FIG. 6.

ppg1-encoded peptide pheromone of S. macrospora is secreted into the culture medium. (A) Halo assays with fungal culture supernatants. To measure the peptide pheromone which is secreted into the culture medium, 20 μl of culture medium of wild-type S. macrospora and an S. macrospora ppg1::hph disruption mutant (strain S52063) was spotted on an S. cerevisiae tester lawn (MATa Δste2 sst2-1) expressing the S. macrospora pheromone receptor PRE2. Analysis of halo formation was performed after 24 to 48 h of incubation of plates at 30°C. (B) Analysis of EGFP secretion mediated by N-terminal signal sequences of PPG1. Fluorescence microscopy. Hyphae of S. macrospora transformants carrying either pSM1 (a), pSppg1-1 (b), pSppg1-2 (c), pSetp1 (d), or pSetp2 (e) were analyzed by fluorescence microscopy. Scale bars represent 5 μm. (C) Western blot. Equal amounts of extracellular cell-free culture medium were analyzed from wild-type S. macrospora (wt) and transformants containing either pSM1 (TpSM1), pSppg1-1 (TpSppg1-1), pSppg1-2 (TpSppg1-2), pSetp1 (TpSetp1), or pSetp2 (TpSetp2). Culture medium was fractionated on a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted on a polyvinylidene difluoride membrane. Western blot detection of EGFP protein was carried out with a polyclonal GFP antibody. Size markers were low-range polypeptides (Bio-Rad), and their sizes are indicated at the left.