Unraveling the Molecular Pathways of Cellular Genetic Transfer

Agrobacterium transfers single-stranded T-DNA (T-strands) and virulence effector proteins into plant cells. VirE2, a key effector protein, enters the plant cell and is thought to bind T-strands, protecting them from nuclease degradation and guiding them to the nucleus. However, the intracellular trafficking mechanisms of VirE2 remain unclear. Using bimolecular fluorescence complementation, in vitro pull-down, yeast two-hybrid, and in vivo co-immunoprecipitation assays, we found that VirE2 binds directly to the cargo-binding domains (CBDs) of several myosin VIII family members and to myosin XI-K. We observed reduced susceptibility to Agrobacterium-mediated transformation of several Arabidopsis actin mutants and in a myosin VIII-1/2/a/b quadruple mutant. Expression of the CBDs of myosin VIII-1, VIII-2, VIII-A, or VIII-B in transgenic plants will inhibit Arabidopsis root transformation. However, none of the myosin VIII proteins contributes to the intracellular trafficking of VirE2. Expression of myosin VIII-2, VIII-A, and VIII-B, but not VIII-1, cDNAs in the myosin VIII-1/2/a/b mutant can partially restore transformation efficiency. Furthermore, functional fluorescent protein-tagged VirE2 synthesized in plant cells relocalizes from the cell periphery to the cytoplasm following T-strand delivery from Agrobacterium. Surprisingly, an Arabidopsis myosin XI-k mutant, transgenic plants expressing the myosin XI-K CBD, and plants subjected to RNAi targeting myosin XI-k all remain highly transformable, even though VirE2 movement along actin filaments is blocked. We hypothesize that myosin VIII proteins facilitate VirE2 tethering to the plasma membrane and are required for its efficient localization to membrane sites where it binds incoming T-strands, whereas myosin XI-K is important for VirE2 movement through the cytoplasm toward the nucleus.