State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China.
Methods Mol Biol. 2024;2771:99-110. doi: 10.1007/978-1-0716-3702-9_14.
Pattern-triggered immunity is the first line of defense against infection by pathogens such as bacteria and fungi in plants, and this mechanism remains poorly defined in plant viruses. Double-stranded RNA (dsRNA) is an intermediate in the replication of plant RNA viruses, and is considered to be a conserved structure of plant viruses similar to pathogen-associated molecular pattern. Whether dsRNA is the elicitor that activates plant immunity in response to virus infection remains obscure. In this method, we use the cDNA of turnip mosaic virus genome as the template to in vitro synthesis of viral dsRNA and examine whether viral dsRNA could activate plant immunity in Arabidopsis thaliana, including MAPK kinase cascade and reactive oxygen burst. In order to provide some references for researchers studying dsRNA in terms of research methodology and experimental methods, we use western blot to measure MAPK kinase cascade and luminol-based assay to measure ROS burst in Arabidopsis thaliana treated by viral dsRNA.
模式触发免疫是植物抵御细菌和真菌等病原体感染的第一道防线，而这种机制在植物病毒中仍未得到很好的定义。双链 RNA (dsRNA) 是植物 RNA 病毒复制的中间产物，被认为是类似于病原体相关分子模式的植物病毒的保守结构。dsRNA 是否是植物对病毒感染产生免疫反应的激发剂尚不清楚。在本方法中，我们使用芜菁花叶病毒基因组的 cDNA 作为模板，体外合成病毒 dsRNA，并研究病毒 dsRNA 是否能在拟南芥中激活植物免疫，包括 MAPK 激酶级联反应和活性氧爆发。为了为研究 dsRNA 的研究人员在研究方法和实验方法方面提供一些参考，我们使用 Western blot 来测量 MAPK 激酶级联反应，并用发光氨法来测量拟南芥中 ROS 爆发。
