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Fungal sexual reproduction requires complex cellular differentiation processes of hyphal cells. The plant pathogenic fungus Fusarium graminearum produces fruiting bodies called perithecia via sexual reproduction, and perithecia forcibly discharge ascospores into the air for disease initiation and propagation. Lipid metabolism and accumulation are closely related to perithecium formation, yet the molecular mechanisms that regulate these processes are largely unknown. Here, we report that a novel fungal specific bZIP transcription factor, F. graminearum perithecium overproducing 1 (Fpo1), plays a role as a global transcriptional repressor during perithecium production and maturation in F. graminearum. Deletion of FPO1 resulted in reduced vegetative growth, asexual sporulation and virulence and overproduced perithecium, which reached maturity earlier, compared with the wild type. Intriguingly, the hyphae of the fpo1 mutant accumulated excess lipids during perithecium production. Using a combination of molecular biological, transcriptomic and biochemical approaches, we demonstrate that repression of FPO1 after sexual induction leads to reprogramming of carbon metabolism, particularly fatty acid production, which affects sexual reproduction of this fungus. This is the first report of a perithecium-overproducing F. graminearum mutant, and the findings provide comprehensive insight into the role of modulation of carbon metabolism in the sexual reproduction of fungi.

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.

Genetically encoded Ca2+ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca2+ signaling in fungi, ranging from documenting Ca2+ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca2+ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca2+ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca2+ indicator. Wild-type and three Ca2+ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

Endophytic fungi are great resources for the identification of useful natural products such as antimicrobial agents. In this study, we performed the antifungal screening of various plant endophytic fungi against the dollar spot pathogen Sclerotinia homoeocarpa and finally selected Humicola sp. JS-0112 as a potential biocontrol agent. The bioactive compound produced by the strain JS-0112 was identified as monorden known as an inhibitor of heat shock protein 90 (Hsp90). Monorden exhibited strong antagonistic activity against most tested plant pathogenic fungi particularly against tree pathogens and oomycetes with the minimum inhibitory concentration values less than 2.5 μg mL–1. Extensive in planta assays revealed that monorden effectively suppressed the development of several important plant diseases such as rice blast, rice sheath blight, wheat leaf rust, creeping bentgrass dollar spot, and cucumber damping-off. Especially, it showed much stronger disease control efficacy against cucumber damping-off than a synthetic fungicide chlorothalonil. Subsequent molecular genetic analysis of fission yeast and Fusarium graminearum suggested that Hsp90 is a major inhibitory target of monorden, and sequence variation among fungal Hsp90 is a determinant for the dissimilar monorden sensitivity of fungi. This is the first report dealing with the disease control efficacy and antifungal mechanism of monorden against fungal plant diseases and we believe that monorden can be used as a lead molecule for developing novel fungicides with new action mechanism for the control of plant diseases caused by fungi and oomycetes.

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca2+ in fungi. Such changes are referred as the Ca2+ signature and control cellular responses by modulating the activity or location of diverse Ca2+-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca2+ signaling, therefore, characterization of how Ca2+ moves to and from the cytoplasm to create Ca2+ signatures under different conditions is fundamental. Three genes encoding plasma membrane Ca2+ channels in a Fusarium graminearum strain that expresses a fluorescent protein-based Ca2+ indicator in the cytoplasm were mutagenized to investigate their roles in the generation of Ca2+ signatures under different growth conditions and genetic backgrounds. The genes disrupted include CCH1 and MID1, which encode a high affinity Ca2+ uptake system, and FIG1, encoding a low affinity Ca2+ channel. Resulting mutants were also analyzed for growth, development, pathogenicity and mycotoxin production to determine how loss of each of the genes alters these traits. To investigate whether individual genes influence the function and expression of other genes, phenotypes and Ca2+ signatures of their double and triple mutants, as well as their expression patterns, were analyzed.

