ROLE OF THE AGROBACTERIUM-TUMEFACIENS VIRD2 PROTEIN IN T-DNA TRANSFERAND 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 virulen ce. Previous results from our laboratory indicated that the C-terminal , bipartite NLS and the omega region are not essential for nuclear upt ake of T-DNA, and further suggested that the omega domain may be requi red for efficient integration of T-DNA into the plant genome. In this study we took two approaches to investigate the importance of the omeg a domain in T-DNA integration. Using the first approach, we constructe d a T-DNA binary vector containing a promoterless gusA-intron gene jus t 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. Approximate ly 0.4% of the tobacco cell clusters infected by a wild-type A. tumefa ciens strain harboring this vector stained blue with 5-bromo-4-chloro- 3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mut ant A. tumefaciens strain harboring the same binary vector, we did not detest any blue staining. Using the second approach, we directly demo nstrated that more T-DNA is integrated irate high-molecular-weight pla ne DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega dele tion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determin e whether the use of the T-DNA right border is altered in those few tu mors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that t he right border was used tee integrate the T-DNA in a similar manner r egardless of whether the VirD2 protein encoded by the inciting A. tume faciens was wild-type or contained are omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active i n cleaving a T-DNA border in vitro as was the wild-type protein. Final ly, we investigated the role of various amino acids of the omega and b ipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the om ega domain, or mutation of the 10-amino-acid region between the two co mponents of the bipartite NLS, had little effect upon the nuclear targ eting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protei n to the nucleus.