Visualization and Quantification of Apoplastic ROS by Plant Immune NADPH Oxidase Utilizing Chemiluminescence

Reactive oxygen species (ROS) are implicated in plant innate immunity. When plants recognize pathogens, apoplastic superoxide (O₂^-) is produced by a plasma membrane NADPH oxidase (RBOH; Respiratory Burst Oxidase Homolog), which is immediately dismutated to hydrogen peroxide (H₂O₂). Nicotiana benthamiana NbRBOHB is responsible for both the transient pattern-triggered immunity (PTI)-ROS burst and the robust effector-triggered immunity (ETI)-ROS burst. Potato (Solanum tuberosum) StRBOHB and StRBOHC are phosphorylated by potato calcium-dependent protein kinases, StCDPK4 and StCDPK5, resulting in the ROS burst. The ROS contribute to the potato resistance to a virulent isolate of late blight pathogen, Phytophthora infestans. Here, we introduce an apoplastic ROS detection protocol utilizing L-012-derived chemiluminescence in response to Agrobacterium-mediated co-expression of StRBOHC and StCDPK5 in N. benthamiana leaves.