Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis.
Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis.
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DOI：
10.21769/bioprotoc.3911
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发表时间：
2021-02
期刊：
影响因子：
0.8
通讯作者：
Florian Dunker;Bernhard Lederer;Arne Weiberg
中科院分区：
文献类型：
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作者：
Florian Dunker;Bernhard Lederer;Arne Weiberg
在过去的十年中，已经注意到微生物病原体和害虫将小RNA（sRNA）效应物递送到它们的宿主植物中以操纵植物生理和免疫以进行感染，称为跨界RNA干扰。在这个过程中，真菌和卵菌寄生虫sRNA劫持植物ARGONAUTE（AGO）/RNA诱导沉默复合物，使宿主靶基因转录后沉默。我们在此描述了我们如何在其宿主植物拟南芥感染期间回收卵菌病原体阿拉伯透明霜霉的跨界sRNA效应子的方法学细节。本生物实验方案包括两个部分：第一，详细描述了Illumina高通量测序分析中植物AGO/sRNA共免疫纯化和sRNA回收的过程。其次，我们解释了如何使用Galaxy服务器对sRNA序列进行生物信息学分析。原则上，该方案适用于研究来自不同宿主植物和植物相互作用（微生物）生物体的AGO结合的sRNA。
Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.