Hyphal coil morphology and its relationship to thromboplerous hyphae in Rhizopogon roseolus.

While observing mycelial features of the ectomycorrhizal fungus Rhizopogon roseolus, we noted the formation of hyphal coils in laboratory cultures. The coiled hyphae initially formed at the hyphal tip in the presence or absence of the plant host (Pinus thunbergii), on both rich and poor modified Melin-Norkrans media. Hyphal coils formed from the hyphal tips toward the middle of the hyphae, with rope-like hyphal strands fused as extensive circular coils. Hyphal coils were generally oval and circular. Coils were composed of 1-5 layers of hyphae. Presence of the host had a significant impact on the diameter of the coil loops, which was consistently wider without the host than with the host present on both media. In addition, the terminal part of the mature coils was melanized and separated from the unmelanized coil by a septum. The melanized coils resembled thick condensed hyphae and were detached and scattered throughout the fungal colony. The observation of morphological characteristics suggested that mature coils detached into the mycelia were thromboplerous hyphae. This is the first report on hyphal coil morphogenesis and its potential relationship to the initial development of thromboplerous hyphae.

Basidium formation of Rhizopogon roseolus with Pinus thunbergii in agar medium.

The edible ectomycorrhizal (ECM) mushroom Rhizopogon roseolus usually develops basidium and basidiospores in the gleba of its basidiomata. Here, we report a novel production of basidia in laboratory cultures of the edible ECM mushroom. The basidium with sterigma was observed on the old mantle structure (> six months) of the ECM between R. roseolus and Pinus thunbergii in a modified Melin-Norkrans medium that was subjected to a temperature shock from 25 °C to 4 °C. The basidia were cylindrical to clavate, with prominent sterigmata and no basidiospores. The absence of basidiospores might indicate partial development of the basidium structure as a response to environmental stress and incomplete life cycle of R. roseolus. In addition, branched cystidia were evident in two or three clavate-to-ovoid cells. This study suggests the possibility of obtaining the primary mycelium of R. roseolus from pure cultures and may be an alternative genetic source for cultivation purposes. Further observations are required to induce basidiosporogenesis of R. roseolus basidia in an agar medium focusing on manipulation of temperature.

The impact of host plant (Pinus thunbergii) on the mycelial features of the ectomycorrhizal fungus Rhizopogon roseolus.

The impact of host plant on the mycelial features of mycorrhizal symbiont and its characteristics has been poorly investigated. This study aimed to compare and quantify (statistically tested) some of the mycelial features of an ectomycorrhiza (ECM)-forming fungus with and without the ECM host. The ECM-forming fungus, Rhizopogon roseolus, inoculated with or without Pinus thunbergii on both rich and poor nutrient media, was observed under laboratory conditions. On rich medium, fungi with the host had the highest colony diameter and consistently produced the highest hyphal length relative to fungi on other media. Thus, the host had a significant impact on the mycelium production of R. roseolus in both rich and poor media. Further, fungi without the host had a higher number of hyphal anastomoses per hyphal length on both poor and rich media than fungi with the host in the same medium. Anastomosis, which refers to the fusion of two parallel hyphae, was evident in all experiments. However, there was no significant impact of the host on the number of hyphal anastomoses. In addition, fungi without the host had more frequent hyphal branching on both rich and poor media than fungi with the host. The occurrence of a host only had a significant impact on the formation of the hyphal branch on poor medium. Further, a chlamydospore-like structure was identified, which had a higher diameter when formed with the host on both rich and poor media. The present data provide new insights into the host plant's impact on the mycelial features of ECM-forming fungi.

Detection of autophagy-related structures in fruiting bodies of edible mushroom, Pleurotus ostreatus.

