The role of fungal Argonautes and other types of RNA-binding proteins in Botrytis-plant cross-kingdom RNAi

灰葡萄孢（Botrytis cinerea）是一种侵袭性植物病原真菌。对于成功感染，B。灰霉病菌分泌跨界小RNA（ck-sRNA）进入植物细胞，劫持植物Argonaute/RNA诱导沉默复合物（AGO/RISC）以抑制重要的宿主防御基因。这种迷人的毒力策略被称为跨王国RNA干扰（ck-RNAi）。拟南芥作为灰霉病菌的寄主植物，也将其ck-sRNA转移到真菌中以抑制病原基因。尽管宿主诱导的基因沉默和ck-sRNA运输的潜在机制在植物保护方面具有很大的潜力，但不幸的是，它们在很大程度上是未知的。通过这个子项目，我们建议阐明Botrytis BcAGO和其他类型的RNA结合蛋白（RBP）在双向植物-真菌ck-RNAi中的作用。i）我们的目的是鉴定与BcAGO结合并指导宿主诱导的基因沉默的番茄ck-sRNA。我们将对番茄ck-sRNAs的主要病原体靶基因进行表征，这将为宿主诱导的基因沉默机制和有希望的真菌靶基因提供信息，以改善植物保护。ii）我们建议研究BcAGO在细胞外RNA转运的ck-sRNA分选中的作用。在植物和动物中，AGO蛋白与内质网和多泡体的内源性膜结构相关。我们的目的是阐明BcAGO与内源性分泌途径的相互联系。这将拓宽我们对ck-sRNA的分选和分泌机制的理解。此外，细胞外囊泡（EV）被认为是介导RNA运输的植物-真菌相互作用。我们的实验室最近发现，葡萄孢EV含有ck-sRNA和推定的RBP。iii）我们建议研究EV-RBP在ck-sRNA转运过程中的作用和功能。这些信息对于实施现有的RNA介导的植物保护策略以提高效率是非常有价值的，这是RU 5116的主要目标。该子项目获得的知识与RU 5116的其他项目具有高度互补性，不仅提供了关于ckRNAi机制的新信息，而且还提供了可转化为创新的基于RNA的植物保护策略的有价值信息。

Botrytis cinerea is an aggressive fungal plant pathogen. For successful infection, B. cinerea secretes cross-kingdom small RNAs (ck-sRNAs) into plant cells that hijack the plant Argonaute/RNA-induced silencing complex (AGO/RISC) to suppress important host defence genes. This fascinating virulence strategy is called cross-kingdom RNA interference (ck-RNAi). Arabidopsis thaliana, host plant of Botrytis, transfers its ck-sRNAs into the fungus to suppress pathogen genes, too. Although the underlying mechanisms of host-induced gene silencing and ck-sRNA transport bear great potential for plant protection purposes, they are unfortunately, largely unknown. With this subproject, we propose to shed light on the roles of the Botrytis BcAGOs and other types of RNA-binding proteins (RBPs) in bidirectional plant-fungal ck-RNAi. i) We aim to identify tomato ck-sRNAs that bind to BcAGOs and direct host-induced gene silencing. We will characterize main pathogen target genes of tomato ck-sRNAs, which will inform on the host-induced gene silencing mechanism and on promising fungal target genes to improve plant protection. ii) We propose to investigate the role BcAGOs in sorting ck-sRNAs for extracellular RNA transport. In plants and animals, AGO proteins associate with endogenous membrane structures of the Endoplasmic Reticulum and multivesicular bodies. We aim to elucidate the inter-connection of BcAGOs with endogenous secretory pathways. This will broaden our understanding of the sorting and secretion mechanism of ck-sRNAs. Moreover, extracellular vesicles (EVs) are suggested to mediate RNA transport in plant-fungal interaction. Our laboratory recently discovered that Botrytis EVs contain ck-sRNAs and putative RBPs. iii) We propose to investigate the role and function of EV-RBPs in the ck-sRNA transport process. This information is highly valuable for implementation into existing RNA-mediated plant protection strategies to improve efficiency, a major goal of the RU5116. Knowledge gained by this subproject is highly complementary to the other projects of the RU5116, not only by providing new information on the ckRNAi mechanism, but also by delivering valuable information that can be translated into innovative RNA-based plant protection strategies.