Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level.

Shi Yu,Guillaume Pilot

doi:10.3389/fpls.2014.00622

Abstract

Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants.

Highlights

MicroRNAs are a class of small noncoding RNAs of 20–24 nucleotides in length that regulate target gene expression at the post-transcriptional level in eukaryotes (Brodersen and Voinnet, 2009; Rogers and Chen, 2013). miRNAs are processed from longer precursor transcripts to a stable hairpin structure with two complementary short RNA strands, which are further processed to miRNA:miRNA∗ duplexes, by RNaseIII enzymes
In contrast to most animal miRNAs, most plant miRNAs show perfect or near-perfect complementarity to their targets, mRNA cleavage is deemed to be the dominant mode of action in plants (Brodersen and Voinnet, 2009; Huntzinger and Izaurralde, 2011)
ANALYSIS OF amiRNAs TARGETING LOG2 In an earlier report, we found that the LOG2 ubiquitin ligase interacted with the GLUTAMINE DUMPER 1 (GDU1) protein, and that knockdown or knockout of LOG2 in Arabidopsis suppressed the phenotype caused by the over-expression of GDU1 (Pratelli et al, 2012)

Summary

Introduction

MicroRNAs (miRNAs) are a class of small noncoding RNAs of 20–24 nucleotides in length that regulate target gene expression at the post-transcriptional level in eukaryotes (Brodersen and Voinnet, 2009; Rogers and Chen, 2013). miRNAs are processed from longer precursor transcripts to a stable hairpin structure with two complementary short RNA strands, which are further processed to miRNA:miRNA∗ duplexes, by RNaseIII enzymes (miRNA∗ being the passenger strand). In contrast to most animal miRNAs, most plant miRNAs show perfect or near-perfect complementarity to their targets, mRNA cleavage is deemed to be the dominant mode of action in plants (Brodersen and Voinnet, 2009; Huntzinger and Izaurralde, 2011) In disagreement with this postulate, evidence from recent reports suggests that translational repression plays a vital role in regulating target gene expression in plants (Aukerman and Sakai, 2003; Chen, 2004; Gandikota et al, 2007; Brodersen et al, 2008; Dugas and Bartel, 2008; Lanet et al, 2009; Zhu et al, 2009; Beauclair et al, 2010; Zhu and Helliwell, 2011; Alonso-Peral et al, 2012; Li et al, 2013b; Ma et al, 2013; Meijer et al, 2013). The action mode of miRNAs on gene expression appears more diverse than initially thought, with effects on target mRNA cleavage, translation inhibition and DNA methylation, possibly exerted concomitantly (Pillai et al, 2007; Voinnet, 2009)

Methods

Results

Conclusion