Immunogold Labeling Locates the Site of Disassembly and Transient Gene Expression of Tobacco Mosaic Virus-Like Pseudovirus Particles In Vivo. Kitty A. Plaskitt. Department of Cell Biology, John Innes Institute, AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, England. Peter A. C. Watkins(2), David E. Sleat(2), Daniel R. Gallie(2), John G. Shaw(3), and T. Michael A. Wilson(2). Departments of (1) Cell Biology and (2) Virus Research, John Innes Institute, AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, England, and (3) Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546, U.S.A.. MPMI 1:10-16. Accepted 15 July 1987. Copyright 1987 The American Phytopathological Society.

Recombinant in vitro transcripts with a 5’ open reading frame for bacterial chloramphenicol acetyltransferase (CAT) and a 3'-proximal copy of the origin-of-assembly sequence from tobacco mosaic virus (TMV) RNA can be assembled in vitro into ribonucleoprotein particles (“pseudoviruses”) resembling TMV. When rubbed on the lower surfaces of leaves of Nicotiana tabacum cv. Xanthi, both these pseudoviruses and true TMV particles were located predominantly within the cytoplasm of the epidermal cells. Cells in mock (buffer)- inoculated leaf panels, excised and fixed immediately or after 15 or 120 min, showed little or no gold-labeling when probed with polyclonal rabbit antisera to TMV coat protein, CAT, or the 126,000 Da (126 kDa) polypeptide encoded by TMV RNA. In contrast, by 15 min, the cytoplasm of epidermal cells from TMV-inoculated (control) leaf panels was strongly labeled by serum against the 126 kDa protein but not by CAT antiserum. Conversely, sections from CAT pseudovirus-inoculated leaf panels, left for 15 or 120 min, revealed particles distributed throughout the epidermal cell cytoplasm, specifically labeled by CAT antiserum. Intracellular TMV or pseudovirus particles, and those attached end-on to the outer (cuticular) surface of the cell, could be labeled with anti-TMV coat protein antiserum. At each of the three sampling times, abrasive wounds were visible in the outer walls of some epidermal cells. Clumps of TMV or pseudovirus particles had entered the cell via these wounds and, by 15 min or less, were dispersed throughout the cytoplasm. Cells in which the clumps failed to disperse, or cells that were irreparably wounded, showed little gold-labeling. Translationally active TMV or CAT mRNA remained associated with coat protein. We propose that these gold-labeled, cytoplasmic complexes may be the so-called striposomes, which can also be labeled with L-[³⁵S]methionine and isolated from epidermal cells by Cs₂SO₄ gradient centrifugation (J. G. Shaw, K. A. Plaskitt, and T. M. A. Wilson, Virology 148:326-336, 1986).