Plant cells are surrounded by cell walls, and their plasma membranes do not touch. For direct intercellular communication plant cells use plasmodesmata (PD), thin cytoplasmic bridges that span the intervening cell wall material. Microscopy as well as measurements of the size exclusion limit (SEL) predict that only small molecules up to 1 kDa can effectively pass through PD during normal plant growth. However, many plant viruses use PD as their major route of passage during infection. This latter observation implies that PD also may transport endogenous large plant molecules, such as proteins, during plant development and differentiation. Thus, the perturbation of PD structure and function by plant viruses is a useful model system to understand plant cell intercellular communication via PD. The tobacco mosaic virus (TMV) movement protein (TMV-MP) potentiates movement of the viral nucleic acid through PD. To do this TMV-MP 1) binds single strand nucleic acids and shapes them into a thin protein-nucleic acid complexes compatible with the narrow dimensions of PD, 2) increases the SEL of PD, and 3) itself likely moves through PD.The proposed research will further utilize the TMV-MP paradigm to investigate the dynamics of PD transport to characterize: 1) PD transport of proteins and protein-nucleic acid complexes; and 2) cytoplasmic transport to PD, focused on the interaction between actin and TMV-MP.
The first aim represents a continuation of ongoing studies. The rationale for the second aim derives from the simple fact that TMV-MP (or its complexes with nucleic acids) must move from their site(s) of synthesis in the cytoplasm to PD for subsequent transport. While the cytoskeleton is emerging as a major track for subcellular transport of macromolecules and organelles, its role in transport to and through PD has not been examined. Since actin is a major component of the cytoskeleton, and preliminary results support an interaction between actin and TMV-MP, the role of actin in cytoplasmic transport to PD will be assessed.
The first aim will be primarily approached using microinjection and high resolution fluorescence microscopy. Biochemically pure TMV-MP (labeled or coinjected with fluorescent dyes) will be directly microinjected into plant cells to determine: 1) the kinetics of transport through PD, 2) the mechanics of TMV-MP mediated movement of nucleic acids through PD, 3) factors that enhance or inhibit PD transport, and 4) the domain(s) of TMV- MP responsible for targeting to, gating of, and movement through PD. Also, TMV-MP (or its domains) will be used as probes in biochemical studies to isolate and characterize components of PD.
The second aim primarily will use actin affinity chromatography in vitro, and immunofluorescence microscopy of TMV-MP and actin in vivo.
Both aims represent new and unstudied areas of investigation that should provide fundamental information critical to understanding intra- and intercellular communication in plants. These studies also should have general relevance to signal and communication pathways in non plant, medically important, systems.
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