Signaling pathways can regulate biological responses by the transcriptional regulation of target genes. In yeast, multiple signaling pathways control filamentous growth, a morphogenetic response that occurs in many species including fungal pathogens. Here, we examine the role of signaling pathways that control filamentous growth in regulating adhesion-dependent surface responses, including mat formation and colony patterning. Expression profiling and mutant phenotype analysis showed that the major pathways that regulate filamentous growth [filamentous growth MAPK (fMAPK), RAS, retrograde (RTG), RIM101, RPD3, ELP, SNF1, and PHO85] also regulated mat formation and colony patterning. The chromatin remodeling complex, SAGA, also regulated these responses. We also show that the RAS and RTG pathways coregulated a common set of target genes, and that SAGA regulated target genes known to be controlled by the fMAPK, RAS, and RTG pathways. Analysis of surface growth-specific targets identified genes that respond to low oxygen, high temperature, and desiccation stresses. We also explore the question of why cells make adhesive contacts in colonies. Cell adhesion contacts mediated by the coregulated target and adhesion molecule, Flo11p, deterred entry into colonies by macroscopic predators and impacted colony temperature regulation. The identification of new regulators (e.g., SAGA), and targets of surface growth in yeast may provide insights into fungal pathogenesis in settings where surface growth and adhesion contributes to virulence.

MAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.

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1MAPK pathways regulate different responses yet can share a subset of common components. In 2 this study, a genome-wide screen was performed to identify genes that, when overexpressed, 3 induce a growth reporter (FUS1-HIS3) that responds to ERK-type MAPK pathways 4 (Mating/filamentous growth or fMAPK) but not p38-type MAPK pathways (HOG) in yeast. 5Approximately 4,500 plasmids overexpressing individual yeast genes were introduced into 6 strains containing the FUS1-HIS3 reporter by high-throughput transformation. Candidate genes 7 were identified by measuring the degree of growth, which was a reflection of reporter activity. 8Of fourteen genes identified and validated by re-testing, two were metabolic controls (HIS3 and 9 ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, 10 MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, 11 KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR 12 pathway that plays an established role in nutrient signaling. MRS6 overexpression stimulated 13 filamentous/invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. 14 Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the 15 p38/HOG pathway MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 16 by two-hybrid analysis. Collectively, the data indicate that Mrs6 may function to selectively 17 propagate an ERK-dependent signal. Generally speaking, the identification of new MAPK 18 pathway regulators by genetic screening in yeast may be a useful resource for understanding 19 signaling pathway regulation. 20 21 PC7386 MATa trpl-901PC7444 MATa ura3-52 leu2 ssk1 pRS316 [d] This study PC7445 MATa ura3-52 leu2 ssk1 pGAL-BMH1 [e] This study PC7446 MATa ura3-52 leu2 ssk1 pGAL-BMH2 [e] This study PC7447 MATa ura3-52 leu2 ssk1 pGAL-MRS6 [e] This study PC7448 MATa ura3-52 leu2 ssk1 pGAL-KAR2 [e] This study [a] Strains are in the ∑1287b background unless otherwise indicated.