Interaction Between Movement Protein Of Cucumber Mosaic Virus And Microfilaments: Mechanism Of Microfilament-mediated Regulation Of Plasmodesmata Permeability

The cell-to-cell movement of macromolecules such as nuclear acids and proteins, through the plasmodesmata, play important roles in plant cell differentiation, plant morphogenesis and plant development. The viral movement proteins (MPs) have been reported to increase the plasmodesmata permeability and mediate the transport of viral nucleoproteins between plant cells. More and more studies suggested that viral MPs may take over or activate the intrinsic mechanism for macromolecule translocation in plant cells to transport their own viruses. Therefore, the study of the molecular and biochemical mechanism for viral MP to gate plasmodesmata facilitates our comprehensive understanding of the regulatory mechanism for plasmodesmata permeability and endogenous macromolecules transportation between plant cells. The cytoskeleton is involved in intracellular trafficking, driving and targetting macromolecules to the plasmodesmata. In addition, the cytoskeleton elements are reportedly the components of the plasmodesmata, and might function in the regulation of plasmodesmata permeability. In present study, we investigated the interaction between MP from Cucumber Mosaic Virus (CMV) and the microfilaments, and microfilament-mediated mechanism for regulating plasmodesmata permeability.To elucidate the molecular basis for the interaction between CMV MP and microfilaments, CMV MP was expressed in transgenic E.coli, and the recombinant protein was purified from the bacterial cells. Actin was purified from rabbit muscle. The association of CMV MP with G-actin and microfilaments was examined by dot western blotting, co-sedimentation and fluorescent double label in vitro. The experiment results showed that CMV MP formed dimmers, as well as protein aggregats in actin polymerized buffer. The formation of dimmers may be regulated by ATP. The experiment results of co-sedimentation, fluorescent measurements, and confocal, CCD and electron microscopy observations demonstrate that CMV MP bind to microfilaments, and have concentration-dependent effects on actin polymerization. 100 nm/L of CMV MP shifted the critical concentration for actin polymerization from 0.2 um/L to 0.12μm/L, however, at higher concentration of 1.7 um/L CMV MP the critical concentration was increased from 0.2μm/L to 0.4 um/L. In addition, CMV MP enhanced actin polymerization at low concentrations but inhibited actin polymerization at high concentrations, too. Further investigation showed that CMV MP at high concentrations severed microfilaments obviously, while no remarkable depolymerization of microfilaments was observed at low concentrations of CMV MP. The effects of CMV MP were abolished when microfilaments were stabilized by phalloidin.Previous works of our lab and reported investigations suggested that the gating of plasmodesmata by MP is related to microfilaments. MP can not open plasmodesmata when microfilaments were stabilized by phalloidin, however, the increasment of plasmodesmata permeability by MP was enhanced when the microfilament was depolymerized by drags. In this study, we demonstrated that CMV MP bind to microfilaments and disrupt the microfilaments in a concentration-dependent manner. It is suggested that MP in the cytoplasm may not cause disruption of the actin cytoskeleton because of its low concentration in the cytoplasm. However, when concentrated at the plasmodesmata, MP maycause the disruption of the microfilaments to increase the permeability of the plasmodesmata.