Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA

Abstract

Viral resistance can be effectively induced in transgenic plants through their silencing machinery. Thus, we designed nine short hairpin RNAs (shRNA) constructs to target nuclear inclusion protein b (NIb), helper component proteinase (HC-Pro), cylindrical inclusion protein (CI) and viral protein genome linked (VPg) genes of Potato virus Y (PVYN) and Tobacco etch virus (TEV-SD1). The shRNAs were completely complementary to the genes of PVYN, and contained 1–3 nt mismatches to the genes of TEV-SD1. To study the specificity of gene silencing in shRNA-mediated viral resistance, the constructs were introduced into tobacco plants. The results of viral resistance assay revealed that these nine kinds of transgenic tobacco plants can effectively induce viral resistance against both PVYN and TEV-SD1, and the shRNA construct targeting the NIb gene showed higher silencing efficiency. Northern blot and short interfering RNA (siRNA) analyses demonstrated that the viral resistance can be attributed to the degradation of the target RNA through the RNA silencing system. Correlation analysis of siRNA sequence characteristics with its activity suggested that the secondary structure stability of the antisense strand did not influence siRNA activity; 1 to 3 nt 5’ end of the sense strand caused a significant effect on siRNA activity where the first base such as U was favourable for silencing; the base mismatch between the siRNA and the target gene may be more tolerated in the 5’ end.