Shi Jinxia, Jia Yijuan, Fang Di, He Shidan, Zhang Peng, Guo Yushuang, Qiao Yongli
Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University.
Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University; College of Agriculture, Yangtze University.
J Vis Exp. 2020 Feb 3(156). doi: 10.3791/60697.
RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.
RNA沉默是真核生物中一种进化上保守的、序列特异性的基因调控机制。几种植物病原体已经进化出具有抑制宿主植物RNA沉默途径能力的蛋白质。与病毒效应蛋白不同，只有几种分泌型效应蛋白显示出能够抑制细菌、卵菌和真菌病原体中的RNA沉默，而大多数效应蛋白的分子功能仍然很大程度上未知。在这里，我们详细描述了一种共浸润试验的略微修改版本，该版本可作为观察RNA沉默和表征植物病原体分泌的效应蛋白的通用方法。该方法的关键步骤是选择健康且发育完全的叶片，将细菌培养物调整到600nm处合适的光密度（OD），并在浸润叶片上的最佳时间观察绿色荧光蛋白（GFP）荧光，以避免遗漏具有弱抑制活性的效应蛋白。这种改进的方案将有助于快速、准确和广泛地筛选RNA沉默抑制因子，并作为研究这些蛋白质分子功能的良好起点。
