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1.
Gene ; 763: 145061, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32818595

RESUMO

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.


Assuntos
Cordyceps/genética , Carpóforos/genética , Transcriptoma , Cordyceps/crescimento & desenvolvimento , Cordyceps/metabolismo , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micélio/genética , Micélio/crescimento & desenvolvimento , Feromônios/metabolismo
2.
Gene ; 743: 144563, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32165290

RESUMO

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.


Assuntos
Cordyceps/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Termotolerância/genética , Temperatura Baixa/efeitos adversos , Cordyceps/crescimento & desenvolvimento , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Duplicação Gênica , Genes Fúngicos/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Filogenia
3.
Virol J ; 16(1): 118, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31623644

RESUMO

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.


Assuntos
Basidiomycota/virologia , Micélio/patogenicidade , Pinus/microbiologia , Doenças das Plantas/microbiologia , RNA de Cadeia Dupla/fisiologia , Totiviridae/fisiologia , Basidiomycota/genética , Basidiomycota/crescimento & desenvolvimento , Basidiomycota/patogenicidade , Genoma Viral/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/virologia , Filogenia , Pinus/classificação , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/genética , RNA Viral/genética , Totiviridae/classificação , Totiviridae/genética , Transcrição Genética , Proteínas Virais/genética , Virulência
4.
Appl Microbiol Biotechnol ; 103(21-22): 8923-8935, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520132

RESUMO

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65 µg L-1) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.


Assuntos
Alternaria/metabolismo , Antineoplásicos/metabolismo , Aspergillus fumigatus/metabolismo , Paclitaxel/biossíntese , Alginatos/química , Alternaria/química , Alternaria/genética , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Fermentação , Microbiologia Industrial , Micélio/química , Micélio/genética , Micélio/metabolismo
5.
Genes (Basel) ; 10(9)2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514481

RESUMO

Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30-60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes-including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases-were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.


Assuntos
Micélio/genética , Pleurotus/genética , Transcriptoma , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Micélio/crescimento & desenvolvimento , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Pleurotus/crescimento & desenvolvimento
6.
Folia Microbiol (Praha) ; 64(6): 835-844, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31372834

RESUMO

Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2 mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5 mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8 µmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.


Assuntos
Cádmio/metabolismo , Proteínas Fúngicas/metabolismo , Hebeloma/metabolismo , Metalotioneína/metabolismo , Zinco/metabolismo , Basidiomycota/genética , Vesículas Citoplasmáticas/metabolismo , Proteínas Fúngicas/genética , Hebeloma/genética , Hebeloma/crescimento & desenvolvimento , Metalotioneína/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Micorrizas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
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
8.
Am J Trop Med Hyg ; 101(3): 716-723, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31287042

RESUMO

This article describes, for the first time, the role of the nasal mucosa (NM) as the initial site for the Histoplasma capsulatum mycelial-to-yeast transition. The results highlight that yeasts may arrive to the cervical lymph nodes (CLN) via phagocytes. Bats and mice were intranasally infected with H. capsulatum mycelial propagules and they were killed 10, 20, and 40 minutes and 1, 2, and 3 hours after infection. The NM and the CLN were monitored for fungal presence. Yeasts compatible with H. capsulatum were detected within the NM and the CLN dendritic cells (DCs) 2-3 hours postinfection, using immunohistochemistry. Histoplasma capsulatum was re-isolated by culturing at 28°C from the CLN of both mammalian hosts 2-3 hours postinfection. Reverse transcription-polymerase chain reaction assays were designed to identify fungal dimorphism, using mycelial-specific (MS8) and yeast-specific (YPS3) gene expression. This strategy supported fast fungal dimorphism in vivo, which began in the NM 1 hour postinfection (a time point when MS8 and YPS3 genes were expressed) and it was completed at 3 hours (a time point when only the YPS3 transcripts were detected) in both bats and mice. The presence of intracellular yeasts in the nasal-associated lymphoid tissue (NALT), in the NM nonassociated with the NALT, and within the interdigitating DCs of the CLN suggests early fungal dissemination via the lymph vessels.


