Identification of a plasmodesmal transport signal: application in the generation of virally resistant plants

Viral spread in plants is essentially dependant on virus encoded movement proteins that mediate transport of the virus from an infected cell to neighboring healthy cells. Cell-cell transport occurs through plasmodesmata, small channels that interconnect all plant cells. The main goal of this project is to study the cell-to-cell transport process of tobacco mosaic virus (TMV), which is mediated by its movement protein TMV-MP. In the course of the project, a novel host specific mechanism for inactivation of TMV-MP activity was described. Furthermore, a novel host plant protein involved in viral spread was identified and characterized. These findings potentially form the basis for the development of antiviral strategies. TMV-MP transports the viral genome via a targeting step towards plasmodesmal channels, enlarges those channels and mediates passage of the viral genome into the neighboring cell. The increase in plasmodesmal channel size interferes with intercellular communication and metabolism of the plant. Consequently, plants have likely evolved mechanisms that inactivate viral MPs to ensure their own survival during viral infection. Indeed, we were able to show that phosphorylation of TMV-MP by a plant kinase blocks cell-to-cell transport of TMV in a host dependant manner. In N. tabacum, a host that survives TMV infections very well, TMV-MP is inactivated by phosphorylation, while in the highly sensitive host N. benthamiana, that is severely damaged during TMV infection, no inactivation occurs. Further studies with several hosts support this correlation. Potentially, host dependant regulation of TMV-MP activity by phosphorylation is the underlying cause for the differential sensitivity of host plants to TMV infections. To elucidate the mechanism of the phosphorylation based inactivation of TMV-MP, so called "movement profiles" were developed. Movement profiles allow detailed comparison of the cell-to-cell transport capacity of various mutants, and reveal at which step of the transport process an inactive mutant is blocked. With this method, two inactive TMV-MP mutants that were blocked at different steps in the transport pathway were identified. These two mutants will be used to generate transgenic plants potentially resistant against TMV infections. Furthermore, we have identified plant endogenous interaction partners of TMV-MP in the host plant N. tabacum. One of these interaction partners, MPB2C, was analyzed in detail. MPB2C localizes at the plant cytoskeleton, and anchors TMV-MP to the cytoskeleton thereby blocking intracellular targeting of TMV-MP towards plasmodesmata. As a consequence, cell-to-cell movement is abolished. This result implicates MPB2C in negative regulation of the transport process, or in an antiviral function. Based on potential application in antiviral strategies, interaction of MPB2C and TMV-MP was patented.