JPWO2021231791A5 - - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C.(s) 119(e) of U.S. Provisional Application No. 63/024,133 and U.S. Provisional Application No. 63/106,614, which are incorporated herein by reference in their entireties.

Agricultural crops are often subject to insect attack. According to the Food and Agriculture Organization of the United Nations, farmers worldwide lose 30 to 40% of their crops to pests and diseases. Crop maintenance and crop health are essential for crop yield and quality, and ultimately they require long-term strategies to minimize pest and disease occurrence. The annual cost of 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.

Although chemical pesticides have been one solution for eradicating pest infestations , alternative, more environmentally safe solutions are needed. Chemical pesticides can be harmful to the environment and lack specificity or selectivity, ultimately resulting in non-target effects. In addition, chemical pesticides are metabolized slowly, leading to their accumulation and the development of resistance. Thus, there has been a long-felt need for more selective, environmentally safe, biodegradable, and more environmentally friendly methods for controlling or eradicating insect infestations .

The present embodiments relate to the control of lepidopteran pests, particularly pests of economic or agricultural importance. In various embodiments, the lepidopteran pest is at least one selected from the group consisting of Spodoptera spp (e.g., S. frugiperda, also known as fall armyworm) and Plutella spp (e.g., P. xylostella, also known as diamondback moth).

The compositions and methods described herein include recombinant polynucleotide molecules, e.g., recombinant DNA constructs for rendering transgenic plants resistant to infestation by lepidopteran pests, and single- or double-stranded DNA or RNA molecules (referred to herein as "triggers") useful for controlling or preventing infestation of plants by lepidopteran pests. In some embodiments, polynucleotide triggers are provided as topically applied agents for controlling or preventing infestation of plants by lepidopteran pests. In some embodiments, crop plants having improved resistance to infestation by lepidopteran pests are provided, such as transgenic crop plants (including reproductive parts such as seeds or tubers) expressing a polynucleotide trigger. In some embodiments, crop plants (including reproductive parts such as seeds or tubers) that have been topically treated with a composition comprising a polynucleotide trigger (e.g., a crop plant sprayed with a solution of a dsRNA molecule) are provided. Also provided are polynucleotide-containing compositions that are topically applied to lepidopteran pests or plants, plant parts, or seeds to be protected from infestation by lepidopteran pests.

Some embodiments relate to the suppression of target genes in lepidopteran pests by polynucleotide triggers. Some embodiments relate to methods 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 suppression are genes that are unique and non-redundant in the lepidopteran pest genome, i.e., genes that have low nucleotide diversity or are evolutionarily or functionally constrained to have more synonymous (Ks) than non-synonymous (Ka) nucleotide changes. Provided herein are nucleotide sequences, referred to herein as "target gene sequence families," the sequences of which consist of SEQ ID NOs: 1-114, 229-361, 495-511, 530-535, 542-822, 1104-1333, and 1564-1623. Also provided are nucleotide sequences referred to herein as "trigger sequences," consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683. The SEQ ID NOs relate to the sequences provided in the sequence listing provided herein.

In one aspect, a method for controlling lepidopteran infestation of a plant comprises contacting a lepidopteran pest with a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity (e.g., a segment of 21 consecutive nucleotides having a sequence of 100% identity) to a corresponding fragment of DNA having a sequence selected from the target gene sequence group or trigger sequence group, or a complementary DNA thereof. In one embodiment, the method for controlling lepidopteran infestation of a plant comprises a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity to a corresponding fragment of DNA having a sequence selected from the target gene sequence group or trigger sequence group, or a complementary DNA thereof. 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 method includes contacting a lepidopteran pest with a polynucleotide comprising a nucleotide sequence that is complementary to at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: ...

Some embodiments relate to a method for controlling lepidopteran infestation of a plant by providing in the diet of a lepidopteran pest an agent comprising a polynucleotide having at least one segment of DNA complementary to either of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a corresponding DNA fragment having a sequence selected from a target gene sequence group, a trigger sequence group (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity), and wherein the agent functions to inhibit a biological function in the lepidopteran pest upon ingestion by the lepidopteran pest, thereby controlling the infestation of the lepidopteran pest. In one embodiment, the method for controlling lepidopteran infestation of plants comprises the steps of: 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 method includes providing a polynucleotide 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 an RNA transcribed from said target gene in the diet of a lepidopteran pest. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group consisting of trigger sequences. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the agent comprising the polynucleotide is formulated, for example, as a sprayable solution or emulsion, a tank mix, or a powder, for application to a field of a crop plant. In some embodiments, the agent is produced biologically, for example, in the form of a microbial fermentation product or expressed in a transgenic plant cell.

In another aspect, a method for causing death or growth inhibition in lepidopteran pest larvae is provided. In some embodiments, at least one RNA comprising at least one silencing element is provided in the diet of lepidopteran pest larvae, where ingestion of the RNA by the lepidopteran pest larvae results in death or growth inhibition of the lepidopteran pest larvae. In some embodiments, the silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence of lepidopteran pest larvae, where the target gene is selected from the group consisting of genes in the group of target gene sequences. In one embodiment, the method for causing death or growth inhibition in lepidopteran pest larvae comprises providing at least one polynucleotide comprising at least one silencing element 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 to the larvae diet. In specific embodiments, the target gene is selected from the group consisting of 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 , 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 is RNA transcribed from the target gene. In some embodiments, the silencing element comprises one or more nucleotide sequences selected from the group consisting of trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments relate to a method of causing death or lower fecundity in a lepidopteran pest comprising providing in the diet of a lepidopteran pest at least one RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of a lepidopteran pest larva, wherein inoculation of the RNA by the lepidopteran pest results in death and lower fecundity in the lepidopteran pest. In some embodiments, the target gene is selected from the group consisting of genes in the group of target gene sequences. In some embodiments, the method causes a decrease in metamorphosis rate or a decrease in feeding activity. In some embodiments, the method is useful for providing plants with increased resistance to infestation by lepidopteran pests.

Some embodiments relate to a method of providing improved resistance to lepidopteran pest infestations comprising topically applying to a plant a composition comprising at least one segment of DNA having 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a corresponding fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or complementary thereto (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity). In one embodiment, the method of providing a plant with improved resistance to lepidopteran pest infestation comprises the step of selecting from 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, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589 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 , 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623. In one embodiment, a method of providing a plant with improved resistance to lepidopteran pest infestation comprises topically applying to the plant a composition comprising at least one polynucleotide comprising at least 18 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 301, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 36 , 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 , 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 group consisting of trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments relate to compositions comprising the polynucleotide that are formulated, for example, as a sprayable solution or emulsion, tank mix, or dust, for application to a field of a crop plant.

Some embodiments relate to insecticidal compositions for controlling lepidopteran pests comprising an insecticidally effective amount of at least one polynucleotide molecule comprising at least one segment of DNA complementary to either or 18 or more contiguous nucleotides that are essentially identical to or complementary to a corresponding fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or either (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity). In some embodiments, the polynucleotide molecule is selected from the group consisting of 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, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 599, 598, 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 , 1263, 1264, 1265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or RNA transcribed from said target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group consisting of trigger sequences. 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.Related embodiments include pesticidal compositions comprising the polynucleotide molecule formulated for application to fields of crop plants, for example in a sprayable solution or emulsion, tank mix, or dust, and optionally comprising one or more additional components such as carrier agents, surfactants, cationic lipids, organosilicones, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides, safeners, and insect growth regulators.

Some embodiments relate to a method of providing a plant with improved resistance to lepidopteran pest infestation, comprising expressing in the plant at least one polynucleotide comprising at least one segment of DNA (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity) that is essentially identical to or complementary to a corresponding fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences , or complementary to either of them. 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.

Some embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one segment of DNA (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity) or a sequence of 18 or more contiguous nucleotides having about 95% to about 100% identity to a corresponding fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or complementary to either of them. In some embodiments, the DNA element encodes a double-stranded RNA. In some embodiments, the double-stranded RNA comprises one or more nucleotide sequences selected from the group of trigger sequences. Related embodiments include a plant chromosome or plastid or a recombinant plant viral vector, a recombinant baculoviral vector comprising the recombinant DNA construct, or a DNA element without a heterologous promoter.

Some embodiments relate to a transgenic crop plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in a lepidopteran pest that contacts or ingests the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more contiguous nucleotides that is complementary to a fragment of a 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 in its genome a recombinant DNA encoding an 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.

Some embodiments relate to an isolated recombinant RNA molecule that causes death or growth inhibition in a lepidopteran pest when ingested or contacted by the lepidopteran pest, the recombinant RNA molecule comprising 18 or more contiguous nucleotides that are essentially 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 (e.g., a segment of 21 contiguous nucleotides having 100% complementarity to the fragment). In some embodiments, the recombinant RNA molecule is double-stranded RNA. Specific embodiments 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, 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 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a combination thereof. Another embodiment relates to an isolated recombinant double-stranded RNA molecule having a strand having 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.

Some embodiments relate to a method of providing a plant with improved resistance to lepidopteran pest infestation, comprising providing to the plant at least one polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of a target gene selected from the group of target gene sequences (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity to the fragment). In one embodiment, the method of providing a plant with improved resistance to lepidopteran pest infestation comprises providing to the plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 contiguous nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene is selected from the group consisting 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.

Some embodiments relate to a method for controlling infestation of lepidopteran pests in plants, comprising contacting the lepidopteran pests with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides 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 (e.g., a segment of 21 contiguous nucleotides with 100% identity or complementary sequence). In some embodiments, the polynucleotide is double-stranded RNA. Some embodiments relate to a method for selecting target genes for RNAi-mediated silencing from a plant genome or from an animal genome (e.g., insects and arthropods). In various embodiments, the method provides a subset of target genes that are present in a single or low copy number (non-repetitive and non-redundant) in a specific genome, or have low nucleotide diversity, or have a ratio of synonymous (Ks) to nonsynonymous (Ka) nucleotide changes where Ks>>Ka.

Some embodiments relate to artificial compositions comprising at least one polynucleotide described herein. In some embodiments, formulations useful for topical application to plants or materials requiring protection from infestation by lepidopteran pests are provided. In some embodiments, recombinant constructs and vectors useful for producing transgenic crop plant cells and transgenic crop plants are provided. In some embodiments, formulations and coatings useful for treating crop plants, seeds or reproductive parts of crop plants, such as tubers, are provided. In some embodiments, commercial products and foodstuffs produced from such crop plants, seeds or reproductive parts treated with or containing the polynucleotides described herein, particularly commercial products and foodstuffs having detectable amounts of the polynucleotides described herein, are provided. Some embodiments relate to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the group of target gene sequences. Another aspect relates to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the group of trigger sequences, or to their complements. Such antibodies are made by routine methods known to those skilled in the art.

In various embodiments described herein, the plant may be any plant subject to infestation by lepidopteran pests. Of particular interest are embodiments in which the plant is [plant genus name]. Examples include plants selected from the group consisting of [crop plants whose names are specified].
Embodiments include those in which the plant is an ungerminated seed of a crop plant, a crop plant in a vegetative growth stage, or a crop plant in a reproductive stage.

Other aspects and specific embodiments of the present invention are disclosed in the detailed description below.

DETAILED DESCRIPTION I. Definitions Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a term is provided in the singular, the inventor also intends that aspect of the invention be described by the plural of that term. In the event of a discrepancy in 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 their ordinary meaning in the field in which they are used, as exemplified in dictionaries specific to various fields, such as, for example, "The American Heritage® 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 any mechanism or mode of action, and references are provided for illustrative purposes only.

Unless otherwise indicated, nucleic acid sequences in the text of 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 for the substitution of uracil (U) nucleotides for thymine (T) nucleotides in the DNA. 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 includes RNA sequences that meet these criteria, except for the substitution of uracil nucleotides for thymine nucleotides. A given first polynucleotide sequence, whether DNA or RNA, further defines a second polynucleotide that is its exact complementary sequence (which may be DNA or RNA) and hybridizes perfectly to the first polynucleotide by forming Watson-Crick base pairs. When the DNA is a DNA duplex (hybridized strand), the base pairs are adenine:thymine or guanine:cytosine; when the DNA is an RNA duplex, 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 unambiguously defined by providing the nucleotide sequence of one strand, whether provided as DNA or RNA. "Essentially identical" or "essentially complementary" to a target gene or a fragment of a target gene means that the polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to a target gene or a fragment of a target gene, or a transcript of a target gene or a fragment of a target gene (generally under physiological conditions such as those found in living plant or animal cells); one skilled in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity. A first nucleic acid sequence is "operably connected" or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.

The term "polynucleotide" generally refers to a DNA or RNA molecule that contains multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules of 18 to 25 nucleotides in length) and longer polynucleotides of 26 or more. 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, contain at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base pairs), which are essentially identical to or complementary to a DNA-equivalent fragment of a target gene or an RNA transcript of a target gene. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including all integer points therebetween." Thus, embodiments of the present invention include oligonucleotides having a length of 18 to 25 nucleotides (18 mers, 19 mers, 20 mers, 21 mers, 22 mers, 23 mers, 24 mers, or 25 mers), or medium-length polynucleotides having a length of 26 nucleotides or more (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 29 nucleotides, about 0, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e.g., between about 300 and about 400 nucleotides, between about 400 and about 500 nucleotides, between about 500 and about 600 nucleotides, between about 600 and about 700 nucleotides, between about 700 and about 800 nucleotides, between about 800 and about 900 nucleotides, between about 900 and about 1000 nucleotides, between about 300 and about 500 nucleotides, between about 300 and about 600 nucleotides, between about 300 and about 700 nucleotides, between about 300 and about 800 nucleotides, between about 300 and about 900 nucleotides, about 1000 nucleotides in length, or polynucleotides of about 1000 or more nucleotides in length, e.g., coding or non-coding portions of a target gene, or up to the entire length of a target gene including both coding and non-coding portions). If a polynucleotide is double-stranded, its length can similarly be described in terms of base pairs.

The polynucleotides described herein can be single-stranded (ss) or double-stranded (ds). "Double-stranded" generally refers to base pairing that occurs between sufficiently complementary, antiparallel strands of nucleic acid to form a double-stranded nucleic acid structure under physiologically relevant 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 hybrid, antisense ssDNA, or antisense ssRNA; mixtures of any of these types of polynucleotides can be used. In some embodiments, the polynucleotide is a double-stranded RNA that is longer than the typical length of naturally occurring regulatory small RNAs, such as endogenously produced siRNAs and mature miRNAs. In some embodiments, the polynucleotide is a double-stranded RNA that is at least about 30 contiguous base pairs in length. In some embodiments, the polynucleotide is a double-stranded RNA that has a length of about 50 to about 500 base pairs. In some embodiments, a polynucleotide can contain components other than standard ribonucleotides, for example, one embodiment is RNA that contains terminal deoxyribonucleotides.

The embodiments provide protection for crop plants against lepidopteran pests. The crop plants include cereal crop plants (e.g., wheat, oats, barley, corn, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (e.g., forage grasses and forage dicotyledons, including alfalfa, vetch, clover, etc.); oilseed crop plants (e.g., cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (e.g., walnuts, cashews, hazelnuts, pecans, almonds, etc.); sugarcane, coconuts, dates, olives, sugarcane, tea, coffee; wood and pulp producing trees; vegetable crop plants such as legumes (e.g., beans, peas, lentils, alfalfa, peanuts), lettuce, asparagus, artichokes, celery, carrots, daikon, etc.). edible parts of the Brassica family (e.g., cabbage, kale, mustard, other leafy rapeseeds, broccoli, cauliflower, Brussels sprouts, turnips, kohlrabi), edible melons (e.g., cucumbers, melons, summer squash, winter squash), edible alliums (e.g., onion, garlic, leeks, shallots, chives), Solanaceae (e.g., tomatoes, eggplants, potatoes, peppers, ground cherries), and Chenopodiaceae (e.g., beets, chard, spinach, quinoa, amaranth); fruit crop plants such as apples, pears, citrus fruits (e.g., oranges, limes, lemons, grapefruit, etc.), stone fruits (e.g., apricots, peaches, plums, nectarines), bananas, pineapples, grapes, kiwifruit, papayas, avocados, and berries.

In various embodiments, the polynucleotides described herein include naturally occurring nucleotides such as those present in DNA and RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides (e.g., a synthetic polynucleotide consisting primarily of ribonucleotides but having one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides, or a synthetic polynucleotide consisting primarily 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 Publication Nos. 2011/0171287, 2011/0171176, 2011/0152353, 2011/0152346, and 2011/0160082, which are incorporated herein by reference. Illustrative examples include, but are not limited to, the phosphodiester backbones of naturally occurring oligonucleotides or polynucleotides, which can be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with fluorescent moieties (e.g., fluorescein or rhodamine) or other labels (e.g., biotin).

Some embodiments relate to polynucleotides comprising at least one segment of DNA or RNA complementary to any of the above, or an RNA transcript of either, or 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences. In some embodiments, the number of consecutive nucleotides is at least 18, e.g., between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or more. In some embodiments, the contiguous nucleotides are more than 18 in number, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 consecutive nucleotides. In some embodiments, the polynucleotide comprises at least 18, 19, 20, or 21 (as used throughout, reference to at least 18, 19, 20, or 21 means that any of these lower limits of the group can be individualized) consecutive nucleotides having 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of any of the above, or at least one segment of DNA or RNA complementary to any of the foregoing. In some 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 having 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of RNA transcript of either, or DNA or RNA complementary to either of the above; such double-stranded nucleic acid expressed as base pairs contains at least 18 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of RNA transcript of either, or DNA or RNA complementary to either of the above. In some embodiments, the length of each segment contained in the polynucleotide is longer than the length typical of naturally occurring small regulatory RNAs, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less 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 Table 1A, Table 1B, and Table 1C. In an embodiment, the polynucleotide expressed in the plant is selected from the group consisting of 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, 507, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 57 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, 1 196, 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 a complement thereof. In some embodiments, the polynucleotide is expressed in the plant. In some embodiments, the polynucleotide is provided locally to the surface of the plant or lepidopteran pest.

Some embodiments relate to polynucleotides designed to modulate expression by inducing regulation or repression of a lepidopteran pest target gene. In some embodiments, the polynucleotide is designed to have a nucleotide sequence that is essentially identical or essentially complementary to the nucleotide sequence of a lepidopteran pest target gene or cDNA (e.g., the target gene sequences), or to the sequence of an RNA transcribed from a lepidopteran pest target gene, which may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules that modulate expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers."

Any effective size polynucleotide 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 generate a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for generating transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used; in such cases, the polynucleotide triggers may be directed to a single target gene, or to multiple target genes.

As used herein, the term "isolated" refers to a molecule separated from other molecules with which it is associated in its native or natural state. Thus, the term "isolated" may refer to a DNA molecule that is separated from other DNA molecules with which it is normally associated in its native or natural state. Such DNA molecules may exist in a recombinant state, such as recombinant DNA molecules. Thus, a DNA molecule that is fused to control or coding sequences with which it is not normally associated, for example as a result of recombinant techniques, is considered to be isolated even if it is integrated into the chromosome of a cell as a transgene or is present together with other DNA molecules.

As used herein, the term "target gene sequence group" refers to a 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 a group of sequences consisting of SEQ ID NOs: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683.

In various embodiments, the lepidopteran pest is at least one insect selected from the group consisting of the genus Spodoptera and the tribe Plutella. Examples of Spodoptera species include S. frugiperda (fall armyworm). Examples of Plutella species include P. xylostella (diamondback moth). The lepidopteran pest may be at any developmental stage.

Some embodiments relate to polynucleotides designed to suppress one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides for expression of a transcript or that encodes a transcript. A "gene" can include, but is not limited to, a promoter region, a 5' untranslated region, a transcript coding region, which can include an intron region, a 3' untranslated region, or a combination of these regions. In some embodiments, the target gene can include coding or non-coding sequences, or both. In other embodiments, the target gene has a sequence identical or complementary to a messenger RNA, for example, in some embodiments, the target gene is a cDNA. In specific embodiments, the polynucleotide is designed to suppress one or more target genes, each of which is encoded by a DNA sequence selected from the group of target gene sequences. In various embodiments, the polynucleotide is designed to suppress one or more target genes, each of which is encoded by a sequence selected from the group of target gene sequences, and can be designed to suppress 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 with 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of either, or a DNA or RNA complementary to either. In such cases, each segment can be identical or different in size or sequence and can be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide comprises multiple segments in a tandem or repeat arrangement, where each segment comprises 21 consecutive nucleotides with 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of either, or a DNA or RNA complementary to either. In some embodiments, the segments can be derived from different regions of the target gene, for example, the segments can correspond to different 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 the segments.

Further definitions are provided in the following sections:

II. Controlling Lepidoptera Infestation by Contacting with a Polynucleotide Provided herein is a method for controlling lepidopteran infestation of a plant by contacting the plant with a polynucleotide comprising at least a segment of 18 or more contiguous nucleotides having about 95% to about 100% identity or complementarity to a DNA or equivalent length fragment of a target gene having a sequence selected from the group consisting of the target gene sequences or the trigger sequences, or an RNA transcript of either, or a DNA or RNA complementary to either of the foregoing. In one embodiment, the method for controlling infestation of a plant with lepidopteran pests comprises the step of administering to the plant a compound selected from the group consisting of 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, 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 includes contacting a lepidopteran pest with a polynucleotide comprising at least 18 consecutive nucleotides having 100% identity to a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of 1008, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or an RNA transcript of any thereof, or a DNA or RNA complementary to any of the foregoing. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized or produced by expression in a microorganism or expression in a plant cell. Embodiments include those in which the polynucleotide is a dsRNA comprising a strand having a sequence selected from the group of trigger sequences. The polynucleotides used in the method can be designed against multiple target genes. Related aspects of the invention include the isolated polynucleotides used in the method and the plants with modified lepidopteran resistance provided by the method. Specific embodiments are wherein the polynucleotide is selected from the group consisting of 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, 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 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 complement thereof. Other specific embodiments include those 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.

