The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically 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 VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.Agrobacterium tumefaciens has the capacity to transfer DNA from its Ti plasmid into plant cells, where the DNA is integrated and transcribed (for reviews, see references 5, 22, 58, and 63). In the case of wild-type tumor-inducing (Ti) plasmids, the transferred DNA (T-DNA) encodes two types of proteins: those that synthesize novel amino acid and sugar conjugates (opines) and those that affect the accumulation of plant hormones responsible for the tumorous growth of the transformed cells. The virulence (vir) genes of the Ti plasmid are necessary for the production and transfer of the T-DNA. vir gene expression is activated by plant wound-released phenolics and sugars, resulting in the accumulation of Vir proteins responsible for T-DNA processing and transfer (58). One of these proteins, VirD2, produces a single-stranded nick at the 25-bp direct repeat border sequences of the Ti plasmid. This is followed by release of a single strand (the T strand) due, apparently, to replacement strand synthesis (45,49,50). The T strand is covalently attached to VirD2 and can associate with the singlestranded DNA-binding protein VirE2 (9-11, 15, 39), resulting in the formation of the T (transfer) complex which is thought to be the transferred intermediate (22,48,61,63). In addition to the T-DNA on Ti plasmids, DNA on plasmids that can replicate in Agrobacterium cells and carry the border sequences of a Ti plasmid, often termed binary vectors, can be mobilized into plant cells by strains of Agro...

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecularweight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.The plant pathogen Agrobacterium tumefaciens causes crown galls on most dicotyledonous plants. These tumors result from a stable genetic transformation of plant cells at a wound site by DNA mobilized from a tumor-inducing (Ti) plasmid located in the virulent bacterium. Genes on the transferred DNA (T-DNA) encode enzymes responsible for the biosynthesis of plant growth factors which cause the uncontrolled proliferation of the plant cells (for a review, see reference 30). The transformation process is mediated in trans by the products of the virulence (vir) genes located on the Ti plasmid and the chromosomal virulence (chv) genes. Vir proteins initiate the excision of the T-DNA from the Ti plasmid, and a single-stranded DNA intermediate which is covalently capped at the 5Ј end by VirD2 is formed. A single-stranded DNA binding protein, VirE2, coats the DNA sometime during the transfer process, forming the T complex, although this interaction may occur after the T strand and VirE2 have been independently transferred to the plant cell (9,16,42,55).Many of the early events leading to the transformation of the plant cell by Agrobacterium are similar to DNA processing and transfer in bacterial conjugation. Moreover, there are homologies between the virulence proteins involved in processing and movement of the T-DNA and the proteins involved in conjugal DNA transfer (30,40,53,70). Of the 11 VirB proteins, each, except VirB1, is essential for virulence (7,8,16,19,27,49,61). The VirB proteins have been proposed to be components of a membrane-bound complex capable of transferring DNA to plant cells. Additionally, extracellular complementation assays demonstrated that all of the VirB proteins so far tested are required for independent VirE2 movement i...