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The amount of virus in infected plant cells increases exponentially after the eclipse period, and eventually reaches a plateau. The level of virus accumulation at the plateau varies depending on the virus or its strain, the host and environmental conditions. The concentration of tobacco mosaic virus (TMV) in the leaf cells of systemically infected tobacco plants is very high at the plateau and remains at this level for the life of the leaf without marked deleterious effect on the cells. There must therefore be a mechanism by which the upper limit of virus accumulation is controlled, however, this is not yet completely understood4). We investigated using an electron microscope whether other challenge-inoculated viruses are able to multiply in the tobacco leaf cells in which the TMV level has reached a plateau. Nicotiana tabacum cv. Xanthi plants were grown in a glasshouse (25-28•Ž), and their lower, well-expanded leaves were inoculated with the OM strain of TMV when five leaves had appeared. Yellow-greeen areas in the upper non-inoculated leaves showing typical mosaic symptoms 30 days after TMV-inoculation were used for challenge inoculation with other viruses as TMV level in these areas rises to a plateau and remains high for 20 days (unpublished data). These yellow-green areas were challenge-inoculated mechanically with potato virus X (O strain), potato virus Y or cucumber mosaic virus (Y strain). Samples were collected from these areas 4, 6, and 8 days after challenge-inoculation. Comparable samples from single virus-inoculated leaves were also processed. These leaf tissues were cut into small pieces and fixed in 2.5% glutaraldehyde in 0.1M sodium phosphate buffer (pH7.4) and then in 1% OsO4. They were dehydrated in the graded ethanol series and embedded in Epon 812 resin. Thin sections were cut with a glass knife using a Porter-Blum MT-1 ultramicrotome, and stained with uranyl acetate and lead citrate. They were examined by a JEM-100-C electron microscope (JEOL, Ltd.). In parenchymal cells of the yellow green areas of tobacco leaves showing mosaic symptoms infected with TMV alone, TMV particles were observed as one or more large crystalline aggregates in the cytoplasm (Plate I-1). Electron microsocpy of the areas challenge-inoculated with other viruses revealed a multiplication of the challenge virus as follows. Challenge inoculation with PVX: PVX particles could be distinguished from TMV particles in the same cells because the former were undulating randomly and were narrower than the latter. PVX particles were frequently found to accumulate as aggregates between TMV crystalline aggregates and the cytosol, organelles such as the nucleus, chloroplasts, or the tonoplast (Plate I-2, 3, 4). In these areas, profiles of masses of PVX particles seemed to demonstrate that these particles were formed in the cytosol and then issued from there and accumulated in the above areas (Plate I-2, 3, 4). In addition, a characteristic laminated inclusion component (LIC) that is known to be induced by PVX infection5) was frequently observed in the cytoplasm (Plate II-1). As infection progressed, PVX aggregates increased in size as well as inoculation with PVX alone. PVX particles were not found inside the crystalline

nippon shokubutsu byori gakkaiho/nihon shokubutsu byouri gakkaihou

Electron Microscopy of Multiplication and Distribution of Other Challenge-inoculated Viruses in Cells in Which Level of Tobacco Mosaic Virus Reached a Plateau.