Plant cell death system
View Patent ↗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 solanaceous plant, wherein cells of the solanaceous plant have within their genome a chimeric gene, the expression of which gene causes plant cell cytotoxicity by ribosome inactivation at a desired target site within the plant body, comprising
(i) transforming plant cells with a chimeric gene comprising (a) a promoter, which promoter is induced at and/or adjacent to the target site, operably linked to (b) a nucleic acid molecule which binds to SEQ. ID. No.:2 in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mm EDTA at 65° C. followed by washing in 0.1×SSC, 0.1% SDS at 68° C., wherein said nucleic acid molecule encodes a mature maize ribosome inactivating protein comprising an α domain and a β domain arranged contiguously and having type 3 ribosome inactivating activity; and
(ii) the selective expression of the ribosome inactivating protein at the target site for induction of plant cell death at that target site.
2. A method of producing a transformed plant according to claim 1 , wherein the nucleic acid molecule comprises SEQ. ID. No.: 2.
3. A method of producing a transformed plant according to claim 1 , wherein said chimeric gene further comprises a 3′ untranslated terminator sequence.
4. A method of producing a transformed plant according to claim 3 , wherein said 3′ untranslated terminator sequence is from plant, bacterial or viral genes.
5. A method of producing a transformed plant according to claim 3 , wherein said 3′ untranslated terminator sequence is selected from the group consisting of 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.
6. A method of producing a transformed plant according to claim 1 , wherein said chimeric gene further comprises a transcriptional or translational enhancer sequence intracellular targeting sequences and introns, or nucleotide sequences operable to facilitate the transformation process and stable expression of said chimeric gene or combinations thereof.
7. A plant transformed with a chimeric gene according to the method of 1 .
8. A plant cell comprising within its genome a chimeric gene, the expression of which gene causes plant cytotoxicity, said chimeric gene comprising (i) a promoter, which promoter is induced at and/or adjacent to a target site, operably linked to (ii) a nucleic acid molecule which binds to the nucleic acid molecule of SEQ ID NO.: 2 in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mm EDTA at 65° C. followed by washing in 0.1×SSC, 0.1% SDS at 68° C., wherein said nucleic acid molecule encodes a mature maize ribosome inactivating protein comprising an α domain and a βdomain arranged contiguously and having type 3 ribosome inactivating activity.
9. A method of producing a transgenic solanaceous plant, comprising growing plants from a seed wherein said seeds have within their genome a chimeric gene, the expression of which gene causes plant cytotoxicity at a target site, said chimeric gene comprising (a) a promoter, which promoter is induced at and/or adjacent to the target site, operably linked to (b) a nucleic acid molecule which binds to SEQ. ID. No.:2 in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1mm EDTA at 65° C. followed by washing in 0.1×SSC, 0.1% SDS at 68° C., wherein said nucleic acid molecule encodes a protein having type 3 ribosome inactivating activity wherein said nucleic acid molecule encodes a mature maize ribosome inactivating protein comprising an α domain and a β domain arranged contiguously and having type 3 ribosome inactivating activity.
10. A nucleic acid molecule comprising (a) a promoter, which promoter is induced at andlor adjacent to the target site, operably linked to (b) a nucleic acid molecule which binds to SEQ. ID. No.:2 in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mm EDTA at 65° C. followed by washing in 0.1×SSC, 0.1% SDS at 68° C., wherein said nucleic acid molecule encodes a protein having type 3 ribosome inactivating activity wherein said nucleic acid molecule encodes a mature maize ribosome inactivating protein comprising an α domain and a β domain arranged contiguously and having type 3 ribosome inactivating activity.
11. A vector comprising the nucleic acid of claim 10 .