Bin Z. He

Quantifying survival is a common and critical task in yeast research. LIVE/DEAD stains based on plasma membrane integrity provide a rapid and high-throughput assay for yeast survival when coupled with flow cytometry. However, variations in staining buffer, dye concentration, incubation time, and flow cytometry settings can impact data quality and reproducibility. This protocol presents a standardized LIVE/DEAD assay for post-stress survival quantification in yeast using flow cytometry. After treating Candida glabrata, an opportunistic yeast pathogen, with different doses of hydrogen peroxide, the post-stress samples were stained with a two-component LIVE/DEAD stain consisting of SYTO 9 and propidium iodide (PI). Flow cytometry was used to distinguish live, damaged, and dead cell populations and quantify their percentages in each sample. Survival estimates based on the percent live statistic were compared to the Colony Forming Unit (CFU) result on the same sample. The two methods yielded consistent results for the mock- and lethal dose (1 M H2O2)-treated samples. At the sublethal dose of 100 mM H2O2, SYTO 9/PI estimated a higher survival rate than CFU, reflecting a key difference between the two, where the protocol presented here evaluates cell survival immediately after the stress, while CFU quantifies the percent of cells able to recover and reproduce. Hence, this protocol measures viability at an earlier stage of the cell-death process. In summary, the protocol described here provides a fast and scalable alternative to CFU for post-stress survival quantification in yeast. Its results provide complementary information to CFU by evaluating survival at an earlier stage and distinguishing between dead and damaged cells.

Quantifying survival is a common and critical task in yeast research. LIVE/DEAD stains based on plasma membrane integrity provide a rapid and high-throughput assay for yeast survival when coupled with flow cytometry. However, variations in staining buffer, dye concentration, incubation time, and flow cytometry settings can impact data quality and reproducibility. This protocol presents a standardized LIVE/DEAD assay for post-stress survival quantification in yeast using flow cytometry. After treating Candida glabrata, an opportunistic yeast pathogen, with different doses of hydrogen peroxide, the post-stress samples were stained with a two-component LIVE/DEAD stain consisting of SYTO 9 and propidium iodide (PI). Flow cytometry was used to distinguish live, damaged, and dead cell populations and quantify their percentages in each sample. Survival estimates based on the percent live statistic were compared to the Colony Forming Unit (CFU) result on the same sample. The two methods yielded consistent results for the mock- and lethal dose (1 M H2O2)-treated samples. At the sublethal dose of 100 mM H2O2, SYTO 9/PI estimated a higher survival rate than CFU, reflecting a key difference between the two, where the protocol presented here evaluates cell survival immediately after the stress, while CFU quantifies the percent of cells able to recover and reproduce. Hence, this protocol measures viability at an earlier stage of the cell-death process. In summary, the protocol described here provides a fast and scalable alternative to CFU for post-stress survival quantification in yeast. Its results provide complementary information to CFU by evaluating survival at an earlier stage and distinguishing between dead and damaged cells.

Combinatorial control by transcription factors (TFs) is central to eukaryotic gene regulation, yet its mechanism, evolution, and regulatory impact are not well understood. Here we use natural variation in the yeast phosphate starvation (PHO) response to examine the genetic basis and species variation in TF interdependence. In Saccharomyces cerevisiae, the main TF Pho4 relies on the co-TF Pho2 to regulate ~28 genes, whereas in the related pathogen Candida glabrata, Pho4 has reduced Pho2 dependence and regulates ~70 genes. We found C. glabrata Pho4 (CgPho4) binds the same motif with 3–4 fold higher affinity. Machine learning and yeast one-hybrid assay identify two intrinsically disordered regions (IDRs) in CgPho4 that boost its activation domain’s activity. In ScPho4, an IDR next to the DNA binding domain both allows for enhanced activity with Pho2 and inhibits activity without Pho2. This study reveals how IDR divergence drives TF interdependence evolution by influencing activation potential and autoinhibition.

