A new strategy for generating geminivirus resistant plants using a DNA betasatellite/split barnase construct.

The betasatellite DNA associated with cotton leaf curl disease contains a single ORF, βC1, which is a pathogenicity determinant. Deletion of the βC1 ORF showed that it was not required for betasatellite replication in the presence of Tomato leaf curl virus-Australia (TLCV-Au). A series of betasatellite/split mutant barnase gene constructs, in which a direct repeat of the Bacillus amyloliquefaciens barnase gene flanked the betasatellite, were shown to replicate in tobacco in the presence of TLCV-Au. A betasatellite/split intact barnase gene construct, with the optimal direct repeat unit of the barnase gene, was introduced into Nicotiana tabacum plants. Approximately one third of the transgenic lines containing the betasatellite/split barnase gene constructs were shown to be completely resistant to the TLCV-Au infection. The betasatellite/split intact barnase gene cassette ensures that there is no expression of the barnase in the absence of TLCV-Au, but upon infection of the cell with the virus, release of the betasatellite/split barnase cassette as a replicating molecule resulting in the reconstitution and expression of an active barnase gene and the destruction of the infected cell. This system offers the potential to provide resistance in a variety of plant species against geminiviruses that support the replication of betasatellite.