Improve tobacco rattle virus-based microRNA silencing by special viral RNA interference suppressor
Jinping Zhao: Texas A&M University, AgriLife Research Center at Dallas
<div><sub>Virus-based microRNA silencing (VbMS) is a powerful tool to dissect the function of plant endogenous microRNAs (miRNA). Target mimic (TM) molecules expressed from virus backbones block the function of convergent miRNAs by knocking down miRNA targets and several members of the miRNA family. To date, various VbMS systems have been developed in tomato, <em>Nicotiana benthamiana</em>, and cotton. The tobacco rattle virus (TRV) based vectors has been the most applied approach for virus induced gene silencing (VIGS), while the TRV-based VbMS system so far has not been widely used and needs improvement for higher efficiency. Here, we report an improved TRV-based VbMS system by integrating special viral RNA interference suppressors (VSRs) into the virus backbone. In this system, two geminivirus VSRs under the control of TRV expression promoter could increase the level of TM molecules. Expression of the artificial designed short tandem target mimics (STTM) of miR165/166 from the improved TRV vectors blocked the function of endogenous miR165/166, causing distinct developmental defects in <em>N. benthamiana</em>, such as ectopic growth of leaf lamina on the abaxial leaf surface along vein and midrib. Molecular analysis confirmed the suppression of endogenous miR165/166 and the increase of mRNA transcripts of the miR165/166 target <em>HDZIP like </em>gene. The improved TRV VbMS system presented approximately 100% efficiency and very mild viral symptom in inoculated plants. Since the TRV VIGS system possesses wider host range than other systems, this improved TRV VbMS system could be applied to a wider range of plant species for functional characterization of miRNAs. </sub></div>
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