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1.
J Agric Food Chem ; 67(32): 8875-8883, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347830

RESUMO

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.


Assuntos
Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Grifola/enzimologia , Micélio/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Grifola/genética , Grifola/crescimento & desenvolvimento , Grifola/metabolismo , Micélio/enzimologia , Micélio/genética , Micélio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
2.
Antonie Van Leeuwenhoek ; 112(7): 1095-1104, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30725325

RESUMO

Rubber anthracnose caused by Colletotrichum gloeosporioides leads to huge economic loss in the natural rubber industry every year. Conidia of C. gloeosporioides are a major infection source but little is known about molecular mechanisms underlying conidial development and infection. In this study, the C. gloeosporioide C2H2 zinc-finger protein transcription factor gene CgAzf1 is shown to be involved in melanin production, conidial development and infection. Deletion of CgAzf1 resulted in decreased melanin production and hydrophilicity of aerial mycelium was increased. The mutants also showed reduced conidiation, low germination rate, and the formation of appressorium lagged too. Virulence assays showed that the CgAzf1 deletion strain could not infect intact rubber tree leaves and had an attenuated virulence on the wounded leaves. Quantitative RT-PCR showed that CgAzf1 regulates expression of genes involved in the MAPK, cAMP-PKA and melanin biosynthesis pathways.


Assuntos
Colletotrichum/metabolismo , Colletotrichum/patogenicidade , Proteínas Fúngicas/metabolismo , Hevea/microbiologia , Melaninas/metabolismo , Doenças das Plantas/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Colletotrichum/genética , Colletotrichum/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Micélio/química , Micélio/genética , Micélio/metabolismo , Micélio/patogenicidade , Filogenia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Fatores de Transcrição/genética , Virulência
3.
Mol Genet Genomics ; 294(3): 663-677, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30778675

RESUMO

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.


Assuntos
Agaricales/genética , Agrocybe/genética , Regulação Fúngica da Expressão Gênica , Transformação Genética , Agaricales/efeitos dos fármacos , Agrocybe/efeitos dos fármacos , Carboxina/farmacologia , Farmacorresistência Fúngica/genética , Carpóforos/efeitos dos fármacos , Carpóforos/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Genoma Fúngico/genética , Íntrons/genética , Mutação , Micélio/efeitos dos fármacos , Micélio/genética , Plasmídeos/genética , Succinato Desidrogenase/genética
4.
BMC Genomics ; 20(1): 121, 2019 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736734

RESUMO

BACKGROUND: Lentinula edodes is one of the most popular edible mushroom species in the world and contains useful medicinal components, such as lentinan. The light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process for ensuring the quantity and quality of this edible mushroom. To understand the molecular mechanisms underlying this critical developmental process in L. edodes, we characterized the morphological phenotypic changes in a strain, Chamaram, associated with abnormal brown film formation and compared its genome-wide transcriptional features. RESULTS: In the present study, we performed genome-wide transcriptome analyses of different vegetative mycelium growth phenotypes, namely, early white, normal brown, and defective dark yellow partial brown films phenotypes which were exposed to different light conditions. The analysis revealed the identification of clusters of genes specific to the light-induced brown film phenotypes. These genes were significantly associated with light sensing via photoreceptors such as FMN- and FAD-bindings, signal transduction by kinases and GPCRs, melanogenesis via activation of tyrosinases, and cell wall degradation by glucanases, chitinases, and laccases, which suggests these processes are involved in the formation of mycelial browning in L. edodes. Interestingly, hydrophobin genes such as SC1 and SC3 exhibited divergent expression levels in the normal and abnormal brown mycelial films, indicating the ability of these genes to act in fruiting body initiation and formation of dikaryotic mycelia. Furthermore, we identified the up-regulation of glycoside hydrolase domain-containing genes in the normal brown film but not in the abnormal film phenotype, suggesting that cell wall degradation in the normal brown film phenotype is crucial in the developmental processes related to the initiation and formation of fruiting bodies. CONCLUSIONS: This study systematically analysed the expression patterns of light-induced browning-related genes in L. edodes. Our findings provide information for further investigations of browning formation mechanisms in L. edodes and a foundation for future L. edodes breeding.


