Quantifying Subtilisin-Induced ROS in Plants

The generation of ROS during subtilisin infiltration was determined using a commercial plant ROS enzyme-linked immnunosorbent assay (ELISA) kit (Chundubio). Three or four upper leaves without infiltration of N. benthamiana were harvested at various times after infiltration with subtilisin, 0.1 g was fully ground in liquid nitrogen, and phosphate-buffered saline (pH 7.4) was added. The plant tissue was homogenized with a low-temperature homogenizer, centrifuged twice for about 20 min, and then the precipitate was discarded for use in ELISA. The standard curve was used to determine the amount in each unknown sample. OD₄₅₀ values were measured with an ELISA reader (Promega). Meanwhile, ROS generation was detected using 3,3′-diaminobenzidine (DAB) solution (Solarbio) as described previously (Yang et al., 2018 (link)). Leaves were then decolorized in boiling ethanol (90%) for 30 min and were photographed by camera (Zhao et al., 2018 (link)).