A Gemini Virus Attenuation Vector for Episomal Plant Gene Therapy – supporting data

Plant viruses inflict billions of dollars in global crop yield loss with limited treatment strategies beyond plant destruction. Herein, we describe a viral attenuation strategy that enables modulation of a ‘gene therapy’ viral vector designed for crop protection. This attenuation vector targets its homologous native viral counterpart, where the A-component of the bipartite Tomato Mottle Virus viral vector delivers siRNA constructs targeting native viral sequences for which its own genome has been synthetically modified to avoid silencing. A target deconstructed viral vector was engineered with a Nano-luciferase reporter gene to quantify effectiveness based on light emission. 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. Luminescence 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.