Fluorescent brightener 28

Cells were prepared as described in the “High-Life Experiments” section above, except diluted to 100 cells/μL prior to loading on a plate. After 0, 8, and 24 hours, all cells from a single plate were transferred to a 50 mL conical tube, and pelleted at 1000xg for 3 minutes. The supernatant was aspirated, and the cells were resuspended in 900 μL of sterile water and transferred to a 1.5 mL tube. 100 μL of 1 mg/mL Fluorescent Brightener 28 (Sigma F3543) in water was added, and the solution was incubated at room temperature in the dark for 5 minutes. Next, the solution was pelleted at 13000xg for 30 seconds, the supernatant was aspirated, and the cells were resuspended in 1 mL sterile water. This wash step was then repeated once. Finally, the cells were resuspended in 10 μL sterile water, and stored on ice in the dark until imaged. Z-stack brightfield and fluorescent images with 0.2 μm spacing were acquired for each sample on a confocal microscope. For green-fluorescent mother cells, the number of bud scars in the blue fluorescent channel were counted manually.

To test the cell wall integrity of ΔperA, three cell wall perturbing agents, Calcofluor white (CFW, Fluorescent brightener 28, Sigma F3543), Congo red (CR, Sigma C6277), and caspofungin (Cancidas, MERCK & CO., INC.) were added to 25 mL of GMM plates in appropriate concentrations. CFW stock solution was made in 10 mg ml⁻¹ in 25 mM NaOH solution, and added to GMM at 5 μg ml⁻¹ for the final concentration. CR stock solution was made at 10 mg ml⁻¹ in sterile H₂O and added to GMM at 0.25 mg ml⁻¹ for the final concentration. Caspofungin stock solution (5 mg ml⁻¹ in PBS) was added to GMM to make 0.5 μg ml⁻¹ for the final concentration. Conidia in 5 μl of sterile H₂O were inoculated onto GMM by serial dilutions and cultured at 37 °C for 2 days.