Antisense RNA Inhibition of the β Subunit of the Dictyostelium discoideum Mitochondrial Processing Peptidase Induces the Expression of Mitochondrial Proteins

We cloned and characterized a cDNA encoding the Dictyostelium discoideum β subunit of mitochondrial processing peptidase (Ddβ-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddβ-MPP is located in the mitochondrial matrix and membrane, whereas Ddα-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddβ-MPP mRNA is down-regulated during early development, the level of the Ddβ-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the β-MPP gene, unexpectedly, the β-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, α-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the β-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from the mitochondria to the nucleus.     

Levels of physiologically active indole derivatives in the fruiting bodies of some edible mushrooms (Basidiomycota) before and after thermal processing

Indole compounds were found in the fruiting bodies of selected mushroom species both before and after thermal processing. On the basis of HPLC analyses the following indole compounds were detected: l-tryptophan, 5-hydroxytryptophan, 5-methyltryptophan, tryptamine, 5-methyltryptamine, serotonin, indole and melatonin. The compound that was present in the largest amounts in the methanolic extracts from unprocessed mushrooms was 5-hydroxytryptophan in the amount of 7.32 mg/100 g DW in the case of the fruiting bodies of Auricularia polytricha; 15.83 in Suillus bovinus; 22.94 in Macrolepiota procera, and 24.83 in Lentinula edodes. In the methanolic extracts from thermally processed mushrooms the amount of 5-hydroxytryptophan was: 3.52 mg/100 g DW in the case of A. polytricha; 5.65 for Leccinum scabrum and 10.11 for M. procera. In addition, serotonin was found in unprocessed fruiting bodies of two mushroom species: 1.03 mg/100 g DW in L. edodes and 13.99 in L. scabrum, and also in thermally processed mushrooms.     

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.     

Occurrence ofPseudomonas tolaasii on fruiting bodies ofLentinula edodes formed onQuercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.Paper No. 301 of the Tottori Mycological Institute.     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the functions and evolution of fungal mitochondrial DNA.     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Lentinula edodes as a Source of Bioelements Released into Artificial Digestive Juices and Potential Anti-inflammatory Material

Lentinula edodes (shiitake), an edible and medicinal mushroom, was chosen for this study with the aim of evaluating the possibility of release of bioelements into artificial digestive juices and analyzing the anti-inflammatory properties. The extracts were prepared from fruiting bodies and biomass enriched with copper (Cu), zinc (Zn), and selenium (Se). The content of bioelements was analyzed by total reflection X-ray fluorescence method. Relatively low content of elements was observed in the fruiting bodies: Cu–1.6, Zn–7.6, and Se–0.12 mg/100 g d.w. compared to mycelial cultures. The anti-inflammatory properties were evaluated in RAW 264.7 cells. Based on the levels of cyclooxygenase 2 protein, nuclear factor erythroid 2-related factor 2, and peroxisome proliferator-activated receptor γ determined using Western blot technique, it was found that the addition of bioelements enhanced the anti-inflammatory properties of mycelium. This indicates that L. edodes cultured on a suitable medium may be used as a potential component of anti-inflammatory products.

     

Relationship between the Carbohydrate Specificity of Lectins and the Carbohydrate Composition of the <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) Sing [<Emphasis Type="Italic">Lentinula edodes</Emphasis> (Berk.) Pegler] Mycelium at Different Stages of Its Morphogenesis

The specificity of lectins from the basidiomycete Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] during solid-state cultivation was found to be maximum to carbohydrates detected in the pyri-dine-soluble fraction of mycelium during the brown mycelial film stage. The carbohydrate composition of the mycelium (i.e., its content of maltose, rhamnose, mannitol, and inositol) was found to be different upon normal fruiting body formation and when the nonpigmented mycelium produced defective fruiting bodies.     

Effects of different levels of wheat bran, rice bran and maize powder supplementation with saw dust on the production of shiitake mushroom (Lentinus edodes (Berk.) Singer)

The cultivation of shiitake mushroom (Lentinus edodes) is increasing rapidly in Bangladesh due to its nutritional and medicinal importance with excellent flavor and longer shelf life. With the aim of increased production, we have cultivated L. edodes on saw dust (SD) supplemented with different levels (10%, 15%, 20%, 25%, 30%, 35% and 40%) of wheat bran (WB), rice bran (RB), maize powder (MP) and their combination (WB+RB+MP = 1:1:1) to investigate the growth, yield and quality of this mushroom. Most of the growth, yield and quality parameters varied significantly when mushrooms were cultivated with different levels of supplementation. The yield of mushroom was increased with the level of each supplementation upto a certain level, and then decreased. SD supplemented with 25% WB produced the highest number of fruiting bodies (34.8/500 g packet), highest biological yield (153.3/500 g packet), and biological efficiency (76.6%) of L. edodes. But the yield of the best quality mushroom was observed on SD with 40% WB supplementation; however, the qualities were not always supplementation dose dependent. In this study, we report that 25% WB supplementation with SD may be very effective for higher yield and 40% WB supplementation for better quality of L. edodes.     

