Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-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. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM 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 beta-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.     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

Intercellular proteins and β-1,3-glucanase activity associated with leaf rust resistance in wheat

To investigate biochemical aspects of resistance conferred by the Lr35 gene for adult-plant resistance in wheat ( Triticum aestivum L.) to leaf rust, pathogen development was related to intercellular protein composition and β -1,3-glucanase (EC 3.2.1.39) activities at three growth stages in infected and uninfected resistant (RL6082 [Thatcher/ Lr35 ]) and susceptible (Thatcher) plants. Leaf rust symptoms produced by pathotype UVPrt9 of Puccinia recondita f. sp. tritici showed that resistance conferred by Lr35 was most effective at the flag leaf stage. Furthermore, fluorescence microscopy indicated that resistance was strongly associated with hypersensitive cell death of invaded tissue. According to polypeptide profiles, intercellular proteins with molecular masses of 35, 33, 31 and 26 kDa were constitutively present at higher levels in resistant than in susceptible plants at the flag leaf stage. Four intercellular proteins (35, 33, 32 and 31 kDa) serologically related to β -1,3-glucanase were present in resistant and susceptible genotypes during all stages of plant growth. Resistance was associated with high constitutive levels of β -1,3-glucanase activity. Susceptibility on the other hand was associated with low constitutive levels of β -1,3-glucanase, while high levels were induced by infection during more advanced stages of colonization. Our results suggest that β -1,3-glucanase is involved in the defense response controlled by the Lr35 gene.     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Development of beta-1,3-glucanase activity in germinated tomato seeds

Laminarin-hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS-PAGE. It was specific for linear beta-1,3-glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo-action pattern. Thus, the enzyme was identified as beta-1,3- endoglucanase (EC 3.2.1.39). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of beta-1,3-glucanase activity. Thus the post-germinative beta-1, 3-glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.     

Optimization of RQRT-pCR protocols to measure beta-1,3-glucanase mRNA levels in infected tissues of rubber tree (Hevea brasiliensis)

RQRT-PCR technique was evaluated for its validity as an alternative to Northern blotting for quantification of plant gene expression in diseased tissues of Hevea. Reliable RT-PCR results could be obtained by co-amplification of housekeeping actin gene as the internal control along with the gene of interest. The product of interest was quantified relative to that of the internal control by measuring net intensity of bands. Expression levels of defense-related beta-1,3-glucanase gene was studied in the pathogen infected tissues of rubber. The beta-1,3-glucanase gene was found to be induced in infected leaf tissues and reached a peak at 48 h after inoculation. The beta-1,3-glucanase gene expression during pathogen infection was determined through Northern blot hybridization also, using 18S RNA as the internal control. RQRT-PCR and Northern hybridization showed almost similar results, thereby validating the use of this technique to study the gene expression in rubber.     

Accumulation of beta-Fructosidase in the Cell Walls of Tomato Roots following Infection by a Fungal Wilt Pathogen

Active defense in plants is associated with marked metabolic alterations, but little is known about the exact role of the reported changes in specific activity of several enzymes in infected plant tissues. β-Fructosidase (invertase), the enzyme that converts sucrose into glucose and fructose, increases upon infection by fungi and bacteria. To understand the relationship between fungal growth and β-fructosidase accumulation, we used an antiserum raised against a purified deglycosylated carrot cell wall β-fructosidase to study by immunogold labeling the spatial and temporal distribution of the enzyme in susceptible and resistant tomato (Lycopersicon esculentum) root tissues infected with the necrotrophic fungus, Fusarium oxysporum f. sp. racidis-lycopersici. In susceptible plants, the enzyme started to accumulate in host cell walls about 72 hours after inoculation. Accumulation occurred only in colonized cells and was mainly restricted to areas where the walls of both partners contacted each other. In resistant plants, accumulation of β-fructosidase was noticeable as soon as 48 hours after inoculation and appeared to reach an optimum by 72 hours after inoculation. Increase in wall-bound β-fructosidase was not restricted to infected cells but occurred also, to a large extent, in tissues that remained uncolonized during the infection process. The enzyme also accumulated in wall appositions (papillae) and intercellular spaces. This pattern of enzyme distribution suggests that induction of β-fructosidase upon fungal infection is part of the plant's defense response. The possible physiological role(s) of this enzyme in infected tomato plants is discussed in relation to the high demand in energy and carbon sources during pathogenesis.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Subcellular Localization of Chitinase and of Its Potential Substrate in Tomato Root Tissues Infected by Fusarium oxysporum f. sp. radicis-lycopersici