In some embodiments, the consecutive nucleotides have a sequence identity of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% to a DNA or target gene having a sequence selected from the target gene sequence group, or an RNA transcript of either, or a fragment of equivalent length of DNA or RNA complementary to either. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an RNA transcript of either, or a fragment of equivalent length of DNA or RNA complementary to either. In some embodiments, the polynucleotide has about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the entire sequence of a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or a fragment of equivalent length of DNA or RNA complementary to either.

In one embodiment, the polynucleotide is selected from the group consisting of 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, 1270, 1275, 1279, 12 The polynucleotide comprises at least one segment of 21 contiguous nucleotides having 100% identity to 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 of either, or a DNA or RNA complementary to any of the foregoing. In some embodiments, the polynucleotide comprises "neutral" sequences (sequences having no sequence identity or complementarity to the target gene) in addition to one or more segments of 21 contiguous nucleotides having 100% identity to a corresponding fragment of the target gene, and thus the polynucleotide as a whole has much less overall sequence identity to the target gene.

The total length of the polynucleotide used in this method may be longer than 18 contiguous nucleotides and may include contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group consisting of the target gene sequence group or the trigger sequence group, or an RNA transcript of either, or a 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 a polynucleotide designed to inhibit one or more target genes, each target gene having a DNA sequence selected from the group consisting of the target gene sequence group. For example, the polynucleotide may have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that inhibits the target gene, or may include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, the 3' end, or at both the 5' and 3' ends. In one embodiment, the polynucleotide can include additional nucleotides that are not specifically associated with (have a sequence that is not complementary or identical to) the target gene sequence group, the trigger sequence group, or the RNA transcript of either, or its complementary DNA or RNA, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the polynucleotide can include additional nucleotides located immediately adjacent to one or more segments of 18 or more consecutive nucleotides that have about 95% to about 100% identity to a fragment of equivalent length of a sequence selected from the group consisting of the target gene sequence group, the trigger sequence group, or the RNA transcript of either, or its complementary DNA or RNA. In one embodiment, the polynucleotide includes one such segment with 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 that includes additional nucleotides to form an overhang, such as a dsRNA that includes 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 the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or the DNA complementary to any of the above. For example, in some embodiments, the polynucleotide comprises at least two segments of 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 the DNA complementary to any 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 are present in the DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or the DNA complementary to any of the above.

The polynucleotides used in the method are provided by any suitable means known to those of skill in the art, and embodiments include those in which the polynucleotides are chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

In some embodiments, the polynucleotides used in the method are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides used in the method are not part of an expression construct and lack additional elements such as promoter or terminator sequences. Such polynucleotides can be relatively short, for example, between about 18 and about 300 or between about 50 and about 500 nucleotides (for single-stranded polynucleotides), or between about 18 and about 300 or about 50 and about 500 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA of any of the dsRNA trigger lengths disclosed in Table 1A, Table 1B, and Table 1C. Alternatively, the polynucleotides can be provided in more complex constructs, for example, as part of a recombinant expression construct, or can be included in, for example, a recombinant vector, for example, 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 a gene of interest (e.g., an insecticidal protein).

Some embodiments relate to a method for controlling infestations of lepidopteran pests of plants, comprising contacting a lepidopteran pest with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA of a target gene selected from the group consisting of genes identified in the group of target gene sequences. In some embodiments, the polynucleotide comprises a dsRNA having a strand with a sequence selected from the group consisting of the group of trigger sequences. In some embodiments, the present invention provides a method for controlling infestations of lepidopteran pests of plants, comprising contacting a lepidopteran pest with an effective amount of a solution comprising double-stranded RNA from a group of trigger sequences, the solution further comprising one or more components selected from the group consisting of an organosilicone surfactant or a cationic lipid.

In various embodiments of the method, the contacting includes application of a suitable composition comprising the polynucleotide used in the method to the surface of the lepidopteran pest; such compositions can be provided, for example, as a solid, liquid (including homogeneous mixtures such as solutions and non-homogeneous 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. The contacting can be in the form of a seed treatment or in the form of a treatment of "seed potato" tubers or tuber fragments (e.g., by immersing, coating, or spraying the seed potatoes). Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of pesticide formulation and seed treatment, can optionally be included in the composition. In some embodiments, the contacting comprises providing the polynucleotide in a composition further comprising one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (e.g., those disclosed in Example 18 of U.S. Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), an organosilicone, an organosilicone surfactant , a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide insecticide, a safener, and an insect growth regulator. In some embodiments, the contacting comprises providing the polynucleotide in a composition further comprising at least one insecticide selected from the group consisting of patatin, a plant lectin, a plant ecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In one embodiment, said contacting comprises providing the polynucleotide in a composition that can be ingested or otherwise internalized by the lepidopteran pest.

It is expected that the combination of the specific polynucleotides used in this method (e.g., the polynucleotide triggers described in the Examples) with one or more non-polynucleotide insecticides will result in enhanced improvements in the prevention or control of lepidopteran pest infestations compared to the effects obtained with the polynucleotide alone or the non-polynucleotide insecticide alone.In one embodiment, it is found that a composition containing one or more polynucleotides and one or more non-polynucleotide insecticides 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 will result in improved prevention or control of lepidopteran pest infestations .

III. Control of Lepidoptera Infestations by Providing Dietary Polynucleotides
Another aspect of the present invention is a method for controlling lepidopteran pest infestation of plants, comprising providing in the diet of a lepidopteran pest an agent comprising a polynucleotide having at least one segment of DNA, or a complementary DNA, having about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group consisting of the target gene sequence group, the trigger sequence group, or 18 or more consecutive nucleotides, wherein the agent functions to inhibit a biological function in the lepidopteran pest upon ingestion by the lepidopteran pest, thereby controlling the infestation by the lepidopteran pest. The polynucleotide can be longer than one or more segments that the polynucleotide comprises, although the length of each polynucleotide segment and the corresponding DNA fragment is equivalent. The polynucleotides used in the method can be designed against multiple target genes. In an embodiment, the agent comprises a dsRNA comprising a strand having a sequence selected from the group of trigger sequences or a complementary strand thereof, or a sequence selected from the group of trigger sequences, comprising 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, 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. In one embodiment, the method for controlling lepidopteran infestation of plants comprises the steps of: , 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, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or a polynucleotide comprising a nucleotide sequence complementary to at least 18 consecutive nucleotides of RNA transcribed from said target gene. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized or produced by expression in a microorganism or by expression in a plant cell. Related aspects of the invention include the isolated polynucleotides used in the methods and the modified lepidopteran-resistant plants provided by the methods.

In various embodiments, the agent comprising a polynucleotide comprises or is produced in a microbial cell. For example, the agent can comprise or be produced in a bacterial or yeast cell. In other embodiments, the agent comprising a polynucleotide comprises or is produced in a transgenic plant cell (e.g., a plant cell that transiently expresses the polynucleotide); such a plant cell can be a cell in a plant or a cell grown in tissue culture or cell suspension.

In various embodiments, the agent comprising a polynucleotide can be provided for feeding by lepidopteran pests in a form suitable for ingestion, for example, as a solid, liquid (including homogeneous mixtures such as solutions and non-homogeneous 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. The agent comprising a polynucleotide can be provided for feeding by lepidopteran pests by applying the agent to a plant subject to infestation by lepidopteran pests, or by applying the agent to the seed of the plant, for example by spraying, dusting, coating the plant, or by applying a soil drench, or in an artificial diet. The agent comprising a polynucleotide can be provided in an artificial diet formulated to meet the specific nutritional requirements for the maintenance of a lepidopteran pest for feeding, where the artificial diet is supplemented with an amount of polynucleotide obtained from another source, such as chemical synthesis, or purified from microbial fermentation; this embodiment can be useful, for example, for determining the timing and amount of an effective polynucleotide treatment regime. In some embodiments, the agent comprising a polynucleotide is provided in the form of a plant cell, or in a plant cell component, or in a microorganism (e.g., bacteria or yeast) or a microbial fermentation product, or in a synthetic or artificial diet, for feeding by a lepidopteran pest. In one embodiment, the agent comprising a polynucleotide is provided in the form of a feed ingested by a lepidopteran pest. The agent comprising a polynucleotide can be provided in the form of a seed treatment or in the form of a treatment of "seed potato" tubers or tuber fragments (e.g., by soaking, coating, or spraying seed potatoes) for feeding by a lepidopteran pest. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of pesticide formulation and seed treatment, may be included in the formulation. In some embodiments, the formulation containing a polynucleotide further comprises one or more components selected from the group consisting of carrier agents, surfactants, cationic lipids (e.g., those disclosed in Example 18 of US Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), organosilicon, organosilicon surfactants , polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides, safeners, and insect growth regulators. In some embodiments, the agent comprising a polynucleotide further comprises at least one insecticide selected from the group consisting of patatin, a plant lectin, a plant ecdysteroid (pecdysteroidhyto), a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a 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 implantable formulation selected from the group consisting of a particle, a pellet, or a capsule implanted in a plant; in such embodiments, the method comprises implanting the implantable formulation in a plant. In some embodiments, the agent comprising a polynucleotide comprises at least one in-furrow formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or drench, or any other form suitable for application in a furrow; in such embodiments, the method comprises in-furrow treatment with an in-furrow formulation. In some embodiments, the method comprises treatment of solanaceous seeds, potato tubers, or potato tuber fragments with the agent.

It is expected that the combination of the specific polynucleotide used in the agent used in this method (e.g., the polynucleotide trigger described in the Examples) with one or more non-polynucleotide insecticides will result in enhanced improvement in preventing or controlling the infestation of lepidopteran pests compared to the effect obtained with the polynucleotide alone or the non-polynucleotide insecticide 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 proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins and one or more non-polynucleotide insecticides is found to have improved effectiveness in preventing or controlling the infestation of lepidopteran pests when provided in the diet of lepidopteran pests.

In some embodiments, the polynucleotide used in the method is a dsRNA or a segment having a sequence selected from the group of trigger sequences, or a complementary segment thereof, wherein the polynucleotide is selected from SEQ ID NOs: 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 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.

In some embodiments, the consecutive nucleotides have a sequence that is at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an equivalent length fragment of DNA or RNA complementary to any of the above. In some embodiments, the consecutive nucleotides are exactly (100%) identical to a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an equivalent length fragment of DNA or RNA complementary to any of the above. In some embodiments, the polynucleotide has at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the entire sequence of a DNA or target gene having a sequence selected from the target gene sequence group or the trigger sequence group, or an equivalent length fragment of DNA or RNA complementary to any of the above. In one embodiment, the polynucleotide is selected from the group consisting of 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, 113 1 to a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of: 1.2, 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. At least one segment of 21 contiguous nucleotides or its complementary DNA having 100% identity to the corresponding fragment of the target gene; in some embodiments, the polynucleotide contains a segment of 21 contiguous nucleotides having 100% identity to the corresponding fragment of the target gene plus "neutral" sequences (sequences having no sequence identity or complementarity to the target gene), so that the polynucleotide as a whole has much less overall sequence identity to the target gene.

The polynucleotides used in this method are generally designed to suppress one or more genes ("target genes"). In other embodiments, the target genes have sequences identical or complementary to messenger RNA, for example, in some embodiments, the target genes are cDNA. In certain embodiments, the polynucleotides are designed to suppress 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 polynucleotides are designed to suppress one or more target genes, each of which has a DNA sequence selected from the group consisting of the target gene sequences, and can be designed to suppress 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 contiguous nucleotides having a sequence of 100% identity to a DNA or target gene having a sequence selected from the target gene sequences or the trigger sequence group, or an RNA transcript of either, or a fragment of equivalent length of DNA or RNA complementary to any of the above. In such cases, each segment can be identical or different in size or sequence, and can be sense or antisense to the target gene. For example, in one embodiment, the polynucleotide comprises multiple segments in tandem or repeat arrangement, where each segment comprises 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of either, or DNA or RNA complementary to either of the above; the segments can be derived from different regions of the target gene, e.g., can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the segments.

The total length of the polynucleotide used in this method may be longer than 18 contiguous nucleotides and may include contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of an equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of either, or a 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 a polynucleotide designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences or the group of trigger sequences. For example, the polynucleotide may have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that suppresses the target gene, or may include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, 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 a sequence that is not complementary or identical to) the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or the DNA complementary to either, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the polynucleotide may include additional nucleotides located immediately adjacent to one or more segments of 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or the DNA complementary to either. In one embodiment, the polynucleotide includes one such segment with 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 that includes additional nucleotides to form an overhang, such as a dsRNA that includes 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 the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or consecutive nucleotides in the target gene, or the DNA complementary to either. For example, in some embodiments, the polynucleotide comprises at least two segments of 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either, 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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either.

The polynucleotides used in the method are provided by any suitable means known to those of skill in the art. Embodiments include those in which the polynucleotides are chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

In some embodiments, the polynucleotides used in the method are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides used in the method are not part of an expression construct and lack additional elements such as promoter or terminator sequences. Such polynucleotides can be relatively short, for example, between about 18 and about 300 or between about 50 and about 500 nucleotides (for single-stranded polynucleotides), or between about 18 and about 300 or about 50 and about 500 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA of any of the dsRNA trigger lengths disclosed in Table 1A, Table 1B, and Table 1C. Alternatively, the polynucleotides can be provided in more complex constructs, for example, as part of a recombinant expression construct, or can be included in, for example, a recombinant vector, for example, 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 a gene of interest (e.g., an insecticidal protein).

IV. Control of Lepidoptera Infestations by Providing Dietary RNA
Another aspect of the present invention provides a method for causing death or growth inhibition in lepidopteran pest larvae by providing at least one polynucleotide comprising at least one silencing element comprising at least 18, 19, 20, or 21 consecutive nucleotides complementary to a target gene having a nucleotide sequence selected from the group of target gene sequences, or RNA transcribed from the target gene, to the larval diet. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized, or produced by expression in a microorganism or expression in a plant cell. In one embodiment, there is provided a method of causing death or growth inhibition in lepidopteran pest larvae, comprising providing to the diet of lepidopteran pest larvae at least one RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the lepidopteran pest larvae, wherein the target gene sequence is selected from the group of target gene sequences, and wherein ingestion of the RNA by the lepidopteran pest larvae results in death or growth inhibition of the lepidopteran pest larvae. A related aspect of the invention is an RNA comprising at least one silencing element, wherein the at least one silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence of the lepidopteran pest larvae, and wherein the target gene sequence is an RNA selected from the group of target gene sequences.
The RNA can be longer than the silencing element or silencing elements it contains, although the length of each silencing element and the corresponding fragment of the target gene sequence are comparable. The RNA used in the method can be designed against multiple target genes. An embodiment includes dsRNA, where the RNA has a strand with a sequence selected from the group of trigger sequences. In a related aspect, a method is provided for causing death or lower fecundity in a lepidopteran pest, comprising providing in the diet of a lepidopteran pest at least one RNA containing at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of a lepidopteran pest larva, wherein the target gene sequence is selected from the group of target gene sequences, the group of trigger sequences, or DNA complementary to either, and wherein ingestion of the RNA by the lepidopteran pest results in death or lower fecundity in the lepidopteran pest. Related aspects of the invention include the isolated RNA used in the method, and the modified lepidopteran-resistant plants provided by the method.

In various embodiments, the RNA-providing food comprises or is produced in a microbial cell. For example, the RNA-providing food may comprise or be produced in a bacterial or yeast cell. In similar embodiments, the RNA-providing food comprises or is produced in a transgenic plant cell (e.g., a plant cell that transiently expresses a polynucleotide); such a plant cell may be a plant cell or a cell grown in tissue culture or cell suspension.

In one embodiment, the feed providing the RNA is provided in the form of any plant subject to infestation by lepidopteran pests, where the RNA is contained in or on the plant. Such plants are stable transgenic plants expressing the RNA, or non-transgenic plants expressing the RNA transiently, or non-transgenic plants treated with the RNA, for example by spraying or coating. Generally, stable transgenic plants generally contain a recombinant structure encoding the RNA integrated into their genome. Of particular interest are embodiments in which the plant is a crop plant.

In various embodiments, the RNA-providing diet is provided in a form suitable for ingestion by the lepidopteran pest, for example, as a solid, a liquid (including homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, a spray, an encapsulated or microencapsulated formulation, in or on a microbead or other carrier particle, a film or coating, on or in a matrix, or as a seed treatment. The RNA-providing diet may be provided by applying the diet to a plant subject to infestation by the lepidopteran pest, for example, by spraying, dusting, coating, by applying a soil drench, or in an artificial diet. In one embodiment, the recombinant RNA-providing diet is provided in the form of a food that is ingested by the lepidopteran pest. The diet providing the RNA is an artificial diet formulated to meet the specific nutritional requirements for the maintenance of lepidopteran pests, where the artificial diet can be supplemented with an amount of RNA obtained from another source, such as chemical synthesis, or purified from microbial fermentation; this embodiment can be useful, for example, for determining the timing and amount of an effective polynucleotide treatment regime. In some embodiments, the agent comprising a polynucleotide is provided 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 a synthetic or artificial diet, for feeding by lepidopteran pests. In one example, the agent comprising a polynucleotide is provided in the form of a bait ingested by lepidopteran pests, for feeding by lepidopteran pests. The agent comprising a polynucleotide can be provided in the form of a seed treatment, or in the form of a treatment of seed tubers or tuber fragments (e.g., by soaking, coating, or spraying seed tubers) for feeding by lepidopteran pests. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of pesticide formulation and seed treatment, may be included in the feed. In some embodiments, the feed providing the RNA further comprises one or more components selected from the group consisting of carrier agents, surfactants, cationic lipids (e.g., those disclosed in Example 18 of US Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), organosilicones, organosilicon surfactants , polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides, safeners, 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 implantable formulation selected from the group consisting of particles, pellets, or capsules implanted into the plant; in such embodiments, the method comprises implanting the implantable formulation into the plant. In some embodiments, the RNA-providing diet comprises at least one in-furrow formulation selected from the group consisting of powders, granules, pellets, capsules, sprays, or drenches, or any other form suitable for application in furrows; in such embodiments, the method comprises in-furrow treatment using an in-furrow formulation. In some embodiments, the method comprises treating solanaceous seeds, potato tubers, or potato tuber fragments with the agent.

It is expected that the combination of the specific RNA (e.g., the dsRNA trigger described in the Examples) used in this method with one or more non-polynucleotide insecticides will result in enhanced improvement in preventing or controlling the infestation of lepidopteran pests compared to the effect obtained with the RNA alone or the non-polynucleotide insecticide 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 proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins is found to have improved effectiveness in preventing or controlling the infestation of lepidopteran pests.

The RNA used in the method can be single-stranded (ss) or double-stranded (ds). In an embodiment of the method, the RNA can 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 a mixture of these types; a mixture of any of these types can also be used. In one embodiment, the RNA is a double-stranded DNA/RNA hybrid. The RNA can include components other than standard ribonucleotides, for example, one embodiment is an RNA that includes terminal deoxyribonucleotides.

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 pest 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 has at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity or complementarity to a fragment of equivalent length of DNA 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 equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a DNA complementary to any of them. In some embodiments, the RNA comprising the silencing element has a sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or complementary to a fragment of DNA having a sequence selected from the group of target gene sequences.

In some embodiments, the silencing element comprises at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of the target gene. In some embodiments, the silencing element comprises at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or a DNA complementary to either of them. In some embodiments, the silencing element comprises at least a segment of 18 or more contiguous nucleotides, such as between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or even more. In some embodiments, the silencing elements are more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 consecutive nucleotides. In certain embodiments, the silencing element comprises at least one segment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a sequence of at least 18, 19, 20, or 21 consecutive nucleotides having 100% identity to a fragment of equivalent length of the target gene, or complementary to any of them. In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having a single strand comprising at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of the target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary to any of them; such double-stranded nucleic acid expressed as base pairs comprises at least 18, 19, 20, or 21 consecutive perfectly matched base pairs to a fragment of equivalent length of the target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary to any of them. In certain embodiments, the length of each silencing element contained in the RNA is longer than typical for naturally occurring small regulatory RNAs, e.g., the length of each segment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the RNA or each silencing element contained in the RNA is less than the total length of the sequence of interest (the DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences). In some embodiments, the total length of the RNA is between about 50 and about 500 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the RNA is a dsRNA of about 100 to about 500 base pairs, for example, a dsRNA of any of the lengths of the dsRNA triggers disclosed in Table 1A, Table 1B, and Table 1C. In an embodiment, the RNA is selected from the group consisting of 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, 1417, 142 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, 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.

The RNA used in this method is generally designed to inhibit one or more genes ("target genes"). In some embodiments, the target gene can include coding or non-coding sequences, or both. In other embodiments, the target gene has a sequence identical or complementary to a messenger RNA, for example, in some embodiments, the target gene is a cDNA. In certain embodiments, the RNA is designed to inhibit one or more target genes, each of which has a DNA sequence selected from the target gene sequence group or the trigger sequence group. In various embodiments, the RNA is designed to inhibit one or more genes, each of which has a sequence selected from the target gene sequence group or the trigger sequence group, and can be designed to inhibit 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 of which comprises at least one segment of DNA complementary to 21 contiguous nucleotides having a sequence having 100% identity or 100% complementarity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or either. In such a case, each silencing element can be identical or different in size or sequence, and can be sense or antisense to the target gene. For example, in one embodiment, the RNA can include multiple silencing elements in tandem or repeat sequences, where each silencing element includes 21 contiguous nucleotides having a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary thereto; the segments can be derived from different regions of the target gene, e.g., the segments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the segments.

