Virus-Based MicroRNA Expression for Gene Functional Analysis in Plants    

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.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…To test whether FoMV VIGS vector is able to induce gene silencing in foxtail millet, we silenced three foxtail millet genes SiPDS, Cloroplastos alterados1 (SiCLA1), and SiIspH. CLA1 encodes 1-deoxy-D-xylulose-5-P synthase (Mandel et al, 1996;Estévez et al, 2001;Tang et al, 2010) and IspH is for an isopentenyl/dimethylallyl diphosphate synthase involved in isoprenoid biosynthesis. Their null mutations or VIGS resulted in albino phenotypes (Rohdich et al, 2002;Hsieh and Goodman, 2005;Yin et al, 2015).…”

“…Bean pot mottle virus-derived vectors have been developed to induce VIGS in soybean (Glycine max) and common bean (Phaseolus vulgaris; Zhang and Ghabrial, 2006;Zhang et al, 2010). Cabbage leaf curl virus-derived vector has already been developed to trigger VIGS for siRNA-mediated silencing in Arabidopsis (Turnage et al, 2002) and miRNA-mediated silencing in tobacco (Nicotiana benthamiana; Tang et al, 2010).…”

Virus-Based MicroRNA Silencing in Plants

“…Therefore, these technologies have the potential to become an attractive and quick approach to uncover miRNA functions in plants, especially in those difficult for genetic transformation. Indeed, we have developed a cabbage leaf curl virus-based vector for the overexpression of miRNAs in plants (Tang et al, 2010). However, virusbased miRNA inactivation has not been reported.…”

MicroRNAs in tomato plants

“…Surprisingly, a viral miRNA expression system could be used to study the function of endogenous miRNA genes by agro-infiltration in Nicotiana benthamiana plants [78]. The modified CbLCV vector may be useful in high-throughput screening of miRNAs in N. benthamiana.…”