Asexual Spore Formation Research Articles

Functional characterization of extracellular and intracellular catalase-peroxidases involved in virulence of the fungal wheat pathogen Zymoseptoria tritici.

Understanding how pathogens defend themselves against host defence mechanisms, such as hydrogen peroxide (H2O2) production, is crucial for comprehending fungal infections. H2O2 poses a significant threat to invading fungi due to its potent oxidizing properties. Our research focuses on the hemibiotrophic fungal wheat pathogen Zymoseptoria tritici, enabling us to investigate host-pathogen interactions. We examined two catalase-peroxidase (CP) genes, ZtCpx1 and ZtCpx2, to elucidate how Z. tritici deals with host-generated H2O2 during infection. Our analysis revealed that ZtCpx1 was up-regulated during biotrophic growth and asexual spore formation invitro, while ZtCpx2 showed increased expression during the transition from biotrophic to necrotrophic growth and in-vitro vegetative growth. Deleting ZtCpx1 increased the mutant's sensitivity to exogenously added H2O2 and significantly reduced virulence, as evidenced by decreased Septoria tritici blotch symptom severity and fungal biomass production. Reintroducing the wild-type ZtCpx1 allele with its native promoter into the mutant strain restored the observed phenotypes. While ZtCpx2 was not essential for full virulence, the ZtCpx2 mutants exhibited reduced fungal biomass development during the transition from biotrophic to necrotrophic growth. Moreover, both CP genes act synergistically, as the double knock-out mutant displayed a more pronounced reduced virulence compared to ΔZtCpx1. Microscopic analysis using fluorescent proteins revealed that ZtCpx1 was localized in the peroxisome, indicating its potential role in managing host-generated reactive oxygen species during infection. In conclusion, our research sheds light on the crucial roles of CP genes ZtCpx1 and ZtCpx2 in the defence mechanism of Z. tritici against host-generated hydrogen peroxide.

Roles of Three FgPel Genes in the Development and Pathogenicity Regulation of Fusarium graminearum.

Fusarium head blight (FHB) is a devastating fungal disease caused by Fusarium graminearum. Pectin lyase, a pectinase, acts on the α-1,4-glycosidic linkage of galacturonic acid primarily by β-elimination. In this study, three pectin lyase genes (FgPel1, 2, 3) in F. graminearum were selected, and deletion mutants (ΔFgPel1, 2, 3) were constructed by homologous recombination for functional characterization. The gene deletions affected the morphology and growth rate of F. graminearum on pectin medium at various concentrations, with the growth rate of ΔFgPel1 being more significant. The growth of ΔFgPel1 colonies slowed at pH 4, with optimal growth at pH 6.5, whereas ΔFgPel2 and ΔFgPel3 exhibited greater inhibition at pH 8. Colony morphology and diameter of the deletion mutants showed no significant differences compared to the wild-type strain PH-1, and there was no effect on conidial production or germination rate. Pathogenicity assays demonstrated that gene deletion significantly reduced the ability of F. graminearum to infest corn silks and wheat ears, and that ΔFgPel2 showed a more pronounced reduction in pathogenicity on wheat spikes. In summary, the pectin lyase genes (FgPel1, 2, 3) are involved in pectin utilization and are influenced by external pH conditions, which attenuate the pathogenicity of F. graminearum without affecting its vegetative growth or asexual spore formation. These findings elucidate the roles of these genes and provide a basis for controlling FHB.

Role of reactive oxygen species (ROS) signaling pathway in the wound-induced spore formation of Pyropia yezoensis

