RESUMO
The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, ß-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including ß-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.
Assuntos
Aureobasidium/metabolismo , Agentes de Controle Biológico/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Sequência de Aminoácidos , Antibiose , Aureobasidium/fisiologia , Agentes de Controle Biológico/química , Agentes de Controle Biológico/metabolismo , Agentes de Controle Biológico/farmacologia , Quitinases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Fungicidas Industriais/química , Fungicidas Industriais/isolamento & purificação , Fungicidas Industriais/metabolismo , Fungicidas Industriais/farmacologia , Geotrichum/efeitos dos fármacos , Glucana 1,3-beta-Glucosidase/metabolismo , Penicillium/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , ProteóliseRESUMO
The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.
Assuntos
Glucana 1,3-beta-Glucosidase/química , Glicosídeo Hidrolases/química , beta-Glucanas/química , Sequência de Aminoácidos/genética , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Glucana 1,3-beta-Glucosidase/metabolismo , Glucanos/química , Glicosídeos/química , Modelos Moleculares , Especificidade por Substrato/fisiologiaRESUMO
Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.
Assuntos
Sordariales/enzimologia , Glucana 1,3-beta-Glucosidase/química , Temperatura , Estabilidade Enzimática , Celulases , Glucana 1,3-beta-Glucosidase/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas em Tandem , Ensaios Enzimáticos , Concentração de Íons de HidrogênioRESUMO
Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes ß-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5â¯min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5â¯min, followed by incubation with PDZ for 20â¯min and exposure to LED light (660â¯nm, 50â¯J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZâ¯+â¯DNase, light only, lightâ¯+â¯DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (pâ¯=â¯0.000) and eDNA amounts (pâ¯≤â¯0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT.
Assuntos
Candida albicans/efeitos dos fármacos , Desoxirribonucleases/farmacologia , Glucana 1,3-beta-Glucosidase/farmacologia , Glucosamina/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Biofilmes , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Glucosamina/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Fatores de TempoRESUMO
Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and ß-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and ß-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and ß-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.
Assuntos
Bioensaio , Resistência à Doença/genética , Glucana 1,3-beta-Glucosidase/genética , Nicotiana/genética , Nicotiana/microbiologia , Serina Endopeptidases/genética , Ambiente Controlado , Expressão Gênica , Fenótipo , Plantas Geneticamente Modificadas , Nicotiana/imunologiaRESUMO
The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of ß-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of ß-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12 hr. During this period, 9.26 generations were obtained, with 1 hr and 29 min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr-1). The entire microorganism growth process was monitored during ß-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1 hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.
Assuntos
Glucana 1,3-beta-Glucosidase/metabolismo , Rhodotorula/enzimologia , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Cinética , Rhodotorula/crescimento & desenvolvimento , Rhodotorula/metabolismo , Especificidade por SubstratoRESUMO
Pythium insidiosum is an important oomycete due to its ability to infect humans and animals. It causes pythiosis, a disease of difficult treatment that occurs more frequently in humans in Thailand and in horses in Brazil. Since cell-wall components are frequently related to host shifts, we decided here to use sequences from the exo-1,3-ß-glucanase gene (exo1), which encodes an immunodominant protein putatively involved in cell wall remodeling, to investigate the microevolutionary relationships of Brazilian and Thai isolates of P. insidiosum. After neutrality ratification, the phylogenetic analyses performed through Maximum parsimony (MP), Neighbor-joining (NJ), Maximum likelihood (ML), and Bayesian analysis (BA) strongly supported Thai isolates being paraphyletic in relation to those from Brazil. The structure recovered by these analyses, as well as by Spatial Analysis of Molecular Variance (SAMOVA), suggests the subdivision of P. insidiosum into three clades or population groups, which are able to explain almost 81% of the variation encountered for exo1. Moreover, the two identified Thai clades were almost as strongly differentiated between each other, as they were from the Brazilian clade, suggesting an ancient Asian subdivision. The derived positioning in the phylogenetic tree, linked to the lower diversity values and the recent expansion signs detected for the Brazilian clade, further support this clade as derived in relation to the Asian populations. Thus, although some patterns presented here are compatible with those recovered with different molecular markers, exo1 was revealed to be a good marker for studying evolution in Pythium, providing robust and strongly supported results with regard to the patterns of origin and diversification of P. insidiosum.
