Tobacco Mosaic Virus Coat Protein and Reporter Gene Transcripts Containing the TMV Origin-Of-Assembly Sequence Do Not Interact in Double-Transgenic Tobacco Plants: Implications for Coat Protein-Mediated Protection. Jane K. Osbourn. Department of Virus Research, John Innes Institute and AFRC Institute of Plant Science Research, Norwich NR4 7UH, U.K.. Kitty A. Plaskitt(2), John W. Watts(2), and T. Michael A. Wilson(1). Departments of (1)Virus Research and (2)Cell Biology, John Innes Institute and AFRC Institute of Plant Science Research, Norwich NR4 7UH, U.K. . MPMI 2:340-345. Accepted 20 July 1989. Copyright 1989 The American Phytopathological Society.

The ability of tobacco mosaic virus (TMV) coat protein (CP), expressed in transgenic tobacco plants, to encapsidate RNA was measured by introducing a second reporter gene whose transcripts contained a copy of the TMV origin-of-assembly sequence (OAS). Electrofusion of transgenic mesophyll protoplasts or cross-pollination of singly transformed plants were used to create heterokaryons or plants bearing both transgenes in which complementation between TMV CP and the OAS⁺ mRNAs could be assessed. Electron microscopy of immune-trapped protoplast or leaf extracts and assays for reporter enzyme activity showed that the endogenous CP was present in insufficient quantities, or was unavailable, to encapsidate significant amounts of OAS⁺ mRNA and thereby to affect its expression. Immunogold labeling of fixed, thin cryosections of young leaves from the homozygous CP-transgenic parent suggested that the endogenous CP was located almost exclusively in chloroplast-associated electron-dense bodies. Double-transgenic tobacco plants retained the ability to resist infection by TMV, measured by the delayed appearance of symptoms and the absence of progeny virions in samples of leaf sap. We conclude that substantial reencapsidation of uncoated RNA from challenge virus by endogenous CP in the cytoplasm of TMV CP-transgenic plants is not likely to account for “CP-mediated protection” (P. Powell Abel, R. S. Nelson, B. De, N. Hoffmann, S. G. Rogers, R. T. Fraley, and R. N. Beachy, Science 232:738-743, 1986).