Organic Nitrogen Supplementation Increases Vegetative and Reproductive Biomass in a Versatile White Rot Fungus.

The Black Poplar Mushroom Cyclocybe aegerita (syn. Agrocybe aegerita) is a white-rot fungus that naturally fruits from woody substrates, including buried wood. It is known for its substrate versatility and is equipped with a respective carbohydrate-active enzyme repertoire being intermediate between typical white-rot fungi and plant litter decomposers. Given relative nitrogen scarcity in wood, mobilization of nitrogen from surrounding litter is known as a way to meet nitrogen requirements for cellular homeostasis and reproduction of wood decay fungi. However, the effect of added nitrogen on vegetative and reproductive biomass has not yet been studied in a uniform minimalistic laboratory setup. For C. aegerita, such a growth and fruiting setup has been developed. In the present study, this white-rot fungus has been grown with and without additional ß-adenosine, an organic nitrogen source present in plant litter. Elevated ß-adenosine levels increased aerial mycelium weight by 30% (1 × ß-adenosine) and 55% (10 × ß-adenosine), reproductive biomass by 75% (1 × ß-adenosine) and by 100% (10 × ß-adenosine), number of primordia by 127% (10 × ß-adenosine) and accelerated primordium formation by 1.6 days (10 × ß-adenosine), compared to the control treatment. These findings imply that C. aegerita invests additional organic nitrogen resources into direct vegetative and reproductive biomass build-up at the same time. Colonization of niches with accessory nitrogen sources, like buried wood, which is near the plant litter layer, may thus provide an evolutionary fitness advantage. Globally anthropogenically altered nitrogen dynamics may affect hyphal-driven processes as well as fruit body-driven food webs.

Transcriptional response of mushrooms to artificial sun exposure.

Climate change causes increased tree mortality leading to canopy loss and thus sun-exposed forest floors. Sun exposure creates extreme temperatures and radiation, with potentially more drastic effects on forest organisms than the current increase in mean temperature. Such conditions might potentially negatively affect the maturation of mushrooms of forest fungi. A failure of reaching maturation would mean no sexual spore release and, thus, entail a loss of genetic diversity. However, we currently have a limited understanding of the quality and quantity of mushroom-specific molecular responses caused by sun exposure. Thus, to understand the short-term responses toward enhanced sun exposure, we exposed mushrooms of the wood-inhabiting forest species Lentinula edodes, while still attached to their mycelium and substrate, to artificial solar light (ca. 30°C and 100,000 lux) for 5, 30, and 60 min. We found significant differentially expressed genes at 30 and 60 min. Eukaryotic Orthologous Groups (KOG) class enrichment pointed to defense mechanisms. The 20 most significant differentially expressed genes showed the expression of heat-shock proteins, an important family of proteins under heat stress. Although preliminary, our results suggest mushroom-specific molecular responses to tolerate enhanced sun exposure as expected under climate change. Whether mushroom-specific molecular responses are able to maintain fungal fitness under opening forest canopies remains to be tested.

The Pacific Tree-Parasitic Fungus Cyclocybe parasitica Exhibits Monokaryotic Fruiting, Showing Phenotypes Known from Bracket Fungi and from Cyclocybe aegerita.

Cyclocybe parasitica is a wood-destroying parasitic edible mushroom growing on diverse broad-leafed trees in New Zealand and other Pacific areas. Recent molecular systematics of European Cyclocybe aegerita, a newly delimited Asian phylum and of related species, corroborated the distinction of the chiefly saprobic cultivated edible mushroom C. aegerita from C. parasitica. Here, we show that C. parasitica exhibits a morpho-physiological trait characteristic to its European cousin, i.e., monokaryotic fruiting sensu stricto (basidiome formation without mating). Monokaryotic fruiting structures formed by C. parasitica ICMP 11668-derived monokaryons were categorized into four phenotypes. One of them displays ulcer-like structures previously reported from bracket fungi. Histology of dikaryotic and monokaryotic C. parasitica fruiting structures revealed anatomical commonalities and differences between them, and towards monokaryotic fruiting structures of C. aegerita. Mating experiments with C. parasitica strains representative of each fruiting phenotype identified compatible sibling monokaryons. Given reports on hypothetically monokaryotic basidiome field populations of 'C. aegerita sensu lato', it seems worthwhile to prospectively investigate whether monokaryotic fruiting s.str. occurs in nature. Sampling from such populations including karyotyping, comparative -omics, and competition assays may help to answer this question and provide evidence whether this trait may confer competitive advantages to a species capable of it.

