Tvbgn3, a beta-1,6-glucanase from the biocontrol fungus Trichoderma virens, is involved in mycoparasitism and control of Pythium ultimum

Even though beta-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a beta-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of beta-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.     

Cloning and characterization of bgn16.3, coding for a beta-1,6-glucanase expressed during Trichoderma harzianum mycoparasitism

AIMS: To clone and characterize the gene coding for BGN16.3, a beta-1,6-glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi. METHODS AND RESULTS: Using degenerate primed PCR and cDNA library screening, we have cloned the cDNA coding BGN16.3. bgn16.3 showed a significant sequence identity (50%) to bgn16.1; however, they both have low identity to the previously cloned bgn16.2, allowing the identification of amino acid sequences putatively involved in the common catalytic activity of the three proteins. bgn16.3 is a single-copy gene and highly homologous sequences are present in all tested Trichoderma species. bgn16.3 expression pattern is analysed by Northern blot, finding that it is expressed during the interaction of T. harzianum CECT 2413 with Botrytis cinerea, supporting the implication of the enzyme in the mycoparasitic process. CONCLUSIONS: The cloned bgn16.3 completes the knowledge on the beta-1,6-glucanase isozyme system from T. harzianum CECT 2413. A highly homologous gene is present in all analysed Trichoderma strains. bgn16.3 is expressed under few specific conditions, including the mycoparasitic process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the knowledge of beta-1,6-glucanases. It implicates this group of enzymes in the mycoparasitism by some biocontrol agents such as T. harzianum.     

Analysis of the β-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Production of Extracellular Proteins by the Biocontrol Fungus Gliocladium virens

Gliocladium virens is a common saprophytic fungus that is mycoparasitic on a large number of fungi. Responses of G. virens toward its environment were examined by monitoring the presence of extracellular proteins in culture fluid during time course experiments. Culture fluid of G. virens grown on glucose, washed cell walls of Rhizoctonia solani (one of its hosts), olive oil, or chitin contained β-glucanase, N-acetylglucosaminidase, lipase, and proteinase activities. There were relatively minor amounts of other enzymatic activities tested. Levels of extracellular enzyme activity varied with the age of the culture and the substrate used as the carbon source. Substrate-associated differences in enzyme activities were detected as early as 8 h after transfer of mycelia from stationary-phase cultures to fresh media. When G. virens was grown on host cell wall material, β-glucanase had the greatest specific activity of any enzyme tested at 8 h. This result suggests that β-glucanase may be the first enzyme important in the G. virens-R. solani interaction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that some of the polypeptides were present in the culture fluid at relatively constant amounts and others accumulated early, at intermediate times, or late in the 8-day incubation test period. Several of the polypeptides present in the culture fluid during the first 24 h disappeared completely by 48 h. Consequently, it appears that extracellular proteins in cultures of G. virens are regulated by a combination of gene regulation and protein degradation.     

Functional analysis of a beta-1,6-glucanase gene from the grass endophytic fungus Epichloë festucae

β-1,6-glucanases degrade the polysaccharide β-1,6-glucan, a cell wall component in some filamentous fungi. A single copy of a β-1,6-glucanase gene, designated gcnA, was identified in each of the grass endophytic fungi Neotyphodium lolii and Epichloë festucae. Phylogenetic analysis indicates that the GcnA protein is a member of glycosyl hydrolase family 5, and is closely related to fungal β-1,6-glucanases implicated in mycoparasitism. The E. festucae gcnA gene was expressed in mycelium grown in culture and in both vegetative and reproductive tissues of perennial ryegrass. A gcnA replacement mutant had reduced β-1,6-glucanase activity when grown in media containing pustulan as the major carbon source. β-1,6-glucanase activity was restored in the replacement mutant by introducing multiple copies of the gcnA gene. Growth of ΔgcnA and gcnA-overexpressing strains in vegetative grass tissues was indistinguishable from wild type strains.     

Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing β-1,3- and β-1,6-glucanase genes

Evidence for the role of chitinases, proteases and β-1,3- and β-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which β-1,3- and β-1,6-glucanase genes, TvBgn2 and TvBgn3 , respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total β-1,3- and β-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum , R. solani and Rhizopus oryzae . Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol.     

