ROS Burst Assay

Apoplastic reactive oxygen species (ROS) were quantified through a fast and robust bioassay, as described by Smith and Heese (2014). Leaf discs (0.2 cm²) from the second leaf of 2.5- to 3-week-old plants were placed individually into wells of a 96-well microplate containing 200 µL of SDW and incubated overnight at constant light and 22°C to reduce the wounding response. After incubation, SDW was replaced with 100 µL of the elicitation solution composed of 5.38 units of Horseradish Peroxidase (MilliporeSigma, Burlington, MA, USA) and 34 µg of Luminol (MilliporeSigma, Burlington, MA, USA) per mL of SDW with or without 5 x 10⁸ CFU/mL of E. coli O157:H7 or S. Typhimurium 14028s. The elicitation solution containing bacteria was prepared with heat-killed bacterial suspensions (incubated at 100°C for 10 minutes) to avoid possible inhibition of ROS production by any unknown virulence factor produced by live bacteria in contact with leaf tissue. After adding the elicitation solution to the wells, plates were immediately inserted in a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, Biotek, Winooski, VT, USA) to measure luminescence and estimate ROS production every 2 minutes between 0 and 90 minutes. For each treatment, 24 leaf discs were collected from six different plants. The experiment was repeated five times with independent batches of plants.