Myosin VIII and XI isoforms interact withAgrobacteriumVirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

Abstract

ABSTRACTVirulentAgrobacteriumstrains 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 vitropull-down, yeast two-hybrid, andin vivoco-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 severalArabidopsisactin mutants and a myosinVIII-1/2/a/bquadruple mutant toAgrobacterium-mediated transformation. Expression of cargo binding domains of myosin VIII-1, VIII-2, VIII-A, or VIII-B in transgenic plants inhibitsArabidopsisroot transformation. However, none of the myosin VIII proteins contribute to the intracellular trafficking of VirE2. Expression of myosinVIII-2,-A,-B, but notVIII-1, cDNAs in the myosinVIII-1/2/a/bmutant 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 fromAgrobacterium. Surprisingly, mutation of myosinXI-kand 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.