Abstract
Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Besides short interfering RNAs, microRNAs (miRNAs) have also been shown to regulate gene expression by RNA silencing in various organisms. However, plant virus-based miRNA silencing has not been reported. In addition, a number of plant miRNAs have been identified or predicted, while their functions are largely unknown. Thus, there is an urgent need for the development of new technologies to study miRNA function. Here, we report that a modified cabbage leaf-curl geminivirus vector can be used to express artificial and endogenous miRNAs in plants. Using this viral miRNA expression system, we demonstrate that VIGS using artificial miRNAs, dubbed as "MIR VIGS," was effective to silence the expression of endogenous genes, including PDS, Su, CLA1, and SGT1, in Nicotiana benthamiana. Silencing of SGT1 led to the loss of N-mediated resistance to Tobacco mosaic virus. Furthermore, using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves. These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized not only to specifically silence genes involved in general metabolism and defense but also to investigate the function of endogenous miRNAs in plants.
Highlights
Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics
We used the miRNA target search tool included in Web MicroRNA Designer to look for the putative targets of miR156 and miR165 and found that miR156 could target at least five genes, including two putative Squamosa promoter-binding protein-like (SPL) genes (TC7909 and TC9706) in N. benthamiana, and miR165 could target a gene (TC12847) that encodes a class III HD-Zip transcription factor in Nicotiana tabacum
We monitored the expression of the above two putative SPL genes and the N. benthamiana homolog of class III HD-ZIP gene (NbHD-ZIPIII) by semiquantitative reverse transcription (RT)-PCR in order to evaluate the silencing effect of miR156 and miR165 that were processed from transiently expressed native miRNA precursors
Summary
Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized to silence genes involved in general metabolism and defense and to investigate the function of endogenous miRNAs in plants. Traditional virus-induced gene silencing (VIGS) is a siRNA-mediated silencing approach and uses viral vectors carrying a fragment of a gene of interest to generate long double-stranded RNAs, which are processed by Dicer to produce siRNAs for the silencing of the target gene This traditional siRNA-mediated VIGS, dubbed as “SIR VIGS” hereafter, has been exploited as an effective and rapid gene “knockdown” technology and widely used to define gene functions (Robertson, 2004). There is a need to develop a toolbox for the induction of miRNA-mediated gene silencing and for functional analyses of miRNAs in plants
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