Multiple virus resistance in plants

Abstract

The present invention provides gene targets, constructs and methods for the genetic control of plant disease caused by multiple plant viruses. The present invention relates to achieving a plant protective effect through the identification of target coding sequences and the use of recombinant DNA technologies for post-transcriptionally repressing or inhibiting expression of the target coding sequences of plant-parasitic viruses. Protein-expression based approaches may also be utilized to augment phenotype resistance. Thus, transcription of a single transgenic event comprising one or more plant expression cassettes can allow for broad spectrum resistance of a plant to multiple plant viral strains and species among the geminiviruses, tospoviruses, and potexviruses.

1 . A tomato plant comprising resistance to a plurality of plant virus species, wherein the resistance is provided by at least two different modes of action selected from the group consisting of dsRNA, miRNA, and inhibition of virion assembly. 
     
     
         2 . The plant of  claim 1 , wherein:
 (a) the resistance is provided by at least three different modes of action;   (b) wherein the resistance comprises resistance against a begomovirus, tospovirus or potexvirus;   (c) wherein the resistance provided to at least one of the plant virus species is provided by expression of a nucleic acid construct that produces dsRNA;   (d) wherein resistance provided to at least one of the plant virus species is provided by expression of a dsRNA fusion construct or wherein resistance provided to at least one of the plant virus species is provided by expression of a nucleic acid construct that produces dsRNA; or   (e) wherein resistance is provided against a begomovirus or tospovirus by a sequence encoded by a stacked miRNA expression cassette.   
     
     
         3 - 5 . (canceled) 
     
     
         6 . The plant of  claim 2 , wherein the dsRNA interferes with expression of a virus coat protein gene, a virus movement protein gene or a virus replication gene; or wherein the miRNA interferes with expression of a virus coat protein gene, a virus movement protein gene or a virus replication gene. 
     
     
         7 . The plant of  claim 2 , wherein the nucleic acid construct which produces dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:379-455. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The plant of  claim 2 , wherein the miRNA comprises a sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141. 
     
     
         12 . The plant of  claim 1 , wherein:
 (a) resistance against a begomovirus is provided by expression of dsRNA which interferes with expression of a begomovirus replication gene;   (b) resistance against a tospovirus or potexvirus is provided by expression of a dsRNA which interferes with expression of a virus coat protein gene or virus movement protein gene;   (c) resistance against a potexvirus is provided by expression of a nucleic acid construct which produces miRNA; or   (d) resistance against a begomovirus or tospovirus is provided by a sequence encoded by a stacked miRNA expression cassette.   
     
     
         13 . The plant of  claim 1 , wherein resistance provided to at least one of the plant virus species is provided by expression of a tospovirus genome segment terminal sequence that inhibits virion assembly. 
     
     
         14 . The plant of  claim 1 , wherein resistance provided to at least one of the plant virus species is provided by inhibiting virion assembly, wherein virion assembly is inhibited by a sequence comprised within a nucleic acid construct comprising a first nucleic acid segment and a second nucleic acid segment, wherein the first and second segments are substantially inverted repeats of each other and are linked together by a third nucleic acid segment, and wherein the third segment comprises at least one terminal sequence of a tospovirus genome segment that inhibits virion assembly. 
     
     
         15 . The plant of  claim 14 , wherein the third nucleic acid comprises a tospovirus genome terminal sequence selected from the group consisting of: a terminal sequence of a CaCV or GBNV L genome segment, a terminal sequence of a CaCV or GBNV M genome segment, a terminal sequence of a CaCV or GBNV S genome segment, a tospovirus genome terminal repeat sequence, a nucleic acid sequence comprising SEQ ID NO: 167, a nucleic acid sequence comprising SEQ ID NO:168, a nucleic acid sequence comprising SEQ ID NO:376, a nucleic acid sequence comprising SEQ ID NO:377, a nucleic acid sequence comprising SEQ ID NO:378, and a nucleic acid sequence comprising SEQ ID NO: 455. 
     
     
         16 . The plant of  claim 15 , wherein the tospovirus genome segment terminal repeat sequence comprises SEQ ID NO:167 or SEQ ID NO:168. 
     
