Assessing Xoo-induced H2O2 Accumulation in Rice

For observation of the water-soaked/hypersensitive reaction, leaves of 5- to 6-week-old IR24 rice were infiltrated with different Xoo suspensions with an optical density at 600 nm (OD₆₀₀) of 0.5 using a needleless syringe, as previously described (Streubel et al. 2013 (link), Yang and Bogdanove 2013 (link)). The water-soaked symptoms were scored and photographed 3 dpi. For H₂O₂ accumulation assays, rice leaves infiltrated with different Xoo suspensions at the indicated time points (1 and 3 dpi) were examined by 3,3′-diaminobenzidene (DAB) staining following the methods described previously (Girija et al. 2017 (link), Sathe et al. 2019 (link)). Then, H₂O₂ accumulation was visualized by visible light microscopy using a 10× objective. To measure H₂O₂ levels in rice leaves, a Hydrogen Peroxide Assay Kit (Solarbio, Beijing, China) was used according to the instruction manual. All the rice inoculations with Xoo were biologically repeated at least three times, and each involved three replicates.