RESUMEN
Cap expansion in agaricoid mushroom species is an important event for sexual reproduction because meiosis occurs in basidia under the cap, and basidiospores can be released by opening the cap. However, molecular mechanisms underlying cap expansion in basidiomycetes remain poorly understood. We aimed to elucidate the molecular mechanisms of cap expansion in basidiomycetes by analyzing the unique cap-expansionless UV mutant #13 (exp2-1) in Coprinopsis cinerea. Linkage analysis and consequent genome sequence analysis revealed that the gene responsible for the mutant phenotypes encodes a putative transcription factor with two C2H2 zinc finger motifs. The mutant that was genome-edited to lack exp2 exhibited an expansionless phenotype. Some of the genes encoding cell wall degradation-related enzymes showed decreased expression during cap expansion and autolysis in the exp2 UV and genome-edited mutant. The exp2 gene is widely conserved in Agaricomycetes, suggesting that Exp2 homologs regulate fruiting body maturation in Agaricomycetes, especially cap expansion in Agaricoid-type mushroom-forming fungi. Therefore, exp2 homologs could be a target for mushroom breeding to maintain shape after harvest for some cultivating mushrooms, presenting a promising avenue for further research in breeding techniques.
Asunto(s)
Agaricales , Basidiomycota , Cuerpos Fructíferos de los Hongos/genética , Agaricales/genética , Dedos de Zinc/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.
Asunto(s)
Arabidopsis , Oryza , Celulosa , Cisteína , Proteínas Fúngicas/genética , Oryza/genética , XilanosRESUMEN
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.
Asunto(s)
Quitina Sintasa/genética , Vectores Genéticos/genética , Monofenol Monooxigenasa/genética , Regiones Promotoras Genéticas/genética , Hongos Shiitake/genética , Transformación Genética , Cuerpos Fructíferos de los Hongos/genética , Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Cuerpos Fructíferos de los Hongos/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Silenciador del Gen/fisiología , Monofenol Monooxigenasa/metabolismo , Micelio/genética , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Organismos Modificados Genéticamente , Hongos Shiitake/enzimología , Hongos Shiitake/crecimiento & desarrollo , Transformación Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
Euglena produces paramylon as a storage polysaccharide, and is thought to require ß-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate ß-1,3-glucan degradation in Euglena, endo-1,3-ß-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS-PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards ß-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0-5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble ß-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-ß-glucanase to be identified from E. gracilis.
Asunto(s)
Euglena gracilis/enzimología , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Euglena gracilis/química , Euglena gracilis/genética , Glucano Endo-1,3-beta-D-Glucosidasa/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Peso Molecular , beta-Glucanos/análisisRESUMEN
BACKGROUND: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that ß-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular ß-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose. RESULTS: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked ß-1,4, ß-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7. CONCLUSION: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.
RESUMEN
MAIN CONCLUSION: Physical properties of wheat coleoptile segments decreased after treatment with hemicellulose-degrading enzymes, indicating that hemicellulosic polysaccharides function to control the strength of primary cell walls. Changes in the physical properties of plant cell walls, a viscoelastic structure, are thought to be one of the growth-limiting factors for plants and one of the infection-affecting factors for fungi. To study the significance of hemicellulosic polysaccharides that form cross-bridges between cellulose microfibrils in controlling cell wall strength in monocot plants, the effects of hemicellulose degradation by recombinant Magnaporthe oryzae xylanase and 1,3-1,4-ß-glucanase, and recombinant Aspergillus oryzae xyloglucanase on the physical properties and polysaccharide solubilization were investigated using wheat (Triticum aestivum L.) coleoptiles. Treatments with xylanase or 1,3-1,4-ß-glucanase significantly decreased the viscosity and elasticity of wheat coleoptile segments. In addition, xyloglucanase treatment slightly decreased the viscoelasticity. Furthermore, 1,3-1,4-ß-glucan polymer was solubilized during hydrolysis with xylanase and xyloglucanase, even though neither enzyme had hydrolytic activity towards 1,3-1,4-ß-glucan. These results suggest that xylan and xyloglucan interact with 1,3-1,4-ß-glucan and that the composites and hemicellulosic polysaccharides form inter-molecular bridges. Degradation of these bridges causes decreases in the physical properties, resulting in increased extensibility of the cell walls. These findings provide a testable model in which wheat coleoptile cell walls are loosened by the degradation of hemicellulosic polysaccharides and hemicellulose-degrading enzymes play a significant role in loosening the walls during fungal infection.
Asunto(s)
Pared Celular/metabolismo , Glicósido Hidrolasas/metabolismo , Polisacáridos/metabolismo , Aspergillus oryzae/enzimología , Aspergillus oryzae/metabolismo , Glucanos/metabolismo , Magnaporthe/enzimología , Magnaporthe/metabolismo , Xilanos/metabolismoRESUMEN
Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-ß-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-ß-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-ß-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.