Fusarium graminearum is a prominent plant pathogenic fungus causing Fusarium head blight in major cereal crops worldwide. To understand the molecular mechanisms underlying fungal development and virulence, large collections of F. graminearum mutants have been constructed. Cytochrome P450 monooxygenases (P450s) are widely distributed in organisms and are involved in a diverse array of molecular/metabolic processes; however, no systematic functional analysis of P450s has been attempted in filamentous fungi. In this study, we constructed a genome-wide deletion mutant set covering 102 P450s and analyzed these mutants for changes in 38 phenotypic categories, including fungal development, stress responses and responses to several xenobiotics, to build a comprehensive phenotypic dataset. Most P450 mutants showing defective phenotypes were impaired in a single phenotypic trait, demonstrating that our mutant library is a good genetic resource for further fungal genetic studies. In particular, we identified novel P450s specifically involved in virulence (5) and both asexual (1) and sexual development (2). Most P450s seem to play redundant roles in the degradation of xenobiotics in F. graminearum. This study is the first phenome-based functional analysis of P450s, and it provides a valuable genetic resource for further basic and applied biological research in filamentous fungi and other plant pathogens.

Arsenite-resistance protein 2 (Ars2) is an important nuclear protein involved in various RNA metabolisms in animals and plants, but no Ars2 ortholog has been studied in filamentous fungi. Although it is an essential gene in most model eukaryotes, FgARS2 null mutants were viable in the plant pathogenic fungus Fusarium graminearum. The deletion of FgARS2 resulted in pleiotropic defects in various fungal developmental processes. Fgars2 mutants were irregular in nuclear division, and conidial germination was significantly retarded, causing the fungus to manifest its hypersensitive phenotypes under DNA damage stress. While FgARS2 deletion caused abnormal morphologies of ascospores and defective ascospore discharge, our data revealed that FgARS2 was not closely involved in small-non-coding RNA production in F. graminearum. The dominant nuclear localization of FgArs2-green fluorescent proteins (GFP) and abnormal nuclear division in FgARS2 deletion mutant implicated that FgArs2 functions in the nucleus. Intriguingly, we found that FgArs2 established a robust physical interaction with the cap binding complex (CBC) to form a tertiary complex CBC-Ars2 (CBCA), and disruption of any CBCA complex subunit drastically attenuated the virulence of F. graminearum. The results of the study indicate that Ars2 regulates fungal development, stress response, and pathogenesis via interaction with CBC in F. graminearum and provide a novel insight into understanding of the biological functions of Ars2 in filamentous fungi.

Sodium is an abundant cation required for protein function and maintenance of cellular osmotic homeostasis. High concentrations of sodium are toxic, and fungi have evolved efficient sodium efflux systems. In this study, we characterized a novel sodium tolerance mechanism in the plant pathogen Fusarium graminearum. Fusarium graminearum sodium sensitive 1 (Fss1) is a nuclear transcription factor with a Zn(II)2 Cys6 fungal-type DNA-binding domain required for sodium tolerance. RNA-seq and genetic studies revealed that a P-type ATPase pump, exitus natru (Latin: exit sodium) 1 (FgEna5), mediates the phenotypic defects of FSS1 mutants. A homologue of PACC (PAC1) was required for FgEna5-dependent sodium and lithium tolerance independent of Fss1. The results of this study revealed that F. graminearum has a distinct and novel pathway for sodium tolerance not present in other model fungi.

