A Simple Protoplast-Based Method for Screening Potent Artificial miRNA for Maximal Gene Silencing in Arabidopsis

The unpredictability of in planta silencing efficiency of artificial micro RNAs (amiRNAs) has greatly limited the application of the amiRNA technology in basic and applied plant research. This unit describes a simple and robust method called the epitope-tagged protein-based amiRNA (ETPamir) screen for identifying the most effective amiRNA for silencing a particular Arabidopsis gene. After selecting three to four amiRNA candidates designed by the WMD3 web server for the target gene, the silencing efficiencies of the amiRNA candidates can be compared by co-expressing individual amiRNAs with the target mRNA encoding an epitope-tagged target protein in Arabidopsis mesophyll protoplasts. By monitoring target protein levels through immunoblotting with commercial "anti-tag" antibodies, the most potent amiRNA can be easily identified as the one with minimal target protein accumulation. This empirical method bypasses the complexity of amiRNA silencing mechanisms and the scarcity of plant specific antibodies, and can be applied to a broad range of plant species. © 2017 by John Wiley & Sons, Inc.