     
         17 . The plant of  claim 1 , wherein the plant comprises resistance to viruses of at least two of the Geminiviridae, Bunyaviridae and Flexiviridae families; or wherein the viruses are selected from the genera  Potexvirus, Tospovirus,  and  Begomovirus.    
     
     
         18 . (canceled) 
     
     
         19 . The plant of  claim 18 , wherein the viruses are selected from the group consisting of:
 a) at least one of TYLCV, ToSLCV, ToLCNDV, PHYVV, PepGMV;   b) one or more of TSWV, GBNV, CaCV; and   c) PepMV.   
     
     
         20 . The plant of  claim 17 , wherein the Potexvirus is  Pepino mosaic virus;  the begomovirus is TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV; or the tospovirus is CaCV, GBNV, or TSWV. 
     
     
         21 . The plant of  claim 18 , wherein the begomovirus is TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV. 
     
     
         22 . The plant of  claim 18 , wherein the topovirus is CaCV, GBNV, or TSWV. 
     
     
         23 . The plant of  claim 18 , wherein the begomovirus is TYLCV and the Potexvirus is  Pepino mosaic virus;  or the tospovirus is TSWV and the potexvirus is  Pepino mosaic virus;  or the wherein the begomovirus is TYLCV, the potexvirus is  Pepino mosaic virus,  and the tospovirus is TSWV. 
     
     
         24 . The plant of  claim 1 , further comprising a sequence selected from the group consisting of SEQ ID NOs:156, 158, 160, 162, 164, 166, and 363-375; or further comprising a sequence selected from the group consisting of: SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, 141, and 379-454. 
     
     
         25 . (canceled) 
     
     
         26 . The plant of  claim 1 , comprising
 (a) at least one sequence selected from the group consisting of SEQ ID NOs:379-454 and at least one sequence selected from the group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455;   (b) at least one sequence selected from the group consisting of SEQ ID NOs:379-454 and at least one sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141;   or   (c) at least one sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141 and at least one sequence selected from the group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455.   
     
     
         27 . The plant of  claim 1 , wherein the plant comprises at least one heterologous nucleic acid sequence that confers viral resistance selected from the group consisting of
 a) a nucleic acid sequence that encodes an RNA sequence that is complementary to all or a part of a first target gene;   b) a nucleic acid sequence that comprises multiple copies of at least one anti-sense DNA segment that is anti-sense to at least one segment of said at least one first target gene;   c) a nucleic acid sequence that comprises a sense DNA segment from at least one target gene;   d) a nucleic acid sequence that comprises multiple copies of at least one sense DNA segment of a target gene;   e) a nucleic acid sequence that transcribes to RNA for suppressing a target gene by forming double-stranded RNA and that comprises at least one segment that is anti-sense to all or a portion of the target gene and at least one sense DNA segment that comprises a segment of said target gene;   f) a nucleic acid sequence that transcribes to RNA for suppressing a target gene by forming a single double-stranded RNA that comprises multiple serial anti-sense DNA segments that are anti-sense to at least one segment of the target gene and multiple serial sense DNA segments that comprise at least one segment of said target gene;   g) a nucleic acid sequence that transcribes to RNA for suppressing a target gene by forming multiple double strands of RNA and comprises multiple segments that are anti-sense to at least one segment of said target gene and multiple sense DNA segments of the target gene, and wherein said multiple anti-sense DNA segments and said multiple sense DNA segments are arranged in a series of inverted repeats;   h) a nucleic acid sequence that comprises nucleotides derived from a plant miRNA; and   i) a nucleic acid sequence encoding at least one tospovirus terminal sequence that interferes with virion assembly.   
     
     
         28 . The plant of  claim 27 , further comprising a non-transgenic plant virus resistance trait. 
     
     
         29 . The plant of  claim 27 , wherein expression of the at least one heterologous nucleic acid sequence results in resistance to two or more viruses selected from the group consisting of: tospoviruses, begomoviruses, and potexviruses. 
     
     
         30 . A transgenic seed or cell of the plant of  claim 1 . 
     
