Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process.

Abstract

Induction of Agrobacterium tumefaciens vir gene expression by wounded plant cells results in production of a free transferable single-stranded (ss) copy of T-DNA, the T-strand. One of the Vir proteins, the VirE2 polypeptide, is a ssDNA-binding protein. In the present work, interaction of nopaline-specific VirE2 protein (Mr 69,000) with ssDNA was studied by using nitrocellulose filter binding, gel retardation, and electron microscopy techniques. The VirE2 protein was found to bind to ssDNA molecules with strong cooperativity, forming VirE2-ssDNA complexes with a binding site of 28-30 nucleotides. The VirE2-ssDNA complexes are stable at high salt concentrations and resistant to exonucleolytic activity. When examined under the electron microscope, the VirE2 protein converted collapsed free ssDNA molecules into unfolded and extended structures. The structure and properties of VirE2-ssDNA complexes predict possible functions in Agrobacterium virulence to (i) protect the T-strands from cellular nucleases and (ii) facilitate transfer of the T-strands through bacterial membranes possibly by specific interaction with putative membrane pores formed in plant-induced Agrobacterium cells.