Fusarium head blight (FHB) caused by infection with Fusarium graminearum leads to enormous losses to crop growers, and may contaminate grains with a number of Fusarium mycotoxins that pose serious risks to human and animal health. Antagonistic bacteria that are used to prevent FHB offer attractive alternatives or supplements to synthetic fungicides for controlling FHB without the negative effects of chemical management. Out of 500 bacterial strains isolated from soil, Bacillus amyloliquefaciens JCK-12 showed strong antifungal activity and was considered a potential source for control strategies to reduce FHB. B. amyloliquefaciens JCK-12 produces several cyclic lipopeptides (CLPs) including iturin A, fengycin, and surfactin. Iturin A inhibits spore germination of F. graminearum. Fengycin or surfactin alone did not display any inhibitory activity against spore germination at concentrations less than 30 μg/ml, but a mixture of iturin A, fengycin, and surfactin showed a remarkable synergistic inhibitory effect on F. graminearum spore germination. The fermentation broth and formulation of B. amyloliquefaciens JCK-12 strain reduced the disease incidence of FHB in wheat. Furthermore, co-application of B. amyloliquefaciens JCK-12 and chemical fungicides resulted in synergistic in vitro antifungal effects and significant disease control efficacy against FHB under greenhouse and field conditions, suggesting that B. amyloliquefaciens JCK-12 has a strong chemosensitizing effect. The synergistic antifungal effect of B. amyloliquefaciens JCK-12 and chemical fungicides in combination may result from the cell wall damage and altered cell membrane permeability in the phytopathogenic fungi caused by the CLP mixtures and subsequent increased sensitivity of F. graminearum to fungicides. In addition, B. amyloliquefaciens JCK-12 showed the potential to reduce trichothecenes mycotoxin production. The results of this study indicate that B. amyloliquefaciens JCK-12 could be used as an available biocontrol agent or as a chemosensitizer to chemical fungicides for controlling FHB disease and as a strategy for preventing the contamination of harvested crops with mycotoxins.

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3-1.8 × 108 CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.

Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.

The survival of cellular organisms depends on the faithful replication and transmission of DNA. Regulatory factor X (RFX) transcription factors are well conserved in animals and fungi, but their functions are diverse, ranging from the DNA damage response to ciliary gene regulation. We investigated the role of the sole RFX transcription factor, RFX1, in the plant-pathogenic fungus Fusarium graminearum . Deletion of rfx1 resulted in multiple defects in hyphal growth, conidiation, virulence, and sexual development. Deletion mutants of rfx1 were more sensitive to various types of DNA damage than the wild-type strain. Septum formation was inhibited and micronuclei were produced in the rfx1 deletion mutants. The results of the neutral comet assay demonstrated that disruption of rfx1 function caused spontaneous DNA double-strand breaks (DSBs). The transcript levels of genes involved in DNA DSB repair were upregulated in the rfx1 deletion mutants. DNA DSBs produced micronuclei and delayed septum formation in F. graminearum . Green fluorescent protein (GFP)-tagged RFX1 localized in nuclei and exhibited high expression levels in growing hyphae and conidiophores, where nuclear division was actively occurring. RNA-sequencing-based transcriptomic analysis revealed that RFX1 suppressed the expression of many genes, including those required for the repair of DNA damage. Taken together, these findings indicate that the transcriptional repressor rfx1 performs crucial roles during normal cell growth by maintaining genome integrity.

The ascomycete fungus Fusarium graminearum is a devastating plant pathogen for major cereal crops. Ascospores are produced via sexual reproduction and forcibly discharged from mature perithecia, which function as the primary inocula. Perithecium development involves complex cellular processes and is under polygenic control. In this study, a novel gene, GEA1, was found to be required for ascus wall development in F. graminearum. GEA1 deletion mutants produced normal-shaped perithecia and ascospores, yet ascospores were observed to precociously germinate inside the perithecium. Moreover, GEA1 deletions resulted in abnormal ascus walls that collapsed prior to ascospore discharge. Based on localization of GEA1 to plasma membrane, GEA1 may be directly involved in ascus wall biogenesis. This is the first report to identify a unique gene required for ascus wall development in F. graminearum.

Fungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungus Gibberella zeae . In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring in G. zeae . We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in an ACS1 deletion mutant. Moreover, deletion of CAT1 and/or CAT2 resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development in G. zeae . Taking these data together, we concluded that G. zeae CATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages.

