Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

A lipase from Lacticaseibacillus rhamnosus IDCC 3201 with thermostability and pH resistance for use as a detergent additive.

Lipases are important biocatalysts and ubiquitous in plants, animals, and microorganisms. The high growth rates of microorganisms with low production costs have enabled the wide application of microbial lipases in detergent, food, and cosmetic industries. Herein, a novel lipase from Lacticaseibacillus rhamnosus IDCC 3201 (Lac-Rh) was isolated and its activity analyzed under a range of reaction conditions to evaluate its potential industrial application. The isolated Lac-Rh showed a molecular weight of 24 kDa and a maximum activity of 3438.5 ± 1.8 U/mg protein at 60 °C and pH 8. Additionally, Lac-Rh retained activity in alkaline conditions and in 10% v/v concentrations of organic solvents, including glycerol and acetone. Interestingly, after pre-incubation in the presence of multiple commercial detergents, Lac-Rh maintained over 80% of its activity and the stains from cotton were successfully removed under a simulated laundry  setting. Overall, the purified lipase from L. rhamnosus IDCC 3201 has potential for use as a detergent in industrial applications. KEY POINTS: • A novel lipase (Lac-Rh) was isolated from Lacticaseibacillus rhamnosus IDCC 3201 • Purified Lac-Rh exhibited its highest activity at a temperature of 60 °C and a pH of 8, respectively • Lac-Rh remains stable in commercial laundry detergent and enhances washing performance.

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

High molecular weight genomic DNA mini-prep for filamentous fungi.

Purification of high quality genomic DNA (gDNA) from filamentous fungi suitable for whole genome sequencing has previously involved many steps. Here, we report a simple and easy-to-follow mini-preparation protocol for high molecular weight (∼20kb) gDNA from filamentous fungi including Aspergillus and Eurotium. This comprehensive protocol includes graphic step-by-step instructions for inoculation, homogenization, and purification of gDNA. The most critical step is a thorough 3-5min homogenization of the freeze-dried mycelium using a motorized hand-held homogenizer with a mini spatula inserted. Approximately 20mg of the fine mycelial powder is then subjected to a modified procedure for the DNeasy Plant Mini Kit (Qiagen). This Qiagen spin column protocol avoids precipitation, dryness, and resuspension of gDNA, which can cause shearing and loss of gDNA. Final gDNA yields from ∼20mg of fine mycelial powder are 8 to 20µg with a consistent 260/280nm absorbance ratio of ∼1.9. All 30 gDNA samples we purified using our method were of high molecular weight (∼20kb). Whole genome sequencing of these DNA samples resulted in 160-260 X coverage with 2×150 reads using NextSeq 500. These gDNAs are also of a suitable quality for Southern blotting and PCR-based amplification of various genes in filamentous fungi.

Diversity, Application, and Synthetic Biology of Industrially Important Aspergillus Fungi.

The filamentous fungal genus Aspergillus consists of over 340 officially recognized species. A handful of these Aspergillus fungi are predominantly used for food fermentation and large-scale production of enzymes, organic acids, and bioactive compounds. These industrially important Aspergilli primarily belong to the two major Aspergillus sections, Nigri and Flavi. Aspergillus oryzae (section Flavi) is the most commonly used mold for the fermentation of soybeans, rice, grains, and potatoes. Aspergillus niger (section Nigri) is used in the industrial production of various enzymes and organic acids, including 99% (1.4 million tons per year) of citric acid produced worldwide. Better understanding of the genomes and the signaling mechanisms of key Aspergillus species can help identify novel approaches to enhance these commercially significant strains. This review summarizes the diversity, current applications, key products, and synthetic biology of Aspergillus fungi commonly used in industry.

In Vitro Activities of LCB 01-0648, a Novel Oxazolidinone, against Gram-Positive Bacteria.

Oxazolidinones are a novel class of synthetic antibacterial agents that inhibit bacterial protein synthesis. Here, we synthesized and tested a series of oxazolidinone compounds containing cyclic amidrazone. Among these compounds, we further investigated the antibacterial activities of LCB01-0648 against drug-susceptible or resistant Gram-positive cocci in comparison with those of six reference compounds. LCB01-0648 showed the most potent antimicrobial activities against clinically isolated Gram-positive bacteria. Against the methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCNS) isolates, LCB01-0648 showed the lowest MIC90s (0.5 mg/L) among the tested compounds. In addition, LCB01-0648 had the lowest minimum inhibitory concentrations (MICs) against the four linezolid-resistant S. aureus (LRSA) strains (range 2-4 mg/L). The results of the time-kill studies demonstrated that LCB01-0648 at a concentration 8× the (MIC) showed bactericidal activity against methicillin-susceptible Staphylococcus aureus MSSA or MRSA, but showed a bacteriostatic effect against LRSA. These results indicate that LCB01-0648 could be a good antibacterial candidate against multidrug-resistant (MDR) Gram-positive cocci.

