Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungus Trichoderma atroviride reveals a role in conidiation and antagonism.

Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T. atroviride interactions with the prey fungi Botrytis cinerea or R. solani. Phenotypic analysis of agl1 deletion strains (Δagl1) showed reduced conidiation compared to T. atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δagl1 strains display reduced antagonism towards B. cinerea and R. solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T. atroviride.

Lanostane Triterpenoids from the Fruiting Bodies of Fomes officinalis and Their Anti-Inflammatory Activities.

In the current study, further chemical investigation of the fruiting bodies of Fomes officinalis led to isolate seven new 24-methyl-lanostane triterpenoids, named officimalonic acids I-O (1-7). Their structures were elucidated based on the analysis of spectroscopic data (HR-MS, 1D and 2D NMR, UV, IR). Compounds 1-3 possessed an unusual C-23 spirostructure moiety, while compounds 4-7 had 23,26-lactone unit. Anti-inflammatory assay revealed that compounds 3 and 5 exhibited significant inhibitory activities against NO production in LPS-induced RAW 264.7 cells and cyclooxygenase (COX-2).

Selenium biofortification in Hericium erinaceus (Lion's Mane mushroom) and its in vitro bioaccessibility.

Hericium erinaceus is a traditional edible mushroom. Selenium (Se) is an essential trace element for humans and other mammals. To develop a Se biofortification strategy for H. erinaceus, the effects of selenate, selenite, and selenomethionine (SeMet) on Se uptake and mushroom growth were investigated. Selenium bioaccessibility and the major Se species present in Se-enriched H. erinaceus were tested in vitro . The H. erinaceus growth was efficiently affected by SeMet than by selenite and selenate. Selenium concentrations in fruiting bodies increased with substrate Se concentration and disturbed accumulation of other microelements. Substrate Se was absorbed and transformed into organic forms. The major Se species in Se-enriched fruiting bodies was SeMet (>63.9%). During in vitro gastrointestinal digestion tests, 51% of total Se was released, and selenocystine (SeCys2 ) (90%) and Se-methylselenocysteine (MeSeCys) (76%) were more easily digested than SeMet (51%). H. erinaceus is suggested as a novel dietary source of supplemental bioavailable Se.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

Comparative Transcriptomics of Flammulina filiformis Suggests a High CO2 Concentration Inhibits Early Pileus Expansion by Decreasing Cell Division Control Pathways.

Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO2 is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO2 concentrations. In comparison to the low CO2 concentration (0.05%), the pileus expansion rates were significantly lower under a high CO2 concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO2 concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO2 conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO2. Transcriptome and qRT-PCR analyses demonstrated that a high CO2 concentration had an adverse effect on gene expression of the ubiquitin-proteasome system and cell cycle-yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO2, which could provide a theoretical basis for a CO2 management strategy in mushroom cultivation.

Selenium biofortification and its effect on multi-element change in Auricularia auricular.

Auricularia auricular could be useful as a candidate for human selenium supplementation. This study examined the effects of exogenous Se on the growth, yield, nutritive value, and mineral accumulation of A. auricular. Selenate or selenite (0.5-40.0 µg g-1) had no effect on mycelium morphology or the yield of fruiting bodies. In some cases, they affected the accumulation of inter-elements and significantly decreased the concentrations of copper, iron, and chromium in the Se-enriched fruiting bodies compared to that with control treatments. The polysaccharide (116.5-131.6 µg g-1) and protein (105.2-113.4 µg g-1) content in Se-enriched fruiting bodies were not significantly different from those observed in the controls (polysaccharide, 114.1 µg g-1; protein, 105.6 µg g-1). Thus, A. auricular can absorb inorganic Se from the substrate and convert it to organic Se compounds (selenocystine (≥4.1%), selenomethionine (≥91.9%), and Se-methylselenocysteine (≥2.3%)).

