Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing

Summary Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER ‐ LIKE ( DCL ) copies, thus enabling functional diversification. In Arabidopsis, DCL 2 and DCL 4 process double‐stranded RNA into 22 and 21 nucleotide small interfering (si) RNA s, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL 2 and DCL 4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL 2 stimulates transitivity and secondary si RNA production through DCL 4 while being sufficient for silencing on its own. Notably, silencing of 35S‐driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL 4 mostly obscures DCL 2 in wild‐type plants. Nonetheless, in a dcl4 mutant compromised in phloem‐originating silencing, ectopically expressed DCL 2 allows restoration of silencing, suggesting that DCL 2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL 2 contribution to phloem‐originating silencing is dependent on the activity of RNA ‐ DEPENDENT RNA POLYMERASE 6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL 2 and DCL 4 mostly act redundantly yet hierarchically when present simultaneously.