Showing papers on "Chitinase published in 1989"

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

Abstract: Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.

Effects of Chitosan, Pectic Acid, Lysozyme, and Chitinase on the Growth of Several Phytopathogens

Abstract: used as the basal culture medium (pH 5.6), Each of 18 phytopathogens was grown on the PDA mediurn containing a test materiai in a concentration of O,Ol, O.1, or 1.0 mgr'ml in a Petri dish (diameter 8.Scm). A control was run with the PDA medium conlaining no test matcrials, The test materials used ttre the lactate salt of highmolecular-weight (HMW)-chitosan [crab shell, MW 400,OOO, the degree of substitution O.95 for NAcl,

A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.

Abstract: Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.

Degradation of Fungal Cell Walls by Lytic Enzymes of Trichoderma harzianum

Abstract: SUMMARY: In in vitro tests, two strains of Trichoderma harzianum failed to parasitize colonies of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis. However, these strains were strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum. When grown in liquid cultures containing laminarin, chitin or fungal cell walls as sole carbon sources, both strains of T. harzianum released, 3-β-glucanase and chitinase into the medium. Higher levels of these enzymes were induced in strain T-203 than in T-35 by hyphal cell walls of F. oxysporum. When the lytic enzymes produced by T-35 were incubated with hyphal cell walls of the test fungi, more glucose and N-acetyl-d-glucosamine was released from cell walls of R. solani and Sclerotium rolfsii than from those of F. oxysporum. Treatment of F. oxysporum cell walls with 2 m-NaOH, protease or trypsin prior to their incubation with the lytic enzymes of T. harzianum significantly increased the release of glucose and N-acetyl-d-glucosamine. The effect of these treatments on R. solani and S. rolfsii cell walls was much lower. These results suggest that proteins in the cell walls of F. oxysporum may make these walls more resistant than those of R. solani or S. rolfsii to degradation by extracellular enzymes of T. harzianum.

Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-β-Glucanases and Chitinases

Abstract: Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [Mr] 35 kilodaltons [kD], isoelectric point [pI] ∼6.4) showed 1,3-β-glucanase activity, while the other one (Mr26 kD, pI ∼6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (Mr33 kD) and three additional chitinases (Mr 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.

Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase

Abstract: An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Abstract: Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli

Abstract: When Sclerotium rolfsii was sprayed with partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was found to be effective in reduction of disease incidence caused by S. rolfsii in beans and Rhizoctonia solani in cotton under greenhouse conditions (62% disease reduction in both diseases). A similar effect was obtained when we used viable cells of E. coli containing the plasmid pLCHIA. However, E. coli carrying the plasmid lacking the pL promotor did not have any effect. These results suggest a role for chitinase in biological control of plant pathogenic fungi

Chitinase in roots of mycorrhizal Allium porrum: regulation and localization

Abstract: Chitinase (EC 3.2.1.14) activity was measured in roots of Allium prorrum L. (leek) during development of a vesicular-arbuscular mycorrhizal symbiosis with Glomus versiforme (Karst.) Berch. During the early stages of infection, between 10 and 20 d after inoculation, the specific activity of chitinase was higher in mycorrhizal roots than in the uninfected controls. However, 60-90 d after inoculation, when the symbiosis was fully established, the mycorrhizal roots contained much less chitinase than control roots. Chitinase was purified from A. porrum roots. An antiserum against beanleaf chitinase was found to cross-react specifically with chitinase in the extracts from non-mycorrhizal and mycorrhizal A. porrum roots. This antiserum was used for the immunocytochemical localization of the enzyme with fluorescent and gold-labelled probes. Chitinase was localized in the vacuoles and in the extracellular spaces of non-mycorrhizal and mycorrhizal roots. There was no immunolabelling on the fungal cell walls in the intercellular or the intracellular phases. It is concluded that the chitin in the fungal walls is inaccessible to plant chitinase. This casts doubts on the possible involvement of this hydrolase in the development of the mycorrhizal fungus. However, fungal penetration does appear to cause a typical defense response in the first stages that is later depressed.

Induced systemic resistance to blue mold: early induction and accumulation of β-1,3-glucanases, chitinases, and other pathogenesis-related proteins (b-proteins) in immunized tobacco

Abstract: Increases in b-proteins were detected in controls only 6 days after challenge. A basal level of chitinases was always detected, but increases in chitinases above this level in immunized plants followed a profile similar to that of the β-1,3-glucanases and other b-proteins. It is likely that β-1,3-glucanases, chitinases, and other b-proteins are coordinately regulated in tobacco. The increases in these proteins coincided with the onset of immunization in plants injected with P. tabacina, and the levels were maintained during the period after challenge, when the development of P. tabacina was restricted

Chitinase production by myrothecium verrucaria and its significance for fungal mycelia degradation

Abstract: Myrothecium verrucaria produced high levels of chitinases in a medium containing chitin used as a sole carbon source. Adding 0.03% urea increased the enzyme yield 4-fold in 7 days compared to the control. Adding oxgall (0.1%) to the growth medium gave the maximum activity (acid-swollen chitin-degrading activity, 2.0IU/ml) in 7 days. The biochemical characterization of the chitinase revealed its broader temperature (25-55°C) and pH (4.0-6.5) profiles of activity which showed its potential application in fungal mycelia degradation. Compared to commercial lytic enzyme preparations (NovoZym 234 and Onozuka R-10), M, verrucaria culture filtrate had 5-6 times more chitinase activity. And this produced significantly higher levels of N-acetyl-D-glucosamine from the fungal mycelia preparations under study.

The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies.

Abstract: A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

Distribution of chitinase and chitobiase in bacillus

Abstract: Sixty strains representing 29 taxospecies ofBacillus were assayed for their ability to hydrolyze colloidal chitin. A qualitative estimation of chitinolysis was made from the clear zone produced around colonies in the conventional agar plate method and chitobiase activity by use of the fluorescence of 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide.

Chitinase Activity from Candida albicans and its Inhibition by Allosamidin

Abstract: Candida albicans chitinase isolated using the Dyno-Mill disruption technique was characterized using an improved radiometric assay procedure. The enzyme had apparent temperature and pH optima of 45 degrees C and 6.5, respectively. The preparation yielded an apparent Km of 3.9 mg chitin ml-1 [17.6 mM-N-acetylglucosamine (GlcNAc) equivalents] and V of 2.3 nmol GlcNAc formed min-1 (mg protein)-1. The potential of the streptomycete antibiotic allosamidin as an antifungal agent is discussed in view of its dose-dependent inhibition of C. albicans chitinase activity (IC50 = 0.3 microM). Allosamidin was a potent competitive inhibitor of enzyme activity (Ki = 0.23 microM).

Amino acid sequence of chitinase from Streptomyces erythraeus.

Abstract: The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.

Bacterial chitinase is modified and secreted in transgenic tobacco.

Abstract: The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.

Purification and characterization of chitinase produced by Streptomyces erythraeus.

Abstract: A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.

Overexpression of chitinase in transgenic plants

Abstract: The preparation of novel recombinant DNA constructs and their use in transforming plants to achieve overexpression of chitinase and thereby conferring resistance to various plant pathogenic fungi.