beta-1,3-Endoglucanase from Soybean Releases Elicitor-Active Carbohydrates from Fungus Cell Walls

Two enzymes from soybean (Glycine max L. Merr. cv Harosoy 63) cotyledons released elicitor-active carbohydrates from cell walls of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea. They were identified as isoenzymes of β-1,3-endoglucanase (EC 3.2.1.39) with isoelectric points of pH 8.7 and 10.5. The pI 10.5 enzyme was extracted in the greatest amount and was isolated as a homogeneous protein of about 33,000 daltons as determined by gel filtration and sodium dodecyl sulfategel electrophoresis. The purified enzymes hydrolyzed several β-1,3-glucans in a strictly random manner, but degraded neither β-1,6- nor β-1,4-glucans.     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Hormonal regulation of beta1,3-glucanase messenger RNA levels in cultured tobacco tissues

We describe the isolation of a cDNA clone of β1,3-glucanase mRNA from Nicotiana tabacum L. cv. `Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single ˜1.6 kb-sized β1,3-glucanase mRNA. The levels of β1,3-glucanase and β1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block β1,3-glucanase production at the level of the mRNA.     

Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection

The acidic, extracellular, glucan endo-1,3-β-glucosidases (EC 3.2.1.39; β-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of β-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the β-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of β-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.     

Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

in-silico characterization of β-(1, 3)-endoglucanase (ENGL1) from Aspergillus fumigatus by homology modeling and docking studies

During the past few years a significant rise in aspergillosis caused by filamentous fungus Aspergillus fumigatus has been recorded particularly in immunocompromised patients. At present, there are limited numbers of antifungal agents to combat these infections and the situation has become more complex due to emergence of antifungal resistance and side-effects of antifungal drugs. These situations have increased the demand for novel drug targets. Recent studies have revealed that the β-1,3-endoglucanase (ENGL1) plays an essential role in cell wall remodeling that is absolutely required during growth and morphogenesis of filamentous fungi and thus is a promising target for the development of antifungal agents. Unfortunately no structural information of fungal β- glucanases has yet been available in the Protein Databank (PDB). Therefore in the present study, 3D structure of β-(1,3)- endoglucanase (ENGL1) was modeled by using I-TASSER server and validated with PROCHECK and VERIFY 3D. The best model was selected, energy minimized and used to analyze structure function relationship with substrate β-(1,3)-glucan by C-DOCKER (Accelrys DS 2.0). The results indicated that amino acids (GLU 380, GLN 383, ASP 384, TYR 395, SER 712, and ARG 713) present in β-1,3-endoglucanase receptor are of core importance for binding activities and these residues are having strong hydrogen bond interactions with β-(1,3)-glucan. The predicted model and docking studies permits initial inferences about the unexplored 3D structure of the β-(1,3)-endoglucanase and may be promote in relational designing of molecules for structure-function studies.     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Cloning, Sequencing, and Expression in Escherichia coli of the New Gene Encoding β-1,3-Xylanase from a Marine Bacterium, Vibrio sp. Strain XY-214

The Vibrio sp. strain XY-214 β-1,3-xylanase gene cloned in Escherichia coli DH5α consisted of an open reading frame of 1,383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed β-1,3-xylan to produce several xylooligosaccharides.     

Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption

The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic β-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, β-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther β-1,3-glucanase is serologically unrelated to the basic β-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as β-1,3-glucanase are specific to each process.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

A single pulse of O₃ (0.15 microliter per liter, 5 hours) induced β-1,3-glucanase and chitinase activities in O₃-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O₃-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for β-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O₃-tolerant cultivar Bel B, β-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O₃ concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in β-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O₃ treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of β-1,3-glucanase and chitinase activities following O₃ treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O₃ treatment increased the amount of the β-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O₃ levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.     

Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Ethylene induced chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and -1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [³⁵S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the -1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native -1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and -1,3-glucanase. A putative -1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of -1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for -1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and -1,3-glucanase are regulated co-ordinately at the level of mRNA.Abbreviations poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

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

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-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 β-1,3-glucanases upon induction with laminarin, a soluble β-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 β-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-β-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.     

A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation

Soybean membrane preparations specifically bound [¹⁴C]mycolaminaran, a branched β-1,3-glucan produced by Phytophthora sp. which elicits production of the phytoalexin glyceollin in soybean tissues. A Scatchard plot of the binding data disclosed the presence of a single affinity class of binding sites with a K_d value of 11.5 micromolar for the glucan. To assess the physiologic importance of mycolaminaran binding in phytoalexin elicitation, several derivatives of mycolaminaran were prepared. Reduced mycolaminaran had slightly greater elicitor activity and binding affinity than the native substance, while periodinated mycolaminaran was virtually devoid of either elicitor activity orbinding capability. Phosphorylated mycolaminaran, on the other hand, gave values for both elicitor activity and membrane binding which were intermediate between the native and periodinated preparations. No other tested carbohydrates competed with the binding of [¹⁴C]mycolaminaran. Soybean membrane preparations contained β-1,3-endoglucanase activity that degraded mycolaminaran and reduced both its efficiency as a phytoalexin elicitor and its membrane binding at temperatures above 0°C. Once [¹⁴C]mycolaminaran bound to membranes, however, it was not appreciably susceptible to glucanase attack and could not be displaced with excess unlabeled ligand. Taken collectively, the observations suggest that the membrane binding sites are mycolaminaran-specific receptors which are physiologically involved in the initiation of phytoalexin production in soybean cotyledons. Because the binding of mycolaminaran to membranes was abolished by heat and proteolytic enzymes, the receptor is probably a protein(s) or glycoprotein(s).