     
         31 . A method for conferring resistance in a tomato plant to a plurality of plant virus species, the method comprising expressing in the plant at least two nucleic acid sequences that collectively provide resistance to said plurality of plant virus species, wherein at least 2 different modes of action are utilized to provide such resistance, comprising expression of at least two sequences selected from the group consisting of: dsRNA, miRNA, and a sequence which interferes with virion assembly. 
     
     
         32 . The method of  claim 31 , wherein:
 (a) the resistance comprises resistance against a begomovirus, tospovirus or potexvirus;   (b) the resistance provided to at least one of the plant virus species is provided by expression of a nucleic acid construct that produces dsRNA;   (c) the resistance provided to at least one of the plant virus species is provided by expression of a dsRNA fusion construct;   (d) the resistance provided to at least one of the plant virus species is provided by expression of a nucleic acid construct that produces miRNA;   (e) the resistance against a begomovirus or tospovirus is provided by a sequence encoded by a stacked miRNA expression cassette; or   (f) the resistance provided to at least one of the plant virus species is provided by expression of a tospovirus genome segment terminal sequence that inhibits virion assembly.   
     
     
         33 - 34 . (canceled) 
     
     
         35 . The method of  claim 32 , wherein the dsRNA interferes with expression of a virus coat protein gene, a virus movement protein gene or a virus replication gene; or wherein the nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NOs:379-455. 
     
     
         36 .- 38 . (canceled) 
     
     
         39 . The method of  claim 32 , wherein the miRNA interferes with expression of a virus coat protein gene, a virus movement protein gene or a virus replication gene; or wherein resistance provided to at least one of the plant virus species is provided by expression of a nucleic acid construct that produces miRNA. 
     
     
         40 . (canceled) 
     
     
         41 . The method of  claim 31 , wherein:
 (a) resistance against a begomovirus is provided by expression of dsRNA which interferes with expression of a begomovirus replication gene;   (b) resistance against a tospovirus or potexvirus is provided by expression of a dsRNA which interferes with expression of a virus coat protein gene or virus movement protein gene;   (c) resistance against a potexvirus is provided by expression of a nucleic acid construct which produces miRNA; or   (d) resistance against a begomovirus or tospovirus is provided by a sequence encoded by a stacked miRNA expression cassette.   
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 31 , wherein resistance provided to at least one of said plant virus species is provided by inhibiting virion assembly, wherein virion assembly is inhibited by a sequence comprised within a nucleic acid construct comprising a first nucleic acid segment and a second nucleic acid segment, wherein the first and second segments are substantially inverted repeats of each other and are linked together by a third nucleic acid segment, and wherein the third segment comprises at least one terminal sequence of a tospovirus genome segment, expression of which inhibits virion assembly. 
     
     
         44 . The method of  claim 43 , wherein the third nucleic acid comprises a tospovirus genome terminal sequence selected from the group consisting of: a terminal sequence of a CaCV or GBNV L genome segment, a terminal sequence of a CaCV or GBNV M genome segment, a terminal sequence of a CaCV or GBNV S genome segment, and a tospovirus genome terminal repeat sequence. 
     
     
         45 . The method of  claim 44 , wherein the terminal sequence or terminal repeat sequence comprises SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:376, SEQ ID NO:377, or SEQ ID NO: 378. 
     
     
         46 . The method of  claim 31 , wherein the plurality of plant virus species are selected from at least two of the Geminiviridae, Bunyaviridae and Flexiviridae families; or wherein the viruses are selected from the genera  Potexvirus, Tospovirus,  and  Begomovirus.    
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 47 , wherein the viruses are selected from the group consisting of:
 a) one or more of TYLCV, ToSLCV, ToLCNDV, PHYVV, PepGMV;   b) one or more of TSWV, GBNV, CaCV; and   c) PepMV.   
     
     
         49 . The method of  claim 31 , wherein the nucleic acid sequence comprises at least one gene suppression element for suppressing at least one first target gene. 
     
     
         50 . The method of  claim 31 , comprising expressing in the plant:
 a) at least one sequence selected from the group consisting of SEQ ID NOs:379-454 and at least one sequence selected from the group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455;   (b) at least one sequence selected from the group consisting of SEQ ID NOs:379-454 and at least one sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141;   or   (c) at least one sequence selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141 and at least one sequence selected from the group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455.   
     
     
         51 . (canceled)