Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress.

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens.

Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans.

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Aspergillus nidulans.

Photoactivated localization microscopy (PALM), one of the super resolution microscopy methods improving the resolution limit to 20 nm, allows the detection of single molecules in complex protein structures in living cells. Microtubule-organizing centres (MTOCs) are large, multisubunit protein complexes, required for microtubule polymerization. The prominent MTOC in higher eukaryotes is the centrosome, and its functional ortholog in fungi is the spindle-pole body (SPB). There is ample evidence that besides centrosomes other MTOCs are important in eukaryotic cells. The filamentous ascomycetous fungus Aspergillus nidulans is a model organism, with hyphae consisting of multinucleate compartments separated by septa. In A. nidulans, besides the SPBs, a second type of MTOCs was discovered at septa (called septal MTOCs, sMTOC). All the MTOC components appear as big dots at SPBs and sMTOCs when tagged with a fluorescent protein and observed with conventional fluorescence microscopy due to the diffraction barrier. In this chapter, we describe the application of PALM in quantifying the numbers of individual proteins at both MTOC sites in A. nidulans and provide evidence that the composition of MTOCs is highly dynamic and dramatically changes during the cell cycle.

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy.

After growing on surfaces, including those of medical and industrial importance, fungal biofilms self-generate internal microenvironments. We previously reported that gaseous microenvironments around founder Aspergillus nidulans cells change during biofilm formation causing microtubules to disassemble under control of the hypoxic transcription factor SrbA. Here we investigate if biofilm formation might also promote changes to structures involved in exocytosis and endocytosis. During biofilm formation, the endoplasmic reticulum (ER) remained intact but ER exit sites and the Golgi apparatus were modified as were endocytic actin patches. The biofilm-driven changes required the SrbA hypoxic transcription factor and could be triggered by nitric oxide, further implicating gaseous regulation of biofilm cellular architecture. By tracking green fluorescent protein (GFP)-Atg8 dynamics, biofilm founder cells were also observed to undergo autophagy. Most notably, biofilm cells that had undergone autophagy were triggered into further autophagy by spinning disk confocal light. Our findings indicate that fungal biofilm formation modifies the secretory and endocytic apparatus and show that biofilm cells can also undergo autophagy that is reactivated by light. The findings provide new insights into the changes occurring in fungal biofilm cell biology that potentially impact their unique characteristics, including antifungal drug resistance.

Hülle Cells of Aspergillus nidulans with Nuclear Storage and Developmental Backup Functions Are Reminiscent of Multipotent Stem Cells.

Some aspergilli are among the most cosmopolitan and ecologically dominant fungal species. One pillar of their success is their complex life cycle, which creates specialized cell types for versatile dispersal and regenesis. One of these cell types is unique to aspergilli-the Hülle cells. Despite being known for over a century, the biological and ecological roles of Hülle cells remain largely speculative. Previously reported data on in vivo Hülle cell formation and localization have been conflicting. Our quantification reveals that Hülle cells can occur at all locations on hyphae and that they show cellular activity similar to that seen with adjacent hyphae, indicating that they develop as intricate parts of hyphal tissue. In addition, we show that during sexual development associated with two parental strains, the typically multinucleate Hülle cells can inherit nuclei from both parents, indicating that they may serve as genetic backups. We provide an easy, reproducible method to study Hülle cell biology and germination with which we investigate the 90-year-old puzzle of whether and how Hülle cells germinate. We present clear evidence for the germination of Hülle cells, and we show that Hülle cells grow hyphae that develop into a spore-producing colony. Finally, we show that Hülle cell-derived colonies produce conidiospores faster than spore-derived colonies, providing evidence for an as-yet-undescribed developmental shortcut program in Aspergillus nidulans We propose that Hülle cells represent a unique cell type as specialized hypha-derived sexual tissue with a nucleus storage function and may act as fungal backup stem cells under highly destructive conditions.IMPORTANCE The in vivo identification of Hülle cells in cases of aspergillosis infections in animals and humans illustrates their biological relevance and suggests that they might be involved in pathogenicity. It is striking that aspergilli have developed and maintained a multinucleate nurse cell that is presumably energy-intensive to produce and is usually found only in higher eukaryotes. Our findings shed light on how the understudied Hülle cells might contribute to the success of aspergilli by acting not only as nurse cells under detrimental conditions (sexual development) but also as fungal backup stem cells with the capacity to produce genetically diverse spores in an accelerated manner, thereby substantially contributing to survival in response to predator attack or under otherwise severely destructive conditions. Our study solved the 90-year-old puzzle of Hülle cell germination and provides easy, reproducible methods that will facilitate future studies on biological and ecological roles of Hülle cells in aspergilli.

