Ascomycete Aspergillus Nidulans Research Articles

Interconnections between the Cation/Alkaline pH-Responsive Slt and the Ambient pH Response of PacC/Pal Pathways in Aspergillus nidulans.

In the filamentous ascomycete Aspergillus nidulans, at least three high hierarchy transcription factors are required for growth at extracellular alkaline pH: SltA, PacC and CrzA. Transcriptomic profiles depending on alkaline pH and SltA function showed that pacC expression might be under SltA regulation. Additional transcriptional studies of PacC and the only pH-regulated pal gene, palF, confirmed both the strong dependence on ambient pH and the function of SltA. The regulation of pacC expression is dependent on the activity of the zinc binuclear (C6) cluster transcription factor PacX. However, we found that the ablation of sltA in the pacX- mutant background specifically prevents the increase in pacC expression levels without affecting PacC protein levels, showing a novel specific function of the PacX factor. The loss of sltA function causes the anomalous proteolytic processing of PacC and a reduction in the post-translational modifications of PalF. At alkaline pH, in a null sltA background, PacC72kDa accumulates, detection of the intermediate PacC53kDa form is extremely low and the final processed form of 27 kDa shows altered electrophoretic mobility. Constitutive ubiquitination of PalF or the presence of alkalinity-mimicking mutations in pacC, such as pacCc14 and pacCc700, resembling PacC53kDa and PacC27kDa, respectively, allowed the normal processing of PacC but did not rescue the alkaline pH-sensitive phenotype caused by the null sltA allele. Overall, data show that Slt and PacC/Pal pathways are interconnected, but the transcription factor SltA is on a higher hierarchical level than PacC on regulating the tolerance to the ambient alkalinity in A. nidulans.

The Forkhead Gene fkhB is Necessary for Proper Development in Aspergillus nidulans.

The forkhead domain genes are important for development and morphogenesis in fungi. Six forkhead genes fkhA-fkhF have been found in the genome of the model filamentous Ascomycete Aspergillus nidulans. To identify the fkh gene(s) associated with fungal development, we examined mRNA levels of these six genes and found that the level of fkhB and fkhD mRNA was significantly elevated during asexual development and in conidia. To investigate the roles of FkhB and FkhD, we generated fkhB and fkhD deletion mutants and complemented strains and investigated their phenotypes. The deletion of fkhB, but not fkhD, affected fungal growth and both sexual and asexual development. The fkhB deletion mutant exhibited decreased colony size with distinctly pigmented (reddish) asexual spores and a significantly lower number of conidia compared with these features in the wild type (WT), although the level of sterigmatocystin was unaffected by the absence of fkhB. Furthermore, the fkhB deletion mutant produced sexual fruiting bodies (cleistothecia) smaller than those of WT, implying that the fkhB gene is involved in both asexual and sexual development. In addition, fkhB deletion reduced fungal tolerance to heat stress and decreased trehalose accumulation in conidia. Overall, these results suggest that fkhB plays a key role in proper fungal growth, development, and conidial stress tolerance in A. nidulans.

Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces-Aspergillus interaction.

Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and above a certain concentration, provokes the production of ORS. In addition, several other secondary metabolites were induced by polaramycin B. Our genome-wide transcriptome analysis showed that polaramycin B treatment causes downregulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included upregulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the upregulation of genes necessary to compensate for the cellular damage caused by polaramycin B.

The sexual spore pigment asperthecin is required for normal ascospore production and protection from UV light in Aspergillus nidulans.

Many fungi develop both asexual and sexual spores that serve as propagules for dissemination and/or recombination of genetic traits. Asexual spores are often heavily pigmented and this pigmentation provides protection from UV light. However, little is known about any purpose pigmentation that may serve for sexual spores. The model Ascomycete Aspergillus nidulans produces both green pigmented asexual spores (conidia) and red pigmented sexual spores (ascospores). Here we find that the previously characterized red pigment, asperthecin, is the A. nidulans ascospore pigment. The asperthecin biosynthetic gene cluster is composed of three genes: aptA, aptB, and aptC, where deletion of either aptA (encoding a polyketide synthase) or aptB (encoding a thioesterase) yields small, mishappen hyaline ascospores; while deletion of aptC (encoding a monooxygenase) yields morphologically normal but purple ascospores. ∆aptA and ∆aptB but not ∆aptC or wild type ascospores are extremely sensitive to UV light. We find that two historical ascospore color mutants, clA6 and clB1, possess mutations in aptA and aptB sequences, respectively.

