The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

White collar‐1 (WC‐1) and white collar‐2 (WC‐2) are essential for light‐mediated responses in Neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and putative photoreceptors remain poorly characterized. Unsupervised hierarchical clustering of genome‐wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. Evidence is shown for photoreception in the absence of the dominant, and here confirmed, white collar complex (WCC) that regulates both types of light responses. VVD primarily modulates late responses, whereas light‐responsive submerged protoperithecia‐1 (SUB‐1), a GATA family transcription factor, is essential for most late light gene expression. After a 15‐min light stimulus, the WCC directly binds the sub‐1 promoter. Bioinformatics analysis detects many early light response elements (ELREs), as well as identifying a late light response element (LLRE) required for wild‐type activity of late light response promoters. The data provide a global picture of transcriptional response to light, as well as illuminating the cis‐ and trans‐acting elements comprising the regulatory signalling cascade that governs the photobiological response.

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.A well-recognized model for fungal genetics, photobiology, and circadian systems, Neurospora is the established model for nearly all aspects of growth and metabolism among the filamentous fungi. The core clock of Neurospora crassa has been well studied; its oscillator comprises a transcription-translation feedback loop involving a complex of five core proteins, White Collar 1 (WC-1), White Collar 2 (WC-2), Frequency (FRQ), Frequency Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK1), as well as several ancillary factors. Transcription factors (TFs) WC-1 and WC-2 form the white collar complex (WCC), which drives the rhythmic expression of FRQ. FRQ binds to FRH to form the FRQ/FRH complex (FFC), which then acts with CK1 on the WCC to inhibit its activity, thus closing the loop. Throughout the circadian cycle, FRQ interacts with many partners that affect the number and location of its posttranslational modifications as well as its stability; it is these posttranslational modifications of FRQ that set the length of the circadian period (1-3).Although historically much interest has focused on clock mechanism, it is through circadian control of transc...

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to acccomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.

Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR, with its broad ligand binding specificity, is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.