Assuntos
Perfilação da Expressão Gênica , Lentinula/genética , Lentinula/metabolismo , Micélio/genética , Micélio/metabolismo , Pigmentação/genética , Genes Fúngicos/genética , Lentinula/efeitos da radiação , Luz , Micélio/efeitos da radiação , Fenótipo , Pigmentação/efeitos da radiação
5.
J Biosci Bioeng ; 128(1): 1-7, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30683592

RESUMO

Tyrosinase is an industrially useful enzyme, however, it causes gill browning of Lentinula edodes fruiting bodies during preservation. In this study, we constructed two vectors, pChG-gTs and pChG-gTa, expressing sense and antisense tyrosinase gene of L. edodes, respectively, using promoters derived from the glyceraldehyde-3-phosphate dehydrogenase gene. The host strain SR-1 of L. edodes was selected because of its fast growth, high protoplast yield, and high regeneration rate. Upon transformation of the host strain SR-1 with the pChG-gTs vector, a clone with 3.6-fold and 14.5-fold higher tyrosinase activity in vegetative mycelia and in fresh gills, respectively, than that of the host strain was obtained from nine transformants. Similarly, two clones containing the pChG-gTa vector with effectively repressed tyrosinase gene expression in vegetative mycelia and gills during the late stage of post-harvest preservation of fruiting bodies were obtained from 10 transformants. However, it remained unclear whether repression of the tyrosinase gene prevented gill browning, as the host strain also showed less browning than a commercial strain. Thus, this study highlights the usefulness of the pChG vector in expressing homologous enzyme coding genes in the vegetative mycelia and fruiting bodies of L. edodes.


Assuntos
Quitina Sintase/genética , Vetores Genéticos/genética , Monofenol Mono-Oxigenase/genética , Regiões Promotoras Genéticas/genética , Cogumelos Shiitake/genética , Transformação Genética , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Carpóforos/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Inativação Gênica/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Organismos Geneticamente Modificados , Cogumelos Shiitake/enzimologia , Cogumelos Shiitake/crescimento & desenvolvimento , Transformação Genética/genética , Regulação para Cima/genética
6.
Biosci Biotechnol Biochem ; 83(4): 774-780, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30585121

RESUMO

Cyclooxygenases are responsible for the production of prostaglandin H2 (PGH2) from arachidonic acid. PGH2 can be converted into some bioactive prostaglandins, including prostaglandin F2α (PGF2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.


Assuntos
Proteínas de Algas/genética , Ácido Araquidônico/metabolismo , Dinoprosta/biossíntese , Gracilaria/química , Engenharia Metabólica/métodos , Mortierella/genética , Prostaglandina-Endoperóxido Sintases/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Algas/metabolismo , Meios de Cultura/química , Expressão Gênica , Gracilaria/genética , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Mortierella/metabolismo , Micélio/genética , Micélio/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Transformação Genética , Transgenes
7.
J Microbiol ; 57(2): 127-137, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30552631

RESUMO

Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.


Assuntos
Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Lacase/biossíntese , Lacase/genética , Interações Microbianas/fisiologia , Polyporaceae/enzimologia , Polyporaceae/genética , Técnicas de Cocultura , Genes Fúngicos/genética , Micélio/enzimologia , Micélio/genética , Micélio/fisiologia , Nutrientes , Estresse Oxidativo , Pleurotus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Trametes/fisiologia , Transcriptoma
8.
Viruses ; 10(12)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558121

RESUMO

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S. sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production.


