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In order to determine compounds present in the aqueous extract of Cinnamomum kanehirae Hay (CWE) that promote the asexual sporulation of Antrodia cinnamomea in submerged fermentation. First, CWE was isolated by alcohol precipitation and fractional extraction with different organic solvents. The influence of the obtained fractions on the asexual sporulation of A. cinnamomea was investigated. It was showed that the chloroform (at 30 μg/mL) and ethyl acetate (at 50 μg/mL) extracts of the supernatant after ethanol precipitation of CWE presented remarkable promoting effects on the sporulation of A. cinnamomea, and the effect of LFE was significantly more pronounced than that of YZE, indicating that both LFE and YZE contained compounds that promote the sporulation of A. cinnamomea. Subsequently, liquid chromatography-mass spectrometry (LC-MS) was used to analyze the chemical components of the fractions obtained from CWE, and erythritol was considered as the major component that promotes the sporulation of A. cinnamomea. Finally, the effect of erythritol with a purity of 98% on the fermentation performance of A. cinnamomead was investigated. The result showed that erythritol did significantly promote the sporulation of A. cinnamomea and increased the spore production by 55.17% compared with the control group at the optimal concentration of 1.0 μg/mL. Meanwhile, 1.0 μg/mL erythritol significantly promoted the mycelial growth and synthesis of intracellular polysaccharides (IPS) of A. cinnamomea in submerged fermentation and increased the biomass and the yield of IPS by 18.65% and 260.13%, respectively. However, erythritol had no significant effect on the synthesis of triterpenes in A. cinnamomea.

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Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline‐pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two‐hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research. [ABSTRACT FROM AUTHOR]

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC–MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pulldown mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a precious edible and medicinal mushroom with various biological activities, such as hepatoprotection, antitumor, antivirus, immunoregulation, and intestinal flora regulation. However, the wild fruiting bodies of A. cinnamomea are scarce and expensive. Submerged fermentation based on spore inoculation has become the most efficient and popular artificial culture method for A. cinnamomea. In order to complement the mechanism of asexual sporulation of A. cinnamomea in submerged fermentation, and provide a theoretical basis to further improve the sporulation, comparative transcriptomics analysis using RNA-seq and RT-qPCR were conducted on A. cinnamomea mycelia cultured under different nutritional conditions to reveal the regulatory mechanism underlying the asexual sporulation induced by nutrient limitation. The obtained mechanism is as follows: under nitrogen starvation, the corresponding sensors transmit signals to genes, such as areA and tmpA, and promote their expression. Among these genes, AreA has a direct or indirect effect on flbD and promotes its expression, further enhancing the expression of brlA. Meanwhile, TmpA has a direct or indirect effect on brlA and promotes its expression; under carbon starvation, transport protein Rco-3, as a glucose sensor, directly or indirectly transmits signals to brlA and promotes its expression. BrlA promotes the expression of abaA gene, which further enhances the expression of wetA gene, and wetA then directly leads to asexual sporulation and promotes spore maturation; meanwhile, gulC can also promote cell autolysis, which provides energy and raw materials for sporulation. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation.

Filamentous fungi reproduce sexually or asexually, and the developmental processes are strictly regulated by a variety of transcription factors. In this study, we characterized a zinc finger transcription factor, called AcrpnR, in Aspergillus cristatus (GME2916). The ∆AcrpnR strain exhibited decreased asexual reproduction and increased cleistothecium production. The complementation strain showed restoration of these phenotypic differences. Overexpression of AcrpnR resulted in enhanced asexual development and delayed and inhibited sexual reproduction, suggesting that AcrpnR is required for proper asexual and sexual development in A. cristatus. In addition, AcrpnR positively regulated the expression of genes of the central regulatory pathway of conidiation and negatively regulated the expression of sex‐related genes. Overall, these results demonstrate that AcrpnR is essential for maintaining a balance between asexual and sexual development. [ABSTRACT FROM AUTHOR]

