Subpopulations of hyphae secrete proteins or resist heat stress in Aspergillus oryzae colonies.

Hyphae at the outer part of colonies of Aspergillus niger and Aspergillus oryzae are heterogeneous with respect to transcriptional and translational activity. This heterogeneity is maintained by Woronin body mediated closure of septal pores that block interhyphal mixing of cytoplasm. Indeed, heterogeneity between hyphae is abolished in ΔhexA strains that lack Woronin bodies. The subpopulation of hyphae with high transcriptional and translational activity secretes enzymes that degrade the substrate resulting in breakdown products that serve as nutrients. The role of hyphae with low transcriptional and translational activity was not yet known. Here, we show that this subpopulation is more resistant to environmental stress in A. oryzae, in particular to temperature stress, when compared to hyphae with high transcriptional and translational activity. Notably, all hyphae of the ΔhexA strain of A. oryzae were sensitive to heat stress explained by the reduced heterogeneity in this strain. Together, we show that different subpopulations of hypha secrete proteins and resist heat stress showing the complexity of a fungal mycelium.

The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans.

One of the drawbacks during second-generation biofuel production from plant lignocellulosic biomass is the accumulation of glucose, the preferred carbon source of microorganisms, which causes the repression of hydrolytic enzyme secretion by industrially relevant filamentous fungi. Glucose sensing, subsequent transport and cellular signalling pathways have been barely elucidated in these organisms. This study therefore characterized the transcriptional response of the filamentous fungus Aspergillus nidulans to the presence of high and low glucose concentrations under continuous chemostat cultivation with the aim to identify novel factors involved in glucose sensing and signalling. Several transcription factor- and transporter-encoding genes were identified as being differentially regulated, including the previously characterized glucose and xylose transporter HxtB. HxtB was confirmed to be a low affinity glucose transporter, localizing to the plasma membrane under low- and high-glucose conditions. Furthermore, HxtB was shown to be involved in conidiation-related processes and may play a role in downstream glucose signalling. A gene predicted to encode the protein kinase PskA was also identified as being important for glucose metabolism. This study identified several proteins with predicted roles in glucose metabolic processes and provides a foundation for further investigation into the response of biotechnologically important filamentous fungi to glucose.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger.

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in ß-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

The Cell Factory Aspergillus Enters the Big Data Era: Opportunities and Challenges for Optimising Product Formation.

Living with limits. Getting more from less. Producing commodities and high-value products from renewable resources including waste. What is the driving force and quintessence of bioeconomy outlines the lifestyle and product portfolio of Aspergillus, a saprophytic genus, to which some of the top-performing microbial cell factories belong: Aspergillus niger, Aspergillus oryzae and Aspergillus terreus. What makes them so interesting for exploitation in biotechnology and how can they help us to address key challenges of the twenty-first century? How can these strains become trimmed for better growth on second-generation feedstocks and how can we enlarge their product portfolio by genetic and metabolic engineering to get more from less? On the other hand, what makes it so challenging to deduce biological meaning from the wealth of Aspergillus -omics data? And which hurdles hinder us to model and engineer industrial strains for higher productivity and better rheological performance under industrial cultivation conditions? In this review, we will address these issues by highlighting most recent findings from the Aspergillus research with a focus on fungal growth, physiology, morphology and product formation. Indeed, the last years brought us many surprising insights into model and industrial strains. They clearly told us that similar is not the same: there are different ways to make a hypha, there are more protein secretion routes than anticipated and there are different molecular and physical mechanisms which control polar growth and the development of hyphal networks. We will discuss new conceptual frameworks derived from these insights and the future scientific advances necessary to create value from Aspergillus Big Data.

Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA.

BACKGROUND: The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis. RESULTS: In this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced. CONCLUSION: The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development.

The capacity of Aspergillus niger to sense and respond to cell wall stress requires at least three transcription factors: RlmA, MsnA and CrzA.

