Hashimoto Masayoshi, Yamaji Yasuyuki, Komatsu Ken
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
Methods Mol Biol. 2019;2028:85-95. doi: 10.1007/978-1-4939-9635-3_4.
Owing to the absence of antiviral chemicals, strategies to deploy antiviral resistance in plants are desirable. Deciphering antiviral resistance mechanisms has been extensively promoted by virus-induced gene silencing (VIGS) technique, which decreases the transcriptional level of the gene of interest via RNA silencing machinery triggered by the partial gene fragment inserted into a viral vector. This technique has contributed to addressing the function of a number of host genes, which are involved in signaling pathways leading to Resistance (R) protein-mediated resistance and the viral disease symptoms such as systemic necrosis. Here we describe the general VIGS protocol and its tips to analyze antiviral resistance mechanism using Tobacco rattle virus (TRV)-based VIGS vector in tobacco plant Nicotiana benthamiana. In most cases, the knockdown of host genes by TRV vector is highly efficient and uniform at the whole plant level but is not associated with any severe symptoms. Using this method together with inoculation of a challenge virus and transient overexpression of a viral elicitor or another host signaling component by agroinfiltration, as well as measurement of hallmarks of antiviral responses, we can address the role of the host factors and the epistatic relationship between several host factors in the resistance signaling pathway against plant virus infection. The protocol described here using the highly susceptible host N. benthamiana to a variety of plant viruses provides the opportunity to study resistance mechanisms underlying many host-virus interactions.
由于缺乏抗病毒化学物质，在植物中部署抗病毒抗性的策略是很有必要的。病毒诱导的基因沉默（VIGS）技术极大地促进了解码抗病毒抗性机制，该技术通过插入病毒载体的部分基因片段触发RNA沉默机制，降低目标基因的转录水平。这项技术有助于阐明许多宿主基因的功能，这些基因参与导致抗性（R）蛋白介导的抗性的信号通路以及诸如系统性坏死等病毒病症状。在这里，我们描述了使用基于烟草脆裂病毒（TRV）的VIGS载体在本氏烟草植株中分析抗病毒抗性机制的一般VIGS方案及其技巧。在大多数情况下，TRV载体对宿主基因的敲低在整株水平上高效且一致，但不伴有任何严重症状。通过将这种方法与接种挑战病毒、通过农杆菌浸润瞬时过表达病毒激发子或另一种宿主信号成分以及测量抗病毒反应的标志相结合，我们可以确定宿主因子的作用以及抗性信号通路中几个宿主因子之间的上位关系，以应对植物病毒感染。这里描述的使用对多种植物病毒高度敏感的宿主本氏烟草的方案，为研究许多宿主 - 病毒相互作用背后的抗性机制提供了机会。
