Figure 2. Tac1-regulated Ifu5 is required for cell wall integrity.

(a) Expression analysis of IFU5 and CDR1 in tac1Δ/Δ and TAC1^HA cells. (b) Expression analysis of IFU5 and CDR1 upon fluphenazine (20·μg·ml^-1, 30 min) treatment in indicated strains. (c) Fivefold serial dilution of cell suspensions was spotted onto YEPD plates supplemented with indicated concentrations of drugs and incubated at 30°C for 48 hr. (d) Representative images of transmission electron microscopy analysis for the cell walls of wild type strain SC5314 and ifu5Δ/Δ. Arrow indicates the mannofibril layer. (e) Analysis of carbohydrate content of cell wall. Cell walls of the wild type, ifu5Δ/Δ, and the reconstituted strain were acid hydrolysed, and released monosaccharide was detected by HPAEC-PAD using a CarboPac PA10 analytical column. Results are expressed as a percentage of dried cell wall (μg°mg^-1). Shown is the mole percentage average from four or five replicates analysed over two experiments and two-tailed, unpaired t test was used to determine the statistical relevance. **p < .01, ***p < .001. (f) Flocculation assay was done by measuring optical density of the cultures directly after vortex mixing followed by a resting period of 15 min. Values shown represent the ratio of the OD_final/OD_initial. Values are means ± SD and are derived from three independent cultures. (g) Mitogen-activated protein kinase activation in indicated strains grown in YEPD medium at 37°C was performed. Anti-phospho-p44/p42 MAPK (Thr202/Tyr204) antibody was used to detect dually phosphorylated Mkc1 and Cek1 MAPKs. (h) Expression of PMT1 in ifu5Δ/Δ. Fold change in (a), (b), and (h; mutant/wild type or treated/untreated) is calculated by 2^-ΔΔCT, normalised to ACT1 (endogenous control), with untreated strain as calibrator. Values are means ± SD and derived from three independent RNA preparations. Two-tailed, unpaired t test was used to determine the statistical relevance. **p < .01, ***p < .001