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

近年来，对病毒基因组结构及其表达策略的研究取得了显著进展。然而，关于病毒基因组在宿主细胞中复制的信息仍然不完整。在本研究中，我们利用电子显微镜水平上的原位杂交技术检测烟草花叶病毒(TMV)和马铃薯X病毒(PVX)的RNA分子在烟草原生质体中的细胞内分布，以获得更好地确定病毒基因组复制机制的方法。电子显微镜水平上的原位杂交可以用两种不同的方法进行：包埋前和包埋后。然而，这些方法并没有在感染病毒的植物细胞中建立。为此，我们首先确定了光学技术条件，并获得了如下最佳结果。感染烟草花叶病毒或PVX的原生质体在4ﾟC的4%多聚甲醛+0.5%戊二醛中固定2小时，包埋在Lowicryl K4M中，在紫外光照射下进行聚合反应。制备超薄切片，与地高辛标记的核酸探针在45ﾟC过夜杂交，分别用羊抗地高辛抗体和兔抗羊免疫球蛋白抗体与胶体金标记的抗体进行杂交检测。在感染TMV的原生质体中，杂交信号定位于细胞质中电子密度稍高的小区域。在其他细胞器上没有观察到标记。在感染PVX的原生质体中，金标记也定位于细胞质中的小区域。在未来的工作中，将有可能同时获得病毒RNA的原位杂交和病毒蛋白的免疫金检测，并确定病毒复制的位置。

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.