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An Optimized LIVE/DEAD Assay Coupled with Flow Cytometry for Quantifying Post-Stress Survival in Yeast Cells

Quantifying survival is a common and critical task in yeast research. LIVE/DEAD stains based on plasma membrane integrity provide a rapid and ...

University of Iowa

Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of  yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In , loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, , as a thiamine phosphatase. We further determine that the 81% identical paralog  does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources.

Dynamic Changes in Yeast Phosphatase Families Allow for Specialization in Phosphate and Thiamine Starvation.

Recent studies have revealed thousands of A-to-I RNA editing events in primates, but the origination and general functions of these events are not well addressed.

Evolutionarily significant A-to-I RNA editing events originated through G-to-A mutations in primates.

Opportunistic yeast pathogens arose multiple times in the Saccharomycetes class, including the recently emerged, multidrug-resistant (MDR) 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 are 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.

Parallel expansion and divergence of an adhesin family in pathogenic yeasts.

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  and protects it against a severe HO stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, . 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.

Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast.

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.

Divergence of TORC1-mediated stress response leads to novel acquired stress resistance in a pathogenic yeast.

Animals typically respond to their reflection as a conspecific and will respond as if the reflection were another animal that they could interact with, either fearfully or aggressively. We investigated how a modified reflective environment of a standard glass aquarium affects the aggressive and fearful behaviors of the crayfish  , based on pre-determined behavior criteria. We found that the crayfish were both increasingly aggressive and slightly fearful in the reflective environment compared to minimal behavioral changes in the control non-reflective environment. Thus, our findings support that crayfish recognize their mirror image as a conspecific.

Reflective environment heightens crayfish aggressive and fearful behaviors.

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 , main TF Pho4 relies on the co-TF Pho2 to regulate ~28 genes, whereas in the related pathogen , Pho4 has reduced Pho2 dependence and regulates ~70 genes. We found  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.

Divergence in a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions.

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 , , and  after hydrogen peroxide treatment and in  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.

An Optimized LIVE/DEAD Assay Using Flow Cytometry to Quantify Post-Stress and Antifungal-Treatment Survival in Diverse Yeasts.

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.

Article	Year
Dynamic Changes in Yeast Phosphatase Families Allow for Specialization in Phosphate and Thiamine Starvation. G3 (Bethesda, Md.)\| PubMed ID: 29748198	2018
Evolutionarily significant A-to-I RNA editing events originated through G-to-A mutations in primates. Genome biology\| PubMed ID: 30712515	2019
Parallel expansion and divergence of an adhesin family in pathogenic yeasts. Genetics\| PubMed ID: 36794645	2023
Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast. bioRxiv : the preprint server for biology\| PubMed ID: 37781605	2023
Divergence of TORC1-mediated stress response leads to novel acquired stress resistance in a pathogenic yeast. PLoS pathogens\| PubMed ID: 37871123	2023
Reflective environment heightens crayfish aggressive and fearful behaviors. microPublication biology\| PubMed ID: 38854633	2024
Divergence in a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions. bioRxiv : the preprint server for biology\| PubMed ID: 39253425	2025
An Optimized LIVE/DEAD Assay Using Flow Cytometry to Quantify Post-Stress and Antifungal-Treatment Survival in Diverse Yeasts. bioRxiv : the preprint server for biology\| PubMed ID: 40502067	2025
Divergence in a eukaryotic transcription factor's co-TF dependence involves multiple intrinsically disordered regions. Nature communications\| PubMed ID: 40533454	2025

Bin Z. He

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