Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand—protein complex: Tight association of VirD2 with the 5′ ends of T-strands

The T-DNA transfer process ofAgrobacterium is activated following induction of expression of the Ti plasmid virulence (vir ) genes. ThevirD1 andvirD2 gene products are required for the production of nicks at the T-DNA borders and for the generation of free linear single-stranded copies of the T-DNA region, T-strands. T-strands are complexed with proteins invir -induced bacteria, since T-strands partition to the aqueous/phenol interface in non-Pronasetreated total cell extracts. To determine whether the proteins are tightly associated with T-strands, DNA-protein complexes were purified away from bulk proteins by adsorption to glass beads. A 58-kDa protein was specifically released fromvir -induced DNA-protein complexes after treatment with S1 nuclease to digest single-stranded DNA. The 58-kDa protein was identified as VirD2 by using VirD2-specific antibodies. The tight association of VirD2 with T-strands was shown directly by using VirD2-specific antibody to isolate T-strands. The 5′ side of the border nick sites on the Ti plasmid was also shown to be tightly associated with protein. The data suggest that after VirD1/VirD2-dependent nicking at the T-DNA borders, the VirD2 protein remains bound to the 5′ end of the nick, and the VirD2 protein continues to bind tightly to this 5′ end during unwinding (or displacement) of the T-strand from the Ti plasmid T-DNA region. The tight binding of VirD2 to T-strands suggests that this protein has additional functions in T-strand generation and potentially in the later steps of T-DNA transfer.