Induction Of Chitinase Research Articles

Chitinases of fungi and plants: their involvement in morphogenesis and host-parasite interaction

Abstract There has been a considerable amount of recent research aimed at elucidating the roles of chitinase in fungi and plants. In filamentous fungi and yeasts, chitinase is involved integrally in cell wall morphogenesis. Chitinase is also involved in the early events of host-parasite interactions of biotrophic and necrotrophic mycoparasites, entomopathogenic fungi and vesicular arbuscular mycorrhizal fungi. In plants, induction of chitinase and other hydrolytic enzymes is one of a coordinated, often complex and multifaceted defense mechanism triggered in response to phytopathogen attack. Chitinase induction in plants is not considered solely as an antifungal resistance mechanism. Plant chitinases can be induced by various abiotic factors as well and there is some circumstantial evidence to suggest a morphogenetic role despite the apparent absence of the substrate in plant cells. Finally, some chitinases and other chitin-binding proteins including some plant lectins share chitin-binding domains as part of their molecular structure and provide fuel for the so-called ‘lectin-chitinase’ debate and speculation for the origin of chitinase in plants.

Chitinases of fungi and plants: their involvement in morphogenesis and host-parasite interaction

There has been a considerable amount of recent research aimed at elucidating the roles of chitinase in fungi and plants. In filamentous fungi and yeasts, chitinase is involved integrally in cell wall morphogenesis. Chitinase is also involved in the early events of host—parasite interactions of biotrophic and necrotrophic mycoparasites, entomopathogenic fungi and vesicular arbuscular mycorrhizal fungi. In plants, induction of chitinase and other hydrolytic enzymes is one of a coordinated, often complex and multifaceted defense mechanism triggered in response to phytopathogen attack. Chitinase induction in plants is not considered solely as an antifungal resistance mechanism. Plant chitinases can be induced by various abiotic factors as well and there is some circumstantial evidence to suggest a morphogenetic role despite the apparent absence of the substrate in plant cells. Finally, some chitinases and other chitin-binding proteins including some plant lectins share chitin-binding domains as part of their molecular structure and provide fuel for the so-called ‘lectin-chitinase’ debate and speculation for the origin of chitinase in plants.

Chitinase induction in onion tissue cultures

Chitinase activity in Allium cepa (onion) tissue cultures was investigated using a radioactive assay and polyacrylamide gel electrophoresis (PAGE). Increased chitinase activity was detected in callus cultured on agar-solidified media for 72 h with 1 mM salicylic acid or 0.05 M ethrel, and in callus cultured in liquid suspension for 72 h with 0.1 mM salicylic acid or 0.1 M ethrel. Non-denaturing PAGE analysis of callus suspension cultures treated with salicylic acid revealed a new chitinase activity which accumulated in the fluid of the suspension cultures.

Plant chitinases

Plant chitinases

Calcium Requirement for Ethylene-Dependent Responses.

Ethylene, a gaseous plant hormone, plays a role in plant development, defense, and climacteric fruit ripening. Both genetic and biochemical evidence suggest that the response of plants to ethylene is mediated by a specific ethylene receptor. The signal emanating from the receptor-effector complex is then presumably transduced via an unknown cascade pathway. We have used the plant pathogenesis response, exemplified by the induction of the pathogenesis-related gene chitinase, as a paradigm to investigate ethylene-dependent signal transduction in the plant cell. We showed that calcium is necessarily involved in the ethylene-mediated pathogenesis response. Blocking calcium fluxes with chelators inhibited ethylene-dependent induction of chitinase accumulation, but not ethylene independent induction. Artificially increasing cytosolic calcium levels by treatments with the calcium ionophore ionomycin or the calcium pump blocker thapsigargin stimulated chitinase accumulation. Plants grown in calcium-poor soil showed a 10-fold reduction in leaf extractable calcium. Their leaves exhibited a reduced pathogenesis reaction to ethylene and were impaired in another hormone response mediated by calcium, i.e., abscisic acid-controlled closure of guard cells. The addition of calcium to leaves excised from calcium-deficient plants restored their sensitivity to ethylene. Ethylene participates in the control of seedling growth, promoting the so-called "triple response" that results in distinct morphological development, such as hypocotyl hook formation. This effect, similar to the ethylene-promoted pathogenesis response, was found to be calcium dependent. The results indicate that calcium is required for a variety of ethylene-dependent processes.

