Showing papers on "Chitinase published in 2001"

Induction of systemic resistance in rice against sheath blight disease by Pseudomonas fluorescens

Abstract: Two Pseudomonas fluorescens strains viz., PF1 and FP7 which inhibited the mycelial growth of sheath blight fungus Rhizoctonia solani and increased the seedling vigour of rice plants in vitro were selected for assessing induced systemic resistance (ISR) against R. solani in rice. The Pseudomonas application as a bacterial suspension or a talc-based formulation through seed, root, soil and foliar application either alone or in combination (seed+root+soil+foliar) effectively reduced sheath blight disease incidence, promoted plant growth and ultimately increased yields under glasshouse or field conditions. Efficacy of Pseudomonas strains against R. solani was comparable to that of the fungicide carbendazim, which is normally used in the field to manage the disease. Pseudomonas treatment of rice cv IR50 led to induction of systemic resistance against R. solani , as a result of increase in chitinase and peroxidase activity. However, the extent of increase varied between treatments, Pseudomonas strains used and their duration. Though two chitinase isoforms (35 and 28 kDa) and five peroxidase isozymes (PO1–PO5) were found to be associated with the ISR, 35 kDa chitinase and three peroxidase isozymes (PO3–PO5) were established as the major determinants of ISR. Although a single application of a Pseudomonas strain resulted in ISR, the combined application through all of the four (seed, root, soil and foliar) methods increased the durability of ISR in rice plants. In addition, the Pseudomonas strains produced chitinase in the culture medium. It is presumed that the induced chitinase, peroxidase and bacterial chitinase may be either directly or indirectly involved in the reduction of sheath blight disease development in rice.

Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Abstract: Of 24 Trichoderma isolates, T. harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and β-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa β-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (β-1,3-glucanase). Km and Kcat were 3.8 g l–1 and 0.71 s–1 for the chitinase (chitin) and 1.1 g l–1 and 52 s–1 for the β-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the β-1,3-glucanase was specific for β-1,3-linkages in polysaccharides. Both enzymes were stable at 30°C, while at 60°C the chitinase and the β-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 µg/ml.

Heterologous Expression of Genes Mediating Enhanced Fungal Resistance in Transgenic Wheat

Abstract: Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with nontransgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm 2 .

Isolation and characterization of novel cDNA clones of acidic chitinases and β-1,3-glucanases from wheat spikes infected by Fusarium graminearum

Abstract: Chitinases and β-1,3-glucanases are important components of plant defense in response to attack by pathogens. To identify specific chitinases and β-1,3-glucanases, we constructed a cDNA library using mRNA from wheat spikelets inoculated with conidia of Fusarium graminearum. Two chitinase and two β-1,3-glucanase clones were isolated using a rice chitinase Ia gene and barley cDNA clones for chitinase II and β-1,3-glucanase as probes. Sequence analysis showed that the cDNA clone SM194 encodes an acidic isoform of class-VII chitinase, the cDNA clone SM383 encodes a class-IV chitinase and the cDNA clones SM289 and SM638 encode two different acidic isoforms of β-1,3-glucanases. Nulli-tetrasomic analysis indicated that SM194 and SM383 were located on all of the group-2 chromosomes of wheat. Genetic mapping showed that at least three copies of class-IV and/or class-VII chitinase genes were clustered on the long arm of chromosome 2D of Aegilops tauschii and that they mapped genetically close to the centromere. SM289 and SM638 were located on all of the group 3 chromosomes of wheat by nulli-tetrasomic analysis, and to the β-1,3-glucanase clusters in the 3BL and 3DL chromosome arms of wheat by genetic mapping. Northern blot hybridization showed that the expression of these genes is induced upon infection with Fusarium graminearum. The accumulation of transcripts for these PR-proteins was more rapid in the resistant variety Sumai 3 than in its susceptible mutant during the first 24 h. This is the first report of the induction of class-IV and class-VII chitinases in cereals by a fungal pathogen.

Salicylic Acid and Ethylene Pathways Are Differentially Activated in Melon Cotyledons by Active or Heat-Denatured Cellulase from Trichoderma longibrachiatum

Abstract: Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3.

