OsREIW1 regulates OsWRKY31 stability. A to C) OsREIW1 interacts with OsWRKY31. A) OsREIW1 and PUB6 as a control were fused to the Gal4 DNA-binding domain (bait). The prey-OsWRKY31 was from Fig. 1. Yeast cells were incubated in SD medium without Leu and Trp (-LW) or lacking Leu, Trp, His, and adenine (-LWHA), and photographed 3 d after plating. Yeast cells containing AD-T7 plus BD-53 and AD-T7 plus BD-Lam plasmids were used as the positive and negative controls, respectively. B) Purified GST-REIW1-3myc and GST-WRKY31-3flag protein (each about 1 μg) were mixed and incubated at 4 °C for 2 h in immunoprecipitation buffer. The protein mixture was precipitated with anti-c-Myc agarose affinity gels, separated on 10% SDS-PAGE gels, and detected by immunoblots with αMyc and αFlag antibodies. C) Agrobacteria harboring 35S:REIW1^H56Y-YFP^C, 35S:WRKY31-YFP^N or 35S:WRKY76.1-YFP^N plasmid were combined accordingly and infiltrated into the leaves of N. benthamiana. Confocal images were taken at 2 d after the infiltrations. Red fluorescence signals indicative of the nuclei were from co-infiltrated 35S:dsRed-NLS (NLS: nuclear localization signal). Bar = 10 μm. D) Ubiquitination of OsWRKY31 by OsREIW1. OsWRKY31 recombinant protein GST-W31-3flag and its phosphomimetic GST-W31^DD-3flag and phosphonull GST-W31^AA-3flag were incubated with E1, E2, and ubiquitin in the presence of GST-REIW1-3myc. The reaction mixture (30 μL) contained 50 ng wheat ubiquitin-activating enzyme E1, 100 ng human ubiquitin-binding enzyme E2 (UBCH5b), 5 µg Arabidopsis ubiquitin (Ub), 500 ng GST-REIW1-3myc, and 2 μg OsWRKY31 or its mutant protein in buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 2 mM DTT, and 5 mM ATP) and incubated at 30 °C for 1 h. The reaction was stopped by adding the 5× SDS loading buffer. Samples were separated by 10% SDS-PAGE gels and analyzed by immunoblots using αUb or αFlag antibody. Relative intensity (numbers in the gel) of ubiquitinated bands was analyzed using ImageJ software. E and F) Cell-free degradation of OsWRKY31. Total proteins were extracted from the leaves of Ubi:REIW1-3myc, reiw1ko, and ZH17 seedlings grown hydroponically for 1 wk. Protein extracts (about 500 μg) were incubated with GST-WRKY31-3flag (2 μg) for various time points at 25 °C. The protein samples were separated and analyzed by immunoblot with αFlag antibody. Representative data from three independent experiments. Quantification of treatments at 0 min was set as 1. Values are mean ± SD (n = 3). G) Segments of leaves from 7-d-old seedlings cultured hydroponically were treated with 100 µM MG132 or DMSO solvent for 1 h and then sampled for protein isolation. The protein mixture was separated on 10% SDS-PAGE gels and detected by immunoblots with αMyc and αFlag antibodies. Representative results from three independent experiments. CBB, Coomassie brilliant blue staining for loading control. H and I) Eighteen-day-old plants were inoculated with M. oryzae SZ spores (5 × 10⁵ conidia mL⁻¹) by foliar spraying. Disease symptoms H) and lesion areas I) were conducted at 7 d after the inoculation. Representative data from three independent experiments. Significance was evaluated by comparing with ZH17 or each other as bracketed using Student's t-test (*, P < 0.05; **, P < 0.01). Bar = 2 cm.