Assuntos
Glucana 1,3-beta-Glucosidase/genética , Pythium/genética , Brasil , Evolução Molecular , Variação Genética , Filogenia , Pythium/enzimologia , TailândiaRESUMO
Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and ß-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and ß-1,3-glucanase, respectively. In vitro production of chitinase and ß-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30°C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the ß-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and ß-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.
Assuntos
Quitinases/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Microbiologia do Solo , Trichoderma/enzimologia , Trichoderma/crescimento & desenvolvimento , Basidiomycota/metabolismo , Carbono/metabolismo , Parede Celular/metabolismo , Quitina/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , Rizosfera , Temperatura , Nicotiana , Trichoderma/isolamento & purificaçãoRESUMO
Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent against many fungal phytopathogens. The aim of this study was to characterize the proteins secreted by T. harzianum ALL42 when its spores were inoculated and incubated for 48 h in culture media supplemented with glucose (GLU) or with cell walls from Fusarium solani (FSCW), a phytopathogen that causes severe losses in common bean and soy crops in Brazil, as well as other crop diseases around the world. Trichoderma harzianum was able to grow in Trichoderma Liquid Enzyme Production medium (TLE) and Minimal medium (MM) supplemented with FSCW and in TLE+GLU, but was unable to grow in MM+GLU medium. Protein quantification showed that TLE+FSCW and MM+FSCW had 45- and 30- fold, respectively, higher protein concentration on supernatant when compared to TLE+GLU, and this difference was observable on 2D gel electrophoresis (2DE). A total of 94 out of 105 proteins excised from 2DE maps were identified. The only protein observed in all three conditions was epl1. In the media supplemented with FSCW, different hydrolases such as chitinases, ß-1,3-glucanases, glucoamylases, α-1,3-glucanases and proteases were identified, along with other proteins with no known functions in mycoparasitism, such as npp1 and cys. Trichoderma harzianum showed a complex and diverse arsenal of proteins that are secreted in response to the presence of FSCW, with novel proteins not previously described in mycoparasitic-related studies.
Assuntos
Parede Celular/química , Proteínas Fúngicas/metabolismo , Fusarium/química , Glucose/farmacologia , Trichoderma/metabolismo , Antibiose , Agentes de Controle Biológico , Parede Celular/metabolismo , Quitinases/genética , Quitinases/metabolismo , Misturas Complexas/metabolismo , Misturas Complexas/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/genética , Fusarium/patogenicidade , Expressão Gênica , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Anotação de Sequência Molecular , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Glycine max/microbiologia , Trichoderma/efeitos dos fármacos , Trichoderma/genética , Trichoderma/crescimento & desenvolvimentoRESUMO
The bacterial spot of tomato, caused by Xanthomonas spp., is a very important disease, especially in the hot and humid periods of the year. The chemical control of the disease has not been very effective for a number of reasons. This study aimed to evaluate, under greenhouse conditions, the efficacy of leaf-spraying chemicals (acibenzolar-S-methyl (ASM) (0.025 g.L(-1)), fluazinam (0.25 g.L(-1)), pyraclostrobin (0.08 g.L(-1)), pyraclostrobin + methiran (0.02 g.L(-1) + 2.2 g.L(-1)), copper oxychloride (1.50 g.L(-1)), mancozeb + copper oxychloride (0.88 g.L(-1) + 0.60 g.L(-1)), and oxytetracycline (0.40 g.L(-1))) on control of bacterial spot. Tomatoes Santa Clara and Gisele cultivars were pulverized 3 days before inoculation with Xanthomonas perforans. The production of enzymes associated with resistance induction (peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, ß-1,3-glucanase, and protease) was quantified from leaf samples collected 24 hours before and 24 hours after chemical spraying and at 1, 2, 4, 6, and 8 days after bacterial inoculation. All products tested controlled bacterial spot, but only ASM, pyraclostrobin, and pyraclostrobin + metiram increased the production of peroxidase in the leaves of the two tomato cultivars, and increased the production of polyphenol oxidase and ß-1,3-glucanase in the Santa Clara cultivar.