In Vivo Activity of LCB 01-0699, a Prodrug of LCB 01-0648, against Staphylococcus aureus.

LCB01-0648 is a novel oxazolidinone compound that shows potent antibacterial activities against most Gram-positive cocci, including the multi-drug resistant Staphylococcusaureus. In this study, in vivo activity of LCB01-0699, a LCB01-0648 prodrug, against S.aureus was evaluated in comparison with that of Linezolid. The results of the systemic infection study demonstrated that LCB01-0699 was more potent than Linezolid against methicillin-susceptible and -resistant S. aureus strains. The in vivo efficacy of LCB01-0699 against methicillin-susceptible and -resistant S. aureus strains in a skin infection model showed more potent activity than Linezolid. LCB01-0699 shows potent in vivo activity against methicillin-susceptible and -resistant S. aureus strains, suggesting that LCB01-0699 would be a novel candidate for the treatment of these infectious diseases caused by S. aureus.

The velvet family of fungal regulators contains a DNA-binding domain structurally similar to NF-κB.

Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.

Antimicrobial Activity of Zabofloxacin against Clinically Isolated Streptococcus pneumoniae.

Zabofloxacin is a novel fluoroquinolone agent that has potent activity against gram-positive pathogens. In this study, we confirmed that zabofloxacin showed the most potent in vitro and in vivo activities against drug-resistant Streptococcus pneumoniae. Among the fluoroquinolone compounds, zabofloxacin showed the most potent in vitro activity against clinical isolates of penicillin-sensitive S. pneumoniae (minimum inhibitory concentration, MIC90: 0.03 mg/L) and penicillin-resistant S. pneumoniae (MIC90: 0.03 mg/L). Against quinolone-resistant S. pneumoniae, zabofloxacin (MIC90: 1 mg/L) was more active than ciprofloxacin, sparfloxacin, and moxifloxacin; however, its activity was the same as that of gemifloxacin. The in vivo activity of zabofloxacin was most potent among the quinolone compounds tested against the systemic infection and respiratory tract infection models in mice.

SscA is required for fungal development, aflatoxin production, and pathogenicity in Aspergillus flavus.

Fungal spores are specialized dormant cells that act as primary reproductive biological particles and exhibit strong viability under extremely harsh conditions. They contaminate a variety of crops and foods, causing severe health hazards to humans and animals. Previous studies demonstrated that a spore-specific transcription factor SscA plays pivotal roles in the conidiogenesis of the model organism Aspergillus nidulans. In this study, we investigated the biological and genetic functions of SscA in the aflatoxin-producing fungus A. flavus. Deletion of sscA showed reduced conidia formation, lost long-term viability, and exhibited more sensitivity to thermal, oxidative, and radiative stresses. The sscA-deficient strain showed increased aflatoxin B1 production in conidia as well as mycelia. Importantly, the absence of sscA affected fungal pathogenicity on crops. Further transcriptomic and phenotypic studies suggested that SscA coordinates conidial wall structures. Overall, SscA is important for conidial formation, maturation and dormancy, mycotoxin production, and pathogenicity in A. flavus.

The MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans.

Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.

Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus.

Asexual spores, called conidia, are key reproductive fungal particles that enable survival in harsh environmental conditions or host systems. The conidia can infect humans, animals, and plants to cause various fungal diseases. Transcription factors, including VosA, WetA, and SscA, have key roles in conidia formation and long-term survival in Aspergillus nidulans. Herein, we report the pleiotropic functions of SscA in the conidia of the human pathogen A. fumigatus. The deletion of sscA increased conidia formation despite decreased fungal growth. Absence of sscA impaired long-term survival and reduced spore resistance to various stresses, including heat, UV, and oxidation. Transcriptomic analyses showed that SscA involved the mRNA expression of cell wall organisation-related genes. Importantly, the sscA deletion mutant conidia contained an increased amount of ß-glucan and chitin compared to wild type conidia. In addition, conidial gliotoxin production was decreased in the sscA deletion strain. Overall, SscA has pleiotropic roles in conidia formation, maturation and dormancy and mycotoxin production in A. fumigatus.

Phylogenomics analysis of velvet regulators in the fungal kingdom.