BACKGROUND: Cell wall integrity, vesicle transport and protein secretion are key factors contributing to the vitality and productivity of filamentous fungal cell factories such as Aspergillus niger. In order to pioneer rational strain improvement programs, fundamental knowledge on the genetic basis of these processes is required. The aim of the present study was thus to unravel survival strategies of A. niger when challenged with compounds interfering directly or indirectly with its cell wall integrity: calcofluor white, caspofungin, aureobasidin A, FK506 and fenpropimorph. RESULTS: Transcriptomics signatures of A. niger and phenotypic analyses of selected null mutant strains were used to predict regulator proteins mediating the survival responses against these stressors. This integrated approach allowed us to reconstruct a model for the cell wall salvage gene network of A. niger that ensures survival of the fungus upon cell surface stress. The model predicts that (i) caspofungin and aureobasidin A induce the cell wall integrity pathway as a main compensatory response via induction of RhoB and RhoD, respectively, eventually activating the mitogen-activated protein kinase kinase MkkA and the transcription factor RlmA. (ii) RlmA is the main transcription factor required for the protection against calcofluor white but it cooperates with MsnA and CrzA to ensure survival of A. niger when challenged with caspofungin and aureobasidin A. (iii) Membrane stress provoked by aureobasidin A via disturbance of sphingolipid synthesis induces cell wall stress, whereas fenpropimorph-induced disturbance of ergosterol synthesis does not. CONCLUSION: The present work uncovered a sophisticated defence system of A. niger which employs at least three transcription factors - RlmA, MsnA and CrzA - to protect itself against cell wall stress. The transcriptomic data furthermore predicts a fourth transfactor, SrbA, which seems to be specifically important to survive fenpropimorph-induced cell membrane stress. Future studies will disclose how these regulators are interlocked in different signaling pathways to secure survival of A. niger under different cell wall stress conditions.

The transcriptional repressor TupA in Aspergillus niger is involved in controlling gene expression related to cell wall biosynthesis, development, and nitrogen source availability.

The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the ΔtupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37°C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism.

Chitinases CtcB and CfcI modify the cell wall in sporulating aerial mycelium of Aspergillus niger.

Sporulation is an essential part of the life cycle of the industrially important filamentous fungus Aspergillus niger. The formation of conidiophores, spore-bearing structures, requires remodelling of the fungal cell wall, as demonstrated by the differences in carbohydrate composition of cell walls of vegetative mycelium and spores. Glycoside hydrolases that are involved in this process have so far remained unidentified. Using transcriptome analysis, we have identified genes encoding putative cell-wall-modifying proteins with enhanced expression in sporulating aerial mycelium compared to vegetative mycelium. Among the most strongly induced genes were those encoding a protein consisting of a putative chitin binding module (CBM14) and the chitinolytic enzymes NagA, CfcI and CtcB. Reporter studies showed that the N-acetyl-ß-hexosaminidase gene nagA was expressed both in vegetative hyphae and in aerial structures (aerial hyphae, conidiophores and conidia) upon starvation. In contrast, promoter activities of the chitinase genes ctcB and cfcI were specifically localized in the conidiophores and conidia. CtcB is an endo-chitinase and CfcI releases monomers from chitin oligosaccharides: together these enzymes have the potential to degrade chitin of the fungal cell wall. Inactivation of both the cfcI and ctcB genes affected neither radial growth rate, nor formation and germination of spores. The amount of chitin in the spore walls of a ΔcfcIΔctcB double deletion strain, however, was significantly increased compared with the wild-type, thus indicating that CfcI and CtcB indeed modify the A. niger cell walls during sporulation. These novel insights in the sporulation process in aspergilli are of strong scientific relevance, and also may aid industrial strain engineering.

The transcriptomic signature of RacA activation and inactivation provides new insights into the morphogenetic network of Aspergillus niger.

RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. Both deletion and dominant activation of RacA (Rac(G18V)) provoke an actin localization defect and thereby loss of polarized tip extension, resulting in frequent dichotomous branching in the ΔracA strain and an apolar growing phenotype for Rac(G18V). In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, Rac(G18V), ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.

Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger.

