Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

Synthesis, Characterization, and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus.

Aspergillus flavus contamination in agriculture and food industries poses threats to human health, leading to a requirement of a safe and effective method to control fungal contamination. Chitosan-based nitrogen-containing derivatives have attracted much attention due to their safety and enhanced antimicrobial applications. Herein, a new benzimidazole-grafted chitosan (BAC) was synthesized by linking the chitosan (CS) with a simple benzimidazole compound, 2-benzimidazolepropionic acid (BA). The characterization of BAC was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H and 13C NMR). Then, the efficiency of BAC against A. flavus ACCC 32656 was investigated in terms of spore germination, mycelial growth, and aflatoxin production. BAC showed a much better antifungal effect than CS and BA. The minimum inhibitory concentration (MIC) value was 1.25 mg/mL for BAC, while the highest solubility of CS (16.0 mg/mL) or BA (4.0 mg/mL) could not completely inhibit the growth of A. flavus. Furthermore, results showed that BAC inhibited spore germination and elongation by affecting ergosterol biosynthesis and the cell membrane integrity, leading to the permeabilization of the plasma membrane and leakage of intracellular content. The production of aflatoxin was also inhibited when treated with BAC. These findings indicate that benzimidazole-derived natural CS has the potential to be used as an ideal antifungal agent for food preservation.

Nucleoside Diphosphate Kinase FgNdpk Is Required for DON Production and Pathogenicity by Regulating the Growth and Toxisome Formation of Fusarium graminearum.

Nucleoside diphosphate kinases (NDPKs) are nucleotide metabolism enzymes that play different physiological functions in different species. However, the roles of NDPK in phytopathogen and mycotoxin production are not well understood. In this study, we showed that Fusarium graminearum FgNdpk is important for vegetative growth, conidiation, sexual development, and pathogenicity. Furthermore, FgNdpk is required for deoxynivalenol (DON) production; deletion of FgNDPK downregulates the expression of DON biosynthesis genes and disrupts the formation of FgTri4-GFP-labeled toxisomes, while overexpression of FgNDPK significantly increases DON production. Interestingly, FgNdpk colocalizes with the DON biosynthesis proteins FgTri1 and FgTri4 in the toxisome, and coimmunoprecipitation (Co-IP) assays show that FgNdpk associates with FgTri1 and FgTri4 in vivo and regulates their localizations and expressions, respectively. Taken together, these data demonstrate that FgNdpk is important for vegetative growth, conidiation, and pathogenicity and acts as a key protein that regulates toxisome formation and DON biosynthesis in F. graminearum.

The catalytic subunit of type 2A protein phosphatase negatively regulates conidiation and melanin biosynthesis in Setosphaeria turcica.

Northern corn leaf blight caused by Setosphaeria turcica is a major fungal disease responsible for significant reductions in maize yield worldwide. Eukaryotic type 2A protein phosphatase (PP2A) influences growth and virulence in a number of pathogenic fungi, but little is known about its roles in S. turcica. Here, we functionally characterized S. turcica StPP2A-C, which encodes the catalytic C subunit of StPP2A. StPP2A-C deletion slowed colony growth, conidial germination, and appressorium formation but increased conidiation, melanin biosynthesis, glycerol content, and disease lesion size on maize. These effects were associated with expression changes in genes related to calcium signaling, conidiation, laccase activity, and melanin and glycerol biosynthesis, as well as changes in intra- and extracellular laccase activity. A pull-down screen for candidate StPP2A-c interactors revealed an interaction between StPP2A-c and StLac1. Theoretical modeling and yeast two-hybrid experiments confirmed that StPP2A-c interacted specifically with the copper ion binding domain of StLac1 and that Cys267 of StPP2A-c was required for this interaction. StPP2A-C expression thus appears to promote hyphal growth and reduce pathogenicity in S. turcica, at least in part by altering melanin synthesis and laccase activity; these insights may ultimately support the development of novel strategies for biological management of S. turcica.

Regulation of sexual differentiation initiation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.

Study on the virulence of Metarhizium anisopliae against Spodoptera frugiperda (J. E. Smith, 1797).

