The S769E substitution in the Cas5 DNA-binding domain phenocopies CAS5 deletion. a Schematic showing the position of S769 in the Cas5 zinc finger domain and the alignment of Cas5 orthologs in related fungal species. The alignment was performed using the Candida Genome Database (CGD), and the phosphorylation site-prediction score was generated using NetPhos. b The phosphomimetic S769E substitution in Cas5 confers hypersensitivity to caspofungin. Caspofungin susceptibility assays were conducted in YPD medium. Growth was measured by absorbance at 600 nm after 48 h at 30 °C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with color using Treeview (see color bar in Fig. 2). c The S769E substitution blocks induction of cell wall genes in response to caspofungin treatment. The transcript levels of ECM331 and PGA13 were monitored by qRT-PCR and normalized to GPD1. Plotted are the fold-changes in transcript levels of ECM331 or PGA13 in response to caspofungin relative to untreated. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. The fold change in gene expression for each mutant strain was compared with the wild-type strain using one-way ANOVA in GraphPad Prism (**P < 0.01). d Phosphomutations in CAS5 do not affect band shifts associated with activation of the cell wall stress response, as observed upon caspofungin treatment. Cas5 was monitored by western blot and detected using an α-HA antibody. Full blots are shown in Supplementary Fig. 2f