Assessing immune responses and cell death in Nicotiana benthamiana leaf agro-infiltration assays is a powerful and widely used experimental approach in molecular plant pathology. Here, we describe a reliable high-throughput protocol to quantify strong, macroscopically visible cell death responses in N. benthamiana agro-infiltration assays. The method relies on measuring the reduction of leaf autofluorescence in the red spectrum upon cell death induction and provides quantitative data suitable for straightforward statistical analysis. Two different well-established model nucleotide-binding and leucine-rich repeat domain proteins (NLRs) were used to ensure the genericity of the approach. Its accuracy and versatility were compared to visual scoring of the cell death response and standard methods commonly used to characterize NLR activities in N. benthamiana. A discussion of the advantages and limitations of our method compared to other protocols demonstrates its robustness and versatility and provides an effective means to select the best-suited protocol for a defined experiment.

中文翻译：

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一种通过荧光成像测量本氏烟草叶片细胞死亡的新型稳健和高通量方法

在本氏烟草叶片农业渗透测定中评估免疫反应和细胞死亡是分子植物病理学中一种强大且广泛使用的实验方法。在这里，我们描述了一种可靠的高通量协议来量化本氏烟草中强烈的、宏观可见的细胞死亡反应农业渗透测定。该方法依赖于测量细胞死亡诱导后红色光谱中叶片自发荧光的减少，并提供适用于直接统计分析的定量数据。使用两种不同的成熟模型核苷酸结合和富含亮氨酸的重复结构域蛋白 (NLR) 来确保该方法的通用性。其准确性和多功能性与细胞死亡反应的视觉评分和通常用于表征本氏烟草NLR 活动的标准方法进行了比较。与其他协议相比，我们的方法的优点和局限性的讨论证明了它的稳健性和多功能性，并提供了一种有效的方法来为定义的实验选择最适合的协议。