Induction and purification of chitinase in Brassica napus L. ssp. oleifera infected with Phoma lingam

A pathogen-induced chitinase (EC 3.2.1.14) was isolated from cotyledons of oilseed rape (Brassica napus cv. Bienvenu) 8 d after inoculation with Phoma lingam. The purified chitinase has a molecular weight of 30 kDa, and an isoelectric point of approx. 9.1. A partial amino-acid sequence obtained after tryptic digestion of the protein shows high sequence similarity to basic chitinases from bean, tobacco, potato, Arabidopsis, barley and rice, as well as to acidic chitinases from tobacco and petunia. A close serological relationship was found between the chitinase isoenzyme and an isoenzyme from sugar-beet (Beta vulgaris L.). When resistant and susceptible cultivars were inoculated with P. lingam there was a significant difference in the increase in chitinase activity during the early stage after inoculation. The resistant cultivars showed a rapid increase in chitinase activity, in contrast to susceptible cultivars where an increase in activity was delayed until 24 h after infection. By measuring the chitinase activity from the mycelium of P. lingam, it was concluded that the increase in chitinase activity found in infected plants was of plant origin. The chitinase activity was found to be restricted to the site of pathogen attack and was not systemically induced in other parts of the plant.

Temporal and spatial patterns of 1, 3‐β‐glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

SummaryInfection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3‐β‐glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3‐β‐glucanase induction were studied in further detail by immunohistochemical and in‐situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non‐infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3‐β‐glucanase mRNA, phenyl‐alanine ammonia‐lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar‐race‐specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3‐β‐glucanase and chitinase is presumably not involved in determining this specificity.

Induction by chromium ions of chitinases and polyamines in barley ( Hordeum vulgare L.) and rape ( Brassica napus L. ssp. oleifera)

Barley and rape seedlings were grown in hydroponic culture with increasing concentrations of CrO 3 (Cr(VI)) of CrCl 3 (Cr(III)). The chitinase activity and the concentrations of putrescine, spermidine and spermine were determined in the third leaf of barley seedlings and in the second leaf of rape seedlings. With increasing concentrations of Cr(VI) (30–100 μg/l) a 5–10-fold increase in chitinase activity and 4–5-fold increase in putrescine concentrations were found in barley and rape seedlings after 4 days. Levels of spermidine and spermine were not consistently influenced by exposure to Cr(VI). Cr(III) at concentrations of 10–50 μg/ml did not significantly alter the concentrations of polyamines nor the chitinase activities in either species, however, at 100 μg/ml in barley Cr(III) induced an increase in putrescine concentration after 6 days of exposure. The induction of chitinases and the increases in the putrescine level caused by Cr(VI) but not by Cr(III) (10–50 μg/ml) exposure is similar in the two species suggesting an analogous defense system in both mono- and di-cotyledonous plants. The anionic form of Cr(VI) seems to be more potent than the cationic form of Cr(III). Grain of barley plants grown in enriched peat and watered with increasing concentrations of Cr(VI) (250, 1250, 6250 or 10 000 μg/ml) for 4 days from 10 days post anthesis (dpa) showed increasing contents of chitinases.

Induction Patterns of Chitinases in Yam Callus by Inoculation with Autoclaved Fusarium oxysporum, Ethylene, and Chitin and Chitosan Oligosaccharides

Chitinase involved in plant self-defense was investigated by inoculation of yam (Dioscorea opposita Thunb) callus with autoclaved mycelia of Fusarium oxysporum Schl. f. sp. raphani Kend. et Snyd., ethylene, and chitin and chitosan oligosaccharides. The induction of chitinase was followed by measuring the enzyme activity, and the isozyme pattern was also analyzed by activity staining and immunoblotting after disc- and SDS-PAGE. Autoclaved Fusarium oxysporum induced acidic chitinases named El and E3 with MW 33, 500 in the callus, while ethylene and oligosaccharides of chitin and chitosan induced some chitinases more basic than E1 and E3, in addition to E1 and E3. These included large chitinases with MW 38, 000 and immunologically different chitinases from E1 and E3. In total, nine chitinases were induced. Isozyme patterns of chitinase induced were different among the elicitors used, although E1 and E3 were commonly induced in all cases. Oligosaccharides of chitin and chitosan showed different chain-length d...