As the dominant fungus during the fermentation of Fuzhuan brick tea, Aspergillus cristatus is easily induced to undergo a sexual cycle under low-salt stress. However, the underlying regulatory mechanism of sexual reproduction is unclear. Here, we report a P53-like transcription factor AcndtA , which encodes an NDT80 DNA binding protein and regulates fungal reproduction, pigmentation and the stress response. Both insertion and deletion mutants of AcndtA exhibited a complete blockade of cleistothecium formation, and overexpressing AcndtA strains (OE: AcndtA) exhibited significantly reduced cleistothecium production, indicating that AcndtA plays a vital role in sexual development. Osmotic stress tests showed that overexpression of AcndtA had a negative impact on growth and conidia production. Additionally, AcndtA insertion, deletion and overexpression mutants exhibited reduced pigment formation. All the above developmental defects were reversed by the re-introduction of the AcndtA gene in Δ AcndtA. Moreover, the growth of AcndtA mutants in carbon-limited medium was better than that of the WT and OE: AcndtA strains, indicating that AcndtA is involved in carbon metabolism. Transcriptional profiling data showed that AcndtA regulated the expression of several genes related to development, osmotic stress and carbon metabolism. • AcndtA plays a vital role in the sexual development of Aspergillus cristatus. • Overexpression of AcndtA reduced growth and conidia production in response to osmotic stress. • Deletion and overexpression of the AcndtA gene leads to reduced pigmentation. • The expression of several genes related to development, osmotic stress and carbon metabolism is affected by AcndtA. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a precious edible and medicinal mushroom with various biological activities, such as hepatoprotection, antitumor, antivirus, immunoregulation, and intestinal flora regulation. However, the wild fruiting bodies of A. cinnamomea are scarce and expensive. Submerged fermentation based on spore inoculation has become the most efficient and popular artificial culture method for A. cinnamomea. In order to complement the mechanism of asexual sporulation of A. cinnamomea in submerged fermentation, and provide a theoretical basis to further improve the sporulation, comparative transcriptomics analysis using RNA-seq and RT-qPCR were conducted on A. cinnamomea mycelia cultured under different nutritional conditions to reveal the regulatory mechanism underlying the asexual sporulation induced by nutrient limitation. The obtained mechanism is as follows: under nitrogen starvation, the corresponding sensors transmit signals to genes, such as areA and tmpA, and promote their expression. Among these genes, AreA has a direct or indirect effect on flbD and promotes its expression, further enhancing the expression of brlA. Meanwhile, TmpA has a direct or indirect effect on brlA and promotes its expression; under carbon starvation, transport protein Rco-3, as a glucose sensor, directly or indirectly transmits signals to brlA and promotes its expression. BrlA promotes the expression of abaA gene, which further enhances the expression of wetA gene, and wetA then directly leads to asexual sporulation and promotes spore maturation; meanwhile, gulC can also promote cell autolysis, which provides energy and raw materials for sporulation.

Identification of aspergilli based on comparative of their diagnostic features in different growth media is a common manner between most of mycologists. Asexual sporulation in Aspergillus species is very complex processes which include numerous morphologically distinct stages. In the present study, asexual reproductive structures of Aspergillus terreus and Aspergillus versicolor have been tracked and the development of conidiophores have been compared in two types of growth media MEA (Malt Extract Agar) and YES (Yeast Extract Sucrose) which considered the most common growth media in identification of aspergilli. Interestingly, YES agar medium induce A. terreus to retard the asexual sporulation process in a distinctive manner. Unusually, the early conidiophore of A. terreus distends to form infertile (immature) vesicle which then produces a second conidiophore. The later conidiophore expands to form fertile (mature) vesicle which differentiates into conidial head. The development of one or more conidiophores from the immature vesicles of A. terreus instead the foot cells or areal hyphae remain mostly uncharacterized by many researchers. On the other hand, the asexual sporulation in both Aspergillus species was dramatically decreased on YES agar medium when compared by MEA medium. Moreover, the accessory conidia in A. terreus and reduced Penicillium-like structures that produce another type of phialedic conidia in A. versicolor were mostly not observed on YES agar medium.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans , a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism. [ABSTRACT FROM AUTHOR]

In 2018, we conducted a cross-sectional study to investigate azole resistance in environmental Aspergillus fumigatus isolates obtained from different agricultural fields in China. Using 63 soil cores, we cultured for azole-resistant A. fumigatus and characterized isolates by their cyp51A gene type, short tandem repeat genotype, and mating type. Of 206 A. fumigatus isolates, 21 (10.2%) were azole resistant. Nineteen of 21 had mutations in their cyp51A gene (5 TR34/L98H, 8 TR34/L98H/S297T/F495I, 6 TR46/Y121F/T289A). Eighteen were cultured from soil samples acquired from strawberry fields, suggesting this soil type is a potential hotspot for azole resistance selection. Twenty resistant isolates were mating type MAT1-1, suggesting asexual sporulation contributed to their evolution. Prochloraz, difenoconazole, and tebuconazole were the most frequently detected fungicides in soil samples with azole-resistant fungus. Our study results suggest that managing the fungicides used in agriculture will help contain the problem of antifungal drug resistance in clinics. [ABSTRACT FROM AUTHOR]

