MAPK pathways are drivers of morphogenesis and stress response in eukaryotes. A major function of MAPK pathways is the transcriptional induction of target genes, which produce proteins that collectively generate a cellular response. One approach to comprehensively understand how MAPK pathways regulate cellular responses is to characterize the individual functions of their transcriptional targets. Here, by examining uncharacterized targets of the MAPK pathway that positively regulates filamentous growth in Saccharomyces cerevisiae (fMAPK pathway), we identified a new role for the pathway in negatively regulating invasive growth. Specifically, four targets were identified that had an inhibitory role in invasive growth: RPI1, RGD2, TIP1, and NFG1/YLR042c. NFG1 was a highly induced unknown open reading frame that Negatively regulated the Filamentous Growth MAPK pathway. We also identified SFG1, which encodes a transcription factor, as a target of the fMAPK pathway. Sfg1p promoted cell adhesion independently from the fMAPK pathway target and major cell adhesion flocculin Flo11p, by repressing genes encoding presumptive cell wall degrading enzymes. Sfg1p also contributed to FLO11 expression. Sfg1p and Flo11p regulated different aspects of cell adhesion, and their roles varied based on the environment. Sfg1p also induced an elongated cell morphology, presumably through a cell-cycle delay. Thus, the fMAPK pathway coordinates positive and negative regulatory proteins to fine-tune filamentous growth resulting in a nuanced response. Functional analysis of other pathways' targets may lead to a more comprehensive understanding of how signaling cascades generate biological responses.

Phenotypes can change during exposure to different environments through the regulation of signaling pathways that operate in integrated networks. How signaling networks produce different phenotypes in different settings is not fully understood. Here, Gene by Environment Interactions (GEIs) were used to explore the regulatory network that controls filamentous/invasive growth in the yeast Saccharomyces cerevisiae. GEI analysis revealed that the regulation of invasive growth is decentralized and varies extensively across environments. Different regulatory pathways were critical or dispensable depending on the environment, microenvironment, or time point tested, and the pathway that made the strongest contribution changed depending on the environment. Some regulators even showed conditional role reversals. Ranking pathways’ roles across environments revealed an under-appreciated pathway (OPI1) as the single strongest regulator among the major pathways tested (RAS, RIM101, and MAPK). One mechanism that may explain the high degree of regulatory plasticity observed was conditional pathway interactions, such as conditional redundancy and conditional cross-pathway regulation. Another mechanism was that different pathways conditionally and differentially regulated gene expression, such as target genes that control separate cell adhesion mechanisms (FLO11 and SFG1). An exception to decentralized regulation of invasive growth was that morphogenetic changes (cell elongation and budding pattern) were primarily regulated by one pathway (MAPK). GEI analysis also uncovered a round-cell invasion phenotype. Our work suggests that GEI analysis is a simple and powerful approach to define the regulatory basis of complex phenotypes and may be applicable to many systems.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs indcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed thatdcp2Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased bydcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs indcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated bydcp2Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

MAPK signaling pathways are conserved drivers of cell differentiation and stress responses. In many fungi including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model yeast Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was employed, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Different stimuli revealed different modes of pathway signaling. For example, combinations of inducers (galactose and ethanol) stimulated the pathway to near maximal levels, which showed dispensability of several fMAPK pathway components (e.g. mucin sensor, PAK), but not others (e.g. adaptor, MAPKKK) and required the Ras2-PKA pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically-relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments.

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