Abstract: Stenotrophomonas maltophilia strain C3, a biocontrol agent of Bipolaris sorokiniana in turfgrass, produced chitinases in broth media containing chitin. Chitinases were partially purified from culture fluid by ammonium sulfate precipitation and chitin affinity chromatography. The chromatography fraction with the highest specific chitinase activity was inhibitory to conidial germination and germ-tube elongation of B. sorokiniana, but it was less inhibitory than the protein fraction or the raw culture filtrate. The fraction exhibited strong exochitinase and weak endo-chitinase activity. Optimum temperature and pH for chitinase activity were 45 to 50 degrees C and 4.5 to 5.0, respectively. Chitinase activity was inhibited by Hg(2+) and Fe(3+), but not by other metal ions or enzyme inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the chromatography fraction revealed the presence of five protein bands of 25, 32, 48, 65, and 75 kDa. Partial amino acid sequences of the 32-, 65-, and 75-kDa proteins indicated that they are homologous to known bacterial chitinases. There was no homology found in the partial amino acid sequences of the 25- and 48-kDa proteins to any known chitinases. Five chitinase-active proteins were detected in the protein and chromatography fractions by activity gels, but when each protein was extracted and re-electrophoresed separately under denaturing conditions, only 32- or 48-kDa proteins were revealed. It was concluded that strain C3 produces at least two chitinases that are antifungal.

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence

Abstract: β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.

Purification of a thermostable endochitinase from Streptomyces RC1071 isolated from a cerrado soil and its antagonism against phytopathogenic fungi.

Abstract: Aims: The chitinolytic activity of an actinomycete, isolated from a tropical acidic ferrasol (FAO) under cerrado (savanna) vegetation, is reported. Methods and Results: Selection of the strain was based on spot inoculation on solid colloidal chitin medium. The use of chemotaxonomic, morphological and physiological procedures placed it in the Streptomyces genus, but identification to species level could not be achieved. A protein with endochitinase activity was isolated and purified from the supernatant fluid by concentration, precipitation, hydrophobic interaction, gel filtration and adsorption procedures. The molecular size of the purified chitinase was estimated by gel filtration to be 70 kDa, and its pI was 6·1. The enzyme had temperature and pH optima of 40°C and 8·0, respectively, and showed thermal (30–70°C) and pH (4–9) stabilities. Antifungal activity of the selected strain was observed following in vitro experiments using growing cells, crude extract or the purified endochitinase, and by detecting growth inhibition of the tested phytopathogenic fungi. Conclusions: Strain Streptomyces RC 1071 could not be placed into any known species, suggesting a new taxon. The purified endochitinase presented similar molecular weight, optimum temperature and pH activity, and stability of other endochitinolytic enzymes reported in the literature. In all three in vitro experiments performed, inhibition of growth of the phytopathogenic fungi used as test organisms was observed. Significance and Impact of the Study: Some of the endochitinase characteristics such as thermal stability, as well as pH tolerance, are very interesting for biotechnological purposes. In addition, due to its antifungal activity, Streptomyces RC 1071 seems promising for use in biological control.

Chitinase from Bacillus thuringiensis subsp. pakistani

Abstract: The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.

Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum.

Abstract: We investigated the regulation of chit33 expression in Trichoderma harzianum CECT 2413. This gene encodes the Chit33 endochitinase, which is a major component of the fungus' chitinolytic enzyme system and is important for biocontrol. To this end, both Northern analysis and reporter gene fusions of a 1.4-kb fragment of the 5'-upstream sequences of chit33 to the Aspergillus niger goxA gene (encoding glucose oxidase) and the Aquorea victoria green fluorescent protein were used. Northern analysis and data obtained with the reporter systems were compatible, thus showing that the 1.4-kb fragment bears all necessary information for the regulation of chit33 gene expression. chit33 is weakly expressed during growth on chitin and Rhizoctonia solani cell walls. The addition of N-acetylglucosamine transiently induced chit33 expression in resting cells of the fungus. The addition of either glucose or glycerol prevented induction of chit33 gene expression by chitin or cell walls. Incubation of T. harzianum in the presence of low concentrations (0.1%, w/v) of glucose and high concentrations (38 mM) of ammonium sulfate, or in the presence of high concentrations (1%, w/v) of glucose and low concentrations (0.38 mM) of ammonium sulfate also stimulated chit33-mRNA accumulation, although to a lower degree than induction by N-acetylglucosamine. Transfer of T. harzianum cultures to either 40 degrees C or 4 degrees C initiated a very rapid expression of chit33 in the absence of an inducer, yet only at very low levels (5%) of the induced control. Confrontation experiments, using the gfp gene as a reporter and R. solani as a host, showed that chit33 is expressed only during but not before the stage of overgrowth on R. solani. These data show that Chit33 is an enzyme involved in mycoparasitism; and its formation is controlled by induction, by either carbon or nitrogen starvation and, to a low degree, also under conditions of temperature stress.

Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential in antifungal biocontrol

Abstract: A novel, highly chitinolytic strain of Alcaligenes xylosoxydans was isolated which showed potential for use as an antifungal biocontrol agent for the control of two fungal plant pathogens It could degrade and utilize dead mycelia of Rhizoctonia bataticola and Fusarium sp (fungal plant pathogens of Cajanus cajan) In vitro it could inhibit the growth of Fusarium sp and R bataticola Chitin at 10–15 g/l was found to be good carbon and nitrogen source Alcaligenes xylosoxydans showed optimum chitinase production at 72 h, pH optima at 8 and growth peak at 120 h Yeast extract, arabinose, Tween 20 and several other surfactants enhanced chitinase production

A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407

Abstract: Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 degrees C, and was inhibited strongly by Hg2+, Pb2+, Ag+, Fe2+, Mn2+ and Zn2+. The enzyme was stable over a broad pH range 4-8 and below 45 degrees C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg.mL-1 and 1.84 mg.mL-1, respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endo- and exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum

Abstract: Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.

A class I chitinase from soybean seed coat

Abstract: Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.

Cloning, Sequences, and Characterization of Two Chitinase Genes from the Antarctic Arthrobacter sp. Strain TAD20: Isolation and Partial Characterization of the Enzymes

Abstract: Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA andArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (881 amino acids [aa]) and ArChiB (578 aa). ArChiA andArChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain ofArChiA exhibits 55% identity with a chitodextrinase fromVibrio furnissii. The ArChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans. The ArChiA chitin-binding domains are homologous to the chitin-binding domain of ArChiB.ArChiA and ArChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of ArChiA,ArChiB, and chitinase A of Serratia marcescenswas studied using differential scanning calorimetry.ArChiA and ArChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.

Interaction of ammonium, glucose, and chitin regulates the expression of cell wall-degrading enzymes in Trichoderma atroviride strain P1.

Abstract: Chitinolytic and glucanolytic fungal cell wall-degrading enzymes have been suggested to be primary determinants of biocontrol by Trichoderma spp. We examined the effects of ammonium, glucose, chitin, and chito-oligomers on transcription of specific genes and secretion of fungal cell wall-degrading enzymes. The genes ech42, nag1, and gluc78 were examined, as were the enzymes they encode (endochitinase CHIT42, N-acetylhexosaminidase CHIT73, and glucan exo-1,3-β-glucanase GLUC78, respectively). gluc78 could be induced by nitrogen starvation alone, while both ech42 and nag1 required nitrogen starvation and the presence of chitin for induction. Starvation for both ammonium and glucose resulted in very early expression and secretion of all cell wall-degrading enzymes examined. In the presence of low levels of ammonium (10 mM), both chito-oligomers and chitin triggered CHIT42 and CHIT40 (chitobiosidase) production. CHIT73 secretion occurred in the presence of N-acetylglucosamine and chito-oligomers, while chitin was less effective. The presence of different chito-oligomers resulted in secretion of specific N-acetylhexosaminidases, of which CHIT73 is one. Our results indicate that the expression and secretion of cell wall-degrading enzymes is nitrogen repressed, that effects of carbon and nitrogen nutrition are interactive, and that especially for chitinolytic enzymes, the inductive effect of chitin is altered by the level of ammonium or glucose in the medium.

Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385.

Abstract: A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.

β‐1,3‐Glucanase and Chitinase Activities and the Resistance Response of Wheat to Leaf Rust

Abstract: To investigate the role of β-1,3-glucanases (EC 3.2.1.39) and chitinases (EC 3.2.1.14) in the resistance response of wheat (Triticum aestivum L.) to leaf rust (Puccinia recondita f. sp. tritici), their intercellular activities were studied in resistant (Karee/Lr35) and susceptible (Karee) near-isogenic wheat lines under conditions of infection and non-infection. Resistance was associated with high constitutively expressed chitinase activity and induced β-1,3-glucanase activity. At 168 hours post-inoculation several infection sites on Karee/Lr35 displayed necrosis, confirming a hypersensitive response, whereas similar necrotic lesions were observed only for a limited number of infection sites on Karee at that sampling stage. The possible role of chitinases and β-1,3-glucanases in the hypersensitive defence response of wheat to leaf rust is discussed.