Fusarium head blight caused by Gibberella zeae is a prominent disease of cereal crops that poses serious human health concerns due to the contamination of grains with mycotoxins. In this study, we deleted an orthologue of areA, which is a global nitrogen regulator in filamentous fungi, to characterize its functions in G. zeae. The areA deletion resulted in an inability to use nitrate as a sole nitrogen source, whereas urea utilization was partially available. The virulence of ΔareA strains on wheat heads was markedly reduced compared with the wild-type strain. The areA mutation triggered loss of trichothecene biosynthesis but did not affect zearalenone biosynthesis. The ΔareA strains showed immaturity of asci and did not produce mature ascospores. Chemical complementation by urea restored normal sexual development, whereas the virulence and trichothecene production were not affected by urea addition. GFP-AreA fusion protein was localized to nuclei, and its expression increased in response to nitrogen-limiting conditions. These results suggest that areA-dependent regulation of nitrogen metabolism is required for vegetative growth, sexual development, trichothecene biosynthesis, and virulence in G. zeae.

The plant pathogenic fungus Fusarium graminearum (teleomorph: Gibberella zeae) is a crucial pathogen of major cereal crops and the fungus has taken on greater importance throughout the world. Taking advantage of rapid progress on fungal molecular techniques, we have studied biosynthetic pathways of harmful secondary metabolites of F. graminearum such as trichothecenes, zearalenone, and aurofusarin. We revealed that both Tri13 and Tri7 genes are requried for the chemotype determination of trichothecenes for nivalenol and 4 -acetyl-nivalenol production, respectively. We also constructed efficient chemotype determination system by utilizing distrupted Tri7 gene sequences of deoxinivalenol producers. Forward and reverse genetic approaches enabled us to characterize the gene clusters responsible for the biosynthesis of two polyketide compounds, zearalenone and aurofusarin. Both gene clusters cover genes encoding polyketide synthases and transcription activators. We also characterized an ABC transporter, ZRA 1, related with zearaleone production and constructed a zearalenone conditional gene expression system from the microarray analyses. These results increased our understanding on Fusarium genetics and mycotoxicology. Our future works will focus on revealing the regulatory mechanisms of toxin production and biological functions of these secondary metabolites.

Department of Agricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921,Korea(Received on September 17, 2011; Accepted on October 10, 2011)The ascomycete fungus Fusarium graminearum is animportant plant pathogen responsible for Fusariumhead blight in small grains and ear rot on maize. Thisfungus also produces the estrogenic metabolite, zearalenone(ZEA) that causes estrogenic disorders in humans andanimals. Previously, we developed a conditional geneexpression system for this fungus using a ZEA-induciblepromoter (Pzear). In the present study, four other estro-genic compounds, including β-estradiol, estriol, estrone,and secoisolariciresinol, were screened as possiblesubstitutes for ZEA in this system. Among them, β-estradiol was able to successfully induce the expressionof a gene controlled by Pzear, while estrone was onlyable to partially induce its expression but the other twocompounds were not effective. In combination, theseresults demonstrate that β-estradiol can replace ZEAin this conditional gene expression system, therebyeliminating the need to use the more expensive reagent,ZEA, and facilitating high-throughput functionalanalyses of F. graminearum in future studies.Keywords :conditional gene expression, estrogens, estradiol,Gibberella zeae, zearalenoneThe plant pathogenic fungus Fusarium graminearum(teleomorph: Gibberella zeae) is responsible for causingFusarium head blight (FHB) on small grains and ear rot onmaize (Leslie and Summerell, 2006). Unfortunately, both ofthese diseases have recently been reported in the rice andmaize fields of Korea (Lee et al., 2009, 2010c). In additionto direct yield losses as a result of disease on heads, thisfungus also produces various mycotoxins, including tri-chothecenes and zearalenone (ZEA) (Desjardins, 2006).Trichothecenes inhibit eukaryotic protein biosynthesis,which leads to food refusal, diarrhea, and contact dermatitisin animals, while trichothecenes represent virulence factorsin plants. ZEA [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcyclic acid lactone] is a polyketide metabolite andis responsible for estrogenic disorders in rats and swine dueto the similarity of its structure with animal estrogens(Desjardins, 2006).The ZEA biosynthesis gene cluster of F. graminearumincludes four genes, a reducing polyketide synthase (PKS4),non-reducing polyketide synthase (PKS13), isoamyl alcoholoxidase (ZEB1), and transcription factor (ZEB2) (Gaffoorand Trail, 2006; Kim et al., 2005; Lysoe et al., 2006). Thetwo PKS genes synthesize a polyketide backbone throughthe head-to-tail condensation of acetate units, and ZEB1plays an important role for an oxidation step which convertsβ-zearalenol (β-ZOL) to ZEA (Kim et al., 2005). ZEB2contains a putative bZIP DNA-binding domain and is atranscriptional activator of the other three genes in thecluster. Recently, a putative ABC transporter gene, ZRA1,has been identified to be required for ZEA production inthis fungus even though its biological function was notelucidated yet (Lee et al., 2011b). These functional studiesrevealed the ZEA biosynthetic pathway, but biologicalfunctions of ZEA in plants and fungi have not yet beencharacterized.Since the whole genome sequences of F. graminearumwere publicly released by the Broad Institute in 2003 (http://www.broadinstitute.org/), many research groups haveperformed functional studies to identify genes related topathogenicity (Ding et al., 2009; Seong et al., 2005), sexualand asexual development (Harris, 2005; Min et al., 2010;Lee et al., 2009, 2010a; Son et al., 2011), and secondarymetabolite biosynthesis pathways (Gaffoor and Trail, 2006;Kim et al., 2005, 2006; Lysoe et al., 2006). While molecularmanipulation tools such as targeted gene deletion and geneover-expression have been well developed, conditional geneexpression systems that allow the expression of certaingenes under desired conditions have been limited in F.graminearum. Currently, the only conditional gene ex-pression system for F. graminearum involves a ZEA-inducible promoter we previously developed (Lee et al.

