Agrobacterium VirD2-Binding Protein Is Involved in Tumorigenesis and Redundantly Encoded in Conjugative Transfer Gene Clusters

Abstract

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.

Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus

“…Previously, we identified a VirD2-binding protein VBP1, which can bind VirD2 directly and is involved in Agrobacterium tumorigenesis (17). A. tumefaciens contains two additional genes encoding proteins highly homologous to VBP1, which are designated VBP2 and VBP3 [supporting information (SI) Table 1].…”

“…A. tumefaciens contains two additional genes encoding proteins highly homologous to VBP1, which are designated VBP2 and VBP3 [supporting information (SI) Table 1]. All of the three VBP proteins can bind to VirD2 (17). To determine whether the VBP-VirD2 interaction is important for T-DNA transfer, we created vbp1 point-mutations by site-directed mutagenesis using PCR.…”

“…We then tested the functionality of these VBP mutant proteins, after introducing the corresponding genes into the vbp triple mutant GMV123 (lacking any functional vbp). The bacterial cells were used to test the function to transfer T-DNA, as detected by the tumorigenesis assay; the crude cell extracts were used in a pull-down assay to assess the function for VBP1-VirD2 interaction, because previous experiments have shown that purified VBP1 can bind to purified VirD2 (17). Mutations at S52 and Y139 did not affect the VBP1-VirD2 interaction (Fig.…”

“…VBP has three functionally redundant homologs; one of them is encoded on the cryptic plasmid (17). We further determined whether VBP is important for plasmid conjugation, because T-DNA transfer is mechanistically related to plasmid conjugation (1, 4, 22 23).…”

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

“…Free GFP and His 6 -tagged Agrobacterium VirD2-binding protein (Vbp1) (Guo et al, 2007) were used as controls. Higher quantities of free GFP than GFP-VirD2 were bound to the beads, as reflected in the quantities of GFPs recovered from the beads (Fig.…”