Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a sy stem which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernat ant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two k nown periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenico l acetyl transferase, and a VirE2::green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture super natant did not require any Ti plasmid genes, except for VirE1, a specific c haperone for VirE2. The levels of the VirE2 and VirD2 proteins in the super natant increased significantly when cells were grown at 19 degrees C as com pared with 28 degrees C. When Agrobacterium expressed the oncogenic suppres sive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly redu ced. However, oncogenic suppressive activity protein did not block the accu mulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2. Vi rE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacte rium may take place in two steps, the first mediated by an unidentified pat hway and the second by the virB system.