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Showing papers on "Chitinase published in 1992"


Journal ArticleDOI
TL;DR: This review article deals with recent developments in molecular and physiological aspects of chitinases from plants, fungi, bacteria, insects and fishes.
Abstract: This review article deals with recent developments in molecular and physiological aspects of chitinases from plants, fungi, bacteria, insects and fishes

343 citations


Journal ArticleDOI
TL;DR: The results demonstrate that near-ambient O(3) levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.
Abstract: 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.

282 citations


Journal ArticleDOI
TL;DR: Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with Chitin as the sole carbon source, suggesting that each protein is encoded by a different gene.
Abstract: Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n= 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n= 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.

265 citations


Journal ArticleDOI
TL;DR: Comparison of the amino acid sequences of maize seed chitinases with those of previously published chit inases from monocot and dicot plants indicates that maize seed Chitinase A and B have diverged significantly from other chitInases.

173 citations


Journal ArticleDOI
TL;DR: The results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted and cannot be explained by simple Mendelian inheritance.
Abstract: The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozygous for a chimeric tobacco (Nicotiana tabacum) chitinase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-transformed plants. Unexpectedly, some transgenic plants accumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, approximately 25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Silencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-dependent silencing in which expression was uniform within individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was examined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheritance. Taken together, the results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppression, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated with cytosine methylation of the transgene at the five restriction sites investigated.

159 citations


Book ChapterDOI
01 Jan 1992
TL;DR: It has been proposed that s-1,3-glucanases may have a role in fruit ripening, pollen tube growth, coleoptile growth, regulation of transport through vascular tissues, cellulose biosynthesis and cell division.
Abstract: The endo-type glucanohydrolases s-1,3-glucanase (E.C. 3.2.1.39) and chitinase (E.C. 3.2.1.14) are abundant proteins widely distributed in seedplant species (Clarke and Stone, 1962; Ballance and Manners, 1978; Powning and Irzykiewicz, 1965). The physiological functions of s-1,3- glucanase and chitinase are not known. Based on the distribution of the enzyme and its putative substrates such as callose, it has been proposed that s-1,3-glucanases may have a role in fruit ripening (Hinton and Pressey, 1980), pollen tube growth (Roggen and Stanley, 1969; Ori et al., 1990), coleoptile growth (Masuda and Wada, 1967), regulation of transport through vascular tissues (Clarke and Stone, 1962), cellulose biosynthesis (Meier et al., 1981) and cell division (Waterkeyn, 1967; Fulcher et al., 1976). Although the existence of other substrates has not been ruled out, chitin, the known substrate of chitinase, is not found in higher plants.

152 citations


Journal ArticleDOI
TL;DR: It is proposed that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chit inases serve as biotic elicitors of defense-related responses in rice.
Abstract: Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched β-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice.

152 citations


Journal ArticleDOI
Takehiko Watanabe1, W Oyanagi1, Kazushi Suzuki1, K. Ohnishi1, H Tanaka1 
TL;DR: A 73-amino-acid segment located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitInases and class III higher-plant chit inases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit.
Abstract: The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed.

139 citations


Journal ArticleDOI
TL;DR: The results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting, such as demethylallosamidin, a much better protector against lysis.
Abstract: Summary: Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

138 citations


Journal ArticleDOI
TL;DR: The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress and Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of β-1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chit inase and ‘pathogenesis-related’ (PR) 1b mRNA levels.
Abstract: Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 microliter/l, 5 h) markedly increased the mRNA level of basic beta-1,3-glucanase and to a lower degree that of basic chitinase. The increase of beta-1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the beta-1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of beta-1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of beta-1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of beta-1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and 'pathogenesis-related' (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.

125 citations


Journal ArticleDOI
TL;DR: Chitinase (EC 3.2.1.14), from the culture filtrate of Trichoderma harzianum, was successively purified by precipitation with ammonium sulfate followed by ion-exchange chromatography on Q-Sepharose, gel filtration on Sephadex G-100, and hydrophobic interaction on Phenyl- Sepharose CL-4B.

Journal ArticleDOI
TL;DR: Inhibition has only been observed of spore germination, of cell separation in budding yeasts and of the action of a yeast toxin, and not of apical extension or branching, but the finding that chitinase activities in situ have different properties to those in cell homogenates, and appear to be protected from environmental stresses is explained.
Abstract: Filamentous fungi with chitin as a major component of their cell walls produce chitinases at all stages of active growth, i.e. during spore germination, exponential growth and mycelial development. The roles of chitinases in these processes have been investigated by assessing the effects of treatment with the inhibitor, allosamidin. Inhibition has only been observed, however, of spore germination, of cell separation in budding yeasts and of the action of a yeast toxin, and not of apical extension or branching. This may be explained by the finding that chitinase activities in situ have different properties to those in cell homogenates, and appear to be protected from environmental stresses.