Asexual spore formation of Pyropia yezoensis significantly impacts yield. After mechanical sectioning of leafy thalli into fragments, somatic cells rapidly develop into a large number of wound-induced spores (WIS, a form of asexual spores). Recent studies on P. yezoensis have demonstrated a notable increase in reactive oxygen species (ROS) during this process; however, the regulatory mechanism underlying this ROS signaling remains largely unclear. This paper shows that in P. yezoensis, ROS signals play a crucial role in WIS formation. When treated with the ROS-producing enzyme NADPH oxidase inhibitor diphenyleneiodonium chloride, P. yezoensis thalli fragments strongly repressed WIS formation, accompanied by a significant decrease of ROS levels, malondialdehyde contents, and activities of NADPH oxidase and antioxidant enzymes. The expression levels of respiratory burst oxidase homolog genes, aquaporin protein genes, superoxide dismutase genes, glutathione S-transferase genes, and glutathioneperoxidase genes were also decreased, thus inhibiting WIS formation. As an upstream signal, ROS promote ion exchange and metabolism both inside and outside of cells within the first 12 h of wound stress. Following 12 h of wound stress, transcriptional activation of specific genes associated with cell cycle progression—which promotes cell division and dedifferentiation under normal conditions—was also blocked by diphenyleneiodonium chloride. In addition, the generated ROS signals also regulated the expression of mitogen-activated protein kinase family genes under wound stress. Overall, this study provides novel insights into the molecular mechanism underlying the ROS signaling regulation of WIS formation in P. yezoensis.

Mannitol improves Monascus pigment biosynthesis with rice bran as a substrate in Monascus purpureus.

To reduce the production cost of Monascus pigments (MPs), the utilization of rice bran (RB), an agricultural waste product, as a substrate in submerged fermentation was conducted in this study. To improve MP production, different nutritional ingredients including mannitol (Man), NH4NO3 (AN), ZnSO4 (Zn), and optimization (Opti), which was a synthesis of the three above ones, were added in rice bran (RB) medium. The yields of MPs, pigment constituents, and growth and development of Monascus purpureus M9 were investigated in this study. Man had the maximum color value of 3,532 U/g, which was 18.69 times more than that of RB and reached up to 76.65% of the value of rice (Rice) fermentation. Man significantly increased the production of two orange pigments, monascorubrin and rubropunctatin, of which the yields were 69.49 and 95.36% of the counterpart of Rice. The biomass and colony diameter of Opti presented the maximum value among different groups. AN and RB induced more asexual spore formation, whereas Opti and Man promoted sexual spore production. Comparative transcriptomic analysis showed that different nutritional ingredients led to changes in pigment production, promoting the growth and development of M. purpureus M9 through the regulation of related gene expression. Man and Opti improved MP production by regulating the primary metabolism, including the Embden-Meyerhof pathway (EMP), the pentose phosphate (PP) pathway, the tricarboxylic (TCA) cycle, fatty acid degradation (FAD), fatty acid biosynthesis (FAB), amino acid metabolism (AAM), and fructose and mannose metabolism (FMM), to provide the precursors (acetyl-CoA and malonyl-CoA) for MP biosynthesis. This study presents a low-cost method for increasing MP production and explains the molecular mechanisms of different nutritional ingredients for enhancing MP biosynthesis.

Fungal spore swelling and germination are restricted by the macrophage phagolysosome

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.

Ultrastructure and Physiological Characterization of Morchella Mitospores and Their Relevance in the Understanding of the Morel Life Cycle.

Morels, which belong to the Ascomycete genus Morchella, are highly valued edible fungi treasured by gourmet chefs worldwide. Some species are saprotrophic and others are able to form facultative mycorrhizal-like associations with plant roots without establishing true ectomycorrhizal symbioses. In general, it is considered that the formation of asexual spores, or mitospores, is an important step in the life cycle of morels. However, ultrastructure characterization and physiological attributes of morel mitospores have received little attention. In this contribution, the mitospores of M. sextelata were successfully induced under laboratory conditions and their ultrastructure, occurrence, germination, physiological characteristics and mating type gene structure were studied. Mitospore production was closely related to aeration, nutrition and humidity conditions. The average germination rate of mitospores on different media and under various induction stimuli was very low, with an average of 1/100,000. Based on the ultrastructure characterization, low germination rate, growth rate decline, rapid aging and mating genotyping, it was concluded that the mitospores of M. sextelata had lost their conventional function as conidia and might act more as mate sperm-like (gamete) structures. Thus, this study contributed to a deeper understanding of the life cycle of the economically and ecologically important morel fungal group.

Soil fungi invest into asexual sporulation under resource scarcity, but trait spaces of individual isolates are unique.