The total length of the RNA can be more than 18 consecutive nucleotides and can include nucleotides in addition to a silencing element having a sequence of about 95% to about 100% identity or complementarity to the target gene sequence group, DNA having a sequence selected from the trigger sequence group, or a fragment of equivalent length of the target gene, or DNA complementary to either. In other words, the total length of the RNA can be longer than the length of a silencing element designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the target gene sequence group, for example, the RNA can have nucleotides adjacent to the "active" silencing element of at least one segment of 18 or more consecutive nucleotides that suppresses the target gene, or can include "spacer" nucleotides between active segments, or can 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 comprises additional nucleotides that are not specifically associated with (have a sequence that is not complementary or identical to) the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or a DNA complementary to either of them, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the RNA comprises 18 or more consecutive nucleotides that have a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or an additional nucleotide located immediately adjacent to one or more silencing elements. In one embodiment, the RNA comprises one such silencing element that has 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 that includes additional nucleotides to form an overhang, such as a dsRNA that includes two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a segment of consecutive nucleotides in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or a DNA complementary to either. For example, in some embodiments, the RNA comprises at least two silencing elements, each of 21 consecutive nucleotides having a sequence of 100% identity to a segment of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or a DNA complementary to either, where (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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or a DNA complementary to either.

In some embodiments, the RNA is composed of naturally occurring ribonucleotides. In certain embodiments, the RNA includes components other than ribonucleotides, such as synthetic RNA that is composed primarily 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.

The RNA used in the method is provided by any suitable means known to those of skill in the art. Embodiments include those in which the RNA is chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or cell culture (such as plant or insect cells grown in culture), produced by expression in plant cells, or produced by microbial fermentation.

In some embodiments, the RNA is provided as isolated RNA that is not part of an expression construct and lacks additional elements such as promoter or terminator sequences. Such RNA is relatively short, e.g., from about 18 to about 300, or from about 50 to about 500 (in the case of single-stranded RNA), or from about 18 to about 300, or from about 50 to about 500 base pairs (in the case of double-stranded RNA), of single- or double-stranded RNA nucleotides.
Alternatively, the RNA may be provided in a more complex construct, for example as part of a recombinant expression construct, or may be included in, for example, 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 including additional RNA encoding an aptamer or ribozyme, or an expression cassette for expressing a gene of interest (e.g., an insecticidal protein).

V. Methods of Providing Plants with Improved Resistance to Lepidopteran Infestation, and the Plants, Plant Parts, and Seeds So Provided Some embodiments relate to methods of providing plants with improved resistance to lepidopteran pest infestation , comprising providing a plant with at least one polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of a target gene selected from the group consisting of genes identified in the group of target gene sequences. In one embodiment, the present invention provides a method of providing a plant with improved resistance to lepidopteran pest infestation , comprising providing a plant with at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 contiguous nucleotides of a target gene or RNA transcribed from a target gene, wherein the target gene is selected from the group of genes identified in the group of target gene sequences or RNA transcribed from a target gene. Embodiments of gene targeting sequences include sequences that target related genes, including genes having sequences selected from the group consisting of the target gene sequences identified by the SEQ ID NOs in Table 1A, Table 1B and Table 1C, and related genes including orthologs from related pests, e.g., from other lepidopteran pests. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized or produced by expression in a microorganism or expression in a plant cell. In some embodiments, the polynucleotide comprises at least one segment of DNA that is essentially identical to or complementary to the target gene sequence, the trigger sequence, or 18 or more contiguous nucleotides that are complementary to either of the sequences. In some embodiments, the polynucleotide is a dsRNA having a strand with a sequence selected from the trigger sequence, or a complement thereof. In some embodiments, the polynucleotide comprises a dsRNA having a strand with a sequence selected from the trigger sequence.

In a related aspect, the invention relates to a plant having improved resistance to lepidopteran pest infestation provided by expressing in the 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 equivalent length of a target gene selected from the group consisting of the genes identified in said target gene sequence group, wherein the resulting plant has improved resistance to lepidopteran pest infestation compared to a control plant in which the polynucleotide is not expressed. In a related aspect, the invention relates to a plant having improved resistance to lepidopteran pest infestation provided by expressing in the plant at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that has a sequence of about 95% to about 100% identity or complementarity to a fragment of a target gene selected from the group consisting of the genes identified in said target gene sequence group, wherein the resulting plant has improved resistance to lepidopteran pest infestation compared to a control plant in which the polynucleotide is not expressed.

In yet another aspect, the invention relates to seeds or propagules (particularly transgenic progeny seeds or propagules) produced by the plants having improved resistance to lepidopteran pest infestation provided by this method. Also contemplated are commodity products produced by the plants having improved resistance to lepidopteran pest infestation provided by this method, and commodity products produced from transgenic progeny seeds or propagules of such plants.

Another aspect of the present invention provides a method for providing plants with improved resistance to lepidopteran pest infestation , comprising topically applying to the plant a composition comprising at least one polynucleotide having at least one segment of DNA complementary to a target gene or a fragment of DNA having a sequence of about 95% to about 100% identity to said target gene sequence group, said trigger sequence group, or 18 or more contiguous nucleotides that are essentially identical to a fragment of equivalent length of DNA having a sequence selected from said target gene sequence group, said trigger sequence group, or at least one segment of DNA complementary to said fragment, in such a manner that plants treated with the polynucleotide-containing composition exhibit improved resistance to lepidopteran pest infestation compared to untreated plants. In one embodiment, said at least one polynucleotide comprises at least one segment of DNA complementary to said target gene sequence group, said trigger sequence group, or 18 or more contiguous nucleotides that are essentially identical to a fragment of equivalent length of DNA having a sequence selected from said target gene sequence group, said trigger sequence group. The polynucleotides are equivalent in length to each segment of the target gene and the corresponding fragment, but may be longer than said segment or the segments that they contain. In one embodiment, the present invention provides the nucleic acid sequences represented by 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, 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 a plant with improved resistance to infestation of lepidopteran pests comprising topically applying to the plant a composition comprising at least one polynucleotide comprising at least 18 contiguous nucleotide sequences of a target gene, or RNA transcribed from a target gene, having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 318, 1320, 1564, 1565, 1569, 1622, and 1623. In one embodiment, the present invention provides a method for providing a plant with improved resistance to infestation of lepidopteran pests comprising topically applying to the plant a composition comprising at least one polynucleotide in such a manner that an effective amount of the polynucleotide is ingested by lepidopteran pests feeding on the plant, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 2, 5, 7, 9, 13, 21 , 26, 27, 29, 36, 37, 39, 44, 47, 87, 95, 99, 103, 30, 318, 1320, 1564, 1565, 1569, 1622, and 1623. 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, 1 196, 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.

The polynucleotides used in the method can be designed against multiple target genes. Embodiments include those in which the composition comprises a dsRNA having a strand with a sequence selected from the group consisting of the trigger sequences. Related aspects of the invention include compositions for topical application and isolated polynucleotides used in the method, and plants with improved lepidopteran resistance provided by the method.

As used throughout the specification, "topical application" refers to application to the surface or exterior of a subject, such as application to the surface or exterior of a plant, e.g., application to the surface of a part of a plant, e.g., leaves, stems, flowers, fruits, shoots, roots, seeds, tubers, flowers, anthers, pollen, or application to the entire plant, or application to the above-ground or underground parts of a plant. Topical application can be performed to a non-living surface, such as application to soil, or to a surface or matrix by which a lepidopteran pest can contact the polynucleotide. In various embodiments of the method, a composition comprising at least one polynucleotide is applied to a plant locally in a suitable form, e.g., as a solid, liquid (including homogeneous mixtures such as solutions and non-homogeneous 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 method, the polynucleotide-containing composition is applied topically to the above-ground parts of the plant, for example, by spraying or dusting the leaves, stems, and flowering parts of the plant.

Embodiments of the method include localized application as a foliar spray (e.g., spraying the leaves of a solanaceous plant with a liquid polynucleotide-containing composition) or foliar dusting (e.g., dusting the crop with a polynucleotide-containing composition in the form of a powder or carrier particles). In other embodiments, the polynucleotide-containing composition is locally applied to underground portions of the plant, such as the roots, for example by means of a soil drench. In other embodiments, the polynucleotide-containing composition is locally applied to seeds grown in the plant. The localized application can be in the form of a localized treatment of the fruit of the crop plant or seeds obtained from the fruit of the crop plant, or of tubers or tuber pieces (e.g., by immersing, coating, or dusting the seed potatoes). The polynucleotide-containing composition can optionally include suitable binders, inert carriers, surfactants, and the like, known to those skilled in the art of pesticide formulation and seed treatment.

In some embodiments, the polynucleotide-containing composition is at least one locally implantable formulation selected from the group consisting of particles, pellets, or capsules locally implanted in the plant; in such embodiments, the method comprises locally implanting the locally implantable formulation in the plant. In some embodiments, the polynucleotide-containing composition is at least one in-furrow formulation selected from the group consisting of powders, granules, pellets, capsules, sprays, or drenches, or any other form suitable for topical application in furrows; in such embodiments, the method comprises in-furrow treatment with an in-furrow formulation. In one embodiment, the polynucleotide-containing composition can be ingested or otherwise internally absorbed by the lepidopteran pest. For example, the polynucleotide-containing composition can be in the form of a bait. In some embodiments, the polynucleotide-containing composition further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (e.g., as disclosed in Example 18 of U.S. Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), an organosilicone, an organosilicone surfactant , a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide insecticide, a safener, and an insect growth regulator. In one embodiment, the composition further comprises a nonionic organosilicone surfactant, such as a SILWET brand surfactant, e.g., SILWET L-77 brand surfactant, having CAS No. 27306-78-1 and EPA No. CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. 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 proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins. Alternatively, such additional components or insecticides can be provided separately, for example, by separate topical application or transgenic expression in the plant. Alternatively, the plant is topically treated with a separate (before, after, or during) application of a substance that improves the efficacy of the polynucleotide-containing composition in addition to the polynucleotide-containing composition. For example, a plant may be sprayed with a first, topical application of a solution containing a non-ionic organosilicone surfactant such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, followed by a second, topical application of a composition comprising the polynucleotide, or vice versa.

It is expected that the combination of certain polynucleotides useful in the polynucleotide-containing compositions (e.g., the polynucleotide triggers described in the examples) with one or more non-polynucleotide insecticides will result in enhanced improvements in the prevention or control of lepidopteran pest infestations compared to the effects obtained with the polynucleotide alone or the non-polynucleotide insecticide alone . In one embodiment, the polynucleotide-containing compositions are provided as transgenic plants expressing one or more polynucleotides and one or more genes encoding a non-polynucleotide 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, wherein the transgenic plants are found to exhibit improved resistance to lepidopteran pest infestations .

Polynucleotides useful in the polynucleotide-containing compositions are provided by any suitable means known to those of skill in the art. Embodiments include those in which the polynucleotides are chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or cell culture (e.g., plant or insect cells grown in culture), produced by expression in plant cells, or produced by microbial fermentation.

In many embodiments, the polynucleotides useful in the polynucleotide-containing compositions are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides useful in the polynucleotide-containing compositions are not part of an expression construct and lack additional elements such as promoter or terminator sequences. Such polynucleotides can be relatively short, for example, single- or double-stranded polynucleotides of about 18 to about 300, or about 50 to about 500 nucleotides (for single-stranded polynucleotides), or about 18 to about 300, or about 50 to about 500 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA of any of the dsRNA trigger lengths disclosed in Table 1A, Table 1B, and Table 1C. Alternatively, the polynucleotides can be provided in more complex constructs, for example, as part of a recombinant expression construct, or can be included in, for example, 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 a gene of interest (e.g., an insecticidal protein).

The polynucleotides useful in the polynucleotide-containing compositions have at least 18 or more consecutive nucleotides that have about 95% to about 100% identity to a fragment of equivalent length 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. In one embodiment, the polynucleotide comprises at least 18 or more consecutive nucleotides that are essentially identical or complementary to a fragment of equivalent 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. In some embodiments, the consecutive nucleotides have about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence to the fragment of DNA having the target gene sequence group, the trigger sequence group, or a fragment of equivalent length of DNA complementary to either. In some embodiments, the consecutive nucleotides are exactly (100%) identical to the fragment of equivalent length of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a fragment of DNA complementary to either. In some embodiments, the polynucleotide has a sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a fragment of DNA complementary to either of them.

Polynucleotides useful in the polynucleotide-containing compositions include at least one segment of 18 or more contiguous nucleotides, e.g., 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 include more than 18 contiguous segments, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 consecutive nucleotides. In certain embodiments, the polynucleotide comprises at least one segment of at least 18, 19, 20, or 21 contiguous nucleotides having 100% identity to a fragment of equivalent length of a target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a DNA complementary to any of them. 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 any of the target gene sequences, DNA having a sequence selected from the trigger sequence group, or target gene, expressed as base pairs such that the double-stranded nucleic acid contains at least one segment of at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of the target gene sequences, DNA having a sequence selected from the trigger sequence group, or target gene, or DNA complementary to any of the target gene sequences. In certain embodiments, the length of each segment contained in the polynucleotide is longer than the length typical of naturally occurring small regulatory RNAs, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less than the total length of the sequence of interest (a DNA having a sequence selected from the group consisting of target gene sequences or a target gene). 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 of between about 100 and about 500 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers disclosed in Table 1A, Table 1B, and Table 1C. In some embodiments, the polynucleotide is selected from the group consisting of 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, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 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, 1 422, 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, 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.

A locally applied polynucleotide is generally designed to inhibit one or more genes ("target genes"). Such target genes may include coding or non-coding sequences, or both. In certain embodiments, the polynucleotide is designed to inhibit 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 locally applied polynucleotide is designed to inhibit one or more genes, each of which has a sequence selected from the group consisting of the target gene sequences, and may be designed to inhibit 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 at least one segment of DNA complementary to the target gene sequences, 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the trigger sequence group, or to any 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 locally applied polynucleotide can include multiple sections in a tandem or repeat sequence, where each section includes at least one segment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from a group of target gene sequences.

The total length of the locally applied polynucleotide can be longer than 18 or more contiguous nucleotides and can include contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or nucleotides in addition to DNA complementary to either. In other words, the total length of the locally applied polynucleotide can be longer than the length of a section or segment of a polynucleotide designed to inhibit one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences. For example, the locally applied polynucleotide can have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that inhibits a target gene, or can include "spacer" nucleotides between active segments, or can have additional nucleotides at the 5' end, 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 (have a non-complementary or non-identical sequence) to the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or DNA complementary to either of them, such as nucleotides that provide a stabilizing second structure or that provide convenience in cloning or production.

In one embodiment, the locally applied polynucleotide comprises additional nucleotides located adjacent to one or more segments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group consisting of the target gene sequence group. In one embodiment, the locally applied polynucleotide comprises 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 that includes additional nucleotides to form an overhang, e.g., a dsRNA that includes 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 the contiguous nucleotides in the target gene sequence group, DNA having a sequence selected from the trigger sequence group, or a fragment of DNA complementary to either of them in the target gene sequence group. For example, in some embodiments, the locally applied polynucleotide comprises at least two segments of 21 contiguous nucleotides each having a sequence of 100% identity to a fragment of the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or a DNA complementary to either of them, 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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or a DNA complementary to either of them.

In a related aspect, the invention relates to a plant having improved resistance to lepidopteran pest infestation provided by this method comprising topically applying to the plant a composition comprising a polynucleotide having at least one segment of DNA complementary to any of 18 or more consecutive nucleotides having a sequence that is about 95% to about 100% identical to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, whereby plants treated with the polynucleotide composition exhibit improved resistance to lepidopteran pest infestation compared to untreated plants.

In one embodiment, a dsRNA trigger having a sequence selected from the group of trigger sequences or a complement thereof, or a sequence selected from 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, 461, 471, 481, 491, 501, 512, 521, 531, 541, 542, 551, 552, 553, 561, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 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 and a crop plant having improved resistance to lepidopteran infestation compared to a control plant, the crop plant being provided by topically applying a dsRNA trigger encoded by a sequence selected from the group consisting of: 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 aspect, the present invention relates to seeds or reproductive parts (particularly transgenic progeny seeds or reproductive parts) produced by the plants having improved resistance to lepidopteran pest infestation provided by this method. Also contemplated are commodity products produced by the plants having improved resistance to lepidopteran pest infestation provided by this method, and commodity products produced from transgenic progeny seeds or reproductive parts of such plants.

VI. Insecticidal Compositions for Controlling Lepidopteran Pests Another aspect of the present invention provides an insecticidal composition for controlling lepidopteran pests, comprising an insecticidally effective amount of at least one RNA comprising at least one segment of DNA complementary to at least 18 consecutive nucleotides that are essentially identical to or complementary to a target gene or a fragment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or any of the above. In this context, " controlling " includes, but is not limited to, inducing physiological or behavioral changes in lepidopteran pests (adults or larvae), such as inhibiting growth, increasing mortality, reducing reproductive capacity, reducing or halting feeding or movement, or reducing or halting development of metamorphic stages. "Insecticidally effective" means, but is not limited to, effective in inducing physiological or behavioral changes in lepidopteran pests (adults or larvae), such as inhibiting growth, increasing mortality, reducing or halting reproductive capacity, reducing or halting feeding or movement, or reducing or halting development of metamorphic stages. In some embodiments, application of an insecticidally effective amount of RNA to a plant improves the plant's resistance to infestation by lepidopteran pests. The RNA can be longer than the segment or segments it contains, although the length of each segment and corresponding fragment of the target gene is comparable. The RNA used in the method can be designed against multiple target genes.
An embodiment is where the pesticidal composition comprises an insecticidally effective amount of a polynucleotide comprising at least 18, 19, 20, or 21 contiguous 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;
or an insecticidally effective amount of at least one polynucleotide comprising at least one silencing element that is complementary to at least 21 consecutive nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a nucleotide sequence selected from said group of target gene sequences;
Or, SEQ 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、１２６５、１２７０、１２７５、１２７９、１２８３、１２９０、１３０８、１３ an insecticidally effective amount of at least one RNA comprising at least one segment identical to or complementary to at least 18, 19, 20, 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of 10, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or an RNA transcribed from said target gene;
or an RNA molecule which, when ingested or contacted by a lepidopteran pest, causes death or growth inhibition in the lepidopteran pest, wherein the RNA molecule is selected from the group consisting of 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, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 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, or comprises at least 18, 19, 20, or 21 contiguous nucleotides that are complementary to 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 an RNA transcribed from said target gene;
or an insecticidal double-stranded RNA molecule which, when ingested or contacted by a lepidopteran pest, causes death or growth inhibition in the lepidopteran pest, wherein at least one strand of the insecticidal double-stranded RNA molecule comprises 21 consecutive nucleotides that are complementary to a target gene or RNA transcribed from the target gene, wherein the target gene is selected from the group consisting of SEQ ID NOs: 22, 5, 7, 9, 13, 21, 26, 27, 29, 36, 37, 39, 44, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, , 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, or has 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;
or a sequence selected from the group of trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized, produced by expression in a microorganism, or by expression in a plant cell. The embodiment includes a sequence selected from the group of trigger sequences (target sequences), or, in more specific examples, 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, 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 also includes an insecticidal composition comprising a 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.

In various embodiments, the insecticidal composition for controlling lepidopteran pests is present in at least one form selected from the group consisting of solids, liquids (including homogeneous mixtures such as solutions, and non-homogeneous mixtures such as suspensions, colloids, micelles, emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, in or on microbeads or other carrier particles, in films or coatings, on or in matrices, or as seed treatments. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of insecticide formulation and seed treatment, may optionally be included in the polynucleotide-containing composition. The lepidopteran pests to be controlled are generally pests that parasitize plants. In some embodiments, the insecticidal composition comprises at least one implantable formulation selected from the group consisting of particles, pellets, or capsules implanted in a plant; in such embodiments, the method comprises implanting the implantable formulation in a plant. In some embodiments, the insecticidal composition is at least one in-furrow formulation selected from the group consisting of a powder, a granule, a pellet, a capsule, a spray, or a drench, or any other form suitable for application in furrows; in such embodiments, the method includes in-furrow treatment with an in-furrow formulation. In one embodiment, the insecticidal composition can be ingested or otherwise internally absorbed by a lepidopteran pest. For example, the insecticidal composition can be in the form of a bait. In some embodiments, the insecticidal composition further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (e.g., as disclosed in Example 18 of U.S. Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), an organosilicone, an organosilicone surfactant , a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide insecticide, a safener, and an insect growth regulator. In one embodiment, the insecticidal composition further comprises a nonionic organosilicone surfactant, such as SILWET® brand surfactants, e.g., SILWET L-77® brand surfactant, having CAS No. 27306-78-1 and EPA No. CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. In some embodiments, the insecticidal composition further comprises at least one insecticide selected from the group consisting of patatin, plant lectins, phytoecdysteroids, Bacillus thuringiensis insecticidal proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins. Alternatively, such additional components or insecticides can be provided separately, for example, by separate topical application or by transgenic expression in the plant. Alternatively, the plant is treated topically with the insecticidal composition as well as with a separate application (before, after, or during) of a substance that improves the effectiveness of the insecticidal composition. For example, a plant can be sprayed with a first topical application of a solution containing a non-ionic organosilicone surfactant such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, and then sprayed with a second topical application of the insecticidal composition (or vice versa).

