Huang Yu, Yang Zhirui, Li Yi
State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Peking University, Beijing, China.
State Key Laboratory of Plant Environmental Resilience, Frontiers Science Center for Molecular Design Breeding, College of Biological Sciences, China Agricultural University, Beijing, China.
Bio Protoc. 2025 Jul 20;15(14):e5383. doi: 10.21769/BioProtoc.5383.
Rice (), a staple crop sustaining half of humanity's caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV-host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening. Key features • The protocol purifies virus particles of RSV with strong infection capacity, which can directly infect rice protoplasts when co-transfected with exogenous plasmids for functional studies. • This transgene-independent approach accelerates antiviral determinant profiling from several months to three days.
水稻是维持全球半数人口热量摄入的主要作物，却受到多种昆虫传播疾病的威胁，比如由水稻条纹病毒（RSV）引起的水稻条纹病。大多数关于植物抗病毒防御机制的基因研究依赖于自然或人工感染以及转基因方法，而这些方法需要数月的植物转化时间。在此，我们展示了一种简化方案，能够在三天内快速分析RSV与宿主的相互作用。该方法包括三个关键阶段：（1）基于聚乙二醇（PEG）从受感染植物组织中沉淀RSV病毒粒子；（2）通过优化甘油垫层的差速超速离心进行连续纯化；（3）通过PEG介导的递送将纯化的病毒粒子高效转染到水稻原生质体中。使用针对病毒RNA的RT-qPCR和RSV核衣壳蛋白特异性单克隆抗体进行免疫印迹法定量评估病毒复制。这种方法消除了对稳定转基因系的依赖，允许同时引入外源质粒进行功能研究。与需要数月时间生成转基因植物的传统方法相比，我们的方案在三天内就能得出可分析的结果，显著加快了抗病毒机制探索和抗性基因筛选的速度。关键特性 • 该方案可纯化具有强感染能力的RSV病毒粒子，在与外源质粒共转染进行功能研究时能直接感染水稻原生质体。 • 这种不依赖转基因的方法将抗病毒决定因素分析从数月缩短至三天。
