2.6. ROS Burst Measurements

The ROS burst was carried out using a luminol-based assay according to the protocol of Albert et al., 2015, with minor modifications. Plants were treated as explained previously and placed back in the growth chamber. Leaf disks were punched at 16 h (6 h after priming) from the source leaves (similar to infected leaves) of plants using a 3.5 mm cork-borer and a plastic rack with holes slightly larger than the cork-borer. Leaf disks were then transferred to a white flat-bottom 96-well plate (Greiner Bio-One) containing 150 µL sterile ddH₂O, covered with aluminum foil and incubated at 21 °C for 16 h to recover. The water was then replaced with 100 µL incubation solution containing 20 µM luminol L-012 (Wako Chemicals, Richmond, VA, USA), and 1 µg/mL horseradish peroxidase (AppliChem, Darmstadt, Germany) prepared in ddH₂O. Background luminescence was measured immediately for 30 min using a GloMax^®-Multi Detection System (Promega, Madison, WI, USA). Using a multipipette, 100 µL of assay buffer consisting of incubation solution and elicitor (at double the final concentration) was then added to start the reaction. For flg22, a final concentration of 100 nM [46] was used and for OGs a concentration of 0.2 mg/mL [47]. OGs were prepared as described previously [19]. For NADPH-oxidase inhibitor assays, diphenyleneiodonium (DPI) was added to both incubation and assay solutions to a final concentration of 5 µM. For the direct effect of fructans on the ROS burst, elicitors were replaced by either inulin or LOS at concentrations of 0.05, 0.5, and 5 mg/mL in the assay solution. Luminescence was then measured for 60 min at 2 min intervals and a 0.5 s integration time. Luminescence readout is given as relative light emitting units (RLU). Background readings were subtracted from the samples to obtain the elicitor-induced response.