Small RNA from Arabidopsis thaliana with RNA silencing directed to Chalcone Synthase (At5G13930) by a hairpin RNA, a 21-nt artificial miRNA and a 22-nt artificial miRNA in the pap1-D over-expressing background. Arabidopsis thaliana

22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger trans-acting phased small interfering RNA (tphasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA) mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis 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 hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA 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 POLYMERASE6 mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families. Overall design: sRNA sequencing from aerial tissues of 3-week old plants grown on MS media