• AwSclB is involved in regulating asexual sporulation and secondary metabolism. • AwSclB regulates asexual spore formation by activating the BrlA-AbaA-WetA pathway. • In the absence of AwSclB , light no longer promotes the formation of conidia. • AwSclB gene contributes to the light response of Aspergillus westerdijkiae by activating ConJ. As a prevalent pathogenic fungus, Aspergillus westerdijkiae poses a threat to both food safety and human health. The fungal growth, conidia production and ochratoxin A (OTA) in A. weterdijkiae are regulated by many factors especially transcription factors. In this study, a transcription factor AwSclB in A. westerdijkiae was identified and its function in asexual sporulation and OTA biosynthesis was investigated. In addition, the effect of light control on AwSclB regulation was also tested. The deletion of AwSclB gene could reduce conidia production by down-regulation of conidia genes and increase OTA biosynthesis by up-regulation of cluster genes, regardless under light or dark conditions. It is worth to note that the inhibitory effect of light on OTA biosynthesis was reversed by the knockout of AwSclB gene. The yeast one-hybrid assay indicated that AwSclB could interact with the promoters of BrlA , ConJ and OtaR1 genes. This result suggests that AwSclB in A. westerdijkiae can directly regulate asexual conidia formation by activating the central developmental pathway BrlA-AbaA-WetA through up-regulating the expression of AwBrlA , and promote the light response of the strain by activating ConJ. However, AwSclB itself is unable to respond to light regulation. This finding will deepen our understanding of the molecular regulation of A. westerdijkiae development and secondary metabolism, and provide potential targets for the development of new fungicides. [ABSTRACT FROM AUTHOR]

Zymoseptoria tritici is the causal agent of septoria tritici blotch, a devastating fungal disease of wheat which can cause up to 40% yield loss. One of the ways in which Z. tritici spreads in the field is via rain splash-dispersed asexual pycnidiospores, however there is currently limited understanding of the genetic mechanisms governing the development of these propagules. In order to explore whether the existing models for conidiation in ascomycete fungi apply to Z. tritici, homologs to the well-characterized Aspergillus nidulans genes abacus (abaA), bristle (brlA), fluffy B (flbB), fluffy C (flbC), and stunted (stuA) were identified and knocked-out by Agrobacterium-mediated transformation. Although deletion of the ZtAbaA, ZtBrlA1, and ZtFlbB genes had no apparent effect on Z. tritici asexual sporulation or on pathogenicity, deletion of ZtFlbC or ZtBrlA2 resulted in mutants with reduced pycnidiospore production compared to the parental IPO323 strain. Deletion of ZtStuA gave non-pigmented mutants with altered vegetative growth and eliminated asexual sporulation and pathogenicity. These findings suggest that the well-established A. nidulans model of asexual sporulation is only partially applicable to Z. tritici, and that this pathogen likely uses additional, as yet uncharacterized genes to control asexual sporulation.

Identification of aspergilli based on comparative of their diagnostic features in different growth media is a common manner between most of mycologists. Asexual sporulation in Aspergillus species is very complex processes which include numerous morphologically distinct stages. In the present study, asexual reproductive structures of Aspergillus terreus and Aspergillus versicolor have been tracked and the development of conidiophores have been compared in two types of growth media MEA (Malt Extract Agar) and YES (Yeast Extract Sucrose) which considered the most common growth media in identification of aspergilli. Interestingly, YES agar medium induce A. terreus to retard the asexual sporulation process in a distinctive manner. Unusually, the early conidiophore of A. terreus distends to form infertile (immature) vesicle which then produces a second conidiophore. The later conidiophore expands to form fertile (mature) vesicle which differentiates into conidial head. The development of one or more conidiophores from the immature vesicles of A. terreus instead the foot cells or areal hyphae remain mostly uncharacterized by many researchers. On the other hand, the asexual sporulation in both Aspergillus species was dramatically decreased on YES agar medium when compared by MEA medium. Moreover, the accessory conidia in A. terreus and reduced Penicillium-like structures that produce another type of phialedic conidia in A. versicolor were mostly not observed on YES agar medium. [ABSTRACT FROM AUTHOR]

