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Measurement of reactive oxygen species (ROS) burst and pathogen assay

BM Bharat Mishra
YS Yali Sun
HA Hadia Ahmed
XL Xiaoyu Liu
MM M. Shahid Mukhtar
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Oxidative burst in Arabidopsis leaves was detected using a luminol-based assay as described previously by Macho et al.85. Briefly, 4-mm leaf discs from 4-week-old Arabidopsis plants were incubated in 100 μl sterile distilled water overnight at room temperature. Immediately before treatment, 100 μl water was replaced with equal amount of luminol/peroxidase working solution containing 1 μM flg22 (Sigma), 34 μg/ml luminol (Sigma) and 20 μg/ml horseradish peroxidase (Sigma). Luminescence was measured on a 96-well plate reader immediately after treatment and recorded every 2 min until 36 min. Four-week-old plants were syringe-infiltrated with DC3000hrcC − bacterial solution OD600 nm = 0.0002 in 10 mM MgCl2. Four leaves per plant and five plants per genotype were used for pathogen quantification through serial dilution.

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