The interaction of plants with viruses in susceptible hosts depends on intricate mechanisms in which host and viral functions involved in virus replication and cell-to-cell spread are balanced by plant host defense reponses that target viral proteins and viral RNA for degradation. Our laboratory investigates the cellular events by which the RNA genome of Tobacco mosaic virus is transported into adjacent cells. Recent cell biological studies in various laboratories (including ours) have revealed important insights into the interactions of viral movement protein (MP, required for the movement of viral RNA) and viral replicase with cellular components, and in vivo studies are aimed at elucidating the functional role of these association during the movement process. Although the viral replicase is primarily involved in virus replication, it is also functionally involved in virus movement and in the suppression of RNA silencing. The replicase of TMV thus represents an additional example for proteins that involved in movement that turn out to have silencing suppressor function. Our laboratory has collected evidence that also the viral MP intercepts with RNA silencing pathways. On the basis of these new functions for replicase and MP it becomes pertinent to investigate whether their known associations with host cellular components are actually involved in defense reponses between the virus and the host rather than in viral RNA movement and replication. Thus, the project proposed here will use state-of-the-art tools to discern which of the associations of MP and replicase with subcellular components are involved in host defense responses such as proteasome-mediated degradation and RNA silencing. To investigate the intracellular location of the ubiquitinylation event and to follow the fate of ubiquitinylated MP versus non-modified MP in livining infected cells, we will use a novel technique that involves Bimolecular Fluorescence Complementation (BiFC). To investigate the role of MP and replicase and their cellular associations in RNA silencing, in vivo cell biological studies using fluorescent proteins to visualize viral factors and RNA silencing pathway components will be combined with molecular and genetic analyses of RNA silencing using both Nicotiana benthamiana and Arabidopsis as hosts. To address the cellular events in Arabidopsis, we will use viral constructs based on Oilseed rape mosaic virus (ORMV), a TMV-related tobamovirus. This research will provide important insights into the mechanisms involved in plant:pathogen interactions, providing new targets for crop improvement. Moreover, by illuminating both virus movement and anti-viral defense, the project will reveal new insight into fundamental biology, such as cell-to-cell communication and RNA silencing.