Autophagy is a well-conserved catabolic process constitutively active in eukaryotes that is involved in maintaining cellular homeostasis by the targeting of cytoplasmic content and organelles to vacuoles. Autophagy is strongly induced by the limitation of nutrients including carbon, nitrogen, and oxygen and is clearly associated with cell death. It has been demonstrated that the accumulation of empty hyphal compartments and cryptic growth in carbon-starved submerged cultures of the filamentous fungus Aspergillus niger is accompanied by a joint transcriptional induction of autophagy genes. This study examines the role of autophagy by deleting the atg1, atg8, and atg17 orthologs in A. niger and phenotypically analyzing the deletion mutants in surface and submerged cultures. The results indicate that atg1 and atg8 are essential for efficient autophagy, whereas deletion of atg17 has little to no effect on autophagy in A. niger. Depending on the kind of oxidative stress confronted with, autophagy deficiency renders A. niger either more resistant (menadione) or more sensitive (H2O2) to oxidative stress. Fluorescence microscopy showed that mitochondrial turnover upon carbon depletion in submerged cultures is severely blocked in autophagy-impaired A. niger mutants. Furthermore, automated image analysis demonstrated that autophagy promotes survival in maintained carbon-starved cultures of A. niger. Taken together, the results suggest that besides its function in nutrient recycling, autophagy plays important roles in physiological adaptation by organelle turnover and protection against cell death upon carbon depletion in submerged cultures.

Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger.

Aspergillus niger is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain ΔflbA that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the ΔbrlA strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown ΔflbA colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of ΔflbA colonies. Of these, 18 proteins had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of flbA results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain ΔflbA has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve A. niger as a cell factory.

The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger.

BACKGROUND: Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. RESULTS: An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. CONCLUSION: We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins.

Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger reveals a coordinated cellular response to counteract ER stress.

BACKGROUND: HacA/Xbp1 is a conserved bZIP transcription factor in eukaryotic cells which regulates gene expression in response to various forms of secretion stress and as part of secretory cell differentiation. In the present study, we replaced the endogenous hacA gene of an Aspergillus niger strain with a gene encoding a constitutively active form of the HacA transcription factor (HacACA). The impact of constitutive HacA activity during exponential growth was explored in bioreactor controlled cultures using transcriptomic analysis to identify affected genes and processes. RESULTS: Transcription profiles for the wild-type strain (HacAWT) and the HacACA strain were obtained using Affymetrix GeneChip analysis of three replicate batch cultures of each strain. In addition to the well known HacA targets such as the ER resident foldases and chaperones, GO enrichment analysis revealed up-regulation of genes involved in protein glycosylation, phospholipid biosynthesis, intracellular protein transport, exocytosis and protein complex assembly in the HacACA mutant. Biological processes over-represented in the down-regulated genes include those belonging to central metabolic pathways, translation and transcription. A remarkable transcriptional response in the HacACA strain was the down-regulation of the AmyR transcription factor and its target genes. CONCLUSIONS: The results indicate that the constitutive activation of the HacA leads to a coordinated regulation of the folding and secretion capacity of the cell, but with consequences on growth and fungal physiology to reduce secretion stress.

The carbon starvation response of Aspergillus niger during submerged cultivation: insights from the transcriptome and secretome.

BACKGROUND: Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from endogenous resources becomes mobilized to fuel maintenance and self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes. RESULTS: This study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential gene expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry. CONCLUSIONS: This study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved.

The use of open source bioinformatics tools to dissect transcriptomic data.

Microarrays are a valuable technology to study fungal physiology on a transcriptomic level. Various microarray platforms are available comprising both single and two channel arrays. Despite different technologies, preprocessing of microarray data generally includes quality control, background correction, normalization, and summarization of probe level data. Subsequently, depending on the experimental design, diverse statistical analysis can be performed, including the identification of differentially expressed genes and the construction of gene coexpression networks.We describe how Bioconductor, a collection of open source and open development packages for the statistical programming language R, can be used for dissecting microarray data. We provide fundamental details that facilitate the process of getting started with R and Bioconductor. Using two publicly available microarray datasets from Aspergillus niger, we give detailed protocols on how to identify differentially expressed genes and how to construct gene coexpression networks.

New resources for functional analysis of omics data for the genus Aspergillus.