Assuntos
Adaptação Fisiológica , Quirópteros/microbiologia , Histoplasma/fisiologia , Micélio/fisiologia , Mucosa Nasal/microbiologia , Animais , Células Dendríticas/microbiologia , Feminino , Histoplasma/genética , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Micélio/genética , Fagocitose , Infecções Respiratórias/microbiologia
9.
Genes (Basel) ; 10(6)2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248134

RESUMO

The establishment of genetic transformation method is crucial for the functional genomics research in filamentous fungi. Although the transformation method has been developed in several types of fungi, a highly efficient and convenient transformation system is desperately needed in Lentinula edodes. Present work established the Agrobacterium-mediated transformation (ATMT) of basidiomycete L. edodes in both monokaryon and dikaryon mycelia by using constructed binary plasmid pCAMBIA-1300-GFP. Then, the transformation efficiency of ATMT was evaluated by using different mediums for recipient incubation and different varieties of L. edodes. The results showed that in dikaryon strain W1, the positive hygromycin-resistant transformants was observed in all medium with the positive frequency of selected transformants that ranged from 0 to 30%. While in the monokaryon strain W1-26, only the millet medium group obtained positive transformants with a positive frequency of 75.48%. Moreover, three dikaryotic wild strains (YS55, YS3334, and YS3357) and two dikaryotic cultivated strains (W1 and S606) showed the highest transformation efficiency, with 32.96% of the germination frequency, and 85.12% of positive frequency for hygromycin-resistant transformants. This work demonstrated that Agrobacterium-mediated transformation was successfully performed in L. edodes, and the genotype of recipients as well as the medium for mycelial incubation were suggested to play key roles in determining the transformation efficiency. These findings may provide new avenues for the genetic modification of edible mushroom and may extend the cognition of DNA-mediated transformation in filamentous fungi.


Assuntos
Agaricales/genética , Agrobacterium tumefaciens/genética , Cogumelos Shiitake/genética , Transformação Genética , Basidiomycota/genética , Patrimônio Genético , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Micélio/genética , Plasmídeos/genética
10.
PLoS One ; 14(5): e0216161, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31136583

RESUMO

Understanding the epidemiology of infectious diseases in a host population is a major challenge in forestry. Radiata pine plantations in New Zealand are impacted by a foliar disease, red needle cast (RNC), caused by Phytophthora pluvialis. This pathogen is dispersed by water splash with polycyclic infection affecting the lower part of the tree canopy. In this study, we extended an SI (Susceptible-Infectious) model presented for RNC to analyse the key epidemiological drivers. We conducted two experiments to empirically fit the extended model: a detached-needle assay and an in vivo inoculation. We used the detached-needle assay data to compare resistant and susceptible genotypes, and the in vivo inoculation data was used to inform sustained infection of the whole plant. We also compared isolations and real-time quantitative PCR (qPCR) to assess P. pluvialis infection. The primary infection rate and the incubation time were similar for susceptible and resistant genotypes. The pathogen death rate was 2.5 times higher for resistant than susceptible genotypes. Further, external proliferation of mycelium and sporangia were only observed on 28% of the resistant ramets compared to 90% of the susceptible ones. Detection methods were the single most important factor influencing parameter estimates of the model, giving qualitatively different epidemic outputs. In the early stages of infection, qPCR proved to be more efficient than isolations but the reverse was true at later points in time. Isolations were not influenced by the presence of lesions in the needles, while 19% of lesioned needle maximized qPCR detection. A primary infection peak identified via qPCR occurred at 4 days after inoculation (dai) with a secondary peak observed 22 dai. Our results have important implications to the management of RNC, by highlighting the main differences in the response of susceptible and resistant genotypes, and comparing the most common assessment methods to detect RNC epidemics.


