A virus-based miRNA suppression (VbMS) system for miRNA loss-of-function analysis in plants

Abstract

MicroRNAs (miRNAs) play key roles in plant development and defense against pathogens. To establish the function of individual miRNAs, gain-of-function analysis is usually done by overexpressing a specific miRNA in transgenic plants and has proved very effective. Loss-of-function analysis by the target mimic method is now also increasingly being used. The mimics expressed in the transgenic plants sequester a specific miRNA and lead to changed phenotypes that elucidate miRNA function. However, it takes time to obtain the transgenic plants. To avoid using transgenic plants, we have developed a virus-based miRNA suppression system (VbMS) based on a Tobacco rattle virus vector. The target mimic sequences of miR156, miR319, or miR164 were introduced into the viral genomic RNA, which was then inoculated to Arabidopsis thaliana plants. The resulting phenotypes were consistent with previous reports from transgenic plants, and the expression of targets of the miRNAs was also increased showing that the activity of the miRNAs had been inhibited. VbMS developed here is validated for loss-of-function analysis of miRNA in plants. Moreover, since only simple agroinfiltration rather than transformation is needed, VbMS is suitable for large-scale approaches to miRNA function analysis in plants.

MicroRNA414c affects salt tolerance of cotton by regulating reactive oxygen species metabolism under salinity stress

“…The increased sensitivity to salinity stress shown by the GhFSD1silenced plants strongly suggested a role for this gene in the tolerance of cotton to salt stress, although the mechanism is still unknown. Many studies have reported that plant virus-based vectors could be used to study miRNA functions in plants [33][34][35]. When the virus-based vectors carrying precursor sequences of endogenous miRNAs infects the plants, using this viral miRNA expression system, the plant displays the phenotype resulting from the overexpression of that miRNA to investigate the function of miRNAs in plants [36,37].…”

Specificity of plant microRNA target MIMICs: Cross-targeting of miR159 and miR319

“…Hence, MIMs can inhibit miRNA activity causing loss-of-function miRNA effects (Franco-Zorrilla et al, 2007). Artificial MIMs, and variations thereof, have now been designed as tools to target miRNAs of interest, being stably introduced into the plant via Agrobacterium transformation (Todesco et al, 2010;Yan et al, 2012) or transiently with the use of viral vectors (Sha et al, 2014, Yan et al, 2014, enabling the in vivo functional roles of miRNAs to be elucidated.…”

Suppression of nbe‐miR166h‐p5 attenuates leaf yellowing symptoms of potato virus X on Nicotiana benthamiana and reduces virus accumulation

“…To investigate the association of nbe‐miR166h‐p5 with PVX infection, the expression of this miRNA was suppressed using the Tobacco rattle virus (TRV)‐based miRNA suppression (VbMS) system (Yan et al ., ), and the progress of PVX infection was monitored. For VbMS, a target mimic (TM) sequence that would sequester nbe‐miR166h‐p5 was designed according to the methods described previously and introduced into the TRV vector, producing TRV:TM (Yan et al ., ). TRV:TM or TRV:00 (empty vector control) was inoculated onto plants by agroinfiltration.…”