Showing papers on "Chitinase published in 1994"

Enhanced Protection Against Fungal Attack by Constitutive Co–expression of Chitinase and Glucanase Genes in Transgenic Tobacco

Abstract: Plants respond to pathogen attack by the induction of a battery of defenses, suggesting that different protective mechanisms may have complementary roles in the overall expression of disease resistance. We have investigated possible functional interactions between two different hydrolytic enzymes, chitinase and glucanase, by constitutive co-expression in transgenic tobacco of genes encoding the rice RCH10 basic chitinase and the alfalfa AGLU1 acidic glucanase. Hybrid plants were generated by crossing transgenic parental lines exhibiting strong constitutive expression of cauliflower mosaic virus (CaMV) 35S enhancer / RCH10 and CaMV 35S double promoter / AGLU1 gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous for each transgene, and in homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone. Productive interactions between chitinase and glucanase transgenes in vivo point to combinatorial expression of antimicrobial genes as an effective approach to engineering enhanced crop protection against fungal disease.

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi

Abstract: Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

Abstract: The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin. Images

Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism.

Abstract: A gene (ech-42; previously named ThEn-42) coding for one of the endochitinases produced by the biocontrol agent Trichoderma harzianum IMI206040 was cloned and characterized. Expression of the cDNA clone in Escherichia coli resulted in bacteria with chitinase activity. This chitinase has been shown to have lytic activity on Botrytis cinerea cell walls in vitro. The ech-42 gene was assigned to a double chromosomal band (chromosome V or VI) upon electrophoretic separation and Southern analysis of the chromosomes. Primer extension analysis indicated that transcription of the gene begins preferentially 109 bp upstream of the translation initiation codon. Expression of ech-42 was strongly enhanced during direct interaction of the mycoparasite with a phytopathogenic fungus when confronted in vitro and by growing it in minimal medium containing chitin as sole carbon source. Similarly, light-induced sporulation resulted in high levels of transcript, suggesting developmental regulation of the gene. The implications of these findings are discussed.

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

Abstract: Summary A novel chitinese gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinaeas. Comparison of the chitinese class V paptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratla marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is Induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinsse class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Suparose chromatography and gel filtration. In vitro assays demonstrated that class V chitinaeas have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitlnase acts synergistically with tobacco class I 1~-l,3-glucanase agalnat Fusarlum solani germlings.

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots

Abstract: Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

Abstract: A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.

The linkage of (1-3)-beta-glucan to chitin during cell-wall assembly in saccharomyces-cerevisiae

Abstract: Pulse-chase experiments with [14C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1-3)-beta-glucan serves as a precursor for alkali-insoluble (1-3)-beta-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan-chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.

Production of Chitinases and β-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

Abstract: The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO3. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium. Images

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-beta-glucanase in potato.

Abstract: Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3-beta-glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3-beta-glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3-beta-glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3-beta-glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3-beta-glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3-beta-glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3-beta-glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3-beta-glucanase are encoded by gene families of considerable complexity.

Mutation analysis of the C-terminal vacuolar targeting peptide of tobacco chitinase: low specificity of the sorting system, and gradual transition between intracellular retention and secretion into the extracellular space.

Abstract: The C-terminal propeptide of tobacco (Nicotiana tabacum) chitinase A has been shown to be necessary and sufficient for targeting of chitinases to the plant vacuole. The sequence specificity of this vacuolar targeting peptide (VTP) has now been analysed using transient expression of chitinases in Nicotiana plumbaginifolia protoplasts. An extracellular cucumber chitinase, previously used as a secreted reporter protein in transgenic tobacco, was also secreted into the incubation medium by the transiently transformed protoplasts. Addition of six to seven amino acids at the C-terminus to generate the VTP of tobacco chitinase A were sufficient to cause retention of most of the cucumber chitinase within the protoplasts. The chitinase A itself, as well as a mutant lacking the N-terminal chitin-binding domain, were retained to 80% in the protoplasts when low concentrations of the plasmid were used in the transient expression system. At high concentrations of plasmid, causing high levels of transiently expressed chitinase, retention was reduced, indicating saturation of the sorting system. Deletion of the C-terminal methionine did not affect the intracellular location, but deletion of even a single internal amino acid of the VTP caused predominantly secretion of tobacco chitinase A. In contrast, exchanges of amino acids in the VTP as well as substitution of the VTP with random sequences had intermediary effects that covered the whole range from retention to secretion. The results suggest that the sorting system responsible for the diversion of secretory proteins to the vacuole has a low specificity for the sequence of C-terminal targeting peptides, and that sequence changes in the VTP allow a gradual transition from vacuolar retention to secretion.

