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

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dc.contributor.authorYu, Shien
dc.contributor.authorPilot, Guillaumeen
dc.contributor.departmentSchool of Plant and Environmental Sciencesen
dc.date.accessioned2019-05-01T14:16:47Zen
dc.date.available2019-05-01T14:16:47Zen
dc.date.issued2014-11-19en
dc.description.abstractArtificial 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.en
dc.description.notesThe authors thank Kasia Dinkeloo and Dr. Rejane Pratelli for critical reading of the manuscript, Dr. Glenda Gillaspy (Virginia Tech) for providing the MIPS1 and SnRK1.1 constructs and the corresponding transgenic lines, Dr. Filip Rolland for kindly allowing us to use the snrk1.1 knock-down lines, and the College of Agriculture and Life Sciences (Virginia Tech) for providing plant growing facilities. This work was supported by The National Science Foundation (No. MCB-1052048 to Guillaume Pilot) and by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture.en
dc.description.sponsorshipNational Science Foundation [MCB-1052048]en
dc.description.sponsorshipVirginia Agricultural Experiment Stationen
dc.description.sponsorshipNational Institute of Food and Agriculture, U.S. Department of Agricultureen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.3389/fpls.2014.00622en
dc.identifier.eissn1664-462Xen
dc.identifier.other622en
dc.identifier.pmid25477887en
dc.identifier.urihttp://hdl.handle.net/10919/89327en
dc.identifier.volume5en
dc.language.isoenen
dc.publisherFrontiersen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectArabidopsisen
dc.subjectNicotiana benthamianaen
dc.subjectartificial microRNAen
dc.subjecttranslational inhibitionen
dc.subjectmRNA stabilityen
dc.subjecttransient expressionen
dc.titleTesting the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational levelen
dc.title.serialFrontiers In Plant Scienceen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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