Condition‐specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response

Cells must quickly respond and efficiently adapt to environmental changes. The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program. The later is regulated by two transcription factors, Msn2 and Msn4, that integrate information from upstream pathways to produce fast, tunable, and robust response to different environmental changes. To understand this integration, we employed a systematic approach to genetically dissect the contribution of various cellular pathways to Msn2/4 regulation under a range of stress and growth conditions. We established a high‐throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single‐knockout and 1,566 double‐knockout strains that carry an HSP12‐GFP allele as a reporter for Msn2/4 activity. Based on the expression of this Msn2/4 reporter in five different conditions, we identified numerous genetic and epistatic interactions between different components in the network upstream to Msn2/4. Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three‐way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway. Synopsis Genetic dissection of the yeast general stress response network, by measuring a fluorescent Msn2/4 reporter in 68 single and 1,566 double mutants under multiple growth and stress conditions, identifies components and modes of action in this complex network. The effects of single‐gene deletions and genetic interactions are characterized upon the activation of Msn2/4 transcription factors and the general stress response. A methodology for analyzing complex genetic interaction data is established. These analyses provide new insights into the functional specialization of RAS paralogs and their crosstalk with the Mediator complex, the HOG MAPK pathway and the cAMP/PKA pathway. An emerging regulatory principle that ensures sharp transitions between tightly repressed and highly induced states is revealed. Graphical Abstract Genetic dissection of the yeast general stress response network, by measuring a fluorescent Msn2/4 reporter in 68 single and 1,566 double mutants under multiple growth and stress conditions, identifies components and modes of action in this complex network.