Filamentous Fungus Aspergillus Nidulans Research Articles

Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis

Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane. Analyses of these mutants highlight the importance of ER-exit as the primary control point in transporter trafficking via Golgi-bypass. Most mutants isolated concerned mutations within the uapA gene, albeit we also obtained uapA extragenetic mutants affecting secretion and growth pleiotropically or leading or apparent activation of an efflux transporter related to purine-detoxification. Our work paves the way to use genetic approaches targeting specifically trafficking mutations affecting Golgi-bypass.

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an invivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

Composite Recycling with Biocatalytic Thermoset Reforming.

Carbon fiber reinforced polymers (CFRPs, or composites) are increasingly replacing traditional manufacturing materials used in the automobile, aerospace, and energy sectors. With this shift, it is vital to develop end-of-life processes for CFRPs that retain the value of both the carbon fibers and the polymer matrix. Here we demonstrate a strategy to upcycle pre- and postconsumer polystyrene-containing CFRPs, cross-linked with unsaturated polyesters or vinyl esters, to benzoic acid. The thermoset matrix is upgraded via biocatalysis utilizing an engineered strain of the filamentous fungus Aspergillus nidulans, which gives access to valuable secondary metabolites in high yields, exemplified here by (2Z,4Z,6E)-octa-2,4,6-trienoic acid. Reactions are engineered to preserve the carbon fibers with much of their sizing so that the isolated carbon fiber plies are manufactured into new composite coupons that exhibit mechanical properties comparable to those of virgin manufacturing substrates. In sum, this represents the first system to reclaim a high value from both the fiber fabric and polymer matrix of a CFRP.

Mutagenic effects of a commercial glyphosate-based herbicide formulation on the soil filamentous fungus Aspergillus nidulans depending on the mode of exposure.

Glyphosate-based herbicides (GBH) are the most used pesticides worldwide. This widespread dissemination raises the question of non-target effects on a wide range of organisms, including soil micro-organisms. Despite a large body of scientific studies reporting the harmful effects of GBHs, the health and environmental safety of glyphosate and its commercial formulations remains controversial. In particular, contradictory results have been obtained on the possible genotoxicity of these herbicides depending on the organisms or biological systems tested, the modes and durations of exposure and the sensitivity of the detection technique used. We previously showed that the well-characterized soil filamentous fungus Aspergillus nidulans was highly affected by a commercial GBH formulation containing 450g/L of glyphosate (R450), even when used at doses far below the agricultural application rate. In the present study, we analysed the possible mutagenicity of R450 in A. nidulans by screening for specific mutants after different modes of exposure to the herbicide. R450 was found to exert a mutagenic effect only after repeated exposure during growth on agar-medium, and depending on the metabolic status of the tested strain. The nature of some mutants and their ability to tolerate the herbicide better than did the wild-type strain suggested that their emergence may reflect an adaptive response of the fungus to offset the herbicide effects. The use of a non-selective molecular approach, the quantitative random amplified polymorphic DNA (RAPD-qPCR), showed that R450 could also exert a mutagenic effect after a one-shot overnight exposure during growth in liquid culture. However, this effect was subtle and no longer detectable when the fungus had previously been repeatedly exposed to the herbicide on a solid medium. This indicated an elevation of the sensitivity threshold of A. nidulans to the R450 mutagenicity, and thus confirmed the adaptive capacity of the fungus to the herbicide.

Aspergillus SUMOylation mutants exhibit chromosome segregation defects including chromatin bridges.

Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans.

The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

Comprehensive analysis of the composition of the major phospholipids during the asexual life cycle of the filamentous fungus Aspergillus nidulans

Filamentous fungi undergo significant cellular morphological changes during their life cycle. It has recently been reported that deletions of genes that are involved in phospholipid synthesis led to abnormal hyphal morphology and differentiation in filamentous fungi. Although these results suggest the importance of phospholipid balance in their life cycle, comprehensive analyses of cellular phospholipids are limited. Here, we performed lipidomic analysis of A. nidulans during morphological changes in a liquid medium and of colonies on a solid medium. We observed that the phospholipid composition and transcription of the genes involved in phospholipid synthesis changed dynamically during the life cycle. Specifically, the levels of phosphatidylethanolamine, and highly unsaturated phospholipids increased during the establishment of polarity. Furthermore, we demonstrated that the phospholipid composition in the hyphae at colony margins is similar to that during conidial germination. Furthermore, we demonstrated that common and characteristic phospholipid changes occurred during germination in A. nidulans and A. oryzae, and that species-specific changes also occurred. These results suggest that the exquisite regulation of phospholipid composition is crucial for the growth and differentiation of filamentous fungi.

