Calcofluor white cfw

Fungal strains used in this study are listed in Table 1. Liquid and solid yeast extract-peptone-dextrose (YPD; Difco, Sparks, MD) media were used for general cultures of C. neoformans. To assay thermo-tolerance, fungal cells grown overnight at 30° were 10-fold diluted, spotted on YPD plates, and then cultured at distinct temperatures (30, 37, 38, and 39°). To examine susceptibility to other stresses, 2.5–5 μl of cultured cells grown in liquid YPD medium overnight were ten-fold serially diluted and spotted on YPD medium containing the indicated concentration of the following compounds; Congo red (CR; Sigma, St. Louis, MO) and sodium dodecyl sulfate (SDS; Fisher, Fair Lawn, NJ) for membrane destabilizing stress; dithiothreitol (DTT, Sigma) for reducing stress; calcofluor white (CFW; Sigma) for chitin synthesis inhibition, which results in cell wall stress, NaCl (Fisher); and KCl (Fisher) for salt stresses. Fungal cells were incubated at 30° and photographed post-treatment from d 2 to d 3.

For analysis of trap formation induced by nematodes, conidia harvested from 7-day-old CMY cultures were resuspended to 1 × 10⁵ conidia per ml in sterile water. A fifty microliter conidial suspension of the WT and mutants was inoculated on separate WA plates and incubated at 28°C for 2 days (Xie et al., 2019 (link)). Arthrobotrys oligospora mycelia and ΔAolatg4 mutants were added to ~200 nematodes on each plate. Trap formation was observed at the time intervals, 12, 24, and 36 h under a light microscopy (Olympus, Tokyo, Japan). Staining with 0.1% Calcofluor White (CFW, Sigma-Aldrich, United States) was carried out to observe trap formation induced by nematodes (Xie et al., 2020 (link)). Trap production and nematode death rate were quantified as the total number of traps and nematodes present per unit area of the plates in WT and ΔAolatg4 mutants.

To assess how the fungal strains respond to cell wall stressors, 10 µL of a solution containing 10⁶ conidia/mL from the WT and Δepl2 strains of T. reesei were inoculated on the center of minimal media (MM) plates (1 g/L MgSO₄·7H₂O (Synth, Diadema, SP, Brazil), 10 g/L KH₂PO₄ (Synth, Brazil), 6 g/L (NH₄)₂SO₄ (Synth, Diadema, SP, Brazil), 3 g/L Na₃C₆H₅O₇ (Synth, Diadema, SP, Brazil), 20 mL/L trace elements, and 20 g/L bacteriological agar (Kasvi, Madrid, Spain)) with 2% glucose (Synth, Diadema, SP, Brazil) in the absence or presence of different concentrations of Calcofluor White (CFW) (Sigma-Aldrich, St. Louis, MO, USA) (20 and 40 µg/mL) or Congo Red (CR) (Sigma-Aldrich, St. Louis, MO, USA) (100 and 200 µg/mL). After incubation for 3 days, the mycelia diameter under the indicated conditions was recorded. Three replicates of each experiment were conducted. Statistical tests were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test (available in Prism software v8.0) for comparing the growth of the parental and mutant strains. CFW and CR stains were used for chitin and β-1,3-glucan; the former interacts preferentially with chitin, and both interfere with cell wall assembly and integrity [34 (link)]. The use of these stressors allows the study of fungi strains with a role in cell wall maintenance.

The WT and mutant (ΔAosnf1, ΔAogal83, and ΔAosnf4) strains were inoculated on PDA and TG plates at 28°C for 6 days, respectively, and we recorded colony growth and measured colony diameter. By use of Czapek-Dox agar medium as the base medium, various carbon sources (glycerol, sucrose, and glucose) were added or not to test the carbon source utilization capacity of the WT and mutant strains, and the colony diameter was measured after 6 days of incubation (12 (link)). After 14 days of incubation on CMY, 20 mL of sterile double-distilled water (ddH₂O) was added to elute and collect the spore suspension, followed by counting under a microscope (63 (link)). The spores were stained with 20 μg/mL calcofluor white (CFW; Sigma-Aldrich), and the morphology of the conidia was visualized under a light microscope (64 (link)).

The fungal cell wall and hyphal septum were visualized by staining with 20 μg/mL calcofluor white (CFW) (Sigma-Aldrich, St. Louis, MO, USA), and mycelial cell nuclei were visualized by staining with 20 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) and 20 μg/mL CFW (63 (link)). As previously described, lipid droplets (LDs) were stained with 10 μg/mL boron dipyrromethene (BODIPY) (Thermo Fisher Scientific, Waltham, MA, USA) (29 (link)). In addition, autophagy of the hyphae was detected by 100 μg/mL monodansylcadaverine (MDC) staining.
For TEM analysis, WT and mutant strains were harvested from colonies cultured on PD broth for 3 days. Then, the mycelia were collected and fixed with 2.5% glutaraldehyde for TEM observation (30 (link)).

Wild-type A. fumigatus strain ATCC 46645 and the corresponding pigmentless pksP mutant (14 (link)) were cultivated on Aspergillus minimal medium (AMM) agar plates as described elsewhere (58 (link)). Conidia were grown for 5 days at 37°C and harvested with 0.9% (wt/vol) NaCl–0.01% (vol/vol) Tween 20. Conidia were labeled with calcofluor white (CFW; Sigma-Aldrich) or fluorescein isothiocyanate (FITC; Sigma-Aldrich) as previously described (13 (link)). To achieve swollen conidia, 4 × 10⁸A. fumigatus wild-type conidia were inoculated in 10 mL RPMI 1640 for 5 h at 200 rpm and 37°C. Subsequently, the conidial suspension was centrifuged at 1,000 × g for 5 min. The supernatant was discarded and the swollen conidia were resuspended in 3.7% formaldehyde in phosphate-buffered saline (PBS) and fixed for 30 min at 4°C. After fixation, the conidia were washed three times by centrifugation at 1,000 × g for 5 min and resuspension in PBS.

Both the WT and mutant strains were inoculated in three media—PDA, TG, and TYGA—and the colony diameters were recorded for a comparison of the mycelial growth rates at 24 h intervals [34 (link)]. The mycelia were collected from the PDA culture for 5 days at 28 °C; 20 μg/mL calcofluor white (CFW, Sigma-Aldrich, St. Louis, MO, USA) was used for the staining to observe the mycelial morphology and septa. The mycelia were stained with 20 μg/mL 4’,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) for 15 min and observed using inverted fluorescence microscopy (Carl Zeiss, Oberkochen, Germany) [35 (link)]; 50 photos were randomly taken for cell nucleus counting.