Studies on the multiplication mechanism of plant viruses by electron microscopic in situ hybridization

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In recent years, remarkable progress has been made in the understanding of viral genome structures and their expression strategy. However, information concerning the replication of viral genome in host cells is still fragmentary. In this study, we thus examine the intracellular distribution of RNA molecules of tobacco mosaic virus (TMV) and potato virus X (PVX) in tobacco protoplasts using in situ hybridization at the electron microscopic level to gain the beter approach to define the mechanism of repllication of viral genome.In situ hybridization at the electron microscopic level can be carried out using two different methohs : Pre-embedding method and post-embedding method. However, these methods were not established in plant cells infected with viruses. Therefore, we first determind the optical technical conditions and obtained the best result as follow. The protoplasts infected with TMV or PVX were fixed in 4% paraformaldehyde plus 0.5% glutaraldehyde for 2 hr at 4ﾟC and embedded in Lowicryl K4M.Polymerization of Lowicryl K4M was carried out with irradiation by UV light. Ultrathin sections were prepared and hybridized with digoxigenin-labelled RNA probes for over night at 45ﾟC.The detection of hybrids was carried out using successively sheep anti-digoxigenin antibody and rabbit anti-sheep IgG antibody conjugated to colloidal gold. In protoplasts infected with TMV,hybridization signals were localized over the slightly electron-dense small areas of the cytoplasm. No labelling was observed on other organelles. In protoplasts infected with PVX,gold labelling were also localized over the small areas in the cytoplasm. However, specific structures were not found in these areas.In future work, it should be possible to obtain simultanous in situ, hybridization of viral RNA and immunogold detection of viral proteins and to define a site of virus replication.