Asunto(s)
Celulasa/química , Celulasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Polisacáridos/metabolismo , Hongos Shiitake/enzimología , Xilanos/metabolismo , Biocatálisis , Celulasa/genética , Proteínas Fúngicas/genética , Glucanos/química , Hongos Shiitake/química , Hongos Shiitake/genética , Especificidad por Sustrato , Xilanos/químicaRESUMEN
OBJECT: In this paper, the authors' goal was to report their novel presurgical simulation method applying interactive virtual simulation (IVS) using 3D computer graphics (CG) data and microscopic observation of color-printed plaster models based on these CG data in surgery for skull base and deep tumors. METHODS: For 25 operations in 23 patients with skull base or deep intracranial tumors (meningiomas, schwannomas, epidermoid tumors, chordomas, and others), the authors carried out presurgical simulation based on 3D CG data created by image analysis for radiological data. Interactive virtual simulation was performed by modifying the 3D CG data to imitate various surgical procedures, such as bone drilling, brain retraction, and tumor removal, with manipulation of a haptic device. The authors also produced color-printed plaster models of modified 3D CG data by a selective laser sintering method and observed them under the operative microscope. RESULTS: In all patients, IVS provided detailed and realistic surgical perspectives of sufficient quality, thereby allowing surgeons to determine an appropriate and feasible surgical approach. Surgeons agreed that in 44% of the 25 operations IVS showed high utility (as indicated by a rating of "prominent") in comprehending 3D microsurgical anatomies for which reconstruction using only 2D images was complicated. Microscopic observation of color-printed plaster models in 12 patients provided further utility in confirming realistic surgical anatomies. CONCLUSIONS: The authors' presurgical simulation method applying advanced 3D imaging and modeling techniques provided a realistic environment for practicing microsurgical procedures virtually and enabled the authors to ascertain complex microsurgical anatomy, to determine the optimal surgical strategies, and also to efficiently educate neurosurgical trainees, especially during surgery for skull base and deep tumors.
Asunto(s)
Simulación por Computador , Imagenología Tridimensional/métodos , Neoplasias Meníngeas/cirugía , Meningioma/cirugía , Cuidados Preoperatorios/métodos , Neoplasias de la Base del Cráneo/cirugía , Adolescente , Adulto , Anciano , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Cordoma/diagnóstico por imagen , Cordoma/patología , Cordoma/cirugía , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Neoplasias Meníngeas/diagnóstico por imagen , Neoplasias Meníngeas/patología , Meningioma/diagnóstico por imagen , Meningioma/patología , Persona de Mediana Edad , Modelos Anatómicos , Neurilemoma/diagnóstico por imagen , Neurilemoma/patología , Neurilemoma/cirugía , Neoplasias de la Base del Cráneo/diagnóstico por imagen , Neoplasias de la Base del Cráneo/patología , Programas Informáticos , Tomografía Computarizada por Rayos X , Interfaz Usuario-Computador , Adulto JovenRESUMEN
BACKGROUND: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. RESULTS: A novel endotransglucosylase catalyzes cleavage and transfer of ß-glucans and decreases the physical strength of plant cell walls. CONCLUSION: Endotransglucosylation causes depolymerization and polymerization of ß-glucans, depending on substrate molecular size. SIGNIFICANCE: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-ß-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing ß-1,4-linkages, in which 1,3-1,4-ß-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into ß-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of ß-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-ß-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize ß-glucans, depending on the size of the acceptor substrate.
Asunto(s)
Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Magnaporthe/enzimología , beta-Glucanos/metabolismo , Conformación de Carbohidratos , Pared Celular/química , Celulosa/biosíntesis , Clonación Molecular , Cotiledón/química , Cotiledón/citología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hidrólisis , Oligosacáridos/química , Oryza/microbiología , Hojas de la Planta/microbiología , Especificidad por Sustrato , Transcripción Genética , Triticum/química , Triticum/citologíaRESUMEN
A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative ß-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 ß-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward ß-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify ß-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of ß-1,3-glucan after its release in the extracellular medium.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Ustilago/enzimología , Ustilago/metabolismo , beta-Glucanos/metabolismo , Aspergillus oryzae/genética , Cromatografía , Cartilla de ADN/genética , Expresión Génica , Hidrólisis , Espectrometría de Masas , Reacción en Cadena de la Polimerasa , ProteoglicanosRESUMEN
Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 ß-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 ß-glucosidase, a gene encoding a putative GH3 ß-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 ß-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from ß-1,3-, ß-1,4-, and ß-1,6-linked oligosaccharides, and from 1,3-1,4-ß-glucan and laminarin polysaccharides, indicating that UeBgl3A is a ß-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.
Asunto(s)
Ustilago/enzimología , beta-Glucosidasa/metabolismo , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Glucanos , Peso Molecular , Filogenia , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masas en Tándem , Ustilago/genética , beta-Glucosidasa/química , beta-Glucosidasa/genéticaRESUMEN
ß-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0-5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on ß-1,3-linked oligosaccharides than on ß-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-ß-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-ß-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-ß-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.
Asunto(s)
Glucosidasas/metabolismo , Magnaporthe/enzimología , beta-Glucanos/metabolismo , Estabilidad de Enzimas , Glucosidasas/química , Concentración de Iones de Hidrógeno , Hidrólisis , Especificidad por Sustrato , TemperaturaRESUMEN
We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.
Asunto(s)
Celulasa/genética , Celulasa/metabolismo , Proteínas Fúngicas/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Magnaporthe/enzimología , beta-Glucanos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Brevibacillus/enzimología , Brevibacillus/genética , Pared Celular/metabolismo , Celulasa/química , Clonación Molecular , ADN de Hongos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Glicósido Hidrolasas/química , Hidrolasas/genética , Magnaporthe/genética , Magnaporthe/metabolismo , Polisacáridos/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Transducción de Señal , Especificidad por Sustrato , beta-Glucanos/químicaRESUMEN
Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), ß-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Magnaporthe/enzimología , Celobiosa/metabolismo , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/química , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Cromatografía de Afinidad , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Oligosacáridos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , beta-Glucanos/metabolismoRESUMEN
The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.
Asunto(s)
Conservación de Alimentos , Cuerpos Fructíferos de los Hongos/enzimología , Regulación Fúngica de la Expresión Génica , Monofenol Monooxigenasa/genética , Hongos Shiitake/enzimología , Hongos Shiitake/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cuerpos Fructíferos de los Hongos/genética , Datos de Secuencia Molecular , Monofenol Monooxigenasa/química , Hongos Shiitake/citologíaRESUMEN
Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited beta-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.