Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants

MicroRNAs (miRNAs) processed from nuclear-encoded transcripts control expression of target transcripts by directing cleavage or translational inhibition. Artificial miRNAs (amiRNAs) that exploit this endogenous gene silencing mechanism can be designed to target any gene of interest and provide a highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants. Here, using Arabidopsis thaliana miR159a, miR167b and miR171a precursors as backbones, we designed two types of amiRNA targeting sequence that encode the silencing suppressor HC-Pro of Potato virus Y (PVY) and the TGBp1/p25 (p25) of Potato virus X (PVX). The detected amiRNAs efficiently inhibited HC-Pro and p25 gene expression and conferred highly specific resistance against PVY or PVX infection in transgenic Nicotiana tabacum; this resistance was also maintained under conditions of increased viral pressure. Moreover, resistance was strongly influenced by the complementarity between the target sequence and amiRNA, and was well correlated to amiRNA expression level; the expression level of amiRNAs was also well related to the precursor backbones. We further showed that transgenic N. tabacum developed highly effective resistance to both PVY and PVX through expression of a dimeric amiRNA precursor. Together, our findings indicate that transgenic plants with multiple virus-specific resistance can be obtained through co-expression of several amiRNAs targeting multiple viruses.