4.5. DAB Staining, H2O2 Treatment, and H2O2 Content Assay

To detect H₂O₂ accumulation in the rice leaves infected to Xoo, the detached leaves at inoculation sites were immediately immersed into 1% (w/v) 3,3′-diaminobenzidine (DAB) (Sangon, Sangno Biotech) in 10 mmol/L 2-(N-morpholino) ethanesulfonic acid (Sangon, Sangno Biotech, Shanghai, China) buffer (pH 6.5), and then were vacuum-infiltrated for 30 min. Afterwards, the leaves were incubated at room temperature for 2 days in absence of light. Stained leaves were bleached by ethanol until a clear background was obtained [59]. The stained leaves were photographed by scientific scanner (Image Scanner III, GE, Sweden). To semi-quantify the intensity of H₂O₂ accumulation at infiltrating inoculation sites, the digitized images of stained inoculation site were analyzed by ImageJ software (version 1.47 t, NIH, WA, USA). DAB color of each inoculation sites was processed by Colour Deconvolution plugin 1.0, then was measured to obtain the mean gray value. The 255 divided by mean gray value was used to indicate the intensity of H₂O₂ accumulation [60].

For evaluating the influence of in vitro H₂O₂ on the rice resistance, the inoculated leaves were sprayed by 25 mM H₂O₂ solution lasting for 5 min on 8:00 am, 12:00 am, and 16:00 pm every day from day 1 to day 14 after leaf-clipping inoculation.

Quantitative measurement of H₂O₂ content was performed using the Amplex Red hydrogen peroxide/peroxidase assay kit (A22188, Molecular Probes, ThermoFisher SCIENTIFIC, CA, USA) according to the manufacturer’s instruction. Briefly, 3-cm long detached leaf fragments next to leaf-clipping inoculation site were immediately immersed into liquid nitrogen and stored in −70 °C. H₂O₂ was extracted from leaves and measured in a 96-well plate by luminescence spectrometer (Infinite M200, Tecan, Switzerland) according to the method described previously [61].

The DAB staining, H₂O₂ treatment, and H₂O₂ content assay were biologically repeated at least twice with similar results, and one replicate was shown.