The Sainsbury Laboratory, Norwich Research Park, University of East Anglia, Norwich, United Kingdom.
Curr Protoc. 2022 Aug;2(8):e523. doi: 10.1002/cpz1.523.
The blast fungus, Magnaporthe oryzae, is a devastating plant pathogen that threatens global food security. The social and economic importance of blast disease has contributed to this filamentous fungus becoming a model organism for the study of host-pathogen interactions. Availability of the complete genome sequences of many strains of the pathogen, as well as rice and wheat cultivars, coupled with the tractability of M. oryzae to classical and molecular genetic manipulation have contributed to its widespread study. Although M. oryzae has been extensively investigated for the past two decades, procedures for storing, maintaining, and manipulating the blast fungus in the laboratory had not been compiled and updated. As a consequence, there is considerable disparity in how the fungus is stored and manipulated between studies. In this article, we present a collection of protocols providing clear explanations of how to preserve filter stocks of M. oryzae; how to grow the fungus in both liquid and solid media; how to extract genomic DNA from fungal mycelium; how to induce appressorium formation on coverslips for visualization and tissue collection; and how to perform two distinct types of plant infection assay for virulence assessment. By sharing our most used laboratory procedures, we aim to address some of the knowledge gaps in current M. oryzae protocols and contribute to uniformity and robustness in studies by the Magnaporthe research community. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Storage of M. oryzae strains Basic Protocol 2: Revival and regular maintenance of M. oryzae cultures in solid medium Alternate Protocol 1: Regular maintenance of M. oryzae cultures in liquid medium Basic Protocol 3: Genomic DNA extraction from M. oryzae mycelium Alternate Protocol 2: Quick DNA extraction from M. oryzae mycelium Basic Protocol 4: M. oryzae induction of appressorium development on glass coverslips for microscopy Alternate Protocol 3: M. oryzae induction of appressorium development on glass coverslips for tissue collection Basic Protocol 5: M. oryzae rice infection assay through spray inoculation Alternate Protocol 4: M. oryzae leaf-drop plant infection assay.
稻瘟病菌，稻瘟霉，是一种毁灭性的植物病原体，威胁着全球粮食安全。该丝状真菌已成为研究宿主-病原体相互作用的模式生物，这归因于稻瘟病的社会和经济重要性。许多病原体菌株、水稻和小麦品种的全基因组序列的可用性，以及稻瘟霉对经典和分子遗传操作的易处理性，都促进了对其的广泛研究。尽管过去二十年对稻瘟霉进行了广泛的研究，但尚未编写和更新用于在实验室中储存、维持和操作该真菌的程序。因此，在不同的研究中，真菌的储存和操作方式存在相当大的差异。在本文中，我们提供了一系列协议，这些协议提供了清晰的解释，说明如何保存稻瘟霉的过滤库存；如何在液体和固体培养基中培养真菌；如何从真菌菌丝体中提取基因组 DNA；如何在载玻片上诱导附着胞形成以进行可视化和组织收集；以及如何进行两种不同类型的植物侵染测定以评估毒力。通过分享我们最常用的实验室程序，我们旨在解决当前稻瘟霉方案中的一些知识空白，并为稻瘟霉研究界的研究提供一致性和稳健性。 © 2022 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：稻瘟霉菌株的储存基本方案 2：在固体培养基中恢复和定期维持稻瘟霉培养物的方法备选方案 1：在液体培养基中定期维持稻瘟霉培养物基本方案 3：从稻瘟霉菌丝体中提取基因组 DNA备选方案 2：从稻瘟霉菌丝体中快速提取 DNA 基本方案 4：在玻璃载玻片上诱导稻瘟霉附着胞发育以进行显微镜观察备选方案 3：在玻璃载玻片上诱导稻瘟霉附着胞发育以进行组织收集基本方案 5：通过喷雾接种进行稻瘟霉水稻感染测定备选方案 4：稻瘟霉叶片坠落植物感染测定。
