ABSTRACT: In filamentous strains of S. cerevisiae, nitrogen stress elicits a complex morphogenetic program resulting in the transition to a filamentous form of growth. The transcription factors Flo8p and Mss11p are both required for this filamentous growth transition, in that homozygous diploid flo8(delta/delta) and mss11(delta/delta) strains do not undergo filamentation under conditions of nitrogen stress. To identify genes that are regulated either directly or indirectly by the Flo8p and Mss11p transcription factors, we have implemented DNA microarray analysis to profile changes in mRNA levels in homozygous diploid strains of the filamentous (Sigma)1278b genetic background deleted for FLO8 and MSS11, respectively, under conditions of nitrogen stress. The transcriptional profiles will identify genes with altered transcriptional levels in the deletion mutants, serving as a means to identify cellular processes and signaling pathways regulated by Flo8p and Mss11p function. Deletion mutants were constructed using a one-step polymerase chain reaction (PCR)-based gene disruption strategy with the G418 resistance cassette from plasmid pFA6a-KanMX6. After the haploid mutants were confirmed via PCR, the strains were allowed to mate on YPD+G418 plates for approximately 20 hours at 30 degrees Celsius. Mated cells were then streaked on SC-Trp-Leu plates to select for Y825 x HLY337 diploids. Yeast transformations were performed according to standard lithium acetate-mediated protocols with modifications. Yeast strains (wild-type and deletion mutants) were cultured under conditions of nitrogen stress (low ammonium media), and RNA was prepared using the Poly(A) Purist kit (Ambion, Austin, TX). RNA concentration and purity were determined spectrophotometrically and by gel electrophoresis. Microarray hybridization was performed with the Yeast Genome S98 Array using standard protocols (Affymetrix, Inc, Santa Clara, CA). Differentially expressed genes were identified by significance analysis of microarrays (SAM). SAM computed a nonparametric score for each gene by dividing the between-group difference of (normalized log) gene expression levels and the within-group difference of gene expression levels. The score was then compared with random permutation scores. The random permutation scores for a gene were computed in the same manner as the original score, but based on randomly sampled gene expressions. If the difference between the original score and the random permutation score was larger than a chosen threshold value, the corresponding change in gene expression was claimed to be significant. Each threshold value corresponded to a false discovery rate (the percentage of genes identified as being significant by chance alone). For all deletion comparisons in this study, we used SAM’s two-class unpaired analysis function, with significance thresholds selected so that the corresponding false discovery rate was 0.
Project description:To mine gene expression data sets effectively, analysis frameworks need to incorporate methods that identify intergenic relationships within enriched biologically relevant subpathways. For this purpose, we developed the Topology Enrichment Analysis frameworK (TEAK). TEAK employs a novel in-house algorithm and a tailor-made Clique Percolation Method to extract linear and nonlinear KEGG subpathways, respectively. TEAK scores subpathways using the Bayesian Information Criterion for context specific data and the Kullback-Leibler divergence for case-control data. In this article, we utilized TEAK with experimental studies to analyze microarray data sets profiling stress responses in the model eukaryote Saccharomyces cerevisiae. Using a public microarray data set, we identified via TEAK linear sphingolipid metabolic subpathways activated during the yeast response to nitrogen stress, and phenotypic analyses of the corresponding deletion strains indicated previously unreported fitness defects for the dpl1? and lag1? mutants under conditions of nitrogen limitation. In addition, we studied the yeast filamentous response to nitrogen stress by profiling changes in transcript levels upon deletion of two key filamentous growth transcription factors, FLO8 and MSS11. Via TEAK we identified a nonlinear glycerophospholipid metabolism subpathway involving the SLC1 gene, which we found via mutational analysis to be required for yeast filamentous growth.
Project description:Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Delta rrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Delta rrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Delta rrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Delta rrg-1 strains. OS-2-phosphate levels are also low in Delta rrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1(D921N) allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway.
Project description:The ability to switch between different morphological forms is an important feature of Candida albicans and is relevant to its pathogenesis. Many conserved positive and negative transcription factors are involved in morphogenetic regulation of the two dimorphic fungi Candida albicans and Saccharomyces cerevisiae. In S. cerevisiae, the transcriptional repressor Sfl1 and the activator Flo8 function antagonistically in invasive and filamentous growth. We have previously reported that Candida albicans Flo8 is a transcription factor essential for hyphal development and virulence in C. albicans. To determine whether a similar negative factor exists in C. albicans, we identified Candida albicans Sfl1 as a functional homolog of the S. cerevisiae sfl1 mutant. Sfl1 is a negative regulator of hyphal development in C. albicans. Deletion of C. albicans SFL1 enhanced filamentous growth and hypha-specific gene expression in several media and at several growth temperatures. Overexpression of the SFL1 led to a significant reduction of filament formation. Both deletion and overexpression of the SFL1 attenuated virulence of C. albicans in a mouse model. Deleting FLO8 in an sfl1/sfl1 mutant completely blocked hyphal development in various growth conditions examined, suggesting that C. albicans Sfl1 may act as a negative regulator of filamentous growth by antagonizing Flo8 functions. We suggest that, similar to the case for S. cerevisiae, a combination of dual control by activation and repression of Flo8 and Sfl1 may contribute to the fine regulatory network in C. albicans morphogenesis responding to different environmental cues.