Homeobox proteins are essential for fungal differentiation and secondary metabolism in Aspergillus nidulans.

The homeobox domain-containing transcription factors play an important role in the growth, development, and secondary metabolism in fungi and other eukaryotes. In this study, we characterized the roles of the genes coding for homeobox-type proteins in the model organism Aspergillus nidulans. To examine their roles in A. nidulans, the deletion mutant strains for each gene coding for homeobox-type protein were generated, and their phenotypes were examined. Phenotypic analyses revealed that two homeobox proteins, HbxA and HbxB, were required for conidia production. Deletion of hbxA caused abnormal conidiophore production, decreased the number of conidia in both light and dark conditions, and decreased the size of cleistothecia structures. Overexpressing hbxA enhanced the production of asexual spores and formation of conidiophore under the liquid submerged conditions. The hbxB deletion mutant strains exhibited decreased asexual spore production but increased cleistothecia production. The absence of hbxB decreased the trehalose content in asexual spores and increased their sensitivity against thermal and oxidative stresses. The ΔhbxA strains produced more sterigmatocystin, which was decreased in the ΔhbxB strain. Overall, our results show that HbxA and HbxB play crucial roles in the differentiation and secondary metabolism of the fungus A. nidulans.

Molecular origin of the Raman signal from Aspergillus nidulans conidia and observation of fluorescence vibrational structure at room temperature.

Successful approaches to identification and/or biological characterization of fungal specimens through Raman spectroscopy may require the determination of the molecular origin of the Raman response as well as its separation from the background fluorescence. The presence of fluorescence can interfere with Raman detection and is virtually impossible to avoid. Fluorescence leads to a multiplicity of problems: one is noise, while another is "fake" spectral structure that can easily be confused for spontaneous Raman peaks. One solution for these problems is Shifted Excitation Raman Difference Spectroscopy (SERDS), in which a tunable light source generates two spectra with different excitation frequencies in order to eliminate fluorescence from the measured signal. We combine a SERDS technique with genetic breeding of mutant populations and demonstrate that the Raman signal from Aspergillus nidulans conidia originates in pigment molecules within the cell wall. In addition, we observe unambiguous vibrational fine-structure in the fluorescence response at room temperature. We hypothesize that the vibrational fine-structure in the fluorescence results from the formation of flexible, long-lived molecular cages in the bio-polymer matrix of the cell wall that partially shield target molecules from the immediate environment and also constrain their degrees of freedom.

Alternative transcription start sites of the enolase-encoding gene enoA are stringently used in glycolytic/gluconeogenic conditions in Aspergillus oryzae.

Gene expression using alternative transcription start sites (TSSs) is an important transcriptional regulatory mechanism for environmental responses in eukaryotes. Here, we identify two alternative TSSs in the enolase-encoding gene (enoA) in Aspergillus oryzae, an industrially important filamentous fungus. TSS use in enoA is strictly dependent on the difference in glycolytic and gluconeogenic carbon sources. Transcription from the upstream TSS (uTSS) or downstream TSS (dTSS) predominantly occurs under gluconeogenic or glycolytic conditions, respectively. In addition to enoA, most glycolytic genes involved in reversible reactions possess alternative TSSs. The fbaA gene, which encodes fructose-bisphosphate aldolase, also shows stringent alternative TSS selection, similar to enoA. Alignment of promoter sequences of enolase-encoding genes in Aspergillus predicted two conserved regions that contain a putative cis-element required for enoA transcription from each TSS. However, uTSS-mediated transcription of the acuN gene, an enoA ortholog in Aspergillus nidulans, is not strictly dependent on carbon source, unlike enoA. Furthermore, enoA transcript levels in glycolytic conditions are higher than in gluconeogenic conditions. Conversely, acuN is more highly transcribed in gluconeogenic conditions. This suggests that the stringent usage of alternative TSSs and higher transcription in glycolytic conditions in enoA may reflect that the A. oryzae evolutionary genetic background was domesticated by exclusive growth in starch-rich environments. These findings provide novel insights into the complexity and diversity of transcriptional regulation of glycolytic/gluconeogenic genes among Aspergilli.

Characterizing the role of Zn cluster family transcription factor ZcfA in governing development in two Aspergillus species.

Filamentous fungi reproduce asexually or sexually, and the processes of asexual and sexual development are tightly regulated by a variety of transcription factors. In this study, we characterized a Zn2Cys6 transcription factor in two Aspergillus species, A. nidulans (AN5859) and A. flavus (AFLA_046870). AN5859 encodes a Zn2Cys6 transcription factor, called ZcfA. In A. nidulans, ΔzcfA mutants exhibit decreased fungal growth, a reduction in cleistothecia production, and increased asexual reproduction. Overexpression of zcfA results in increased conidial production, suggesting that ZcfA is required for proper asexual and sexual development in A. nidulans. In conidia, deletion of zcfA causes decreased trehalose levels and decreased spore viability but increased thermal sensitivity. In A. flavus, the deletion of the zcfA homolog AFLA_046870 causes increased conidial production but decreased sclerotia production; these effects are similar to those of zcfA deletion in A. nidulans development. Overall, these results demonstrate that ZcfA is essential for maintaining a balance between asexual and sexual development and that some roles of ZcfA are conserved in Aspergillus spp.

