of at least one antisense DNA segment that is antisense with respect to at least one segment of the gene to be repressed; (c) DNA comprising at least one sense DNA segment which is at least one segment of the gene to be suppressed; (d) DNA comprising multiple copies of at least one sense DNA segment which is at least one segment of the gene to be suppressed; (e) DNA transcribed into RNA for repressing the gene that is repressed by forming double-stranded RNA, comprising at least one antisense DNA segment which is antisense to at least one segment of the gene that is repressed, and at least one sense DNA segment which is at least one segment of the gene that is repressed; (f) DNA for transcribing into RNA for repressing a gene that is repressed by forming a single double-stranded RNA, comprising a plurality of consecutive antisense DNA segments that are antisense to at least one segment of the gene to be repressed, and a plurality of consecutive sense DNA segments that are at least one segment of the gene to be repressed. (g) DNA for transcribing into RNA for repressing a gene that is repressed by forming multiple double strands of RNA, comprising a plurality of antisense DNA segments which are antisense to at least one segment of the gene to be repressed, and a plurality of sense DNA segments which are at least one segment of the gene to be repressed, wherein the plurality of antisense DNA segments and the plurality of sense DNA segments are arranged in a series of reverse repeats; (h) DNA containing nucleotides derived from plant miRNA; (i) DNA containing siRNA nucleotides; (j) DNA that is transcribed into an RNA aptamer capable of binding to a ligand; and (k) DNA transcribed into an RNA aptamer capable of binding to a ligand, and DNA transcribed into a regulatory RNA capable of regulating the expression of the repressed gene, wherein the regulation depends on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer. Includes one or more of the following.

[0178] In some embodiments, introns are used to deliver gene repressor elements in the absence of any protein-coding exons (coding sequences). In one example, an intron, such as an intron that promotes expression, is intercepted by embedding a gene repressor element within the intron, where the gene repressor element is then excised from the intron during transcription. Therefore, protein-coding exons do not need to provide the gene repression function of the recombinant DNA components disclosed herein.

[0179] XXIV. Transcriptional Regulators In some embodiments, the recombinant DNA components or polynucleotides of the present invention include DNA encoding transcriptional regulators. These transcriptional regulators include elements that modulate the expression level of the recombinant DNA components of the present invention (compared to expression in the absence of such regulators). Examples of suitable transcriptional regulators include riboswitches (cis or trans), transcript stabilization sequences, and miRNA recognition sites, as detailed in particular in U.S. Patent Application Publication No. 2006 / 0200878, incorporated herein by reference.

[0180] XXIV. Production and Utilization of Transgenic Plant Cells and Transgenic Plants Plant transformation may include any known method and composition. Suitable methods for plant transformation include virtually any method that can introduce DNA into cells. One method of plant transformation is irradiation with a particulate gun, as exemplified in U.S. Patent Nos. 5,015,580 (Soybeans), 5,538,880 (Maize), 5,550,318 (Maize), 5,914,451 (Soybeans), 6,153,812 (Wheat), 6,160,208 (Maize), 6,288,312 (Rice), 6,365,807 (Rice), 6,399,861 (Maize), and 6,403,865 (Maize), any of which are incorporated as references to enable the production of transgenic plants.

[0181] Another useful method of plant transformation is Agrobacterium-mediated transformation by Agrobacterium containing a two-component Ti plasmid system, where Agrobacterium contains a first Ti plasmid and a second chimeric plasmid containing at least one T-DNA boundary of a wild-type Ti plasmid, which functions in the transformed plant cell and holds a promoter operably ligated to the polynucleotide or recombinant DNA component of the present invention. See, for example, the two-component system described in U.S. Patent No. 5,159,135 incorporated as reference. Also see De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smaller plasmid containing one or more T-DNA boundaries can be conveniently constructed and manipulated in a suitable alternative host such as E. coli, and then transferred to Agrobacterium.

