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 (TNTs) 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 plant viruses, the transport of which via Pd represents a conceptual paradigm for intercellular traffic of macromolecules. Tobacco mosaic virus (TMV), whose Pd transport is mediated by its movement protein (MP), is used as tool to study the mechanisms of this transport. Pd transport comprises 3 sequential pathways (that remain obscure even 35 years after identification of MP as the first Pd-moving protein): targeting to Pd, gating of Pd, and inactivation of the Pd-gating factor to prevent further interference with intercellular communication. This work focuses on these processes, using the following model: (1) In the cytoplasm, MP Pd localization signal (PLS) interacts with SYTA, a synaptotagmin that mediates contacts between the ER and plasma membrane (PM), and MP Pd-gating domain (PDG) interacts with ANK, another plant factor required for viral movement. (2) SYTA directs MP to PM and (3) hands MP to a Pd-associated protein PDLP4, resulting in Pd localization. (4) ANK (or ANK-MP complex) activates Pd-associated ß-1,3 glucanase (PBG), which (5) relaxes the Pd callose sphincter and elevates permeability. (6) MP then is destabilized by the ubiquitin/proteasome system. Aim 1. Mapping protein interactions in Pd targeting and gating. It is known that MP PLS interacts with SYTA and PDLP4, and MP PGD interacts with ANK, which, in turn, interacts with PBG. But it is not known whether these interactions occur in a single complex with all or some of these proteins, or whether they are mutually exclusive. These interactions will be mapped in vitro, in yeast, and in planta to define the functional pathway for MP targeting to Pd in preparation for their gating. Aim 2. Pd gating mechanism. The hypothesis will be tested that MP redirects ANK from the cytoplasm to Pd, where ANK (or ANK-MP complexes) activates PBG and gates Pd. These experiments will define the functional pathway for MP gaiting of Pd. Aim 3. UPS-mediated down-regulation of MP. Challenge with viruses or bacteria induces expression of the plant defense-related F-box protein VBF. Among its pathogen-encoded substrates, VBF recognizes MP. The hypothesis will be tested that VBF targets MP to proteasomal degradation via the SCFVBF pathway. The expected outcomes of the proposed research will define basic concepts and molecular pathways that underly intercellular transport of macromolecules.