All organisms must respond to stressful stimuli that result from external environmental changes or internal defects caused by mutation and disease. Decades of research have characterized the mechanisms for surviving individual stresses, by mapping downstream protection systems as well as upstream signaling pathways that mediate these responses 
. However, much less is known about the effects of combinatorial stress treatments and how cells defend against compound stresses. For example, stressful environmental changes in nature likely occur together, either simultaneously or in close succession, especially for microbes living in natural conditions. How the mechanisms of stress defense differ when cells experience successive stresses rather than a single insult is poorly understood.
Successive stress treatments can cause cells to acquire resistance to a severe (‘secondary’) stress after experiencing an initial mild (‘primary’) dose of stress. Acquired stress resistance can occur if the mild and severe treatments represent the same stressor but also across different mild and severe stresses (known as ‘cross-stress’ protection). Acquired stress resistance has been observed in diverse organisms, including yeast, bacteria, archaea, plants, flies, and mammals including mice and humans 
. A better understanding of how cells are able to increase their resistance to further insults has potential medical application for decreasing cell death and improving human recovery from stressful events such as chemotherapy treatments and ischemia following heart attack or stroke 
In yeast, it had been suggested that acquired stress resistance in general, and cross-stress protection specifically, may be due to activation of the Environmental Stress Response (ESR) 
. The ESR is a gene expression response commonly activated by a wide variety of stressful conditions 
. It includes induced expression of ~300 genes involved in stress defense, and reduced expression of ~600 genes broadly involved in protein synthesis and growth. However, we previously showed that ESR activation alone is insufficient to explain cross-stress protection 
. Moreover, the ‘general-stress’ transcription factors MSN2
are conditionally required for acquired stress resistance, depending on the precise combination of mild and severe stress treatments 
. These results revealed that the mechanism of acquired stress resistance is more complex than previously suspected and suggested that the response occurs through different mechanisms depending on the mild stress pretreatment.
Many studies have identified genes required to survive a single dose of oxidative stress, and several studies characterized increased tolerance after preconditioning (reviewed in 
). The majority of these studies used single-gene approaches, though several used the yeast deletion collection to interrogate the entire genome 
. Kelley et al
. (2009) identified genes required to survive an acute dose of H2
and genes necessary to acquire H2
resistance following a mild H2
pretreatment. They found that the genes required for acquisition of H2
tolerance only partially overlapped the genes required to survive the acute dose alone, indicating that the mechanism of acquired H2
tolerance is distinct from the mechanism of basal H2
. The mechanism of cross-stress protection, in which the mild pretreatment is a different stressor than the subsequent severe stress, is largely unexplored.
Here, we leveraged the power of yeast genetics and high-throughput analysis to identify genes and processes important for acquired resistance to severe H2
stress after each of three mild pretreatments (mild NaCl, heat shock, or DTT treatment). We used the pooled yeast deletion collection 
, including ~4,800 homozygous diploid nonessential genes (homozygous profiling), ~1,300 heterozygous diploid essential genes (haploinsufficiency profiling), and 1,140 strains harboring DAmP alleles of the essential genes (in which the transcript is destabilized due to insertion of a drug marker into the 3' UTR 
) to query the vast majority of the yeast genome in a single experiment. We found that, although each pretreatment provided similar levels of subsequent H2
resistance, different genes and processes were required depending on the mild stress used. Functional analysis of the genes required during each pretreatment provided new insights into the relationships between regulators and processes. Acquired stress resistance thus serves as a unique phenotype through which to uncover new insights into stress biology.