Silencing P25, HC-Pro and Brp1 of Potato Virus (Viroid) Using Artificial microRNA Confers Resistance to PVX, PVY and PSTVd in Transgenic Potato

Virus infection is the key constraint to potato cultivation worldwide. Especially, coinfection by multiple viruses could exacerbate the yield loss. Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to viruses. In this study, three amiRNAs containing Arabidopsis miR159 as a backbone, expressing genes targeting P25, HC-Pro and Brp1 of potato virus X (PVX), potato virus Y (PVY) and potato spindle tuber viroid (PSTVd), were constructed. amiR-159(P25), amiR-159(HCPro) and amiR-159(Brp1) were cloned into the plant expression vector pCAMBIA1301 with a CaMV35S promoter, producing the p1301-pre-amiR(P25-HCPro-Brp1) vector. Twenty-three transgenic plants (Solanum tuberosum cv. 'Youjin') were obtained by Agrobacterium tumefaciens-mediated transformation, and ten PCR-positive transplants were chosen for further analysis. Quantitative real-time PCR results indicated that 10 transgenic plants could express amiRNAs successfully. Southern blotting hybridization proved that amiR-159(P25-HCPro-Brp1) had integrated into potato genome in transgenic lines. Viral (viroid) challenge assays revealed that these transgenic plants demonstrated resistance against PVX, PVY and PSTVd coinfection simultaneously, whereas the untransformed controls developed severe symptoms. This study demonstrates a novel amiRNA-based mechanism that may have the potential to develop multiple viral resistance strategies in potato.