Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance.

Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.