[0182] Detailed procedures for Agrobacterium-mediated transformation of plants, particularly crop plants, are described in U.S. Patents Nos. 5,004,863, 5,159,135, 5,518,908 (Cotton); U.S. Patents Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (Soybean); U.S. Patents Nos. 5,591,616 and 5,981,840 (Maize); No. 5 This includes procedures disclosed in U.S. Patent Nos. 463,174 (Canola and other rapeseed), No. 7,026,528 (Wheat), and No. 6,329,571 (Rice), and U.S. Patent Publication Nos. 2004 / 0244075 (Maize) and 2001 / 0042257A1 (Sugar beet), all of which are specifically incorporated by reference to enable the production of transgenic plants. U.S. Patent Publication No. 2011 / 0296555 discloses in Example 5 a transformation vector (including the vector sequence) and a detailed protocol for transforming maize, soybeans, canola, cotton, and sugarcane, which are specifically incorporated by reference to enable the production of transgenic plants. Similar methods have been reported for many plant species, both dicotyledonous and monocotyledonous, including peanuts (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA, 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio / Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379); wheat (Vasil et al. (1992) Bio / Technology, 10:667; Becker et al. (1994) Plant J., 5:299); and alfalfa (Masoud et al. (1996) Transgen. This includes Res., 5:313; and tomatoes (Sun et al. (2006) Plant Cell Physiol., 47:426-431).See also U.S. Patent Application Publication No. 2003 / 0167537A1, incorporated by reference, for a vector of transformed Arabidopsis thaliana plants in which a transcription factor is constitutively expressed by a CaMV35S promoter, a method of transformation, and a description of production. Transformation methods particularly useful for Solanaceae plants are well known in this field, for example, tomato (Sharma et al. (2009), J. Biosci., 34:423-433), J. Biosci., 34:423-433), eggplant (Arpaia et al. (1997) Theor. Appl. Genet., 95:329-334), potato (Bannerjee et al. (2006) Plant Sci., 170:732-738; Chakravarty et al. (2007) Amer. J. Potato Res., 84:301-311; S. Millam “Agrobacterium-mediated transformation of potato.” Chapter 19 (pp. 257-270), “Transgenic Crops of the World: Essential Protocols”, Ian S. Curtis (editor), Springer, See the publicly described transformation methods for peppers (Li et al. (2003) Plant Cell Reports, 21: 785-788), 2004.Stable transgenic potatoes, tomatoes, and eggplants have been commercially introduced in various fields; see, for example, K. Redenbaugh et al. “Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FLAVR SAVR Tomato”, CRC Press, Boca Raton, 1992, and for documents on commercially transgenic crops in the extensive publicly available GM crop database; CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC, available electronically at cera-gmc.org / ?action=gm_crop_database.Various methods of transgenicizing other plant species are known in the art, for example, see the encyclopedia reference “Compendium of Transgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (Available electronically at mrw.interscience.wiley.com / emrw / 9781405181099 / hpt / toc) includes cereals and fodder grasses (rice, maize, wheat, barley, oats, sorghum, pearl millet, finger millet, cold-season fodder grasses, and bahia grass), oilseed crops (soybeans, rapeseed, sunflowers, peanuts, flax, sesame, safflower), legumes and fodder (kidney beans, cowpeas, peas, broad beans, lentils, teparry beans, Asian beans, pigeon peas, vetch, chickpeas, fan beans, alfalfa, and clover), temperate fruits and nuts (apples, pears, peas, plums, berries, cherries, Transformation procedures are described for grapes, olives, almonds, Persian walnuts, tropical and subtropical fruits and nuts (citrus fruits, grapefruit, banana plantains, pineapples, papayas, mangoes, avocados, kiwifruit, passionfruit, and persimmons), vegetable crops (tomatoes, eggplants, bell peppers, rapeseed, radishes, carrots, melons, leeks, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beets, stevia, potatoes, sweet potatoes, cassava, and cotton), plantation crops, ornamental plants, and turfgrasses (tobacco, coffee, cocoa, tea, rubber trees, medicinal plants, ornamental plants, and turfgrasses), and forest tree species.

