In 1986 this laboratory reported (Science 232:738-743) that transgenic tobacco plants that express a gene encoding the capsid protein (CP) of tobacco mosaic virus (TMV) are substantially protected against infection by TMV. This type of resistance, """"""""coat-protein mediated resistance (CP-MR)"""""""", has been used to develop resistance against many different types of viruses in a number of types of plants. Although the successes of CP-MR are well known, and advanced stages of testing and evaluation of transgenic plant lines are in progress, the cellular and molecular mechanisms of resistance are not yet known. The system best-suited for in-depth study is TMV and transgenic tobacco and tomato plants. Published information of the crystal structure of TMV-CP and x-ray fiber diffraction studies of TMV will be used to create mutant proteins to investigate the role of protein structure, protein:protein, and protein:RNA interactions on CP-MR. The effects of wild-type and mutant CPs on virus uncoating, establishment of infection, and local and systemic spread of virus in transgenic plants and synchronously infected protoplasts will be determined. To study early events in virus uncoating in CP-MR an epitope-tagging approach will be taken to follow virus disassembly and protein exchange reactions. To monitor localized spread of the infection in CP-MR, virus replication will be followed in real time by imaging and recording the expression of a luciferase gene that has been incorporated into the virus. To explore the role that expression of the TMV-CP gene in specific cell types has on resistance, transgenic plant lines will be developed that express the CP gene under control of inducible, tightly regulated promoters. Promoters controlled by wounding or added reagents and promoters expressed specifically in epidermal and vascular xylem cells, vascular phloem cells, and in chloroplast containing cells will be used singly or in combination to examine the role of CP in various aspects of virus infection and disease development. Interfacing structural chemistry, protein modeling, molecular genetics, cell biology, and virology as described here represents a novel approach to studies of viral pathogenesis. It will also lead to better understanding of CP-MR and further develop transgenic resistance for use in food production to decrease the often severe virus infections that limit food availability and exacerbate hunger in many parts of the world.
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