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

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.

Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection

“…The presence of transcript in knockdown lines indicated that the miRNA regulates expression by repressing the translation of the Bd IRI transcripts as has been seen with other miRNAs in plants [49–51]. The loss of ice-binding activity in transgenic lines was observed in crude cell extracts containing both intracellular and extracellular components, but also in the apoplastic extracts of IBP knockdowns (Fig 10).…”

Loss of Arabidopsis β-COP Function Affects Golgi Structure, Plant Growth and Tolerance to Salt Stress

“…According to qPCR analysis, the mRNA levels of β1-COP and β2-COP were reduced by 20-25% and 60-75%, respectively. The different growth phenotype among the two single null mutants and the amiR-β1/β2-cop plants may be explained by the fact that plant amiRNAs have also been shown to have an effect at the translational level (Yu and Pilot, 2014). When grown in the presence of NaCl, the amiR-β1/β2-cop mutant was even more sensitive than the single KO mutants.…”

Synthetic Botany

“…Artificial microRNAs (amiRNAs), for example, exploit the endogenous silencing machinery to repress gene expression. The extent of amiRNA-mediated repression can be tuned by altering their expression level (Yu and Pilot 2014). Systems for optimizing the specificity and the efficiency of the amiRNAs (Li et al 2013) as well as for obtaining inducible multigene silencing (Goh et al 2012) have also been implemented.…”