Minisymposium 25: Pathogen virulence mechanisms

Abs # 46002: SCF^VirF-mediated targeted proteolysis of the Agrobacterium VirE2 protein and its plant cell interactor, VIP1

Presenter:	Tzfira, Tzvi , ttzfira@ms.cc.sunysb.edu
Authors	Tzfira, Tzvi  (A)   Li, Jianxiong  (A)   Vaidya, Manjusha  (A)   Citovsky, Vitaly  (A)
Affiliations:	(A): Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY

Plant genetic transformation by Agrobacterium represents a unique case of trans-kingdom DNA transfer. This process occurs via a nucleoprotein intermediate, the T-complex, composed of a single-stranded T-DNA copy (T-strand) associated with two virulence proteins, VirD2 and VirE2; a single molecule of VirD2 is bound to the 5’ end of the T-strand whereas multiple molecules of VirE2, the major protein component of the T-complex, coat the entire length of the T-strand. Import of the T-complex into the plant cell nucleus is facilitated by the host VirE2-binding protein VIP1. Once inside the nucleus, the T-strand must be uncoated from its chaperones, VirE2 and VIP1, prior to integration into the host genome. We hypothesized that this uncoating is mediated by another virulence protein, VirF, which is known to be exported into the host cell, contain an F-box motif, and interact with ASK1, a member of the E3 ubiquitin ligase or SCF complex. Our studies using expression of GFP-tagged VirF in plant cells revealed its nuclear accumulation, suggesting that VirF functions within the host cell nucleus. We discovered that VirF specifically interacted with VIP1, but not with VirE2, in the yeast two-hybrid system. Using a bimolecular fluorescence complementation (BiFC) assay, in which the fluorescence of the two halves of YFP is restored when they are brought together as fusions with interacting proteins, we directly demonstrated the VirF-VIP1 interaction in planta. To analyze the function of VirF in targeted proteolysis, we designed a yeast-based protein degradation assay in which VirF significantly reduced the stability of both VIP1 and VirE2, but not of VirD2. In addition, coexpression of VirF and GFP-tagged VIP1 in tobacco and onion cells resulted in specific destabilization of VIP1, directly indicating the role of VirF in targeted proteolysis of VIP1 in planta. That VirF-induced destabilization of VIP1 and VirE2 occurred via the SCF pathway was confirmed using a yeast mutant in a key component of the SCF complex, Skp1; in this mutant, VirF was unable to destabilize VIP1. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of the expression of T-DNA, but not of the microbombarded DNA, by a proteasomal inhibitor MG132. Based on these data, we suggest that SCF^VirFcomplexes function to uncoat the Agrobacterium T-strand prior to or during its integration into the host genome.