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...

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The onr (origin of traer) seqe and mob (mobilization) genes of pla d RSF1010 can unctnly replace transfer DNA (T-DNA) borders to generate an RSF1010 intermediate transferable to plants trgh activities of the tumor-inducing (Tiplid uence (vir) (5)(6)(7)(8) and is capped at its 5' end by the VirD2 protein (9-11) to form the "T-complex" (8). Evidence of functional similarity between these DNA transfer systems comes from the elegant experiments of . These workers demonstrated that in an Agrobacterium strain harboring a disarmed Ti plasmid (a Ti plasmid containing the vir genes but not the T-DNA or its borders), the conjugal origin of transfer sequence (oriT and mobilization (mob) genes of the small wide-host-range plasmid RSF1010 can functionally replace T-DNA borders and generate an RSF1010 intermediate transferable to plants by the vir gene transfer machinery. This strongly supports a conjugal model of T-DNA transfer and further suggests that at least some of the Ti plasmid vir gene products are functionally equivalent to the transfer (tra) gene products of conjugal plasmids. With the Escherichia coli conjugative F plasmid, at least 15 plasmid tra gene products are involved in the assembly of a pilus structure used to initiate binding to the recipient cell and in the formation of a proposed membrane-spanning ssDNA transfer channel at the pilus base (13). In the Agrobacterium plant transformation process, however, the mechanism by which the T-DNA is transported across both the bacterial and plant cell membranes and walls is poorly understood. Attachment of Agrobacterium to plant cells is mediated by bacterial chromosomal gene products (14-16), and extracellular pili have not been implicated in virulence (2). By analogy with plasmid conjugal systems, however, T-complex export might likely be mediated by a vir-specified membrane-pore structure.Products of the Ti plasmid virB operon are the best candidates to form a membrane-associated T-DNA transport apparatus. This hypothesis is based in part on the membrane localization ofthe 11 virB gene products predicted by the virB DNA sequence analysis (17-21) and confirmed in the case of several VirB proteins by cellular fractionation experiments (22-24). In addition, recent genetic studies have shown that at least three virE genes, virB9, virB10, and virBil, are essential for tumorigenicity (25). Because virB functions are not necessary for T-DNA processing reactions (26), this finding suggests that virB gene products mediate a late step (or steps) in the T-DNA transfer process. Interestingly, the predicted virBiW gene product shares amino acid sequence similarity with ComG 1, a protein required for the uptake and passage of DNA through the cell wall of competent Bacillis subtilis cells (23,27 9350The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the -9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virBII, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHol insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirBll-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A.tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virBIl genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virBII, the virB9 and virB10 genes are required for tumorigenicity.Infection of susceptible plants by the phytopathogenic bacterium Agrobacterium tumefaciens results in crown gall tumors. The unique disease mechanism involves the transfer of a specific DNA molecule (the T-DNA) from a large bacterial tumor-inducing (Ti) plasmid into the plant cell genome (reviewed in references 3 and 58). Expression of T-DNA genes encoding plant growth hormones results in unregulated plant cell growth and subsequent tumor formation. The proteins which mediate most of the steps in T-DNA processing and mobilization are supplied in trans by a separate, nontransferred section of the Ti plasmid termed the virulence (vir) region. In octopine-type Ti plasmids, the vir region consists of a contiguous ==35-kilobase-pair (kbp) segment containing at least seven transcriptional loci. Mutations in these loci either completely abolish virulence (virA, -B, -D, and -G) or cause attenuated virulence (virC, -E, and -F) (26,27,40).Expression of the vir genes is inducible by phenolic compounds, such as acetosyringone, present in wounded plant tissue. A regulatory system consisting of VirA (a transmembrane sensor) and VirG (a transcriptional activator) regulate vir gene expression (24, 25,44). Following vir induction, the VirDl and VirD2 proteins provide topoisomerase (19) and endonuclease (42, 56) functions that initiate T-DNA processing by introducing a site-and strand-specific nick within conserved 25-bp cis-acting sequences, termed borders, that delineate the ends of the T-DNA molec...