Journal ArticleDOI
Kay A. Lawton1, Eric R. Ward1, George Payne1, Mary B. Moyer1, John Ryals1 
TL;DR: The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.
Abstract: Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.

Journal ArticleDOI
TL;DR: It is suggested that the growth inhibition of Trichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.
Abstract: Plant chitinases and β-1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and β-1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.

Journal ArticleDOI
TL;DR: Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and it is concluded that these chit inases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the pro sequences are located at the C terminal.
Abstract: Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.

Journal ArticleDOI
TL;DR: It is suggested that direct repeat sequences present in other chitinase genes recently characterized may be involved in repression and induction for this entire class of catabolite-controlled genes.
Abstract: We report the identification and partial characterization of the promoters for two chitinase genes from Streptomyces plicatus. Chitinases are a family of enzymes made by Streptomyces and other soil microbes to digest chitin, an abundant source of carbon and nitrogen in the soil. The promoter regions of two chitinases were defined by using transcriptional fusions to the xylE reporter gene. Transcription was shown to be glucose-sensitive and chitin-dependent. Each promoter contains a putative RNA polymerase binding site with a recognition sequence very similar to that observed in many eubacterial vegetatively expressed genes. In both promoters, a pair of 12-base-pair direct repeat sequences overlap the putative RNA polymerase binding sites. Further analysis of one of the promoters revealed that a single-base change within the direct repeat sequences resulted in glucose-resistant, chitin-independent expression in vivo. In addition, the promoter region that includes the direct repeat sequences was shown to interact with a sequence-specific DNA binding factor in vitro. Similar direct repeat sequences are present in other chitinase genes recently characterized, and we suggest that these repeats may be involved in repression and induction for this entire class of catabolite-controlled genes.

Journal ArticleDOI
TL;DR: This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitIn-binding domain and a fusion of the chit in-binding domains to a structurally unrelated domain, the chItinase domain.


Journal ArticleDOI
TL;DR: Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity that exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro.
Abstract: Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium.

Journal ArticleDOI
TL;DR: One major additional band with chitinase activity could be detected in Allium porrum (leek) root extracts after colonization by Glomus versiforme or GlomUS intraradix when compared to control root extracts by using a two-dimensional polyacrylamide gel electrophoresis (PAGE) system.

Journal ArticleDOI
TL;DR: Southern hybridization analysis indicates that the chitinase is encoded by a small family of genes.
Abstract: A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. Oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24 amino acid N-terminal signal peptide, a cysteine-rich domain and a catalytic domain. The primary structure of the mature ChB4 shows a low degree of identity with class I and II chitinases, 43–48% and 35% respectively. In contrast, ChB4 shows 62% identity to a basic sugar-beet chitinase and 63% identity to an acidic chitinase from bean. The expression of chitinase messenger RNA (mRNA) in response to infection with Phoma lingam (Tode ex. Fr.) Desm. was examined by northern hybridization and scintilation counting. A differential induction was seen between resistant and susceptible cultivars where 3-fold higher chitinase transcript levels were estimated one day after inoculation in resistant as compared to susceptible cultivar. This difference diminished eight days after inoculation. Southern hybridization analysis indicates that the chitinase is encoded by a small family of genes.

Journal ArticleDOI
01 Jun 1992-Planta
TL;DR: It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.
Abstract: Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In "effective", nitrogen-fixing nodules, colonized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascorbate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. "Ineffective" nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of "ineffective" nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an antiserum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of Mr 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.

Journal ArticleDOI
TL;DR: The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed.
Abstract: A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.