The velvet Regulator VosA Governs Survival and Secondary Metabolism of Sexual Spores in Aspergillus nidulans.

The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans. In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A. nidulans. During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans.

Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms.

The accessibility to next-generation sequencing (NGS) techniques has enabled the sequencing of hundreds of genomes of species representing all kingdoms. In the case of fungi, genomes of more than a thousand of species are publicly available. This is far from covering the number of 2.2-3.8 million fungal species estimated to populate the world but has significantly improved the resolution of the fungal tree of life. Furthermore, it has boosted systematic evolutionary analyses, the development of faster and more accurate diagnostic analyses of pathogenic strains or the improvement of several biotechnological processes. Nevertheless, the diversification of the nature of fungal species used as model has also weakened research in other species that were traditionally used as reference in the pre-genomic era. In this context, and after more than 65 years since the first works published by Pontecorvo, Aspergillus nidulans remains as one of the most referential model filamentous fungus in research fields such as hyphal morphogenesis, intracellular transport, developmental programs, secondary metabolism, or stress response. This mini-review summarizes how A. nidulans has contributed to the progress in these fields during the last years, and discusses how it could contribute in the future, assisted by NGS and new-generation molecular, microscopy, or cellular tools.

Identification and Characterization of Aspergillus nidulans Mutants Impaired in Asexual Development under Phosphate Stress.

The transcription factor BrlA plays a central role in the production of asexual spores (conidia) in the fungus Aspergillus nidulans. BrlA levels are controlled by signal transducers known collectively as UDAs. Furthermore, it governs the expression of CDP regulators, which control most of the morphological transitions leading to the production of conidia. In response to the emergence of fungal cells in the air, the main stimulus triggering conidiation, UDA mutants such as the flbB deletant fail to induce brlA expression. Nevertheless, ΔflbB colonies conidiate profusely when they are cultured on a medium containing high H2PO4- concentrations, suggesting that the need for FlbB activity is bypassed. We used this phenotypic trait and an UV-mutagenesis procedure to isolate ΔflbB mutants unable to conidiate under these stress conditions. Transformation of mutant FLIP166 with a wild-type genomic library led to the identification of the putative transcription factor SocA as a multicopy suppressor of the FLIP (Fluffy, aconidial, In Phosphate) phenotype. Deregulation of socA altered both growth and developmental patterns. Sequencing of the FLIP166 genome enabled the identification and characterization of PmtCP282L as the recessive mutant form responsible for the FLIP phenotype. Overall, results validate this strategy for identifying genes/mutations related to the control of conidiation.

GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Light in the Fungal World: From Photoreception to Gene Transcription and Beyond.

Fungi see light of different colors by using photoreceptors such as the White Collar proteins and cryptochromes for blue light, opsins for green light, and phytochromes for red light. Light regulates fungal development, promotes the accumulation of protective pigments and proteins, and regulates tropic growth. The White Collar complex (WCC) is a photoreceptor and a transcription factor that is responsible for regulating transcription after exposure to blue light. In Neurospora crassa, light promotes the interaction of WCCs and their binding to the promoters to activate transcription. In Aspergillus nidulans, the WCC and the phytochrome interact to coordinate gene transcription and other responses, but the contribution of these photoreceptors to fungal photobiology varies across fungal species. Ultimately, the effect of light on fungal biology is the result of the coordinated transcriptional regulation and activation of signal transduction pathways.

CARS spectroscopy of Aspergillus nidulans spores.

Coherent Anti-Stokes Raman Spectroscopy (CARS) is performed on single spores (conidia) of the fungus Aspergillus nidulans in order to establish a baseline measurement for fungal spores. Chemical maps of single spores are generated and spectral differentiation between the cell wall and the cytoplasm is achieved. Principal Component Analysis of the measured spectra is then completed as a means to quantify spore heterogeneity. Applications range from the quick and accurate diagnosis of public health concerns to real-time agricultural and environmental sensing of fungal symbionts and pathogens.

Antiproliferative Activity and Characterization of Metabolites of Aspergillus nidulans: An Endophytic Fungus from Nyctanthes arbor-tristis Linn. Against Three Human Cancer Cell Lines.