Project description:The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.
Project description:Pseudohyphal growth is a developmental pathway seen in some strains of yeast in which cells form multicellular filaments in response to environmental stresses. We used multiplexed transposon "Calling Cards" to record the genome-wide binding patterns of 28 transcription factors (TFs) in nitrogen-starved yeast. We identified TF targets relevant for pseudohyphal growth, producing a detailed map of its regulatory network. Using tools from graph theory, we identified 14 TFs that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA-binding preferences for these key TFs were unknown. Using Calling Card data, we predicted the in vivo DNA-binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them more rapidly to execute the filamentous growth program when nitrogen starved if they had been previously exposed to this condition.
Project description:Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.
Project description:The genome of the filamentous fungus Neurospora crassa contains a single gene encoding a heterotrimeric G-protein beta subunit, gnb-1. The predicted GNB-1 protein sequence is most identical to G beta proteins from the filamentous fungi Cryphonectria parasitica and Aspergillus nidulans. N. crassa GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to G beta proteins from budding and fission yeasts. Previous studies in animal and fungal systems have elucidated phenotypes of G beta null mutants, but little is known about the effects of G beta loss on G alpha levels. In this study, we analyzed a gnb-1 deletion mutant for cellular phenotypes and levels of the three G alpha proteins. Delta gnb-1 strains are female-sterile, with production of aberrant fertilized reproductive structures. Delta gnb-1 strains conidiate more profusely and have altered mass on solid medium. Loss of gnb-1 leads to inappropriate conidiation and expression of a conidiation-specific gene during growth in submerged culture. Intracellular cyclic AMP levels are reduced by 60% in vegetative plate cultures of delta gnb-1 mutants. Loss of gnb-1 leads to lower levels of the three G alpha proteins under a variety of conditions. Analysis of transcript levels for the gna-1 and gna-2 G alpha genes in submerged cultures indicates that regulation of G alpha protein levels by gnb-1 is posttranscriptional. The results suggest that GNB-1 directly regulates apical extension rate and mass accumulation. In contrast, many other delta gnb-1 phenotypes, including female sterility and defective conidiation, can be explained by altered levels of the three N. crassa G alpha proteins.
Project description:Heterotrimeric G proteins, consisting of alpha, beta and gamma subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein alpha subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of delta gna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a delta gna-1 strain accentuates the delta gna-1 phenotypes. delta gna-1 delta gna-2 strains have a slower rate of hyphal apical extension than delta gna-1 strains on hyperosmotic media. Moreover, delta gna-1 delta gna-2 mutants have more pronounced defects in female fertility than delta gna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein alpha subunits with overlapping functions in eukaryotic microbes.
Project description:The transcription factor Flo8 is essential for filamentous growth in Saccharomyces cerevisiae and is regulated under the cAMP/protein kinase A (PKA) pathway. To determine whether a similar pathway/regulation exists in Candida albicans, we have cloned C. albicans FLO8 by its ability to complement S. cerevisiae flo8. Deleting FLO8 in C. albicans blocked hyphal development and hypha-specific gene expression. The flo8/flo8 mutant is avirulent in a mouse model of systemic infection. Genome-wide transcription profiling of efg1/efg1 and flo8/flo8 using a C. albicans DNA microarray suggests that Flo8 controls subsets of Efg1-regulated genes. Most of these genes are hypha specific, including HGC1 and IHD1. We also show that Flo8 interacts with Efg1 in yeast and hyphal cells by in vivo immunoprecipitation. Similar to efg1/efg1, flo8/flo8 and cdc35/cdc35 show enhanced hyphal growth under an embedded growth condition. Our results suggest that Flo8 may function downstream of the cAMP/PKA pathway, and together with Efg1, regulates the expression of hypha-specific genes and genes that are important for the virulence of C. albicans.
Project description:Physiological levels of CO(2) have a profound impact on prominent biological attributes of the major fungal pathogen of humans, Candida albicans. Elevated CO(2) induces filamentous growth and promotes white-to-opaque switching. However, the underlying molecular mechanisms of CO(2) sensing in C. albicans are insufficiently understood. Here we identify the transcription factor Flo8 as a key regulator of CO(2)-induced morphogenesis in C. albicans by screening a gene null mutant library. We show that Flo8 is required for CO(2)-induced white-to-opaque switching, as well as for filamentous growth. Ectopic expression of FLO8 hypersensitizes C. albicans cells to the elevated CO(2) levels. Furthermore, we demonstrate that CO(2) signaling in C. albicans involves two pathways: the already reported cAMP/protein kinase A and another major one that is unidentified. The two pathways converge on the transcription factor Flo8, which is the master regulator of CO(2) sensing in C. albicans and plays a critical role in regulation of white-to-opaque switching and filamentous growth. Our findings provide new insights into the understanding of CO(2) sensing in pathogenic fungi that have important implications for higher organisms.