Compositions and methods for the suppression of target polynucleotides from lepidoptera

Abstract

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a pest from the Lepidoptera order, they are capable of decreasing the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant. The present invention provides target polynucleotides encoding polypeptides from specific protein families and various target polynucleotides set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or active variants and fragments thereof, wherein a decrease in expression of one or more the sequences in the target pest controls the pest (i.e., has insecticidal activity). Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is Spodoptera frugiperda . Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

That which is claimed: 
     
         1 . A plant cell having stably incorporated into its genome a heterologous polynucleotide comprising a silencing, wherein said silencing element comprises a fragment of at least 20 consecutive nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50,
 wherein said silencing element, when ingested by a pest from the Lepidoptera order, reduces the level of a target sequence in said pest and thereby controls the pest from the Lepidoptera order.   
     
     
         2 . The plant cell of  claim 1 , wherein said pest comprises  Spodoptera frugiperda.    
     
     
         3 . The plant cell of  claim 1 , wherein said silencing element comprises
 a) a polynucleotide comprising the sense or antisense sequence of the sequence set forth in SEQ ID NO: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 415, or 418;   b) a polynucleotide comprising the sense or antisense sequence of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 415, or 418;   c) a polynucleotide comprising the sequence set forth in SEQ ID NO: 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214, 215, 217, 218, 220, 221, 223, 224, 226, 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250, 251, 253, 254, 256, 257, 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289, 290, 292, 293, 295, 296, 298, 299, 301, 302, 304, 305, 307, 308, 310, 311, 313, 314, 316, 317, 319, 320, 322, 323, 325, 326, 328, 329, 331, 332, 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349, 350, 352, 353, 355, 356, 358, 359, 361, 362, 364, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 382, 383, 385, 386, 388, 389, 391, 392, 394, 395, 397, 398, 400, 401, 403, 404, 406, 407, 409, 410, 412, 413, 416, 417, 419, or 420   d) a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214, 215, 217, 218, 220, 221, 223, 224, 226, 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250, 251, 253, 254, 256, 257, 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289, 290, 292, 293, 295, 296, 298, 299, 301, 302, 304, 305, 307, 308, 310, 311, 313, 314, 316, 317, 319, 320, 322, 323, 325, 326, 328, 329, 331, 332, 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349, 350, 352, 353, 355, 356, 358, 359, 361, 362, 364, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 382, 383, 385, 386, 388, 389, 391, 392, 394, 395, 397, 398, 400, 401, 403, 404, 406, 407, 409, 410, 412, 413, 416, 417, 419, or 420.   
     
     
         4 . The plant cell of  claim 1 , wherein said silencing element comprises a hairpin RNA. 
     
     
         5 . The plant cell of  claim 4 , wherein said polynucleotide comprising the silencing element comprises, in the following order, a first segment, a second segment, and a third segment, wherein
 a) said first segment comprises at least about 20 nucleotides having at least 90% sequence complementarity to a target sequence set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50;   b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,   c) said third segment comprises at least about 20 nucleotides having at least 85% complementarity to the first segment.   
     
     
         6 . The plant cell of  claim 1 , wherein said silencing element is operably linked to a heterologous promoter. 
     
     
         7 . The plant cell of  claim 1 , wherein said plant cell has stably incorporated into its genome a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof, wherein the combined expression of the silencing element and the suppressor enhancer element increases the concentration of an inhibitory RNAi specific for the pest target sequence in said plant cell. 
     
     
         8 . The plant cell of  claim 1 , wherein said plant cell is from a monocot. 
     
     
         9 . The plant cell of  claim 8 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         10 . The plant cell of  claim 1 , wherein said plant cell is from a dicot. 
     
     
         11 . The plant cell of  claim 10 , wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis , or cotton. 
     
     
         12 . A plant or plant part comprising a plant cell of  claim 1 . 
     
     
         13 . The plant or plant part of  claim 7 , wherein the combined expression of said silencing element and the suppressor enhancer element increases the concentration of an inhibitory RNA specific for the pest target sequence in the phloem of said plant or plant part. 
     
     
         14 . A transgenic seed from the plant of  claim 12 . 
     
     
         15 . A method for controlling Lepidoptera comprising feeding to a Lepidoptera a composition comprising a silencing element, wherein said silencing element, when ingested by said Lepidoptera, reduces the level of a target Lepidoptera sequence and thereby controls the Lepidoptera and said silencing element comprises a fragment of at least 20 consecutive nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 
     
     
         16 . The method of  claim 15 , wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide comprising said silencing element. 
     
     
         17 . The method of  claim 15 , wherein said pest comprises  Spodoptera frugiperda.    
     
     
         18 . The method of  claim 15 , wherein said silencing element comprises
 a) a polynucleotide comprising the sense or antisense sequence of the sequence set forth in SEQ ID NO: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 415, or 418;   b) a polynucleotide comprising the sense or antisense sequence of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 415, or 418;   c) a polynucleotide comprising the sequence set forth in SEQ ID NO: 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214, 215, 217, 218, 220, 221, 223, 224, 226, 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250, 251, 253, 254, 256, 257, 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289, 290, 292, 293, 295, 296, 298, 299, 301, 302, 304, 305, 307, 308, 310, 311, 313, 314, 316, 317, 319, 320, 322, 323, 325, 326, 328, 329, 331, 332, 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349, 350, 352, 353, 355, 356, 358, 359, 361, 362, 364, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 382, 383, 385, 386, 388, 389, 391, 392, 394, 395, 397, 398, 400, 401, 403, 404, 406, 407, 409, 410, 412, 413, 416, 417, 419, or 420;   d) a polynucleotide comprising the sequence set forth in SEQ ID NO: 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214, 215, 217, 218, 220, 221, 223, 224, 226, 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250, 251, 253, 254, 256, 257, 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289, 290, 292, 293, 295, 296, 298, 299, 301, 302, 304, 305, 307, 308, 310, 311, 313, 314, 316, 317, 319, 320, 322, 323, 325, 326, 328, 329, 331, 332, 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349, 350, 352, 353, 355, 356, 358, 359, 361, 362, 364, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 382, 383, 385, 386, 388, 389, 391, 392, 394, 395, 397, 398, 400, 401, 403, 404, 406, 407, 409, 410, 412, 413, 416, 417, 419, or 420.   
     
     
         19 . The method of  claim 15 , wherein said silencing element comprises a hairpin RNA. 
     
     
         20 . The method of  claim 19  wherein said polynucleotide comprising the silencing element comprises, in the following order, a first segment, a second segment, and a third segment, wherein
 a) said first segment comprises at least about 20 nucleotides having at least 90% sequence complementarity to the target polynucleotide; 
 b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and, 
 c) said third segment comprises at least about 20 nucleotides having at least 85% complementarity to the first segment. 
 
     
     
         21 . The method of  claim 15 , wherein said silencing element is operably linked to a heterologous promoter. 
     
     
         22 . The method of  claim 16 , wherein said plant or plant part has stably incorporated into its genome a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof, wherein the combined expression of the silencing element and the suppressor enhancer element increases the concentration of an inhibitory RNAi specific for the pest target sequence in said plant cell. 
     
     
         23 . The method of  claim 15 , wherein said plant is a monocot. 
     
     
         24 . The method of  claim 23 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         25 . The method of  claim 15 , wherein said plant is a dicot. 
     
     
         26 . The method of  claim 25 , wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis , or cotton.