Characterization of Viruses in Phloem by Correlative X-Ray Microtomography (μCT)-Volume Electron Microscopy (vEM) Imaging.

Abstract

Studying virus-infected phloem is of significant importance, as it not only enhances our understanding of viral pathogenesis but also leverages viruses as tools to expand knowledge about plant phloem physiology. The uneven distribution pattern of phloem-infecting viruses poses methodological challenges for such studies-requiring both large field of view (FOV) and high-resolution imaging. A comprehensive anatomical analysis of the phloem necessitates global visualization, while resolving viral structures demands local high-resolution observation. This chapter describes a method, the X-ray microtomography (μCT)-volume electron microscopy (vEM) correlative imaging technique, which effectively addresses these methodological requirements, where μCT provides the large FOV for identification of regions of interest, followed by vEM acquisition of high-resolution images. It is a six-step protocol, including: (1) sample preparation, (2) flaw detection, (3) overview imaging by μCT, (4) identifying viral infection regions, (5) high-resolution imaging by vEM, and (6) image processing and analysis. In this workflow, the steps of sample preparation and identification of viral infection regions are critical. This protocol was originally established for investigating Southern rice black-streaked dwarf virus (SRBSDV) infection in rice phloem, with parameters optimized for plant reoviruses. We provide advice on how to adapt the approach for studying other viral infections.

Plant phenotyping relevance

植物のウイルス感染部位と師部構造をμCT・vEM相関イメージングで取得・解析する6段階プロトコルが中心であり、植物状態の画像ベース計測法に該当する。

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