During the last few decades, a plethora of sequencing studies provided insight into fungal community composition under various environmental conditions. Still, the mechanisms of species assembly and fungal spread in soil remain largely unknown. While mycelial growth patterns are studied extensively, the abundant formation of asexual spores is often overlooked, though representing a substantial part of the fungal life cycle relevant for survival and dispersal. Here, we explore asexual sporulation (spore abundance, size and shape) in 32 co-occurring soil fungal isolates under varying resource conditions, to answer the question whether resource limitation triggers or inhibits fungal investment into reproduction. We further hypothesized that trade-offs exist in fungal investment towards growth, spore production and size. The results revealed overall increased fungal investment into spore production under resource limitations; however, effect sizes and response types varied strongly among fungal isolates. Such isolate-specific effects were apparent in all measured traits, resulting in unique trait spaces of individual isolates. This comprehensive dataset also elucidated variability in sporulation strategies and trade-offs with fungal growth and reproduction under resource scarcity, as only predicted by theoretical models before. The observed isolate-specific strategies likely underpin mechanisms of co-existence in this diverse group of saprobic soil fungi.

Dual Regulatory Role of Chromatin Remodeler ISW1 in Coordinating Cellulase and Secondary Metabolite Biosynthesis in Trichoderma reesei.

ABSTRACTThe saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers isolated from nature. T. reesei also produces a typical yellow pigment identified as sorbicillinoids during cultivation. Here, we identified an evolutionarily conserved histone remodeling factor, ISW1, in T. reesei that simultaneously participates in regulating cellulase and the yellow pigment biosynthesis. Trisw1 deletion almost abolished vegetable growth, asexual spore formation, and cellulase gene expression. However, its absence significantly enhanced the production of the yellow pigment. The observed dual regulatory role of TrISW1 was dependent on its ATPase activity. We demonstrated that Trisw1 disruption elevated the transcription of ypr1 coding for the transcriptional activator of sor genes encoding the polyketide synthases catalyzing the biosynthesis of sorbicillinoids but compromised that of xyr1 encoding the key transcriptional activator of cellulase genes. Discrete T. reesei homologous ISW1 accessory factors were also found to exert differential effects on the expression of these two types of genes. Further analyses showed that TrISW1 was recruited to cellulase gene promoters, and its absence interfered with loss of histone H4 at the cbh1 and eg1 promoters upon cellulose induction. To the contrary, Trisw1 deletion facilitated loss of H4 at the sor locus. These data indicate that TrISW1 represents an important chromatin remodeler with a dual role in coordinating the cellulolytic response and biosynthesis of the major secondary metabolite in T. reesei.

Functions of PUF Family RNA-Binding Proteins in Aspergillus nidulans.

RNA-binding proteins are involved in RNA metabolism and posttranscriptional regulation of various fundamental biological processes. The PUF family of RNA-binding proteins is highly conserved in eukaryotes, and its members regulate gene expression, mitochondrial biogenesis, and RNA processing. However, their biological functions in Aspergillus species remain mostly unknown in filamentous fungi. Here we have characterized the puf genes in the model organism Aspergillus nidulans. We generated deletion mutant strains for the five putative puf genes present in the A. nidulans genome and investigated their developmental phenotypes. Deletion of pufA or pufE affected fungal growth and asexual development. pufA mutants exhibited decreased production of asexual spores and reduced mRNA expression of genes regulating asexual development. The pufE deletion reduced colony growth, increased formation of asexual spores, and delayed production of sexual fruiting bodies. In addition, the absence of pufE reduced both sterigmatocystin production and the mRNA levels of genes in the sterigmatocystin cluster. Finally, pufE deletion mutants showed reduced trehalose production and lower resistance to thermal stress. Overall, these results demonstrate that PufA and PufE play roles in the development and sterigmatocystin metabolism in A. nidulans.

The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides.

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine-stabilised (CS)αβ defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore-associated defX gene products delay the growth of Gram-positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore-associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a 'virulence school' for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co-infecting microorganisms to break colonisation resistance at host surfaces.

Dynamic Regulation of Peroxisomes and Mitochondria during Fungal Development.