We identified two syntaxin-like SNARE genes, named GzSYN1 and GzSYN2, from the plant pathogenic ascomycete Gibberella zeae, and characterized the functions and cellular localization of these genes. The GzSYN1 deletion mutant (Deltagzsyn1) had 71% reduced hyphal growth compared to the wild-type strain, but produced perithecia with normal ascospores. Deltagzsyn2 had the same hyphal growth rate as the wild-type, but completely lost both self and female fertility. When Deltagzsyn2 was spermatized for Deltamat1-1 or Deltamat1-2 strains, it retained its male fertility, but the ascus shape was abnormal and ascospore delimitation was delayed. The Deltagzsyn1 and Deltagzsyn2 virulence on barley was reduced by 67% and 75%, respectively, compared to the wild-type. The GFP::GzSYN1 fusion protein was localized in vesicles, vacuoles, plasma membranes, and septa, whereas GFP::GzSYN2 was found only in plasma membranes and septa. These results suggest that syntaxins have key roles in fungal development and virulence in G. zeae.

We previously characterized members of the Myb protein family, MYT1 and MYT2, in Fusarium graminearum. MYT1 and MYT2 are involved in female fertility and perithecium size, respectively. To expand knowledge of Myb proteins in F. graminearum, in this study, we characterized the functions of the MYT3 gene, which encodes a putative Myb-like transcription factor containing two Myb DNA-binding domains and is conserved in the subphylum Pezizomycotina of Ascomycota. MYT3 proteins were localized in nuclei during most developmental stages, suggesting the role of MYT3 as a transcriptional regulator. Deletion of MYT3 resulted in impairment of conidiation, germination, and vegetative growth compared to the wild type, whereas complementation of MYT3 restored the wild-type phenotype. Additionally, the Δmyt3 strain grew poorly on nitrogen-limited media; however, the mutant grew robustly on minimal media supplemented with ammonium. Moreover, expression level of nitrate reductase gene in the Δmyt3 strain was decreased in comparison to the wild type and complemented strain. On flowering wheat heads, the Δmyt3 strain exhibited reduced pathogenicity, which corresponded with significant reductions in trichothecene production and transcript levels of trichothecene biosynthetic genes. When the mutant was selfed, mated as a female, or mated as a male for sexual development, perithecia were not observed on the cultures, indicating that the Δmyt3 strain lost both male and female fertility. Taken together, these results demonstrate that MYT3 is required for pathogenesis and sexual development in F. graminearum, and will provide a robust foundation to establish the regulatory networks for all Myb-like proteins in F. graminearum.

