ROSBurstAssay.md

ROS burst assay

The ROS burst assay was based on previously published protocols.33,85 In brief, a 96-well plate containing water and plant material (as described above) was incubated overnight at RT to remove ROS that had resulted from mechanical damage to the tissue during preparation. The next day, the water was replaced with 100 μL of fresh Milli-Q water containing 20 μg·mL-1 horseradish peroxidase (Sigma-Aldrich, Taufkirchen, Germany) and 20 μM L-012 (Wako Chemicals, Neuss, Germany). After a short incubation period (∼15 min), 100 μL of double-concentrated elicitor solutions were added to the wells. All elicitors were dissolved in Milli-Q water without additional treatment. Measurements of elicitor-triggered apoplastic ROS production were started immediately and performed continuously with an integration time of 450 ms in a TECAN SPARK 10 M multiwell plate reader (Männedorf, Switzerland).