Plant cell death system

Abstract

This invention seeks to provide means by which transgenic plants harbour within their genome a chimaeric gene which when expressed produces a protein having a cytotoxic effect. Cytotoxicity is achieved by way of a protein or part thereof capable of blocking cellular protein synthesis, such as a maize ribosome inactivating protein or part thereof. The chimaeric gene may comprise a promoter operably linked to a sequence encoding a protein capable of blocking cellular protein synthesis such that the promoter is inducible at and/or adjacent to a target site.

1 . A method of producing a transgenic plant which harbours within the genome of the plant a chimaeric gene, the expression of which gene causes plant cytotoxicity at a target site, wherein a plant is transformed with a chimaeric gene comprising a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to a coding sequence, which coding sequence encodes a maize ribosome inactivating protein or a part thereof.  
     
     
         2 . A method of producing a transgenic plant according to  claim 1 , wherein the coding sequence of said maize ribosome inactivating protein comprises a pro-maize ribosome inactivating protein comprising the N-terminal, the α domain, the central spacer peptide, the β domain and the C-terminal peptide.  
     
     
         3 . A method of producing a transgenic plant according to  claim 1 , wherein the coding sequence of said maize ribosome inactivating protein comprises a recombinant mature RIP comprising an α domain and a β domain arranged contiguously.  
     
     
         4 . A method of producing a transgenic plant according to  claim 1 , wherein the coding sequence of said maize ribosome inactivating protein comprises a recombinant RIP comprising as the active part the α domain only of a maize ribosome inactivating protein.  
     
     
         5 . A method of producing a transformed plant according to  claim 1 , wherein the coding sequence of said ribosome inactivating protein comprises as the active part a recombinant RIP comprising the β domain only of a maize ribosome inactivating protein.  
     
     
         6 . A method of producing a transformed plant according to claims  1  or  2 , wherein said coding sequence comprises the coding sequence identified as SEQ. ID. No.: 1, or a coding sequence which is homologous thereto.  
     
     
         7 . A method of producing a transformed plant according to claims  1  or  3 , wherein said coding sequence comprises the sequence identified in SEQ. ID. No.: 2 or a coding sequence which is homologous thereto.  
     
     
         8 . A method of producing a transformed plant according to  claim 4 , wherein said coding sequence comprises the sequence identified in SEQ. ID. No.: 3 or a coding sequence homologous thereto.  
     
     
         9 . A method of producing a transformed plant according to  claim 5 , wherein said coding sequence comprises the sequence identified in SEQ. ID. No.: 4 or a coding sequence homologous thereto.  
     
     
         10 . A method of producing a transformed plant according to claims  6 ,  7 ,  8  or  9 , wherein said homologous coding sequence has at least 70% with SEQ. ID. No.: 1; SEQ. ID. No.: 2; SEQ. ID. No.: 3; or SEQ. ID. No.: 4; respectively.  
     
     
         11 . A method of producing a transformed plant according to  claim 10 , wherein said homologous coding sequence has at least 80% homology with SEQ. ID. No.: 1; SEQ. ID. No.: 2; SEQ. ID. No.: 3; or SEQ. ID. No.: 4; respectively.  
     
     
         12 . A method of producing a transformed plant according to  claim 10  or  11 , wherein said homologous coding sequence has at least 85% homology with SEQ. ID. No.: 1; SEQ. ID. No.: 2; SEQ. ID. No.: 3; or SEQ. ID. No.: 4; respectively.  
     
     
         13 . A method of producing a transformed plant according to  claim 10 ,  11  or  12 , wherein said homologous coding sequence has at least 90% homology with SEQ. ID. No.: 1; SEQ. ID. No.: 2; SEQ. ID. No.: 3; or SEQ. ID. No.: 4; respectively.  
     
     
         14 . A method of producing a transformed plant according to any one of the preceding claims, wherein said chimaeric gene further comprises a 3′ untranslated terminator sequence.  
     
     
         15 . A method of producing a transformed plant according to  claim 14 , wherein said 3′ untranslated terminator sequence is from plant, bacterial or viral genes.  
     
     
         16 . A method of producing a transformed plant according to any one of claims  14  or  15 , wherein said 3′ untranslated terminator sequence is selected from the group comprising the pea rbcS E9 terminator sequence, the nos terminator sequence derived from the nopaline synthase gene of  Agrobacterium tumefaciens  and the 35S terminator sequence from cauliflower mosaic virus.  
     
     
         17 . A method of producing a transformed plant according to any one of the preceding claims, wherein said chimaeric gene comprises a transcriptional or translational enhancer sequence and/or intracellular targeting sequences and introns, and/or nucleotide sequences operable to facilitate the transformation process and the stable expression of said chimaeric gene.  
     
     
         18 . A method of producing a transformed plant according to any one of the preceding claims, wherein said chimaeric gene comprises a first promoter operably linked to an α domain of a maize ribosome inhibiting protein and a second promoter operably linked to a β domain of a maize ribosome inhibiting promoter.  
     
     
         19 . A method according to  claim 18 , wherein said first and second promoters express at one overlapping target site.  
     
     
         20 . A method according to  claim 18  or  19 , wherein said first promoter is the KNT1 promoter and the second promoter is the KNT2 promoter (SEQ. ID. No.: 18).  
     
     
         21 . A plant transformed with a chimaeric gene according to the method of any one of claims  1 - 20 , wherein said chimaeric gene comprises a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to a coding sequence, which coding sequence encodes a maize ribosome inactivating protein or part thereof.  
     
     
         22 . A plant cell transformed with a chimaeric gene according to the method of any one of claims  1 - 20 , wherein said chimaeric gene comprises a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to a coding sequence, which coding sequence encodes a maize ribosome inactivating protein or a part thereof.  
     
     
         23 . A DNA isolate of a chimaeric gene comprising a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to a coding sequence, which coding sequence encodes a maize ribosome inactivating protein or a part thereof.  
     
     
         24 . A DNA isolate of a chimaeric gene comprising a first promoter operably linked to a first domain of a maize ribosome inactivating protein and a second promoter operably linked to a second domain of a maize ribosome inactivating protein.  
     
     
         25 . A DNA isolate of a chimaeric gene according to  claim 24 , said first and second promoters having one overlapping expression site.  
     
     
         26 . A biologically functional expression vehicle containing a chimaeric gene comprising a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to a coding sequence, which coding sequence encodes a maize ribosome inactivating protein or a part thereof.  
     
     
         27 . A biologically functional expression vehicle according to  claim 26 , said expression vehicle further comprising a first promoter operably linked to a first domain of a maize ribosome inactivating protein and a second promoter operably linked to a second domain of a maize ribosome inactivating protein.