A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

McHale, Marcus, Eamens, Andrew L., Finnegan, E. Jean, & Waterhouse, Peter M. (2013) A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis. The Plant Journal, 76(3), pp. 519-529.

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Abstract

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. 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.

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12 citations in Web of Science®

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ID Code: 65908
Item Type: Journal Article
Refereed: Yes
Additional URLs:
Keywords: 22 nucleotide, Arabidopsis, artificial microRNA, asymmetric, CHALCONE SYNTHASE, phased small-interfering RNA, RNA silencing, technical advance, Small-interfering rnas, Technical advances, Genes, Histology, Nucleotides, Plants (botany), Seed, Tissue, RNA, Arabidopsis thaliana
DOI: 10.1111/tpj.12306
ISSN: 0960-7412
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > PLANT BIOLOGY (060700)
Divisions: Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2013 Wiley-Blackwell Publishing Ltd.
Deposited On: 09 Jan 2014 01:39
Last Modified: 02 Jul 2014 23:33

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