Assuntos
Phytophthora/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Epidemias , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Micélio/genética , Agulhas , Nova Zelândia , Pinus/genética , Pinus/parasitologia , Esporângios/genética
11.
Genes (Basel) ; 10(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832255

RESUMO

Gloeostereum incarnatum is a precious edible mushroom that is widely grown in Asia and known for its useful medicinal properties. Here, we present a high-quality genome of G. incarnatum using the single-molecule real-time (SMRT) sequencing platform. The G. incarnatum genome, which is the first complete genome to be sequenced in the family Cyphellaceae, was 38.67 Mbp, with an N50 of 3.5 Mbp, encoding 15,251 proteins. Based on our phylogenetic analysis, the Cyphellaceae diverged ~174 million years ago. Several genes and gene clusters associated with lignocellulose degradation, secondary metabolites, and polysaccharide biosynthesis were identified in G. incarnatum, and compared with other medicinal mushrooms. In particular, we identified two terpenoid-associated gene clusters, each containing a gene encoding a sesterterpenoid synthase adjacent to a gene encoding a cytochrome P450 enzyme. These clusters might participate in the biosynthesis of incarnal, a known bioactive sesterterpenoid produced by G. incarnatum. Through a transcriptomic analysis comparing the G. incarnatum mycelium and fruiting body, we also demonstrated that the genes associated with terpenoid biosynthesis were generally upregulated in the mycelium, while those associated with polysaccharide biosynthesis were generally upregulated in the fruiting body. This study provides insights into the genetic basis of the medicinal properties of G. incarnatum, laying a framework for future characterization of bioactive proteins and pharmaceutical uses of this fungus.


Assuntos
Agaricales/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento Completo do Genoma/métodos , Agaricales/classificação , Carpóforos/genética , Regulação Fúngica da Expressão Gênica , Tamanho do Genoma , Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala , Família Multigênica , Micélio/genética , Filogenia , Imagem Individual de Molécula
12.
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
13.
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
14.
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
15.
Sci Justice ; 59(1): 102-108, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30654963

RESUMO

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1 µm and width 6.4 µm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.


Assuntos
Alucinógenos/análise , Micélio/genética , Psilocybe/classificação , Psilocibina/análogos & derivados , Chile , DNA Fúngico/análise , Tráfico de Drogas , Genética Forense , Cromatografia Gasosa-Espectrometria de Massas , Microscopia Eletrônica de Varredura , Psilocibina/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de RNA , Esporos/genética
16.
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
17.
Genomics ; 111(1): 50-58, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29288711

RESUMO

Heimuer, Auricularia heimuer, is one of the most famous traditional Chinese foods and medicines, and it is the third most important cultivated mushroom worldwide. The aim of this study is to develop genomic resources for A. heimuer to furnish tools that can be used to study its secondary metabolite production capability, wood degradation ability and biosynthesis of polysaccharides. The genome was obtained from single spore mycelia of the strain Dai 13782 by using combined high-throughput Illumina HiSeq 4000 system with the PacBio RSII long-read sequencing platform. Functional annotation was accomplished by blasting protein sequences with different public available databases to obtain their corresponding annotations. It is 49.76Mb in size with a N50 scaffold size of 1,350,668bp and encodes 16,244 putative predicted genes. This is the first genome-scale assembly and annotation for A. heimuer, which is the third sequenced species in Auricularia.


Assuntos
Agaricales/genética , Basidiomycota/genética , Genoma Fúngico , Análise de Sequência de DNA , Sequenciamento Completo do Genoma , Basidiomycota/metabolismo , Bases de Dados de Proteínas , Carpóforos/genética , Micélio/genética , Polissacarídeos/biossíntese , Metabolismo Secundário , Esporos/genética , Sequências de Repetição em Tandem
18.
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
19.
Folia Microbiol (Praha) ; 64(1): 33-39, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29938299

RESUMO

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.


Assuntos
Acremonium/genética , Eletroporação/métodos , Transformação Genética , Acremonium/efeitos dos fármacos , Acremonium/metabolismo , Cefalosporinas/biossíntese , Farmacorresistência Bacteriana , Viabilidade Microbiana , Micélio/efeitos dos fármacos , Micélio/genética , Micélio/metabolismo , Fleomicinas/farmacologia , Protoplastos/fisiologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
20.
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
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