Peroxisomes and mitochondria are organelles that perform major functions in the cell and whose activity is very closely associated. In fungi, the function of these organelles is critical for many developmental processes. Recent studies have disclosed that, additionally, fungal development comprises a dynamic regulation of the activity of these organelles, which involves a developmental regulation of organelle assembly, as well as a dynamic modulation of the abundance, distribution, and morphology of these organelles. Furthermore, for many of these processes, the dynamics of peroxisomes and mitochondria are governed by common factors. Notably, intense research has revealed that the process that drives the division of mitochondria and peroxisomes contributes to several developmental processes—including the formation of asexual spores, the differentiation of infective structures by pathogenic fungi, and sexual development—and that these processes rely on selective removal of these organelles via autophagy. Furthermore, evidence has been obtained suggesting a coordinated regulation of organelle assembly and dynamics during development and supporting the existence of regulatory systems controlling fungal development in response to mitochondrial activity. Gathered information underscores an important role for mitochondrial and peroxisome dynamics in fungal development and suggests that this process involves the concerted activity of these organelles.

The velvet repressed vidA gene plays a key role in governing development in Aspergillus nidulans.

Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a C2H2 zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and β-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.

Nodulation Induces Systemic Resistance of Medicago truncatula and Pisum sativum Against Erysiphe pisi and Primes for Powdery Mildew-Triggered Salicylic Acid Accumulation.

Plants encounter beneficial and detrimental microorganisms both above- and belowground and the health status of the plant depends on the composition of this pan-microbiome. Beneficial microorganisms contribute to plant nutrition or systemically or locally protect plants against pathogens, thus facilitating adaptation to a variety of environments. Induced systemic resistance, caused by root-associated microbes, manifests as aboveground resistance against necrotrophic pathogens and is mediated by jasmonic acid/ethylene-dependent signaling. By contrast, systemic acquired resistance relies on salicylic acid (SA) signaling and confers resistance against secondary infection by (hemi)biotrophic pathogens. To investigate whether symbiotic rhizobia that are ubiquitously found in natural ecosystems are able to modulate resistance against biotrophs, we tested the impact of preestablished nodulation of Medicago truncatula and pea (Pisum sativum) plants against infection by the powdery mildew fungus Erysiphe pisi. We found that root symbiosis interfered with fungal penetration of M. truncatula and reduced asexual spore formation on pea leaves independently of symbiotic nitrogen fixation. Improved resistance of nodulated plants correlated with elevated levels of free SA and SA-dependent marker gene expression upon powdery mildew infection. Our results suggest that nodulation primes the plants systemically for E. pisi-triggered SA accumulation and defense gene expression, resulting in increased resistance.

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

[This corrects the article DOI: 10.1371/journal.pgen.1007511.].

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.

Structure, kinetics, molecular and redox properties of a cytosolic and developmentally regulated fungal catalase-peroxidase

CAT-2, a cytosolic catalase-peroxidase (CP) from Neurospora crassa, which is induced during asexual spore formation, was heterologously expressed and characterized. CAT-2 had the Met-Tyr-Trp (M-Y-W) adduct required for catalase activity. Its KM for H2O2 was micromolar for peroxidase and millimolar for catalase activity. A Em = −158 mV reduction potential value was obtained and the Soret band shift suggested a mixture of low and high spin ferric iron. CAT-2 EPR spectrum at 10 K indicated an axial and a rhombic component. With peroxyacetic acid (PAA), formation of Compound I* was observed with EPR. CAT-2 homodimer crystallographic structure contained two K+ ions; Glu107 residues were displaced to bind them. CAT-2 showed the essential amino acid residues for activity in similar positions to other CPs. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. A perhydroxy modification of the indole nitrogen of Trp90 was oriented toward the catalytic His91. In contrast to cytochrome c peroxidase and ascorbate peroxidase, the catalase-peroxidase heme propionates are not exposed to the solvent. Together with other N. crassa enzymes that utilize H2O2 as a substrate, CAT-2 has many tryptophan and proline residues at its surface, probably related to H2O2 selection in water.

