Isolation and characterization of acDNA encoding a plant phosphatase implicated in nuclear import of the Agrobacterium VirD2 protein

Agrobacterium tumefaciens is a widespread soil bacterium that elicits tumors on most dicots and some monocots through an interkingdom transfer of genetic information--T(transfer)-DNA. The T-DNA transfer process in planta includes crossing the plant cell wall, nuclear translocation, and chromosome integration. This process is presumably mediated by Agrobacterium vir gene products and plant factors. Information concerning the plant factors and the interaction with Agrobacterium vir gene products is, unfortunately, very limited. The covalent association of VirD2 protein with the T-DNA strand, and the plant-functional bipartite nuclear localization sequence (NLS) in the VirD2 protein suggest that VirD2 may play a role in nuclear import of T-DNA through interaction with plant factors. Therefore, I set up an interaction trap and isolated 28 families of cDNA clones that encode VirD2 NLS region binding proteins. One of the strongly interacting clones, designated DIG3, specifically interacts with the VirD2 NLS. Sequence analysis suggests that DIG3 is a novel type 2C serine/threonine protein phosphatase 2C (PP2C) with 30% to 36% amino acid identity to PP2Cs from other organisms, including the A. thaliana ABI1 protein. Interestingly, in my experiments, the A. thaliana abi1 mutant showed a 2- to 4-fold greater sensitivity in response to Agrobacterium infection than did the wild-type plant. Overexpression of the DIG3 gene in tobacco BY2 protoplasts partially blocked nuclear import of a GUS-VirD2 NLS fusion protein. These data suggest that DIG3 is involved in nuclear import of VirD2 protein and, possibly, the T-DNA. My experiments demonstrated that the VirD2 NLS region was phosphorylated in vitro by protein kinase C and in vivo in tobacco protoplasts, suggesting that the serine...