Modified recipe to inhibit fruiting body formation for living fungal biomaterial manufacture.

Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium.

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.

Selenium Biofortification and Antioxidant Activity in Cordyceps militaris Supplied with Selenate, Selenite, or Selenomethionine.

Selenium (Se) is an essential trace element with multiple functions that may help mitigate adverse health conditions. Cordyceps militaris is an edible mushroom with medicinal properties. The experiment was conducted under artificial cultivation, with five Se concentrations (0, 5, 10, 20, and 40 µg g-1) and three forms of Se (selenate, selenite, and selenomethionine). C. militaris can absorb inorganic from the substrate and convert it to organic Se compounds (selenocystine, selenomethionine, and an unknown species) in fruiting bodies. Compared with the control treatment, Se applications (40 µg g-1 selenate and selenite) significantly increased the Se concentration in fruiting bodies by 130.9 and 128.1 µg g-1, respectively. The biofortification with selenate and selenite did not affect fruiting body production, in some case, but did enhance the biological efficiency. Moreover, the abundance of cordycepin and adenosine increased, while the amino acid contents remained relatively stable. Meanwhile, Se-biofortified C. militaris showed effective antioxidant activities. These results suggest that Se-biofortified C. militaris fruiting bodies may enhance human and animal health when it was included as part of a healthy diet or used as Se supplements.

Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Genetic and Functional Analyses of the DKxanthene Biosynthetic Gene Cluster from Myxococcus stipitatus DSM 14675.

DKxanthenes are a class of yellow secondary metabolites produced by myxobacterial genera Myxococcus and Stigmatella. We identified a putative 49.5 kb DKxanthene biosynthetic gene cluster from Myxococcus stipitatus DSM 14675 by genomic sequence and mutational analysis. The cluster was comprisedof 15 genes (MYSTI_06004-MYSTI_06018) encoding polyketide synthases, non-ribosomal peptide synthases, and proteins with unknown functions. Disruption of the genes by plasmid insertion resulted in defects in the production of yellow pigments. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis indicated that the yellow pigments produced by M. stipitatus DSM 14675 might be noble DKxanthene derivatives. M. stipitatus did not require DKxanthenes for the formation of heat-resistant viable spores, unlike Myxococcus xanthus. Furthermore, DKxanthenes showed growth inhibitory activity against the fungi Aspergillus niger, Candida albicans, and Rhizopus stolonifer.

Baseline sensitivity and biochemical responses of Valsa mali to propamidine.

In the current study, baseline sensitivity of Valsa mali to propamidine was determined using 80 strains collected from apple orchards in Shaanxi Province, China. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.086 to 0.852 µg/mL, with a mean of 0.405 ±â€¯0.137 µg/mL. After treated with propamidine, mycelia were contorted with an increased number of branches, loss of fruiting body production, and decreased cell membrane permeability. Moreover, the enzyme activities of the complexes I, II, IV and ATPase in the mitochondrial respiratory chain were increased significantly, while the enzyme activities of complexes III decreased. Importantly, both on detached leaves and branches of apple trees, propamidine applied at 100 µg/mL exhibited over 75% protective and curative efficacies, which were even better than the efficacies obtained by carbendazim at the same concentration. These results indicated that propamidine could be used as an alternative compound in controlling Valsa canker and mitochondrial respiratory chains might be correlated with the action mode of propamidine. This study encourages further investigation for the action mechanism of propamidine against plant pathogens and the information could be valuable for synthesis of new antifungal drugs with novel modes of action.

Development of antler-type fruiting bodies of Ganoderma lucidum and determination of its biochemical properties.