[0183] Transformation methods for providing transgenic plant cells and transgenic plants containing stably incorporated recombinant DNA are preferably carried out in tissue culture on a culture medium and in a controlled environment. “Culture medium” refers to a mixture of various nutrients used to grow cells in vitro, i.e., in an intact in vivo environment. Target recipient cells include, but are not limited to, meristem cells, callus, immature embryos or embryonic parts, and gamete cells such as microspores, pollen, sperm, and egg cells. Any cell capable of regenerating a reproductively viable plant is intended to be a useful recipient cell for carrying out the present invention. Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or embryonic parts, seedling apical meristem, and microspores. These cells, capable of growing as callus, can function as recipient cells for genetic transformation. Practical transformation methods and materials for producing transgenic plants of the present invention (e.g., transformation of target cells of various culture media and recipients, immature embryos, and subsequent regeneration of fertile transgenic plants) are specifically incorporated by reference, for example, in U.S. Patent No. 6,194,636 and U.S. Patent Application Publication No. 2004 / 0216189.

[0184] In typical transformation procedures, DNA is introduced into target cells in small quantities during each transformation experiment. Generally, marker genes are used to provide an efficient system for identifying cells that are stably transformed by receiving and integrating transgenic DNA components into their genome. Preferred marker genes provide selective markers that confer resistance to selective agents such as antibiotics or herbicides. Any antibiotic or herbicide to which plant cells are resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of viable cells, the target cells are generally those to which the resistance-constituting gene is integrated and expressed at a level sufficient to allow cell survival. Cells can be further tested to confirm stable integration of recombinant DNA. Commonly used selective marker genes include those that confer resistance to antibiotics such as kanamycin or paromomycin (nptll), hygromycin B (aphIV), and gentamicin (aac3 and aacC4), or to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selectors are exemplified in U.S. Patents 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters, such as markers that provide the ability to visually identify transformants, can also be used. Examples of useful screenable markers include, for example, genes that produce a detectable color by acting on a chromogenic substrate (e.g., β-glucuronidase (GUS) (uidA) or luciferase (luc)) or genes that express proteins that are themselves detectable, such as green tendency protein (GFP) (gfp) or immunogenic molecules. Those skilled in the art will ...

Claims

1. below: (a) Contacting the lepidopteran pest with at least one polynucleotide that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from said target gene, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (b) Providing the lepidopteran pest with the diet of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from the target gene, at least one polynucleotide sequence that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (c) Inducing death or inhibiting the growth of the larvae of a lepidopteran pest by providing the lepidopteran pest with at least one polynucleotide containing a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from said target gene, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (d) Applying topically to the plant a composition comprising at least one polynucleotide that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from said target gene, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (e) Locally applying at least one polypeptide to the plant in such a manner that an effective amount of the polynucleotide is ingested by lepidopterans feeding on at least one polynucleotide containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from the target gene, which is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (f) Expressing in the plant at least one polynucleotide that is essentially identical or complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of DNA having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or that is essentially identical or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (g) A method for preventing the invasion of a lepidopteran pest into a plant, comprising contacting the lepidopteran pest with at least one double-stranded or single-stranded effective amount of DNA having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, which is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides, or at least complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides.

2. The method according to claim 1, wherein the at least one polynucleotide is a double-stranded RNA.

3. The method according to claim 2, wherein the double-stranded RNA is synthesized chemically or enzymatically, or produced by expression in a microorganism or in a plant cell.

4. The method of claim 2, wherein the double-stranded RNA is sequence numbers 115, 116, 119, 121, 123, 127, 135, 140, 141, 143, 150, 151, 153, 158, 161, 201, 209, 213, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044 ,1047,1089,1090,1091,1095,1096,1098,1099,1102,1103,1334,1335,1336,1341,1343,1346,1351,1353,1354,1362,1363,1377,1386,1387,1393,1396,1417,1420,1422,1425,1426,1446,1447,1470,1473,1481,1493,149 A method comprising at least about 50 consecutive nucleotides, at least about 100, at least about 150, or at least about 200 consecutive nucleotides of a sequence selected from the group consisting of 4, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, plus one chain complementary to at least about 100, at least about 150, or at least about 200 consecutive nucleotides.

