RESUMO
Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.
Assuntos
Quitinases , Resistência à Doença , Fusarium , Doenças das Plantas , Triticum , Quitinases/genética , Quitinases/metabolismo , Resistência à Doença/genética , Fusarium/genética , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Irã (Geográfico) , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Triticum/genética , Triticum/metabolismo , Triticum/microbiologiaRESUMO
BACKGROUND: Many phytopathogens secrete a large number of cell wall degrading enzymes (CWDEs) to decompose host cell walls in order to penetrate the host, obtain nutrients and accelerate colonization. There is a wide variety of CWDEs produced by plant pathogens, including glycoside hydrolases (GHs), which determine the virulence, pathogenicity, and host specificity of phytopathogens. The specific molecular mechanisms by which pathogens suppress host immunity remain obscure. RESULT: In this study, we found that CgEC124 encodes a glycosyl hydrolase with a signal peptide and a conserved Glyco_hydro_cc domain which belongs to glycoside hydrolase 128 family. The expression of CgEC124 was significantly induced in the early stage of Colletotrichum graminicola infection, especially at 12 hpi. Furthermore, CgEC124 positively regulated the pathogenicity, but it did not impact the vegetative growth of mycelia. Ecotopic transient expression of CgEC124 decreased the disease resistance and callose deposition in maize. Moreover, CgEC124 exhibited the ß-1,3-glucanase activity and suppresses glucan-induced ROS burst in maize leaves. CONCLUSIONS: Our results indicate that CgEC124 is required for full virulence of C. graminicola but not for vegetative growth. CgEC124 increases maize susceptibility by inhibiting host reactive oxygen species burst as well as callose deposition. Meanwhile, our data suggests that CgEC124 explores its ß-1,3-glucanase activity to prevent induction of host defenses.
Assuntos
Colletotrichum , Doenças das Plantas , Imunidade Vegetal , Zea mays , Colletotrichum/patogenicidade , Resistência à Doença , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Glucana 1,3-beta-Glucosidase/genética , Glucanos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Espécies Reativas de Oxigênio/metabolismo , Zea mays/imunologia , Zea mays/microbiologiaRESUMO
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
In this study, an endophytic Bacillus sp. strain (K7) was isolated from the medicinally important ornamental plant, Jasminum officinale. Biochemical analyses were conducted to evaluate the nature of the extracted product, which displayed strong anticandidal activity against Candida albicans (CA) SC5314, as evident from the results obtained in agar-cup diffusion tests, phase-contrast microscopy, scanning electron microscopy and minimum inhibitory concentration assays. After confirming the presence of the gene clusters encoding the lipopeptides iturins and fengycin in the genome of K7, their corresponding molecular ions were identified using MALDI-TOF-MS. 3D structures of the lipopeptides were downloaded from specific databases and molecular docking was performed against a vital CA enzyme, exo-1,3-beta-glucanase, involved in cell wall remodelling, adhesion to polymer materials and biofilm formation. The docking score of iturins was found to be -8·6 and -8·2 kcal mol-1 and for fengycin it was -9·4 kcal mol-1 , indicating a strong affinity of these cyclic lipopeptides towards exo-1,3-beta-glucanase. The combined in vitro and in silico anticandidal studies suggested that these secreted lipopeptides from Bacillus sp. may be used as potential therapeutics against opportunistic and complicated infections of CA.
Assuntos
Bacillus , Bacillus/metabolismo , Candida albicans/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Lipopeptídeos/farmacologia , Simulação de Acoplamento Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
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
Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and ß-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and ß-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.
Assuntos
Resistência à Doença/imunologia , Peroxissomos/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Potyviridae , Triticum/imunologia , Triticum/virologia , Quitinases/metabolismo , Clorofila/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Oxirredução , Peroxidases/metabolismo , Peroxissomos/genética , Peroxissomos/virologia , Fenótipo , Folhas de Planta/imunologia , Folhas de Planta/virologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D, a putative sterol carrier protein gene from elongating cotton (Gossypium hirsutum) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D-suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D, which encodes a PD-targeting ß-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs, leading to the switch from symplasmic to apoplasmic pathways.
Assuntos
Proteínas de Transporte/genética , Fibra de Algodão , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Gossypium/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , Plasmodesmos/metabolismo , Proteínas de Transporte/metabolismo , Regulação para Baixo/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Gossypium/ultraestrutura , Hexoses/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Família Multigênica , Permeabilidade , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Plasmodesmos/ultraestrutura , Plântula/metabolismo , Homologia de Sequência de Aminoácidos , Esteróis/biossíntese , Esteróis/metabolismo , Sacarose/metabolismo , Supressão GenéticaRESUMO
ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.
