Thompson Natalie, Kandaswamy Rekha, Anwar Aliya Fathima, Polston Jane, Sunter Garry, Curtis Wayne R
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802, USA.
Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA.
Sci Rep. 2025 Jul 11;15(1):25085. doi: 10.1038/s41598-025-09038-3.
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.
尽管植物病毒在全球范围内造成了数十亿美元的作物产量损失，但它们也编码可用于作物保护的功能。开发了一种病毒载体减毒策略，可对用于保护性基因传递的“基因治疗”病毒载体进行调控。这种减毒载体靶向其同源的天然病毒对应物，其中二分体番茄斑驳病毒（ToMoV）病毒载体的A组分递送靶向天然病毒序列的siRNA构建体，其自身基因组已进行合成修饰以避免沉默。用纳米荧光素酶报告基因构建了一种靶向外壳蛋白替代病毒载体，以基于生物发光来量化有效性。为了沉默目标病毒，设计了针对反式激活蛋白（TrAP）的siRNA构建体，该蛋白负责激活驱动天然外壳蛋白转录本的启动子。将该siRNA置于减毒载体中，通过修饰其自身的TrAP序列来避免自我靶向。使用农杆菌DNA递送在烟草中引发病毒感染。浸润后3天、6天和9天的测量结果显示，减毒处理后的发光显著降低，表明目标病毒转基因表达受到抑制。这项工作为一系列可迅速应用于应对各种作物威胁的病毒减毒研究奠定了基础。
