Fig. 4

NPR3g, a direct downstream gene of WRKY40, negatively regulates PM resistance by repressing PR1 expression. A The position of the W-box element in the promoter and its mutated sequence are displayed. Y1H analysis indicates that WRKY40 specifically binds to the W-box element of the NPR3g promoter. Selection medium contains SD/-Leu medium supplemented with 0 and 100 ng/mL aureobasidin A. B EMSA analysis demonstrates the specific binding of WRKY40 to the W-box element of the NPR3g promoter. Competitor refers to the unlabeled biotin probe. The mutated W-box element sequence corresponds to that in Figure A. C and D LUC analysis indicates that WRKY40 positively regulates the activity of the NPR3g promoter-driven LUC. The mutated W-box element sequence corresponds to that in Figure A. E Expression levels of NPR3g in WRKY40 transgenic plants. Ri-WRKY40 represents RNAi-silenced WRKY40 plants, OE-WRKY40 represents WRKY40-overexpressing plants, and EV represents empty vector control plants. F Observations of the overall powdery mildew lesions on leaf surfaces were made 7 days after PM inoculation. Subsequently, leaves were stained with trypan blue and examined for spores and mycelia using a super-depth stereomicroscope. Ri-NPR3g represents RNAi-silenced NPR3g plants, OE-NPR3g represents NPR3g-overexpressing plants, and EV represents empty vector control plants. Black and white scale bars represent 1 cm and 1 mm, respectively. G Spore counts and leaf area measurements were taken for all leaves of the entire plant 7 days after PM inoculation to calculate spore density per unit leaf area. H RT-qPCR indicates that the expression of PR1 in NPR3g transgenic lines compared to EV after PM attack. The data represent the means and standard deviations of three independent replicate experiments. Asterisks (*) indicate significant differences from the control (Student’s t test, **P < 0.01)