High-degree and broad-spectrum resistance mediated by a combination of NIb siRNA and miRNA suppresses replication of necrotic and common strains of potato virus Y

In plants, viral replication can be inhibited through gene silencing, which is mediated by short interfering RNA (siRNA) or microRNA (miRNA). However, under natural conditions, viruses are extremely susceptible to mutations that may decrease the efficiency of cleavage of these small RNAs (sRNAs). Therefore, a single sRNA may not provide a sufficient degree of viral resistance to transgenic plants. Potato virus Y necrotic strain (PVYN) and Potato virus Y common strain (PVYO) are the two major PVY strains that cause systemic necrosis and mottling, respectively, in tobacco. In this study, we designed specific siRNAs and miRNAs to target two regions of the PVYO replicase gene (NIb). Eight plant expression vectors containing one or two sRNAs were constructed. Luciferase activity assays showed that the designed sRNAs successfully cleaved the NIb gene of PVYO and PVYN, and the vector carrying a combined siRNA- and miRNA-based short hairpin RNA (shRNA) demonstrated the strongest inhibitory effect. These effects were confirmed through the acquisition of PVYO and PVYN resistance in transgenic sRNA-expressing Nicotiana tabacum plants. This phenomenon could be related to a plant defense mechanism in which siRNA and miRNA pathways are complementary and interact to achieve gene silencing. Furthermore, there is a tendency for the homologous small RNA sequences (PVYO) to be more effective in conferring resistance than those with imperfect homology (PVYN). Overall, these findings confirm that the use of a combined siRNA- and miRNA-based shRNAs is a promising approach for introducing viral resistance to plants through genetic engineering.