Plant viral expression vectors and use of same for generating genotypic variations in plant genomes

Abstract

A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.

What is claimed is: 
     
         1 . A plant viral expression vector comprising a pTRV backbone and comprising a nucleic acid sequence encoding a heterologous polypeptide, said nucleic acid sequence being devoid of the 2b sequence as set forth in SEQ ID NO: 43. 
     
     
         2 . The plant viral expression vector of  claim 1 , wherein said heterologous polypeptide comprises a meganuclease. 
     
     
         3 . The plant viral expression vector of  claim 1 , wherein said heterologous polypeptide comprises a chimeric nuclease. 
     
     
         4 . The plant viral expression vector of  claim 2 , wherein said chimeric nuclease comprises a DNA binding domain and a nuclease. 
     
     
         5 . The plant viral expression vector of  claim 4 , wherein said nuclease is selected from the group consisting of a restriction enzyme, a topoisomerase, a recombinase, an integrase and a DNAse. 
     
     
         6 . The plant viral expression vector of  claim 4 , wherein said nuclease comprises a cleavage domain of a type II restriction endonuclease. 
     
     
         7 . The plant viral expression vector of  claim 4 , wherein said nuclease is selected from the group consisting of FokI, I-SceI, I-CeuI, homing nuclease, DNA gyrase, eukaryotic topoisomerase II, bacterial topoisomerase IV, topoisomerase VI, Cre recombinase, Hin recombinase, Rad51/RecA, deoxyribonuclease I, deoxyribonuclease II and micrococcal nuclease. 
     
     
         8 . The plant viral expression vector of  claim 4 , wherein said DNA binding domain is selected from the group consisting of a helix-turn-helix (pfam 01381), a leucine zipper (ZIP) domain, a winged helix (WH) domain, a winged helix turn helix domain (wHTH), a helix-loop-helix and a zinc finger domain. 
     
     
         9 . The plant viral expression vector of  claim 4 , wherein said chimeric nuclease further comprises a localization signal to a DNA-containing organelle. 
     
     
         10 . The plant viral expression vector of  claim 2 , wherein said meganuclease further comprises a localization signal to a DNA-containing organelle. 
     
     
         11 . The plant viral expression vector of  claim 1 , wherein said nucleic acid sequence encodes two heterologous polypeptides. 
     
     
         12 . The plant viral expression vector of  claim 1 , wherein the pTRV is a pTRV2. 
     
     
         13 . The plant viral expression vector of  claim 11 , wherein said nucleic acid sequence comprises two separate sub genomic promoters (sgPs) for regulating transcription of said two heterologous polypeptides. 
     
     
         14 . The plant viral expression vector of  claim 11 , wherein said two heterologous polypeptides are separated by nucleic acid sequence encoding a cleavage domain. 
     
     
         15 . The plant viral expression vector of  claim 14 , wherein said cleavage domain comprises a T2A-like protein sequence as set forth in SEQ ID NO: 40. 
     
     
         16 . A plant cell comprising the plant viral expression vector of  claim 1 . 
     
     
         17 . A plant comprising the plant viral expression vector of  claim 1 . 
     
     
         18 . A method of generating a plant, the method comprising introducing into one or more cells of the plant at least one plant viral expression vector of  claim 1 . 
     
     
         19 . A method of generating genotypic variation in a genome of a plant, the method comprising introducing into the plant at least one plant viral expression vector of  claim 4 , wherein said DNA binding domain mediates specific targeting of said nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant. 
     
     
         20 . The method of  claim 19 , further comprising introducing into the plant a DNA vector.