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

Yumin Tao, Purdue University

Abstract

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$\sp{394}$ residue is the phosphorylation target site. Alteration of this serine residue to either alanine or aspartate decreased the nuclear localization activity of the VirD2 NLS regardless of whether DIG3 is overexpressed in the same cell. Thus, the negative role of DIG3 in VirD2 nuclear import appears to be achieved by phosphorylation and dephosphorylation of the Ser$\sp{394}$ residue in the VirD2 NLS. As far as I am aware, this is the first example in which the nuclear import of a protein/nucleic acid complex is regulated by phosphorylation of the protein component of the complex.

Degree

Ph.D.

Advisors

Gelvin, Purdue University.

Subject Area

Plant pathology|Molecular biology|Environmental science|Ecology|Botany|Microbiology

COinS