Assuntos
Sizofirano/química , beta-Glucanas/química , Varredura Diferencial de Calorimetria , Configuração de Carboidratos , Cromatografia em Gel , Difusão Dinâmica da Luz , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/metabolismo , Peso Molecular , Proteoglicanas , Cloreto de Sódio , Soluções/química , Termodinâmica , Água/químicaRESUMO
The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), ß-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Frutas/microbiologia , Rhizopus/efeitos dos fármacos , Rhizopus/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Quitinases/metabolismo , Frutas/enzimologia , Glucana 1,3-beta-Glucosidase/metabolismo , Solanum lycopersicum/enzimologia , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Doenças das Plantas/microbiologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Longevidade , S-Adenosilmetionina/metabolismo , Trifosfato de Adenosina/metabolismo , Restrição Calórica , Epistasia Genética , Genes Dominantes , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Redes e Vias Metabólicas , Metionina/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Fungal cell walls contain ß-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of ß-glucan by host cells through at least two mechanisms: concealment of ß-glucans beneath α-glucans and enzymatic removal of any exposed ß-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed ß-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-ß1,3-glucanase and Eng1 is an endo-ß1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for ß1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host ß-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two ß1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall ß-glucans to minimize host detection of Histoplasma yeasts.
Assuntos
Glucana 1,3-beta-Glucosidase/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Histoplasma/enzimologia , Histoplasmose/microbiologia , Histoplasma/metabolismo , Histoplasma/patogenicidade , Humanos , Especificidade por Substrato , beta-Glucanas/metabolismoRESUMO
BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of â¼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of â¼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Glucana 1,3-beta-Glucosidase/química , Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Secundária de ProteínaRESUMO
Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two ß-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Glucanos/metabolismo , Metais Pesados/toxicidade , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Permeabilidade/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Plasmodesmos/efeitos dos fármacos , Plasmodesmos/metabolismoRESUMO
Gall-making Cynipidae manipulate the leaves of host plant to form galls where offspring find shelter and food. The relationship between oak gallwasp and biochemical mechanisms of galls still requires a better understanding. So, in this research, protein and phenolic compound contents, as well as the activity of antioxidative enzymes and pathogenesis-related (PR) proteins were determined. Galls caused by asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model were used. All cynipid species modified the protein levels of gall tissues, but they cannot be treated as protein sinks. Significantly higher levels of phenols were observed in the galled leaves and galls of all cynipid species when compared with the control tissues. Peroxidase and polyphenol oxidase activity was usually low or showed no activity in galled tissues of all species. PR proteins, such as chitinase and ß-1,3-glucanase, had a similar activity profile. Their activity significantly increased in the leaves with galls of all cynipid species, especially those infested with C. quercusfolii. Data generated in this study clearly indicate that galling Cynipidae manipulate the biochemical machinery of the galls for their own needs. However, the pattern of the biochemical features of leaves with galls and galled tissues depends on gall-making species.
Assuntos
Tumores de Planta/parasitologia , Quercus/parasitologia , Árvores/parasitologia , Vespas/fisiologia , Animais , Catecol Oxidase/metabolismo , Quitinases/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Peroxidase/metabolismo , Folhas de Planta/metabolismo , Quercus/metabolismo , Reprodução Assexuada/fisiologia , Árvores/metabolismoRESUMO
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
MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.
