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1996 Fiscal Year Final Research Report Summary

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

Research Project

Project/Area Number 07660054
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 植物保護
Research InstitutionTokyo University of Agriculture and Technology

Principal Investigator

HOSOKAWA Daijiro  Tokyo University of Agriculture and Technology, Faculty of Agriculture, Prf., 農学部, 教授 (50014957)

Project Period (FY) 1995 – 1996
Keywordstobacco mosaic virus / potato virus X / antibody-colloidal gold / In situ hybridization / electron microscopy / digoxigenin-labelled riboprobe
Research Abstract

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.

  • Research Products

    (2 results)

All Other

All Publications (2 results)

  • [Publications] 張 飛雲・渡辺真理子・細川大二郎: "電子顕微鏡オートラジオグラフィー法によるタバコプロトプラストにおけるタバコモザイクウイルスのRNA複製部位の観察" 平成9年度 日本植物病理学会大会. (発表予定).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] ZHANG,F.Y., M.WATANABE and D.HOSOKAWA: "Electron microscopic autoradiography of tobacco protoplasts infected with tobacco mosaic virus" Annals of the Phytopathological Society of Japan. (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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