5. The method of claim 1, wherein the method comprises topically applying to a plant a composition comprising at least one polynucleotide having a nucleotide sequence complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene or RNA transcribed from the target gene, or complementary to nucleotides having at least 90%, 95%, or 98% identity of said consecutive nucleotides, wherein the target gene is Sequence ID No. 2, 5, 7 ,9,13,21,26,27,29,36,37,39,44,47,87,95,99,103,301,309,318,319,320,321,323,324,328,341,343,346,356,358,359,503,504,505,506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672 ,717,730,731,736,737,738,740,741,742,743,749,757,760,761,763,766,808,809,810,814,815,817,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251,1263,1264,126 A method comprising having a nucleotide sequence selected from the group consisting of 5, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, wherein the composition further optionally comprises one or more components selected from the group consisting of carriers, surfactants, cationic lipids, organosilicones, organosilicone surfactants, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, toxicity mitigators, and insect growth regulators.

6. A method according to claim 1, wherein the method comprises contacting an effective amount of a solution containing double-stranded RNA with a lepidopteran pest, wherein at least one strand of the double-stranded RNA is sequence numbers 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 50 9, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817 ,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166,1187,1190,1192,1195,1196,1216,1217,1240,1243,1251,1263,1264,1265,1270,1275,1279,1283,1290,1308,1310,1316,1318,1320,1 A method comprising a sequence selected from the group consisting of 564, 1565, 1569, 1622, and 1623, which is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a gene, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity of said consecutive nucleotides, wherein RNA interference is induced, resulting in death by lepidopteran insects.

7. The method according to claim 6, wherein the solution further comprises one or more components selected from the group consisting of organosilicon surfactants or cationic lipids.

8. A method according to claim 1, wherein the DNA in (a), (b), (c), (d), (e), or (f) has a sequence selected from the group consisting of SEQ ID NOs: 7, 9, 21, 26, 27, 29, 87, 95, or 99, or wherein the double strand of (g) comprises a sequence selected from the group consisting of SEQ ID NOs: 121, 123, 135, 140, 141, 143, 201, 209, and 213, or a single strand complementary to at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 consecutive nucleotides of a sequence having at least 90%, at least 95%, or 98% identity with respect to the selected sequence or to fragments thereof of equivalent length to one strand.

9. A method according to claim 1, wherein the lepidopteran pest is selected from the group consisting of Spodptera frugiperda and Plutella xylostellaika.

10. A method according to any one of claims 1 to 4, 6, or 7, wherein the at least one double-stranded RNA comprises a single strand complementary to at least 50 consecutive nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 115 to 228, 362 to 494, 512 to 529, 536 to 541, 823 to 1103, 1334 to 1563, and 1624 to 1683, or a sequence having at least 90%, at least 95%, or 98% identity with respect to the selected sequence or fragments thereof of equivalent length to the single strand.

11. A method according to claim 10, wherein the one chain comprises a sequence complementary to at least about 100 consecutive nucleotides of a sequence selected from the group.

12. A method according to any one of claims 1 to 4, 6 or 7, wherein the double-stranded RNA comprises at least one sequence selected from the group consisting of SEQ ID NOs: 115 to 228, 362 to 494, 512 to 529, 536 to 541, 823 to 1103, 1334 to 1563, and 1624 to 1683, or a fragment thereof of at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 consecutive nucleotides, or a sequence having at least 90%, at least 95%, or 98% identity with the double-stranded RNA or the fragment thereof.

13. The method of claim 10 or 11, wherein the at least one double strand is sequence numbers 115, 116, 119, 127, 150, 151, 153, 158, 161, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334 ,1335,1336,1341,1343,1346,1351,1353,1354,1362,1363,1377,1386,1387,1393,1396,1417,1420,1422,1425,1426,1446,1447,1470,1473,1481,1493,1494,1495,1500,1505,1509,1513,1520,1538,1540,1546,1548,1550,1624,1625,16 A method comprising a sequence having at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 nucleotides or at least about 200 consecutive nucleotides, to a sequence selected from the group consisting of 29, 1682, and 1683, or a sequence having at least 90%, at least 95%, or 98% identity to the sequence or to fragments of the same length as the one strand.

