Given that Cas5 is known to regulate cell wall integrity22, we sought to further evaluate the impact of Cas5 on cell wall homeostasis. We observed that Cas5 has a profound impact on RNA PolII binding to genes with cell wall functions; GO terms from process ontology revealed that 23 of the 163 genes with Cas5-dependent increases in RNA PolII binding in response to caspofungin encode proteins involved in cell wall organization and biogenesis (P-value 7.45e–06, Bonferroni Correction) (Supplementary Data 3). Among these genes were WSC1 and WSC2, which encode transmembrane sensors that respond to cell wall perturbations, yet have not been previously linked to Cas5. We confirmed the induction of these transcripts in response to caspofungin was dependent on Cas5 by quantitative RT-PCR (qRT-PCR) (Fig. 3a). We next evaluated the impact of Cas5 on tolerating cell wall stress in a wild-type strain and an echinocandin-resistant mutant. We measured susceptibility of a cas5Δ/cas5Δ mutant to two cell wall perturbing agents, caspofungin and calcofluor white. The cas5Δ/cas5Δ mutant was hypersensitive to both cell wall stressors, and the hypersensitivity phenotype was complemented by re-introduction of a wild-type allele (Fig. 3b). Given that the most common mechanism of resistance to echinocandins is mutations in the drug target gene, FKS1, we tested whether Cas5 also enables Fks-dependent caspofungin resistance. Homozygous deletion of CAS5 reduced resistance of a strain carrying the Fks1F641S substitution, suggesting Cas5 is a key regulator of target-mediated echinocandin resistance (Fig. 3b). Thus, Cas5 regulates cell wall stability during periods of cell wall stress, with a profound impact on drug resistance.Fig. 3Cas5 regulates cell wall stability under basal and cell wall stress conditions. a WSC1 and WSC2, transmembrane sensors that respond to cell wall perturbation, require Cas5 for upregulation in response to caspofungin. The transcript level of WSC1 and WSC2 were monitored by qRT-PCR and normalized to TEF1. Plotted are the log2 fold-changes in transcript levels upon caspofungin treatment relative to untreated conditions in both the wildtype (black) and cas5∆/cas5∆ mutant (gray). Error bars represent standard deviation (s.d.) from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (**P < 0.01). b Cas5 is required for tolerance and resistance to cell wall stress. Caspofungin or calcofluor white 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). c Deletion of CAS5 leads to the activation of the cell wall integrity pathway in the absence of cell wall stress. A SN95 wild-type strain and a cas5Δ/cas5Δ mutant were left untreated (−) or treated for 1 h with 125 ng/ml of caspofungin (+), as indicated. Phosphorylated Mkc1 was monitored by western blot and detected with α-p44/42 antibody. Full blots are shown in Supplementary Fig. 2a. Cdc28 was detected with α-PSTAIRE antibody and used as a loading control. d RLM1, a transcription factor downstream of Mkc1 in the cell wall integrity pathway, is induced in a cas5Δ/cas5Δ mutant in the absence of cell wall tress. The transcript level of RLM1 was monitored by qRT-PCR and normalized to TEF1. Differences in transcript level upon caspofungin treatment relative to untreated conditions are plotted. Error bars represent s.d. from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (***P < 0.001)