Nrg1 activates hyphal growth in S. japonicus and is repressed by QS and the SAPK pathway.

(A) Venn diagrams indicating the number of differentially expressed up- and downregulated genes during unperturbed growth in sty1Δ and atf1Δ mutants with respect to wild type cells. (B) Heatmap of the subset of differentially expressed genes (up- and downregulated) during unperturbed growth in sty1Δ and atf1Δ mutants with respect to wild type cells. Green indicates decrease and red indicates increase in gene expression. (C) nrg1⁺ mRNA levels were measured by qPCR from total RNA extracted from cell samples corresponding to S. japonicus wild type, sty1Δ, atf1Δ, pyp1Δ, and pka1Δ strains growing exponentially in YES medium. Results are shown as relative fold expression (mean ± SD) from three biological repeats. **, P<0.005; ns, not significant, as calculated by unpaired Student´s t test. (D) nrg1⁺ mRNA levels measured by qPCR from total RNA extracted from cell samples from wild type, sty1Δ and atf1Δ strains growing exponentially (time 0h), incubated for 3h in the presence of 0.2 μM CPT, or for 3h in the presence of 0.2 μM CPT plus 0.5 mM phenylethanol (PE). Results are shown as relative fold expression (mean ± SD) from three biological repeats. *, P<0.05; **, P<0.005; as calculated by unpaired Student´s t test. (E) Cell length represented as box and whisker plots in S. japonicus wild type, and nrg1Δ mutants growing exponentially in high glucose (6%) YES medium for 6h in the presence of 0.2 μM CPT. Experiment was performed per triplicate (n≥ 300 cells) and quantification of one is shown. ****, P<0.0001, as calculated by unpaired Student´s t test. (F) Cells from log-phase cultures of the indicated strains growing in YES medium (2.10⁶) were spotted on YEMA plates, incubated at 30°C for 7 days, and then photographed. The total area of mycelial expansion (expressed as relative units) was measured for each strain (n≥6) and is represented as scatter plot. *, P<0.05; **, P<0.005; ***, P<0.001; as calculated by unpaired Student´s t test.