Import of agrobacterium T-DNA into plant nuclei: Two distinct functions ofVirD2 and VirE2 proteins

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDN A) were tested for import into plant nuclei in vitro. Import of these compl exes was fast and efficient end could be inhibited by a competitor, a nucle ar localization signal (NLS) coupled to BSA. For import of short ssDNA, Vir D2 was sufficient, whereas import of long ssDNA additionally required VirE2 . A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were import ed into nuclei, once bound to ssDNA they were not imported, implying that w hen complexed to DNA, the NLSs of VirE2 are not exposed and thus do not fun ction. RecA, another ssDNA binding protein, could substitute for VirE2 in t he nuclear import of T-DNA but not in earlier events of T-DNA transfer to p lant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore , by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is ac cepted for translocation into the nucleus.