INHIBITION OF VIRB-MEDIATED TRANSFER OF DIVERSE SUBSTRATES FROM AGROBACTERIUM-TUMEFACIENS BY THE INCQ PLASMID RSF1010

The transfer of DNA from Agrobacterium tumefaciens into a plant cell r equires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediat e is a single-stranded DNA (T strand), covalently attached to the VirD 2 protein and coated with the single stranded DNA-binding protein, Vir E2. The movement of this intermediate out of Agrobacterium cells and i nto plant cells requires the expression of the virB operon, which enco des 11 proteins that localize to the membrane system. Our earlier stud ies showed that the IncQ broad-host-range plasmid RSF1010, which can b e transferred from Agrobacterium cells to plant cells, inhibits the tr ansfer of T-DNA from pTiA6 in a fashion that is reversed by overexpres sion of virB9 virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. B y using extracellular complementation assays, the effects of RSF1010 o n movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drasticall y inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of V irE2 and the uncoated T strand as assayed by extracellular complementa tion. Our results are consistent with a model in which the RSF1010 pla smid, or intermediates from it, compete with the T strand and VirE2 fo r a common transport site.