Fig. 4: ROS-induced mitophagy is blocked in the ΔPsAF5 mutant.

a–c Immunoblot analysis of mitophagy markers in the wild-type P6497 and the ΔPsAF5 mutant mycelium under H₂O₂ treatment. The analysis of total proteins used His-tag antibodies against AIF B (a mitochondrial matrix protein) (a) and mitochondrial carrier (MC) (an inner mitochondrial membrane protein) (b), and using anti-Flag antibodies against Cytc (a mitochondrial matrix protein) (c). The tagged proteins were expressed in P6497 and ΔPsAF5 by ectopic over-expression. ACTB antibody was used as a loading control. A representative example of three replicates is shown for (a–c). d–g The relative number of mitochondria was quantified by using qPCR to determine the ratio of mitochondrial DNA to nuclear DNA in each strain, which were subjected to treatment with 0 or 1 mM H₂O₂ for 36 h with or without 20 nM bafilomycin A1 (an autophagy inhibitor). Two-tailed Student’s t test was used (n = 3 biologically independent samples, mean ± SEM; ns non-significant). h, i The relative number of mitochondria was measured by the ratio of mitochondrial DNA to nuclear DNA in the indicated strains treated with pyraclostrobin (2 μg/mL) or ametoctradin (4 μg/mL), n = 3 biologically independent samples. j, k The sensitivities of ΔPsAF5 mutants to mitophagy inducers were significantly increased. The effect of 2 mg/L pyraclostrobin or 4 mg/L ametoctradin on hyphal growth of the wild-type P6497 and ΔPsAF5 mutants were assessed after 7 d post-inoculation (n = 3 biologically independent samples). Experiments in (d–k) were independently repeated twice with similar results. Bar plots with mean ± SEM, ordinary one-way ANOVA and Dunnett’s multiple comparisons test were used; ns non-significant. Source data are provided as a Source Data file.