Strict recruitment of an RNA-dependent RNA polymerase in plant siRNA amplification

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an ARGONAUTE (AGO) protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNAs and AGO1 triggers siRNA amplification. In this pathway the cleavage fragment is converted into a double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and the dsRNA is processed into siRNAs by Dicer proteins. Because non-specific RDR6 recruitment causes siRNA production from non-targets, it is critical that RDR6 is strictly recruited to an RNA that serves as a template of dsRNA formation. Here, we established an in vitro system for secondary siRNA production, and using this system, reveal that RDR6 is recruited to a cleavage fragment by 22-nt siRNA containing AGO1-RISCs by coordination of SUPPRESSOR OF GENE SILENCING 3 and SILENCING DEFECTIVE 5.