Zymoseptoria tritici is the causal agent of septoria tritici blotch, a devastating fungal disease of wheat which can cause up to 40%yield loss. One of the ways in which Z. tritici spreads in the field is via rain splash-dispersed asexual pycnidiospores, however there is currently limited understanding of the genetic mechanisms governing the development of these propagules. In order to explore whether the existing models for conidiation in ascomycete fungi apply to Z. tritici, homologs to the well-characterized Aspergillus nidulans genes abacus (abaA), bristle (brlA), fluffy B (flbB), fluffy C (flbC), and stunted (stuA) were identified and knocked-out by Agrobacterium-mediated transformation. Although deletion of the ZtAbaA, ZtBrlA1, and ZtFlbB genes had no apparent effect on Z. tritici asexual sporulation or on pathogenicity, deletion of ZtFlbC or ZtBrlA2 resulted in mutants with reduced pycnidiospore production compared to the parental IPO323 strain. Deletion of ZtStuA gave non-pigmented mutants with altered vegetative growth and eliminated asexual sporulation and pathogenicity. These findings suggest that the well-established A. nidulans model of asexual sporulation is only partially applicable to Z. tritici, and that this pathogen likely uses additional, as yet uncharacterized genes to control asexual sporulation. [ABSTRACT FROM AUTHOR]

Understanding the occurrence and spread of azole resistance in Aspergillus fumigatus is crucial for public health. It has been hypothesized that asexual sporulation, which is abundant in nature, is essential for phenotypic expression of azole resistance mutations in A. fumigatus facilitating subsequent spread through natural selection. Furthermore, the disease aspergilloma is associated with asexual sporulation within the lungs of patients and the emergence of azole resistance. This study assessed the evolutionary advantage of asexual sporulation by growing the fungus under pressure of one of five different azole fungicides over seven weeks and by comparing the rate of adaptation between scenarios of culturing with and without asexual sporulation. Results unequivocally show that asexual sporulation facilitates adaptation. This can be explained by the combination of more effective selection because of the transition from a multicellular to a unicellular stage, and by increased mutation supply due to the production of spores, which involves numerous mitotic divisions. Insights from this study are essential to unravel the resistance mechanisms of sporulating pathogens to chemical compounds and disease agents in general, and for designing strategies that prevent or overcome the emerging threat of azole resistance in particular. [ABSTRACT FROM AUTHOR]

Background: RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants. Results: Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. Conclusion: This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model. [ABSTRACT FROM AUTHOR]

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A . cristatus . However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1 , which is homologous to hog1 from Saccharomyces cerevisiae , in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that Δ Achog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of Δ Achog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A . cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in Δ Achog1 . Pull-down and LC-MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pull-down mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A . cristatus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shao, Tan, Song, Wang, Liu, Huang, Ren and Liu.)

Ganoderma lucidum differentiates in liquid static culture by forming aerial mycelia and asexual spores, and this differentiation process is accompanied by higher production of anti-tumor compounds ganoderic acids. To gain an insight into the molecular events during asexual sporulation of G. lucidum, comparative transcriptome analysis using suppression subtractive hybridization (SSH) technique was performed to identify preferentially expressed genes in liquid static culture vs . in traditional shaking culture. After macroarray analysis of 1920 cDNAs from SSH library, 147 unigenes which exhibited high expression in static culture were identified. Among these sequences, putative translations of 88 unigenes possessed much similarity to known proteins involved in cell organization, signal transduction, cell metabolism, protein biosynthesis and transcription regulation; 13 had significant similarity to hypothetical proteins; the remaining 46 showed little or no similarity to GenBank sequences. RT-qPCR analysis confirmed increases in transcripts of selected genes under liquid static culture condition. The results of this study present the useful application of EST analysis on G. lucidum and provide preliminary indication of gene expression putatively involved in asexual sporulation process. [ABSTRACT FROM AUTHOR]

Background: It has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development. Results: G-protein Gγ and Gβ subunits, named Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayed significantly slower growth in planta. Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered. Conclusion: This study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected. [ABSTRACT FROM AUTHOR]