Following the importance of antler-type fruiting bodies of Ganoderma lucidum, in this study, the impact of main growth parameters such as ventilation and light on the development of antler-type fruiting bodies has been investigated together with the determination of physico-chemical properties of antler fruiting bodies. For this, the primordia bags of G. lucidum were kept under controlled ventilation to adjust the CO2 produced by the mushrooms owing to its respiration under light and dark conditions. The bioactive compounds such as phenolics, flavonoids, water-soluble polysaccharides and ganoderic acid showed a two-fold increase in the antler-type fruiting bodies as compared to normal kidney-shaped fruiting bodies. It is assumed from this study that the antler type fruiting bodies are developed due to restricted ventilation which causes an increase in the level of CO2 gas in the air as a result of respiration of mushroom. The shape and colour of antler fruiting bodies again dependent on the light provided in the growth chamber. This study also proves that with the manipulation of light and ventilation antler-type fruiting bodies of G. lucidum could be developed with higher quantity of bioactive compounds and with higher antioxidant potential.

Chitosan nanoparticles-loaded Citrus aurantium essential oil: a novel delivery system for preserving the postharvest quality of Agaricus bisporus.

BACKGROUND: One of the main problems in the button mushroom industry is the rapid deterioration of fruit bodies after harvest. Today, nanotechnology has become a more reliable technique to improve the quality of products in food packaging. In the present study, the effectiveness of chitosan nanoparticles containing Citrus aurantium essential oil on postharvest quality of white button mushroom was examined and compared to essential oil fumigation and control treatments. RESULTS: Based on high-resolution transmission electron microscopy and dynamic light scattering, nanoparticles exhibited a spherical shape of 20-60 nm diameter. The results revealed that the application of chitosan nanoparticles loaded with C. aurantium oil significantly decelerated the rate of color change, weight loss and firmness compared to fumigation with essential oil and control treatments. Treatment of fruit bodies with chitosan nanoparticles loaded with C. aurantium oil promoted the accumulation of phenolic compounds and ascorbic acid, and resulted in increases in catalase and superoxide dismutase and a decrease in polyphenol oxidase activities, as the highest antioxidant capacity was observed after 15 days of cold storage. CONCLUSION: This present research demonstrates that gradual release of C. aurantium essential oil from chitosan nanoparticles could be an effective and practical method for extending the shelf life of white button mushroom up to 15 days without significant decrease in antioxidant capacity. © 2018 Society of Chemical Industry.

A Sphingosine-type cerebroside in Clavicorona pyxidata induce fruit body formation.

Clavicorona pyxidata is a wild edible and medicinal mushroom that is rich in bioactive natural products and has thus been extensively used as traditional medicine in China. The present study has determined that the organic crude extract prepared from a fermented culture of C. pyxidata imparted auto-inhibitory effects on mycelial growth and then induced the formation of fruiting bodies. By monitoring bioactivity, one compound was isolated via successive chromatography over silica gel, Sephadex LH-20, and Cl8-reversed phase silica gel and was identified as a known sphingosine-type cerebroside by nuclear magnetic resonance (NMR) and physicochemical data, namely, (4E, 8E)-N-D-2'-hydroxypalmitoyl-1-O-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine. The application of this cerebroside at a concentration of 200 µg/disc paper resulted in the inhibition of aerial hyphal growth of C. pyxidata. The findings of the present study indicated that this C. pyxidata cerebroside is a fruiting body-inducing substance (FIS).

Kojic acid-mediated damage responses induce mycelial regeneration in the basidiomycete Hypsizygus marmoreus.

Mechanical damage can induce fruiting body production in fungi. In this study, the antioxidant kojic acid (KA) was found to enhance injured mycelial regeneration and increase fruiting body production in Hypsizygus marmoreus. KA reduced the level of reactive oxygen species (ROS), which are harmful to mycelia when excessively generated by mechanical damage. Moreover, KA increased catalase and superoxide dismutase activities and glutathione and ascorbic acid contents by up-regulating antioxidant gene expression. These results suggest that KA promotes mycelial regeneration in response to damage by activating a "stress signal" and enhances the ability of H. marmoreus to resist oxidative damage by invoking the antioxidant system. In addition, KA increased the content of extracellular ATP, which serves as a "stress signal" in response to injury, and modulated ROS signaling, decreasing NADPH oxidase gene expression and ROS levels in the mycelial-regeneration stage. KA treatment also up-regulated the MAPK, Ca2+ and oxylipin pathways, suggesting their involvement in the damage response. Furthermore, laccase and cellulase activities were stimulated by KA at different developmental stages. These results demonstrate that KA regulates gene expression and activates pathways for mycelial wound healing, regeneration of damaged mycelia and reproductive structure formation in the basidiomycete H. marmoreus.