It is expected that the combination of the specific RNA (e.g., the dsRNA trigger described in the Examples) used in this method with one or more non-polynucleotide insecticides will result in enhanced improvement in preventing or controlling the infestation of lepidopteran pests compared to the effect obtained with the RNA alone or the non-polynucleotide insecticide 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 proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins, is found to have improved effectiveness in preventing or controlling the infestation of lepidopteran pests.

In various embodiments, the insecticidal composition comprises or is produced in a microbial cell. For example, the insecticidal composition can comprise or be produced in a bacterial or yeast cell. In similar embodiments, the insecticidal composition comprises or is produced in a transgenic plant cell (e.g., a plant cell that transiently expresses a polynucleotide), which can be a cell in a plant or a cell grown in tissue culture or cell suspension.

The insecticidal composition can be provided for feeding by lepidopteran pests by applying the composition to a plant or surface subject to infestation by the lepidopteran pest, for example by spraying, dusting, or coating the plant, plant seed, or seed potato, or by applying a soil drench, or by in-furrow treatment, or by providing in an artificial diet. The insecticidal composition can be provided for feeding by lepidopteran pests in an artificial diet formulated to meet the specific nutritional requirements for maintaining the lepidopteran pest, where the artificial diet is supplemented with an amount of RNA obtained from another source, such as chemical synthesis, or purified from microbial fermentation; this embodiment can be useful, for example, to determine the timing and amount of effective RNA treatment regimes. In some embodiments, the insecticidal composition is 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 bait. In one embodiment, the insecticidal composition is provided in the form of a bait for ingestion by lepidopteran pests. The insecticidal composition may be provided in the form of a seed (or seed potato) treatment for ingestion by lepidopteran pests.

In one embodiment, the insecticidal composition is provided in the form of any plant subject to infestation by lepidopteran pests, wherein the RNA is contained in or on the plant. Such plants are stable transgenic plants expressing the RNA, or non-transgenic plants that transiently express the RNA or are treated with the RNA, for example by spraying or coating. Stable transgenic plants generally contain a recombinant construct that codes for the RNA integrated into their genome.

RNA useful in the insecticidal composition can be single-stranded (ss) or double-stranded (ds). Embodiments include those 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 can be used. In one embodiment, a double-stranded DNA/RNA hybrid is used. The RNA can contain components other than standard ribonucleotides (e.g., in one embodiment, RNA containing terminal deoxyribonucleotides).

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

The RNA in the insecticidal composition comprises at least one segment of DNA that is complementary to 18 or more consecutive nucleotides having about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or any of the above. In some embodiments, the RNA comprises at least one of 18 or more consecutive nucleotides, e.g., between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or more. In some embodiments, the segments are 18 or more, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 At least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 consecutive nucleotides. In certain embodiments, the RNA comprises at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of the target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a DNA complementary to any of them. 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 with a sequence of 100% identity to a fragment of equivalent length of the target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary to any of them; such double-stranded nucleic acid, expressed as base pairs, contains at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of the target gene having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary to any of them. In certain embodiments, the length of each segment contained in the RNA is longer than typical for naturally occurring small regulatory RNAs, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the RNA, or the length of each segment contained in the RNA, is less than the total length of the sequence of interest (a DNA or a 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 between about 50 and about 500 nucleotides in length.

The RNA in the insecticidal composition is generally designed to inhibit one or more genes ("target genes"). Such target genes may include coding or non-coding sequences, or both. In certain embodiments, the RNA is designed to inhibit 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 inhibit 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 inhibit 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 of which comprises at least one segment of DNA complementary to the target gene sequences, 21 contiguous nucleotides having 100% identity to a fragment of equivalent length of DNA having a sequence selected from the trigger sequence group, or to any of the above. In such cases, each section can be identical or different in size or sequence and can be sense or antisense to the target gene. For example, in one embodiment, the RNA can include multiple sections in tandem or repeat sequences, where each section includes at least one segment of DNA that is complementary to the target gene sequence group, 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the trigger sequence group, or any of the above; the segments can be derived from different regions of the target gene, e.g., the segments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the segments.

The total length of the RNA in the insecticidal composition can be longer than 18 consecutive nucleotides and can include consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or nucleotides in addition to DNA complementary to either. In other words, the total length of the RNA can be longer than the length of a section or segment of RNA designed to inhibit one or more target genes, each target gene having a DNA sequence selected from the group consisting of the target gene sequence group and a trigger sequence. For example, the RNA can have nucleotides adjacent to the "active" segment of at least one segment of 18 or more consecutive nucleotides that inhibits the target gene, or can include "spacer" nucleotides between active segments, or can have additional nucleotides at the 5' end, the 3' end, or at both the 5' and 3' ends. In one embodiment, the RNA comprises additional nucleotides that are not specifically related (have a sequence that is not complementary or identical to) the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or the DNA complementary to either, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the RNA comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene, or the DNA complementary to either. In one embodiment, the RNA comprises one such segment with 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 that comprises additional nucleotides to form an overhang, such as a dsRNA that comprises two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a segment of consecutive nucleotides in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the target gene. For example, in some embodiments, the RNA comprises at least two segments of 21 consecutive nucleotides having a sequence that is 100% identical to a segment of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or a DNA complementary to either of them, 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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either of them.

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

In some embodiments, the RNA is provided as an isolated RNA that is not part of an expression construct. In some embodiments, the RNA is provided as an isolated RNA that lacks additional elements such as promoter or terminator sequences. Such RNA can be relatively short, for example, single-stranded or double-stranded RNA, between about 18 and about 300, or between about 50 and about 500 nucleotides (for single-stranded RNA), or between about 18 and about 300, 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 can be included in, for example, 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 additional RNA encoding an aptamer or ribozyme, or an expression cassette for expressing a gene of interest (e.g., an insecticidal protein).
VII. Methods for Providing Plants with Improved Resistance to Lepidopteran Pest Infestations , and Plants and Seeds So Applied

Another aspect of the invention relates to a method for providing a plant with improved resistance to lepidopteran pest infestation, comprising expressing in the plant at least one polynucleotide comprising at least 18 consecutive nucleotides essentially identical or complementary to a target gene or a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences , or at least one segment of DNA complementary thereto, whereby the resulting plant has 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 the plant at least one polynucleotide comprising at least 18 consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of a target gene or DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary thereto. In one embodiment, the present invention relates to a nucleic acid sequence encoding a nucleic acid sequence represented by 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, 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 method for providing a plant with improved resistance to lepidopteran pest infestation, comprising expressing in the plant at least one polynucleotide comprising at least one segment identical to 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", e.g., expressing in a plant an RNA comprising a ribonucleotide sequence that is antisense or essentially complementary to at least one fragment of a target gene or DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a DNA complementary to either of them. Embodiments include those in which the polynucleotide expressed in the plant is an RNA comprising at least one segment having a sequence selected from the group of trigger sequences, or a complement thereof. However, the polynucleotide expressed in the plant can also be DNA (e.g., DNA produced in the plant during genome replication), or RNA encoded by such DNA. Related aspects of the invention include isolated polynucleotides used in the methods and modified lepidoptera-resistant plants provided by the methods.

The method includes expressing at least one polynucleotide in a plant, wherein the polynucleotide comprises 18 or more contiguous nucleotides that are essentially identical to or complementary to a target gene or a 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. In some embodiments, a first polynucleotide is provided to a plant in the form of DNA (e.g., in the form of an isolated DNA molecule, or as an expression construct, 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 integrating the polynucleotide into the genome of the plant from where it can be expressed in one or more cells of the plant. In one embodiment, a first polynucleotide (e.g., a recombinant DNA construct comprising a promoter operably linked to DNA comprising at least one segment of DNA complementary to 18 or more contiguous nucleotides essentially identical to or complementary to a target gene or a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences) is stably integrated into the genome of the plant, from which a second produced polynucleotide (e.g., an RNA transcript comprising a transformant of at least one segment of DNA complementary to 18 or more contiguous nucleotides essentially identical to or complementary to a target gene or a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences) is expressed in one or more cells of the plant. Methods for providing stably transformed plants are provided in the section entitled "Making and Using Transgenic Plant Cells and Transgenic Plants".

In another embodiment, the polynucleotide expressed in the plant is expressed by transient expression (i.e., expression that does not result from stable integration of the sequence into the genome of the plant). In such an embodiment, the method can include 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 infiltrating plant leaves with a polynucleotide solution using a needleless syringe.

In some embodiments, where 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 the second polynucleotide produced subsequently is transiently expressed in the plant. In some embodiments, the first polynucleotide is selected from one or more of: (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 comprising a modified PolIII gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule comprising 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 the plant by topical application of a polynucleotide-containing composition to the plant in a suitable form, such as a solid, liquid (including homogeneous mixtures such as solutions and non-homogeneous 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 in the form of a seed treatment or seed potato treatment of a crop plant. Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of pesticide formulation and seed treatment, can optionally be included in the composition. In such embodiments, the polynucleotide-containing composition may further comprise one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (e.g., as disclosed in Example 18 of U.S. Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide insecticide, a safener, and an insect growth regulator; in one embodiment, the composition further comprises a nonionic organosilicone surfactant, such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, having CAS No. 27306-78-1 and EPA No. CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. 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 proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins. Alternatively, such additional components or insecticides can be provided separately, for example, by separate topical application or transgenic expression in the plant. Alternatively, the plant is topically treated with a separate (before, after, or during) application of a substance that improves the efficacy of the polynucleotide-containing composition in addition to the polynucleotide-containing composition. For example, the plant may be sprayed with a first, topical application of a solution containing a non-ionic organosilicone surfactant, such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, followed by a second, topical application of a composition comprising the polynucleotide, or vice versa.

It is expected that the combination of certain polypeptides used in this method (e.g., the polynucleotide triggers described in the Examples) with one or more non-polynucleotide insecticides will result in enhanced improvements in the prevention or control of lepidopteran pest infestations compared to the effects obtained with the polynucleotide alone or the non-polynucleotide insecticide alone. In one embodiment, transgenic plants expressing at least one polynucleotide comprising 18 or more contiguous nucleotides essentially identical to or complementary to a target gene or a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences (e.g., the polynucleotide triggers described in the Examples), or at least one segment of DNA complementary to either, and one or more genes encoding a non-polynucleotide insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein, are found to exhibit improved resistance to lepidopteran pest infestation .

In some embodiments where the polynucleotides expressed in the plant are 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 secondly is transiently expressed in the plant; the site of application of the first polynucleotide need not 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 onto the leaves or by injection into the stem, and the second polynucleotide can be transiently expressed elsewhere in the plant, e.g., in the roots or throughout the plant. In some embodiments of the method, a composition comprising at least one polynucleotide is applied locally to the above-ground parts of the plant, e.g., sprayed or dusted onto the leaves, stems, and flowering parts of the plant. In other embodiments, a composition comprising at least one polynucleotide is applied locally to the underground parts of the plant, e.g., to the roots, e.g., using a soil trench. In other embodiments, a composition comprising at least one polynucleotide is applied locally to the seeds (or, in the case of potatoes, locally applied to the seed potatoes) that have grown into plants with improved resistance to lepidopteran pest infestation . In some embodiments, the polynucleotide expressed in the plant is RNA, which can be single-stranded (ss) or double-stranded (ds) RNA, or a combination of both.

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 (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 where the polynucleotides are expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and the secondarily produced second polynucleotide is transiently expressed in the plant; in such embodiments, the first polynucleotide is selected from one or more of: (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 comprising a modified PolIII gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule comprising a modified PolIII gene that is transcribed into an RNA molecule, and (i) a double-stranded, hybridized RNA/DNA molecule, or combinations thereof. In such embodiments in which the polynucleotide is expressed by transient expression, the first polynucleotide may consist of naturally occurring nucleotides such as those present 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., comprises chemically modified nucleotides. The first polynucleotide is provided by any suitable means known to those skilled in the art. Embodiments include those in which the first polynucleotide is chemically or enzymatically synthesized (e.g., by in vitro transcription, such as transcription using T7 polymerase or other polymerases), produced by expression in a microorganism or in a cell culture (plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation. The first polynucleotide may be provided as an RNA or DNA fragment. Alternatively, the first polynucleotide can be provided in a more complex construct, for example as part of a recombinant expression construct, or can be included in a recombinant vector, for example a recombinant plant virus vector or a recombinant baculovirus vector; such recombinant expression constructs or vectors can be designed to include additional elements, such as an expression cassette for expressing a gene of interest (e.g., an insecticidal protein).

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

The polynucleotide expressed in the plant has at least 18 consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or the trigger sequence group, or at least one segment of complementary DNA of any of the above. In one embodiment, the polynucleotide expressed in the plant comprises at least 18 consecutive nucleotides that are essentially identical or complementary to a fragment of equivalent length 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 them. In some embodiments, the consecutive nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or a fragment of DNA complementary to either of them. In some embodiments, the contiguous nucleotides are exactly (100%) identical to the target gene sequence, the trigger sequence, or a fragment of comparable length of DNA complementary to either. In some embodiments, the polynucleotide expressed in the plant has all sequences that are about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the target gene sequence, the trigger sequence, or a fragment of DNA complementary to either.

Generally, the polynucleotide expressed in the plant is designed to suppress one or more genes ("target genes"). Such target genes may include coding or non-coding sequences, or both. In certain embodiments, the polynucleotide expressed in the plant is designed to suppress 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 suppress one or more genes, each of which has a sequence selected from the group consisting of the target gene sequences, and may be designed to suppress 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 of which comprises at least one segment of DNA complementary to any of the above, or 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequences or the trigger sequence. 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 can include multiple sections in tandem or repeated sequences, where each section includes at least one segment of 21 contiguous nucleotides having a sequence that is 100% identical to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, and the segments can be derived from different regions of the target gene, e.g., the segments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the segments.

The total length of the polynucleotide expressed in the plant can be longer than 18 or more contiguous nucleotides and can include contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, or nucleotides in addition to DNA complementary to either. In other words, the total length of the polynucleotide expressed in the plant can be longer than the length of a section or segment of a polynucleotide designed to suppress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences. For example, the polynucleotide expressed in the plant can have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that suppresses the target gene, can include "spacer" nucleotides between active segments, or can have additional nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends. In one embodiment, the polynucleotide expressed in the plant comprises additional nucleotides that are not specifically related (i.e., have a non-complementary or non-identical sequence) to the DNA or target gene having a sequence selected from the group of target gene sequences, or to its complementary DNA, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or production. In one embodiment, the polynucleotide expressed in the plant comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more consecutive nucleotides that have a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length 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 comprises one such segment with an additional 5'G or an additional 3'C or both adjacent to the segment. In another embodiment, the polynucleotide expressed in the plant is a double-stranded RNA that includes additional nucleotides to form an overhang, such as a dsRNA that includes two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide expressed in the plant is not 100% identical or complementary to the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the contiguous nucleotides in the target gene, or a fragment of DNA complementary to either of them. For example, in some embodiments, the polynucleotide expressed in the plant comprises at least two segments of each of 21 contiguous nucleotides having 100% identity to the target gene sequence group, the fragment of DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either of them, 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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either of them.

In a related aspect, the invention relates to a plant having improved resistance to infestation by lepidopteran pests, by expressing in the plant at least one polynucleotide comprising at least 18 consecutive nucleotides essentially identical or complementary to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary thereto, whereby the resulting plant has improved resistance to infestation by lepidopteran pests compared to a control plant in which the polynucleotide is not expressed. In a related aspect, the invention relates to a plant having improved resistance to infestation by lepidopteran pests, by expressing in the plant at least one polynucleotide comprising at least 18 consecutive nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or at least one segment of DNA complementary thereto, whereby the resulting plant has improved resistance to infestation by lepidopteran pests compared to a control plant in which the polynucleotide is not expressed. One embodiment is a crop plant having improved resistance to lepidopteran pest infestation compared to a control plant, provided by expressing in said plant an RNA having a sequence selected from the group of trigger sequences, or its complement. In yet another aspect, the invention relates to seeds (particularly transgenic progeny seeds) produced by the plants having improved resistance to lepidopteran pest infestation provided by this method. Also contemplated are commodity products produced by the plants having improved resistance to lepidopteran pest infestation provided by this method, and commodity products produced from transgenic progeny seeds of such plants.

VIII. Recombinant DNA Constructs for Controlling Lepidopteran Pests Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one segment of DNA having a sequence of 18 or more contiguous nucleotides having about 95% to about 100% identity to a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or complementary to either. In some embodiments, the recombinant DNA construct comprises: (a) a sequence selected from the group consisting of 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, 80 8, 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, 1 265, 1270, 1275, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, or a nucleotide sequence that is complementary to at least 18, 19, 20, or 21 consecutive nucleotides of an RNA transcribed from said target gene. DNA; 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, 54 7, 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, or (c) a DNA encoding at least one silencing element that is complementary to at least 18, 19, 20, or 21 consecutive nucleotides of a target gene or RNA transcribed from said target gene, wherein said target gene is selected from the group consisting of 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, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 4, 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, 7 30, 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, (d) a DNA having a sequence selected from the group consisting of 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 (d) the trigger gene. (e) a DNA encoding at least one silencing element comprising at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a target gene selected from a group of gene sequences or a gene in an RNA transcribed from said target gene; or (f) a DNA encoding an RNA comprising at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a nucleotide sequence selected from the group of trigger sequences or its complement, or an orthologous nucleotide sequence from a lepidopteran pest, wherein said orthologous nucleotide sequence has at least 95% sequence identity to a nucleotide sequence selected from the group of trigger sequences, wherein the percent sequence identity is calculated over the same length; or (g) a DNA encoding at least one silencing element comprising at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a nucleotide sequence selected from the group of trigger sequences or its complement, or an orthologous nucleotide sequence from a lepidopteran pest; or (g) a DNA encoding an RNA comprising at least one strand comprising at least 18, 19, 20 or 21 contiguous nucleotides that are complementary to a nucleotide sequence selected from another double-stranded RNA region, the trigger sequence group, or its complement, or an orthologous nucleotide sequence from a lepidopteran pest, wherein the orthologous nucleotide sequence has at least 95% sequence identity to a nucleotide sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either, and wherein the percent sequence identity is calculated over the same length; or (g) a heterologous promoter operably linked to a DNA encoding an RNA comprising 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, 537, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 589, 590, 591, 592, 593, 594, 595, 5 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 , 1473, 1481, 1493, 1494, 1495, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or a complement thereof.

An embodiment includes a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding a dsRNA having a strand with a sequence selected from a group of trigger sequences, or a fragment thereof, or any of its complements. The recombinant DNA construct is useful, for example, for providing a plant with improved resistance to lepidopteran pest infestation by expressing the transcript of such a recombinant DNA construct in the plant. The recombinant DNA construct is also useful for producing a polynucleotide useful for making a composition that can be applied to plants, seeds, reproductive plant parts, soil or fields, or surfaces that require protection from lepidopteran pest infestation . Related aspects of the invention include a composition comprising a recombinant DNA construct; a plant chromosome or plastid or a recombinant plant viral vector or a recombinant baculoviral vector comprising said recombinant DNA construct; a transgenic Solanaceae plant cell carrying said recombinant DNA construct in its genome and optionally comprising DNA encoding at least one insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein, and a transgenic Solanaceae plant comprising such a transgenic Solanaceae plant cell, or a fruit, seed or reproductive part of the transgenic Solanaceae plant; and a plant with improved lepidoptera resistance provided by expression of or treatment with the recombinant DNA construct or RNA encoded thereby.

The recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, or its complementary DNA. In some embodiments, the segment of 18 or more contiguous nucleotides has about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA having a sequence selected from the group of target gene sequences, or its complementary DNA. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, or its complementary DNA. In some embodiments, the DNA has all sequences of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA having a sequence selected from the group of target gene sequences, or its complementary DNA.

Thus, the recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides or complementary DNA thereof designed to suppress expression of a target gene having a sequence selected from a group of target gene sequences. In some embodiments, the DNA comprises at least 18 or more contiguous nucleotides, e.g., between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or more. In some embodiments, the segments are greater than 18 in number, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 consecutive nucleotides. In certain embodiments, the DNA encodes an RNA comprising at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having 100% sequence identity to a DNA having a sequence selected from the target gene sequence group or a fragment of equivalent length of the target gene, or a complementary DNA thereof. In certain embodiments, the DNA encodes a double-stranded nucleic acid (e.g., dsRNA) having a single strand comprising one strand comprising at least 18, 19, 20, or 21 consecutive nucleotides or their complementary DNA with a sequence of 100% identity to a fragment of equivalent length of the DNA or target gene having a sequence selected from the group of target gene sequences; and at least one segment of at least 18, 19, 20, or 21 consecutive perfectly matched base pairs or their complementary DNA corresponding to a fragment of equivalent length of the DNA or target gene having a sequence selected from the group of target gene sequences. In certain embodiments, the length of each segment contained in the DNA is longer than the typical length of naturally occurring small regulatory RNA. In some embodiments, the length of each segment is 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 (the DNA or target gene having a sequence selected from the group consisting of the group of target gene sequences). In some embodiments, the total length of the DNA is between about 50 and about 500 nucleotides in length. In some embodiments, the DNA is selected from the group consisting of 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, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579 , 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, The nucleic acid sequence encoding the RNA is 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 a complement thereof.