Assuntos
Ascomicetos/genética , Ascomicetos/virologia , Micovírus/fisiologia , Pequeno RNA não Traduzido/genética , Transcrição Genética , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Micélio/genética , Micélio/virologia , Filogenia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA de Transferência/genética , Virulência
9.
BMC Genomics ; 19(1): 705, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30253736

RESUMO

BACKGROUND: Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. RESULTS: We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620-6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116-175 genes encoding CSEPs with limited gene amplification. CONCLUSIONS: Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.


Assuntos
Ascomicetos/genética , Genoma Fúngico , Especificidade de Hospedeiro/genética , Doenças das Plantas/microbiologia , Adaptação Fisiológica , Ascomicetos/classificação , Ascomicetos/patogenicidade , Deleção de Genes , Perfilação da Expressão Gênica , Genes Fúngicos , Tamanho do Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Micélio/genética , Micélio/metabolismo , Técnicas de Tipagem Micológica , Poaceae/microbiologia
10.
Int J Mol Sci ; 19(8)2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103374

RESUMO

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.


Assuntos
Farmacorresistência Fúngica/fisiologia , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Micélio/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismo , Receptores Sulfonilureia/metabolismo , Triticum/microbiologia , Antifúngicos/farmacologia , Arabidopsis/microbiologia , Proteínas Fúngicas/genética , Fusarium/genética , Micélio/genética , Receptores Sulfonilureia/genética
11.
Appl Microbiol Biotechnol ; 102(18): 7997-8009, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29959464

RESUMO

Morels are some of the most highly prized edible and medicinal mushrooms, and the outdoor cultivation has been achieved in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and the number of sclerotia indicates the spawn quality during cultivation. However, the sclerotial formation and differentiation mechanisms are poorly understood. In this study, the sclerotial formation process of Morchella importuna and the effects of reactive oxygen species on scerotial formation were studied. Scerotial formation was defined as five distinctive phases, hypha early, hyphal growth, sclerotial initiation, development, and maturation. The mycelia in the sclerotium-forming area were swollen, darkened, and dense with sclerotial formation, but hydrogen peroxide accumulated in the region lacking sclerotial formation. The expression of all six genes for superoxide dismutases tested increased with sclerotial maturation. A difference in hydrogen peroxide concentration of 20 mM could promote the sclerotial initiation and induce expression of sod genes. The MAPK signaling pathway was activated, and they passed the signal from an area of high oxidative stress to a low area to initiate sclerotial formation. An understanding of the sclerotial formation mechanisms in M. importuna may help to understand the life cycle and facilitate the fruiting body cultivation.


Assuntos
Ascomicetos/metabolismo , Hifas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , China , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hifas/genética , Hifas/metabolismo , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Estresse Oxidativo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
12.
Int J Med Mushrooms ; 20(7): 607-621, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30055553

RESUMO

The genus Pleurotus is the third most commonly produced edible fungi in the world. In addition, species of genus Pleurotus have functional properties such as anticancer, antiviral, antimicrobial, anti-inflammatory, and antioxidant activities, which are mainly attributed to phenolic compounds. For these reasons, this study evaluated the productivity and antioxidant activity (AA) of 2 wild strains (white and pink), 2 reconstituted strains (called "BB" and "RR"), and 4 hybrid strains (H1, H2, H3, and H4) of P. djamor from monokaryotic components (neohaplonts). The results showed that the white wild-type strain and the reconstituted strains exhibited the best production potential, expressed as biological efficiency and mycelial growth rate. The carpophores of hybrid strains H1 and H3 had the greatest AA, as evaluated with DPPH radical scavenging and reducing power assays, respectively. The H3 strain had the highest total phenol (TP) content. Pearson correlations led us to conclude that the mycelial growth rate has a regular inverse correlation with TP and a regular direct correlation with AA of methanolic extracts from carpophores and myce-lia. This is, to our knowledge, the first report in the literature about the effect of Pleurotus strain hybridization through a chemical de-dikaryotization process on TP content.