14. The method of claim 1, wherein the target gene of (a), (b), (c), (d), or (e), or the DNA of (f) is sequence numbers 2, 5, 13, 36, 37, 39, 44, 47, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 7 41, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 1187, 1 A method comprising a sequence selected from the group consisting of 190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623.

15. The method according to claim 13 or 14, wherein the lepidopteran pest is Plutella xylostella.

16. A plant having improved resistance to the invasion of lepidopteran pests, or the fruit, seeds, or reproductive parts of said plant, provided by the method of claim 1.

17. A plant according to claim 16, wherein the plant is a crop plant.

18. below: (a) At least one insecticidal amount of polynucleotides comprising a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or RNA transcribed from said target gene, which is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides, or a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides; or (b) At least one insecticidal amount of at least one polynucleotide comprising at least one silencing element complementary to at least 18, 19, 20, 21, or 25 consecutive nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623; or (c) At least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, 1564-1623, or at least one RNA in an insecticidal amount containing at least one segment that is identical to or complementary to the RNA transcribed from the target gene; or (d) RNA molecules that, when ingested or come into contact with by lepidopteran pests, cause death or inhibition of growth of said lepidopteran pests, wherein the RNA molecule has a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or has a nucleotide sequence that is complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity of said consecutive nucleotides; or (e) an insecticidal double-stranded RNA molecule that, when ingested or in contact with by a lepidopteran pest, causes death or inhibits growth in the lepidopteran pest, wherein at least one strand of the insecticidal double-stranded RNA molecule is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene or RNA transcribed from the target gene, or complementary to nucleotides having at least 90%, 95%, or 98% identity of said consecutive nucleotides, wherein the target gene has a sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623; or (f) At least one insecticidal amount of double-stranded RNA containing one strand that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity with said consecutive nucleotides. An insecticidal composition for controlling lepidopteran pests, including [the specified ingredient].

19. An insecticidal composition according to claim 18, wherein the double-stranded RNA molecule of (e) or the double-stranded RNA of (f) comprises a single strand complementary to at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 nucleotides or at least about 200 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or a sequence having at least 90%, at least 95%, or 98% identity to the selected sequence or a fragment thereof of equivalent length to the single strand.

20. An insecticidal composition according to claim 19, wherein the single strand comprises a sequence complementary to at least about 100 consecutive nucleotides of a sequence selected from the group.

21. An insecticidal composition according to claim 18, wherein the double-stranded RNA comprises at least one sequence selected from the group consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or a fragment thereof of at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 consecutive nucleotides, or a sequence having at least 90%, at least 95%, or 98% identity with the double-stranded RNA or a fragment thereof.

22. An insecticidal composition according to any one of claims 19 to 21, wherein the group is sequence numbers 115, 116, 119, 127, 150, 151, 153, 158, 161, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 5 28, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 139 3. An insecticidal composition comprising 1396, 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, and 1683.

23. The insecticidal composition of claim 18, wherein the groups in (a), (b), (c), (d) and (e) are sequence numbers 2, 5, 13, 36, 37, 39, 44, 47, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506 ,509,510,530,532,533,542,545,546,547,549,551,552,553,559,560,561,563,564,570,572,573,581,593,596,602,611,612,620,665,666,672,717,730,731,736,737,738,740 ,741,742,743,749,757,760,761,763,766,808,809,810,814,815,817,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,1157,1163,1166 An insecticidal composition comprising 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623.

24. An insecticidal composition according to claims 22 and 23, wherein the lepidopteran pest is Plutella xylostella.

25. An insecticidal composition according to any one of claims 18 to 24, wherein the insecticidal composition exists in at least one form selected from the group consisting of solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particles, film, matrix, seed treatment, soil drench, transplantable formulation, and furrow formulation.

