Closterovirus vectors and methods

Abstract

The present disclosure relates to the development and use of Closterovirus-based vectors for the delivery of nucleotides to plants. Specifically, the present disclosure provides viral vectors based on Grapevine leafroll-associated virus-2 for the delivery and expression of genes in plants, particularly grape plants. Methods of making and using these vectors, as well as the plants transformed by these vectors, are also contemplated.

What is claimed is: 
     
         1 . A replication-competent plant gene transfer vector comprising a nucleic acid encoding:
 a) viral genes from Grapevine leafroll-associated virus-2 (LR-2) comprising methyltransferase, RNA helicase, and RNA-dependent RNA polymerase;   b) an RNAi suppressor selected from the group consisting of LR-2 p24 and Beet yellow virus p21;   c) LR-2 leader proteases L1 and L2; and   d) a heterologous polynucleotide operably linked to a promoter, wherein the heterologous polynucleotide is expressed in a plant cell, and wherein the vector is capable of infectious replication in the plant cell, and wherein the vector can be used for virus-induced gene silencing.   
     
     
         2 . A conditionally-replicating plant gene transfer vector comprising a nucleic acid encoding:
 a) viral genes from Grapevine leafroll-associated virus-2 (LR-2) comprising methyltransferase, RNA helicase, and RNA-dependent RNA polymerase;   b) an RNAi suppressor selected from the group consisting of LR-2 p24 and Beet yellow virus p21;   c) one or more LR-2 leader proteases selected from the group consisting of L1, L2, and both L1 and L2, wherein at least one of the one or more leader proteases is inactivated such that the vector cannot infectiously replicate independently; and   d) a heterologous polynucleotide operably linked to a promoter, wherein the heterologous polynucleotide is expressed in a plant cell, and wherein the vector is adapted for virus-induced gene silencing.   
     
     
         3 . The vector of  claim 1 , further comprising viral genes from Grapevine leafroll-associated virus-2 (LR-2) that are involved in virion assembly, transport within plants, or both. 
     
     
         4 . The vector of  claim 3 , wherein the viral genes are selected from the group consisting of p6, Hsp70h, p63, CPm, CP and p19 from Grapevine leafroll-associated virus-2 (LR-2). 
     
     
         5 . The vector of  claim 2 , wherein the heterologous polynucleotide encodes at least one polypeptide selected from the group consisting of a reporter molecule, a selectable marker, and a therapeutic gene. 
     
     
         6 . The vector of  claim 5 , wherein the therapeutic gene is antifungal, antibacterial, antiviral or a taste modifier. 
     
     
         7 . The vector of  claim 5 , wherein the therapeutic gene is for the treatment of Pierce's Disease or powdery mildew. 
     
     
         8 . The vector of  claim 7 , wherein the therapeutic gene is a polynucleotide which triggers viral induced gene silencing, a Run1 polynucleotide, or encodes a lysozyme polypeptide. 
     
     
         9 . The vector of  claim 2 , wherein the L2 protease has the amino acid sequence shown in SEQ ID NO: 6. 
     
     
         10 . A plant cell comprising the vector of  claim 1 . 
     
     
         11 . A plant comprising the vector of  claim 2 . 
     
     
         12 . A method for virus-induced gene silencing by expressing a heterologous gene in a plant cell, comprising introducing into the plant cell the vector of  claim 5 . 
     
     
         13 . The method of  claim 12 , wherein introducing the vector into the plant cell comprises agroinoculation. 
     
     
         14 . The method of  claim 12 , wherein the vector is a replication-competent vector, wherein the vector is introduced into a plant cell and subsequently replicates and infects at least one additional plant cell. 
     
     
         15 . The method of  claim 14 , wherein the vector is a replication-competent vector, wherein the vector systemically infects a plant structure selected from the group consisting of tissue, leaf, stem, root, fruit, seed or entire plant. 
     
     
         16 . The method of  claim 12 , wherein introducing the vector into the plant cell comprises grafting a plant part comprising the vector to a plant part that does not comprise the vector. 
     
     
         17 . The method of  claim 12 , further comprising grafting a plant part comprising the plant cell comprising the vector to a plant part that does not comprise the vector. 
     
     
         18 . The vector of  claim 1 , wherein the RNAi suppressor is p24. 
     
     
         19 . The vector of  claim 2 , wherein the RNAi suppressor is p24.