Plant chitinase genes

Abstract: (FM) Friedrich Miescher Institute, Box 2543, CH-4002 Basel, Switzerland (BR) Cen~e National de la Recherche, Scienfifique, Institut de Biologic Moleculaire des Plantes, 12, rue du G6n6rat Zimmer, F-67084 Strasbourg Cedex, France (HL) Institute of Molecular Plant Sciences, Leiden University, Box 9502, NL-2300 RA Leiden, The Netherlands (JM) Biotechnology Research, Maribo Seed, Box 17, Langebrogade 1, DK1001 Copenhagen K, Denmark (J-MN), Botanical Institute, University of Basel, Hebelstrasse 1, CH-4057 Basel, Switzerland (JR) Biotechnology Research, CIBA-Geigy Corporation, Box 12257, Research Triangle Park, NC 27709, USA

Regulation of cucumber class III chitinase gene expression

Abstract: The chromosomal region encoding the acidic class III chitinase from cucumber has been isolated and characterized. As a result of an apparent gene triplication, the pathogen-induced gene (CHI2) is flanked by two closely related genes with complete open reading frames (ORF). The high level of conservation within the three ORFs suggests an essential role for each encoded protein in plant growth and development. The developmental and tissue-specific expression of RNA from each gene was analyzed using both gene-specific probes and RNA-PCR. The expression of each gene in response to various inducing treatments was also characterized. Only transcripts corresponding to CHI2 were detected. Chitinase mRNA abundance increased slightly following cycloheximide application; however, its potent induction by salicylic acid was inhibited by cycloheximide treatment.

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense.

Abstract: We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.

Chitinolytic properties of Bacillus pabuli K1

Abstract: The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied. Chitinase activity was measured as the release of p-nitrophenol from p-nitrophenyl-N,N'-diacetylchitobiose. Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined. The optimum environmental conditions for chitinase production were: 30 degrees C, initial pH 8, initial oxygen 10% and aw > 0.99. Chitinase production was induced when B. pabuli K1 was grown on colloidal chitin. The smallest chito-oligosaccharide able to induce chitinase production was N,N'-diacetylchitobiose, (GlcNAc)2. Production was also induced by (GlcNAc)3 and (GlcNAc)4. When the bacterium was grown on glucose or N-acetylglucosamine, no chitinases were formed. The highest chitinase production observed was obtained with colloidal chitin as substrate. The production of chitinases by B. pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose. The production was also repressed by 0.6% starch, laminarin and beta-glucan from barley and by glycerol. The addition of pectin and carboxymethyl cellulose increased chitinase production.

Characterization of a novel cis-acting element that is responsive to a fungal elicitor in the promoter of a tobacco class I chitinase gene.

Abstract: The expression of tobacco class I chitinase gene is effectively induced by a fungal elicitor in suspension-cultured tobacco cells. To identify cis-acting DNA elements that respond to the elicitor, a series of promoter constructs of the chitinase gene CHN50 fused to β-glucuronidase gene was introduced into tobacco cultured cells. Promoter deletion analysis of the chitinase gene CHN50 in transgenic tobacco calli indicated that the DNA region between positions -788 and -345 from the start site of transcription is required for inducibility by the elictor. A gel mobility shift assay revealed that nuclear factor(s) specifically interacted with the DNA region between positions -574 and -476. Moreover, this novel DNA-binding activity was present in nuclear extracts prepared from elicitor-treated cultured cells but not in extracts from untreated cells. Competitive binding assays and methylation interference experiments showed that the nuclear factor(s) bound specifically to a sequence of 22 bp that extended from positions -539 to -518 and contained a direct repeat of GTCAG spaced by three nucleotides. This motif is a candidate for a cis-acting elicitor-responsive element (EIRE) that is involved in the transcription of the class I chitinase gene.

Immobilization of chitinase on a reversibly soluble–insoluble polymer for chitin hydrolysis

Abstract: Serratia marcescens chitinase was immobilized by covalent binding to a polymer (hydroxypropyl methylcellulose acetate succinate, AS-L) showing reversibly soluble-insoluble characteristics with pH change. The immobilized enzyme (CH-AS) was soluble above pH 5.2 and insoluble below 4.5, which offers advantages in that it can carry out hydrolysis of chitin particles in a soluble form yet be recovered after precipitation at low pH. CH-AS has much higher activity than chitinase immobilized to a water-insoluble carrier. The effects of pH and temperature on the activity and stability of CH-AS, and the adsorption of CH-AS to chitin were studied and compared with those of free chitinase. Following repeated pH cycles between 6.6 and 4.5, CH-AS lost 30% of its enzyme activity during the first cycle due to protein release and enzyme denaturation, but substantially less activity was lost in the following cycles, with minimum enzyme denaturation. Chitin hydrolysis with CH-AS could be carried out in a semi-batch mode with intermittent enzyme precipitation and product removal, this can enhance product yield up to 1.4-fold when compared with batch reaction.

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

Abstract: The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions.

Site-directed Mutagenesis of the Asp-197 and Asp-202 Residues in Chitinase A1 of Bacillus circulans WL-12

Abstract: The contribution of the Asp-197 and Asp-202 residues in chitinase A1 of Bacillus circulans WL-12 to the catalytic reaction was studied by site-directed mutagenesis of these residues. A kinetic analysis of the purified mutant chitinases suggests the involvement of both the Asp-197 and Asp-202 residues in the catalytic events of this enzyme, although the effects of mutations of Asp-197 were less severe than those of the other mutations.