Quantifying post-stress survival in yeasts is crucial for biological, biomedical, and industrial research. Traditional methods like Colony Forming Unit (CFU) assays are labor-intensive and time-consuming. In this study, we systematically characterize a two-dye (SYTO 9 / Propidium Iodide) LIVE/DEAD assay coupled with flow cytometry to rapidly and scalably quantify post-stress survival in diverse yeast species. By optimizing staining buffer, dye concentrations and staining time, we minimized artifacts and improved reproducibility. Notably, we identify an “Intermediate” population containing damaged cells with enhanced SYTO 9 uptake but little propidium iodide (PI) accumulation under sublethal stress, providing finer gradations of cellular damage compared to CFU or PI staining alone. We demonstrate the assay’s applicability across Candida glabrata , Saccharomyces cerevisiae , and Candida albicans after hydrogen peroxide treatment and in C. glabrata after Amphotericin B exposure. While CFU is more sensitive at lower stress levels, the SYTO 9/PI staining effectively distinguishes sublethal from lethal doses, offering a valuable alternative for rapid, high-throughput survival quantification.

Combinatorial control by transcription factors (TFs) is central to eukaryotic gene regulation, yet its mechanism, evolution, and regulatory impact are not well understood. Here we use natural variation in the yeast phosphate starvation (PHO) response to examine the genetic basis and species variation in TF interdependence. In Saccharomyces cerevisiae , main TF Pho4 relies on the co-TF Pho2 to regulate ~28 genes, whereas in the related pathogen Candida glabrata , Pho4 has reduced Pho2 dependence and regulates ~70 genes. We found C. glabrata Pho4 (CgPho4) binds the same motif with 3-4 fold higher affinity. Machine learning and yeast one-hybrid assay identify two intrinsically disordered regions (IDRs) in CgPho4 that boost its activation domain's activity. In ScPho4, an IDR next to the DNA binding domain both allows for enhanced activity with Pho2 and inhibits activity without Pho2. This study reveals how IDR divergence drives TF interdependence evolution by influencing activation potential and autoinhibition.

Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H₂O₂ stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae. This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of-Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1’s response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.

Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H ₂ O ₂ stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae . This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of- Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1’s response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.

Opportunistic yeast pathogens arose multiple times in the Saccharomycetes class, including the recently emerged, multidrug-resistant Candida auris. We show that homologs of a known yeast adhesin family in Candida albicans, the Hyr/Iff-like (Hil) family, are enriched in distinct clades of Candida species as a result of multiple, independent expansions. Following gene duplication, the tandem repeat-rich region in these proteins diverged extremely rapidly and generated large variations in length and β-aggregation potential, both of which were known to directly affect adhesion. The conserved N-terminal effector domain was predicted to adopt a β-helical fold followed by an α-crystallin domain, making it structurally similar to a group of unrelated bacterial adhesins. Evolutionary analyses of the effector domain in C. auris revealed relaxed selective constraint combined with signatures of positive selection, suggesting functional diversification after gene duplication. Lastly, we found the Hil family genes to be enriched at chromosomal ends, which likely contributed to their expansion via ectopic recombination and break-induced replication. Combined, these results suggest that the expansion and diversification of adhesin families generate variation in adhesion and virulence within and between species and are a key step toward the emergence of fungal pathogens.

He BZ, Zhou X, O’Shea EK. 2017. Evolution of reduced co-activator dependence led to target expansion of a starvation response pathway. eLife 6:e25157. doi: 10.7554/eLife.25157.

Opportunistic yeast pathogens evolved multiple times in the Saccharomycetes class, including the recently emerged, multidrug-resistant Candida auris . We show that homologs of a known yeast adhesin family in Candida albicans , the Hyr/Iff-like (Hil) family, are enriched in distinct clades of Candida species as a result of multiple, independent expansions. Following gene duplication, the tandem repeat-rich region in these proteins diverged extremely rapidly and generated large variations in length and β-aggregation potential, both of which were known to directly affect adhesion. The conserved N-terminal effector domain was predicted to adopt a β-helical fold followed by an α-crystallin domain, making it structurally similar to a group of unrelated bacterial adhesins. Nonsynonymous-to-synonymous substitution rate analysis of the effector domain in C. auris revealed relaxed selective constraint and signatures of positive selection, suggesting functional diversification after gene duplication. Lastly, we found the Hil family genes to be enriched at chromosomal ends, which likely contributed to their expansion via ectopic recombination and break-induced replication. We hypothesize that the expansion and diversification of adhesin families are a key step toward the emergence of fungal pathogens and also generate variation in adhesion and virulence within and between species.