Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucans

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.     

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.     

Molecular cloning and characterization of an endo-1,3-beta-D-glucanase from the mollusk Spisula sachalinensis

cDNA encoding the endo-1,3-beta-d-glucanase from Spisula sachalinensis (LIV) was amplified by PCR using oligonucleotides deduced from the N-terminal end peptide sequence. Predicted enzyme structure consists of 444 amino acids with a signal sequence. The mature enzyme has 316 amino acids and its deduced amino acid sequence coincides completely with the N-terminal end (38 amino acids) of the beta-1,3-glucanase (LIV) isolated from the mollusk. The enzyme sequence from Val 121 to Met 441 reveals closest homology with Pacifastacus leniusculus lipopolysaccharide- and beta-1,3-glucan-binding protein and with coelomic cytolytic factors from Lumbricus terrestris. The mollusk glucanase also shows 36% identity and 56% similarity with beta-1,3-glucanase of the sea urchin Strongylocentrotus purpuratus. It is generally considered that invertebrate glucanase-like proteins containing the bacterial glucanase motif have evolved from an ancient beta-1,3-glucanase gene, but most of them lost their glucanase activity in the course of evolution and retained only the glucan-binding activity. A more detailed evaluation of the protein folding elicited very interesting relationships between the active site of LIV and other enzymes, which hydrolyze native glucans.     

Analysis of the beta-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes beta-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of beta-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular beta-1,3-glucanases upon induction with laminarin, a soluble beta-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible beta-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-beta-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa beta-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.     

Induction of β-1,3-glucanase in callus cultures <Emphasis Type="Italic">in vitro</Emphasis>

Sodium salicylate (NaSA) increased induction of both intracellular and extracellular beta-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular beta-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The co-cultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors.     

Exo- and endoglucanases of maize coleoptile cell walls: their interaction and possible regulation

Glucanase-mediated degradation of beta-(1,3)(1,4)-glucans has been attributed to auxin-induced cell wall loosening and thus tissue growth in cereal plants, but the regulatory mechanisms for the auxin-enhancement of glucanase activities in situ are not fully understood. Here, we report evidence for possible mechanisms which might account for auxin-induced changes in glucanase activities. A likely cause for acceleration of wall glucan degradation is the change in the ratio of exo- and endoglucanases. The combined enzymes synergistically promote beta-(1,3)(1,4)-glucan hydrolysis. In addition, these enzyme activities are enhanced by other enzymic and non-enzymic proteins and are also partially stimulated by divalent cations such as Ca(2+) and Mg(2+) at certain pH values. The acceleration of glucan degradation mediated by auxin may be mediated by changes and/or interactions of any of these factors in situ.     

Glucanase,glucan synthase and chitinase activity in barley genotypes susceptible or resistant toErysiphe graminis f.sp.hordei

In barley genotype susceptible toErysiphe graminis f. sp.hordei 1,3-β-glucan synthase activity in whole leaf extracts was higher in comparison with healthy plants. A positive correlation was found between the activity of 1,3-β-glucan synthase and the degree of barley resistance. On the contrary, the 1,3-β-D-glucanase activity in whole leaves was negatively correlated to host plant resistance. This phenomen is evident only in the early phase of plant pathogen interaction. However, in epidermal cells the 1,3-glucanase activity was not significantly changed after attack and the 1,3-glucan synthase activity was practically zero. Chitinase activity in inoculated leaves and epidermis was higher than in healthy ones, but no unambigous correlation was found between the enzyme activity and host resistance.     

Induction of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion (Allium cepa var aggregatum)

The Pseudomonas fluorescens isolate Pfl was found to inhibit the growth of pathogen Alternaria palandui, in vitro. In the present study, foliar application of a talc-based formulation of Pfl significantly reduced the incidence of leaf blight of onion, caused by A. palandui. Induction of defense-related proteins viz., chitinase, beta-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by application of Pfl, was studied against A. palandui infection in resistant (IHR 56) and susceptible (MDUI) onion cultivars. Chitinase in both cultivars, with or without challenge-inoculation of A. palandui revealed changes in the isoform pattern. The Native-PAGE of PO showed induction of PO2 isoform in both the cultivars, in response to inoculation of pathogen. Isoform analysis of PPO also exhibited induction in the Pfl-treated plants challenged with pathogen. Similarly, the activity of beta-1,3-glucanase was greatly induced in Pfl-treated plants, challenged with pathogen as compared to controls. Thus, the P. fluorescens-treated plants showed significant increase in the levels of the defense enzymes, in comparison to the plants challenged with the pathogen.     

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.     

Jasmonic Acid and Salicylic Acid Induce Accumulation of β-1,3-Glucanase and Thaumatin-Like Proteins in Wheat and Enhance Resistance Against Stagonospora nodorum

The effect of application of jasmonic acid (JA) and salicylic acid (SA) on the induction of resistance in wheat to Stagonospora nodorum and on the induction of -1,3-glucanase and thaumatin-like proteins (TLPs) was studied. Western blot analysis revealed that two -1,3-glucanases with apparent molecular masses of 31 and 33 kDa that cross-reacted with a barley glucanase antiserum were induced in wheat leaves after treatment with JA and SA. When wheat plants were treated with SA and JA, a TLP with an apparent molecular mass of 25 kDa and several other isoforms of TLP were induced. Pre-treatment of wheat plants with SA and JA significantly reduced (up to 56 %) the incidence of leaf blotch disease incited by S. nodorum compared with untreated control plants.     

Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.     

Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.     

Salicylic acid induced systemic resistant on peanut against Alternaria alternata

Abstract

The effect of Salicylic Acid (SA) in inducing resistance in groundnut plants against Alternaria alternata was investigated. Foliar application of SA at the concentration of 1 mM significantly reduced the leaf blight disease intensity and increased the pod yield under glasshouse conditions. Changes in the activities of phenylalanine ammonium lyase, chitinase β-1,3 glucanase and in phenolic content on groundnut after application of SA and inoculation with A. alternate were studied. In SA-treated leaves (plants) an increase in phenolic content was observed five days after challenge inoculation with A. alternata in groundnut plants pretreated with SA. There was a marked increase in chitinase and pathogen inoculation in SA-treated leaves. In chitinase, β-1,3 glucanase activities were observed in response to plants with an increase in SA treated leaves. Foliar applications of SA-induced in peroxidase and polyphenol oxidase activities were observed upon challenge inoculation with pathogen.     

Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.