Abstract
Agrobacterium tumefaciens is the causal agent of crown gall disease. The bacterium is capable of transferring a segment of single-stranded DNA (ssDNA) into recipient cells during the transformation process, and it has been widely used as a genetic modification tool for plants and nonplant organisms. Transferred DNA (T-DNA) has been proposed to be escorted by two virulence proteins, VirD2 and VirE2, as a nucleoprotein complex (T-complex) that targets the host nucleus. However, it is not clear how such a proposed large DNA-protein complex is delivered through the host nuclear pore in a natural setting. Here, we studied the natural nuclear import of the Agrobacterium-delivered ssDNA-binding protein VirE2 inside plant cells by using a split-GFP approach with a newly constructed T-DNA-free strain. Our results demonstrate that VirE2 is targeted into the host nucleus in a VirD2- and T-DNA-dependent manner. In contrast with VirD2 that binds to plant importin α for nuclear import, VirE2 directly interacts with the host nuclear pore complex component nucleoporin CG1 to facilitate its nuclear uptake and the transformation process. Our data suggest a cooperative nuclear import model in which T-DNA is guided to the host nuclear pore by VirD2 and passes through the pore with the assistance of interactions between VirE2 and host nucleoporin CG1. We hypothesize that this large linear nucleoprotein complex (T-complex) is targeted to the nucleus by a "head" guide from the VirD2-importin interaction and into the nucleus by a lateral assistance from the VirE2-nucleoporin interaction.