INTERACTION OF THE DNA MODIFYING PROTEINS VIRD1 AND VIRD2 OF AGROBACTERIUM-TUMEFACIENS - ANALYSIS BY SUBCELLULAR-LOCALIZATION IN MAMMALIAN-CELLS

Interaction between Agrobacterium tumefaciens and plants provides a un ique example of interkingdom gene transfer. Agrobacterium, a plant pat hogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA), This process depends, amo ng others, on the specialized bacterial virulence proteins VirD1 and V irD2 that excise the T-DNA from its adjacent sequences. Subsequent to transfer to the plant cell, the virulence protein VirD2, through its n uclear localization signal (NLS), is believed to guide the T-DNA to th e nucleus. The T-DNA then is integrated into the plant genome. Althoug h both of these proteins are essential for bacterial virulence, physic al interaction of them has not been analyzed so far. We studied associ ations between these proteins by expressing them in mammalian cells an d by testing for intracellular localization and colocalization, When e xpressed in human cells [HeLa, human embryo kidney (HEK) 293], the Vir D2 protein homogeneously distributed over the nucleoplasm, The presenc e of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs r endered the protein cytoplasmic, However, this double NLS mutant was t ranslocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2-VirD2 interaction. The VirD1 protein, by itself locali zed in the cytoplasm, moved to the nucleus when coexpressed with the V irD2 protein, suggesting VirD1-VirD2 interaction. This interaction was confirmed by coimmunoprecipitation tests. Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization. The data are discussed in terms of functions of the VirD proteins, In addition, coimport of proteins into nuclei is suggested as a useful sy stem in studying individual protein-protein interactions.