(A) CRG1 gene dose is important for cantharidin tolerance. Wt, crg1Δ/Δ and CRG1-overexpressing crg1Δ/Δ mutants were assessed in the presence of cantharidin in YPD. Dose-response curves were obtained by plotting OD₆₀₀ at saturation point vs. tested drug concentrations. (B) Chemical (left panel) and genetic inhibition (right panel) of protein phosphatases result in the induction of CRG1. Wt, glc7Δ/GLC7 heterozygous, sit4Δ/Δ and ptc1Δ/Δ homozygous deletion mutants grown to mid-exponential phase were incubated with or without cantharidin (250 µM). For each time point, total RNA was extracted, cDNA synthesized and the relative abundance of CRG1 transcript was analyzed by qRT-PCR. Data are the mean of at least three independent experimental replicates, and error bars represent the standard deviation. (C) Point mutations in conserved residues of the Crg1 methyltransferase domain reduce cantharidin tolerance. Site-specific mutations in the conserved motifs of methyltransferase domain are shown with the arrows. Mutated CRG1 ORFs were cloned under GAL1 promoter and transformed into crg1Δ/Δ cells. The transformants were grown in SD-URA with raffinose (2%) to mid-exponential phase and induced with galactose (2%). The fitness of point mutants based on saturation at final OD was assessed in the presence of cantharidin (6 µM). (D) A plot comparing the transcriptome profiles of crg1Δ/Δ and CRG1-overexpressing crg1Δ/Δ mutants. Exponentially grown cells were treated with cantharidin (250 µM) for 1 hour or DMSO, total RNA was extracted and synthesized cDNA was hybridized to Affymetrix Tiling arrays. Significantly different GO Biological processes are listed in Table S1. (E) Diagram showing that the methionine biosynthesis is tightly linked to SAM cycling in methylation reactions coordinated by methyltransferases. The genes that are transcriptionally different between cantharidin-resistant and sensitive mutant are shown in red. STR3 (P-value <0.0057), MET17 (P-value <0.0036), MET6 (P-value <0.012), SAM1 (P-value <0.02), SAH1 (P-value <0.03), MMP1 (P-value <0.04), SAM2 (P-value <0.056) (Table S2).

(A) Tmt1, a sequence homolog of Crg1, modifies small molecule intermediates of TCA cycle (trans-aconitate) and leucine biosynthesis (3-isopropylmalate and isopropylmaleate) to form methyl esters. (B) Hypothetical methylation of cantharidin by Crg1.

(A) Silver stained 12% SDS-PAGE of purified His-tagged Crg1. Wild-type cells (Y258) carrying empty vector BG1805 and BG1805-GAL1-CRG1 were grown in SD-URA and raffinose (2%) to mid-exponential phase. The expression of CRG1 was induced with galactose (2%) for 5 hours. His-tagged Crg1 was purified with Ni-sepharose resin. The diagram (right panel) shows the transfer of [methyl-¹⁴C] from S-adenosyl-[methyl-¹⁴C]methionine to a potential substrate by purified Crg1. (B) In vitro enzymatic reaction mixtures containing cantharidin, S-adenosyl-[methyl-¹⁴C]methionine, and Crg1 were separated by reverse phase chromatography. Radioactivity in the fractions was quantified with a scintillation counter, and a cantharidin and Crg1-dependent peak with a retention time of 18–20 minutes was identified (asterisk). (C) In vitro analysis of the reactions containing cantharidin and mutated forms of Crg1 by measurement of the amount of acid-labile volatile radioactivity (see Materials and Methods for details). (D) Additional in vitro reaction with unlabeled SAM were prepared in a similar manner and analyzed by liquid chromatography-mass spectrometry with positive ionization. The mass spectrum of the peak from the full reaction with an elution time of 18.6–18.8 minutes is shown. (E) The mass spectrum of the peak with an elution time of 19.2 minutes from the complete reaction with cantharidin, SAM and Crg1.

