Visualizing ROS Burst in Plant Leaves

For testing the ROS burst in the leaves by DAB (3,3′-diaminobenzidine) staining, detached leaf samples were collected at 2 d after infiltration of A. tumefaciens strains as previously described (Dong et al., 2011 (link)). Leaves were stained with 1 mg ml^–1 DAB solution (Sigma-Aldrich, USA) at 25 °C for 6 h, then decolorized with boiling alcohol, and kept in 30% glycerin. To determine the role of Al6 in the ROS burst in response to flg22, a luminol-based assay was adopted as previously described (Trujillo, 2016 (link)). After the instantaneous expression of GFP and Al6 in N. benthamiana for 36 h, leaf disks were washed with water and kept overnight in the dark at room temperature. Then, the leaf disks were stained in test buffer containing luminol (Sigma-Aldrich, USA), horseradish peroxidase (Sigma-Aldrich, USA), and 1 μM flg22 (GenScript, China), and luminescence detection was conducted for at least 30 min in the microplate reader (BioTek, China).