26. An insecticidal composition according to any one of claims 18 to 25, further comprising at least one component selected from the group consisting of a carrier, a surfactant, a cationic lipid, an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide insecticide, a toxicity mitigator, and an insect growth regulator.

27. An insecticidal composition according to any one of claims 18 to 26, wherein the insecticidal composition comprises an insecticidal double-stranded RNA molecule that, when ingested or in contact with by a lepidopteran pest, causes death or inhibition of growth in the lepidopteran pest, wherein the insecticidal double-stranded RNA molecule comprises at least one segment complementary to 21 consecutive nucleotides of DNA having a sequence selected from the group consisting of SEQ ID NOs: 1 to 114, 229 to 361, 495 to 511, 530 to 535, 542 to 822, 1104 to 1333, and 1564 to 1623, wherein the length of the double-stranded RNA molecule is at least 50 base pairs long, or between about 100 base pairs and about 500 base pairs long.

28. below: (a) DNA containing a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, or 1564-1623, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity of said consecutive nucleotides, or RNA transcribed from said target gene; or (b) 18 or more consecutive nucleotides having 100% identity with a fragment of equivalent length of DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, 1564-1623, or DNA complementary thereto; or (c) DNA encoding at least one silencing element complementary to at least 18 consecutive nucleotides of a target gene or RNA transcribed from said target gene, wherein the target gene has a sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623; or (d) DNA encoding RNA containing a sequence selected from the group consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683 A recombinant DNA component containing a heterologous promoter that is operably linked to it.

29. A plant chromosome or plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the recombinant DNA component of claim 16.

30. A transgenic crop plant cell having the recombinant DNA component of claim 28 in its genome.

31. A transgenic crop plant cell according to claim 30, wherein the transgenic crop plant cell further comprises DNA encoding at least one insecticide selected from the group consisting of patatin, plant lectin, plant ecdysteroid, Bacillus thuringiensis insecticide protein, Xenorhabdus insecticide protein, Photorhabdus insecticide protein, Bacillus laterosporous insecticide protein, and Bacillus sphaericus insecticide protein.

32. A transgenic Solanaceae plant cell according to claim 30, or a transgenic crop plant comprising the fruit, seeds, or reproductive part of the transgenic crop plant.

33. A method for producing polynucleotides for use in the control of lepidopteran pests, wherein the method is as follows: (a) Incubating in a reaction mixture of cellular ribonucleic acid (RNA) and ribonuclease to produce 5' nucleoside monophosphates (5' NMPs); (b) the step of removing ribonuclease; and (c) Incubate in the reaction mixture or a second reaction mixture containing the 5'NMPs, polyphosphate kinase, polyphosphate, polymerase, and SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 532, 533, 542, 545, 546, 547, 549, 55 1, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749 ,757,760,761,763,766,808,809,810,814,815,817,818,821,822,1104,1105,1106,1111,1113,1116,1121,1123,1124,1132,1133,1147,1156,115 7. Incubate in deoxyribonucleic acid (DNA) encoding an RNA sequence having at least 80% identity to 1163, 1166, 1187, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or a segment containing at least 18 consecutive nucleotides, (here The aforementioned segments are sequence numbers 115, 116, 119, 127, 150, 151, 153, 158, 161, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947,953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, The steps include incubating in a mixture having at least 90% identity to the sequence segments of 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, and generating RNA of interest (optionally, the reaction mixture in step (c) further comprises nucleoside kinase, NMP kinase, and / or NDP kinase), Methods that include...

34. A method according to claim 1, wherein the at least 18 consecutive nucleotides referenced herein in (a) to (g) are at least 18, 19, 20, or 21 consecutive nucleotides.