Journal ArticleDOI
TL;DR: Five extracellular chitinases of 20.5, 30, 47, 70, and 92 kDa purified from the culture filtrate of Streptomyces olivaceoviridis ATCC 11238 found to be resistant to proteolysis by trypsin, papain, and Staphylococcus aureus V8 protease.
Abstract: Five extracellular chitinases of 20.5, 30, 47, 70, and 92 kDa purified from the culture filtrate of Streptomyces olivaceoviridis ATCC 11238 differed in their sequences at the amino termini of the protein chains. In the native state, the chitinases were found to be resistant to proteolysis by trypsin, papain, and Staphylococcus aureus V8 protease. The latter produced several fragments of identical molecular mass from chitinases denaturated with sodium dodecyl sulfate. Five proteases were detected in the protein concentrate from the culture filtrate, and two of them showing ability to cleave chitinases in the native state were purified. One, a protease of 42 kDa, released a 30-kDa protein from the 70-kDa chitinase that reacts with anti-30 kDa chitinase antibodies; the other, a protease of 29 kDa, split the 30-kDa chitinase into 20.5-, 18-, and 16-kDa fragments. From these results, it was deduced that the 70-kDa chitinase is the precursor protein of the 30- and 20.5-kDa chitinases.

Journal ArticleDOI
TL;DR: Genomic clones representing two members of the gene subfamily encoding these enzymes in Nicotiana tabacum showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae.
Abstract: The fungicidal class I chitinases are believed to be important in the induced defense response of plants. We isolated and partially characterized genomic clones representing two members, CHN14 and CHN50, of the gene subfamily encoding these enzymes in Nicotiana tabacum L. cv. Havana 425. The coding sequences of genes CHN14, CHN50, and CHN48, which was cloned earlier, are identical at 79–95% of the positions. Tobacco is an amphidiploid species derived from ancestors most closely related to the present-day species N. sylvestris and N. tomentosiformis. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and partial sequencing of the purified enzymes suggest that the gene pairs CHN48/CHN50 and CHN14/CHN14′ are homeologues. Gene CHN48, which encodes chitinase A (Mr ca. 34 kDa), and gene CHN14 are derived from N. tomentosiformis; whereas gene CHN50, which encodes chitinase B (Mr ca. 32 kDa), and gene CHN14′ are derived from N. sylvestris. Class I chitinases are induced in leaves of plants treated with ethylene or infected with the fungal pathogen Cercospora nicotianae and in cultured cells transferred to medium without added auxin and cytokinin. RNase protection assays show that under these conditions transcripts encoded by the homeologues CHN48 and CHN50 account for > 90% of the total chitinase mRNA. The less abundant transcript, CHN48, consistently showed a greater degree of induction than CHN50. Expression of the homeologues CHN14 and CHN14′ represented < 10% of the total chitinase mRNA. They showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae. Therefore the two sets of homeologues are regulated in the same way by hormones and respond differently to infection by a pathogen.

Journal ArticleDOI
TL;DR: It is suggested that exogenous chitinases cause premature hatch of nematode eggs and could be used as an aid in the control of nem atodes.
Abstract: Chitinases from Phaseolus vulgaris, Serratia marcescens and Streptomyces griseus were compared for their effects on eggs and juveniles of Meloidogyne hapla Chitwood. Increased hatch rates of nematode eggs were observed on incubation with chitinase from all three sources. After treatment with chitinases from S. marcescens and St. griseus many of the eggs remaining became spherical with a concomitant increase in the number of dead juveniles. Egg mortality was observed with purified P. vulgaris chitinase at concentrations of 17 μg protein/ml and above. The results suggest that exogenous chitinases cause premature hatch of nematode eggs and could be used as an aid in the control of nematodes.

Journal ArticleDOI
TL;DR: The localization of chitinase and 1,3-β-glucanase in planta with immunogold labelling techniques is reported and degenerated material, most probably of fungal origin, was observed, which showed strong labelling in incompatible interactions.

Journal ArticleDOI
21 Oct 1992-Gene
TL;DR: The complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chi1 is determined and the Chi1 sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans.

Journal ArticleDOI
31 Jul 1992-Science
TL;DR: Two tobacco chitinases represent a different class of hydroxyproline-containing proteins, which are predominantly extracellular, structural glycoproteins proteins that lack enzymatic activity and contain many hydroxyProline residues.
Abstract: The fungicidal type I chitinases contribute to the defense response of plants against pathogens. Two tobacco chitinases represent a different class of hydroxyproline-containing proteins. Hydroxyproline-rich proteins are predominantly extracellular, structural glycoproteins proteins that lack enzymatic activity and contain many hydroxyproline residues. In contrast, type I chitinases are vacuolar enzymes. They are not glycosylated and contain a small number of hydroxyproline residues restricted to a single, short peptide sequence.

Journal ArticleDOI
TL;DR: Almost all the flower chitinase activity is localized in the stigma and increases about five-fold therein following anther dehiscence, which strongly suggests the plant chit inase has a heretofore unrecognized specific function in the sexual reproduction of higher plants.