MCB-9418700 Abstract How plant viruses move from the initially inoculated cell to neighboring cells and eventually systemically to cause disease is not understood. Red clover necrotic mosaic virus (RCNMV) has been developed as a model system to study plant virus movement. To understand the mechanisms of both cell-to-cell and long distance movement, a series of genetic, biochemical and cell biology experiments are designed to address the following questions: How does the movement protein (MP) facilitate viral spread from cell to cell? Where within the cell does the MP appear to act? Can fuctional domains be distinguished in the MP? What are the roles of the MP and capsid protein (CP) in long distance movement of the virus? I s virion formation requried for long distance movement? Do the movement and capsid protiens interact with one another to achieve long distance movement? What if any, host factors interact with the MP and/or CP to cause a systemic infection? The approaches to answer these questions are outlined in the following three objectives: 1) Generate and characterize alanine scanning mutants of the RCNMV CP. 2) Analyze wild-type and mutant RCNMV MP behavior in transgenic plants. 3) Identify host factors that facilitate viral cell-to-cell and/or long distance movement and characterize the interactions between MP, CP, and host factors. A series of CP alanine scanning mutants will be constructed and analyzed for CP expression, virion formation, and their ability to facilitate long distance movement. Transgenic plants will be generated expressing the wild-type and mutant MPs. These will be used to determine the subcellular localization of MPs as well as for complementation experiments. Wild-type and mutant CPs and MPs over-produced in E.coli will be microinjected into various plant cell types and assayed for the ability to modify plasmodesmata and transport macromoleucles from one cell type to another. The hypothesized interactions betw een MP and CP will be assessed genetically, biochemically, in gel shift assays, by the yeast two hybrid system, and/or by mutational analysis. cDNA expression libraries from RCNMV systemic host plants will be screened for a host factor(s) that specifically interacts with the MP using the yeast two hybrid system, lgt11 colony hybridization, or affinity chromatography. Results from the proposed expeirments will afford a better understanding of the mechanisms by which plant viruses move and ultimately cause disease. They will also serve as a basis for studying how plant viruses usurp normal host functions to achieve infection and how plants communicate from cell to cell. The identification of host factors involved in viral movement will provide insights into protein-protein interactions, host specificity, and the structure and function of plasmodesmata. %%% This proposal seeks to understand how plant RNA viruses move from cell to cell. There are several players in the process, a protein called movement protein (MP) , the protein which comprises the coat of the virus to make up the capsid (CP), and the exquisitely organized channels between plant cells which traverse the cell wall of adjacent cells, the plasmodesmata. Previous work in the Lommel lab has shown that changes in the amino acid sequence (through site- directed mutagenesis) of the movement protein incapacitate viral spread from cell to cell. It has also been shown that CP mutations change the ability of the virus to move over long distances, e.g., from leaf to leaf. There is a postulated interaction of CP with MP and the nature of that interaction will now be explored through controlled mutagenesis of CP, followed by analyzing cell to cell movement and by determining the nature of the interaction of CP with mutant and wild type MP. The nature of the interaction of these proteins with host proteins will also be determined, since it is hypothesized that some host protein is involved in lo ng distance transport. The nature of cell-to-cell transport in plants is one of the main questions confronting plant cell biologists currently and typifies the field in that incremental increases in our basic knowledge will lead to potentially large pay-offs in both fundamental (plasmodesmatal action) and applied (plant disease resistance) areas. ***