Assuntos
Anti-Infecciosos/química , Antioxidantes/química , Fenóis/química , Extratos Vegetais/química , Pleurotus/química , Verduras/química , Anti-Infecciosos/isolamento & purificação , Antioxidantes/isolamento & purificação , Quimera/genética , Quimera/crescimento & desenvolvimento , México , Micélio/química , Micélio/genética , Micélio/crescimento & desenvolvimento , Fenóis/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Verduras/genética , Verduras/crescimento & desenvolvimento
13.
Appl Microbiol Biotechnol ; 102(18): 7849-7863, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30032435

RESUMO

Laccase production and pellet formation of transformants of Coprinopsis cinerea strain FA2222 of C. cinerea laccase gene lcc1 subcloned behind the gpdII-promoter from Agaricus bisporus were compared with a control transformant carrying no extra laccase gene. At the optimum growth temperature of 37 °C, maximal laccase yields of 2.9 U/ml were obtained by the best lcc1 transformant pYSK7-26 in liquid shake flask cultures. Reduction in temperature to 25 °C increased laccase yields up to 9.2 U/ml. The control transformant had no laccase activities at 37 °C but native activity at 25 °C (3.5 U/ml). Changing the temperature had severe effects on the morphology of the mycelial pellets formed during cultivation, but links of distinct pellet morphologies to native or recombinant laccase production could not be established. Automated image analysis was used to characterise pellet formation and morphological parameters (pellet area, diameter, convexity and mycelial structure). Cross sections of selected pellets showed that they differentiated in an outer rind and an inner medulla of loosened hyphae. Pellets at 25 °C had a small and dense outer zone and adopted with time a smooth surface. Pellets at 37 °C had a broader outer zone and a fringy surface due to generation of more and larger protuberances in the rind that when released can serve for production of further pellets.


Assuntos
Agaricales/enzimologia , Agaricales/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Agaricales/genética , Técnicas de Cultura Celular por Lotes , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Micélio/enzimologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Temperatura Ambiente
14.
Int J Mol Sci ; 19(7)2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29932128

RESUMO

Alternaria alternata (Fries) Keissler is a lethal pear pathogen that causes leaf black spot disease of pear in Southern China. Heat-stable activity factor (HSAF) is a polycyclic tetramate macrolactam (PTM) produced by Lysobacter enzymogenes and many other microbes with a broad-spectrum antifungal activity against many filamentous fungi. In this study, we evaluated the antifungal effect of HSAF against A. alternata and proposed its antifungal mechanism in A. alternata. We report that HSAF inhibited the mycelial growth of A. alternata in a dose-dependent manner. Transcriptomics analysis revealed that HSAF treatment resulted in an expression alteration of a wide range of genes, with 3729 genes being up-regulated, and 3640 genes being down-regulated. Furthermore, we observed that HSAF treatment disrupted multiple signaling networks and essential cellular metabolisms in A. alternata, including the AMPK signaling pathway, sphingolipid metabolism and signaling pathway, carbon metabolism and the TCA (tricarboxylic acid) cycle, cell cycle, nitrogen metabolism, cell wall synthesis and a key hub protein phosphatase 2A (PP2A). These observations suggest that HSAF breaches metabolism networks and ultimately induces increased thickness of the cell wall and apoptosis in A. alternata. The improved understanding of the antifungal mechanism of HSAF against filamentous fungi will aid in the future identification of the direct interaction target of HSAF and development of HSAF as a novel bio-fungicide.


Assuntos
Alternaria/genética , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica , Lactamas Macrocíclicas/metabolismo , Alternaria/efeitos dos fármacos , Alternaria/fisiologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/microbiologia , Ontologia Genética , Lactamas Macrocíclicas/farmacologia , Lysobacter/metabolismo , Micélio/efeitos dos fármacos , Micélio/genética , Micélio/fisiologia , Doenças das Plantas/microbiologia , Pyrus/microbiologia
15.
J Basic Microbiol ; 58(8): 698-703, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29873407

