TOR and RAS pathways regulate desiccation tolerance in<i>Saccharomyces cerevisiae</i>

Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas

doi:10.1091/mbc.e12-07-0524

Cited by 48 publications

(36 citation statements)

References 48 publications

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“…To assess percentage relative viability of samples using flow cytometry, the number of cells categorized as viable, based on a lack of fluorescence, was divided by the number of total cells counted and multiplied by 100. The values of culture viability (% relative viability) we observed using the standard plating method were the same as those previously observed in our laboratory: exponential phase wild-type cultures were 0.00004 ± 0.00003% viable, stationary phase wild-type cultures were 40 ± 11% viable and stationary phase petite cells were 0.007 ± 0.007% viable [value ± standard error of the mean (SEM)] (Calahan et al, 2011;Welch et al, 2013). Culture viability as determined by the tadpoling method was not significantly different from plating: exponential phase wild-type cultures were 0.00008 ± 0.00006% viable, stationary phase wild-type cultures were 23 ± 6% viable and stationary phase petite cells were 0.002 ± 0.002% viable.…”

Section: Resultssupporting

confidence: 68%

“…To obtain cultures with reduced viability on which to test these methods, we utilized desiccation. Desiccated cultures have been shown to vary greatly in their survival rates from 20% to 0.0001%, depending on their genetic background or growth conditions (Calahan et al, 2011;Welch et al, 2013). We prepared cultures of cells that have been shown to vary greatly in desiccation tolerancestationary phase and exponential phase wild-type cells, and stationary phase petite cells (mrpl16Δ).…”

Section: Resultsmentioning

confidence: 99%

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A simple colony‐formation assay in liquid medium, termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Welch

Koshland

2013

Yeast

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Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst~10 8 dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space-saving method, amenable to high-throughput screens, for accurately measuring yeast cell viability.

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Section: Resultssupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

A simple colony‐formation assay in liquid medium, termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Welch

Koshland

2013

Yeast

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“…1A). Thus, inactivating intracellular trehalases increased both a cell's ability to accumulate intracellular trehalose and conferred the highest induced desiccation tolerance recorded for logarithmically growing cells (8,9,18).…”

Section: Import Of Extracellular Trehalose Confers Robust Desiccationmentioning

confidence: 97%

Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae

Tapia

Young

Fox³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

154

132

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Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.trehalose | desiccation | yeast | anhydrobiosis | stress

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“…Moreover, the osmolyte accumulation under drought can lead to misfolding and aggregation of proteins [39], and these changes can potentially limit cells' responses to desiccation [40]. Therefore, to better understand the drought resistant mechanism in E. pusillum, we investigated the expression changes of genes commonly involved in the response to stresses in other organisms, such as oxidative stress, osmotic regulation and post-translational processing under our drought treatment.…”

Section: The Expression Changes Of Gene Commonly Involved In Stress Rmentioning

confidence: 99%

Comparative transcriptome analysis of the lichen-forming fungus Endocarpon pusillum elucidates its drought adaptation mechanisms

Wang

Zhang

Zhou

et al. 2014

Sci. China Life Sci.

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The lichen-forming fungus was isolated from the desert lichen Endocarpon pusillum that is extremely drought resistant. To understand the molecular mechanisms of drought resistance in the fungus, we employed RNA-seq and quantitative real-time PCR to compare and characterize the differentially expressed genes in pure culture at two different water levels and with that in desiccated lichen. The comparative transcriptome analysis indicated that a total of 1781 genes were differentially expressed between samples cultured under normal and PEG-induced drought stress conditions. Similar to those in drought resistance plants and non-lichenized fungi, the common drought-resistant mechanisms were differentially expressed in E. pusillum. However, the expression change of genes involved in osmotic regulation in E. pusillum is different, which might be the evidence for the feature of drought adaptation. Interestingly, different from other organisms, some genes involved in drought adaption mechanisms showed significantly different expression patterns between the presence and absence of drought stress in E. pusillum. The expression of 23 candidate stress responsive genes was further confirmed by quantitative real-time PCR using dehydrated E. pusillum lichen thalli. This study provides a valuable resource for future research on lichen-forming fungi and shall facilitate future functional studies of the specific genes related to drought resistance. lichen, mycobiont, dehydration, drought adaption, drought resistant Citation:Wang YY, Zhang XY, Zhou QM, Zhang XL, Wei JC. Comparative transcriptome analysis of the lichen-forming fungus Endocarpon pusillum elucidates its drought adaptation mechanisms.

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TOR and RAS pathways regulate desiccation tolerance inSaccharomyces cerevisiae

Cited by 48 publications

References 48 publications

A simple colony‐formation assay in liquid medium, termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

A simple colony‐formation assay in liquid medium, termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae

Comparative transcriptome analysis of the lichen-forming fungus Endocarpon pusillum elucidates its drought adaptation mechanisms

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