Autophagy is involved in various fungal morphogenetic processes. However, there are limited reports regarding the role of autophagy in mushroom fruiting body formation. The purpose of this study was to reveal the autophagy-related structures in mushroom-forming fungi. The edible mushroom Pleurotus ostreatus was used in this study. Transmission electron microscopy revealed double-membrane bounded structures containing cytoplasmic components in the fruiting bodies of this fungus. Some of these double-membrane structures were observed to interact with the vacuoles. Additionally, curved flat cisternae of various lengths were detected in the cytoplasm. The shape, size and thickness of the limiting membrane of the double-membrane structures and the flat cisternae corresponded well with those of the autophagosomes and the isolation membranes, respectively. Regarding autophagosome formation, a membrane-bound specific zone was detected near the isolation membrane, which appeared to expand along the novel membrane. This is the first detailed report showing autophagy-related structures in P. ostreatus and provides a possible model for autophagosome formation in these filamentous fungi.

Cytological comparison of mycelial aggregates of Rhizopogon roseolus with and without the ectomycorrhiza host.

Most cytology reports on ectomycorrhiza (ECM) have been less focused on the fungal structures, especially on the exploratory organ. This study aimed to evaluate the morphological response of explorative mycelia of ECM forming fungi with and without the occurrence of the ECM host. We assessed the mycelial aggregates of Rhizopogon roseolus which was inoculated with and without Pinus thunbergii under controlled laboratory conditions. The mycelial aggregates with the host produced strikingly complex hyphal strands relative to those without host. Light microscopy revealed that the cytology and plectology of both mycelial aggregates had the approximately similar architecture. The tubular hyphae diameter without the host was consistently larger than that with the host. This study confirmed that the septa diameter of the tubular hyphae conjunction of mycelial aggregates with the host were shorter than those without the host. In addition, partially and completely dissolved septa of the tubular hyphae were evident with and without host. We also described the thromboplerous hyphae, which have rarely been reported in vitro. These hyphae were produced in higher numbers near the fungal inoculum with and without the host. The current findings will add to the current knowledge regarding ECM fungi mycelial aggregate response to the host.

Effect of sodium chloride on basidiospore germination and vegetative mycelial growth of the ectomycorrhizal fungus Rhizopogon roseolus.

Rhizopogon roseolus is a basidiomycetous ectomycorrhizal fungus that inhabits mainly coastal areas. Understanding the response of this fungus to salinity at each stage of its life cycle will lead to elucidation of the strategies for its propagation. This study examined the effect of sodium chloride (NaCl) on basidiospore germination and mycelial growth of both homokaryotic and heterokaryotic strains of R. roseolus, on nutrient agar media with varying concentrations of NaCl (0, 50, 150, and 300 mM). Regardless of the presence of NaCl, R. roseolus basidiospores germinated and the germlings grew, forming compatible fusions. In addition, all multispore strains, including homokaryons and heterokaryons, grew under these NaCl conditions. Most of these strains had an effective concentration inhibiting mycelial growth by 50% value greater than 300 mM of NaCl. These results indicate that R. roseolus can germinate, grow, and mate in the presence of NaCl, allowing it to propagate in saline habitats.

Identification, characterization and expression of A-mating type genes in monokaryons and dikaryons of the edible mushroom Mycoleptodonoides aitchisonii (Bunaharitake).

Identifying the mating-type in Mycoleptodonoides aitchisonii is important for enhancing breeding and cultivation of this edible mushroom. To clarify the molecular mechanisms of the bipolar mating system in M. aitchisonii, the homeodomain protein gene 2 (Mahd2) was characterized. A genomic DNA fragment of Mahd2 in M. aitchisonii 50005-18 strain was 1,851 bp long and encoded a protein of 614 amino acids. Transcriptional analysis revealed that the expression of Mahd2 was higher in monokaryotic strains that produced clamp cells than in those that did not. The highest relative expression level of Mahd2 was observed in monokaryon TUFC 50005-4, which was capable of forming a true clamp. These results suggested that the formation of clamp cells is regulated by A-mating type homeodomain proteins, and the frequency of clamp cell formation might be promoted by high expression of the Mahd2 gene.

Nuclear behavior in Coprinus comatus from Japan and estimation of the lifecycle.