Inhibition of bioluminescence in the living gills of the luminous fungus Mycena chlorophos by trans-4-aminocinnamic acid.

The living gills of the fungus Mycena chlorophos spontaneously emit green light. It was previously reported that trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnnamic acid are essential for the bright light production in the living gills. However, the chemical mechanisms underlying their bioluminescence are unknown. In the present study, trans-4-aminocinnamic acid was found to inhibit light production in the living gills. The concentrations of trans-4-aminocinnamic acid that inhibited the bioluminescence intensity by 50% of initial values for immature and mature gills were 0.07 µM and 4 µM, respectively. Approximately 20% of the bioluminescence intensity of the immature and mature gills was not inhibited by a further increase in the concentration of trans-4-aminocinnamic acid. Moreover, the bioluminescence that was activated by trans-4-hydroxycinnamic acid or trans-3,4-dihydroxycinnamic acid (0.01 mM) was completely inhibited by trans-4-aminocinnamic acid. Therefore, trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid functioned for the bioluminescence that was inhibited by trans-4-aminocinnamic acid. trans-4-Aminocinnamic acid strongly bound to the bioluminescence system(s) and withstood rinsing of the gills with 10 mM phosphate buffer (pH = 7), and high concentrations of trans-4-hydroxycinnamic acid (1 mM) and trans-3,4-dihydroxycinnamic acid (0.1 mM) functioned to displace trans-4-aminocinnamic acid from the bioluminescence system(s) and reactivate bioluminescence. Benzenamine, trans-cinnamic acid, trans-2-aminocinnamic acid, and trans-3-aminocinnamic acid did not inhibit bioluminescence. Therefore, the structure-specific inhibition by trans-4-aminocinnamic acid suggested that the 4-hydroxy group in trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid molecules plays a functional role in the bioluminescence reaction.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr = 9.84 mm day-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22 U g-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

YelA, a putative Dictyostelium translational regulator, acts as antagonist of DIF-1 signaling to control cell-type proportioning.

DIF-1 (differentiation-inducing factor1) is a polyketide produced by Dictyostelium prespore cells which induces initially uncommitted cells to differentiate as prestalk cells. Exposure of cells to DIF-1 causes transitory hypo-phosphorylation of seven serine residues in YelA, a protein with a region of strong homology to the MIF4G domain of the eukaryotic initiation factor eIF4G. Based upon its domain architecture, which in one important aspect closely resembles that of Death-Associated Protein 5 (DAP5), we predict a role in stimulating internal ribosome entry-driven mRNA translation. The two paradigmatic DIF-1 inducible genes are ecmA (extracellular matrixA) and ecmB. In support of a YelA function in DIF-1 signaling, a YelA null strain showed greatly increased expression of ecmA and ecmB in response to DIF-1. Also, during normal development in the null strain, expression of the two genes is accelerated. This is particularly evident for ecmB, a marker of stalk tube and supporting structure differentiation. Mutants in DIF-1 bio-synthesis or signaling display a rudimentary or no basal disc and, conversely, YelA null mutants produce fruiting bodies with a highly enlarged basal disc that ectopically expresses a stalk tube-specific marker. Thus YelA acts as an antagonist of DIF-1 signaling, with a consequent effect on cell type proportioning and it is predicted to act as a translational regulator.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.