The recombinant DNA construct generally comprises a heterologous promoter operably linked to DNA designed to repress one or more genes ("target genes"). Such target genes may comprise coding or non-coding sequences, or both. In certain embodiments, the recombinant DNA construct 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 recombinant DNA construct 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 may be designed to repress multiple genes from this group, or may target different regions of one or more of these genes. In one embodiment, the recombinant DNA construct comprises a heterologous promoter operably linked to multiple sections or segments, each of which comprises at least one segment of 21 contiguous nucleotides or complementary DNA having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequences. 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 recombinant DNA construct can include a heterologous promoter operably linked in tandem or repeat sequences to multiple sections, where each section includes at least one segment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group, or a complementary DNA thereof, where the segments can be derived from different regions of the target gene, e.g., the segments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can be optionally used between or adjacent to the segments.

The recombinant DNA construct may include a heterologous promoter operably linked to DNA that may have a total length greater than 18 contiguous nucleotides, and may include nucleotides in addition to at least one segment of 18 or more contiguous nucleotides or complementary DNA having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences. In other words, the total length of the DNA may be greater than the length of a segment of DNA designed to repress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences. For example, the DNA may have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that represses the target gene, or may include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, the 3' end, or at both the 5' and 3' ends. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from the group consisting of the target gene sequences, or a complementary DNA thereof, or DNA containing additional nucleotides not specifically related (i.e., having a sequence that is not complementary or identical) to the target gene, such as nucleotides that provide a stabilizing secondary structure or provide convenience in cloning or manufacturing. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from the group consisting of the target gene sequences, the group consisting of the trigger sequences, or DNA containing additional nucleotides located immediately adjacent to one or more segments of DNA complementary to either of the 18 or more consecutive nucleotides having a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of the target gene. In one embodiment, the heterologous promoter is operably linked to DNA containing one such segment with an additional 5'G or an additional 3'C or both adjacent to the segment. In another embodiment, the heterologous promoter is operably linked to DNA encoding a double-stranded RNA that includes additional nucleotides to form an overhang; thus, in various embodiments, the heterologous promoter is not 100% identical or complementary to the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or contiguous nucleotides in a target gene, or a fragment of DNA complementary to either. For example, in some embodiments, the heterologous promoter comprises at least two segments of each of 21 contiguous nucleotides having 100% identity to the target gene sequence group, the fragment of DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either, 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 are present in the target gene sequence group, the DNA having a sequence selected from the trigger sequence group, or the DNA complementary to either.

In the recombinant DNA construct, the heterologous promoter is operably linked to DNA encoding a transcript that can be single-stranded (ss) or double-stranded (ds), or a combination of both. Embodiments of the method include those in which the DNA encodes a transcript comprising sense single-stranded RNA (ssRNA), antisense ssRNA, or double-stranded RNA (dsRNA), or any combination thereof.

The recombinant DNA construct may be provided by any suitable means known to those skilled in the art. Embodiments include those in which the recombinant DNA construct is synthesized in vitro, produced by expression in a microorganism or in a cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

The heterologous promoter used in the recombinant DNA construct is selected from the group consisting of a promoter that functions in plants, a promoter that functions in prokaryotes, a promoter that functions in fungal cells, and a baculovirus promoter. Non-limiting examples of promoters are described in the "Promoters" section.

In some embodiments, the recombinant DNA construct also includes a second promoter that is operably linked to the DNA. For example, a DNA comprising at least one segment of 18 or more contiguous nucleotides can be flanked by two promoters arranged such that the promoters transcribe in a convergent manner in opposite directions, producing opposite strand transcripts of the DNA that are complementary to each other and can hybridize to form double-stranded RNA. In one embodiment, the DNA is located between two root-specific promoters, which allow transcription of the DNA in opposite directions, resulting in the formation of dsRNA.

In some embodiments, the recombinant DNA construct comprises other DNA elements in addition to a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides or complementary DNA having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences. 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 a transgene such as an insecticidal protein or a selectable marker) or for expressing non-coding sequences (e.g., for expressing additional inhibitory elements). The inclusion of one or more recognition sites for binding and cleavage by small RNA (e.g., by miRNA or siRNA that is expressed only in specific cells or tissues) allows for a more precise expression pattern in the plant, where expression of the recombinant DNA construct is inhibited where the small RNA is expressed.

In some embodiments, the recombinant DNA construct is provided in a recombinant vector. "Recombinant vector" refers to a recombinant polynucleotide molecule used to transfer genetic information from one cell to another. Suitable embodiments for the present invention include, but are not limited to, recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculoviral vectors. Alternative embodiments include recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculoviral vectors, that contain DNA elements that do not contain a heterologous promoter.

In some embodiments, the recombinant DNA construct is provided in a chromosome or plastid of a plant, for example, in a transgenic plant cell or transgenic plant. Thus, the present invention encompasses a transgenic plant cell having a recombinant DNA construct in its genome, as well as a transgenic or partially transgenic plant comprising such a transgenic plant cell. A partially transgenic plant includes, for example, a non-transgenic scion grafted onto a transgenic rootstock comprising the transgenic plant cell. An embodiment includes a transgenic tomato rootstock comprising the transgenic plant cell. The plant can be any plant that is subject to infestation by lepidopteran pests. Of particular interest are embodiments in which the plant is a crop plant. Embodiments include those in which the plant is an ungerminated crop plant seed, a crop plant in the vegetative growth stage, or a crop plant in the reproductive stage. An embodiment includes those in which 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 aspect, the present invention relates to seeds (particularly transgenic progeny seeds) produced by transgenic plants having in their genome the recombinant DNA constructs described herein. Embodiments also include transgenic seed potatoes having in their genome the recombinant DNA constructs described herein. Also contemplated are commodity products produced by such transgenic plants, and commodity products produced from transgenic progeny seeds of such transgenic plants.

The recombinant DNA construct may be provided in a composition for topical application to the surface of a plant or a plant seed, or to any substrate requiring protection from infestation by lepidopteran pests. Similarly, the recombinant DNA construct may be provided in a composition for topical application to a lepidopteran pest, or in a composition for ingestion by a lepidopteran pest. In various embodiments, such compositions containing the recombinant DNA construct are provided in at least one form selected from the group consisting of solids, liquids (including homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, provided in or on microbeads or other carrier particles, in a film or coating, on or in a matrix, or as a seed treatment. The topical application may be in the form of a topical treatment of the fruit of a solanaceous plant or seeds obtained from the fruit of a solanaceous plant, or in the form of a treatment of tubers or tuber fragments of "seed potatoes" (e.g., by immersing, coating, or dusting the seed potatoes). Suitable binders, inert carriers, surfactants, etc., known to those skilled in the art of pesticide formulation and seed treatment, may optionally be included in the composition. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one locally implantable formulation selected from the group consisting of particles, pellets, or capsules locally implanted in the plant; in such embodiments, the method comprises locally implanting a locally implantable formulation in the plant. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one in-furrow formulation selected from the group consisting of powders, granules, pellets, capsules, sprays, or drenches, or any other form suitable for topical application in furrows; in such embodiments, the method comprises in-furrow treatment with an in-furrow formulation. In one embodiment, the composition for topical application containing the recombinant DNA construct may be ingested or otherwise internalized by a lepidopteran pest. For example, the composition for topical application containing the recombinant DNA construct may be in the form of a bait. In some embodiments, the composition comprising the recombinant DNA construct further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (e.g., as disclosed in Example 18 of U.S. Patent Application Publication No. 2011/0296556, incorporated herein by reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide insecticide, a safener, and an insect growth regulator. In one embodiment, the composition containing the recombinant DNA construct further comprises a nonionic organosilicone surfactant, such as a SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, having CAS No. 27306-78-1 and EPA No. CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. In some embodiments, the composition comprising the recombinant DNA construct further comprises at least one insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

It is expected that the combination of certain recombinant DNA constructs described herein (e.g., recombinant DNA constructs comprising a polynucleotide trigger as described in the Examples), whether by transgenic expression or by topical application, with one or more non-polynucleotide insecticides, whether by transgenic expression or by topical application, will result in enhanced improvements in the prevention or control of lepidopteran pest infestations , compared to the effects obtained with the transgenic DNA construct alone or the non-polynucleotide insecticide alone, whether by transgenic expression or by topical application. In one embodiment, recombinant DNA constructs for expressing one or more polypeptides and one or more polynucleotides encoding a non-polynucleotide insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein are found to provide improved resistance to lepidopteran pest infestations in plants expressing the recombinant DNA construct. One embodiment relates to a recombinant DNA for expressing an RNA comprising a segment comprising a sequence selected from the group of trigger sequences and one or more genes encoding a non-polynucleotide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

The composition containing the recombinant DNA construct can be provided for feeding by lepidopteran pests by applying the composition to a plant or surface subject to infestation by the lepidopteran pest, for example by spraying, dusting or coating the plant, or by applying a soil drench, or by providing in an artificial diet. The composition containing the recombinant DNA construct can be provided for feeding by lepidopteran pests in an artificial diet formulated to meet the specific nutritional requirements for sustaining the lepidopteran pest, where the artificial diet is supplemented with some amount of the recombinant DNA construct obtained from another source, such as in vitro synthesis, or purified from microbial fermentation or other biological sources; this embodiment can be useful, for example, for determining the timing and amount of an effective treatment regime. In some embodiments, the composition containing the recombinant DNA construct is 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 in microbial fermentation products, or in a synthetic diet. In one embodiment, the composition containing the recombinant DNA construct is provided in the form of a bait for ingestion by lepidopteran pests. The composition containing the recombinant DNA construct can be provided in the form of a seed treatment for feeding by lepidopteran pests.

In various embodiments, the composition containing the recombinant DNA construct comprises or is produced in a microbial cell. For example, the composition containing the recombinant DNA construct can comprise or be produced in a bacterial or yeast cell. In similar embodiments, the composition containing the recombinant DNA construct comprises or is produced in a transgenic plant cell (e.g., in a plant cell that transiently expresses the recombinant DNA construct); such a plant cell can be a cell in a plant or a cell grown in tissue culture or cell suspension.

IX. Transgenic Plant Cells Some embodiments relate to transgenic crop plant cells expressing polynucleotides useful in the methods described herein for suppressing expression of a target gene in a lepidopteran pest or for controlling lepidopteran infestation . In one aspect, the present invention provides a transgenic solanaceous plant cell having a recombinant DNA in its genome encoding an RNA comprising at least one segment of DNA having a sequence of about 95% to about 100% identity of 18 or more consecutive nucleotides to a fragment of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a sequence complementary to either of them. In one aspect, the present invention provides a transgenic solanaceous plant cell having a recombinant DNA in its genome encoding an RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of a lepidopteran pest larva, wherein the target gene sequence is selected from the group of target gene sequences, the group of trigger sequences, or a sequence complementary to either of them. In one aspect, the invention provides a transgenic solanaceous plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in a lepidopteran pest that contacts or ingests the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides that is complementary to a fragment of the target gene, and wherein the target gene is selected from the group consisting of genes in the group of target gene sequences. A specific embodiment is a transgenic crop plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in a lepidopteran pest that contacts or ingests the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides that is complementary to a fragment of one or more target gene sequences. In one aspect, the invention provides a transgenic crop plant cell having in its genome a recombinant DNA encoding an RNA having a sequence selected from the group of trigger sequences. Such transgenic crop plant cells are useful for providing transgenic crop plants having improved resistance to infestation by lepidopteran pests compared to control plants lacking such plant cells. The transgenic crop plant cells can be isolated transgenic solanaceous plant cells, or transgenic solanaceous plant cells grown in culture, or transgenic cells of any transgenic crop plant that is subject to infestation by lepidopteran pests.

In one embodiment, the recombinant DNA is stably integrated into the genome of the transgenic crop plant, from which it can be expressed in one or more cells of the transgenic solanaceous plant. Methods for providing stably transformed plants are provided in the section entitled "Making and Using Transgenic Plant Cells and Transgenic Plants."

Some embodiments relate to a transgenic solanaceous plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in a lepidopteran pest that contacts or ingests 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 a complement thereof. In some embodiments, the silencing element comprises at least one 18 or more contiguous nucleotides having a sequence that is about 95% to about 100% complementary to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences, the group of trigger sequences, or a DNA complementary to either of them. In some embodiments, the silencing element comprises at least one 18 or more contiguous nucleotides capable of hybridizing in vivo or under physiological conditions (e.g., physiological conditions typically found in cells of lepidopteran pests) to a fragment of comparable length of DNA having a sequence selected from the target gene sequence group, the trigger sequence group, or DNA complementary to either of them. The number of contiguous nucleotides is at least 18, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or more. In some embodiments, the contiguous nucleotides are more than 18 in number, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., 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 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or more than 500 contiguous nucleotides. In certain embodiments, the silencing element comprises at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of its complementary DNA. In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having one strand comprising at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of its complementary DNA; such double-stranded nucleic acid, expressed as base pairs, comprises at least 18, 19, 20, or 21 consecutive, perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or at least one segment of its complementary DNA. In certain embodiments, the length of each silencing element contained in the RNA is longer than the typical length of naturally occurring small regulatory RNAs. In some embodiments, the length of each segment is at least about 30 contiguous nucleotides (or base pairs). In certain embodiments, the length of the RNA is between about 50 and about 500 nucleotides. In certain embodiments, the RNA has a sequence selected from the group of trigger sequences.

In some embodiments, the transgenic crop plant cell is capable of further expressing additional heterologous DNA sequences. In one embodiment, the transgenic solanaceous plant cell has a genome further comprising recombinant DNA encoding at least one insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In certain embodiments, the transgenic crop plant cell stably integrates into its genome (i) recombinant DNA encoding at least one RNA having a sequence selected from the group of trigger sequences, and (ii) DNA encoding at least one insecticide selected from the group consisting of patatin, plant lectins, phytoecdysteroids, Bacillus thuringiensis insecticidal proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins.

In related aspects, the invention relates to the transgenic crop plant cells, commodity products produced from the transgenic crop plants, and transgenic solanaceous plant seeds or transgenic reproductive parts of transgenic solanaceous plants. Also contemplated are commodity products produced by the transgenic crop plants and commodity products produced from transgenic progeny seeds of such transgenic crop plants.

X. Methods for Selecting 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 present in a single or low copy number (non-repeated and non-redundant) in a particular genome. Such target genes can be genes from plant genomes or genes from animal genomes. In some embodiments, the target genes are genes of invertebrate pests, such as invertebrate pests of plants or invertebrate pests of vertebrates. In some embodiments, the target genes are genes of insect pests of plants or nematode pests of plants. In some embodiments, the target genes are genes of lepidopteran pests. A further aspect includes producing a polynucleotide (e.g., ssRNA or dsRNA triggers, such as dsRNA triggers described in the Examples, or recombinant DNA constructs useful for generating transgenic plants) based on a target gene for RNAi-mediated silencing selected by any of the methods described herein.

In one embodiment, the method comprises identifying single or low copy number genes in a selected genome, or alternatively identifying single or low copy number genes in an ortholog database from related 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 or low copy number 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 has been identified as single or low copy number in the second species. In one embodiment, the identification of single or low copy number genes is performed by applying a computer-implemented algorithm to the set of genes from the first species to identify a subset of single or low copy number 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 or low copy number genes from the first species to identify the corresponding single or low copy number genes from the second species. Single or low copy number genes from the 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 triggers described in the Examples, or recombinant DNA constructs for generating transgenic plants) and methods for their use to prevent or control infestation by the second species. Another embodiment relates to identifying genes that are lethal to the survival of an organism by searching databases containing such information for model species that are orthologous to the target organism.

An embodiment of the method includes the further step of estimating the nucleotide diversity of low/single copy genes in a population of selected organisms and selecting those low/single copy genes that further have the lowest nucleotide diversity. The low/single copy genes that further have 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 organism by searching a database containing such information for model species that are orthologous to the target organism.

An embodiment of the method includes the further step of comparing the ratio of synonymous (Ks) to nonsynonymous (Ka) nucleotide changes as an estimate of functional or evolutionary constraints. In one embodiment, the method includes selecting genes for which Ks is equal to or greater than Ka. In one embodiment, the method includes selecting genes for which Ka>>Ka.

A related aspect of the invention is a set 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 single or low copy number target genes from a larger set of genes from the genome. One embodiment relates to a set of target genes for RNAi-mediated silencing selected from an invertebrate genome by identifying single or low copy number target genes from a larger set of genes from the invertebrate genome. Particular embodiments relate to a set of target genes for RNAi-mediated silencing in lepidopteran pests selected from a lepidopteran genome by identifying single or low copy number target genes from a larger set of genes from the lepidopteran genome. Particular embodiments relate to a set of target genes for RNAi-mediated silencing in lepidopteran pests selected from a lepidopteran genome by identifying single or low copy number target genes from a larger set of genes from the lepidopteran genome.

Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from a genome by estimating the nucleotide diversity for a given set of genes in a population of individuals of a pest carrying said genome and selecting those genes with the lowest nucleotide diversity. One embodiment relates to a set of target genes for RNAi-mediated silencing selected from an invertebrate genome by estimating the nucleotide diversity for a given set of genes in an individual population of said invertebrates carrying said genome and selecting those genomes with said lowest nucleotide diversity. Another embodiment relates to a set of target genes for RNAi-mediated silencing selected from an invertebrate genome by estimating the nucleotide diversity of low/single copy genes in an individual population of invertebrates carrying said genome and selecting those low/single copy genes that also have the lowest nucleotide diversity.

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 nonsynonymous (Ka) nucleotide changes in genes of the genome, comparing the ratio of synonymous (Ks) to nonsynonymous (Ka) nucleotide changes, and selecting genes for which Ks is equal to or greater than Ka. In one embodiment, the set of target genes for RNAi-mediated silencing are genes for which Ks is equal to or greater than Ka. In one embodiment, the set of target genes for RNAi-mediated silencing are genes for 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.

Further aspects of the invention are polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the group of trigger gene sequences, or their complements, and polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the group of trigger sequences and their complements, or their complements; such antibodies are made 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).

XI. Selection of Effective Polynucleotides by "Tiling" The polynucleotides used in the embodiments described herein need not be the full length of the target gene, and in many embodiments will be much shorter than the target gene. An example of a technique useful for selecting effective polynucleotides is "tiling", i.e., the evaluation of polynucleotides that correspond to adjacent or overlapping segments of the target gene.

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

If one is interested in designing a target that is effective in suppressing multiple target genes, the multiple target gene sequences are aligned and a polynucleotide trigger is designed to correspond to a region of high sequence homology common to the multiple targets. Conversely, if one is interested in designing a target that is effective in selectively suppressing one of the multiple target sequences, the multiple target gene sequences are aligned and a polynucleotide trigger is designed to correspond to a region of no or low sequence homology common to the multiple targets.

XII. Thermodynamic Considerations in Selecting Effective Polynucleotides In some embodiments, polynucleotide triggers can be designed or their sequences 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 an individual siRNA) and another nucleic acid strand (e.g., a target gene transcript).

Methods and algorithms for predicting nucleotide sequences that may be effective for RNAi-mediated silencing of target genes are 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: D672-D678 and published at https://www.flyrnai.org/snapdragon; and "E-RNAi," described in Horn et al. (2010) Nucleic Acids Res., 38: W332-W339 and published at https://www.dkfz.de/signaling/e-rnai3/.

XIII . Allowed 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 to an RNA (e.g., a transcript) transcribed from a target gene to suppress expression of the target gene (e.g., resulting in a reduction 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 a target gene or sequence or to an RNA transcribed from a target gene to suppress expression of a target gene (e.g., to result in a reduction in the level or activity of a 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 to or essentially complementary to a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some embodiments, the polynucleotide or portion thereof is designed to be 100% identical or 100% complementary to one or more sequences of 21 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "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 contiguous nucleotides in either the endogenous target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 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 said target gene or RNA transcribed from the target gene.

Sequence identity as used herein in the context of two polynucleotides or polypeptides: the term "sequence identity" or "identity" refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

As used herein, the term "percentage of sequence identity" may refer to a value determined by comparing two optimally aligned sequences of molecules (e.g., nucleic acid sequences or polypeptide sequences) over a comparison window, where the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleotides or amino acid residues occur in both sequences to give 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 at all positions compared to a reference sequence is said to be 100% identical to the reference sequence, and vice versa. The term "about" in reference to numerical values of sequence length means a stated value with a +/- variance of 1 to 5%. For example, about 30 contiguous nucleotides means a range of 27 to 33 contiguous nucleotides, or any range therebetween. The term "about" in reference to percentage sequence identity numbers means the stated value has a variance of +/- 1-3 percent, rounded to the nearest integer. For example, about 90% sequence identity means a range of 87-93%. However, said percentage of sequence identity cannot exceed 100%. Thus, about 98% sequence identity means a range of 95-100%.

Polynucleotides containing mismatches to a target gene or transcript can be used in certain embodiments of the compositions and methods described herein. In some embodiments, the polynucleotide comprises at least 18 or at least 19 or at least 21 contiguous 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 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcript of the target gene can have one or two mismatches to the target gene or transcript (i.e., one or two mismatches between the 21 contiguous nucleotides of the polynucleotide and the 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, 350 or more nucleotides containing a contiguous 18, 19, 20, or 21 or more nucleotide span of identity or complementarity to a sequence of comparable length in the target gene or a transcript of the target gene may have one or more mismatches to the target gene or transcript.

In designing polynucleotides with mismatches to endogenous target genes or to RNA transcribed from said target genes, certain types of mismatches and mismatches at certain positions that are well tolerated 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 highly tolerated 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. The tolerable mismatch can be empirically determined in routine assays, such as in vitro feeding assays against lepidopteran pest larvae.

