Fig. 3

Effects of deletion of CHS3, CHS2, CHS8 or CHS2 and CHS8 on the PHR1 null mutant phenotype. a Upper panel: strains CAI4 (wild type), Myco3 (chs3Δ), C155 (chs2Δ), NGY128 (chs8Δ) and NGY138 (chs2Δ chs8Δ) and in the lower panel their respective phr1Δ derivatives CAS8 (phr1Δ), FP3 (phr1Δ chs3Δ), FP155 (phr1Δ chs2Δ), FP128 (phr1Δ chs8Δ) and FP138 (phr1Δ chs2Δ chs8Δ). Blastospores were induced to switch to hyphal growth in M199-150 mM HEPES-pH 7.5 at 37 °C and 5 hours later aliquots of culture were processed for CW-staining without fixation. Pictures were taken using the same exposure time (2.5 s). For chs3Δ and phr1Δ chs3Δ the corresponding bright field (BF) image is also shown. The arrow indicates a cell in which CW penetrated. b Chitin content at time zero, 3 and 5 hours. The mean value of each strain is expressed as a percentage of the wild type value at time zero [100 %,12.50 μg of GlcNac/mg d.w. (dry weight of cells) ± 0.84 standard deviation (SD)] (n = 3). c Same as in a but cells were collected 24 hours after induction of hyphal growth. Due to permeability of CW into the cells, the image exposure time for phr1Δ chs3Δ cells was reduced to 1 s. The corresponding BF image shows cell ghosts of highly fluorescent cells. Magnification, x 1,300. d Determination of the percentage of dead cells by MB staining in M199-pH 7.5. Data are mean values ± S.D. For the parental strains a single category “dead hyphae” is shown and includes: dead mother cells, hyphae with dead apex or with dead mid-compartments or long dead hyphae. At least 500 hyphae were counted in triplicate and percentage of “dead hyphae” are shown. For the phr1Δ mutants, samples were sonicated for 5 s before MB staining. Blue cells were counted over a total of at least 500 cells