Transcriptome of different fruiting stages in the cultivated mushroom Cyclocybe aegerita suggests a complex regulation of fruiting and reveals enzymes putatively involved in fungal oxylipin biosynthesis.

BACKGROUND: Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages. RESULTS: A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies. CONCLUSIONS: In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.

Identification of Feldin, an Antifungal Polyyne from the Beefsteak Fungus Fistulina hepatica.

Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.

Multilocus phylogeny- and fruiting feature-assisted delimitation of European Cyclocybe aegerita from a new Asian species complex and related species.

Cyclocybe aegerita (synonym: Agrocybe aegerita) is a widely cultivated edible and reportedly almost cosmopolitan mushroom species that serves as a model fungus for basidiome formation and as producer of useful natural products and enzymes. Focusing on strains from different continents, here, we present a phylogenetic analysis of this species and some adjacent taxa that employs four phylogenetic markers. In addition, we tested the strains' capability to fructify on agar media. Our analysis reveals that "C. aegerita sensu lato" splits up into the following two well-supported monophyletic geographic lineages: a European clade and an Asian clade. The European one is closely associated with the Chinese species Cyclocybe salicaceicola. In contrast, the Asian lineage, which we preliminarily designate as Cyclocybe chaxingu agg., may comprise several species (species complex) and clusters with the Pacific species Cyclocybe parasitica (New Zealand). In addition, fruiting properties differ across C. aegerita and its Asian and Pacific relatives; however, strains from the Asian clade and C. parasitica tend to form larger basidiomes with relatively big caps and long stipes and strains from the European clade exhibit a more variable fruiting productivity with the tendency to form more basidiomes, with smaller caps and shorter stipes. Moreover, some strains showed individual fruiting patterns, such as the preference to fruit where they were exposed to injuring stimuli. In conclusion, the delimitation of the newly delimited Asian species complex from our multilocus phylogeny of "C. aegerita sensu lato", which is supported by phenotypic data, depicts an exemplary case of biogeographic diversity within a previously thought homogeneous species of near worldwide distribution.

Volatilomes of Cyclocybe aegerita during different stages of monokaryotic and dikaryotic fruiting.

Volatile organic compounds (VOC) are characteristic for different fungal species. However, little is known about VOC changes during development and their biological role. Therefore, we established a laboratory cultivation system in modified crystallizing dishes for analyzing VOC during fruiting body development of the dikaryotic strain Cyclocybe aegerita AAE-3 as well as four monokaryotic offspring siblings exhibiting different fruiting phenotypes. From these, VOC were extracted directly from the headspace (HS) and analyzed by means of gas chromatography-mass spectrometry (GC-MS). For all tested strains, alcohols and ketones, including oct-1-en-3-ol, 2-methylbutan-1-ol and cyclopentanone, were the dominant substances in the HS of early developmental stages. In the dikaryon, the composition of the VOC altered with ongoing fruiting body development and, even more drastically, during sporulation. At the latter stage, sesquiterpenes, especially Δ6-protoilludene, α-cubebene and δ-cadinene, were the dominant substances. After sporulation, the amount of sesquiterpenes decreased, while additional VOC, mainly octan-3-one, appeared. In the HS of the monokaryons, less VOC were present of which all were detectable in the HS of the dikaryon C. aegerita AAE-3. The results of the present study show that the volatilome of C. aegerita changes considerably depending on the developmental stage of the fruiting body.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

New terpenoids from the fermentation broth of the edible mushroom Cyclocybe aegerita.

The strophariaceous basidiomycete Cyclocybe aegerita (synonyms Agrocybe aegerita and A. cylindracea) is one of the most praised cultivated edible mushrooms and is being cultivated at large scale for food production. Furthermore, the fungus serves as a model organism to study fruiting body formation and the production of secondary metabolites during the life cycle of Basidiomycota. By studying the secondary metabolite profiles of C. aegerita, we found several terpenoids in submerged cultures. Aside from the main metabolite, bovistol (1), two new bovistol derivatives B and C (2, 3) and pasteurestin C as a new protoilludane (4) were isolated by preparative HPLC. Their structures were elucidated by mass spectrometry and NMR spectroscopy. The relative configurations of 2-4 were assigned by ROESY correlations, and 3 J H,H coupling constants in the case of 4. Applying quantitative PCR for gene expression validation, we linked the production of bovistol and its derivatives to the respective biosynthesis gene clusters.

Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

BACKGROUND: Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. RESULTS: Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. CONCLUSIONS: The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

Distinguishing commercially grown Ganoderma lucidum from Ganoderma lingzhi from Europe and East Asia on the basis of morphology, molecular phylogeny, and triterpenic acid profiles.