XIV. Embedding silencing elements in neutral sequences In some embodiments, the silence elements that have sequences corresponding to the target gene and are responsible for the observed suppression of the target gene are embedded in "neutral" sequences, i.e., inserted into additional nucleotides that do not have identical sequence or complementarity to the target gene. Neutral sequences may be desirable, for example, to increase the overall length of the polynucleotide. For example, it may be desirable for a polynucleotide to be of a certain size, for reasons such as stability in manufacturing, cost-effectiveness, or biological activity. In some embodiments, neutral sequences are also useful for forming loops in hairpin triggers, or as spacers between trigger regions.

In another Coleoptera species, Diabrotica virgifera, dsRNAs longer than or equal to about 60 base pairs (bp) have been reported to be required for biological activity in an artificial feeding bioassay; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi:10.1371/journal.pone.0047534. Thus, in one embodiment, a 21-bp dsRNA silencing element that corresponds to a target gene in the group of target gene sequences and was found to provide control of Lepidoptera infestations is embedded in an additional 39-bp neutral sequence, thereby forming a polynucleotide of about 60 base pairs. In some embodiments, the dsRNA trigger comprises a neutral sequence of about 60 to about 500 or about 100 to about 450 base pairs, in which at least one segment of 21 consecutive nucleotides is embedded with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of a target gene having a sequence selected from the group of target gene sequences.In another embodiment, a single 21 base pair silencing element with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of a target gene is found to be effective when embedded in a larger section of neutral sequence (e.g., total polynucleotide length is about 60 to about 300 base pairs). In embodiments in which the polynucleotide comprises a region of neutral sequence, the polynucleotide has a relatively low overall sequence identity compared to the target gene; for example, a dsRNA having an overall length of 210 base pairs containing a single 21 base pair trigger (having 100% identity or complementarity to a 21 nucleotide fragment of the target gene) embedded in an additional 189 base pair neutral sequence has about 10% overall sequence identity to the target gene.

XV. Insecticidal double-stranded RNA molecules Another aspect of the present invention provides insecticidal double-stranded RNA molecules that, when ingested by or contacted by a lepidopteran pest, cause death or growth inhibition in the lepidopteran pest, wherein the insecticidal double-stranded RNA molecule comprises at least one segment of 18 or more contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length of a target gene or DNA (cDNA) 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 in length. In some embodiments, the insecticidal double-stranded RNA molecule comprises at least one segment of at least 30 contiguous nucleotides in length. In some embodiments, the insecticidal double-stranded RNA molecule comprises a plurality of segments of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to segments of equivalent length of a target gene or DNA (cDNA) having a sequence selected from the group of target gene sequences, where the segments are derived from different regions of the target gene (e.g., the segments can correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene can optionally be 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 contiguous nucleotides that are essentially identical or essentially complementary to segments of equivalent length of a DNA (cDNA) having a sequence selected from the group of target gene sequences, where the segments are derived from different regions of the target gene and arranged in the insecticidal double-stranded RNA molecule in an order that is different from the order in which the segments naturally occur in the target gene. In some embodiments, the insecticidal double-stranded RNA molecule comprises multiple segments of 21 consecutive nucleotides each having a sequence of 100% identity or 100% complementarity to a segment of comparable length of a target gene or DNA (cDNA) having a sequence selected from the group of target gene sequences, where the segments are derived from different 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 occur in the target gene. In some embodiments, the insecticidal double-stranded RNA molecule comprises a single strand comprising a sequence selected from the group of trigger sequences, or its complement. The insecticidal double-stranded RNA molecule can be applied topically to plants to control or prevent infestation 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 or 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 for other aspects of the invention.

Some embodiments relate to tank mixes comprising one or more insecticidal polynucleotides and water or other solvent, optionally including a cationic lipid or an organosilicone surfactant, or both. Embodiments include a tank mix formulation of said polynucleotides and optionally at least one insecticide selected from the group consisting of patatin, a plant lectin, a plant ecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. Embodiments of such compositions include those in which the one or more insecticidal polynucleotides are provided in a living or dead microorganism, such as a bacteria, fungus, yeast cell, or provided as a microbial fermentation product, or provided in a living or dead plant cell, or provided as a synthetic recombinant polynucleotide. In one embodiment, the composition comprises a non-pathogenic strain of a microorganism containing a polynucleotide as described herein; ingestion or ingestion 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 viral vector comprising a polynucleotide as described herein; feeding by a lepidopteran pest of a plant treated with the plant viral vector results in inhibition of growth or death of the lepidopteran pest. In one embodiment, the composition comprises a baculoviral vector comprising a polynucleotide as described herein; ingestion or uptake by a vector results in inhibition of growth or death of the lepidopteran pest. In one embodiment, the polynucleotide as described herein is encapsulated in a synthetic matrix, such as a polymer, or attached to a microparticle and applied topically to a surface of a plant; feeding by a lepidopteran pest of the topically treated plant results in inhibition of growth or death of the lepidopteran pest. In one embodiment, the polynucleotide as described herein is provided in the form of a plant cell (e.g., a transgenic Solanaceae plant cell of the invention) expressing the polynucleotide; feeding by a lepidopteran pest of the plant cell or contents of the plant cell results in inhibition of growth or death of the lepidopteran pest.

In some embodiments, one or more polynucleotides described herein are provided with suitable adhesives and wetting agents necessary for efficient leaf coverage, and UV protectants to protect polynucleotides such as dsRNA from UV damage. Such additives are commonly used in the bio-pesticides industry and are known to those skilled in the art. Compositions for soil application can include granular formulations that serve as bait for lepidopteran pest larvae. In some embodiments, one or more polynucleotides described herein are further provided with carrier agents, surfactants, cationic lipids (e.g., those disclosed in Example 18 of US Patent Application Publication No. 2011/0296556, which is incorporated herein by reference), organosilicon, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides , toxic safeners, and insect growth regulators. In some embodiments, the composition further comprises at least one insecticide selected from the group consisting of patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

Such compositions are applied in any convenient manner, such as, for example, by spraying or dusting directly on the lepidopteran pests, or by spraying or dusting the plants or environment in which it is desired to prevent or control infestation by the lepidopteran pests, or by applying a coating to the surface of a plant, or by applying a coating to seeds (or seed potatoes) in preparation for planting the seeds, or by applying a soil drench around the roots of a plant in which it is desired to prevent or control infestation by the lepidopteran pests.

An effective amount of the polynucleotide described herein is an amount sufficient to provide control of lepidopteran pests or prevent infestation by lepidopteran pests; the determination of the effective amount of the polynucleotide is performed using routine assays. There is no upper limit to the concentration and dosage of the insecticidal polynucleotide that may be useful in the methods and compositions provided herein, but lower effective concentrations and dosages are generally required for efficiency and economy. Non-limiting embodiments of an effective amount of polynucleotide include about 10 nanograms to about 100 micrograms of polynucleotide per milliliter in the form of a liquid sprayed on a plant, or about 10 milligrams to about 100 grams of polynucleotide per acre when applied to a field of plants, or about 0.001 micrograms to about 0.1 micrograms of polypeptide per milliliter in artificial food for feeding on lepidopteran pests. When the polynucleotides described herein are applied topically to plants, the concentration can be adjusted to take into account the amount of spray or treatment applied to the surface of the plant foliage or other plant parts, such as petals, stems, tubers, fruits, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using the 25 amino acid long polynucleotides described herein is about 1 nanomole (nmol) of polynucleotide per plant, for example, about 0.05 to 1 nmol of polynucleotide 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 polynucleotide per plant. In certain embodiments, about 40 to about 50 nmol of ssDNA polynucleotide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of dsRNA is applied. In certain embodiments, compositions containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of dsRNA or ssDNA (21 mer) are applied. In certain embodiments, compositions of dsRNA polynucleotides of the invention containing about 0.5 to about 1.5 milligrams per milliliter of about 50 to about 200 or more nucleotides are applied. In certain embodiments, about 1 to about 5 nanomoles of dsRNA of the invention are applied to a plant. In certain embodiments, the polynucleotide composition topically applied to the plant contains at least one polynucleotide of the invention at a concentration of about 0.01 to about 10 milligrams per milliliter, 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 polynucleotide. Lower concentrations may be used when using long dsRNA molecules of the invention that can be processed into multiple oligonucleotides (e.g., multiple triggers encoded by a single recombinant DNA molecule of the invention). Non-limiting examples of effective polynucleotide treatment regimes include treatments of about 0.1 to about 1 nmol of polynucleotide molecule per plant, or about 1 to about 10 nmol of polynucleotide molecule per plant, or about 10 nmol to about 100 nmol of polynucleotide molecule per plant.

In some embodiments, one or more polynucleotides are provided with a "translocation agent," which is an agent that allows the topically applied polynucleotide to enter the cells of an organism. Such a translocation agent can be incorporated as part of a composition comprising the polynucleotide described herein, or can be applied prior to, simultaneously with, or after application of the polynucleotide. In some embodiments, the translocation agent is an agent that improves uptake of the polynucleotides of the invention by lepidopteran pests. In some embodiments, the translocation agent is an agent that conditions the surface of a plant tissue, e.g., a seed, leaf, stem, root, flower, or fruit, for penetration of the polynucleotide into the plant cell. In some embodiments, the translocation agent allows a pathway for the polynucleotide to enter the plant cell through a cuticular wax barrier, a stomata, and/or a cell wall or membrane barrier.

Suitable transfer agents include agents that increase the permeability of the exterior of the organism or increase the permeability of the cells of the organism to the polynucleotide. Suitable transfer agents include chemical agents, physical agents, or combinations thereof. Chemical agents for conditioning or transfer 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, application of the polynucleotide and transfer agent optionally includes an incubation step, a neutralization step (e.g., to neutralize the acid, base, or oxidizing agent or to inactivate the enzyme), a water wash step, or a combination thereof. Suitable transfer agents may be in the form of an emulsion, a reverse emulsion, a liposome, or other micelle-like composition, or may cause the polynucleotide to take the form of an emulsion, a reverse emulsion, a liposome, or other micelle-like composition. Examples of transfer agents include counterions or other molecules known to associate with nucleic acid molecules, such as inorganic ammonium ions, alkyl ammonium ions, lithium ions, spermine, spermidine, or putrescine, and other polyamines, and other cations. Examples of transfer agents include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or solvents that are miscible with water or that dissolve phosphonucleotides in non-aqueous systems, such as those used in synthesis reactions. Examples of transfer agents include oils of natural origin or synthetic origin, such as oils of plant origin, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants and published online in the 9th Compendium of Herbicide Adjuvants), paraffin oils, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines, such as polyethyleneimine or N-pyrrolidine, with or without surfactants or emulsifiers.

Embodiments of the transfer agent include organosilicone formulations. For example, a suitable transfer agent is an organosilicone formulation commercially available as SILWET L-77™ brand surfactant having CAS No. 27306-78-1 and EPA No. CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, N.Y. One embodiment includes a composition comprising a polynucleotide and a transfer agent comprising an organosilicone formulation, such as SILWET L-77, in the range of about 0.015 to about 2% (weight percent) 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 weight percent). One embodiment includes a composition comprising a polynucleotide of the invention and a transfer agent comprising SILWET L-77® brand surfactant in the range of about 0.3 to about 1% (weight percent) or about 0.5 to about 1% (weight percent) by weight.

（化合物Ｉ：ポリアルキレンオキシドヘプタメチルトリシロキサン、平均ｎ＝７．５）
である。
移動剤として有用な有機シリコーン化合物は、例えば、重量で約０．０１５％から約２％（重量パーセント）（例えば、約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重量パーセント）の範囲の新たに作成された濃度で使用される。 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 head group, (b) a covalently linked alkyl linker, including but not limited to an n-propyl linker, (c) a covalently linked polyglycol chain, and (d) an end group. The trisiloxane head group of such organosilicone compounds includes, but is not limited to, heptamethyltrisiloxane. The alkyl linker can 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 can include a mixture providing 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 end group can 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 transfer agent for use in the present invention is Compound I:

(Compound I: Polyalkylene oxide heptamethyltrisiloxane, average n=7.5)
It is.
Organosilicon compounds useful as transfer agents are used, for example, in freshly made concentrations ranging from about 0.015% to about 2% by weight (weight percent) (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 weight percent).

Embodiments of the transfer agent include one or more salts, such as ammonium chloride, tetrabutylphosphonium bromide, ammonium sulfate, etc., provided in or used with compositions comprising polynucleotides. In some embodiments, ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate are used at a concentration 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, compositions comprising polynucleotides include ammonium salts at a concentration of 300 millimolar or greater. In certain embodiments, compositions comprising polynucleotides include an organosilicone 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.

An embodiment of the transfer agent comprises a phosphate salt. Phosphate salts useful in compositions comprising polynucleotides include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, compositions comprising polynucleotides comprise a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprise polynucleotide phosphate salts in the range of about 1 mM to about 25 mM or in the range of about 5 mM to about 25 mM. In certain embodiments, compositions comprise polynucleotide sodium phosphate salts at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate salts at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate salts in the range of about 1 mM to about 25 mM or in the range of about 5 mM to about 25 mM. In certain embodiments, the composition comprising the polynucleotide comprises a sodium phosphate salt in the range of about 10 mM to about 160 mM or in the range of about 20 mM to about 40 mM. In certain embodiments, the composition comprising the polynucleotide comprises a sodium phosphate buffer at a pH of about 6.8.

The embodiment of the transfer agent includes a surfactant and/or an effective molecule contained therein. The surfactant and/or the effective molecule contained therein includes, but is not limited to, sodium or lithium salts of fatty acids (such as tallow, beef tallow fatty acids or phospholipids) and organosilicone surfactants. In certain embodiments, the composition containing the polynucleotide is 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 comprising a polynucleotide are formulated with a non-polynucleotide herbicide, for example, auxin-like benzoic acid herbicides including glyphosate, dicamba, chloramben, and TBA, auxin-like herbicides including glufosinate, phenoxycarboxylic acid herbicides, pyridine carboxylic acid herbicides, quinoline carboxylic acid herbicides, pyrimidine carboxylic acid herbicides, and benazolin ethyl herbicides, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohexandione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibitor herbicides. In certain embodiments, compositions comprising polynucleotides are formulated with non-polynucleotide insecticides, such as 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, compositions comprising polynucleotides and non-polynucleotide insecticides provide enhanced improvements in preventing or controlling lepidopteran pest infestations compared to the effects achieved with the polynucleotide alone or the non-polynucleotide insecticide alone. In some embodiments, a composition comprising a double-stranded RNA having a strand with a sequence selected from the group of trigger sequences is combined with a non-polynucleotide insecticide (patatin, plant lectins, phytoecdysteroids, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, and Bacillus sphaericus insecticidal protein), wherein the combination is found to be effective in improving the prevention or control of lepidopteran pest infestations compared to the effectiveness obtained with the double-stranded RNA alone or the non-polynucleotide insecticide alone.

XVI. Related Art Implementations of the polynucleotides and nucleic acid molecules described herein may include additional elements, such as promoters, small RNA 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 selection markers) or non-coding sequences (e.g., for expressing additional inhibitory elements). For example, one aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to DNA comprising at least one segment of DNA complementary thereto, or 18 or more contiguous nucleotides having about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences or the group of trigger sequences. Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA hairpin having an antisense region having a sequence or a fragment of a sequence selected from the group of trigger sequences. In another embodiment, a recombinant DNA construct comprising (a) an RNA silencing element for silencing a target gene selected from a group of target gene sequences, and (b) a promoter operably linked to DNA encoding an aptamer is stably integrated into the genome of a plant, from where the RNA transcript comprising said RNA aptamer and the RNA silencing element are expressed in the cells of said plant; said aptamer serves to guide said 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 (e.g., by miRNA or siRNA that is expressed only in specific cells or tissues) allows for a more precise expression pattern in the plant, where expression of said recombinant DNA construct is suppressed where the small RNA is expressed. Such additional elements are described below.

XVII. Promoter The promoters used in the present invention are functional in the cells in which the construct is intended to be transcribed. Generally, these promoters are heterologous promoters used in the recombinant constructs, i.e., promoters that are not naturally found operably linked to other nucleic acid elements used in the constructs described herein. In various embodiments, the promoter is selected from the group consisting of constitutive 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, for example, a polII promoter, a polIII promoter, a polV promoter, or a polV promoter.

Non-structural promoters suitable for use with the recombinant DNA constructs of the present invention include spatially specific promoters, temporally 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). Often, seed-specific, embryo-specific, starch-specific, or endosperm-specific promoters are particularly useful. Temporally specific promoters can include promoters that tend to promote expression at particular developmental stages in the plant's growth cycle, at different times of day or night, or during different seasons of the year. Inducible promoters include promoters that are induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e.g., lack of water 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 with temporally specific, spatially specific, or inducible expression patterns; examples of miRNA promoters, as well as methods for identifying miRNA promoters with particular expression patterns, are provided in U.S. Patent Application Publication Nos. 2006/0200878, 2007/0199095, and 2007/0300329, which are specifically incorporated herein by reference. Expression-specific promoters can also include promoters that are generally constitutively expressed, but that differ in the degree or "strength" of expression, including promoters that are generally considered to be "strong promoters" or "weak promoters."

Specific promoters of interest are: the opaline synthase promoter isolated from the T-DNA of Agrobacterium; 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. Pat. Nos. 5,837,848, 6,437,217, and 6,426,446; the maize L3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252; Examples of promoters include a promoter for a plant nuclear gene encoding a plastid-localized aldolase, as disclosed in US Patent Application Publication No. 2004/0216189; a cold-inducible promoter, as disclosed in US Patent Application Publication No. 6,084,089; a salt-inducible promoter, as disclosed in US Patent Application Publication No. 6,140,078; a light-inducible promoter, as disclosed in US Patent Application Publication No. 6,294,714; a pathogen-inducible promoter, as disclosed in US Patent Application Publication No. 6,252,138; and a water-deficient inducible promoter, as disclosed in US Patent Application Publication No. 2004/0123347A1. All of the above patents and patent publications that disclose promoters and their uses, particularly in recombinant DNA constructs that function in plants, are incorporated herein by reference.

Plant vascular or phloem specific promoters of interest include the rolC or rolA promoter of Agrobacterium rhizogenes, the promoter of Agrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RS1 gene promoter, the Commelina yellow spot badna virus promoter, the coconut leaf spot virus promoter, the rice tungro bacillus-like virus promoter, the promoter of the pea glutamine synthetase GS3A gene, the invCD111 and invCD141 promoters of the potato invertase gene, Kertbundit et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, promoters isolated from Arabidopsis thaliana shown to have phloem-specific expression in tobacco; VAHOX1 promoter region, pea cell wall invertase gene promoter, carrot acid invertase gene promoter, sulfate transporter gene Sultr1;3 promoter, plant sucrose synthetase gene promoter, and plant sucrose transporter gene promoter.

Suitable promoters for use with the recombinant DNA constructs or polynucleotides of the invention include polymerase II ("pol II") promoters and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs that are typically less than 400 nucleotides in length and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Thus, Pol II promoters are included in certain embodiments in which short RNA transcripts are produced from the recombinant DNA constructs of the invention. In one embodiment, the recombinant DNA construct includes a pol II promoter and expresses an RNA transcript flanked by self-cleaving ribozyme sequences (e.g., self-cleaving hammerhead ribozymes) to result in processed RNA, such as a single-stranded RNA that binds to the Lepidoptera target gene transcript, with well-defined 5' and 3' ends without potentially interfering adjacent sequences. Another approach uses polIII promoters to produce transcripts with relatively well-defined 5' and 3' ends, i.e., to transcribe RNA with minimal 5' and 3' flanking sequences. In some embodiments, the polIII promoter (e.g., U6 or H1 promoter) is used to add a short AT-rich transcription termination site that results in a two base pair overhang (UU) in the transcribed RNA; this is useful, for example, for the expression of siRNA type constructs. The use of polIII promoters to drive the expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448. Baculovirus promoters, such as the baculovirus polyhedrin and p10 promoters, are known in the art and commercially available; see, for example, Invitrogen's "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", 2002 (Life Technologies, Carlsbad, Calif.) and F. J. Haines et al. "Baculovirus Expression Vectors", undated (Oxford Expression Technologies, Oxford, UK).

The promoter element may comprise a nucleic acid sequence that is not a naturally occurring promoter or promoter element or its homologue but is capable of regulating expression of a gene. Examples of such "gene-independent" control sequences include naturally occurring or artificially designed RNA sequences that contain ligand binding regions or aptamers (see "Aptamers" below) and regulatory regions (capable of acting in cis). See, e.g., 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 a particular spatial or temporal specificity, e.g., to regulate the translation of DNA encoding a silencing element for silencing a lepidopteran target gene only in the presence (or absence) of a given concentration of an appropriate ligand. One example is a riboregulator that responds to an endogenous ligand (e.g., jasmonic acid or salicylic acid) produced by a plant when it is stressed (e.g., abiotic stress such as water, temperature, or biotic stress such as nutritional stress, infestation by pests or pathogens); when stressed, the level of the endogenous ligand increases to a level sufficient for the riboregulator to initiate transcription of DNA encoding a silencing element for silencing a lepidopteran target gene.
XVIII. Recombinase Sites In some embodiments, the recombinant DNA construct or polynucleotide of the invention comprises DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct comprises at least one pair of loxP sites, where site-specific recombination of DNA between the loxP sites is mediated by Cre recombinase. The positions and relative orientations of the loxP sites are selected to achieve the desired recombination; for example, if the loxP sites are in the same orientation, the DNA between the loxP sites is excised into a circular shape. In another embodiment, the recombinant DNA construct comprises DNA encoding one loxP site; in the presence of Cre recombinase and another DNA having a loxP site, the two DNAs are recombined.

