RESUMEN
Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa, and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so/ham-1, and show that it is the absence of this gene-and not of ada-3-that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations-of a largely unclear nature-that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures.
RESUMEN
Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.
Asunto(s)
Proteínas Fúngicas/fisiología , Neurospora crassa/crecimiento & desarrollo , Factores de Transcripción/fisiología , Proteínas Fúngicas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Hifa/genética , Hifa/crecimiento & desarrollo , Neurospora crassa/genética , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Factores de Transcripción/genéticaRESUMEN
Wiley is proud to announce the launch of Health Science Reports. I talked to the Editor in Chief, Dr. Charles Young, about the journal, its philosophy, and its contribution to scientific communication in the health sciences.
RESUMEN
Circadian clocks drive daily oscillations in a variety of biological processes through the coordinate orchestration of precise gene expression programs. Global expression profiling experiments have suggested that a significant fraction of the transcriptome and proteome is under circadian control, and such output rhythms have historically been assumed to rely on the rhythmic transcription of these genes. Recent genome-wide studies, however, have challenged this long-held view and pointed to a major contribution of posttranscriptional regulation in driving oscillations at the messenger RNA (mRNA) level, while others have highlighted extensive clock translational regulation, regardless of mRNA rhythms. There are various examples of genes that are uniformly transcribed throughout the day but that exhibit rhythmic mRNA levels, and of flat mRNAs, with oscillating protein levels, and such observations have largely been considered to result from independent regulation at each step. These studies have thereby obviated any connections, or coupling, that might exist between the different steps of gene expression and the impact that any of them could have on subsequent ones. Here, we argue that due to both biological and technical reasons, the jury is still out on the determination of the relative contributions of each of the different stages of gene expression in regulating output molecular rhythms. In addition, we propose that through a variety of coupling mechanisms, gene transcription (even when apparently arrhythmic) might play a much relevant role in determining oscillations in gene expression than currently estimated, regulating rhythms at downstream steps. Furthermore, we posit that eukaryotic genomes regulate daily RNA polymerase II (RNAPII) recruitment and histone modifications genome-wide, setting the stage for global nascent transcription, but that tissue-specific mechanisms locally specify the different processes under clock control.
Asunto(s)
Ritmo Circadiano/genética , Eucariontes/genética , Regulación de la Expresión Génica/genética , Proteoma , Transcripción Genética , Transcriptoma , Relojes Circadianos , Genoma , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages.
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Relojes Circadianos , Neurospora crassa/citología , Neurospora crassa/fisiología , Animales , Botrytis/fisiología , Proteínas CLOCK/fisiología , Ritmo CircadianoRESUMEN
High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER) is exceeded, a pathway known as the unfolded protein response (UPR) is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Neurospora crassa/crecimiento & desarrollo , Respuesta de Proteína Desplegada/fisiología , Celulosa , Retículo Endoplásmico/fisiología , Proteínas Fúngicas/fisiología , Genes Fúngicos/fisiología , Neurospora crassa/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only â¼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for â¼34% of the â¼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.