Significance Double-stranded RNA (dsRNA) synthesis by RNA-dependent RNA polymerase (RDR) is a critical step in secondary small interfering RNA (siRNA) biogenesis. However, how RDR specifically converts the targets of primary small RNAs into dsRNA intermediates remains unclear. Here, we developed an in vitro system that recapitulates the production of secondary siRNAs that are physiologically important in plants. Leveraging this system, we showed that a combination of four plant factors promotes physical recruitment of RDR6 to the target RNA. Moreover, we found that dsRNA synthesis by RDR6 is enhanced by the removal of the poly(A) tail, which is achieved by cleavage at another small RNA-binding site bearing appropriate mismatches. Our data elucidate the molecular events necessary for secondary siRNA biogenesis in plants.