Minisymposium 6: Cytoskeleton

Add this abstract to my Itinerary

Abs # M0602: Microtubule-assisted movement of the Agrobacterium T-complex

Presenter:	Tzfira, Tzvi       Contact Presenter
Authors	Li, Jianxiong  (B)   Salman, Hanna  (C)   Elbaum, Michael  (C)   Tzfira, Tzvi  (A) (B)
Affiliations:	(A): The University of Michigan (B): State University of New York at Stony Brook (C): Weizmann Institute of Science

On its route to the nucleus, the T-complex must pass the highly dense and contorted structure of the host cell cytoplasm. The large size of the T-complex rules out the possibility that the T-DNA simply diffuses through the cytoplasm, and suggests an active mechanism for its delivery to the nuclear pore. Since transport of large molecules in the cytoplasm occurs largely via mechanisms involving the cytoskeleton and associated motor proteins, we explored the possibility that the T-complex travels along the microtubule network. Indeed, single-particle tracking showed that complexes produced in vitro between ssDNA and a specific VirE2 mutated in the NLS region are actively transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. We therefore identified and isolated Arabidopsis mutants knocked out in various dynein light chain homologues, and tested their susceptibility to Agrobacterium infection. A Dynein Light Chain 3 (dlc3) mutant was found to be resistant to both stable and transient Agrobacterium-mediated genetic transformation, as evidenced using root tumorigenesis and GUS expression assays respectively. DLC3 co-localized with the microtubule network in plant cells, and bound VIP1 in the yeast two-hybrid assay. Furthermore, a GFP fusion of the nuclear protein VIP1 failed to localize to the nucleus of dlc3 Arabidopsis mutant, remaining mostly cytoplasmic. Since VIP1 specifically interacts with VirE2 and assists its nuclear import during the transformation process, we suggest that it is also involved in the cytoplasmic shuttling of the Agrobacterium T-complex.