A highly conserved mechanism for the detoxification and assimilation of the toxic phytoproduct L-azetidine-2-carboxylic acid in Aspergillus nidulans

Plants produce toxic secondary metabolites as defense mechanisms against phytopathogenic microorganisms and predators. L-azetidine-2-carboxylic acid (AZC), a toxic proline analogue produced by members of the Liliaceae and Agavaciae families, is part of such a mechanism. AZC causes a broad range of toxic, inflammatory and degenerative abnormalities in human and animal cells, while it is known that some microorganisms have evolved specialized strategies for AZC resistance. However, the mechanisms underlying these processes are poorly understood. Here, we identify a widespread mechanism for AZC resistance in fungi. We show that the filamentous ascomycete Aspergillus nidulans is able to not only resist AZC toxicity but also utilize it as a nitrogen source via GABA catabolism and the action of the AzhA hydrolase, a member of a large superfamily of detoxifying enzymes, the haloacid dehalogenase-like hydrolase (HAD) superfamily. This detoxification process is further assisted by the NgnA acetyltransferase, orthologue of Mpr1 of Saccharomyces cerevisiae. We additionally show that heterologous expression of AzhA protein can complement the AZC sensitivity of S. cerevisiae. Furthermore, a detailed phylogenetic analysis of AzhA homologues in Fungi, Archaea and Bacteria is provided. Overall, our results unravel a widespread mechanism for AZC resistance among microorganisms, including important human and plant pathogens.

Characterization of Aspergillus nidulansTRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII.

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the ‘Trs33 side’ of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.

AmdS as a dominant recyclable marker in Cryptococcus neoformans

While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds.

The mechanism of peroxisome motility in filamentous fungi

Peroxisomes (POs) are an essential part of the fungal cell’s inventory. They are involved in cellular lipid homeostasis, reactive oxygen metabolism and in the synthesis of secondary metabolites. These functions are thought to require frequent organelle-organelle interactions and the even-distribution of POs in fungal hypha. Recent work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans reveals a crucial role of early endosomes (EEs) in the dynamic behavior of POs required for their cellular distribution and interaction. This article summarizes the main findings, which provided unexpected insight into the mechanism by which fungal cells organize themselves.

Isopod grazing induces down-regulation of Aspergillus nidulans anti-fungivore defence marker genes

Abstract Variation in grazing intensity determines the way in which mycelial growth and physiology is influenced by invertebrate fungivores. Recently, low-intensity grazing by facultative mesofaunal fungivores, small dipteran larvae and collembola, was found to enhance the activity of fungal defensive secondary metabolism. Here, we ask whether higher intensity grazing by macrofaunal fungivores induces a different fungal response. To test this, we quantified changes in the expression of two anti-fungivore chemical defence marker genes, laeA and aflR, in the ascomycete Aspergillus nidulans exposed to grazing pressure exerted by individuals of the isopod Oniscus asellus. Compared to no and low-intensity grazing pressure, higher intensity grazing significantly reduced the transcript levels of these genes. This finding suggests a negative relationship between grazing pressure intensity and the capacity of a saprotrophic fungus to resist fungivory.

Live cell imaging of endosomal trafficking in fungi.

Endosomes are multipurpose membranous carriers important for endocytosis and secretion. During membrane trafficking, endosomes transport lipids, proteins, and even RNAs. In highly polarized cells such as fungal hyphae, they shuttle bidirectionally along microtubules mediated by molecular motors like kinesins and dynein. For in vivo studies of these highly dynamic protein/membrane complexes, advanced fluorescence microscopy is instrumental. In this chapter, we describe live cell imaging of endosomes in two distantly related fungal model systems, the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans. We provide insights into live cell imaging of dynamic endosomal proteins and RNA, dual-color detection for colocalization studies, as well as fluorescence recovery after photobleaching (FRAP) for quantification and photo-activated localization microscopy (PALM) for super-resolution. These methods described in two well-studied fungal model systems are applicable to a broad range of other organisms.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters.