In China and other countries of East Asia, so-called Ling-zhi or Reishi mushrooms are used in traditional medicine since several centuries. Although the common practice to apply the originally European name 'Ganoderma lucidum' to these fungi has been questioned by several taxonomists, this is still generally done in recent publications and with commercially cultivated strains. In the present study, two commercially sold strains of 'G. lucidum', M9720 and M9724 from the company Mycelia bvba (Belgium), are compared for their fruiting body (basidiocarp) morphology combined with molecular phylogenetic analyses, and for their secondary metabolite profile employing an ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESIMS) in combination with a high resolution electrospray ionization mass spectrometry (HR-ESI-MS). According to basidiocarp morphology, the strain M9720 was identified as G. lucidum s.str. whereas M9724 was determined as Ganoderma lingzhi. In molecular phylogenetic analyses, the M9720 ITS and beta-tubulin sequences grouped with sequences of G. lucidum s.str. from Europe whereas those from M9724 clustered with sequences of G. lingzhi from East Asia. We show that an ethanol extract of ground basidiocarps from G. lucidum (M9720) contains much less triterpenic acids than found in the extract of G. lingzhi (M9724). The high amount of triterpenic acids accounts for the bitter taste of the basidiocarps of G. lingzhi (M9724) and of its ethanol extract. Apparently, triterpenic acids of G. lucidum s.str. are analyzed here for the first time. These results demonstrate the importance of taxonomy for commercial use of fungi.

A central role for Ras1 in morphogenesis of the basidiomycete Schizophyllum commune.

Fungi have been used as model systems to define general processes in eukaryotes, for example, the one gene-one enzyme hypothesis, as well as to study polar growth or pathogenesis. Here, we show a central role for the regulator protein Ras in a mushroom-forming, filamentous basidiomycete linking growth, pheromone signaling, sexual development, and meiosis to different signal transduction pathways. ras1 and Ras-specific gap1 mutants were generated and used to modify the intracellular activation state of the Ras module. Transformants containing constitutive ras1 alleles (ras1(G12V) and ras1(Q61L)), as well as their compatible mating interactions, did show strong phenotypes for growth (associated with Cdc42 signaling) and mating (associated with mitogen-activated protein kinase signaling). Normal fruiting bodies with abnormal spores exhibiting a reduced germination rate were produced by outcrossing of these mutant strains. Homozygous Δgap1 primordia, expected to experience increased Ras signaling, showed overlapping phenotypes with a block in basidium development and meiosis. Investigation of cyclic AMP (cAMP)-dependent protein kinase A indicated that constitutively active ras1, as well as Δgap1 mutant strains, exhibit a strong increase in Tpk activity. Ras1-dependent, cAMP-mediated signal transduction is, in addition to the known signaling pathways, involved in fruiting body formation in Schizophyllum commune. To integrate these analyses of Ras signaling, microarray studies were performed. Mutant strains containing constitutively active Ras1, deletion of RasGap1, or constitutively active Cdc42 were characterized and compared. At the transcriptome level, specific regulation highlighting the phenotypic differences of the mutants is clearly visible.

Factors supporting cysteine tolerance and sulfite production in Candida albicans.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Differential interaction of the two related fungal species Candida albicans and Candida dubliniensis with human neutrophils.

Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known, however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host antifungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis. C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived inflammatory and regulatory cytokines and chemokines, such as IL-1α, IL-1ß, IL-1ra, TNF-α, IL-10, G-CSF, and GM-CSF, were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae.

Microbial quorum-sensing molecules induce acrosome loss and cell death in human spermatozoa.

Infertility in men and women is frequently associated with genital contamination by various commensal or uropathogenic microbes. Since many microorganisms are known to release quorum-sensing signals in substantial amounts, we raised the question whether such molecules can directly affect human spermatozoa. Here we show that farnesol and 3-oxododecanoyl-l-homoserine lactone, employed by the opportunistic pathogenic yeast Candida albicans and the gram-negative bacterium Pseudomonas aeruginosa, respectively, induce multiple damage in spermatozoa. A reduction in the motility of spermatozoa coincided in a dose-dependent manner with apoptosis and necrosis at concentrations which were nondeleterious for dendritic cell-like immune cells. Moreover, sublethal doses of both signaling molecules induced premature loss of the acrosome, a cap-like structure of the sperm head which is essential for fertilization. Addressing their mechanism of action, we found that the bacterial molecule, but not the fungal molecule, actively induced the acrosome reaction via a calcium-dependent mechanism. This work uncovers a new facet in the interaction of microorganisms with human gametes and suggests a putative link between microbial communication systems and host infertility.