Comparison of secondary metabolite production in the sterigmatocystin and austinol cluster deletion strains in <i>Aspergillus nidulans</i>

A filamentous fungus Aspergillus nidulans produces a mycotoxin sterigmatocystin (ST), in addition to other secondary metabolites (SMs). SM biosynthetic pathways share common precursor substances. However, how the loss of one SM affects the production of other SMs remains unclear. In this study, we deleted 8 biosynthetic gene clusters (BGCs) or a polyketide synthase gene wA, respectively, and analyzed ST production in those deletion strains. In aus1Δ lacking austinol (AUS) cluster A, ST production was increased compared to the control strain. However, in aus1Δ aus2Δ lacking both AUS cluster A and B, ST production was comparable to the control. In STΔ lacking ST cluster, AUS and dehydroaustinol (DAUS) production was comparable to the control.

CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function.

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Woronin body hitchhiking on early endosomes is dispensable for septal localization in Aspergillus nidulans.

The proper functioning of organelles depends on their intracellular localization, mediated by motor protein-dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungus Aspergillus nidulans. However, the physiological role of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent widespread leakage. Here, we tested whether peroxisome hitchhiking is important for Woronin body motility, distribution, and function in A. nidulans. We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of peroxisome hitchhiking significantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging.

Generation and comprehensive analysis of Synechococcus elongatus–Aspergillus nidulans co-culture system for polyketide production

BackgroundArtificial microbial consortia composed of heterotrophic and photoautotrophic organisms represent a unique strategy for converting light energy and carbon dioxide into high-value bioproducts. Currently, the types of desired bioproducts are still limited, and microbial fitness benefit rendered by paired partner generally needs to be intensified. Exploring novel artificial microbial consortia at a laboratory scale is an essential step towards addressing this unmet need. This study aimed to conduct and analyze an artificial consortium composed of cyanobacterium Synechococcus elongatus FL130 with the filamentous fungus Aspergillus nidulans TWY1.1 for producing fungi-derived secondary metabolite of polyketide neosartoricin B.ResultsPolyketide-producing A. nidulans TWY1.1 substantially ameliorated the growth and the survival of sucrose-secreting cyanobacterium S. elongatus FL130 in salt-stressed environments. Besides sucrose, comparable amounts of other carbohydrates were released from axenically cultured FL130 cells, which could be efficiently consumed by TWY1.1. Relative to axenically cultured FL130, less glycogen was accumulated in FL130 cells co-cultured with TWY1.1, and the glycogen phosphorylase gene catalyzing the first step for glycogen degradation had two-fold expression. Different from axenically cultured filamentous fungi, abundant vacuoles were observed in fungal hyphae of TWY1.1 co-cultured with cyanobacterium FL130. Meanwhile, FL130 cells displayed a characteristic pattern of interacting with its heterotrophic partner, densely dispersing along certain hyphae of TWY1.1. Finally, polyketide neosartoricin B was produced from TWY1.1 in FL130-TWY1.1 co-cultures, which was tightly adjusted by nitrogen level.ConclusionOverall, the results thoroughly proved the concept of pairing cyanobacteria with filamentous fungi to build artificial consortia for producing fungi-derived biomolecules.

Polystyrene Upcycling into Fungal Natural Products and a Biocontrol Agent.

