Myosin VIII and XI isoforms interact with Agrobacterium VirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

Abstract

Virulent Agrobacterium strains transfer single-strand T-DNA (T-strands) and virulence effector proteins into plant cells. VirE2, one of these virulence effectors, enters the plant cell and is thought to bind T-strands, protecting them from nuclease degradation and helping guide them to the nucleus. How VirE2 is trafficked inside the plant cell is not fully understood. 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 of several myosin VIII family members, and to myosin XI-K. We observed reduced susceptibility of several Arabidopsis actin mutants and a myosin VIII-1/2/a/b quadruple mutant to Agrobacterium-mediated transformation. Expression of cargo binding domains of myosin VIII-1, VIII-2, VIII-A, or VIII-B in transgenic plants inhibits Arabidopsis root transformation. However, none of the myosin VIII proteins contribute to the intracellular trafficking of VirE2. Expression of myosin VIII-2, -A, -B, but not VIII-1, cDNAs in the myosin VIII-1/2/a/b mutant partially restored transformation. Furthermore, functional fluorescently-tagged VirE2, synthesized in plant cells, relocalized from the cellular periphery into the cytoplasm after delivery of T-strands from Agrobacterium. Surprisingly, mutation of myosin XI-k and expression of the myosin XI-K cargo binding domain had no effect on transformation, although it blocked VirE2 movement along actin filaments. We hypothesize that myosin VIII proteins facilitate VirE2 tethering to the plasma membrane and are required for efficient localization of VirE2 to membrane sites from which they bind incoming T-strands. Myosin XI-K is important for VirE2 movement through the cytoplasm towards the nucleus.