Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.

The transfer process of T (transfer)-DNA of Agrobacterium tumefaciens is activated after the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products then act to generate a free transferable single-stranded copy of the T-DNA, designated the T-strand. Although some vir proteins are responsible for the synthesis of the T-strand, others may mediate T-strand transfer to plant cells as part of a DNA-protein complex. Here, a novel 69-kilodalton vir-specific single-stranded DNA binding protein is identified in Agrobacterium harboring a nopaline-type Ti plasmid. This protein binds single-stranded but not double-stranded DNA regardless of nucleotide sequence composition. The molecular size of the vir-specific single-stranded DNA binding protein and its relative abundance in acetosyringone-induced Agrobacterium suggested that it might be the product of the virE locus; molecular cloning and expression of the virE region in Escherichia coli confirmed this prediction.

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The T-DNA transfer process of Agrobacterium is activated following induction of expression of the Ti plasmid virulence (vir) genes. The virDI and virD2 gene products are required for the production of nicks at the T-DNA borders and for the generation of free linear single-stranded copies of the T-DNA region, T-strands. T-strands are complexed with proteins in vir-induced bacteria, since T-strands partition to the aqueous/phenol interface in non-Pronasetreated total cell extracts. To determine whether the proteins are tightly associated with T-strands, DNA-protein complexes were purified away from bulk proteins by adsorption to glass beads. A 58-kDa protein was specifically released from virinduced DNA-protein complexes after treatment with S1 nuclease to digest single-stranded DNA. The 58-kDa protein was identified as VirD2 by using VirD2-specific antibodies. The tight association of VirD2 with T-strands was shown directly by using VirD2-specific antibody to isolate T-strands. The 5' side of the borders nick sites on the Ti plasmid was also shown to be tightly associated with protein. The data suggest that after VirDl/VirD2-dependent nicking at the T-DNA borders, the VirD2 protein remains bound to the 5' end of the nick, and the VirD2 protein continues to bind tightly to this 5' end during unwinding (or displacement) of the T-strand from the Ti plasmid T-DNA region. The tight binding ofVirD2 to T-strands suggests that this protein has additional functions in T-strand generation and potentially in the later steps of T-DNA transfer.

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