Regulation of nutrient utilization in filamentous fungi.

Organisms must accurately sense and respond to nutrients to survive. In filamentous fungi, accurate nutrient sensing is important in the establishment of fungal colonies and in continued, rapid growth for the exploitation of environmental resources. To ensure efficient nutrient utilization, fungi have evolved a combination of activating and repressing genetic networks to tightly regulate metabolic pathways and distinguish between preferred nutrients, which require minimal energy and resources to utilize, and nonpreferred nutrients, which have more energy-intensive catabolic requirements. Genes necessary for the utilization of nonpreferred carbon sources are activated by transcription factors that respond to the presence of the specific nutrient and repressed by transcription factors that respond to the presence of preferred carbohydrates. Utilization of nonpreferred nitrogen sources generally requires two transcription factors. Pathway-specific transcription factors respond to the presence of a specific nonpreferred nitrogen source, while another transcription factor activates genes in the absence of preferred nitrogen sources. In this review, we discuss the roles of transcription factors and upstream regulatory genes that respond to preferred and nonpreferred carbon and nitrogen sources and their roles in regulating carbon and nitrogen catabolism. KEY POINTS: • Interplay of activating and repressing transcriptional networks regulates catabolism. • Nutrient-specific activating transcriptional pathways provide metabolic specificity. • Repressing regulatory systems differentiate nutrients in mixed nutrient environments.

Aspects of the Neurospora crassa Sulfur Starvation Response Are Revealed by Transcriptional Profiling and DNA Affinity Purification Sequencing.

Accurate nutrient sensing is important for rapid fungal growth and exploitation of available resources. Sulfur is an important nutrient source found in a number of biological macromolecules, including proteins and lipids. The model filamentous fungus Neurospora crassa is capable of utilizing sulfur found in a variety of sources from amino acids to sulfate. During sulfur starvation, the transcription factor CYS-3 is responsible for upregulation of genes involved in sulfur uptake and assimilation. Using a combination of RNA sequencing and DNA affinity purification sequencing, we performed a global survey of the N. crassa sulfur starvation response and the role of CYS-3 in regulating sulfur-responsive genes. The CYS-3 transcription factor bound the promoters and regulated genes involved in sulfur metabolism. Additionally, CYS-3 directly activated the expression of a number of uncharacterized transporter genes, suggesting that regulation of sulfur import is an important aspect of regulation by CYS-3. CYS-3 also directly regulated the expression of genes involved in mitochondrial electron transfer. During sulfur starvation, genes involved in nitrogen metabolism, such as amino acid and nucleic acid metabolic pathways, along with genes encoding proteases and nucleases that are necessary for scavenging nitrogen, were activated. Sulfur starvation also caused changes in the expression of genes involved in carbohydrate metabolism, such as those encoding glycosyl hydrolases. Thus, our data suggest a connection between sulfur metabolism and other aspects of cellular metabolism. IMPORTANCE Identification of nutrients present in the environment is a challenge common to all organisms. Sulfur is an important nutrient source found in proteins, lipids, and electron carriers that are required for the survival of filamentous fungi such as Neurospora crassa. Here, we transcriptionally profiled the response of N. crassa to characterize the global response to sulfur starvation. We also used DNA affinity purification sequencing to identify the direct downstream targets of the transcription factor responsible for regulating genes involved in sulfur uptake and assimilation. Along with genes involved in sulfur metabolism, this transcription factor regulated a number of uncharacterized transporter genes and genes involved in mitochondrial electron transfer. Our data also suggest a connection between sulfur, nitrogen, and carbon metabolism, indicating that the regulation of a number of metabolic pathways is intertwined.

Developing Functional Genomics Platforms for Fungi.

Fungi are responsible for diseases that result in the deaths of over a million individuals each year and devastating crop infestations that threaten global food supplies. However, outside of a select few model organisms, the majority of fungal genes are uncharacterized. The roles of these genes in the biology of the organism, pathogenesis, and mediating interactions with the environment and other microbes are unknown. Historically, fungal gene characterization has primarily relied on classical genetic screens. However, advances in sequencing technology have enabled more rapid methods of gene functional characterization. Large-scale transcriptional profiling projects are one solution to generating hypotheses about fungal gene function. Together with other 'omics techniques and newer tools that enable massively parallel mutant screens, knowledge of fungal gene function will be substantially improved. Understanding the function of fungal genes will be instrumental in increasing global food security, protecting ecosystems, and improving health outcomes.

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus.

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Network of nutrient-sensing pathways and a conserved kinase cascade integrate osmolarity and carbon sensing in Neurospora crassa.

Identifying nutrients available in the environment and utilizing them in the most efficient manner is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of carbohydrates, from simple sugars to the complex carbohydrates found in plant cell walls. The zinc binuclear cluster transcription factor CLR-1 is necessary for utilization of cellulose, a major, recalcitrant component of the plant cell wall; however, expression of clr-1 in the absence of an inducer is not sufficient to induce cellulase gene expression. We performed a screen for unidentified actors in the cellulose-response pathway and identified a gene encoding a hypothetical protein (clr-3) that is required for repression of CLR-1 activity in the absence of an inducer. Using clr-3 mutants, we implicated the hyperosmotic-response pathway in the tunable regulation of glycosyl hydrolase production in response to changes in osmolarity. The role of the hyperosmotic-response pathway in nutrient sensing may indicate that cells use osmolarity as a proxy for the presence of free sugar in their environment. These signaling pathways form a nutrient-sensing network that allows Ncrassa cells to tightly regulate gene expression in response to environmental conditions.

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

Genetically engineered transvestites reveal novel mating genes in budding yeast.

Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Two haploid cells of opposite mating types mate by signaling to each other using reciprocal pheromones and receptors, polarizing and growing toward each other, and eventually fusing to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating-type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering "transvestite" cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. Strong pheromone secretion is essential for efficient mating, and the weak mating of transvestites can be improved by boosting their pheromone production. Synthetic biology can characterize the factors that control efficiency in biological processes. In yeast, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more and less pheromone. This discrimination comes at a cost: weak mating in situations where all potential partners make less pheromone.