Multiple Argonaute complexes orchestrate epigenetic silencing in the Arabidopsis germline

Abstract Small RNAs (sRNAs) are widespread across diverse flowering plant species and play important roles regulating reproduction and environmental responses. However, how sRNAs exert their function is largely unknown, likely due to their typical origin in non-genic regions, the lack of distinct gene targets, and the sparse characterization of their associated Argonaute (AGO) effector proteins. Here we show that Arabidopsis male reproductive sRNAs associate with AGO4, 5, 6 and 9, all of which are enriched in meiocytes along with RNA Polymerase V (Pol V). This enables these sRNAs to methylate loci with imperfect sequence homology. AGO4, AGO6 and AGO9 predominantly load 24-nucleotide (nt) Pol IV-derived sRNAs and mediate DNA methylation in meiocytes in a dosage-dependent manner. Despite closer homology to microRNA-associated AGO1 and AGO10, AGO5 primarily binds CLSY3- and Pol IV-derived 21-24 nt sRNAs. Transcriptomic and methylomic analyses of meiocytes from single and compound ago mutants demonstrate distinct yet complementary roles of AGO4/6/9 and AGO5 in regulating protein-coding genes and silencing transposons in the germline. These molecular disruptions are accompanied by significant meiotic defects. Together, our findings reveal how AGO4/6/9-mediated DNA methylation and AGO5, likely acting through post-transcriptional gene silencing, work together to promote male germline development.