Dunker Florian, Lederer Bernhard, Weiberg Arne
Faculty of Biology, Genetics, Biocenter Martinsried, LMU Munich, Großhaderner Str. 2-4, 82152 Planegg-Martinsried, Germany.
Bio Protoc. 2021 Feb 5;11(3):e3911. doi: 10.21769/BioProtoc.3911.
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 during infection of its host plant . 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.
在过去十年中，人们注意到微生物病原体和害虫会将小RNA（sRNA）效应子传递到宿主植物中，以操纵植物生理和免疫来实现感染，这被称为跨界RNA干扰。在这个过程中，真菌和卵菌寄生虫的sRNA劫持植物AGO（AGO）/RNA诱导沉默复合体，在转录后沉默宿主靶基因。我们在此描述了在卵菌病原体感染宿主植物过程中，我们如何回收跨界sRNA效应子的方法细节。本生物协议包含两个部分：第一，详细描述植物AGO/sRNA共免疫纯化及用于Illumina高通量测序分析的sRNA回收过程。第二，我们解释如何使用Galaxy服务器对sRNA序列读数进行生物信息学分析。原则上，该协议适用于研究来自不同宿主植物和与植物相互作用的（微）生物的AGO结合sRNA。
