Department of Molecular Life Sciences, Technical University of Munich, Freising, Germany.
Department of General Genetics, Center for Plant Molecular Biology, Eberhard-Karls-University Tübingen, Tübingen, Germany.
Nat Protoc. 2023 Nov;18(11):3173-3193. doi: 10.1038/s41596-023-00879-8. Epub 2023 Sep 11.
Type-III effector proteins are major virulence determinants that most gram-negative bacteria inject into host cells to manipulate cellular processes for infection. Because effector-targeted cells are embedded and underrepresented in infected plant tissues, it is technically challenging to isolate them for focused studies of effector-induced cellular changes. This protocol describes a novel technique, effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), for isolating biotin-labeled nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors by using streptavidin-coated magnetic beads. This protocol is an extension of the existing Nature Protocols Protocol of the INTACT method for the affinity-based purification of nuclei of specific cell types in the context of developmental biology. In a phytopathology scenario, our protocol addresses how to obtain eINTACT transgenic lines and compatible bacterial mutants, verify the eINTACT system and purify nuclei of bacterial effector-recipient cells from infected tissues. Differential analyses of purified nuclei from plants infected by bacteria expressing the effector of interest and those from plants infected by effector-deletion bacterial mutants will reveal the effector-dependent nuclear changes in targeted host cells. Provided that the eINTACT system is available, the infection experiment takes 5 d, and the procedures, from collecting bacteria-infected leaves to obtaining nuclei of effector-targeted cells, can be completed in 4 h. eINTACT is a unique method for isolating high-quality nuclei from bacterial effector-targeted host cells in native infection contexts. This method is adaptable to study the functions of type-III effectors from numerous gram-negative bacteria in host plants that are amenable to transformation.
III 型效应蛋白是大多数革兰氏阴性细菌将其注入宿主细胞以操纵感染过程中细胞过程的主要毒力决定因素。由于效应器靶向细胞嵌入在感染植物组织中且代表性不足,因此从技术上讲,很难将其分离出来,以对效应器诱导的细胞变化进行集中研究。本方案描述了一种新的技术,即效应诱导的特定细胞类型标记核的分离(eINTACT),用于通过链霉亲和素包被的磁珠从已接收黄单胞菌细菌效应器的拟南芥植物细胞中分离生物素标记的核。该方案是现有 INTACT 方法的扩展,该方法用于在发育生物学背景下亲和纯化特定细胞类型的核。在植物病理学情况下,我们的方案解决了如何获得 eINTACT 转基因系和相容的细菌突变体,验证 eINTACT 系统以及从感染组织中纯化细菌效应器受体细胞的核。用表达感兴趣的效应器的细菌感染的植物和用缺失效应器的细菌突变体感染的植物的纯化核的差异分析将揭示靶宿主细胞中依赖效应器的核变化。假设 eINTACT 系统可用,则感染实验需要 5 天,并且从收集细菌感染的叶子到获得效应器靶向细胞的核,整个过程可以在 4 小时内完成。eINTACT 是一种从天然感染环境中的细菌效应器靶向宿主细胞中分离高质量核的独特方法。该方法适用于研究可转化的宿主植物中许多革兰氏阴性细菌的 III 型效应子的功能。