BACKGROUND: Detailed and comprehensive genome annotation can be considered a prerequisite for effective analysis and interpretation of omics data. As such, Gene Ontology (GO) annotation has become a well accepted framework for functional annotation. The genus Aspergillus comprises fungal species that are important model organisms, plant and human pathogens as well as industrial workhorses. However, GO annotation based on both computational predictions and extended manual curation has so far only been available for one of its species, namely A. nidulans. RESULTS: Based on protein homology, we mapped 97% of the 3,498 GO annotated A. nidulans genes to at least one of seven other Aspergillus species: A. niger, A. fumigatus, A. flavus, A. clavatus, A. terreus, A. oryzae and Neosartorya fischeri. GO annotation files compatible with diverse publicly available tools have been generated and deposited online. To further improve their accessibility, we developed a web application for GO enrichment analysis named FetGOat and integrated GO annotations for all Aspergillus species with public genome sequences. Both the annotation files and the web application FetGOat are accessible via the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). To demonstrate the value of those new resources for functional analysis of omics data for the genus Aspergillus, we performed two case studies analyzing microarray data recently published for A. nidulans, A. niger and A. oryzae. CONCLUSIONS: We mapped A. nidulans GO annotation to seven other Aspergilli. By depositing the newly mapped GO annotation online as well as integrating it into the web tool FetGOat, we provide new, valuable and easily accessible resources for omics data analysis and interpretation for the genus Aspergillus. Furthermore, we have given a general example of how a well annotated genome can help improving GO annotation of related species to subsequently facilitate the interpretation of omics data.

Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero.

The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and nearly complete cell retention even at high flow rates. Transcriptomic analysis was used to explore the potential for product formation at very low specific growth rates. The carbon- and energy-limited retentostat cultures were highly reproducible. While the specific growth rate approached zero (<0.005 h(-1)), the growth yield stabilized at a minimum (0.20 g of dry weight per g of maltose). The severe limitation led to asexual differentiation, and the supplied substrate was used for spore formation and secondary metabolism. Three physiologically distinct phases of the retentostat cultures were subjected to genome-wide transcriptomic analysis. The severe substrate limitation and sporulation were clearly reflected in the transcriptome. The transition from vegetative to reproductive growth was characterized by downregulation of genes encoding secreted substrate hydrolases and cell cycle genes and upregulation of many genes encoding secreted small cysteine-rich proteins and secondary metabolism genes. Transcription of known secretory pathway genes suggests that A. niger becomes adapted to secretion of small cysteine-rich proteins. The perspective is that A. niger cultures as they approach a zero growth rate can be used as a cell factory for production of secondary metabolites and cysteine-rich proteins. We propose that the improved retentostat method can be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.

Reconstruction of signaling networks regulating fungal morphogenesis by transcriptomics.

Coordinated control of hyphal elongation and branching is essential for sustaining mycelial growth of filamentous fungi. In order to study the molecular machinery ensuring polarity control in the industrial fungus Aspergillus niger, we took advantage of the temperature-sensitive (ts) apical-branching ramosa-1 mutant. We show here that this strain serves as an excellent model system to study critical steps of polar growth control during mycelial development and report for the first time a transcriptomic fingerprint of apical branching for a filamentous fungus. This fingerprint indicates that several signal transduction pathways, including TORC2, phospholipid, calcium, and cell wall integrity signaling, concertedly act to control apical branching. We furthermore identified the genetic locus affected in the ramosa-1 mutant by complementation of the ts phenotype. Sequence analyses demonstrated that a single amino acid exchange in the RmsA protein is responsible for induced apical branching of the ramosa-1 mutant. Deletion experiments showed that the corresponding rmsA gene is essential for the growth of A. niger, and complementation analyses with Saccharomyces cerevisiae evidenced that RmsA serves as a functional equivalent of the TORC2 component Avo1p. TORC2 signaling is required for actin polarization and cell wall integrity in S. cerevisiae. Congruently, our microscopic investigations showed that polarized actin organization and chitin deposition are disturbed in the ramosa-1 mutant. The integration of the transcriptomic, genetic, and phenotypic data obtained in this study allowed us to reconstruct a model for cellular events involved in apical branching.