P15 hairpin constructs and use

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. The invention further relates to genetically modified TGB-3 viral sequences suitable to induce gene silencing. In particular hairpin constructs based on such sequences proved highly efficient to induce a PTGS mechanism and degradation of the whole of RNA2 thereby. When plants are transformed accordingly the spread of the virus in the plant is significantly reduced or blocked.

1 . A plant or plant cell comprising a modified TGB-3 gene, wherein said TGB-3 gene comprises the sequence of SEQ ID NO: 1 or SEQ ID NO:5. 
     
     
         2 . A genetically modified TGB-3 viral sequence comprising a sequence selected from the group consisting of
 (a) a nucleotide sequence comprising SEQ ID NO: 3 and an antisense sequence of SEQ ID NO: 3;   (b) a nucleotide sequence comprising a fragment of SEQ ID NO: 3 and an antisense sequence of said fragment of SEQ ID NO: 3;   (c) a nucleotide sequence comprising a modified SEQ ID NO: 3 and an antisense sequence of said modified SEQ ID NO: 3; and   (d) a nucleotide sequence comprising modified SEQ ID NO:3 fragment and an antisense sequence of said modified SEQ ID NO: 3 fragment;   wherein said genetically modified TGB-3 viral sequence when transcribed in a cell is capable of forming a double-stranded self-complementing RNA molecule.   
     
     
         3 . The TGB-3 viral sequence of  claim 2 , wherein the sense and antisense sequences are comprised in one nucleic acid sequence. 
     
     
         4 . The TGB-3 viral sequence of  claim 3 , further comprising an intron fragment interspersed between the sense and antisense sequences, wherein the TGB-3 viral sequence when transcribed in a cell is capable of forming a hairpin RNA molecule. 
     
     
         5 . The TGB-3 viral sequence of  claim 4 , wherein the intron fragment is derived from a plant gene. 
     
     
         6 . The TGB-3 viral sequence of  claim 5 , wherein the plant gene is a beet gene. 
     
     
         7 . The TGB-3 viral sequence of  claim 4 , wherein the intron fragment is an intron fragment of highly transcribed genes. 
     
     
         8 . The TGB-3 viral sequence of  claim 7 , wherein the highly transcribed genes are ribosomal RNA genes. 
     
     
         9 . The TGB-3 viral sequence of  claim 7 , wherein the highly transcribed genes are highly transcribed sugar beet genes. 
     
     
         10 . The TGB-3 viral sequence of  claim 2 , wherein in said modified SEQ ID NO:3 sequence a translation start and/or a stop codon of the SEQ ID NO: 3 sequence is/are modified to inhibit translation. 
     
     
         11 . The TGB-3 viral sequence of  claim 2  comprising SEQ ID NO: 9 or SEQ ID NO: 13. 
     
     
         12 . The TGB-3 viral sequence of  claim 2  consisting of SEQ ID NO: 9 or SEQ ID NO: 13. 
     
     
         13 . A vector comprising the genetically modified TGB-3 viral sequence according to  claim 2 . 
     
     
         14 . The vector of  claim 13  operably linked to one or more regulatory sequence(s) active in a plant cell. 
     
     
         15 . A double stranded self-complementary RNA molecule expressed by a vector of  claim 13 . 
     
     
         16 . 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 1  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell, and   transforming a plant cell with the nucleic acid construct, thereby inducing resistance to a virus in a plant or in a plant cell.   
     
     
         17 . 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 2  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell, and   transforming a plant cell with the nucleic acid construct, thereby inducing resistance to a virus in a plant or in a plant cell.   
     
     
         18 . The method according to  claim 17 , 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. 
     
     
         19 . A method for inducing post-transcriptional gene silencing of the whole of RNA2 and more in particular of a TGB-3 movement protein in a plant or a plant cell, comprising the steps of:
 preparing a nucleic acid construct comprising a genetically modified TGB-3 viral sequence according to  claim 2  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell, and   transforming a plant cell with the nucleic acid construct, whereby expression in said plant cells of an RNA molecule that is capable of forming a double-stranded RNA molecule triggers a mechanism of post-transcriptional gene silencing.   
     
     
         20 . The method according to  claim 19 , wherein the plant cell is a stomatal cell. 
     
     
         21 . The method according to  claim 19 , wherein the plant is selected from the group consisting of apple, blueberry, potato, clover, orchid, barley, peanut and sugar beet. 
     
     
         22 . The method according to  claim 19 , further comprising regenerating a transgenic plant from the transformed plant cell. 
     
     
         23 . The method according to  claim 19 , wherein the regulatory sequence comprises a promoter sequence or a terminator sequence active in a plant. 
     
     
         24 . The method according to  claim 19 , wherein the promoter sequence is a constitutive or a foreign promoter sequence. 
     
     
         25 . The method according to  claim 23 , wherein the promoter sequence is selected from the group consisting of the 35S Cauliflower Mosaic Virus promoter and the polyubiquitin  Arabidopsis thaliana  promoter. 
     
     
         26 . The method according to  claim 23 , wherein the promoter sequence is a promoter active in the root tissue of plants. 
     
     
         27 . The method according to  claim 26 , wherein the promoter sequence is a promoter active in the root tissue of beet plants. 
     
     
         28 . The method according to  claim 26 , wherein said promoter active in the root tissue of plants is the par promoter of the haemoglobin gene from  Perosponia andersonii.    
     
     
         29 . A transgenic plant or a transgenic plant cell resistant to a virus and comprising a nucleic acid construct having a genetically modified TGB-3 viral sequence according to  claim 2  operably linked to one or more regulatory sequence(s) active in a plant or a plant cell. 
     
     
         30 . The transgenic plant or transgenic plant cell according to  claim 29 , 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. 
     
     
         31 . The transgenic plant or transgenic plant cell according to  claim 29  selected from the group consisting of apple, blueberry, potato, clover, orchid, barley, peanut and sugar beet. 
     
     
         32 . The transgenic plant or transgenic plant cell according to  claim 29 , wherein the regulatory sequence comprises a promoter sequence and a terminator sequence that are active in a plant. 
     
     
         33 . The transgenic plant of  claim 32 , wherein said promoter is active in the root tissue of plants. 
     
     
         34 . The transgenic plant of  claim 33 , wherein said promoter is a par promoter of the haemoglobin gene from  Perosponia andersonii.    
     
     
         35 . The transgenic plant or transgenic plant cell according to  claim 29 , wherein said transgenic plant is sugar beet and said transgenic plant cell is a sugar beet cell. 
     
     
         36 . The transgenic plant or transgenic plant cell according to  claim 29 , wherein the regulatory sequence(s) comprise a promoter sequence which is a constitutive or a foreign vegetal promoter sequence. 
     
     
         37 . The transgenic plant or transgenic plant cell according to  claim 36 , wherein the promoter is selected from the group consisting of the 35S Cauliflower Mosaic Virus promoter and the polyubiquitin  Arabidopsis thaliana  promoter. 
     
     
         38 . A transgenic plant tissue derived from the transgenic plant cell according to  claim 29 , wherein said tissue is selected from the group consisting of fruit, stem, root, tuber, and seed. 
     
     
         39 . A transgenic reproducible structure obtained from the transgenic plant cell according to  claim 29 , wherein said reproducible structure is selected from the group consisting of calluses, buds or embryos.