Genetic transformation of plants to constitutively express virus coat protein genes has been used to confer protection against 5 different plant viruses. Recently, we completed the first field tests with tomato plants and documented field level production against tobacco mosaic virus (TMV). However, the cellular and molecular bases for the engineered protection are not known. Recently, we demonstrated that protection probably blocks an early stage in virus infection that prevents uncoating of the virus. A second manifestation of protection is that systemic movement of virus is slowed in these transgenic plants. We have also conclusively demonstrated that the capsid protein (rather than it's mRNA) is responsible for conferring protection (Powell Abel et al., manuscript in preparation). We now propose to carry out in vivo and in vitro experiments to: (1) determine the role of specific amino acid sequences or protein structures in the protection reaction by producing mutants that affect the sequence and length of the coat protein; (2) determine the amino acid sequences (or protein domains) that confer strain specificity to the protection reaction by producing chimeric proteins comprised of segments of two strains that do not cross-protect against each other (i.e., the common strain and legume strains of (TMV); (3) characterize the protection against systemic virus movement and so determine the role of specific amino acid sequences in such protection. These experiments will lead to a more complete understanding of genetically engineered (cross) protection, and enable the formulation of rational approaches to improve and/or extend the protection.
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