The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens

McMaster University, Department of Biology and Antimicrobial Research Centre, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada Correspondence Christian Baron baronc{at}mcmaster.ca The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process. Abbreviations: AS, acetosyringone; EM, electron microscopy; immuno-EM, immuno-electron microscopy; T4SS, type IV secretion system; T-DNA, transferred DNA; TEM, transmission electron microscopy Supplementary data are available with the online version of this paper.