Development of resistance to raspberry bushy dwarf virus

Abstract

The present invention relates to isolated Raspberry Bushy Dwarf Virus (RBDV) nucleic acid sequences which encode RBDV coat and movement proteins or polypeptides and mutant or modified forms thereof. The invention further relates to heterologous nucleic acid constructs, vectors, transformation methods, plant cells and plants comprising such RBDV-encoding nucleic acids and methods for inducing resistance to RBDV by transforming plants with a nucleic acid construct comprising RBDV protein or polypeptide-encoding nucleic acid sequences.

It is claimed:  
     
         1 . An isolated raspberry bushy dwarf virus (RBDV) protein or polypeptide selected from the group consisting of: 
 (a) a RBDV coat protein or polypeptide with an amino acid sequence having at least 80% sequence identity to the sequence presented as SEQ ID NO:2;    (b) a RBDV coat protein or polypeptide with an amino acid sequence having at least 90% sequence identity to the sequence presented as SEQ ID NO:2;    (c) a RBDV coat protein or polypeptide with an amino acid sequence having at least 95% sequence identity to the sequence presented as SEQ ID NO:2; and    (d) a RBDV coat protein or polypeptide with the amino acid sequence presented as SEQ ID NO:2.    
     
     
         2 . An isolated raspberry bushy dwarf virus (RBDV) polynucleotide selected from the group consisting of: 
 (a) a polynucleotide with a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV coat protein or polypeptide having at least 80% sequence identity to the sequence presented as SEQ ID NO:2;    (b) a polynucleotide with a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV coat protein or polypeptide having at least 90% sequence identity to the sequence presented as SEQ ID NO:2;    (c) a polynucleotide with a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV coat protein or polypeptide having at least 95% sequence identity to the sequence presented as SEQ ID NO:2;    (d) a polynucleotide with a nucleic acid sequence which encodes or is complementary to a sequence which encodes the RBDV coat protein or polypeptide having the sequence presented as SEQ ID NO:2;    (e) a polynucleotide sequence with the nucleic acid sequence presented as SEQ ID NO:1, which encodes or is complementary to a sequence which encodes a RBDV coat protein or polypeptide; and    (f) a polynucleotide with a nucleic acid sequence that hybridizes, under high stringency conditions to the RBDV coat protein-encoding nucleic acid sequence presented as SEQ ID NO:1, or the complement or a fragment thereof.    
     
     
         3 . An isolated raspberry bushy dwarf virus (RBDV) protein or polypeptide selected from the group consisting of: 
 (a) a RBDV movement protein or polypeptide with an amino acid sequence having at least 80% sequence identity to the sequence presented as SEQ ID NO:4;    (b) a RBDV movement protein or polypeptide with an amino acid sequence having at least 90% sequence identity to the sequence presented as SEQ ID NO:4;    (c) a RBDV movement protein or polypeptide with an amino acid sequence having at least 95% sequence identity to the sequence presented as SEQ ID NO:4; and    (d) a RBDV movement protein or polypeptide with the amino acid sequence presented as SEQ ID NO:4.    
     
     
         4 . An isolated raspberry bushy dwarf virus (RBDV) polynucleotide selected from the group consisting of: 
 (a) a polynucleotide comprising a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV movement protein or polypeptide having at least 80% sequence identity to the sequence presented as SEQ ID NO:4;    (b) a polynucleotide comprising a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV movement protein or polypeptide having at least 90% sequence identity to the sequence presented as SEQ ID NO:4;    (c) a polynucleotide comprising a nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV movement protein or polypeptide having at least 95% sequence identity to the sequence presented as SEQ ID NO:4;    (d) a polynucleotide comprising the nucleic acid sequence which encodes or is complementary to a sequence which encodes a RBDV movement protein or polypeptide having the sequence presented as SEQ ID NO:4;    (e) a polynucleotide sequence with the nucleic acid sequence presented as SEQ ID NO:3, which encodes or is complementary to a sequence which encodes a RBDV movement protein or polypeptide; and    (f) a polynucleotide with a nucleic acid sequence that hybridizes, under high stringency conditions to the RBDV movement protein-encoding nucleic acid sequence presented as SEQ ID NO:3, or the complement or a fragment thereof.    
     
     
         5 . The isolated polynucleotide of  claim 1  or  3 , wherein % identity is calculated using the CLUSTAL-W program in MacVector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.  
     
     
         6 . The isolated polynucleotide of  claim 1  or  3 , wherein high stringency conditions means hybridization at 42° C. in 50% formamide, 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA, followed by washing two times in 2×SSPE and 0.5% SDS at room temperature and two additional times in 0.1×SSPE and 0.5% SDS at 42° C.  
     
     
         7 . An isolated polynucleotide which encodes a raspberry bushy dwarf virus (RBDV) coat or movement protein or polypeptide, wherein expression of said polynucleotide in a transgenic plant is associated with enhanced resistance to infection by RBDV when compared to RBDV infection of a plant which does not express said RBDV coat or movement protein or polypeptide-encoding polynucleotide.  
     
     
         8 . The isolated polynucleotide of  claim 7 , wherein said polynucleotide encodes a wild type RBDV coat or movement protein.  
     
     
         9 . The isolated polynucleotide of  claim 7 , wherein said polynucleotide encodes a mutated or modified RBDV coat or movement protein.  
     
     
         10 . The isolated mutated or modified RBDV movement protein-encoding polynucleotide of  claim 9 , wherein said polynucleotide has a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.  
     
     
         11 . A plant transformation vector comprising an isolated polynucleotide of any one of  claims 7  to  10 .  
     
     
         12 . The plant transformation vector of  claim 11  operably linked to a promoter and additional appropriate control sequences recognized by a host cell transformed with the vector.  
     
     
         13 . A transgenic plant cell comprising the plant transformation vector of  claim 12 .  
     
     
         14 . A transgenic plant comprising the plant cell of  claim 13 , wherein said plant exhibits enhanced resistance to infection by RBDV relative to a non-transgenic plant.  
     
     
         15 . A method for producing a transgenic raspberry plant, comprising the steps of: 
 (i) introducing into progenitor cells of said raspberry plant a plant transformation vector according to  claim 11  to produce transformed progenitor plant cells; and    (ii) growing the transformed progenitor plant cells to produce a transgenic raspberry plant wherein the RBDV-encoding polynucleotide within said vector is expressed and said transgenic plant exhibits enhanced resistance to RBDV infection relative to a raspberry plant which has not been so transformed.    
     
     
         16 . The method according to  claim 15  wherein said plant transformation vector comprises a mutated or modified RBDV movement protein encoding polynucleotide sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.  
     
     
         17 . An RBDV-resistant raspberry plant produced according to the method of  claim 15  or  16 .