Assuntos
Resistência à Doença/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Doenças das Plantas/microbiologia , Solanum lycopersicum/microbiologia , Xanthomonas/crescimento & desenvolvimento , Catecol Oxidase/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/imunologia , Peptídeo Hidrolases/metabolismo , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Doenças das Plantas/imunologia , Xanthomonas/efeitos dos fármacosRESUMO
The bacterial spot of tomato, caused by
Assuntos
Resistência à Doença , Fungicidas Industriais/farmacologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas/crescimento & desenvolvimento , Catecol Oxidase/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/imunologia , Peptídeo Hidrolases/metabolismo , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Doenças das Plantas/imunologia , XanthomonasRESUMO
In previous studies, we first isolated one different protein ß-1,3-glucanase using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from normal wheat (Triticum aestivum L.) and chemical hybridization agent-induced male sterility (CIMS) wheat. In this experiment, ß-1,3-glucanase activity and the expression of a callose deposition-related gene, UDP-glucose phosphorylase (UGPase), were determinate in normal, CIMS, and genetic male sterility (GS) wheat. ß-1,3-glucanase activity was significantly different between the fertile and sterile lines during callose synthesis and degradation, but there was no difference between CIMS and GS wheat. The UGPase gene of callose deposition was highly expressed in the meiophase and sharply decreased in the tetrad stage. However, the expression of the UGPase gene was significantly different between the fertile and sterile lines. These data indicated that ß-1,3-glucanase activity and the expression of the UGPase gene play important roles in the male sterility of wheat. Consequently, pollen mother cells (PMCs) might degenerate at the early meiosis stage, and differences in UGPase gene expression and ß-1,3-glucanase activity might eventually result in complete pollen collapse. In addition, the critical period of anther abortion might be the meiosis stage to the tetrad stage rather than what we previously thought, the mononuclear period.
Assuntos
Regulação da Expressão Gênica de Plantas , Glucana 1,3-beta-Glucosidase/metabolismo , Glucanos/metabolismo , Infertilidade das Plantas/genética , Triticum/enzimologia , Triticum/genética , DNA Complementar/genética , Eletroforese em Gel Bidimensional , Regulação Enzimológica da Expressão Gênica , Genes de Plantas , Nucleotidiltransferases/metabolismo , Proteínas de Plantas , Pólen/metabolismo , Pólen/ultraestrutura , RNA Ribossômico 18S/genética , Triticum/ultraestruturaRESUMO
The effect of the antagonistic yeast XL-1 on resistance-associated enzyme activities in postharvest cantaloupe was studied by inoculating the antagonistic yeast XL-1. Cantaloupes were sterilized, dried in air, and soaked in antagonistic yeast treatment liquid for 30 s. After drying in air, the cantaloupe was stored at room temperature (2°-5°C). The activities of resistance-associated enzymes in cantaloupe like polyphenol oxidase, ß-1,3-glucanase, peroxidase, and superoxide dismutase were measured every 7 days. Our results indicated that the antagonistic yeast XL-1 significantly improved the activity of ß-1,3-glucanase and chitinase to promote the disease resistance of postharvest cantaloupe.
Assuntos
Cucumis melo/enzimologia , Armazenamento de Alimentos/métodos , Proteínas de Plantas/metabolismo , Leveduras/fisiologia , Catecol Oxidase/metabolismo , Quitinases/metabolismo , Cucumis melo/química , Ensaios Enzimáticos , Glucana 1,3-beta-Glucosidase/metabolismo , Peroxidase/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Plant ß-1,3-glucanases are commonly involved in disease resistance. This report describes the cloning and genetic transformation of a ß-1,3-glucanase gene from peanut. The gene was isolated from both the genomic DNA and cDNA of peanut variety Huayu20 by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), respectively. The DNA sequence contained 1471 bp including two exons and one intron, and the coding sequence contained 1047 bp that coded for a 348-amino acid protein with a calculated molecular weight of 38.8 kDa. The sequence was registered in NCBI (GenBank accession No. JQ801335) and was designated as Ah-Glu. As determined by BLAST analysis, the Ah-Glu protein has 42-90% homology with proteins from Oryza sativa (BAC83070.1), Zea mays (NP_001149308), Arabidopsis thaliana (NP_200470.1), Medicago sativa (ABD91577.1), and Glycine max (XP_003530515.1). The over-expression vector pCAMBIA1301-Glu containing Ah-Glu was constructed, confirmed by PCR and restriction enzyme digestion, and transformed into peanut variety Huayu22 by Agrobacterium EHA105-mediated transformation. The putative transformed plants (T0) were confirmed by PCR amplification. RT-PCR analysis and ß-glucuronidase (GUS) staining showed that the transferred Ah-Glu was expressed as mRNA and protein. In a laboratory test, the transgenic plants were found to be more resistant to the fungal pathogen Cercospora personata than the non-transgenic plants were.