Fusarium graminearum , a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in F. graminearum , we functionally characterized the F. graminearum ortholog of Aspergillus nidulans wetA , which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of F. graminearum wetA did not alter mycelial growth, sexual development, or virulence, but the wetA deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the F. graminearum wetA deletion mutants was reduced. The wetA deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidial dormancy by suppressing microcycle conidiation in F. graminearum . Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in F. graminearum conidiogenesis. The conserved and distinct roles identified for WetA in F. graminearum provide new insights into the genetics of conidiation in filamentous fungi.

Fusarium graminearum (teleomorph Gibberella zeae) is a prominent pathogen that infects major cereal crops such as wheat, barley, and maize. Both sexual (ascospores) and asexual (conidia) spores are produced in F. graminearum. Since conidia are responsible for secondary infection in disease development, our objective of the present study was to reveal the molecular mechanisms underlying conidiogenesis in F. graminearum based on the framework previously described in Aspergillus nidulans. In this study, we firstly identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in F. graminearum. Deletion of abaA did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in abaA deletion mutants. Overexpression of abaA resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using A. nidulans AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in F. graminearum as they are in A. nidulans. Results from RNA-sequencing analysis suggest that AbaA plays a pivotal role in conidiation by regulating cell cycle pathways and other conidiation-related genes. Thus, the conserved roles of the AbaA ortholog in both A. nidulans and F. graminearum give new insight into the genetics of conidiation in filamentous fungi.

The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of Fg NOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper -branching. The Delta Fgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development.

Fungi use mannitol to store carbon, balance redox, and mannitol serves as an antioxidant. Several fungi also increase stress tolerance by accumulating mannitol. The results of this study showed that conidia of the cereal head blight fungus Gibberella zeae were readily changed to chlamydospore-like structures (CLS) in cultures supplemented with high amounts of mannitol. CLS cellular features were atypical of chlamydospores, but accumulated high levels of glycogen, lipids, and chitin in the cytoplasm. In addition, CLS exhibited increased tolerance to environmental stresses, including UV, heat, and drought compared to normal conidia. Molecular approaches revealed that several genes associated with lipid metabolism, signal transduction, acetyl-CoA production, and chitin synthesis were involved in CLS formation. This is the first report to characterize conidia modifications similar to chlamydospores in G. zeae applying histological and molecular approaches. The results suggest CLS serve a role in G. zeae survival strategies under hot and dry field conditions.

The homothallic ascomycete fungus Gibberella zeae is a plant pathogen that is found worldwide, causing Fusarium head blight (FHB) in cereal crops and ear rot of maize. Ascospores formed in fruiting bodies (i.e., perithecia) are hypothesized to be the primary inocula for FHB disease. Perithecium development is a complex cellular differentiation process controlled by many developmentally regulated genes. In this study, we selected a previously reported putative transcription factor containing the Myb DNA-binding domain MYT2 for an in-depth study on sexual development. The deletion of MYT2 resulted in a larger perithecium, while its overexpression resulted in a smaller perithecium when compared to the wild-type strain. These data suggest that MYT2 regulates perithecium size differentiation. MYT2 overexpression affected pleiotropic phenotypes including vegetative growth, conidia production, virulence, and mycotoxin production. Nuclear localization of the MYT2 protein supports its role as a transcriptional regulator. Transcriptional analyses of trichothecene synthetic genes suggest that MYT2 additionally functions as a suppressor for trichothecene production. This is the first study characterizing a transcription factor required for perithecium size differentiation in G. zeae, and it provides a novel angle for understanding sexual development in filamentous fungi.