This study examined the impact of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) conidia on the eggs, larvae, pupae, and adults of Spodoptera frugiperda. The results showed that eggs, larvae, pupae, and adults exhibited mortality rates that were dependent on the dose. An increased amount of conidia (1.5 × 109 conidia/mL) was found to be toxic to larvae, pupae, and adults after 9 days of treatment, resulting in a 100% mortality rate in eggs, 98% in larvae, 76% in pupae, and 85% in adults. A study using earthworms as bioindicators found that after 3 days of exposure, M. anisopliae conidia did not cause any harmful effects on the earthworms. In contrast, the chemical treatment (positive control) resulted in 100% mortality at a concentration of 40 ppm. Histopathological studies showed that earthworm gut tissues treated with fungal conidia did not show significant differences compared with those of the negative control. The gut tissues of earthworms treated with monocrotophos exhibited significant damage, and notable differences were observed in the chemical treatment. The treatments with 70 and 100 µg/mL solutions of Eudrilus eugeniae epidermal mucus showed no fungal growth. An analysis of the enzymes at a biochemical level revealed a decrease in the levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase in S. frugiperda larvae after exposure to fungal conidia. This study found that M. anisopliae is effective against S. frugiperda, highlighting the potential of this entomopathogenic fungus in controlling this agricultural insect pest.

Mitochondrial Outer Membrane Translocase MoTom20 Modulates Mitochondrial Morphology and Is Important for Infectious Growth of the Rice Blast Fungus Magnaporthe oryzae.

Mitochondria are highly dynamic organelles that constantly change their morphology to adapt to the cellular environment through fission and fusion, which is critical for a cell to maintain normal cellular functions. Despite the significance of this process in the development and pathogenicity of the rice blast fungus Magnaporthe oryzae, the underlying mechanism remains largely elusive. Here, we identified and characterized a mitochondrial outer membrane translocase, MoTom20, in M. oryzae. Targeted gene deletion revealed that MoTom20 plays an important role in vegetative growth, conidiogenesis, penetration, and infectious growth of M. oryzae. The growth rate, conidial production, appressorium turgor, and pathogenicity are decreased in the ΔMotom20 mutant compared with the wild-type and complemented strains. Further analysis revealed that MoTom20 localizes in mitochondrion and plays a key role in regulating mitochondrial fission and fusion balance, which is critical for infectious growth. Finally, we found that MoTom20 is involved in fatty-acid utilization, and its yeast homolog ScTom20 is able to rescue the defects of ΔMotom20 in mitochondrial morphology and pathogenicity. Overall, our data demonstrate that MoTom20 is a key regulator for mitochondrial morphology maintenance, which is important for infectious growth of the rice blast fungus M. oryzae. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Cdc42 GAP Rga6 promotes monopolar outgrowth of spores.

The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein.

New Insights of Transcriptional Regulator AflR in Aspergillus flavus Physiology.

Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Development of a Rapid Sporulation Method of Fusarium graminearum Using Liquid Cultivation.

Fusarium graminearum is an important fungus causing a variety of maize diseases, including stalk rot, ear rot, and sheath rot. However, conidia of F. graminearum are not easily obtained under normal culture conditions, which seriously affects the identification and pathogenicity assessment of the isolates and screening of resistance sources. This study was undertaken to develop and utilize a rapid sporulation technique of F. graminearum using liquid cultivation, which could meet the needs of various tests. The results show that the optimum conditions for sporulation of F. graminearum were as follows: culture medium, 0.154 mol/liter of saline; temperature, 28 to 30°C; incubation time, 96 h; initial pH, 9 to 10; illumination, continuous ultraviolet light; and shaking speed, 150 rpm. Using this culture method, conidial concentration of tested F. graminearum strains can reach >1.5 × 105 conidia/ml. Compared with the existing methods using mung bean and carboxylmethyl cellulose as matrix, saline is relatively inexpensive, and the culture process, relatively quick. Overall, this study provided a systematic, rapid, and simple method to obtain a large number of conidia of F. graminearum.

Experimental validation under controlled conditions of real time PCR to quantify arbuscular mycorrhizal colonization in root.

Mycorrhizal colonization of roots is traditionally evaluated by empirical methods, such as root microscopy. We compared this method with data from using a real time PCR technique, and determined the correlation between methods, indicating particularities of a promising system for a quick and accurate molecular diagnostic of arbuscular mycorrhization.

The decrotonylase FoSir5 facilitates mitochondrial metabolic state switching in conidial germination of Fusarium oxysporum.

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalyzed by FoSir5, that support conidial germination in F. oxysporum.

Regulatory function of the novel transcription factor CxrC in Penicillium oxalicum.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

Survival of the nematophagous fungus Duddingtonia flagrans to in vitro segments of sheep gastrointestinal tract.