Assuntos
Drosera/enzimologia , Evolução Molecular , Glucana 1,3-beta-Glucosidase/genética , Sequência de Aminoácidos , Simulação por Computador , Drosera/genética , Genes de Plantas , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/metabolismo , Glucuronidase/metabolismo , Motivos de Nucleotídeos , Filogenia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Estresse Fisiológico/genética , Nicotiana/genética , Fatores de Transcrição/metabolismoRESUMO
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
BACKGROUND: Sugarcane smut, which is caused by Sporisorium scitamineum, has been threatening global sugarcane production. Breeding smut resistant sugarcane varieties has been proven to be the most effective method of controlling this particular disease. However, a lack of genome information of sugarcane has hindered the development of genome-assisted resistance breeding programs. Furthermore, the molecular basis of sugarcane response to S. scitamineum infection at the proteome level was incomplete and combining proteomic and transcriptional analysis has not yet been conducted. RESULTS: We identified 273 and 341 differentially expressed proteins in sugarcane smut-resistant (Yacheng05-179) and susceptible (ROC22) genotypes at 48 h after inoculation with S. scitamineum by employing an isobaric tag for relative and absolute quantification (iTRAQ). The proteome quantitative data were then validated by multiple reaction monitoring (MRM). The integrative analysis showed that the correlations between the quantitative proteins and the corresponding genes that was obtained in our previous transcriptome study were poor, which were 0.1502 and 0.2466 in Yacheng05-179 and ROC22, respectively, thereby revealing a post-transcriptional event during Yacheng05-179-S. scitamineum incompatible interaction and ROC22-S. scitamineum compatible interaction. Most differentially expressed proteins were closely related to sugarcane smut resistance such as beta-1,3-glucanase, peroxidase, pathogenesis-related protein 1 (PR1), endo-1,4-beta-xylanase, heat shock protein, and lectin. Ethylene and gibberellic acid pathways, phenylpropanoid metabolism and PRs, such as PR1, PR2, PR5 and PR14, were more active in Yacheng05-179, which suggested of their possible roles in sugarcane smut resistance. However, calcium signaling, reactive oxygen species, nitric oxide, and abscisic acid pathways in Yacheng05-179 were repressed by S. scitamineum and might not be crucial for defense against this particular pathogen. CONCLUSIONS: These results indicated complex resistance-related events in sugarcane-S. scitamineum interaction, and provided novel insights into the molecular mechanism underlying the response of sugarcane to S. scitamineum infection.
Assuntos
Resistência à Doença , Metabolismo Energético , Proteoma , Proteômica , Saccharum/metabolismo , Saccharum/microbiologia , Basidiomycota , Sinalização do Cálcio , Regulação da Expressão Gênica de Plantas , Glucana 1,3-beta-Glucosidase/metabolismo , Interações Hospedeiro-Patógeno , Metabolômica/métodos , Óxido Nítrico/metabolismo , Doenças das Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Axillary buds (AXBs) of hybrid aspen (Populus tremula×P. tremuloides) contain a developing dwarfed shoot that becomes para-dormant at the bud maturation point. Para-dormant AXBs can grow out after stem decapitation, while dormant AXBs pre-require long-term chilling to release them from dormancy. The latter is mediated by gibberellin (GA)-regulated 1,3-ß-glucanases, but it is unknown if GA is also important in the development, activation, and outgrowth of para-dormant AXBs. The present data show that para-dormant AXBs up-regulate GA receptor genes during their maturation, but curtail GA biosynthesis by down-regulating the rate-limiting GIBBERELLIN 3-OXIDASE2 (GA3ox2), which is characteristically expressed in the growing apex. However, decapitation significantly up-regulated GA3ox2 and GA4-responsive 1,3-ß-glucanases (GH17-family; α-clade). In contrast, decapitation down-regulated γ-clade 1,3-ß-glucanases, which were strongly up-regulated in maturing AXBs concomitant with lipid body accumulation. Overexpression of selected GH17 members in hybrid aspen resulted in characteristic branching patterns. The α-clade member induced an acropetal branching pattern, whereas the γ-clade member activated AXBs in recurrent flushes during transient cessation of apex proliferation. The results support a model in which curtailing the final step in GA biosynthesis dwarfs the embryonic shoot, while high levels of GA precursors and GA receptors keep AXBs poised for growth. GA signaling, induced by decapitation, reinvigorates symplasmic supply routes through GA-inducible 1,3-ß-glucanases that hydrolyze callose at sieve plates and plasmodesmata.
Assuntos
Giberelinas/fisiologia , Glucana 1,3-beta-Glucosidase/metabolismo , Brotos de Planta/metabolismo , Populus/metabolismo , Indução Enzimática/fisiologia , Giberelinas/metabolismo , Glucana 1,3-beta-Glucosidase/biossíntese , Glucana 1,3-beta-Glucosidase/genética , Redes e Vias Metabólicas/fisiologia , Dormência de Plantas/fisiologia , Brotos de Planta/enzimologia , Brotos de Planta/crescimento & desenvolvimento , Populus/enzimologia , Populus/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A glycoside hydrolase (GH) family 17 ß-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (â¼20 % identity) with that of other ß-1,3-glucanases,despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 ß-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a ß-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-ß-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.