BACKGROUND: Endophytic fungi are receiving attention as sources of structurally novel bioactive secondary metabolites towards drug discovery from natural products. This study reports the isolation and characterization of secondary metabolites from an endophytic fungus Aspergillus nidulans, associated with Nyctanthes arbor-tristis Linn., a plant which has a traditional use to cure many ailments including cancer. OBJECTIVE: The objective of this study was to evaluate the antiproliferative activity of the metabolites of A. nidulans from N. arbor-tristis on three human cancer cell lines, lung (NCI-H460), breast (MCF-7) and uterine cervix (HeLa), and carry out their characterization. METHODS: The extracts of the endophytic fungus cultured on potato dextrose agar were subjected to various chromatographic techniques. Structures of pure compounds were determined using spectroscopic techniques. The non-polar constituents were analyzed by GC-MS. Antiproliferative activity was determined by sulforhodamine B (SRB) assay. RESULTS: The extracts and fractions showed moderate to good growth inhibition of the aforementioned human cancer cell lines. The broth extract was most potent (IC50 = 10 ± 3.1 µg/mL and LC50= 95 ± 3.9) against HeLa whereas petroleum ether insoluble fraction of mycelium was most active against NCI-H460 and MCF-7 (IC50 = 10 ± 2.1 µg/mL and 18 ± 3.1 µg/mL respectively). GC-MS led to identify 12 compounds in mycelium and 19 compounds in broth. Four pure compounds were isolated and characterized one compound 5, 10-dihydrophenazine-1-carboxylic acid (1) from broth and three 1-hydroxy-3-methylxanthone (2), ergosterol (3) and sterigmatocystin (4) from mycelium. 1 has not been reported earlier as a plant/fungal metabolite while 2-4 are new from this source. Sterigmatocystin exhibited growth inhibitory effect (IC50 = 50 ± 2.5 µM/mL) against only MCF-7 cell line whereas other compounds had IC50 > 100. CONCLUSIONS: In this paper, the cytotoxicity of mycelium and broth constituents of endophytic fungus Aspergillus nidulans from Nyctanthes arbor-tristis is reported for the first time. The study shows that fungus Aspergillus nidulans from Nyctanthes arbor-tristis is capable of producing biologically active natural compounds and provides a scientific rationale for further chemical investigations of endophyte-producing natural products.

Why outcross? The abandon-ship hypothesis in a facultative outcrossing/selfing fungal species.

Most species able to reproduce both sexually and asexually (facultative sexual species) invest more in sexual reproduction in stressful environment conditions. According to the abandon-ship hypothesis, plasticity for investment in sexual reproduction may have been selected in these species, allowing unfit genotypes to generate progeny carrying new advantageous allelic combinations. We tested this hypothesis in Aspergillus nidulans, a fungus able to reproduce asexually, or sexually, by outcrossing and/or haploid selfing (i.e. fusion of genetically identical haploid nuclei, causing immediate genome-wide homozygosity). We crossed various strains of A. nidulans in a non-stressful environment and a stressful environment containing a non-lethal dose of fungicide. Without stress, crosses preferentially generated haploselfed fruiting bodies, whereas stressful conditions significantly increased the outcrossing rate. Our results strongly support the abandon-ship hypothesis and suggest that, for parents with low fitness, the costs of investing in sexual reproduction may be compensated by the production of fitter progeny carrying beneficial allele combinations. Similarly, the progeny generated by outcrossing was less fit than that produced by haploid selfing in non-stressful environments, but fitter in stressful conditions, suggesting that outcrossing may have short-term advantages in stressful environments in A. nidulans.

The Central Stalk Determines the Motility of Mitotic Kinesin-14 Homodimers.

Mitotic kinesin-14 homodimers that contain an N-terminal nonmotor microtubule-binding tail contribute to spindle organization by preferentially crosslinking two different spindle microtubules rather than interacting with a single microtubule to generate processive motility. However, the mechanism underlying such selective motility behavior remains poorly understood. Here, we show that when a flexible polypeptide linker is inserted into the coiled-coil central stalk, two homodimeric mitotic kinesin-14s of distinct motility-the processive plus-end-directed KlpA from Aspergillus nidulans [1] and the nonprocessive minus-end-directed Ncd from Drosophila melanogaster [2]-both switch to become processive minus-end-directed motors. Our results demonstrate that the polypeptide linker introduces greater conformational flexibility into the central stalk. Importantly, we find that the linker insertion significantly weakens the ability of Ncd to preferentially localize between and interact with two microtubules. Collectively, our results reveal that besides the canonical role of enabling dimerization, the central stalk also functions as a mechanical component to determine the motility of homodimeric mitotic kinesin-14 motors. We suggest that the central stalk is an evolutionary design that primes these kinesin-14 motors for nontransport roles within the mitotic spindle.

Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.