Genetic Analysis of Desiccation Tolerance inSaccharomyces cerevisiae

Abstract

Desiccation tolerance, the ability to survive nearly total dehydration, is a rare strategy for survival and reproduction observed in all taxa. However, the mechanism and regulation of this phenomenon are poorly understood. Correlations between desiccation tolerance and potential effectors have been reported in many species, but their physiological significance has not been established in vivo. Although the budding yeast Saccharomyces cerevisiae exhibits extreme desiccation tolerance, its usefulness has been hampered by an inability to reduce tolerance more than a few fold by physiological or genetic perturbations. Here we report that fewer than one in a million yeast cells from low-density logarithmic cultures survive desiccation, while 20-40% of cells from saturated cultures survive. Using this greatly expanded metric, we show that mutants defective in trehalose biosynthesis, hydrophilins, responses to hyperosmolarity, and hypersalinity, reactive oxygen species (ROS) scavenging and DNA damage repair nevertheless retain wild-type levels of desiccation tolerance, suggesting that this trait involves a unique constellation of stress factors. A genome-wide screen for mutants that render stationary cells as sensitive as log phase cells identifies only mutations that block respiration. Respiration as a prerequisite for acquiring desiccation tolerance is corroborated by respiration inhibition and by growth on nonfermentable carbon sources. Suppressors bypassing the respiration requirement for desiccation tolerance reveal at least two pathways, one of which, involving the Mediator transcription complex, is associated with the shift from fermentative to respiratory metabolism. Further study of these regulators and their targets should provide important clues to the sensors and effectors of desiccation tolerance.

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

“…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.…”

“…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).…”

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

“…1A) (8). Wild-type cells constitutively expressing TDH3pr-AGT1 were also extremely sensitive to desiccation when trehalose was not present in the media.…”

“…It is found in extremely high concentrations in most anhydrobiotes, including in the model organism Saccharomyces cerevisiae (6,7). In this yeast, exponentially dividing cells have very low levels of trehalose and are extremely desiccation sensitive (8). However, in saturated cultures, yeast cells accumulate high levels of a number of stress effectors, including extremely high levels of trehalose (up to 15% of dry cell mass) (6,7).…”

“…The correlative evidence for trehalose being sufficient for desiccation tolerance was provided from our previous study comparing desiccation sensitivity of saturated and exponentially dividing cultures of yeast (8). Cells in a saturated culture rapidly lose desiccation tolerance when they divide upon dilution into fresh media.…”