Gibberella zeae is an important pathogen of major cereal crops. The fungus produces ascospores that forcibly discharge from mature fruiting bodies, which serve as the primary inocula for disease epidemics. In this study, we characterized an insertional mutant Z39P105 with a defect in sexual development and identified a gene encoding a putative transcription factor designated as MYT1. This gene contains a Myb DNA-binding domain and is conserved in the subphylum Pezizomycotina of Ascomycota. The MYT1 protein fused with green fluorescence protein localized in nuclei, which supports its role as a transcriptional regulator. The MYT1 deletion mutant showed similar phenotypes to the wild-type strain in vegetative growth, conidia production and germination, virulence, and mycotoxin production, but had defect in female fertility. A mutant overexpressing MYT1 showed earlier germination, faster mycelia growth, and reduced mycotoxin production compared to the wild-type strain, suggesting that improper MYT1 expression affects the expression of genes involved in the cell cycle and secondary metabolite production. This study is the first to characterize a transcription factor containing a Myb DNA-binding domain that is specific to sexual development in G. zeae.

The pyruvate-acetaldehyde-acetate (PAA) pathway has diverse roles in eukaryotes. Our previous study on acetyl-coenzyme A synthetase 1 (ACS1) in Gibberella zeae suggested that the PAA pathway is important for lipid production, which is required for perithecia maturation. In this study, we deleted all three pyruvate decarboxylase (PDC) genes, which encode enzymes that function upstream of ACS1 in the PAA pathway. Results suggest PDC1 is required for lipid accumulation in the aerial mycelia, and deletion of PDC1 resulted in highly wettable mycelia. However, the total amount of lipids in the PDC1 deletion mutants was similar to that of the wild-type strain, likely due to compensatory lipid production processes in the embedded mycelia. PDC1 was expressed both in the aerial and embedded mycelia, whereas ACS1 was observed only in the aerial mycelia in a PDC1-dependent manner. PDC1 is also involved in vegetative growth of embedded mycelia in G. zeae, possibly through initiating the ethanol fermentation pathway. Thus, PDC1 may function as a key metabolic enzyme crucial for lipid production in the aerial mycelia, but play a different role in the embedded mycelia, where it might be involved in energy generation by ethanol fermentation.

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases ( ACS genes ACS1 and ACS2 ), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2 , has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in 657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000 phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have evolved to control various cellular processes at the transcriptional level., Author Summary Large collections of mutant lines allow for identification of gene functions. Here we constructed a mutant library of 657 putative transcription factors (TFs) through homologous recombination in the head blight fungus, Fusarium graminearum, providing a resource for understanding gene regulation in fungus. By screening these mutants in 17 phenotypic categories, we constructed a dataset of over 11,000 phenotypes. This study provides new insight into understanding multiple phenotypes caused by single TF as well as regulation of gene expression at the transcription level in F. graminearum. Furthermore, our TF mutant library will be a valuable resource for fungal studies through the distribution of mutants and easy access to our phenotypic and genetic data.

Adenosine triphosphate (ATP) citrate lyase (ACL) is a key enzyme in the production of cytosolic acetyl-CoA, which is crucial for de novo lipid synthesis and histone acetylation in mammalian cells. In this study, we characterized the mechanistic roles of ACL in the homothallic ascomycete fungus Gibberella zeae, which causes Fusarium head blight in major cereal crops. Deletion of ACL in the fungus resulted in a complete loss of self and female fertility as well as a reduction in asexual reproduction, virulence, and trichothecene production. When the wild-type strain was spermatized with the ACL deletion mutants, they produced viable ascospores, however ascospore delimitation was not properly regulated. Although lipid synthesis was not affected by ACL deletion, histone acetylation was dramatically reduced in the ACL deletion mutants during sexual development, suggesting that the defects in sexual reproduction were caused by the reduction in histone acetylation. This study is the first report demonstrating a link between sexual development and ACL-mediated histone acetylation in fungi.