The genomic region matters when synthesizing dsRNA for plant virus suppression via RNAi
Karol Alves Barroso, J. C. Milagres, Talia Tracton, Teja Shidore, Raja T. T. Muthuramalingam, Rania El-Tanbouly, Márcia Michelle de Queiroz Ambrósio, Washington da Silva
Abstract
Plant viruses are one of the most economically important plant pathogen groups in the world, and there is no viricide available for their control. Therefore, RNA interference (RNAi)-based crop protection has become a promising strategy for the control of viral plant pathogens in agricultural systems. Herein, we aimed to test the hypothesis that exogenously applied dsRNA molecules derived from different viral genomic regions induce different levels of viral suppression by RNAi in plants. We also evaluated the fate and movement of the dsRNA molecules inside tobacco plants. We synthesized dsRNAs from three potato virus Y (PVY) cistrons, helper component-protease (HC-Pro), nuclear inclusion protein b (NIb), and coat protein (CP), and applied them to tobacco leaves to test our hypothesis. Our results indicated that all three dsRNAs applied can provide some level of protection to plants against PVY infection. However, the intensity and longevity of protection depend on the type of dsRNA applied. HC-Pro-dsRNA induced greater protection, entered, and moved faster in tobacco plants than dsRNAs from NIb and CP cistrons. Furthermore, dsRNAs were detected in systemic leaves after 24 h of dsRNA application and remained for at least 14 days, demonstrating that these molecules translocated systemically inside the plant. The synthesis and application of exogenous dsRNAs targeting the HC-Pro genomic region of PVY appear to be a promising strategy for controlling PVY. Moving forward, we are working on developing a delivery system to sustainably provide plants with those molecules to create viricides for practical agricultural applications. Potato virus Y (PVY) causes severe yield and production losses in various Solanaceae family members, e.g., potatoes, tobacco, and tomatoes. In our studies, we conducted virus-derived small interfering RNA (vsiRNA) mapping analyses and chose those genomic regions with highly abundant vsiRNAs, denominated “vsiRNA hotspots”, to guide the selection of regions for dsRNA synthesis. We found that HC-Pro-dsRNA provided greater plant protection against PVY infection than CP-dsRNA and NIb-dsRNA. This can be explained by our finding that HC-Pro-dsRNA enters and moves faster inside the plant than the other dsRNAs tested, indicating that the genomic region matters when synthesizing dsRNA for plant virus suppression via RNAi. Furthermore, we demonstrated that the movement of these molecules inside the plants can occur both in the form of dsRNAs and vsiRNAs. Our study sheds important light on developing RNAi-based technologies to control plant viruses that could translate into field applications.