Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have ...identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:β-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and β-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.
•Recombinant β-1,3-glucanase from alkalophilic bacteria was expressed and purified.•The endo-β-1,3-glucanase is a metalloenzyme and thermostable.•Blg32 could hydrolysed oligosaccharides to produce ...glucose.•The active site can accommodate five degree of polymerization of oligosaccharides.
Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
Endo-1,3-β-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-β-glucanase from ...the scallop
, named L
, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-β-glucanases, L
was expressed in vitro first. Enzymatic analysis demonstrated that L
preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, L
was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of L
was different from that of other marine mollusk endo-1,3-β-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-β-glucanases, which facilitated development of a unique endo-1,3-β-glucanase tool in the synthesis of novel glycosides.
In natural environments, several adverse environmental conditions occur simultaneously constituting a unique stress factor. In this work, physiological parameters and the hormonal regulation of ...Carrizo citrange and Cleopatra mandarin, two citrus genotypes, in response to the combined action of high temperatures and water deprivation were studied. The objective was to characterize particular responses to the stress combination.
Experiments indicated that Carrizo citrange is more tolerant to the stress combination than Cleopatra mandarin. Furthermore, an experimental design spanning 24 h stress duration, heat stress applied alone induced higher stomatal conductance and transpiration in both genotypes whereas combined water deprivation partially counteracted this response. Comparing both genotypes, Carrizo citrange showed higher phostosystem-II efficiency and lower oxidative damage than Cleopatra mandarin. Hormonal profiling in leaves revealed that salicylic acid (SA) accumulated in response to individual stresses but to a higher extent in samples subjected to the combination of heat and drought (showing an additive response). SA accumulation correlated with the up-regulation of pathogenesis-related gene 2 (CsPR2), as a downstream response. On the contrary, abscisic acid (ABA) accumulation was higher in water-stressed plants followed by that observed in plants under stress combination. ABA signaling in these plants was confirmed by the expression of responsive to ABA-related gene 18 (CsRAB18). Modulation of ABA levels was likely carried out by the induction of 9-neoxanthin cis-epoxicarotenoid dioxygenase (CsNCED) and ABA 8'-hydroxylase (CsCYP707A) while conversion to ABA-glycosyl ester (ABAGE) was a less prominent process despite the strong induction of ABA O-glycosyl transferase (CsAOG).
Cleopatra mandarin is more susceptible to the combination of high temperatures and water deprivation than Carrizo citrange. This is likely a result of a higher transpiration rate in Carrizo that could allow a more efficient cooling of leaf surface ensuring optimal CO2 intake. Hence, SA induction in Cleopatra was not sufficient to protect PSII from photoinhibition, resulting in higher malondialdehyde (MDA) build-up. Inhibition of ABA accumulation during heat stress and combined stresses was achieved primarily through the up-regulation of CsCYP707A leading to phaseic acid (PA) and dehydrophaseic acid (DPA) production. To sum up, data indicate that specific physiological responses to the combination of heat and drought exist in citrus. In addition, these responses are differently modulated depending on the particular stress tolerance of citrus genotypes.
A new HPLC method was developed to separate linear from β(1–6)-branched β(1–3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal β(1–6)/β(1–3)-glucan branching ...transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171–3180). Both enzymes release laminaribiose from the reducing end of a β(1–3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked β(1–3;1–6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a β(1–3)-linked product with a β(1–6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched β(1–3)-glucans of the cell wall.
•PaBglu50A is the first β-1,3 glucanase in Glycoside Hydrolase (GH) family 50.•PaBglu50A was expressed in E. coli and biochemically characterized.•PaBglu50A exhibited maximal activity at pH 5.5 and ...45°C, respectively.•PaBglu50A displayed activity towards water-insoluble curdlan and laminarin.•This study provides new insights to further understand GH family 50.
A novel β-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the highest identity of 34% with the β-agarase belonging to glycoside hydrolase (GH) family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The Km and Vmax of PaBglu50A for curdlan and laminarin were 94.4mgml−1 and 23.4μmolmin−1mg−1, 3.65mgml−1 and 8.89μmolmin−1mg−1, respectively. All characterized members of GH family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo-β-1,3-glucanase, making a significant difference from the reported enzymes of GH family 50. These results suggested that PaBglu50A is the first endo-type β-1,3-glucanase (EC 3.2.1.39) in GH family 50.
(CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as ...alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT-either alone or in combination-to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and β-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as
and
. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB.
β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall ...assembly. Using
as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized
and radiolabeled substrate UDP-(
C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the
Δ and
Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage. The double
Δ
Δ mutant showed a drastically sick phenotype. An
Gas1p ortholog, Gel4p from
, also showed dual β-(1,3)-glucan elongating and branching activity. Both
Gas1p and
Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.
The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using
as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double
Δ
Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to
Gas1p, GH72 family
Gas2p and
Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.
The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate ...composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β‐1,3‐glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate‐binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3‐glucans have a higher degree of polymerization and are less branched than the wild‐type strain. The mutant showed significant differences in global patterns of gene expression, a hyper‐branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium‐mediated plant infection.
The fungal cell wall is pivotal for cell protection, determination of cell shape and function and during host infection. This paper details the carbohydrate composition of the rice blast fungus cell wall and analysis of a family of β‐1,3‐glucan glucanosyltransferases (Gels) during infective and vegetative growth. Gel proteins are expressed during infection‐related development and plant infection, and a null mutant Δgel1Δgel3Δgel4, with modified cell wall 1,3‐glucans shows a hyper‐branching phenotype with no sporulation, and is thus non‐pathogenic.
Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass ...degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS.
The maximum activities of cellulases (0.032 U mL-1), endo-1,4-β--xylanase (10.82 U mL-1) and endo-1,3-β glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, β-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and β-glucosidases, were also identified in the secretome.
This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails.
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