Method of genetic modification of a wild type viral sequence

Abstract

The present invention concerns a method of genetic modification of a TGB-3 wild type viral sequence for reducing or suppressing the possible deleterious effects of the agronomic properties of a transformed plant or plant cell by said TGB-3 viral sequence, comprising the following successive steps: submitting said sequence to point mutation(s) which allow the substitution of at least one amino-acid into a different amino-acid; selecting genetically modified TGB-3 wild type viral sequences having said point mutation(s) and which are not able to promote cell-to-cell movement of a mutant virus having a dysfunctional TGB-3 wild type viral sequence, when expressed in trans from a replicon; further selecting among said genetically modified TGB-3 viral sequences, the specifically genetically modified sequence which inhibits infection with a co-inoculated wild type virus when the mutant form was expressed from a replicon; and recovering said specifically genetically modified TGB-3 viral sequence.

1 . A method of identifying mutants in a triple gene block 3 (TGB-3) viral sequence which inhibit infection of a virus into a cell, comprising: 
 mutating said TGB-3 sequence;    selecting TGB-3 mutants which no longer promote cell-to-cell movement of a (TGB-3 minus) mutant virus when expressed in trans from a replicon;    further selecting from the identified mutants those which also inhibit infection with a co-inoculated wild type virus when the mutant TGB-3 is expressed from a replicon;    recovering said mutant TGB-3 viral sequence wherein a mutant TGB-3 sequence recovered from the foregoing selection steps is a mutant TGB-3 viral sequence that inhibits infection of a virus into a cell.    
     
     
         2 . The method according to  claim 1 , wherein the TGB-3 wild type viral sequence is the beet necrotic yellow vein virus (BNYVV) P15 sequence.  
     
     
         3 . A genetically modified TGB-3 viral sequence obtained by the method of  claim 1 .  
     
     
         4 . The genetically modified TGB-3 viral sequence according to  claim 3 , selected from the group consisting of: SEQ ID NOS: 1, 3, and 5.  
     
     
         5 . A vector comprising the genetically modified TGB-3 viral sequence according to  claim 3 .  
     
     
         6 . A method for inducing resistance to a virus in a plant or a plant cell comprising: 
 preparing a nucleic acid construct comprising a genetically modified TGB-3 viral sequence according to  claim 3  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell, and    transforming a plant cell with said nucleic acid construct.    
     
     
         7 . The method according to  claim 6 , wherein the virus is selected from the group consisting of the apple stem pitting virus, the blueberry scorch virus, the potato virus M, the white clover mosaic virus, the  Cymbidium  mosaic virus, the barley stripe mosaic virus, the potato mop top virus, the peanut clump virus, the beet soil-borne virus and the BNYVV virus.  
     
     
         8 . The method according to  claim 6  wherein the plant cell is a stomatal cell.  
     
     
         9 . The method according to  claim 6  wherein the plant is selected from the group consisting of apple, blueberry, potato, clover, orchid, barley, peanut and sugar beet.  
     
     
         10 . The method according to  claim 6 , wherein the regulatory sequence comprises a promoter sequence or a terminator sequence active in a plant.  
     
     
         11 . The method according to  claim 10 , wherein the promoter sequence is a constitutive or a foreign promoter sequence.  
     
     
         12 . A transgenic plant or transgenic plant cell resistant to a virus comprising a nucleic acid construct having a genetically modified TGB-3 viral sequence according to  claim 4  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell.  
     
     
         13 . A transgenic plant or transgenic plant cell according to  claim 12 , wherein the virus is selected from the group consisting of the apple stem pitting virus, the blueberry scorch virus, the potato virus M, the white clover mosaic virus, the  Cymbidium  mosaic virus, the potato virus X, the barley stripe mosaic virus, the potato mop top virus, the peanut clump virus, the beet soil-borne virus and the BNYVV virus.  
     
     
         14 . The transgenic plant or transgenic plant cell according to  claim 12  selected from the group consisting of apple, blueberry, potato, clover, orchid, barley, peanut and sugar beet.  
     
     
         15 . The transgenic plant or transgenic plant cell according to  claim 12 , wherein the regulatory sequence comprises a promoter sequence and a terminator sequence active in a plant.  
     
     
         16 . The transgenic plant or transgenic plant cell according to  claim 12 , wherein the regulatory sequence(s) comprise a promoter sequence which is a constitutive or a foreign vegetal promoter sequence.  
     
     
         17 . The transgenic plant or transgenic plant cell according to  claim 16 , wherein the promoter sequence is selected from the group consisting of the 35S Cauliflower Mosaic Virus promoter, the polyubiquitin  Arabidopsis thaliana  promoter, and both.  
     
     
         18 . The transgenic plant or transgenic plant cell according to  claim 16  wherein promoter sequence is active in root tissues.  
     
     
         19 . The transgenic plant tissue of  claim 12  wherein said tissue is selected from the group consisting of fruit, stem, root, tuber, and seed.  
     
     
         20 . The vector of  claim 5  operably linked to one or more regulatory sequence(s) active in a plant cell.