Antiserum raised against a tomato (Lycopersicon esculentum Mill.) chitinase (molecular mass of 26 kilodaltons) was used as a probe to study the subcellular localization of this enzyme in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici. A time-course experiment revealed that chitinase accumulated earlier in the incompatible interaction than in the compatible one. However, in both systems, chitinase deposition was largely correlated with pathogen distribution. The enzyme was found to accumulate in areas where host walls were in close contact with fungal cells. In contrast, the enzyme could not be detected in vacuoles and intracellular spaces. The substantial amount of chitinase found at the fungus cell surface supports the view of an antifungal activity. However, the preferential association of the enzyme with altered fungal wall areas indicates that chitinase activity is either preceded by the hydrolytic action of other enzymes such as β-1,3-glucanases or coincides with these enzymes. The possibility that fungal glucans released through the action of β-1,3-glucanases may act as elicitors of chitinase production is discussed.     

Activation of Glycosidases as a Consequence of Infection Stress in Fusarium Wilt of Tomato

Changes of β-1,3-glucanase, chitinase, β-1,4-glucosidase and N-acetylglucosaminidase activity have been investigated in relation to the development of symptoms and colonization by the pathogen in roots, stems and leaves of susceptible (‘Improved, Pearson’) and resistant (‘Improved Pearson VF11’) tomato plants infected by Fusarium oxysporum f. sp. lycopersici. Glycosidase activities increased after inoculation to different extents depending on the plant part and cultivar. Increases were always higher in susceptible than in resistant plants. Changes in the β-1,3-glucanase activity after inoculation were particularly large in stems of infected plants. In contrast, chitinase activity increased more in roots than in stems. The β-1,3-glucosidase and chitinase activity decreased slightly from the basal to the apical third of stems. The trend of changes of the glycosidase activity generally were well related with the severity of disease symptoms and the fungal colonization of basal stem segments. There was no evidence that the increase of glycosidase activity after the infection was directly related with the resistance to Fusarium wilt in tomato.     

A study of some biochemical and histopathological responses of wet-stored recalcitrant seeds of Avicennia marina infected by Fusarium moniliforme

Although fungi cause a recognized problem during storage of recalcitrant seeds of many tropical species, there are no data to date on defence strategies of these seeds against fungal attack. To ascertain whether recalcitrant seeds of Avicennia marina elaborate compounds that might suppress fungal proliferation during hydrated storage, the production and efficacy of beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) were studied in relation to histopathological changes. Freshly harvested seeds had low beta-1,3-glucanase and chitinase activities and fluorescence microscopy revealed progressive deterioration of the internal tissues of these seeds associated with fungal infection during hydrated storage. In seeds treated to minimize associated fungi (clean seeds), beta-1,3-glucanase and chitinase activities increased significantly during 10 d of hydrated storage. Similar high levels of activity were observed when these seeds were experimentally infected with Fusarium moniliforme and subjected to further storage. The histopathological observations indicated delayed disease development in the 10-d clean-storage period, although the hypersensitive response was not observed. The results suggest that, although the recalcitrant seeds of A. marina elaborate some antifungal enzymes, there is a lack of effective defence strategies that might lead to successful responses against fungal infections.     

Control of Extracellular beta-1,3-glucanase activity in a basidiomycete species.

The basidiomycete QM 806 excreted large amounts of beta-1,3-glucanase into the culture medium. Synthesis and excretion of the enzyme were triggered by a critically low concentration of carbon source. The extracellular beta-1,3-glucanase exhibited a remarkable stability. Addition of glucose or other carbon sources to a culture after consumption of the initial carbon source led to an inactivation of the extracellular beta-1,3-glucanase by an inactivating system, which could be separated from the cells. The inactivation of beta-1,3-glucanse was prevented by cycloheximide. This indicates the necessity of active protein synthesis for the inactivation process but does not prove that the inactivating system itself is a protein. Marked changes in the electrophoretic mobility and immunological properties of beta-1,3-glucanase indicate rather profound alterations of the enzyme protein in the course of inactivation.     

Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis.

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.     

Ultrastructural changes and location of beta-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of β-1,3-glucanase in callus cells with immunofluorescence labeling. Stronger fluorescence was observed in the extra-cellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in β-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.