Polystyrene (PS) is one of the most used yet infrequently recycled plastics. Although manufactured on the scale of 300 million tons per year globally, current approaches toward PS degradation are energy- and carbon-inefficient, slow, and/or limited in the value that they reclaim. We recently reported a scalable process to degrade post-consumer polyethylene-containing waste streams into carboxylic diacids. Engineered fungal strains then upgrade these diacids biosynthetically to synthesize pharmacologically active secondary metabolites. Herein, we apply a similar reaction to rapidly convert PS to benzoic acid in high yield. Engineered strains of the filamentous fungus Aspergillus nidulans then biosynthetically upgrade PS-derived crude benzoic acid to the structurally diverse secondary metabolites ergothioneine, pleuromutilin, and mutilin. Further, we expand the catalog of plastic-derived products to include spores of the industrially relevant biocontrol agent Aspergillus flavus Af36 from crude PS-derived benzoic acid.

Towards engineering artificial kinesin-14 motors with plus-end-directed processivity.

Kinesins are motor proteins that utilize the chemical energy of ATP hydrolysis to generate movement and force inside cells on the microtubule filaments to play important roles in a variety of essential intracellular processes. Kinesin-14s are a subset of kinesins with C-terminal motor domains and commonly exhibit minus-end-directed motility on the microtubule. Our previous work revealed that KlpA, a kinesin-14 motor from the filamentous fungus Aspergillus nidulans, uniquely exhibits processive plus-end-directed motility on single microtubules as individual homodimers. We hypothesized that KlpA achieves plus-end-directed motility on a single microtubule by adopting a strained cis-configuration whereby the N-terminal tail and the C-terminal motor domains simultaneously bind to the same microtubule. To test this hypothesis, we combined bioinformatic analysis and in vitro single-molecule fluorescence microscopy to search for additional kinesin-14s that exhibit similar plus-end-directed motility as KlpA. We found that AnKinesin-14 from Aspergillus niger has a similar coiled-coil profile as that of KlpA and exhibits processive motility towards the microtubule plus ends (unpublished results). Additionally, we engineered a chimera that replaces the C-terminal motor domains of AnKinesin-14 with those of Ncd (a nonprecessive minus-end-directed kinesin-14) and retains the coiled-coil profile of AnKinesin-14. Our single-molecule motility experiments showed that the chimera moves processively on the microtubule towards the plus ends. Collectively, our findings not only provide strong support of our previous hypothesis for the plus-end-directed motility of KlpA but also lay out a conceptual framework for how to engineer artificial kinesin motors with desired directionality using a cut-and-paste approach.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

CAMP signaling factors regulate carbon catabolite repression of hemicellulase genes in Aspergillus nidulans

Carbon catabolite repression (CCR) enables preferential utilization of easily metabolizable carbon sources, implying the presence of mechanisms to ensure discriminatory gene repression depending on the ambient carbon sources. However, the mechanisms for such hierarchical repression are not precisely understood. In this report, we examined how deletion of pkaA and ganB, which encode cAMP signaling factors, and creA, which encodes a well-characterized repressor of CCR, affects CCR of hemicellulase genes in the filamentous fungus Aspergillus nidulans. β-Xylanase production increased not only in ΔcreA but also in ΔpkaA and ΔganB, with the highest level observed in their double deletants, irrespective of the presence or absence of d-glucose. Expression of the β-xylanase genes in the presence of d-glucose was de-repressed in all the deletion mutants, with significantly higher tolerance against d-glucose repression in ΔpkaA and ΔganB than in ΔcreA. In the presence of galactomannan and d-glucose, partial de-repression of β-mannanase production was detected in ΔcreA, but not in ΔpkaA and ΔganB. The double deletion of creA/pkaA and creA/ganB led to earlier production. Release from d-glucose repression of the β-mannanase genes was partial in the single deletants, while nearly full de-repression was observed in ΔcreAΔpkaA and ΔcreAΔganB. The contribution of PkaA and GanB to CCR by d-xylose of the β-mannanase genes was very minor compared to that of CreA. Consequently, the present study revealed that cAMP signaling plays a major role in CCR of hemicellulase gene expression in a manner that is clearly independent from CreA.

Transmembrane helices 5 and 12 control transport dynamics, substrate affinity, and specificity in the elevator-type UapA transporter.