(A) Experimental scheme for analysis of cantharidin-specific genetic interactors of CRG1. Double deletion mutants crg1ΔxxxΔ generated through Synthetic Genetic Array (SGA) were pooled together and treated with cantharidin (30 µM) for 20 generations in YPD. Genomic DNA was isolated, unique strain-representative barcodes were PCR amplified, and the PCR products were hybridized to TAG4 arrays for the quantitative analysis of fitness of the mutants (see Materials and Methods for details). (B) Scatter plot representing cantharidin-gene interactions obtained from the comparative analysis of ura3ΔxxxΔ (control single deletion pool) and crg1ΔxxxΔ pools. CRG1-dependent interactors are highlighted in the red square. The hits are obtained from the averaged datasets (n = 6 for crg1ΔxxxΔ pools and n = 4 for ura3ΔxxxΔ). Significant negative genetic interactors were categorized according to their biological processes (P-value <0.002 before multiple testing correction) (Table S3). (C) Representative growth curves for the top hits (sensitive and resistant) that genetically interact with CRG1 in the presence of cantharidin. Cells were grown in YPD media with and without cantharidin. met22 and dbf2 deletion strains were treated with cantharidin to test their sensitivity and resistance, respectively.

(A) CRG1 is synthetically lethal with lipid-related genes in the presence of cantharidin. Cells were normalized to an equivalent OD₆₀₀,10-fold diluted, spotted onto synthetic complete defined medium containing cantharidin and incubated at 30°C. (B) Comparative phospholipid profiles of wild type and crg1 mutants in response to cantharidin. Cells grown to mid-exponential phase in YPD were treated with cantharidin (250 µM) for 2 hours. Lipid standards were added to the cells, and extracted lipids were measured using ESI-MS/MS. The quantities of lipid species are expressed as ion intensities relative to the levels in DMSO, and converted to a log2 scale. Data are the average of three samples. Statistical significance in the abundance of lipid species in the presence of cantharidin between wild type and mutants was determined using Kruskal-Wallis test, *P-value <0.05 (Table S4). (C) A simplified diagram of phospholipid biosynthesis linked to sphingolipid biosynthesis. PIs species contribute to biosynthesis of complex sphingolipids, GPI anchors, and PIPs. (D) Comparison of sphingolipid profiles of wt, crg1Δ/Δ and CRG1-overexpressing crg1Δ/Δ mutants in the presence of cantharidin (250 µM). The suffixes -B, -C, and -D on IPC and MIPC denote hydroxylation states, having two, three, or four hydroxyl groups, respectively. Statistical significance in the abundance of lipid species in the presence of cantharidin between wild type and mutants was determined using Kruskal-Wallis test, *P-value <0.05 (Table S4).

(A) Diagram showing how PIP species are involved in Pkc1-dependent changes in actin cytoskeleton. (B) Crg1 is important for actin patch integrity during cantharidin stress. Wt, crg1Δ/Δ mutant and CRG1-overexpressing crg1Δ/Δ cells were grown to mid-exponential phase at 30°C in YPD in the presence of cantharidin (250 µM) or DMSO for 1 hour. Cells fixed with formaldehyde were stained for actin with FITC-phalloidin and visualized by fluorescence microscopy. Bar, 5 µm. The number of actin patches per cell in each sample was quantified. Values are the mean of three independent replicates (n = 270–1000), error bars are the standard deviation; * P-value <0.025, ** P-value <0.0002 (Student's t-test). (C) Cantharidin induces CRG1 transcription via the Cell Wall Integrity (CWI) pathway. Cells grown to mid-exponential phase were treated with cantharidin (250 µM) in YPD for indicated time. Total RNA was extracted, cDNA was prepared and analyzed by qRT-PCR. CRG1 transcript levels were normalized to ACT1. The simplified diagram of CWI pathway is shown. Slt2 is a kinase, Rlm1 is a transcriptional activator governed by the CWI pathway. (D) A preliminary model describing Crg1-cantharidin interaction. See text for details.