Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors