Luminol l 012

The ROS production was determined in V1 plants leaf disks using the luminol assay [100 (link)] in a Multi-Detection Microplate Reader Synergy HT (BioTek). Leaf disks were transferred to a multi-well plate (see Supplementary Figure S1B), containing 200 μL water or 100 μM INA, and they were incubated overnight. Afterwards, the previous solution was removed, and 100 μL per well of a solution containing 20 μM luminol L-012 (Wako) and 100 μg/mL peroxidase from horseradish type VI.A (Sigma, P6782) were added, and incubated in the dark for 30 min. Then, the reactions were started by adding 100 μL of water (Mock condition) as negative control, 100 μL of 2 μM flg22 (flg22 condition) as positive control, 100 μL of 200 μM INA (INA condition), and 100 μL of 2 μM flg22 to those disks previously incubated overnight with 100 μM INA (preINA + flg22). The ROS production, measured as relative luminescent units (RLUs), was measured over 90 min, with an integration time of 0.6 s. Data shown represent mean ± SE from one representative experiment of three independent ones performed with similar results (see Supplementary Figure S1A). The total areas under the kinetic curves were integrated, and the resulting values were statistically analyzed by One way ANOVA (p < 0.05), post hoc Tukey test.

Tomato protoplasts were used to measure reactive oxygen species (ROS) production using a luminol‐based assay (Halter et al., 2014). One hundred to 200 μl of transformed protoplast samples were incubated in W1 buffer without MES (0.5M mannitol, 20 mM KCl) at 20–22°C in the dark for 6–8 h. Before measurement, protoplasts were harvested by centrifugation at 100 g for 1 min and resuspended in 100 μl W5 buffer (18.4 g l⁻¹ CaCl₂ × 2H₂O, 1.0 g l⁻¹ glucose, 9.0 g l⁻¹ NaCl, 0.4 g l⁻¹ KCl) containing 200 μM luminol L‐012 (Wako Chemicals) and 20 μg ml⁻¹ horseradish peroxidase and incubated for an additional 30 min in the dark. After treatment with 500 nM flg22, luminescence was recorded for 30 min using a Mithras LB 940 multiplate reader (Berthold).

Fourth leaves from 3-week-old rice seedlings grown on soil without chitin supplementation were excised into nine leaf discs 0.5 mm in size and floated overnight at 22°C in a well filled with sterilized DW (sDW). COs or CNF elicitation solutions were prepared by suspending COs or CNF in sDW to a final concentration of 0.01% (w/v). Peroxidase from a horseradish root (HRP: Oriental Yeast, Japan) stock solution (500×HRP) and luminol L-012 (L-012; Wako, Tokyo, Japan) stock solution (20 mM) were prepared as previously described (Parada et al., 2018 (link)). Before elicitation, the sDW was carefully removed from each well without tissue damage or desiccation. The elicitation solution was immediately added to each well after removing sDW, and chemiluminescence was measured with a microplate reader (ARVO X3; PerkinElmer Japan, Japan) for 40 min.

ROS analyses were performed as described^{44 (link)} with the following modifications: Each well contained 200 µl of water supplied with 5 µM luminol L-012 (Wako Chemicals), 2 µg horseradish peroxidase (Fluka) and 5 nM Pep-13 or W2A peptide.

Leaf discs (4-mm diameter) were excised from leaves of 6-week-old plants and incubated in water overnight. Leaf discs (n = 6) were placed in a 96-well plate (1 disc/well, Greiner, Kremsmünster, Austria) containing 18 μg/mL luminol L-012 (Wako, Osaka, Japan) and 18 μM horseradish-peroxidase (AppliChem, Darmstadt, Germany). flg22 (Biomatik, Kitchener, Canada) was added to yield indicated concentrations. Luminescence was recorded over time using a TriStar² S LB 942 plate reader (Berthold Technologies, Bad Wildbad, Germany). flg22 peptide was kindly provided by Georg Felix. Peptide was dissolved in water and diluted in a solution containing 0.1% BSA and 0.1 M NaCl.

Twelve leaves from 10–day–old rice or 2–week–old wheat seedlings were used to generate leaf discs of 0.25 cm² using a hole punch and were incubated in water overnight in a 96–well plate to eliminate the effects of wounding. The next morning water was replaced by 100 μL reaction solution containing 200 μm luminol L–012 (Wako, Japan) and 1 μg horseradish peroxidase (Sigma, St. Louis, MO, USA) supplemented with 10 μm OsPep plus 0.05% silwet L–77 or silwet L–77 only (mock). The luminescence was recorded by a Varioskan Lux luminometer (Thermo, Waltham, MA, USA) for a period of 30 min.