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action.

Furocoumarins and coumarins photoinactivate Colletotrichum acutatum and Aspergillus nidulans fungi under solar radiation

The increasing tolerance to currently-used fungicides is a major problem both in clinical and agricultural areas leading to an urgent need for the development of novel antifungal strategies. This study investigated the in vitro antimicrobial photo treatment (APT) of conidia of the plant-pathogenic fungus Colletotrichum acutatum and the ascomycete Aspergillus nidulans with the furocoumarins 8-methoxypsoralen (8-MOP) and isopimpinellin, and a mixture of two coumarins (7-methoxy coumarin and citropten). Subcellular localization of the photosensitizer 8-MOP was also determined in C. acutatum conidia. Additionally, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP (50μM) led to a reduction of approximately 4 logs in the survival of the conidia of both species, and the mixture of the two coumarins (12.5mgL−1) resulted in a reduction of approximately 4 logs for A. nidulans and 3 logs for C. acutatum. Isopimpinellin (50μM) displayed a reduction of 4 logs for A. nidulans but less than 2 logs for C. acutatum. Washing the conidia to remove unbound photosensitizers before light exposure reduced the photodynamic inactivation of C. acutatum both with 8-MOP and the mixture of the two coumarins. The reduction was smaller for A. nidulans. 8-MOP spread throughout the cytoplasm and accumulated in structures such as lipid bodies of C. acutatum conidia. No damage to orange tree leaves was observed after APT with any of the photosensitizers.

Cytoplasmic Dynamics of the General Nuclear Import Machinery in Apically Growing Syncytial Cells

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus.

Transport assays in filamentous fungi: Kinetic characterization of the UapC purine transporter of Aspergillus nidulans

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. Our method makes use of A. nidulans germinating conidiospores at a defined morphological stage in which most transporters show maximal expression, avoiding technical difficulties associated with the use of mycelia. In combination with the ease of construction of genetic null mutants in A. nidulans, our method allows the rigorous characterization of any transporter in genetic backgrounds that are devoid of other transporters of similar specificity. Here, we use this method to characterize the kinetic parameters and the specificity profile of UapC, a uric acid-xanthine transporter present in all ascomycetes and member of the ubiquitous Nucleobase-Ascorbate Transporter family, in specific genetic backgrounds lacking other relevant transporters.

Establishing a versatile Golden Gate cloning system for genetic engineering in fungi

The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules allow not only simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.

Base composition and nucleosome density in exonic and intronic regions in genes of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae

We sequenced nucleosomal DNA fragments of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae and then mapped those sequences on their genomes. We compared the GC content and nucleosome density in the exonic and intronic regions in the genes of A. nidulans and A. oryzae. Although the GC content and nucleosome density in the exonic regions tended to be higher than those in the intronic regions, the difference in the distribution of the GC content was more notable than that of the nucleosome density. Next, we compared the GC content and nucleosome density in the exonic regions of 9616 orthologous gene pairs. In both Aspergillus species, the GC content did not correlate with the nucleosome density. In addition, the Spearman's rank correlation coefficient (ρ=0.51) between the GC content of the exonic regions of the 9616 orthologous gene pairs was higher than that (ρ=0.31) of the nucleosome densities of A. nidulans and A. oryzae. These results strongly suggest that the GC content in the exons of the orthologous gene pairs has been conserved during evolution but the nucleosome density has varied throughout.

Transport Assays in Aspergillus nidulans

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. These assays make use of A. nidulans germinating conidiospores, thus avoiding technical difficulties associated with the use of mycelia. The ease of construction genetic null mutants in this model fungus permits the rigorous characterization of any transporter in the absence of similar transporters with overlapping specificities, a common problem in relevant studies.

Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi

Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.