Induction of Chitinase in Resistant and Susceptible Varieties of Chickpea (Cicer arietinum L.) following Infection with Ascochyta rabiei

Chickpea genome, as revealed by chitinase specific probe, was found to possess 5 copies of chitinase gene. Chitinase activity assayed in Ascochyta resistant (G40), partially resistant (Pusa 261) and susceptible (Pusa 209) varieties of chickpea following infection with Ascochyta rabiei revealed significant induction in the levels of the enzyme in all the three varieties. However, G40 exhibited 3 times higher chitinase activity in comparison to Pusa 209. Chitinase specific mRNA showed a parallel increase upon infection. The results suggest that regulation of chitinase activity upon infection is at the transcriptional level.

Induction of Chitinases in Rice Callus Treated with Chitin Derivatives

Induction of Chitinases in Rice Callus Treated with Chitin Derivatives

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi

Chitinase (EC 3.2.1.14.) activity increased in pea (Pisum sativum L.) leaves inoculated with both virulent and avirulent isolates of Ascochyta pisi Lib. Three basic chitinase isoenzymes were purified: two, A1 and A2, separated by high performance liquid chroma tography, had a relative molecular mass (Mr) of approx. 34 000, with an isoelectric point (pI) of 8.5, and one, B, had an Mr of approx. 25 000, with a pI of 9.0. Elevated levels of chitinase isoenzymes were found in the leaves: thus 150 h after inoculation, there was a ninefold increase for isoenzymes A1 and A2 combined, with a threefold increase for isoenzyme B in susceptible plants, and a sevenfold increase for isoenzymes A1 and A2 combined, with a twofold increase for isoenzyme B in hypersensitive-reacting plants. The N-terminal 15-23 amino-acid residues of the three chitinase isoenzymes were sequenced, and considerable sequence similarity was found to chitinase sequences from tobacco, potato and bean, as well as to hevein and wheat-germ agglutinin. Purified chitinase protein cross-reacted with specific antiserum raised against a chitinase isoenzyme from sugar beet (Beta vulgaris L.). Immunoblots prepared using leaf proteins isolated at different time points following inoculation revealed the accumulation of polypeptides corresponding to at least two of the chitinase isoenzymes.

Induction of Chitinases in Rice Callus Treated with Chitin Derivatives.

Induction of Chitinases in Rice Callus Treated with Chitin Derivatives.

Local and systemic induction of peroxidase, chitinase and resistance in cucumber plants by K 2HPO 4

Spraying the lower surface of the first true leaf (leaf 1) of cucumber plants with 50 m m K 2HPO 4 induced systemic resistance to anthracnose caused by Colletotrichum lagenarium, 2 to 7 days later. Within 16 h of application, the activities of peroxidase and chitinase increased in the induced leaf, and they continued to increase over the next 7 days reaching levels at least ten-fold higher than in leaf 1 of plants treated with water. During this period, the activities of both enzymes were usually two-fold or higher in leaf 2 of plants induced with K 2HPO 4 than in plants treated with water. If the induced plants were not further treated or mock-challenged with water on leaf 2, the enzyme levels increased slightly and levelled out at between two or four-fold higher than in the corresponding control plants. If plants were challenged with C. lagenarium on leaf 2, the enzyme activity at the site of challenge increased in the induced plants prior to rising in the control plants. Large increases in enzyme activity were only evident after the lesions became visible (3–4 days after challenge) in the control plants and this only occurred at the site of challenge. Seven days after challenge, the enzyme activities in the leaf tissue adjacent to the site of inoculation were similar in both the control and induced plants. Correlations were made between peroxidase and chitinase activities induced by several treatments on leaf 1 and the level of protection observed in leaf 2 after challenge with C. lagenarium.

