STK-12 acts as a transcriptional brake to control the expression of cellulase-encoding genes in Neurospora crassa
Fig 6
(A) Phenotype of wild-type (WT), Δigo-1, Pc-NcIGO-1, and Pc-NcIGO-1S47A strains when grown on Avicel medium. Conidia from indicated strains were separately inoculated into Avicel medium and batch cultured for 5 days. Total extracellular protein concentration and endoglucanase activity of culture broth were measured and expressed as a percentage of those in WT. Values represent means of at least three biological replicates, error bars show standard deviation. Statistical significance was determined by two-tailed Student’s t-test (*, P < 0.05; **, P < 0.01; n. s., not significant). (B) Subcellular localization of IGO-1 and its mutant derivative. Pc-NcIGO-1 and Pc-NcIGO-1S47A strains were grown on MM plates for 5 days. (C) Yeast two-hybrid assay demonstrating interaction between STK-12 and NcIGO-1. Yeast cells were grown on SD/-Trp/-Leu or SD/-Trp/-Leu/-His medium for 3 days. (D) Western blot analyses showing that polyclonal antibody specifically recognizes STK-12 protein in WT and Pc-STK-12 strains but not in Δstk-12 mutant. Arrows indicate STK-12 or STK-12-GFP protein band detected by STK-12 polyclonal antibody. (E) Immunoprecipitation assays using STK-12 antiserum showing that STK-12 interacts with IGO-1 in vivo. PI, preimmune serum. (F) stk-12 and igo-1 probably act in same pathway to regulate cellulases. Conidia from WT and stk-12 and igo-1 single (Δstk-12, Δigo-1) and double (Δstk-12Δigo-1) mutants were inoculated into Avicel medium and batch-cultured for 5 days. Total extracellular protein concentration and cellulase activity were measured. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was determined by two-tailed Student’s t-test (**, P < 0.01; n. s., not significant).