Methods to block aphid transmission of poleroviruses and to develop virus management tools
Disclosed herein are plant-based, molecular and diagnostic tools that can be used to block aphid transmission of poleroviruses, including stabilized proteins for expression in transgenic plants and/or in formulations for direct plant delivery. Further disclosed are proteins that can kill aphids and methods to produce these proteins. Antibodies and useful for diagnostic and therapeutic uses against poleroviruses.
1 . An isolated protein comprising the protein sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12.
2 . The isolated protein of claim 1 , further comprising a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag.
3 . A vector comprising a nucleic acid encoding a protein having SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12.
4 . A recombinant potato leaf roll virus comprising the protein of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12.
5 . A modified protein comprising a polypeptide having at least 98% identity to SEQ ID NO: 2 or 98% identity to SEQ ID NO: 3, and wherein the modified protein comprises an amino acid substitution in any one of residues 137, 138, 139, 140, 141, 142, 143, 144, or 145.
6 . The modified protein of claim 5 , wherein the polypeptide has at least 98% identity to SEQ ID NO: 2 and wherein the modified protein comprises an amino acid substitution in any one of residues 137, 138, 139, 140, 141, 142, 143, 144, or 145.
7 . The modified protein of claim 6 , wherein the amino acid substitution comprises an alanine substitution.
8 . The modified protein of claim 5 , wherein the polypeptide has at least 98% identity to SEQ ID NO: 3 and wherein the modified protein comprises an amino acid substitution in any one of residues 137, 138, 139, 140, 141, 142, 143, 144, or 145.
9 . The modified protein of claim 8 , wherein the amino acid substitution comprises an alanine substitution.
10 . A transgenic plant comprising a heterologous nucleic acid encoding SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12.
11 . The transgenic plant of claim 10 , wherein the heterologous nucleic acid is operatively linked to a plant promoter sequence.
12 . A plant comprising a protein having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12.
13 . A method of controlling aphids, comprising the step of:
exposing an aphid to a protein having an amino acid sequence: 1) at least 98% identical to SEQ ID NO: 2 and wherein the protein comprises an amino acid substitution in at least one of residues 137, 138, 139, 140, 141, 142, 143, 144, or 145, and the protein further comprises a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag operably linked to the protein; or 2) at least 98% identical to SEQ ID NO: 3 and wherein the protein comprises an amino acid substitution in any one of residues 137, 138, 139, 140, 141, 142, 143, 144, or 145, and the protein further comprises a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag operably linked to the protein, thereby inducing increased mortality in the aphid.
14 . The method of claim 13 , wherein the protein has an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 12 and the protein further comprises a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag operably linked to the protein.
15 . The method of claim 13 , wherein the exposing step comprises an aphid feeding on a plant containing the protein.
16 . The recombinant potato leaf roll virus of claim 4 , further comprising a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag operably linked to said protein.
17 . The modified protein of claim 5 , further comprising a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FlAsH tag, or polyhistidine (HIS) tag.
18 . The transgenic plant of claim 10 , wherein the heterologous nucleic acid further encodes a green-fluorescent protein (GFP), a yellow fluorescent protein (YFP), strep tag, FLASH tag, or polyhistidine (HIS) tag operably linked to said protein.