In order to elucidate the lifecycle of Coprinus comatus, we examined the number of nuclei in basidiospores, hyphal cells and oidia. Basidiospores isolated from the fruiting bodies of four Japanese strains were binucleate. In both primary and secondary mycelia, most of the cells were binucleate. In addition, oidia and oidiophores were observed for the first time in this mushroom and most of the oidia were binucleate. Based on these results, the lifecycle of C. comatus was inferred to be as follows. A homokaryotic binucleate basidiospore germinates and produces homokaryotic binucleate hyphae. After mating between compatible homokaryotic binucleate hyphae, a heterokaryotic binucleate secondary mycelium is produced. If environmental conditions are suitable for fruiting, homokaryotic binucleate basidiospores in the fruiting body are produced.

Purification, characterization and partial amino acid sequences of thermo-alkali-stable and mercury ion-tolerant xylanase from Thermomyces dupontii KKU-CLD-E2-3.

We investigated the properties of the low molecular weight thermo-alkali-stable and mercury ion-tolerant xylanase production from Thermomyces dupontii KKU-CLD-E2-3. The xylanase was purified to homogeneity by ammonium sulfate, Sephadex G-100 and DEAE-cellulose column chromatography which resulted 27.92-fold purification specific activity of 56.19 U/mg protein and a recovery yield of 2.01%. The purified xylanase showed a molecular weight of 25 kDa by SDS-PAGE and the partial peptide sequence showed maximum sequence homology to the endo-1,4-ß-xylanase. The optimum temperature and pH for its activity were 80 °C and pH 9.0, respectively. Furthermore, the purified xylanase can maintain more than 75% of the original activity in pH range of 7.0-10.0 after incubation at 4 °C for 24 h, and can still maintain more than 70% of original activity after incubating at 70 °C for 90 min. Our purified xylanase was activated by Cu2+ and Hg2+ up to 277% and 235% of initial activity, respectively but inhibited by Co2+, Ag+ and SDS at a concentration of 5 mM. The Km and Vmax values of beechwood xylan were 3.38 mg/mL and 625 µmol/min/mg, respectively. Furthermore, our xylanase had activity specifically to xylan-containing substrates and hydrolyzed beechwood xylan, and the end products mainly were xylotetraose and xylobiose. The results suggested that our purified xylanase has potential to use for pulp bleaching in the pulp and paper industry.

Secondary Metabolites and Their Biological Activity from Aspergillus aculeatus KKU-CT2.

The bioactive compounds of the fungus Aspergillus aculeatus strain KKU-CT2, have been studied. The crude extracts from this fungus showed good antimicrobial activity against human pathogens, including Gram-positive and Gram-negative bacteria and yeast-like fungi. Its chemical components were isolated and purified by chromatographic methods. The structures of the secondary metabolites were elucidated by spectroscopic methods (IR, 1H, and 13C NMR). They were identified as ergosterol peroxide (1), secalonic acid D (2), secalonic acid F (3), variecolin (4), variecolactone (5), and ergosterol (6). Compounds 1 and 4-6 are reported for the first time as fungal metabolites from this species. Compound 1 displayed inhibitory effects on HSV-1 with an IC50 of 11.01 µg/ml. Compounds 3, 4, and 6 exhibited antimalarial activity against Plasmodium falciparum with IC50 of 1.03, 1.47, and 5.31 µg/ml, respectively. Additionally, all compounds from A. aculeatus KKU-CT2 showed unprecedented anticancer activities against human epidermoid carcinoma in the mouth (KB) (compounds 1-6), human breast cancer (MCF-7) (compounds 2, 4, and 5), and human lung cancer cells (NCI-H187) (compounds 1-4 and 6). These results suggest that secondary metabolites from A. aculeatus KKU-CT2 might be interesting for further derivatization, targeting diseases such as cancer.

Expression of α-glucosidase during morphological differentiation in the basidiomycetous fungus Pholiota microspora.