RESUMO

To understand molecular mechanism of cold-induced fruiting in Flammulina velutipes, which is one of most popular edible fungi in east Asia, de novo assembly of the F. velutipes transcriptome was carried out. There were 26,888,494 and 26,275,146 clean reads obtained from mycelium and primordia of F. velutipes, respectively. A total of 20,157 unigenes were de novo assembled and 15,058 of them were annotated. Moreover, 7935 unigenes were differentially expressed between mycelium and primordia, 4025 of them were up-regulated and 3910 were down-regulated. GO and KEGG pathway analysis of the differentially expressed unigenes indicated that functional groups associated with two-component signaling pathway, calcium signaling, mitogen-actived protein kinase pathway, molecular chaperones, cell wall and membrane system, play an important role in cold-induced fruiting of F. velutipes. In this work 643 EST-SSRs were identified in 20,157 unigenes and 1560 EST-SSRs primers pairs were designed. Moreover, 5548 and 5955 SNPs were detected in mycelium and primordia, respectively. Consequently, results of this work can serve as a valuable resource for functional genomics study of cold-induced fruiting in F. velutipes.


Assuntos
Temperatura Baixa , Flammulina/genética , Carpóforos/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Transcriptoma , Etiquetas de Sequências Expressas , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Repetições de Microssatélites , Anotação de Sequência Molecular , Micélio/genética , Micélio/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Transdução de Sinais/genética
16.
Gene ; 666: 108-115, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-29738838

RESUMO

BACKGROUND: Hericium erinaceus, a famous edible mushroom, is also a well-known traditional medicinal fungus. To date, a large number of bioactive metabolites with antitumor, antibacterial, and immune-boosting effects were isolated from the free-living mycelium and fruiting body of H. erinaceus. OBJECTIVE: Here we used the proteomic approach to explore proteins involved in the regulation of bioactive metabolites, including terpenoid, polyketide, sterol and etc. RESULTS: Using mass spectrometry, a total of 2543 unique proteins were identified using H. erinaceus genome, of which 2449, 1855, 1533 and 690 proteins were successfully annotated in Nr, KOG, KEGG and GO databases. Among them, 722 proteins were differentially expressed (528 up- and 194 down-regulated) in fruiting body compared with mycelium. Most of differentially expressed proteins were putatively involved in energy metabolism, molecular signaling, and secondary metabolism. Additionally, numerous proteins involved in terpenoid, polyketide, and sterol biosynthesis were identified. Our data revealed that proteins involved in polyketide biosynthesis were up-regulated in the fruiting body, while some proteins in mevalonate (MEP) pathway from terpenoid biosynthesis were generally up-regulated in mycelium. CONCLUSIONS: The present study suggested that the differential regulation of biosynthesis genes could produce various bioactive metabolites with pharmacological effects in H. erinaceus.


Assuntos
Agaricales/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Agaricales/genética , Carpóforos/genética , Carpóforos/metabolismo , Proteínas Fúngicas/genética , Expressão Gênica , Redes e Vias Metabólicas , Micélio/genética , Micélio/metabolismo , Proteoma/genética
17.
Bioprocess Biosyst Eng ; 41(7): 1029-1038, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29610994

RESUMO

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.


Assuntos
Aspergillus niger , Quitina Sintase/genética , Ácido Cítrico/metabolismo , Proteínas Fúngicas/genética , Inativação Gênica , Aspergillus niger/genética , Aspergillus niger/metabolismo , Quitina Sintase/metabolismo , Proteínas Fúngicas/metabolismo , Micélio/genética , Micélio/metabolismo
18.
J Agric Food Chem ; 66(14): 3716-3725, 2018 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29584419

