RNA-dependent RNA polymerase RDR6 is involved in the biogenesis of plant trans-acting siRNAs. This process is initiated by miRNA-directed and Argonaute (AGO) protein-mediated cleavage of TAS gene transcripts. One of the cleavage products is converted by RDR6 to double-stranded (ds)RNA, the substrate for Dicer-like 4 (DCL4). Interestingly, TAS3 transcript contains two target sites for miR390::AGO7 complexes, 5′-non-cleavable and 3′-cleavable. Here we show that RDR6-mediated synthesis of complementary RNA starts at a third nucleotide of the cleaved TAS3 transcript and is terminated by the miR390::AGO7 complex stably bound to the non-cleavable site. Thus, the resulting dsRNA has a short, 2-nt, 3′-overhang and a long, 220-nt, 5′-overhang of the template strand. The short, but not long, overhang is optimal for DCL4 binding, which ensures dsRNA processing from one end into phased siRNA duplexes with 2-nt 3′-overhang