Isolation and characterization of a mycovirus in Lentinula edodes

A mycovirus was isolated from an edible mushroom, Lentinula edodes, that was suffering from a severe epidemic. Fractionation of the diseased cell extract by isopycnic centrifugation with 50% CsCl revealed that the diseased mushroom was infected by Lentinula edodes spherical virus (LeSV), a new spherical virus with a diameter of 55 nm. The particle of LeSV encapsidated the 12 kb RNA genome by a 120 kDa coat protein. BLAST analysis of the partially sequenced LeSV genome showed 95% sequence identity with a putative RNA-dependent RNA polymerase (RdRp) gene of the mycovirus HKB, which was previously reported as being a double-stranded RNA (dsRNA) element. In contrast to HKB, the RNA genome in LeSV is encapsidated by the 120 kDa coat protein. To confirm that the LeSV coat protein is encoded by the viral genome, the N-terminal amino acid sequence of the coat protein was determined. The resulting N-terminal amino acid sequence, N-SALDVAPVVPELYFXXLEV-C, was found to be located in the middle of the HKB ORF1, suggesting that the LeSV coat protein was indeed encoded by the virus. To detect LeSV in L. edodes, a primer set targeting the RdRp gene was designed based on the partial sequence of the LeSV genome. RT-PCR analysis showed that 56 of the 84 commercially available dikaryotic cultivars carry LeSV. The transmission pattern of the virus was determined by analysing basidiospores from LeSV-infected and LeSV-free fruiting bodies. Nine out of 10 basidiospores from the LeSV-infected cultivars contained the virus while the spores from the LeSV-free parent were free of LeSV, suggesting that vertical transmission is the primary mode of LeSV propagation.     

Isolation, Characterization, and Expression of the Gene Encoding the β Subunit of the Mitochondrial Processing Peptidase from Blastocladiella emersonii

A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the β subunit of the Blastocladiella mitochondrial processing peptidase (β-MPP). The predicted β-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that β-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of β-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.     

Strain typing of shiitake (<Emphasis Type="Italic">Lentinula edodes</Emphasis>) cultivars by AFLP analysis,focusing on a heat-dried fruiting body

To validate strain typing by amplified fragment length polymorphism (AFLP) analysis in shiitake (Lentinula edodes) cultivars, the reproducibility of AFLP markers with DNA extracted from the heat-dried fruiting body was evaluated. DNAs were extracted from three different portions of the heat-dried fruiting body – the stipe, pileus, and gill – and AFLP analysis of all parts was carried out using two combinations of selected amplification primer pairs. AFLP profiles of DNA from the gill tissue of heat-dried fruiting body were almost identical to those of cultured mycelia in the same strains, although it was difficult to detect reproducible AFLP profiles from stipe and pileus DNA. These results indicated that AFLP analysis would be applicable for strain typing with heat-dried fruiting bodies of L. edodes by using the DNA extracted from gills.Contribution No. 364 of the Tottori Mycological Institute     

Fruiting body productivity of protoplast-derived clones inLentinula edodes

More than 100 dikaryotic clones (protoclones) derived from mycelial protoplasts of aLentinula edodes dikaryon were examined for their mycelial growth and fruiting body productivity. These protoclones exhibited a variety of vegetative mycelial growth rates, but no apparent difference in colonial morphology compared with the original (parental) dikaryon. Protoclones were cultivated on wood logs under natural conditions, and they exhibited a very wide range of fruiting body yields. Of the 134 protoclones, four were selected that produced a 30–40% increase in dry weight of fruiting body yield over that of the original dikaryon. This high productivity of fruiting bodies was maintained for at least several years. The present results suggest thatL. edodes protoclones can be practically used in strain improvement to increase the capability of fruiting body formation. Contribution No. 287 from the Tottori Mycological Institute.     

Mitochondrial Effects on the Physiological Characteristics of Lentinula edodes

In the mating of filamentous basidiomycetes, dikaryotic mycelia are generated through the reciprocal movement of nuclei to a monokaryotic cytoplasm where a nucleus of compatible mating type resides, resulting in the establishment of two different dikaryotic strains having the same nuclei but different mitochondria. To better understand the role of mitochondria in mushrooms, we created four sets of dikaryotic strains of Lentinula edodes, including B2 × E13 (B2 side) and B2 × E13 (E13 side), B5 × E13 (B5 side) and B5 × E13 (E13 side), E8 × H3 (E8 side) and E8 × H3 (H3 side), and K3 × H3 (K3 side) and K3 × H3 (H3 side). The karyotypes and mitochondrial types of the dikaryotic strains were successfully identified by the A mating type markers and the mitochondrial variable length tandem repeat markers, respectively. Comparative analyses of the dikaryotic strains on the mycelial growth, substrate browning, fruiting characteristics, and mitochondrial gene expression revealed that certain mitochondria are more effective in the mycelial growth and the production of fruiting body, possibly through the activated energy metabolism. Our findings indicate that mitochondria affect the physiology of dikaryotic strains having the same nuclear information and therefore a selection strategy aimed at mitochondrial function is needed in the development of new mushroom strain.     

Overexpression of the laccase gene,lcc1, in Lentinula edodes using the pChG vector

Lentinula edodes secretes laccase (Lcc: EC 1.10.3.2), an industrially useful enzyme. In this study, we introduced and expressed the L. edodes Lcc gene, lcc1, driven by L. edodes glyceraldehyde-3-phosphate dehydrogenase gene promoter into L. edodes. The resulting transformants showed 2-fold Lcc activity than that of the host strain, and expression of the recombinant lcc1 was confirmed by RT-PCR.