A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection. Botrytis cinerea is a notorious plant pathogen that can only be effectively controlled by chemical fungicides. With the aim to reduce fungicide application, new control strategies are in need. Cross-kingdom RNA interference (RNAi) is an emerging field in plant-pathogen research. Uncovering the key factors to understand the molecular mechanisms and functions in cross-kingdom RNAi is fundamental to develop innovative RNA-based strategies for crop protection. B. cinerea produces extracellular small RNAs to induce natural cross-kingdom RNAi in plant hosts for infection. In this study, we describe the B. cinerea BcRDR1 as a novel pathogenicity factor that is required for small RNA production and cross-kingdom RNAi. Establishing an advanced GFP-based switch-on reporter expressed transgenic plants allowed us to visualize and record the dynamics of cross-kingdom RNAi during infection. We transformed this newly acquired information to design pathogen small RNA-specific target mimics, so-called short tandem target mimics, to express in transgenic plants, which led to reduced disease severity. Our study not only expanded the knowledge of the molecular functions in cross-kingdom RNAi, but also showcased how such gained knowledge can be instrumental to interfere with pathogen cross-kingdom RNAi for a better pathogen control.