XIX. Aptamers In some embodiments, a recombinant DNA construct or polynucleotide of the invention comprises an RNA aptamer, i.e., DNA engineered into an RNA that binds to a ligand via a binding mechanism that is not primarily based on Watson-Crick base pairing (as opposed to base pairing that occurs between complementary antiparallel nucleic acid strands, for example, resulting in the formation of a double-stranded nucleic acid structure). See, e.g., Ellington and Szostak (1990) Nature, 346:818-822. Examples of aptamers are available, for example, online at the public aptamer database, aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100). However, aptamers useful in the invention can be monovalent (binds to a single ligand) or multivalent (binds to two or more individual ligands, e.g., binds one unit to two or more different ligands).

Ligands useful in the present invention include any molecule (or portion of a molecule) that can be recognized by and bind to a nucleic acid secondary structure, primarily through a mechanism based on Watson-Crick base pairing or not. In this way, the recognition and binding of a ligand to an aptamer is similar to that of an antigen to an antibody, or a biological effector to a receptor. A ligand can include a single molecule (or portion of a molecule) or a combination of two or more molecules (or portions of molecules), and can include one or more macromolecular complexes (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 mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, herbicides such as glyphosate and dicamba, proteins such as virus or phage coat proteins and invertebrate epidermal or gut surface proteins, and RNAs such as viral RNA, transfer RNA (t-RNA), ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamers useful in the present invention do not bind ligands but are thermoresponsive, i.e., the conformation of the aptamer is determined by temperature; see, e.g., Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

XX. Transgene Transcription Unit In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises a transgene transcription unit. The transgene transcription unit comprises a DNA sequence encoding a gene of interest, such as a native or heterologous protein. The gene of interest can be any coding or non-coding sequence from any species, including but not limited to non-eukaryotic organisms such as bacteria and viruses; fungi, protists, plants, invertebrates, and vertebrates. Particular genes of interest are genes encoding at least one insecticide selected from the group consisting of patatin, plant lectins, phytoecdysteroids, Bacillus thuringiensis insecticidal proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Bacillus laterosporous insecticidal proteins, and Bacillus sphaericus insecticidal proteins. The transgene transcription unit may further comprise 5' or 3' sequences, or both, required for transcription of the transgene.

XXI. Introns In some embodiments, a recombinant DNA construct or polynucleotide of the invention comprises DNA encoding a spliceable intron. "Intron" generally refers to a segment of DNA (or RNA transcribed from such a segment) located between exons (protein-coding segments of DNA or the corresponding transcribed RNA), where during maturation of messenger RNA, a present intron is enzymatically "spliced out" 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 that are transcribed into an RNA segment that can be spliced out from the maturing RNA transcript, but are not introns found between protein-coding exons. One of these examples are splicable sequences that have the ability to enhance the expression of downstream coding sequences in plants (in some cases, particularly in monocots); these splicable sequences occur naturally in the 5' untranslated regions of some plant genes, as well as in some viral genes (e.g., the tobacco mosaic virus leader sequence or "omega" leader described by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638) as enhancing expression in plant genes. These splicable sequences or "expression enhancing introns" can be artificially inserted into the 5' untranslated regions of plant genes between the promoters (but not before any protein-coding exons). Examples of such expression-promoting introns include, but are not limited to, the maize alcohol dehydrogenase (Zm-Adh1), maize bronze-1 expression-promoting intron, rice actin 1 (Os-Act1) intron, shrunken-1 (Sh-1) intron, maize sucrose synthetase intron, heat shock protein 18 (hsp18) intron, and the 82 kilodalton heat shock protein (hsp82) intron. U.S. Patent Nos. 5,593,874 and 5,859,347, specifically incorporated herein by reference, describe methods of improving recombinant DNA constructs for use in plants by including an expression-promoting intron derived from the 70 kilodalton maize heat shock protein (hsp70) in an untranslated leader located 3' from the gene promoter and 5' from the exon encoding the first protein.

XXII. Ribozymes In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding one or more ribozymes. Particular ribozymes of interest include self-cleaving ribozymes, hammerhead ribozymes, or hairpin ribozymes. In one embodiment, the recombinant DNA construct comprises DNA encoding one or more ribozymes that serve to cleave the transcribed RNA to provide specific segments of RNA, such as silencing elements for silencing target genes in Lepidoptera.

XXIII. Gene Suppression Elements In some embodiments, a recombinant DNA construct or polynucleotide of the invention comprises DNA encoding an additional gene suppression element for suppressing a target gene other than a Lepidoptera target gene. The suppressed target gene may comprise coding or non-coding sequences, or both.

Suitable gene suppression elements are described in detail in U.S. Patent Application Publication No. 2006/0200878, the disclosure of which is specifically incorporated herein by reference, and include the following:
(a) DNA comprising at least one antisense DNA segment that is antisense to at least one segment of the gene to be silenced;
(b) DNA comprising multiple copies of at least one antisense DNA segment that is antisense to at least one segment of said gene to be silenced;
(c) DNA comprising at least one sense DNA segment which is at least a segment of said gene to be silenced;
(d) DNA comprising multiple copies of at least one sense DNA segment which is at least one segment of said gene to be silenced;
(e) DNA for transcribing into RNA for suppressing said gene to be suppressed by forming double stranded RNA, said DNA comprising at least one antisense DNA segment that is antisense to at least one segment of said gene to be suppressed, and at least one sense DNA segment that is at least one segment of said gene to be suppressed;
(f) DNA that transcribes into RNA for suppressing a gene to be suppressed by forming a single double-stranded RNA, the DNA comprising a plurality of contiguous antisense DNA segments that are antisense to at least one segment of the gene to be suppressed, and a plurality of contiguous sense DNA segments that are at least one segment of the gene to be suppressed.
(g) DNA for transcribing into RNA for suppressing a gene to be suppressed by forming multiple duplexes of RNA, the DNA comprising multiple antisense DNA segments that are antisense to at least one segment of the gene to be suppressed, and multiple sense DNA segments that are at least one segment of the gene to be suppressed, and wherein the multiple antisense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats;
(h) DNA comprising nucleotides derived from a plant miRNA;
(i) DNA comprising siRNA nucleotides;
(j) DNA that transcribes into an RNA aptamer capable of binding to a ligand; and (k) DNA that transcribes into an RNA aptamer capable of binding to a ligand, and DNA that transcribes into a regulatory RNA capable of regulating expression of the gene to be silenced, wherein said regulation is dependent on the conformation of the regulatory RNA, and wherein the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.
Includes one or more of the following.

In some embodiments, introns are used to deliver gene suppression elements in the absence of any protein-encoding exons (coding sequences). In one example, an intron, such as an expression-enhancing intron, is interrupted by embedding a gene suppression element within the intron, where the gene suppression element is excised from the intron during transcription. Thus, protein-encoding exons are not required to provide the gene suppression function of the recombinant DNA constructs disclosed herein.

XXIV. Transcriptional Regulatory Elements In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding a transcriptional regulatory element. A transcriptional regulatory element comprises an element that regulates the level of expression of a recombinant DNA construct of the present invention (compared to expression in the absence of such regulatory element). Examples of suitable transcriptional regulatory elements include riboswitches (cis-acting or trans-acting), transcript stabilizing sequences, and miRNA recognition sites, as specifically detailed in U.S. Patent Application Publication No. 2006/0200878, which is incorporated herein by reference.

XXIV. Creation and Use of Transgenic Plant Cells and Transgenic Plants Plant transformation can include any known method and composition. Methods suitable for plant transformation include virtually any method that can introduce DNA into cells. One method of plant transformation is particle bombardment, as exemplified, for example, in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (corn), U.S. Pat. No. 5,550,318 (corn), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (corn), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), U.S. Pat. No. 6,399,861 (corn), and U.S. Pat. No. 6,403,865 (corn), all of which are incorporated by reference to allow for the production of transgenic plants.

Another useful method of plant transformation is Agrobacterium-mediated transformation with Agrobacterium containing a binary Ti plasmid system, in which the Agrobacterium carries a first Ti plasmid and a second chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a promoter that functions in transformed plant cells and is operably linked to a polynucleotide or recombinant DNA construct of the invention. See, for example, the binary system described in U.S. Pat. No. 5,159,135, which is incorporated by reference. See also De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such binary systems, a smaller plasmid containing one or more T-DNA borders can be conveniently constructed and engineered in a suitable alternative host, such as E. coli, and then transferred to Agrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants, particularly crop plants, are described in U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908 (cotton); 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soybean); 5,591,616 and 5,981,840 (corn); Nos. 4,463,174 (rapeseed, including canola), 7,026,528 (wheat), and 6,329,571 (rice), and U.S. Patent Application Publication Nos. 2004/0244075 (corn) and 2001/0042257A1 (sugar beet), all of which are specifically incorporated by reference to enable the production of transgenic plants. U.S. Patent Application Publication No. 2011/0296555 discloses in Example 5 transformation vectors (including vector sequences) and detailed protocols for transforming corn, soybean, 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 peanut (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), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See also the description of vectors, transformation methods, and production of transformed Arabidopsis plants in which transcription factors are constitutively expressed by the CaMV35S promoter in U.S. Patent Application Publication No. 2003/0167537A1, which is incorporated by reference. Transformation methods particularly useful for Solanaceae plants are well known in the art and are described, for example, in 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, 2004), and pepper (Li et al. (2003) Plant Cell Reports, 21: 785-788). Stable transgenic potatoes, tomatoes, and eggplants have been commercially introduced in various regions; 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 a description of commercial transgenic crops in the extensive publicly available GM crop database, see 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 for transformation of other plant species are known in the art and are described, for example, in 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 forage grasses (rice, corn, wheat, barley, oats, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahia grass), oilseed crops (soybean, oilseed rape, sunflower, peanut, flax, sesame, safflower), legume grains and forages (bean, cowpea, pea, broad bean, lentil, tepary bean, Asian bean, pigeon pea, vetch, chickpea, lupin, alfalfa, and clover), temperate fruits and nuts (apple, pear, peach, plum, berries, cherries, Transformation procedures are described for tropical and subtropical fruits and nuts (citrus, grapefruit, banana plantain, pineapple, papaya, mango, avocado, kiwi fruit, passion fruit, and persimmon), vegetable crops (tomato, eggplant, bell pepper, canola, radish, carrot, melon, leek, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turfgrasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turfgrasses), and forest tree species.

Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably performed in tissue culture on media and in a controlled environment. "Media" refers to a number of nutrient mixtures used to grow cells in vitro, i.e., outside an intact living organism. Recipient cell targets include, but are not limited to, meristematic cells, callus, immature embryos or parts of embryos, and gamete cells such as microspores, pollen, sperm, egg cells, etc. Any cell capable of regenerating a reproductive plant is intended to be a recipient cell useful in the practice of the present invention. Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, etc. Those cells capable of growing as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for producing the transgenic plants of the present invention (e.g., transformation of various media and recipient target cells, immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. No. 6,194,636 and U.S. Patent Application Publication No. 2004/0216189, which are specifically incorporated by reference.

In typical transformation practices, DNA is introduced into the target cells in only a small proportion of any one transformation experiment. Typically, marker genes are used to provide an efficient system for identifying cells that have received the transgenic DNA construct and have been stably transformed by integrating it into their genome. A preferred marker gene provides a selective marker that confers resistance to a selective agent, such as an antibiotic or herbicide. Any antibiotic or herbicide to which the plant cell is resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells, typically cells in which the resistance-conferring gene has been integrated and expressed at a sufficient level to allow the cells to survive are of interest. The cells can be further tested to confirm stable integration of the recombinant DNA. Commonly used selectable 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 selectable marker genes and selection agents are exemplified in U.S. Patent Nos. 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 can also be used, such as markers that provide the ability to visually identify transformants. 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 express a protein that is itself detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those skilled in the art will recognize that many other useful markers or reporters are available.

Detecting or measuring transcription of the recombinant DNA construct in the transgenic plant cells can be accomplished by any suitable method, including protein detection methods (e.g., Western blot, ELISA, and other immunochemical methods), enzyme activity measurements, or nucleic acid detection methods (e.g., Southern blot, Northern blot, PCR, RT-PCR, fluorescent in situ hybridization).

Other suitable methods for detecting or measuring transcription in plant cells of recombinant polynucleotides of the invention targeting a target gene in a lepidopteran pest include measuring other traits that directly or surrogately indicate the expression level of the target gene in the lepidopteran pest, such as, for example, growth rate, mortality rate, reproduction rate, or recruitment rate of the lepidopteran pest, compared to the expression level observed in the absence of the recombinant polynucleotide, or measuring injury (e.g., root damage) or yield loss in plants or fields of plants infested with lepidopteran pests. In general, suitable methods for detecting or measuring transcription in plant cells of a recombinant polynucleotide of interest include, for example, macroscopic or microscopic morphological traits, growth rate, yield, reproduction rate or recruitment rate, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e.g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of phenotypic traits or surrogate assays (e.g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance to abiotic stress). Such methods include direct measurements of resistance to invertebrate pests or pathogens (e.g., damage to plant tissue), or surrogate assays (e.g., plant yield assays, or the Western corn rootworm (Diabrotica virgifera virgifera LeConte) larval bioassay described in WO 2005/110068 A2 and U.S. Patent Application Publication No. 2006/0021087 A1, specifically incorporated by reference, or the soybean cyst nematode bioassay described in Steeves et al. (2006) Funct. Plant Biol., 33:991-999S, in which cysts per plant, cysts per gram of roots, eggs per plant, eggs per gram of roots, and eggs per cyst are measured, or the Colorado potato beetle (Lepidopteran decemlineata) bioassay described herein in the Examples.

The recombinant DNA constructs of the present invention can be stacked with other recombinant DNA to confer additional traits (e.g., in the case of transformed plants, traits including herbicide resistance, insect resistance, low temperature germination resistance, water deficit tolerance, etc.) by, for example, expressing or suppressing other genes. Constructs for down-regulating and up-regulating gene expression are disclosed in U.S. Patent Application Publication No. 2004/0126845 A1, specifically incorporated by reference.

The seeds of the fertile transgenic plants can be harvested and used to grow progeny generations, including hybrid generations, of the transgenic plants of the invention that contain the recombinant DNA construct in their genome. Thus, in addition to direct transformation of plants with the recombinant DNA construct of the invention, transgenic plants of the invention can be prepared by crossing a first plant carrying the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line suitable for transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. The transgenic plants of the invention can be crossed with plant lines carrying other recombinant DNA that confers one or more additional traits (e.g., including, but not limited to, herbicide resistance, disease or pest resistance, environmental stress resistance, modified nutrient content, and improved yield) to produce progeny plants carrying recombinant DNA that confers both a given target sequence expression behavior and the additional trait.

In such combined trait breeding, the transgenic plant that provides the additional trait can be the male line (pollinator) and the transgenic plant that retains the base trait can be the female line. The progeny of this cross will segregate such that some plants retain the DNA for the traits of both parents and some retain the DNA for the trait of one parent. Such plants can be identified by markers associated with the parental recombinant DNA, and progeny plants that retain the DNA for the traits of both parents can be crossed back to the female parent line multiple times, e.g., usually six to eight generations, to produce homozygous progeny plants having substantially the same genotype as one original transgenic parent line, as well as the recombinant DNA of the other transgenic parent line.

Yet another aspect of the invention is a transgenic plant grown from a transgenic seed of the invention (in the case of potato, a transgenic seed potato). The invention contemplates transgenic plants grown directly from a transgenic seed containing the recombinant DNA, as well as progeny generation plants including transgenic plants grown directly from a transgenic seed, and inbred or hybrid lines created by crossing a transgenic plant grown directly from a transgenic seed to a second plant that was not grown from a transgenic seed. Crossing can be accomplished, for example, by the following steps:
(a) sowing seeds of a first parent plant (e.g., non-transgenic or transgenic) and a second parent plant that is transgenic according to the invention;
(b) growing seeds of the first and second parent plants into flowering plants;
(c) pollinating flowers of the first parent with pollen of the second parent; and (d) harvesting seeds produced on the parent plant having the pollinated flowers.

It is often desirable to introgress recombinant DNA into an elite variety, e.g., by backcrossing, to transfer a particular desired trait from a source to an inbred or other plant lacking that trait. This can be accomplished, for example, by crossing a first superior inbred line ("A") (the recurrent parent) to a donor inbred line ("B") (the non-recurrent parent) carrying the appropriate gene for the trait of interest (the construct prepared according to the present invention). The first progeny of this cross are selected among the resulting progeny such that the desired trait is transferred from the non-recurrent parent "B", and the selected progeny are then backcrossed to the superior recurrent parent "A". After five or more generations of backcrossing with selection for the desired trait, the progeny are essentially hemizygous for the locus controlling the trait to be transferred, but like the superior parent for most or almost all other genes. The final backcross generation is selfed to provide progeny that are pure breeding for the gene to be transferred, e.g., one or more transformation events.

Through a series of breeding operations, a selected DNA construct can be moved from one line to an entirely different line without the need for further recombinant operations. Thus, inbred plants can be produced that are true breedings of one or more DNA constructs. By crossing different inbred plants, a large number of different hybrids can be produced that have various combinations of DNA constructs. In this way, plants can be produced that have desirable agronomic characteristics often associated with hybrids ("hybrid vigor"), as well as desirable characteristics that are imparted by one or more DNA constructs.

In certain transgenic plant cells and transgenic plants of the invention, it is sometimes desirable to express a gene of interest while also regulating expression of a lepidopteran target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA further comprising a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of the invention is performed with simultaneous transcription of the gene expression element.

In some embodiments, the recombinant DNA constructs of the invention can be transcribed in any plant cell or tissue or in the whole plant at any developmental stage. Transgenic plants can be derived from any monocotyledonous or dicotyledonous plant, including, but not limited to, plants of commercial or agricultural interest, such as crop plants (particularly crop plants used for human food or animal feed), wood or pulp producing trees, vegetable plants, fruit plants, and ornamental plants. Examples of plants of interest include cereal plants (e.g., wheat, oats, barley, corn, rye, triticale, rice, millet, sorghum, quinoa, amaranth, buckwheat, etc.); forage crop plants (e.g., forage grasses and forage dicotyledons, including alfalfa, vetch, clover, etc.); oilseed crop plants (cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanut, and oil palm); tree nuts (e.g., walnuts, cashews, hazelnuts, pecans, almonds, etc.); sugarcane, coconut, dates, olives, sugarcane, tea, coffee; wood and pulp producing trees; vegetable crop plants such as legumes (e.g., beans, peas, lentils, alfalfa, peanuts), lettuce, asparagus, artichoke, celery, carrots, radishes, Brassicas (e.g., cabbage, kale, mustard, other leafy rapeseeds, broccoli, cauliflower, Brussels sprouts, turnips, kohlrabi), edible melons (e.g., cucumber, melon, summer squash, winter squash), edible onions (e.g., onion, garlic, chives, shallots, chives), edible parts of the Solanaceae family (e.g., tomato, eggplant, potato, bell pepper, ground cherry), and edible parts of the Chenopodiaceae family (e.g., beet, chard, spinach, quinoa, amaranth); for example, apple, pear, citrus fruits (e.g., orange, lime, lemon, grapefruit, etc.), stone fruits (e.g., apricot, peaches, plum, nectarine), banana, pineapple, grapes, kiwifruit, papaya, avocado, and berries; biomass or biofuels (e.g., suki, switchgrass, jatropha, oil palm, and Botryococcus eukaryotic microalgae such as B. braunii, Chlorella spp, and Dunaliella spp, and eukaryotic macroalgae such as Gracilaria spp, and Sargassum spp; and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental ground covers, and ornamental grasses.

The present invention also provides commodity products produced from the transgenic plant cells, plants, or seeds of the present invention, including, but not limited to, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of plants, meal, oil, extracts, fermented or digested products, ground or whole grains or seeds of plants, or food or non-food products comprising such commodity products produced from the transgenic plant cells, plants, or seeds of the present invention. Detection of one or more nucleic acid sequences of the recombinant DNA construct of the present invention in one or more of the goods or commodity products contemplated herein is factual evidence that said goods or commodity products contain or are derived from the transgenic plant cells, plants, or seeds of the present invention.

Generally, transgenic plants having a recombinant DNA construct of the present invention in their genome exhibit increased resistance to lepidopteran pest infestations . In various embodiments, for example, when the transgenic plant expresses a recombinant DNA construct of the present invention stacked with other recombinant DNA to confer additional traits, the transgenic plant exhibits the following traits compared to a plant lacking the recombinant DNA construct:
(a) improved abiotic stress tolerance;
(b) improved biotic stress tolerance;
(c) altered primary metabolite composition;
(d) altered secondary metabolite composition;
(e) altered trace element, carotenoid, or vitamin composition;
(f) improved yield;
(g) Improved nitrogen, phosphate, and other nutrient utilization capacity;
(h) modified agronomic characteristics;
(i) modified growth or reproductive characteristics; and (j) improved harvesting, storage or processing qualities.

In some embodiments, the transgenic plants exhibit improved tolerance to abiotic stress (e.g., tolerance to water deficit or drought, heat, cold, suboptimal nutrient or salinity levels, suboptimal light levels) or biotic stress (e.g., crowding, allelopathy, or wounding); improved tolerance to primary metabolites (e.g., fatty acids, oils, amino acids, proteins, sugars, carbohydrates) composition; altered secondary metabolite (e.g., alkaloids, terpenoids, polyketides, nonribosomal peptides, and secondary metabolites derived from mixed biosynthesis) composition; altered trace element (e.g., iron, zinc), carotenoid (e.g., β-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e.g., tocopherol) composition; improved yield (e.g., under non-stress conditions). improved yield under biotic or abiotic stress); improved ability to use nitrogen, phosphorus, or other nutrients; modified agronomic characteristics (e.g., delayed ripening; delayed senescence; earlier or later maturity; modified shade tolerance; improved resistance to lodging of roots or stems; improved resistance to "green snap" of stems; modified photoperiod response); modified growth or reproductive characteristics (e.g., intentional dwarfing; intentional male sterility, e.g., improved utility in hybridization procedures; improved vegetative growth rate; improved germination; improved male and female fertility); improved harvest, storage, or processing qualities (e.g., improved resistance to pests during storage, improved resistance to breakage, improved customer appeal); or a combination of these traits.