In vivo study of toxoplasmic parasitemia using interferon-gamma-deficient mice: absolute cell number of leukocytes, parasite load and cell susceptibility

Toxoplasma gondii infection was induced in wild type (WT) C57BL/6 and BALB/c mice and same-background interferon-γ knockout (GKO) mice by peroral inoculation of Fukaya strain cysts. We studied parasitemia, absolute cell number of leukocytes, and cell susceptibility in peripheral blood leukocyte (PBL) subsets in vivo. Parasitemia was observed in both WT and GKO mice, although the pattern of the parasite load was totally different among them. After infection, the absolute number of neutrophils in peripheral blood increased in both GKO C57BL/6 and GKO BALB/c mice with statistical significance, while it rose up slightly in susceptible WT C57BL/6 mice, but declined moderately in resistant WT BALB/c mice. The absolute number of lymphocytes in both WT and GKO mice decreased significantly after infection. Although leukocyte number per μl decreased significantly in both strains of WT mice, it increased in GKO BALB/c mice. There were no differences in susceptibility of PBL subsets to T. gondii infection as assessed by quantitative competitive polymerase chain reaction in both WT and GKO mice. These results indicate that each of the PBL subsets was infected in vivo. The increase of neutrophils only among immunocompromised or susceptible strains suggests that the neutrophil may be involved in the lethal process of T. gondii infection. The lack of any difference in cell susceptibility per μg gDNA among the PBL subsets examined implies that the neutrophil may contribute to the whole body dissemination process of the parasite in vivo more than other PBL subsets through the increase in number.     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Genetic Transformation of the Biocontrol Fungus Gliocladium virens to Benomyl Resistance

Methodology was developed to isolate and regenerate protoplasts from the biocontrol fungus Gliocladium virens and to transform them to benomyl resistance with a Neurospora crassa β-tubulin gene. Southern blots demonstrated that multiple copies of the vector integrated into the chromosomal DNA of stable biotypes but not of abortive transformants. Analysis of nuclear condition in vegetative and asexual structures demonstrated that no structure of G. virens is dependably uninucleate and thus preferentially suitable for transformation.     

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

Background

Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.

Results

Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.

Conclusions

The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

Feeding of Whitefly on Tobacco Decreases Aphid Performance via Increased Salicylate Signaling

Background

The feeding of Bemisia tabaci nymphs trigger the SA pathway in some plant species. A previous study showed that B. tabaci nymphs induced defense against aphids (Myzus persicae) in tobacco. However, the mechanism underlying this defense response is not well understood.

Methodology/Principal Findings

Here, the effect of activating the SA signaling pathway in tobacco plants through B. tabaci nymph infestation on subsequent M. persicae colonization is investigated. Performance assays showed that B. tabaci nymphs pre-infestation significantly reduced M. persicae survival and fecundity systemically in wild-type (WT) but not salicylate-deficient (NahG) plants compared with respective control. However, pre-infestation had no obvious local effects on subsequent M. persicae in either WT or NahG tobacco. SA quantification results indicated that the highest accumulation of SA was induced by B. tabaci nymphs in WT plants after 15 days of infestation. These levels were 8.45- and 6.14-fold higher in the local and systemic leaves, respectively, than in controls. Meanwhile, no significant changes of SA levels were detected in NahG plants. Further, biochemical analysis of defense enzymes polyphenol oxidase (PPO), peroxidase (POD), β-1,3-glucanase, and chitinase demonstrated that B. tabaci nymph infestation increased these enzymes’ activity locally and systemically in WT plants, and there was more chitinase and β-1, 3-glucanase activity systemically than locally, which was opposite to the changing trends of PPO. However, B. tabaci nymph infestation caused no obvious increase in enzyme activity in any NahG plants except POD.

