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

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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.