The nematophagous fungus Duddingtonia flagrans is used in integrated management of gastrointestinal nematodes in ruminants. The chlamydospores of the fungus, orally administered, pass through the segments of the ruminant digestive tract and, in the feces, capture the nematodes preventing their migration to grasslands. The drastic conditions of the gastrointestinal segments can negatively affect the fungus' biocontrol activity. The aim of this study was to assess the effect of in vitro conditions of the sheep's main gastrointestinal segments on the concentration, viability and nematode predatory ability of D. flagrans chlamydospores. The segments evaluated separately in vitro were the oral cavity, rumen, abomasum, and small intestine. The results showed that chlamydospores concentration was not affected by exposure to the different segments. The viability of the chlamydospores after exposure to the oral cavity (2.53 × 106 CFU/mL) and small intestine (1.24 × 105 CFU/mL) was significantly lower than its control treatment, with values of 6.67 × 106 CFU/mL and 2.31 × 105 CFU/mL respectively. Nematode predatory ability after rumen exposure was reduced by 7% compared to the control treatment, by 25% after abomasum exposure and by 17% after small intestine. This study revealed the individual in vitro effect of each segment of ovine gastrointestinal tract on the integrity of this strain of the fungus D. flagrans affecting its viability and nematode predatory ability under the evaluated conditions. Delivery systems could be designed to protect chlamydospores considering the impact of each gastrointestinal segment.

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins-hydrophobins (HFBs)-that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.

A Fungal Transcription Regulator of Vacuolar Function Modulates Candida albicans Interactions with Host Epithelial Cells.

Microorganisms typically maintain cellular homeostasis despite facing large fluctuations in their surroundings. Microbes that reside on human mucosal surfaces may experience significant variations in nutrient and ion availability as well as pH. Whether the mechanisms employed by these microbial cells to sustain homeostasis directly impact on the interplay with the host's mucosae remains unclear. Here, we report that the previously uncharacterized transcription regulator ZCF8 in the human-associated yeast Candida albicans maintains vacuole homeostasis when the fungus faces fluctuations in nitrogen. Genome-wide identification of genes directly regulated by Zcf8p followed by fluorescence microscopy to define their subcellular localization uncovered the fungal vacuole as a top target of Zcf8p regulation. Deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and luminal pH and rendered the fungus resistant or susceptible to nigericin and brefeldin A, two drugs that impair vacuole and associated functions. Furthermore, we establish that the regulator modulates C. albicans attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Our findings, therefore, suggest that fungal vacuole physiology regulation is intrinsically linked to, and shapes to a significant extent, the physical interactions that Candida cells establish with mammalian mucosal surfaces. IMPORTANCE Candida albicans is a fungus that resides on various human mucosal surfaces. Individuals with debilitated immune systems are prone to develop C. albicans infections, which can range in severity from mucosal disease (e.g., oral thrush in AIDS patients) to life-threatening conditions (e.g., deep-seated, disseminated infections in patients undergoing organ transplants). Understanding the cellular and molecular mechanisms that this eukaryotic microbe employs to colonize different parts of the human body and to cause disease will lay the foundation for the development of novel strategies for preventing and treating C. albicans infections. This report establishes the fungal vacuole, a key organelle to the overall fungal physiology, as a key determinant of the interplay between C. albicans and mammalian mucosal surfaces.

A Histone Deacetylase, Magnaporthe oryzae RPD3, Regulates Reproduction and Pathogenic Development in the Rice Blast Fungus.