35. A method according to claim 1, wherein the method comprises topically applying a composition comprising at least one polynucleotide to a plant in such a manner that an effective amount of the polypeptide is ingested by a lepidopteran pest that feeds on the plant, the polynucleotide comprising at least 18, 19, 20, or 21 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623, or a nucleotide sequence complementary to RNA transcribed from the target gene; wherein the lepidopteran pest is Plutella xylostella; here the target genes are sequence numbers 2, 5, 13, 36, 37, 39, 44, 47, 103, 301, 309, 318, 319, 320, 321, 323, 324, 328, 341, 343, 346, 356, 358, 359, 503, 504, 505, 506, 509, 510, 530, 53 2, 533, 542, 545, 546, 547, 549, 551, 552, 553, 559, 560, 561, 563, 564, 570, 572, 573, 581, 593, 596, 602, 611, 612, 620, 665, 666, 672, 717, 730, 731, 736, 737, 738, 740, 741, 742, 743, 749, 757, 760, 761, 763, 766, 808, 809, 810, 814, 815, 817, 818, 821, 822, 1104, 1105, 1106, 1111, 1113, 1116, 1121, 1123, 1124, 1132, 1133, 1147, 1156, 1157, 1163, 1166, 11 Having the sequence 87, 1190, 1192, 1195, 1196, 1216, 1217, 1240, 1243, 1251, 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, or 1623;Or, here, the polynucleotide in question is sequence numbers 115, 116, 119, 127, 150, 151, 153, 158, 161, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 5 38, 539, 823, 826, 827, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1 023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 1089, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1 A method comprising a single chain having a sequence selected from the group consisting of 387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683.

36. An insecticidal composition for controlling lepidopteran pests, comprising an insecticidal double-stranded RNA molecule that causes death or inhibits growth in lepidopteran pests when ingested or come into contact with them, wherein at least one strand of the insecticidal double-stranded RNA molecule is complementary to a sequence selected from the group consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or contains at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 consecutive nucleotides, or contains a sequence that has at least 90%, at least 95%, or 98% identity with the selected sequence or a fragment thereof of equivalent length to at least one strand.

37. The insecticidal composition of claim 36, wherein the group is sequence numbers 115, 116, 119, 127, 150, 151, 153, 158, 161, 217, 434, 442, 451, 452, 453, 454, 456, 457, 461, 474, 476, 479, 489, 491, 492, 521, 522, 523, 524, 527, 528, 536, 538, 539, 823, 826, 82 7, 828, 830, 832, 833, 834, 840, 841, 842, 844, 845, 851, 853, 854, 862, 874, 877, 883, 892, 893, 901, 946, 947, 953, 998, 1011, 1012, 1017, 1018, 1019, 1021, 1022, 1023, 1024, 1030, 1038, 1041, 1042, 1044, 1047, 10 89, 1090, 1091, 1095, 1096, 1098, 1099, 1102, 1103, 1334, 1335, 1336, 1341, 1343, 1346, 1351, 1353, 1354, 1362, 1363, 1377, 1386, 1387, 1393, 1396, 1417, 1420, 1422, 1425, 1426, 1446, 1447, 1470, 1473, 1481, 14 An insecticidal composition comprising 93, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, and 1683, or an array having at least 90%, at least 95%, or 98% identity with the selected array or fragments thereof of equivalent length to at least one chain.

38. An insecticidal composition according to claim 36, wherein the double-stranded RNA comprises at least about 50 consecutive nucleotides, at least about 100 nucleotides, at least about 150 or at least about 200 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or a single strand complementary to at least about 50 consecutive nucleotides, at least about 100 or at least about 200 consecutive nucleotides, or a sequence having at least 90%, at least 95%, or 98% identity to the selected sequence or a fragment thereof of equivalent length to the single strand.

39. An insecticide composition according to claim 18, wherein the group in (a), (b), (c), (d), and (e) is sequence numbers 1, 2, 13, 36, 37, 39, 47, 103, 302, 340, 346, 349, 1121, 1157, 1178, 1179, 1185, 1310, 1313, 1318, and 1322.

40. An insecticide composition according to claim 18, wherein the group in (f) is sequence numbers 115, 116, 127, 150, 151, 153, 161, 217, 435, 473, 479, 482, 1351, 1387, 1408, 1409, 1415, 1540, 1543, 1548, and 1552.

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