All life forms have evolved to respond appropriately to various environmental and internal cues. In the animal kingdom, the prototypical regulator class of such cellular responses is the Rel homology domain proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Fungi, the close relatives of animals, have also evolved with their own NF-κB-like regulators called velvet family proteins to govern cellular and chemical development. Here, we conducted a detailed investigation of the taxonomic broad presence of velvet proteins. We observed that velvet proteins are widely distributed in the fungal kingdom. Moreover, we have identified and characterized 21 major velvet clades in fungi. We have further revealed that the highly conserved velvet domain is composed of three distinct motifs and acts as an evolutionarily independent domain, which can be shuffled with various functional domains. Such rearrangements of the velvet domain have resulted in the functional and type diversity of the present velvet regulators. Importantly, our in-deep analyses of the primary and 3D structures of the various velvet domains showed that the fungal velvet domains can be divided into two major clans: the VelB and the VosA clans. The 3D structure comparisons revealed a close similarity of the velvet domain with many other eukaryotic DNA-binding proteins, including those of the Rel, Runt, and signal transducer and activator of transcription families, sharing a common ß-sandwich fold. Altogether, this study improves our understanding of velvet regulators in the fungal kingdom.IMPORTANCEFungi are the relatives of animals in Opisthokonta and closely associated with human life by interactive ways such as pathogenicity, food, and secondary metabolites including beneficial ones like penicillin and harmful ones like the carcinogenic aflatoxins. Similar to animals, fungi have also evolved with NF-κB-like velvet family regulators. The velvet proteins constitute a large protein family of fungal transcription factors sharing a common velvet domain and play a key role in coordinating fungal secondary metabolism, developmental and differentiation processes. Our current understanding on velvet regulators is mostly from Ascomycota fungi; however, they remain largely unknown outside Ascomycota. Therefore, this study performed a taxonomic broad investigation of velvet proteins across the fungal kingdom and conducted a detailed analysis on velvet distribution, structure, diversity, and evolution. The results provide a holistic view of velvet regulatory system in the fungal kingdom.

Characterization of the velvet regulators in Aspergillus fumigatus.

Fungal development and secondary metabolism is intimately associated via activities of the fungi-specific velvet family proteins. Here we characterize the four velvet regulators in the opportunistic human pathogen Aspergillus fumigatus. The deletion of AfuvosA, AfuveA and AfuvelB causes hyperactive asexual development (conidiation) and precocious and elevated accumulation of AfubrlA during developmental progression. Moreover, the absence of AfuvosA, AfuveA or AfuvelB results in the abundant formation of conidiophores and highly increased AfubrlA mRNA accumulation in liquid submerged culture, suggesting that they act as repressors of conidiation. The deletion of AfuvosA or AfuvelB causes a reduction in conidial trehalose amount, long-term spore viability, conidial tolerance to oxidative and UV stresses, and accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source, suggesting an interdependent role of them in many aspects of fungal biology. Genetic studies suggest that AfuAbaA activates AfuvosA and AfuvelB expression during the mid to late phase of conidiation. Finally, the AfuveA null mutation can be fully complemented by Aspergillus nidulans VeA, which can physically interact with AfuVelB and AfuLaeA in vivo. A model depicting the similar yet different roles of the velvet regulators governing conidiation and sporogenesis in A. fumigatus is presented.

The putative guanine nucleotide exchange factor RicA mediates upstream signaling for growth and development in Aspergillus.

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.

LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity.

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.

Regulators of the Asexual Life Cycle of Aspergillus nidulans.

The genus Aspergillus, one of the most abundant airborne fungi, is classified into hundreds of species that affect humans, animals, and plants. Among these, Aspergillus nidulans, as a key model organism, has been extensively studied to understand the mechanisms governing growth and development, physiology, and gene regulation in fungi. A. nidulans primarily reproduces by forming millions of asexual spores known as conidia. The asexual life cycle of A. nidulans can be simply divided into growth and asexual development (conidiation). After a certain period of vegetative growth, some vegetative cells (hyphae) develop into specialized asexual structures called conidiophores. Each A. nidulans conidiophore is composed of a foot cell, stalk, vesicle, metulae, phialides, and 12,000 conidia. This vegetative-to-developmental transition requires the activity of various regulators including FLB proteins, BrlA, and AbaA. Asymmetric repetitive mitotic cell division of phialides results in the formation of immature conidia. Subsequent conidial maturation requires multiple regulators such as WetA, VosA, and VelB. Matured conidia maintain cellular integrity and long-term viability against various stresses and desiccation. Under appropriate conditions, the resting conidia germinate and form new colonies, and this process is governed by a myriad of regulators, such as CreA and SocA. To date, a plethora of regulators for each asexual developmental stage have been identified and investigated. This review summarizes our current understanding of the regulators of conidial formation, maturation, dormancy, and germination in A. nidulans.

The novel spore-specific regulator SscA controls Aspergillus conidiogenesis.

IMPORTANCE: Filamentous fungi produce myriads of asexual spores, which are the main reproductive particles that act as infectious or allergenic agents. Although the serial of asexual sporogenesis is coordinated by various genetic regulators, there remain uncharacterized transcription factors in Aspergillus. To understand the underlying mechanism of spore formation, integrity, and viability, we have performed comparative transcriptomic analyses on three Aspergillus species and found a spore-specific transcription factor, SscA. SscA has a major role in conidial formation, maturation and dormancy, and germination in Aspergillus nidulans. Functional studies indicate that SscA coordinates conidial wall integrity, amino acid production, and secondary metabolism in A. nidulans conidia. Furthermore, the roles of SscA are conserved in other Aspergillus species. Our findings that the SscA has broad functions in Aspergillus conidia will help to understand the conidiogenesis of Aspergillus species.

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

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