Plant viruses, during the infection process, face a unique challenge: to move from infected into adjoining healthy cells they must cross a seemingly impregnable barrier - the plant cell wall. Cell walls make it impossible for the virus to enter its host cells by membrane fusion or endocytotic pathways, the mechanisms common for infection by animal viruses. To move between cells, therefore, plant viruses presumably take advantage of natural plant intercellular connections, the plasmodesmata (PD). In recent years evidence has accumulated to suggest that a specific virus-encoded product is involved in the movement process. In the case of tobacco mosaic virus (TMV), it has been shown that the viral P30 protein is responsible for the movement function. Cell-to-cell transport of TMV RNA by the P30 protein will be used as an experimental system to study general mechanisms of PD permeability and function in transmembrane transport of nucleic acids. Three immediate goals will be pursued in the present proposal: (i) development of experimental systems to follow interaction of TMV P30 with PD; (ii) use of purified P30 as a specific probe to isolate and clone PD proteins, and (iii) study of the molecular mechanism(s) of P30 interaction with PD and its role in plant resistance to systemic viral infection. Interaction between P30 and PD will be monitored by three different experimental approaches: (i) direct monitoring of the effect of P30 on cell-to-cell transfer of fluorescent dyes and fluorescein-conjugated proteins co-injected into plant tissues which grow as a filament of single cells (cultured root cells, stamen hairs, etc.); (ii) in vivo cross-linking of P30 to its putative PD receptors in TMV infected plant tissue; and (iii) binding of PD proteins to P30 immobilized on affinity columns. These experimental systems will serve to identify a punitive PD localization signal of P30 that will be useful as a probe to purify the PD proteins. Isolated PD and P30 proteins then will be used to examine the detailed mechanism of their interaction. For example, the potential ability of P30 to modify PD permeability by phosphorylating their protein subunits will be tested. Purified PD proteins will be used to clone their encoding genes. This will be achieved by microsequencing the purified protein and using the complementary oligonucleotides to screen plant cDNA libraries. Alternatively, plant cDNA expression libraries will be screened with antibodies to the purified PD. Isolation of PD and cloning of their genes will lay a foundation for future study of PD physiology and their role in plant development. Also, experimental approaches developed to study cell-- to-cell movement of plant viruses may be used to search for analogous (or homologous) viral-movement trough gap junctions in animal cells.
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