Cell biological studies on mechanisms of cell-to-cell movement of plant viruses

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Cell-to-cell movement of tobacco mosaic virus (TMV) and potato virus X (PVX) were investigated using methods of molecular cell biology. The results are as follows.1. The effects of deletion mutants in 30 k protein (movement protein) gene of TMV on cell-to-cell movement and localization of movement protein in tobacco protoplasts has been investigated. Cell-to-cell movement and localization of 30 k protein in protoplasts of 3 C-terminal amino acids deletion mutant indistiguishable from those of wild-type clone. When 33 C-terminal amino acids were deleted, virus moved slowly and lesser amounts of 30 k protein were found in the cytoplasms of protoplasts. Deletion of 77 C-terminal amino acids and residues from 9 to 142 at N-terminal abolish cell-to-cell movement and 30 k protein are not found in protoplasts, though virus particles aboundantly accumulated in protoplasts.2. We have developed a in situ hybridization method using digoxygenin labelled-RNA probes to monitor the localization of TMV-RNA in tobacco plants and protoplasts. We are carring out studies to investigate behavior of virus RNA during cell-to-cell movement of TMV using this in situ hybridization method.3. The 25 k protein which is part of the triple gene block of PVX are involved in cell-to-cell movement, In this experiment, we have used an E.coli expression system to produce significant amounts of 25 k protein. A polyclonal antiserum were produced using purified 25 k protein. An immunocytochemical analysis of infected tobacco leaves reveals the 25 k protein is exclusively associated with cytoplasmic inclusions in the cytoplasm of infected cells. These results indicate that the triple gene block proteins of PVX constitute a new class of movement protein.