Acetylation and deacetylation of histones are key epigenetic mechanisms for gene regulation in response to environmental stimuli. RPD3 is a well-conserved class I histone deacetylase (HDAC) that is involved in diverse biological processes. Here, we investigated the roles of the Magnaporthe oryzae RPD3 (MoRPD3) gene, an ortholog of Saccharomyces cerevisiae Rpd3, during development and pathogenesis in the model plant-pathogenic fungus Magnaporthe oryzae. We demonstrated that the MoRPD3 gene is able to functionally complement the yeast Rpd3 deletion mutant despite the C-terminal extension of the MoRPD3 protein. MoRPD3 localizes primarily to the nuclei of vegetative hyphae, asexual spores, and invasive hyphae. Deletion of MoRPD3 appears to be lethal. Depletion of MoRPD3 transcripts via gene silencing (MoRPD3kd, where "kd" stands for "knockdown") has opposing effects on asexual and sexual reproduction. Although conidial germination and appressorium formation rates of the mutants were almost comparable to those of the wild type, in-depth analysis revealed that the appressoria of mutants are smaller than those of the wild type. Furthermore, the MoRPD3kd strain shows a significant reduction in pathogenicity, which can be attributed to the delay in appressorium-mediated penetration and impaired invasive growth. Interestingly, MoRPD3 does not regulate potassium transporters, as shown for Rpd3 of S. cerevisiae. However, it functioned in association with the target of rapamycin (TOR) kinase pathway, resulting in the dependency of appressorium formation on hydrophilic surfaces and on TOR's inhibition by MoRPD3. Taken together, our results uncovered distinct and evolutionarily conserved roles of MoRPD3 in regulating fungal reproduction, infection-specific development, and virulence. IMPORTANCE RPD3 is an evolutionarily conserved class I histone deacetylase (HDAC) that plays a pivotal role in diverse cellular processes. In filamentous fungal pathogens, abrogation of the gene encoding RPD3 results in either lethality or severe growth impairment, making subsequent genetic analyses challenging. Magnaporthe oryzae is a causal agent of rice blast disease, which is responsible for significant annual yield losses in rice production. Here, we characterized the RPD3 gene of M. oryzae (MoRPD3) in unprecedented detail using a gene-silencing approach. We provide evidence that MoRPD3 is a bona fide HDAC regulating fungal reproduction and pathogenic development by potentially being involved in the TOR-mediated signaling pathway. To the best of our knowledge, this work is the most comprehensive genetic dissection of RPD3 in filamentous fungal pathogens. Our work extends and deepens our understanding of how an epigenetic factor is implicated in the development and virulence of fungal pathogens of plants.

The Heterotrimeric Transcription Factor CCAAT-Binding Complex and Ca2+-CrzA Signaling Reversely Regulate the Transition between Fungal Hyphal Growth and Asexual Reproduction.

The life cycle of filamentous fungi generally comprises hyphal growth and asexual reproduction. Both growth and propagation processes are critical for invasion growth, spore dissemination, and virulence in fungal pathogens and for the production of secondary metabolites or for biomass accumulation in industrial filamentous fungi. The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor comprising three subunits, HapB, HapC, and HapE, and is highly conserved in fungi. Previous studies revealed that CBC regulates sterol metabolism by repressing several genes in the ergosterol biosynthetic pathway in the human fungal pathogen Aspergillus fumigatus. In the present study, we found dysfunction of CBC caused the abnormal asexual reproduction (conidiation) in submerged liquid culture. CBC suppresses the activation of the brlA gene in the central regulatory pathway for conidiation combined with its upstream regulators fluG, flbD, and flbC by binding to the 5'-CCAAT-3' motif within conidiation gene promoters, and lack of CBC member HapB results in the upregulation of these genes. Furthermore, when the expression of brlA or flbC is repressed, the submerged conidiation does not happen in the hapB mutant. Interestingly, deletion of HapB leads to enhanced transient cytosolic Ca2+ levels and activates conidiation-positive inducer Ca2+-CrzA modules to enhance submerged conidiation, demonstrating that CrzA works with CBC as a reverse regulator of fungal conidiation. To the best of our knowledge, the finding of this study is the first report for the molecular switch mechanism between vegetative hyphal growth and asexual development regulated by CBC, in concert with Ca2+-CrzA signaling in A. fumigatus. IMPORTANCE A precisely timed switch between vegetative hyphal growth and asexual development is a crucial process for the filamentous fungal long-term survival, dissemination, biomass production, and virulence. However, under the submerged culture condition, filamentous fungi would undergo constant vegetative growth whereas asexual conidiation rarely occurs. Knowledge about possible regulators is scarce, and how they could inhibit conidiation in liquid culture is poorly understood. Here, we demonstrated that the transcription factor heterotrimeric CBC dominantly maintains vegetative growth in liquid-submerged cultures by directly suppressing the conidiation-inductive signal. In contrast, calcium and the transcription factor CrzA, are positive inducers of conidiation. Our new insights into the CBC and Ca2+-CrzA regulatory system for transition control in the submerged conidiation of A. fumigatus may have broad repercussions for all filamentous fungi. Moreover, our elucidation of the molecular mechanism for submerged conidiation may support new strategies to precisely control vegetative growth and asexual conidiation in aspergilli used in industry.

Secondary metabolites of Hülle cells mediate protection of fungal reproductive and overwintering structures against fungivorous animals.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.