a, Inhibition of Pkc1 activity with 1NA-PP1 can be reversed to restore septin and gelsolin ring formation. Micrographs showing the cellular localization of Sep5–GFP and gelsolin–GFP at the appressorium pore following Pkc1 blocking at 10 h.p.i. and releasing at 13 h.p.i. Appressoria were imaged at 24 h.p.i. Images are representative of n = 2 replications of the experiment. b, Heat map showing the expression of NOX1, NOX2 and NOXR in an RNA-seq analysis of the PKC1AS-mutant. Mycelium was grown in CM shake cultures for 48 h in the presence or absence of 500 nM 1NA-PP1 at 1, 3, 6, 12 or 24 h.p.i. (n = 3 biological replications of the experiment). The full RNA‐seq dataset from this study can be found at the Gene Expression Omnibus (GEO) under accession number GSE70308. c, Pkc1 interacts with Nox2 and NoxR in a yeast two-hybrid assay. Simultaneous co-transformation of pGBK-PKC (bait vector) and pGAD-Nox2 and pGAD-NoxR (prey vectors) into the Y2H Gold strain resulted in the activation of three reporter genes and growth on medium-stringency medium for Pkc1 and NoxR (−His, −Leu, –Trp, +X-α-gal), and on high-stringency medium for Pkc1 and Nox2 (–His, −Leu, −Trp, −Ade, +X-α-gal). Co-transformation also activates the expression of MEL1, which results in the secretion of α–galactosidase and the hydrolysis of X-α-gal in the medium, turning the yeast colonies blue. Images are representative of n = 3 independent biological replications of the experiment. d, Alignment of a region of the predicted amino acid sequence of NoxR using Muscle40. Sequence conservation is shaded in grey, with a consensus threshold of 75%. The predicted Pkc1 phosphorylation site is marked with a red arrow and is highly conserved; black arrows indicate other potential Pkc1 phosphorylation targets based on the motif S*APS. e, ΔpdeH mutants generate excess appressorium turgor. Percentage of Guy11 and ΔpdeH-mutant appressoria that undergo incipient cytorrhysis after exposure to glycerol solutions of 0–3.5 M. Data are mean ± s.e.m. for n = 3 independent biological replicates; 50 appressoria were counted per replicate. **P < 0.01 (two-tailed unpaired Student’s t-test). f, Cellular localization of Sln1–GFP in appressorium pores of Guy11, with or without hydroxyurea (HU) added at 6 h.p.i and imaged at 24 h.p.i. Images are representative of n = 3 independent biological replicates. Scale bars, 10 µm.