A 22‐nt artificial micro RNA mediates widespread RNA silencing in A rabidopsis

Summary It is known that 22‐nucleotide (nt) micro RNA s (mi RNA s) derived from asymmetric duplexes trigger phased small‐interfering RNA (phasi RNA ) production from complementary targets. Here we investigate the efficacy of 22‐nt artificial mi RNA (ami RNA )‐mediated RNA silencing relative to conventional hairpin RNA (hp RNA ) and 21‐nt ami RNA ‐mediated RNA silencing. CHALCONE SYNTHASE ( CHS ) was selected as a target in A rabidopsis thaliana due to the obvious and non‐lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over‐expression of CHS in the pap1‐D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hp RNA and ami RNA plant expression vectors under the control of an A rabidopsis R u B is C o small subunit 1A promoter ( SSU ). In this system, hp RNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21‐nt ami RNA expression from symmetric mi RNA /mi RNA * duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22‐nt ami RNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA ‐ DEPENDENT RNA POLYMERASE 6‐mediated accumulation of phasi RNA s from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22‐nt ami RNA ‐directed RNA silencing and associated phasi RNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22‐nt ami RNA ‐directed RNA silencing requires little modification of existing ami RNA technology and is expected to be effective in suppressing other genes and/or members of gene families.