Conclusions/Significance

In conclusion, these results underscore the important role that induction of the SA signaling pathway by B. tabaci nymphs plays in defeating aphids. It also indicates that the activity of β-1, 3-glucanase and chitinase may be positively correlated with resistance to aphids.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Regulation of Morphogenesis and Biocontrol Properties in Trichoderma virens by a VELVET Protein,Vel1

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.Trichoderma-based formulation products account for about 60% of the biofungicide market (35). Despite the use of Trichoderma-based biofungicides as an alternative and additive to chemical fungicides, the applications of these preparations are limited because their efficacy is lower than that of fungicides. A lack of understanding of the regulation of biocontrol has limited progress in enhancing the competitiveness of these fungi through genetic manipulation of desired traits. The success of a biocontrol agent also depends on the ability of researchers to develop an effective formulation based on active propagules that survive under the conditions that occur in nature and are effective against the target pathogens. Trichoderma spp. produce two types of propagules, conidia during solid-state fermentation and chlamydospores during liquid fermentation. Both types are used in commercial formulations depending on the growth conditions (17, 35). Thus, understanding how the two sporulation pathways are controlled is critical for obtaining an improved, balanced formulation product. Identification of a global regulator of morphogenesis and biocontrol properties (such as antibiosis and mycoparasitism) would provide an opportunity to manipulate the morphogenetic and antagonistic traits, leading to wider commercial acceptance of Trichoderma spp. in the long run.Trichoderma virens is a commercially formulated biocontrol agent that is effective against soilborne plant pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp.; its major direct mode of action is antibiosis and mycoparasitism (20, 36). This species has also been used as a model system for studies of biocontrol mechanisms, and the genome has recently been sequenced (http://genome.jgi-psf.org/Trive1). The role of beta-glucanases, chitinases, and proteases in biocontrol has been reported previously (2, 8, 29). Some strains of T. virens (designated Q strains) produce copious amounts of the antibiotic gliotoxin that is involved in biocontrol (10, 12, 39). In an attempt to identify regulators of biocontrol properties, the role of a mitogen-activated protein kinase (MAPK) pathway was studied previously (22, 24). Deletion of the TmkA/Tvk1 MAPK gene resulted in derepressed conidiation and different biocontrol behavior for two strains of T. virens; Mukherjee et al. (24) noted the reduced ability of these mutants to parasitize the sclerotia of S. rolfsii and R. solani, while Mendoza-Mendoza et al. (22) found that deletion of this MAPK gene improved the biocontrol activity of T. virens against R solani and P. ultimum. The production of secondary metabolites was not affected by deletion of this gene. To date, no gene that regulates the balance between conidiation or chlamydospore formation, secondary metabolism, and antagonistic or biocontrol properties has been identified in any Trichoderma sp.The Vel1 VELVET protein has been shown to be a regulator of morphogenesis and secondary metabolism in some filamentous fungi (6). In Aspergillus nidulans, VeA physically interacts with VelB and the regulator of secondary metabolism LaeA to form a complex that regulates secondary metabolism and sexual reproduction (3). Deletion of the VeA gene leads to an increase in asexual development (conidiation in the dark) and reduced biosynthesis of sterigmatocystin (the product of a polyketide synthetase [PKS]) and penicillin (the product of a nonribosomal peptide synthetase [NRPS]), while it reduces and delays sexual reproduction (15, 16). VeA is also required for the production of sclerotia and for aflatoxin biosynthesis in Aspergillus parasiticus (7). Deletion of the VeA gene in Neurospora crassa, like deletion of the VeA gene in A. nidulans, results in deregulated conidiation, while in Acremonium chrysogenum, loss of VeA leads to increased hyphal fragmentation and reduced cephalosporin production (4, 9). Deletion of the VeA gene in Fusarium verticilliodes resulted in a loss of hydrophobicity and an increased macroconidium-to-microconidium ratio; these defects could be restored by growing the organism on osmotically stabilized media (18). The mutants were also defective in production of the mycotoxins fumonisin and fusarin (25).To test the hypothesis that Vel1 is a global regulator of gene expression in T. virens, we examined the functions of Vel1 in this organism by using gene knockout and complementation. Here we report that in addition to a role in conidiation and secondary metabolism, Vel1 also regulates conidiophore aggregation, chlamydosporogenesis, mycoparasitism, and biocontrol efficacy in T. virens. Thus, we identified the first master regulator of morphogenesis and antagonistic properties in this economically important fungus.     

Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

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.