RESUMO

In some industrial processes, cold and light stresses are recognized as two important environmental triggers for the transformation of mycelia into fruit-bodies via intermediate primordia in Flammulina velutipes cultivation. To gain insights into the mechanism of regulation of F. velutipes mycelia in response to cold and light stress, proteins expressed abundantly and characteristically at particular stress states were investigated by using the isobaric tags for the relative and absolute quantitation labeling technique. Among the 1046 nonredundant proteins identified with a high degree of confidence, 264 proteins, which were detected as differentially expressed proteins, were associated with 176 specific KEGG pathways. In-depth data analysis revealed that the regulatory network underlying the cold and light response mechanisms of F. velutipes mycelia was complex and multifaceted, as it included varied functions such as rapid energy supply, the biosynthesis of lysine, phenylalanine, tyrosine, and γ-aminobutyric acid, the calcium signal transduction process, dynein-dependent actin and microtubule cytoskeleton formation, autolysis, oxidative stress adaptation, pigment secretion, tissue and organ morphogenesis, and other interesting stress-related processes. Insights into the proteins might shed light on an intuitive understanding of the cold and light stress response mechanism underlying the fruiting processes of F. velutipes. Furthermore, the data might also provide further insights into the stress response mechanism of macro-fungi and valuable information for scientific improvement of some mushroom cultivation techniques in practice.


Assuntos
Flammulina/efeitos da radiação , Proteínas Fúngicas/química , Micélio/metabolismo , Proteoma/química , Proteômica/métodos , Cromatografia Líquida de Alta Pressão , Flammulina/química , Flammulina/genética , Flammulina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Luz , Espectrometria de Massas , Micélio/química , Micélio/genética , Micélio/efeitos da radiação , Proteoma/genética , Proteoma/metabolismo , Temperatura Ambiente
19.
Microbiol Res ; 207: 256-268, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29458862

RESUMO

To explore the number of enzymes engaged by Cerrena unicolor FCL139 for wood degradation, the transcriptomes of the fungus growing on birch, ash, maple sawdust and the control liquid medium were analyzed. Among 12,966 gene models predicted for the C. unicolor genome, 10,396 all-unigenes were detected, of which 9567 were found to be expressed in each of the tested growth media. The highest number (107) of unique transcripts was detected during fungus growth in the control liquid medium, while the lowest number (11) - in the fungal culture comprising maple saw dust. Analysis of C. unicolor transcriptomes identified numerous genes whose expression differed substantially between the mycelia growing in control medium and each of the sawdust media used, with the highest number (828) of upregulated transcripts observed during the fungus growth on the ash medium. Among the 294 genes that were potentially engaged in wood degradation, the expression of 59 was significantly (p < .01) changed in the tested conditions. The transcripts of 37 of those genes were at least four times more abundant in the cells grown in all sawdust media when compared to the control medium. Upregulated genes coding for cellulases and, to a lower extent, hemicellulases predominated during fungus growth on sawdust. Transcripts encoding cellulolytic enzymes were the most abundant in mycelia grown on birch and maple while lower number of such transcripts was detected in fungus growing on ash. The expression pattern of lignolytic activities-coding genes was strongly dependent on the type of sawdust applied for fungus growth medium.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Polyporales/genética , Madeira/metabolismo , Madeira/microbiologia , Celulases/genética , Proteínas Fúngicas/biossíntese , Perfilação da Expressão Gênica , Micélio/genética
20.
Braz J Microbiol ; 49(3): 632-640, 2018 Jul - Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29482997

RESUMO

The present study conducted a genetic characterization and determined growth rate and biomass production in solid and liquid media, using strains obtained from wild edible sporomes of Lyophyllum that grow in high mountains. Vegetative isolation was used to obtain a total of four strains, which were divided into two clades within the section Difformia: Lyophyllum sp. and Lyophyllum aff. shimeji. Growth rate and biomass production were influenced by both the culture media and the strains. In a potato dextrose agar medium, the strains presented a higher growth rate, while in a malt extract-peptone and yeast agar medium, the growth rate was lower, but with a higher biomass production that was equal to that in the malt extract-peptone and yeast liquid medium.


Assuntos
Agaricales/crescimento & desenvolvimento , Agaricales/genética , Agaricales/química , Agaricales/metabolismo , Biomassa , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Cinética , México , Micélio/química , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo
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