Role of <i>Agrobacterium</i> VirD2 protein and plant histone H2A in T -DNA integration
Agrobacterium tumefaciens is a soil borne phytopathogen that causes crown gall tumors on plants. The process of tumor formation involves the transfer and integration of a specific DNA segment (T-DNA) from the bacterium into the plant chromosome. VirD2 is an Agrobacterium protein involved in T-DNA processing and transfer into plants. VirD2 protein covalently attaches to the T-DNA and enters into the plant cell. A C-terminal region of VirD2 called ω is involved in T-DNA integration but not T-DNA transfer. A bacterium containing a particular VirD2 ω mutation will transiently transform cells but tumors will not result. My experiments investigated the role of this ω region in T-DNA integration into the plant chromosome. I used an ω mutant Agrobacterium strain and showed that, unlike the situation with the wild-type bacterium, with this particular mutant there was little T-DNA integrated into the plant chromosome. Plant factors are postulated to be involved in T-DNA transfer and integration. To isolate plant genes that are involved in T-DNA transfer and integration, I identified several Arabidopsis thaliana T-DNA tagged mutants that are deficient in forming crown gall tumors after infection with Agrobacterium. In one of the mutants, I identified a disrupted gene that is involved in the tumorigenesis deficiency phenotype. The mutant gene encodes histone H2A. Complementation analysis indicates that histone H2A is involved in T-DNA transformation, most likely in the T-DNA integration step. I characterized the histone H2A genes in Arabidopsis . There are at least six variants of Arabidopsis histone H2A genes. During the process of complementation I discovered that germ-line transformation of Arabidopsis can bypass some of the steps involved in conventional Agrobacterium root transformation. Using different avirulent Agrobacterium mutants and Arabidopsis ecotypes and/or mutants that are resistant to Agrobacterium root transformation, I showed that germ-line transformation required Agrobacterium vir genes but not several plant genes required for Agrobacterium binding to or T-DNA integration in root tissue. Finally, I developed a potential new strategy to control crown gall disease. By using Arabidopsis as a model plant, I expressed Agrobacterium VirD2 protein in Arabidopsis and showed that transgenic plants expressing VirD2 protein are resistant to Agrobacterium root transformation.
0307: Molecular biology