Fig. 5: LP-induced H₂O₂ enhances OsPHR2 binding affinity to target promoters.

a The schematic diagram of the OsIPS1, OsSPX1, and OsmiR827 genomic region. P1, P2, or P3 of the indicated genes represents the DNA fragments for the ChIP-qPCR assays. Red short lines indicate the positions of the P1BS motif. b, c Dual-luciferase (LUC) reporter gene assays of transcription activation of OsPHR2 and its mutated variants on the promoter of OsIPS1, OsSPX1, and OsmiR827 in leaves of N. benthamiana plants treated with 2 mM H₂O₂ or LP. GFP, OsPHR2-GFP, C140S-GFP, or C377S-GFP was used as effector, and LUC driven by OsIPS1, OsSPX1, or OsmiR827 promoter was used as reporter. The activity of OsIPS1::LUC + GFP, OsSPX1::LUC + GFP, or OsmiR827::LUC + GFP under mock conditions was set to 1. d Enrichment of the indicated DNA fragments in ChIP-qPCR assays. Chromatin from GFP/phr2, GFP/phr2rboh-DH, OsPHR2-GFP/phr2, OsPHR2-GFP/phr2rboh-DH, C140S-GFP/phr2 and C377S-GFP/phr2 plants treated with 2 mM H₂O₂ or LP for an additional 2 days were immunoprecipitated using the anti-GFP antibody and then used for qPCR. For each probe, the expression level in different plants was normalized to GFP/phr2 plants under mock conditions (which was set as 1). e The effects of H₂O₂ on the binding affinity of MBP-OsPHR2 or MBP-C377S to the indicated probes determined by EMSA assay. The EMSA analysis was repeated three times with similar results for (e). Data are means ± SD (n  =  3 replicates for c and d). The different letters above the bars denote significant differences (P  <  0.05) according to a two-sided Duncan’s multiple range test. Source data are provided in the Source Data file.