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences, all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best-studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead not only to increased rate of transport but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding and broadened specificity, leading in specific cases to UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function and how this knowledge can be applied to genetically modify their transport characteristics.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in Aspergillus nidulans.

Nutrient transporters have been shown to translocate to the plasma membrane (PM) of the filamentous fungus Aspergillus nidulans via an unconventional trafficking route that bypasses the Golgi. This finding strongly suggests the existence of distinct COPII vesicle subpopulations, one following Golgi-dependent conventional secretion and the other directed towards the PM. Here, we address whether Golgi-bypass concerns cargoes other than nutrient transporters and whether Golgi-bypass is related to cargo structure, size, abundance, physiological function, or polar vs. non-polar distribution in the PM. To address these questions, we followed the dynamic subcellular localization of two selected membrane cargoes differing in several of the aforementioned aspects. These are the proton-pump ATPase PmaA and the PalI pH signaling component. Our results show that neosynthesized PmaA and PalI are translocated to the PM via Golgi-bypass, similar to nutrient transporters. In addition, we showed that the COPII-dependent exit of PmaA from the ER requires the alternative COPII coat subunit LstA, rather than Sec24, whereas PalI requires the ER cargo adaptor Erv14. These findings strengthen the evidence of distinct cargo-specific COPII subpopulations and extend the concept of Golgi-independent biogenesis to essential transmembrane proteins, other than nutrient transporters. Overall, our findings point to the idea that Golgi-bypass might not constitute a fungal-specific peculiarity, but rather a novel major and cargo-specific sorting route in eukaryotic cells that has been largely ignored.

GalR, GalX and AraR co-regulate d-galactose and l-arabinose utilization in Aspergillus nidulans.

SummaryFilamentous fungi produce a wide variety of enzymes in order to efficiently degrade plant cell wall polysaccharides. The production of these enzymes is controlled by transcriptional regulators, which also control the catabolic pathways that convert the released monosaccharides. Two transcriptional regulators, GalX and GalR, control d‐galactose utilization in the model filamentous fungus Aspergillus nidulans, while the arabinanolytic regulator AraR regulates l‐arabinose catabolism. d‐Galactose and l‐arabinose are commonly found together in polysaccharides, such as arabinogalactan, xylan and rhamnogalacturonan I. Therefore, the catabolic pathways that convert d‐galactose and l‐arabinose are often also likely to be active simultaneously. In this study, we investigated the interaction between GalX, GalR and AraR in d‐galactose and l‐arabinose catabolism. For this, we generated single, double and triple mutants of the three regulators, and analysed their growth and enzyme and gene expression profiles. Our results clearly demonstrated that GalX, GalR and AraR co‐regulate d‐galactose catabolism in A. nidulans. GalX has a prominent role on the regulation of genes of d‐galactose oxido‐reductive pathway, while AraR can compensate for the absence of GalR and/or GalX.

A Transient Receptor Potential-like Calcium Ion Channel in the Filamentous Fungus Aspergillus nidulans.

Transient Receptor Potential (TRP) proteins constitute a superfamily that encodes transmembrane ion channels with highly diverse permeation and gating properties. Filamentous fungi possess putative TRP channel-encoded genes, but their functions remain elusive. Here, we report that a putative TRP-like calcium channel, trpR, in the filamentous fungus Aspergillus nidulans, performs important roles in conidiation and in adapting to cell wall disruption reagents in a high temperature-induced defect-dependent manner, especially under a calcium-limited culture condition. The genetic and functional relationship between TrpR and the previously identified high-affinity calcium channels CchA/MidA indicates that TrpR has an opposite response to CchA/MidA when reacting to cell wall disruption reagents and in regulating calcium transients. However, a considerable addition of calcium can rescue all the defects that occur in TrpR and CchA/MidA, meaning that calcium is able to bypass the necessary requirement. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and the P-type Golgi Ca2+ ATPase PmrA suggests two channels that may work as ion transporters, transferring Ca2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis. Therefore, combined with data for the trpR deletion mutant revealing abnormal cell wall structures, TrpR works as a Golgi membrane calcium ion channel that involves cell wall integration.