The molecular structure of agrobacterium VirE2-single stranded DNA complexes involved in nuclear import

Abstract

Nuclear import of DNA is a central event in genetic transformation of plant cells by Agrobacterium tumefaciens. Agrobacterium elicits tumors on plant hosts by transporting a single-stranded (ss) copy of the bacterial transferred DNA (T-DNA) from its Ti (tumor-inducing) plasmid into the plant cell nucleus. Presumably, the process of T-DNA nuclear import is mediated by two agrobacterium proteins, VirD2 and VirE2, which are thought to directly associate with the transported T-DNA. Both proteins have been shown to contain functional nuclear localizations signals (NLS). Recently, VirE2 alone has been shown to actively transport ssDNA into the plant cell nucleus. To understand the process of DNA nuclear import, it is important to know the structure of the transport intermediate. To this end, complexes of VirE2 and ssDNA were analyzed by scanning transmission electron microscopy (STEM). This analysis suggests that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure. The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import. Detailed mass analysis of VirE2-ssDNA filaments is presented and a structural model is proposed.