The Morphology and Structure of Red Pigment Producing Fungus: Monascus Purpureus

Thered pigment producing, ascospore forming fungus Monascus purpureuswas obtained by monospore isolation and maintained on potato dextrose agar at 32°C for 7 days. M. purpureusproduces compact colonies of mycelia and accumulates large quantities of red pigment. Here we aimed to describe this newly isolated red pigment producing fungus using biochemical and microscopy technique. A newly isolated red pigment producing fungus from local red fermented rice was identified using Microbial Identification System based on fatty acids profiles.The growth, morphology, and structure of M. purpureuswere characterized by Scanning Electron Microscopy (SME). We found that M. purpureusreproduces sexually (by the formation of cleistothecium with ascospores) and asexual (by the formation of conidia). In Monascusspecies, the formation of either asexual or sexual spores appears to be an effective growth strategy. On the basis of biochemical and all morphological investigations it could be concluded that the new strain isolated from red fermented rice belongs to species M. purpureus, labeled as M. purpureus FTCC 5391.

Integration of the catalytic subunit activates deneddylase activity in vivo as final step in fungal COP9 signalosome assembly.

The eight-subunit COP9 signalosome (CSN) is conserved from filamentous fungi to humans and functions at the interface between cellular signalling and protein half-life control. CSN consists of six PCI and two MPN domain proteins and forms a scaffold for additional interacting proteins. CSN controls protein stability in the ubiquitin-proteasome system where the MPN domain CSN5/CsnE subunit inactivates cullin-RING ligases. The CSN5/CsnE isopeptidase functions as deneddylase and removes the ubiquitin-like protein Nedd8. The six PCI domain proteins of human CSN form a horseshoe-like ring and all eight subunits are connected by a bundle of C-terminal α-helices. We show that single deletions of any csn subunit of Aspergillus nidulans resulted in the lack of deneddylase activity and identical defects in the coordination of development and secondary metabolism. The CSN1/CsnA N-terminus is dispensable for deneddylase activity but required for asexual spore formation. Complex analyses in mutant strains revealed the presence of a seven-subunit pre-CSN without catalytic activity. Reconstitution experiments with crude extracts of deletion strains and recombinant proteins allowed the integration of CSN5/CsnE into pre-CSN resulting in an active deneddylase. This supports a stable seven subunit pre-CSN intermediate where deneddylase activation in vivo can be controlled by CSN5/CsnE integration as final assembly step.

Identification and Expression Analysis of Regulatory Genes Induced by Near-Ultraviolet Irradiation in <i>Bipolaris oryzae</i>

Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic response and to identify new genes upregulated by NUV irradiation, suppression subtractive hybridization (SSH) was carried out in B. oryzae. To confirm the differential gene expression in NUV-irradiated mycelia, quantitative real-time PCR (qRT-PCR) analysis was performed among 301 genes arbitrarily chosen from 1170 cDNA clones. The expression of 46 genes (named NUV01 to NUV46) was found to be significantly enhanced (>4-fold) by NUV irradiation. Sequence analysis revealed that 23 out of the 46 sequences (50%) showed significant matches to known fungal genes. The 46 genes were categorized as either BLR1-dependent or BLR1-independent expression groups using the BLR1-deficient mutant, which presumably lacks the blue/UVA-absorbing photoreceptor. This finding demonstrates that NUV irradiation can induce gene regulation, and that this response may be mediated by both a blue/UVA-absorbing photoreceptor and an as-yet-unidentified photoreceptor in B. oryzae.

Interplay of the fungal sumoylation network for control of multicellular development

The role of the complex network of the ubiquitin-like modifier SumO in fungal development was analysed. SumO is not only required for sexual development but also for accurate induction and light stimulation of asexual development. The Aspergillus nidulans COMPASS complex including its subunits CclA and the methyltransferase SetA connects the SumO network to histone modification. SetA is required for correct positioning of aerial hyphae for conidiophore and asexual spore formation. Multicellular fungal development requires sumoylation and desumoylation. This includes the SumO processing enzyme UlpB, the E1 SumO activating enzyme AosA/UbaB, the E2 conjugation enzyme UbcN and UlpA as major SumO isopeptidase. Genetic suppression analysis suggests a connection between the genes for the Nedd8 isopeptidase DenA and the SumO isopeptidase UlpA and therefore a developmental interplay between neddylation and sumoylation in fungi. Biochemical evidence suggests an additional connection of the fungal SumO network with ubiquitination. Members of the cellular SumO network include histone modifiers, components of the transcription, RNA maturation and stress response machinery, or metabolic enzymes. Our data suggest that the SumO network controls specific temporal and spatial steps in fungal differentiation.