Assuntos
Arachis/enzimologia , Resistência à Doença/genética , Glucana 1,3-beta-Glucosidase/genética , Transformação Genética , Glucana 1,3-beta-Glucosidase/metabolismo , Glucuronidase/genética , Dados de Sequência Molecular , Oryza , Plantas Geneticamente Modificadas , RNA Mensageiro/biossínteseRESUMO
The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-ß-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and ß-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or ß-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and ß-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells.
Assuntos
Anti-Infecciosos/farmacologia , Glucana 1,3-beta-Glucosidase/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação/efeitos dos fármacos , Paracoccidioides/crescimento & desenvolvimento , Tunicamicina/farmacologia , alfa-Amilases/metabolismo , Paracoccidioides/citologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/enzimologiaRESUMO
Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.
Assuntos
Capsicum/genética , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Phytophthora/patogenicidade , Doenças das Plantas/genética , Capsicum/microbiologia , China , Genótipo , Glucana 1,3-beta-Glucosidase/metabolismo , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Regulação para CimaRESUMO
Dry bean (Phaseolus vulgaris L.) is an important economic crop in Brazil but its yield can be significantly reduced by white mold, a disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive, and non-host-specific fungus. This fungus secretes numerous cell wall-degrading enzymes such as polygalacturonases, exo-ß-1,3-glucanases, xylanases, and cellulases that have been detected during the early stages of infection. In this study, the activities of these enzymes were detected in all carbon sources tested (citrus pectin, cell wall extract from P. vulgaris, carboxymethyl cellulose, and glucose), but the highest levels were found when using citrus pectin and cell wall extract from P. vulgaris. Regardless of the carbon source, pH decreased throughout the culture time. During pathogenesis in dry bean stems, increased enzyme activities were also observed. Reverse transcriptase-polymerase chain reaction experiments showed that the induction of polygalacturonases (sspg1, sspg3, sspg5, sspg6, and sspg7), exo-ß-1,3-glucanases, and endo-ß-1-4-glucanase in S. sclerotiorum occurred during the early stages of colonization.
Assuntos
Ascomicetos/enzimologia , Celulase/genética , Glucana 1,3-beta-Glucosidase/genética , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Poligalacturonase/genética , Sequência de Bases , Parede Celular , Dados de Sequência Molecular , Pectinas , Phaseolus/enzimologia , Extratos Vegetais/metabolismoRESUMO
In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a ß-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of ß-1,3-1,4-glucanases and presented high protein identity with ß-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with ß-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao ß-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a ß-1,3-1,4-glucanase, in particular against M. perniciosa.
Assuntos
Agaricales/efeitos dos fármacos , Cacau/enzimologia , Glucana 1,3-beta-Glucosidase/farmacologia , Modelos Moleculares , Micélio/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Agaricales/crescimento & desenvolvimento , Cacau/microbiologia , Escherichia coli , Fluorescência , Glucana 1,3-beta-Glucosidase/genética , Espectrometria de Massas , Micélio/crescimento & desenvolvimento , Propídio , Proteínas Recombinantes/genéticaRESUMO
We report the molecular characterization of ß-1,3-glucanase-producing Bacillus amyloliquefaciens-an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the ß-1,3-glucanase gene was found to be 747 bp in length. A homology model of the ß-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target ß-1,3-glucanase protein and the template protein, endo ß-1,3-1,4-glucanase protein (PDB ID: 3o5s), were found to share 94% sequence identity and to have similar secondary and tertiary structures. In the modeled structure, three residues in the active site region of the template-Asn52, Ile157 and Val158-were substituted with Asp, Leu and Ala, respectively. Computer-aided docking studies of the substrate disaccharide (ß-1, 3-glucan) with the target as well as with the template proteins showed that the two protein-substrate complexes were stabilized by three hydrogen bonds and by many van der Waals interactions. Although the binding energies and the number of hydrogen bonds were the same in both complexes, the orientations of the substrate in the active sites of the two proteins were different. These variations might be due to the change in the three amino acids in the active site region of the two proteins. The difference in substrate orientation in the active site could also affect the catalytic potential of the ß-1,3 glucanase enzyme.
Assuntos
Bacillus/enzimologia , Endófitos/enzimologia , Glucana 1,3-beta-Glucosidase/metabolismo , Hevea/microbiologia , Phytophthora/fisiologia , Sequência de Aminoácidos , Bacillus/genética , Bacillus/fisiologia , Sequência de Bases , Endófitos/genética , Endófitos/fisiologia , Genes Bacterianos , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/genética , Hevea/parasitologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and ß-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a ß-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.