In another embodiment, the transgenic seed, or the seed produced by the transgenic plant, has an altered primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, carbohydrate) composition, an altered secondary metabolite composition, an altered trace element, carotenoid, or vitamin composition, an altered harvest, storage, or processing quality, or a combination thereof. In another embodiment, it may be desirable to modify the levels of natural components of the transgenic plant or the seed of the transgenic plant, for example, to reduce the levels of allergenic proteins or glycoproteins or the levels of toxic metabolites.

Typically, screening of a population of transgenic plants regenerated from transgenic plant cells is performed to identify transgenic plant cells that develop into transgenic plants with desired traits. The transgenic plants are assayed to detect enhanced traits, such as enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein, and enhanced seed oil. Screening methods include direct screening of traits in greenhouse or field trials, or screening for alternative traits. Such analyses aim to detect changes in the chemical composition, biomass, physiological characteristics, or morphology of the plants. Changes in chemical composition, such as the nutritional composition of grains, are detected by analysis of seed composition and content of protein, free amino acids, oil, free fatty acids, starch, tocopherol, or other nutrients. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weight, and (in the case of grain-producing plants such as corn, rice, or wheat) ear or seed head length and diameter. Changes in physiological properties are identified by assessing responses to stress conditions, such as assays under stress conditions such as water deficit, nitrogen or phosphate deficiency, low or high temperature growing conditions, pathogen or insect attack, light deficiency, or increased plant density. Other selective traits include days to flowering, days to pollen shedding, days to fruit maturity, fruit or tuber quality or yield, days to silking in corn, leaf elongation rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace root, green hold, stem lodging, root lodging, plant health, fertility, green snap, and pest resistance. Additionally, phenotypic traits of harvested fruits, seeds, or tubers can be assessed; for example, for tomatoes and eggplants, these can include total number or weight of harvested fruits, color, acidity, sugar content, flavor, etc. of such fruits, and for potatoes, these can include number or total weight of harvested tubers, and quality of such tubers, etc.

The following examples are provided for illustrative purposes and should not be construed as limiting.

Identification of target genes The target genes used for RNAi were identified by analyzing the transcriptome of the target lepidopteran pest to identify genes with the desired nucleotide expression profile at the product-related stage, and single and low copy number using publicly available data. These secondary confirmations were performed using literature searches and public databases such as Database of Essential Genes (DEG), iBeetle, Flybase, and Lepbase to evaluate their importance as RNAi targets. These genes were then candidates for the design of dsRNA triggers.

Design of Effective Trigger Sequences The identified target genes were then processed by a proprietary algorithm to divide them into all possible 18-25 bp long segments. These segments were then matched to the RNA sequences of the specific target genes from the respective public transcriptomes by the Burrows Wheeler Aligner (BWA) tool. These matching segments were then analyzed to identify one or more subsequences of the target genes. These subsequences were then input into a combination of a proprietary algorithm and various public dsRNA design tools, such as Snapdragon, E-RNAi, and SiFi21, to design efficient dsRNA triggers. The specificity of the designed triggers was then checked using BLAST and proprietary code for matches to non-target species, and any hits from this analysis were redesigned using the methodology described above.

Identification of RNAi lethal genes in P. xylostella.
Primary Screening Bioassays dsRNA molecules were tested for insecticidal activity against P. xyostella in an artificial diet-based bioassay. Briefly, artificial diets containing unformulated (e.g., naked dsRNA) or formulated (e.g., see section VI. above) dsRNA were placed in 47 mm petri dishes. Five to seven late L1 stage DBM larvae were added per dish using a pencil brush. Each treatment contained five replicates. The insects surviving two days after the start of the assay were considered the total number of insects in the test, and this number was used to calculate mortality. Fresh treated diet was added to each dish on day 7, followed by untreated diet as needed. Larvae were observed for mortality on day 9. Observations continued for 14 days until the larvae developed into pupae. Mortality was corrected by the Henderson-Tilton formula, as reported in Tables 2 and 3. Bioassays were scored as follows:

Scoring data is provided below in Tables 2 and 3. Each dsRNA molecule in Tables 2 and 3 is identified by a GS number which correlates with the Trigger ID and SEQ ID NOs provided in Table 1A. Underlined/italicized SEQ ID NOs represent sequences with a score of ++ or +++ per the data shown in Tables 2 and 3.

Identification of RNAi lethal genes in S. frugiperda.
Primary Screening Bioassay The insecticidal activity of dsRNA molecules against S. frugiperda was tested by an artificial diet-based bioassay. Briefly, artificial diets containing unformulated or formulated dsRNA were placed in 24-well plates. One fall armyworm larva was placed per well using a fine pencil brush. The plates were covered with a commercially available translucent seal. Each treatment contained 16-24 replicates randomized in multiple 24-well plates. On day 5, the insects were re-treated with fresh diet incorporating dsRNA. The insects were observed for mortality and growth inhibition (arrest at L3) for up to 12 days. dsRNA designed to target green fluorescent protein and water were used as negative controls. The bioassays were scored as follows:

Scoring data is provided below in Tables 4 and 5. Each dsRNA molecule in Tables 4 and 5 is identified by a GS number which correlates with the SEQ ID NOs provided in Table 1A. Bold SEQ ID NOs in Table 1A indicate that these sequences have a score of ++ or +++.

Identification of RNAi lethal genes in P. xylostella.
The insecticidal activity of dsRNA molecules against P. xyostella was tested in an artificial diet-based bioassay. Briefly, 50 μL of naked dsRNA was incorporated into artificial diet plugs at 1 mg/mL. Each plug was placed in a 47 mm petri dish. Five late L1 instar larvae were placed per dish. Fresh untreated diet was added on the 7th day. The live insects after 3 days of dsRNA exposure are the total number of insects used to calculate mortality. Insect mortality and growth were monitored for up to 13 days.

The scoring data is provided in Tables 6 and 7 below. Mortality was corrected by the Henderson Tilton formula, and the corrected mortality is: Each dsRNA molecule in Tables 6 and 7 is identified by a GS number, which correlates with the SEQ ID NOs provided in Table 1B. SEQ ID NOs in bold represent sequences with a score of ++ or +++.

Identification of RNAi lethal genes in P. xylostella.
The insecticidal activity of dsRNA molecules against P. xyostella was tested in an artificial diet-based bioassay. Briefly, 50 μL of naked dsRNA was incorporated into artificial diet plugs at 1 mg/mL. Each plug was placed in a 47 mm petri dish. Five late L1 instar larvae were placed per dish. Fresh untreated diet was added on the 7th day. Surviving insects after 3 days of dsRNA exposure were the total number of insects used to calculate mortality. Insect mortality and growth were monitored for up to 14 days. Scoring data is provided in Table 9 below. Mortality was corrected by the Henderson Tilton formula, and the corrected mortality is as follows: Each dsRNA molecule in Table 9 is identified by a GS number, which correlates with the sequence number provided in Table 1C. The bolded sequences in Table 1C represent sequences having a score of ++ or +++, and each such sequence may be used individually in the claimed methods or compositions.

Microinjection Study of dsRNA in P. xylostella DBM Injection Protocol Scoring data is provided in Table 9 below. Each dsRNA molecule in Table 1C is identified by a GS number that correlates with the sequence numbers provided in Tables 1A-1C. Specific sequences that showed greater than 30% mortality in the feeding assay above were tested by microinjection to further identify effective sequences, as shown in Table 9 below. P. xlostella larvae were injected with dsRNA of sequences identified in an injection bioassay similar to that described in Knorr et al. (2018) Scientific Reports 8:2061 at 11 "Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests" along with the species-specific diet. Results are shown in Table 9, with 50%-63% mortality scored as ++ and 64% or greater mortality scored as +++.

Claims (40)

1. A method for controlling lepidopteran pest infestations in plants comprising:
(a) contacting the lepidopteran pest with at least one polynucleotide comprising 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 an RNA transcribed from the target gene, or a nucleotide sequence that is complementary to at least 90%, 95% or 98% identical to the consecutive nucleotides; or (b) providing in the food of the lepidopteran pest at least one polynucleotide comprising 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 an RNA transcribed from the target gene, or a nucleotide sequence that is complementary to at least 90%, 95% or 98% identical to the consecutive nucleotides; or (c) causing death or growth inhibition in larvae of the lepidopteran pest by providing in the food of the larvae at least one polynucleotide comprising at least one silencing element comprising at least 18, 19, 20, 21 or 25 consecutive nucleotides that are complementary to 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 an RNA transcribed from the target gene ; or (d) topically applying to the plant a composition comprising at least one polynucleotide comprising 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 an RNA transcribed from the target gene, or a nucleotide sequence that is complementary to at least 90%, 95% or 98% identity to the consecutive nucleotides; or (e) topically providing a composition comprising at least one polypeptide to the plant in such a manner that an effective amount of the polynucleotide is taken up by a Lepidoptera species growing on the plant, the at least one polynucleotide comprising 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 an RNA transcribed from the target gene, or a nucleotide sequence that is complementary to at least 90%, 95% or 98% identity to the consecutive nucleotides; or (f) expressing in the plant at least one polynucleotide comprising at least one segment essentially identical to or complementary to at least 18, 19, 20, 21, 25, 50, 100, 150 or 200 consecutive nucleotides of a 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, and 1564-1623, or essentially identical to or complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to said consecutive nucleotides; or (g) contacting the lepidopteran pest with an effective amount of at least one double-stranded RNA, wherein one strand of the double-stranded RNA is selected from the group consisting of SEQ ID NOs: 115-228 , 362-494 , 512-529 , or 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: 536-541, 823-1103, 1334-1563, and 1624-1683 , or is complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to said consecutive nucleotides .
A method comprising : The method of claim 1 , wherein the at least one polynucleotide is double-stranded RNA. 3. The method of claim 2 , wherein the double-stranded RNA is chemically or enzymatically synthesized or produced by expression in a microorganism or by expression in a plant cell. The double-stranded RNA is selected from the group consisting of 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, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 28, 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 3. The method of claim 2, wherein the nucleic acid sequence comprises one strand that is complementary to at least about 50 contiguous nucleotides, at least about 100 nucleotides , at least about 150 nucleotides, or at least about 200 contiguous nucleotides of a sequence selected from the group consisting of: 1548, 1550, 1624, 1625, 1629, 1682, or 1683, or complementary to a nucleotide sequence having at least 90%, 95%, or 98% identity to said contiguous nucleotides . The method comprises topically applying to the plant a composition comprising at least one polynucleotide comprising 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 or RNA transcribed from the target gene , or is complementary to nucleotides having at least 90%, 95%, or 98% identity to the consecutive nucleotides, wherein the target gene is selected from the group consisting of SEQ ID NOs: 2, 5, 7, 9, 13, 21, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109, 7, 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, 73 1, 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, 127 5, 1279, 1283, 1290, 1308, 1310, 1316, 1318, 1320, 1564, 1565, 1569, 1622, and 1623, and optionally, wherein the composition further comprises one or more components selected from the group consisting of carrier agents, surfactants, cationic lipids, organosilicon, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non- polynucleotide insecticides, safeners, and insect growth regulators. The method includes contacting the lepidopteran pest with an effective amount of a solution comprising double-stranded RNA, wherein at least one strand of the double -stranded RNA is selected from the group consisting of 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, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 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, 82 2, 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, 2. The method of claim 1, wherein the nucleotide sequence is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150 or 200 consecutive nucleotides of a gene having a sequence selected from the group consisting of 1569, 1622 and 1623, or is complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to said consecutive nucleotides, and wherein RNA interference is induced and death by the lepidopteran pest occurs . 7. The method of claim 6 , wherein the solution further comprises one or more components selected from the group consisting of an organosilicone surfactant or a cationic lipid. 2. The method of claim 1, wherein the target gene of ( a), (b), (c), (d) or (e) or the DNA of (f) has a sequence selected from the group consisting of SEQ ID NOs: 7, 9, 21, 26, 27, 29, 87, 95, or 99, or the double-stranded RNA of (g) comprises a strand complementary to at least about 50 contiguous nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 121 , 123, 135, 140, 141, 143, 201, 209, and 213, or a sequence having at least 90%, at least 95%, or 98% identity to said selected sequence or to a fragment thereof of equivalent length to said strand . 2. The method of claim 1 , wherein the lepidopteran pest is selected from the group consisting of Spodptera frugiperda and Plutella xylostella. 8. The method of any of claims 1-4, 6 or 7, wherein the at least one double-stranded RNA comprises one strand complementary to at least about 50 contiguous nucleotides, at least about 100 nucleotides, at least about 150 nucleotides or at least about 200 contiguous 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 to a fragment thereof of equivalent length to one strand thereof. 11. The method of claim 10 , wherein said one strand comprises a sequence complementary to at least about 100 contiguous nucleotides of a sequence selected from said group. 8. The method of any 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: 115-228, 362-494, 512-529, 536-541, 823-1103, 1334-1563, and 1624-1683, or a fragment thereof of at least about 50 contiguous nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 contiguous nucleotides, or a sequence having at least 90%, at least 95%, or 98% identity to the double-stranded RNA or fragment thereof. The at least one double stranded RNA is selected from the group consisting of SEQ ID NOs: 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, 13 41, 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, 1629, 1682, and 16 12. The method of claim 10 or 11, comprising a strand that is complementary to at least about 50 contiguous nucleotides, at least about 100 nucleotides, at least about 150 nucleotides or at least about 200 contiguous nucleotides of a sequence selected from the group consisting of: 83, or a sequence having at least 90%, at least 95% or 98% identity to the selected sequence or to a fragment thereof of equivalent length to said strand . The target gene of ( a), (b), (c), (d), or (e), or the DNA of (f) is selected from the group consisting of SEQ ID NOs: 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, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 30, 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, 2. The method of claim 1 , having a sequence selected from the group consisting of: 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 of claim 13 or 14 , wherein the lepidopteran pest is Plutella xylostella. 13. A plant, or a fruit, seed, or reproductive part of said plant, having improved resistance to lepidopteran pest infestation provided by the method of claim 1. The plant of claim 16 , wherein the plant is a crop plant. ( a) an insecticidally effective amount of at least one polynucleotide comprising 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 an RNA transcribed from the target gene, or a nucleotide sequence that is complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to the consecutive nucleotides; or (b) an insecticidally effective amount of at least one polynucleotide comprising at least one silencing element that is complementary to at least 18, 19, 20, 21, or 25 consecutive nucleotides of a 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) an insecticidally effective amount of at least one RNA comprising at least one segment identical to or complementary to 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, and 1564-1623, or an RNA transcribed from the target gene ; or (d) an RNA molecule that, when ingested or contacted by a lepidopteran pest, causes death or growth inhibition in the lepidopteran pest, wherein the RNA molecule comprises 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 is complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to the consecutive nucleotides of an RNA transcribed from the target gene; or (e) an insecticidal double-stranded RNA molecule that, when ingested or contacted by a lepidopteran pest, causes death or growth inhibition 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 is complementary to a nucleotide sequence having at least 90%, 95% or 98% identity to the 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) an insecticidally effective amount of at least one double-stranded RNA, wherein one strand of the double-stranded RNA comprises one strand that is complementary to at least 18, 19, 20, 21, 25, 50, 100, 150, or 200 contiguous 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 to said contiguous nucleotides ;
1. An insecticidal composition for controlling lepidopteran pests comprising: The 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 to a fragment thereof of equivalent length to the single strand. 20. The insecticidal composition of claim 19 , wherein said one strand comprises a sequence complementary to at least about 100 contiguous nucleotides of a sequence selected from said group. 19. The insecticidal composition of 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 to the double-stranded RNA or a fragment thereof. The group includes SEQ ID NOs: 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, 104 1, 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, 142 2 , 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. ( a), (b), (c), (d) and (e) are selected from the group consisting of SEQ ID NOs: 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, 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, 19. The insecticidal composition of claim 18, which is 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 claim 22 or 23 , wherein the lepidopteran pest is Plutella xylostella. 25. The insecticidal composition according to any one of claims 18 to 24, in at least one form selected from the group consisting of a solid , a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, a microbead, a carrier particle, a film, a matrix, a seed treatment, a soil drench, a transplantable formulation, and an in furrow formulation. 26. The insecticidal composition according to any one of claims 18 to 25, further comprising at least one component selected from the group consisting of carrier agents, surfactants , cationic lipids, organosilicones, organosilicon surfactants , polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules , non-polynucleotide insecticides, safeners, and insect growth regulators. 27. The 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 contacted by a lepidopteran pest, causes death or growth inhibition in the lepidopteran pest, wherein the insecticidal double-stranded RNA molecule comprises at least one segment that is complementary to 21 consecutive nucleotides of a 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, and 1564-1623, or an RNA transcribed from the DNA, and wherein the double-stranded RNA molecule is at least 50 base pairs in length, or between about 100 base pairs and about 500 base pairs in length . ( a) a DNA comprising 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, and 1564-1623, or an RNA transcribed from the target gene, or a nucleotide sequence that has at least 90%, 95% or 98% identity to the consecutive nucleotides ; or (b) DNA comprising 18 or more consecutive nucleotides having 100% identity to a fragment of equivalent length to a 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, and 1564-1623, or a DNA complement thereof ; or (c) DNA encoding at least one silencing element that is complementary to at least 18 consecutive nucleotides of a target gene or an RNA transcribed from the 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 an RNA comprising 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 construct comprising a heterologous promoter operably linked to a 17. A plant chromosome or plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the recombinant DNA construct of claim 16. 29. A transgenic crop plant cell comprising in its genome the recombinant DNA construct of claim 28. 31. The transgenic crop plant cell of claim 30, further comprising DNA in its genome encoding at least one insecticide selected from the group consisting of patatin , a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein , a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. 31. A transgenic crop plant comprising a transgenic solanaceous plant cell according to claim 30, or a fruit, a seed, or a reproductive part of said transgenic crop plant . 1. A method for producing a polynucleotide for use in controlling lepidopteran pests , comprising the steps of:
( a) incubating cellular ribonucleic acid (RNA) and ribonuclease in a reaction mixture to generate 5' nucleoside-monophosphates (5'NMPs);
(b) removing said ribonuclease; and (c) removing in said reaction mixture or in a second reaction mixture said 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, 357, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 58, 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, 74 0, 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, or a segment having at least 80% identity to the sequences of ... and incubating a deoxyribonucleic acid (DNA) template encoding an RNA sequence comprising the segments of SEQ ID NOs: 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, 1 024, 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, 1629, 1682, or 1683), producing an RNA of interest .
A method comprising :
Optionally, the method wherein the reaction mixture of step (c) further comprises a nucleoside kinase , an NMP kinase, and/or an NDP kinase . 2. The method of claim 1 , wherein the at least 18 contiguous nucleotides described in (a) through (g) are at least 18, 19, 20, or 21 contiguous nucleotides. The method comprises topically applying a composition comprising at least one polynucleotide to the plant in a manner such that an effective amount of the polynucleotide is ingested by a lepidopteran pest growing on the plant, the polynucleotide comprising a nucleotide sequence that is complementary to at least 18, 19, 20, or 21 consecutive nucleotides of a target gene or RNA transcribed from the 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 , wherein the lepidopteran pest is Plutella xylostella , wherein the target gene is selected from the group consisting of SEQ ID NOs: 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, 5 42, 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, 1564, 1565, 1569, 1622, or 1623, or wherein the polynucleotide has the sequence of SEQ ID NO: 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, 8 33, 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, 1 047, 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, 1629 , 1682, or 1683. 1. An insecticidal composition for controlling lepidopteran pests comprising an insecticidal double-stranded RNA molecule which, when ingested or contacted by a lepidopteran pest, causes death or growth inhibition in the lepidopteran pest, wherein at least one strand of the insecticidal double-stranded RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100 , 150 or 200 contiguous nucleotides that are 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 a sequence having at least 90%, at least 95%, or 98% identity to the selected sequence or a fragment thereof of equivalent length to at least one strand thereof . The group includes SEQ ID NOs: 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, 1 095, 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, 149 37. The insecticidal composition of claim 36, wherein the selected sequence is selected from the group consisting of SEQ ID NO:5, 1500, 1505, 1509, 1513, 1520, 1538, 1540, 1546, 1548, 1550, 1624, 1625, 1629, 1682, and 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 at least one strand thereof . 37. The insecticidal composition of claim 36, wherein the double-stranded RNA comprises one strand complementary to at least about 50 contiguous nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 contiguous 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 one strand thereof. 19. The insecticide composition of claim 18 , wherein the groups in ( a), (b), (c), (d) and (e) are SEQ ID NOs: 1, 2, 13, 36, 37, 39, 47, 103, 302, 340, 346, 349, 1121, 1157, 1178, 1179, 1185, 1310, 1313, 1318 and 1322. 19. The insecticide composition of claim 18 , wherein the group in ( f) is SEQ ID NOs: 115, 116, 127, 150, 151, 153, 161, 217, 435, 473, 479, 482, 1351, 1387, 1408, 1409, 1415, 1540, 1543, 1548, and 1552.