The α-glucosidase gene from Pholiota microspora, designated PnGcs, was amplified and characterized. The open reading frame region of PnGcs, from ATG to the stop codon, is 2937 bp and encodes a protein of 979 amino acids with a signal peptide of 20 amino acids at the N-terminus. The predicted amino acid sequence of PnGcs indicated that it is a glycoside hydrolase family 31 protein. Quantitative reverse transcription PCR was used to investigate PnGcs expression in mycelia cultured in minimal medium containing various carbon sources, as well as in tissue during different stages of development of fruiting bodies. When P. microspora was grown in minimal medium supplemented with different carbon sources, PnGcs expression was highest when induced by maltose. During cultivation on sawdust medium, PnGcs expression increased dramatically at the fruiting body formation stage compared with the mycelial growth stage, which implied that PnGcs is closely associated with fruiting body development.

Biosynthesis of integric acid isolated from the wood-decay fungus Xylaria feejeensis 2FB-PPM08M.

The biosynthesis of integric acid, a secondary metabolite of the wood-decay fungus Xylaria feejeensis strain 2FB-PPM08M, has been studied. Labeling experiments using [1-(13)C], [2-(13)C] and [1,2-(13)C2] acetate and L-methionine (methyl-(13)C) were separately performed with fungal culture. The labeling patterns of these metabolites indicated the same origin, and determined that integric acid was formed through the condensation of a sesquiterpene and a polyketide. These experiments showed that side chain of compounds would be synthesized by the polyketide pathway, while the ring carbon indicated the biosynthesis of compounds via the mevalonate pathway.

Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

Gymnopilins, a product of a hallucinogenic mushroom, inhibit the nicotinic acetylcholine receptor.

Gymnopilins are substances produced in fruiting bodies of the hallucinogenic mushroom, Gymnopilus junonius. Although, only a few biological effects of gymnopilins on animal tissues have been reported, it is believed that gymnopilins are a key factor of the G. junonius poisoning. In the present study, we found that gymnopilins inhibited ACh-evoked responses in neuronal cell line, PC12 cell, and determine the underlying mechanism. Gymnopilins were purified from wild fruiting bodies of G. junonius collected in Japan. Ca(2+)-imaging revealed that gymnopilins reduced the amplitude of ACh-evoked [Ca(2+)]i rises by about 50% and abolished the ACh responses remaining in the presence of atropine. Gymnopilins greatly reduced the amplitude of [Ca(2+)]i rises evoked by nicotinic ACh receptor agonists, 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP) and nicotine. In the whole-cell voltage clamp recording, gymnopilins inhibited the DMPP-evoked currents, but did not affect the voltage-gated Ca(2+) channel currents. These results indicate that gymnopilins directly act on nicotinic ACh receptors and inhibit their activity. This biological action of gymnopilins may be one of the causes of the G. junonius poisoning.

Multiple effects of gymnopilin on circulatory system of the rat.

Gymnopilin is one of the substances produced by the hallucinogenic mushroom, Gymnopilus junonius. In this study, we examined effects of gymnopilins purified from wild fruiting bodies of G. junonius on contractile activity of aorta preparations and blood pressure in rats. Gymnopilins at lower concentrations than 5 mg/mL did not evoke contraction of helical strips of the thoracic aorta. In contrast, gymnopilins (5 mg/mL) applied to the aorta strips pre-contracted by norepinephrine (100 nM) caused relaxation. This relaxing action did not depend on the activity of the endothelium cells. The relaxing effect of 5-mg/mL gymnopilins was observed in aorta strips contracted by angiotensin II (10 nM) and the high K+ solution (60 mM). Moreover, the adenylyl cyclase inhibitor, SQ-22536, significantly inhibited the relaxing effect of gymnopilins at 1 mg/mL on the norepinephrine-contracted strips. These results suggested that gymnopilins acted directly on smooth muscle cells of the aorta and activated the cAMP-dependent cascade to cause the vasodilation. Paradoxically, gymnopilins injection into the jugular vein transiently increased blood pressure without affecting the heart rate. This result suggests that gymnopilins increase cardiac output and/or tension of the artery through the excitation of the vasomotor nerve that overcame the direct relaxing effect on the vascular smooth muscle.

Gymnopilin--a substance produced by the hallucinogenic mushroom, Gymnopilus junonius--mobilizes intracellular Ca(2+) in dorsal root ganglion cells.

