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Poster

Session: Session 1 Presenting Authors at Posters (Evens)

P-236 - Correlative imaging of viral replicase to identify viral replication complexes at the ultrastructural level in plant cells

Tuesday, July 15, 2025
09:55 - 11:30  
Location: Confex Hall 3
Plant-virus Interactions (incl. Viromes, Evolution & Vectors)

    Presenting Author(s)

  • SK

    Sakugawa Kagari

    Tokyo University of Agriculture and technology
    Fuchu-city, Tokyo, Japan

    • Author(s)

  • TK

    Takuma Kozono

    Smart-Core-Facility Promotion Organization Tokyo University of Agriculture and Technology
    Fuchu, Tokyo, Japan

  • NS

    Nobumitsu SASAKI

    Professor
    Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

  • TA

    Tsutomu Arie

    Professor
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan
    fuchu, Tokyo, Japan

  • KK

    Ken KOMATSU

    Professor
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

Positive-strand plant RNA viruses assemble host membrane-bound viral replication complexes (VRCs) to replicate their genomic RNA. Identifying VRCs at the ultrastructural level remains challenging due to their small size and poorly understood distribution in the broad field of view of infected cells. To determine the localization of VRCs, which are likely associated with replicases, we have recently developed a novel fluorescent imaging system to observe the functional replicase of plantago asiatica mosaic virus (PlAMV) during infection in Nicotiana benthamiana using frankenbody (FB), an antibody fragment that binds to the human influenza hemagglutinin (HA) epitope. mCherry-fused FB visualized the HA-tagged PlAMV replicase as punctate structures associated with the endoplasmic reticulum and plasmodesmata. In this study, to observe these puncta, which may represent VRCs, at the ultrastructural level, we employed correlative light and electron microscopy (CLEM), seamlessly integrating the two imaging modalities. Since the fluorescence of mCherry was significantly weakened after fixation for CLEM, we tested a HaloTag-fused FB (FB-HaloTag), which covalently binds to a photostable dye. The FB-HaloTag system labeled replicase-associated puncta, similar to FB-mCherry. Notably, it exhibited enhanced brightness and resistance to photobleaching during CLEM analysis. Our CLEM method would be a promising tool for visualizing plant-pathogen interactions at the ultrastructural level.