Induction of chitinase and β-1,3-glucanase in tobacco plants infected with Pseudomonas tabaci and Phytophthora parasitica var. Nicotianae

We have measured by rocket immunoassay the glucan endo-1,3-β-glucosidase (β-1,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14) contents of Nicotiana tabacum cv. “Havana 425” plants following infection with the bacterial pathogen Pseudomonas tabaci and the fungal pathogen Phytophtorara parasitica var. nicotianae. Both enzymes were induced, relative to non-infected controls, in leaves infected with P. tabaci and in stems and leaves infected with P. parasitica. Increases in enzyme content were confined to infected parts of the plant indicating that this response is local rather than systemic. The β-1,3-glucanase- and chitinase-mRNA content of tissues was measured by RNA blot hybridization using the inserts of cDNA clones for the tobacco mRNAs as probes. P. tabaci and P. parasitica infection increased the mRNA content relative to controls indicating that both pathogens act at least in part at the level of mRNA accumulation. As judged by immunological tests and RNA hybridization, the β-1,3-glucanase and chitinase induced by P. tabaci and P. parasitica are the same basic isoforms known to be induced by tobacco mosaic virus. Therefore the induction appears to be a non-specific response of the host to pathogens.

Chitinase isoenzymes induced in carrot cell culture by treatment with ethylene

In cultured carrot cells, treatment with exogenously supplied ethylene or 1-aminocyclopropane-1-carboxylic acid induced chitinase activity. Most of the induced enzymes were secreted into the culture medium. The extracellular chitinases induced by ethylene were resolved by gel-filtration chromatography into four isoenzymes with different molecular sizes. Two of them were exo-hydrolases which liberated chitobiose as a sole product from insoluble chitin. Another two isomers were thought to be endo-chitinase because they yielded chitin oligomers of various sizes. As major molecular species of chitinase found in ethylene-treated carrot cell cultures were different from those induced by fungal walls, it was suggested that ethylene and fungal components act as independent signals in chitinase induction.

Pectic polysaccharides elicit chitinase accumulation in tobacco

Upon infection of leaves of tobacco (Nicotiana tabacum L. ev. Havana) with Erwinia carotovora (Jones) Holl, strain 3912, a phytopathogenic bacterium that secretes pectinolytic enzymes, chitinase (EC 3.2.1.14) levels increased 12‐fold within 48 h. Heat‐killed E. carotovara cells did not induce this response. In young excised tobacco plants supplied with pectic polysaccharides, chitinase activity increased to about the same level as in leaves infected with E. carotovora. The amount of pectic polysaccharides required for half‐maximal induction was about 160 μg (g fresh weight)−1. Using in vivo labeling of plants with [35S]‐cysteine, it could be demonstrated that elicitormediated chitinase induction is due to enhanced de novo synthesis of the enzyme.

Induction of the defense-related glucanohydrolases, β-1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves

Nicotiana tabacum L. cv. Samsun plants carrying the N allele form localized necrotic lesions at the site of infection by tobacco mosaic virus (TMV). Within 3 days following inoculation, the content of β-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) as measured by ‘rocket’ electroimmunoassay increases in the infected leaves by up to 20-fold to approx. 3% of the soluble-protein fraction. This induction was not observed in TMV infected leaves of Samsun nn plants, which do not exhibit the hypersensitive, local lesion response, suggesting that β-1,3-glucanase and chitinase induction is part of the hypersensitive reaction. The content of mRNA in TMV-infected leaves was measured by Northern-blot analysis using inserts of cDNA clones of tobacco chitinase and β-1,3-glucanase as probes. There was a parallel increase in the two mRNAs following TMV infection indicating that chitinase and β-1,3-glucanase are coordinately induced at the mRNA level.

Induction, purification and possible function of chitinase in cultured carrot cells

Chitinase activity was induced in cultured carrot cells when they were treated with insoluble mycelial walls of Chaetomium globosum, or with chitin or chitosan. Laminarin and lichenan were less effective inducers of the enzyme, and N-acetylglucosamine oligomers (hexamer and pentamer) showed a negligible effect. Most of the induced chitinase activity was found in the soluble fraction of carrot cells. Chitinase induced in carrot cells was purified, and resolved into two isoenzymes by column chromatography: their specific activities were increased, respectively, 579- and 976-fold. The purified enzymes released sugar components from insoluble mycelial walls of C. globosum. These partial hydrolysates, when used to treat cultured carrot cells, stimulated an increase in phenylalanine ammonia-lyase activity and the accumulation of phenolic acids. These observations support our hypothesis that fungal wall components, such as chitin and chitosan, induce chitinases in carrot cells and that these enzymes then release wall fragments from the fungus which act as elicitors of lignification in the carrot cells.