Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein–sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

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