A 22‐nt artificial microRNA mediates widespread RNA silencing in Arabidopsis. (3rd October 2013)

• Record Type: Journal Article
• Authors: McHale, Marcus; Eamens, Andrew L.; Finnegan, E. Jean; Waterhouse, Peter M.
• Is Part Of: Plant journal. Volume 76:Number 3(2013:Nov.)
• Page Start: 519
• Page End: 529
• Publication Date: 2013-10-03
• Subjects: Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.12306
• Languages: English
• ISSNs: 0960-7412

Abstract

<abstract abstract-type="main" id="tpj12306-abs-0001"> <title>Summary</title> <p>It is known that 22‐nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small‐interfering RNA (phasiRNA) 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. <italic>CHALCONE SYNTHASE</italic> (<italic>CHS</italic>) was selected as a target in <italic>Arabidopsis thaliana</italic> due to the obvious and non‐lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over‐expression of <italic>CHS</italic> in the <italic>pap1‐D</italic> 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 <italic>Arabidopsis RuBisCo small subunit 1A</italic> promoter (SSU). In this system, hpRNA expression triggered <italic>CHS</italic> silencing in most leaf tissues but not in roots or seed coats. Similarly, 21‐nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered <italic>CHS</italic> silencing in all leaf tissues but not in roots or seed coats. However, 22‐nt amiRNA expression from an asymmetric duplex triggered <italic>CHS</italic> silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread <italic>CHS</italic> silencing required RNA‐DEPENDENT RNA POLYMERASE6‐mediated accumulation of phasiRNAs from the endogenous <italic>CHS</italic> 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.</p> </abstract>