A geminivirus attenuation vector for crop protection using episomal plant gene therapy

Although plant viruses inflict billions of dollars in global crop yield loss, they also encode functions that can be used for crop protection. A viral vector attenuation strategy is developed that enables modulation of a 'gene therapy' viral vector designed for protective gene delivery. This attenuation vector targets its homologous native viral counterpart, where the A-component of the bipartite tomato mottle virus (ToMoV) viral vector delivers siRNA constructs targeting native viral sequences for which its own genome has been synthetically modified to avoid silencing. A target coat protein replacement viral vector was engineered with a Nano-luciferase reporter gene to quantify effectiveness based on bioluminescence. To silence the target virus, siRNA constructs were designed against the transactivating protein (TrAP) which is responsible for activating the promoter driving the native coat protein transcript. This siRNA was placed in the attenuation vector designed to avoid self-targeting by modifying its own TrAP sequence. Viral infections were initiated in tobacco using Agrobacterium DNA delivery. Measurements at 3, 6, and 9 days post infiltration revealed a significantly reduced luminescence in attenuation treatment demonstrating suppression of the target virus transgene expression. This work sets the stage for a breadth of virus attenuation studies that can be rapidly applied to diverse crop threats.