Gymnopilus junonius is a widely spread mushroom in Japan and well known as a hallucinogenic mushroom. Gymnopilin was purified from the fruiting body of G. junonius and was reported to act on the spinal cord and depolarize motoneurons. This is the only evidence that gymnopilin has a biological effect on animals and no mechanism of the action has been determined at all. In this study, we examined effects of gymnopilin on intracellular Ca(2+) concentrations ([Ca(2+)](i)) of cultured cells isolated from the dorsal root ganglion (DRG) of the rat. The cell culture consisted of neurons and non-neuronal cells. Gymnopilin increased [Ca(2+)](i) in both the types of cells. The gymnopilinevoked [Ca(2+)](i) rise in the non-neuronal cells was inhibited by cyclopiazonic acid and U-73122, inhibitors of Ca(2+)-ATPase of the intracellular Ca(2+) store and phospholipase C, respectively, but not by removal of extracellular Ca(2+). These results indicate that gymnopilin activated phospholipase C and mobilize Ca(2+) from the intracellular Ca(2+) store in non-neuronal cells from the DRG. This is the first report to show that gymnopilin directly acts on cells isolated from the mammalian nervous system.

Karyological characterization of meiosis, post-meiotic mitosis and nuclear migration in the ectomycorrhizal fungus Rhizopogon roseolus (= R. rubescens).

Karyological characteristics during basidiosporogenesis of Rhizopogon roseolus, a member of the hypogeous Agaricomycetes, were studied by light and transmission electron microscopy. More than 1000 tissue fragments of young basidiomata were stained with HCl-Giemsa and observed by a light microscopy to evaluate nuclear behavior. Basidium morphology in the hymenium was observed by transmission electron microscopy. Meiosis and post-meiotic mitosis took place in the center of the basidium. Sterigmata appeared when the first meiotic division occurred, and the center of the basidium became constricted when the second meiotic division occurred. Asynchronous nuclear migration from the basidium into the basidiospores occurred after post-meiotic mitosis, producing eight uninucleate basidiospores. The nucleus migrated patchily into basidiospores. The pattern of post-meiotic mitosis of R. roseolus, in which post-meiotic mitosis took place in the center of the basidium, is reported for the first time.

Novel effects of extracts from poisonous mushrooms on expression and function of the human ether-a-go-go-related gene channel.

UNLABELLED: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr), which plays a pivotal role in cardiac action potential repolarization. Inherited mutations of this gene cause Long QT syndrome type 2. hERG expression is altered by several types of drugs as well as by temperature. Heat shock protein 70 (Hsp70) and Heat shock cognate protein 70 (Hsc70) have reciprocal effects on hERG proteins. We examined the effects of poisonous mushrooms on hERG protein expression and its channel function. METHODS: We evaluated the effects of several types of poisonous mushrooms on the expression and function of wild-type hERG by Western blotting, reverse transcription polymerase chain reaction (PCR), and patch clamping in transfected HEK293 cells and mouse HL-1 cardiomyocytes. RESULTS: Extracts of Gymnopilus junonius (junonius) increased expression of both hERG and Hsp70 in HEK293 cells with concomitant decrease in Hsc70, whereas extracts of Amanita ibotengutake (ibotengutake) decreased hERG proteins with increase in Hsc70. Knockdown of Hsp70 and Hsc70 by small interfering RNA abolished the effects of the two mushrooms on hERG, respectively. Certain fractions of junonius increased expression of hERG proteins. hERG currents were increased by extracts of junonius, resulting in shortening of action potential duration (APD). In contrast, hERG currents were decreased and APD was prolonged by extracts of ibotengutake. CONCLUSION: junonius enhanced the expression and function of hERG by increasing Hsp70 and decreasing Hsc70. Ibotengutake decreased hERG expression via increase in Hsc70. Constituents of junonius may have the potential for use in treatment of arrhythmia.

A-mating-type gene expression can drive clamp formation in the bipolar mushroom Pholiota microspora (Pholiota nameko).

In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.