Argonaute (AGO) proteins bind to small RNAs to induce RNA interference (RNAi), a conserved gene regulatory mechanism in animal, plant, and fungal kingdoms. Small RNAs of the fungal plant pathogen Botrytis cinerea were previously shown to translocate into plant cells and to bind to the host AGO, which induced cross-kingdom RNAi to promote infection. However, the role of pathogen AGOs during host infection stayed elusive. In this study, we revealed that members of fungal plant pathogen B. cinerea BcAGO family contribute to plant infection. BcAGO1 binds to both fungal and plant small RNAs during infection and acts in bidirectional cross-kingdom RNAi, from fungus to plant and vice versa. BcAGO2 also binds fungal and plant small RNAs but acts independent from BcAGO1 by regulating distinct genes. Nevertheless, BcAGO2 is important for infection, as it is required for effective pathogen small RNA delivery into host cells and fungal induced cross-kingdom RNAi. Providing these mechanistic insights of pathogen AGOs promises to improve RNAi-based crop protection strategies.
Cross-kingdom RNA interference (RNAi) is an emerging field in plant-microbe research, but the underlying biological processes are barely understood. Argonaute (AGO) proteins are small RNA binding proteins that are key factors in RNAi by inducing gene silencing. The functional role of AGOs in RNA silencing is highly conserved from fungi to plants and humans. In this study, we demonstrate that different members of the AGO family in the fungal plant pathogen Botrytis cinerea act in bidirectional cross-kingdom RNAi during infection of host plants, such as tomato. Uncovering the critical role of fungal AGOs during plant infection and their underlying molecular mechanisms open new avenues to develop novel RNA-based strategies to control pathogens in agricultural relevant crops.