PROJECT SUMMARY Perhaps one of the most intriguing, yet least studied, aspects of macromolecular transport is traffic through intercellular connections. These connections are termed tunneling nanotubes in mammals and plasmodesmata (Pd) in plants. Whereas both mammalian and plant viruses utilize cell-to-cell transport pathways, the first such capability was identified for movement proteins (MPs) of plant viruses, the transport of which via Pd represents a conceptual paradigm for intercellular traffic of macromolecules. Whereas the main research effort in the field, including our lab, has been focused on the mechanism of MP cell-to-cell movement, the important question of how the movement mechanism is controlled remained largely unaddressed. The proposed research program aims to begin filing this gap in our knowledge. Research in my laboratory focuses on three diverse aspects of plant biology and plant-pathogen interactions: (i) cell-to-cell transport of the movement protein (MP) of the model plant virus TMV; (ii) bacterium-to-plant cell transport of proteins and nucleic acids during genetic transformation by Agrobacterium; and (iii) epigenetic regulation of gene expression, including the pathogen response genes, by histone modifications. Recently, all three projects converged on the regulatory roles of ubiquitin writers (E3 ligases) and erasers (deubiquitinases) in the plant life cycle in general, and in the MP cell-to-cell transport in particular. Specifically, our findings suggest that ubiquitin writers and erasers dynamically control two major aspects of the MP cell- to-cell transport process: cargo stability/availability and assembly/disassembly of the Pd transport machinery. The proposed program leverages the wealth of molecular tools and experimental approaches that we have developed along the years to study cell-to-cell transport of plant viruses and roles of protein ubiquitylation/deubiquitylation in plant-pathogen interactions to address a new (and, so far, completely unresearched) level of regulation of intercellular transport through Pd by reversible action of ubiquitin writers and erasers of the plant cell. We believe that this goal is innovative and likely to be transformative also for studies of transport of other proteins, viral and plant, through Pd.