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.     

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.     

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

Pea saponins in the pea-Fusarium solani interaction

Saponin-like compounds isolated fromPisum sativum were tested for antifungal activity, effect on pea tissue, and effect on chitin and chitosan synthesis inFusarium solani. Growth ofFusarium solani f. sp.phaseoli and f. sp.pisi macroconidia was inhibited by saponins at concentrations of 150 and 300 μg/ml, respectively. Pod endocarp tissue treated with saponins showed temporary reduction in cell viability (esterase activity); however, there was no significant reduction in resistance toF. solani f. sp.phaseoli, normally incompatible on peas. Macroconidia germinated in the presence of saponin showed decreased incorporation ofN-[³H]acetylglucosamine into chitin and chitosan at concentrations as low as 32 μg/ml. Thus, a reduction in chitin and chitosan synthesis may be associated with inhibition of fungal growth. Saponins may contribute to the disease resistance of peas     

Molecular characterization of a pea β-1,3-glucanase induced by Fusarium solani and chitosan challenge

-glucanases are prominent proteins in pea endocarp tissue responding to fungal infection. We have cloned and sequenced a partial pea cDNA clone, pPIG312, corresponding to a -1,3-glucanase in pea pods challenged with the incompatible pathogen Fusarium solani f. sp. phaseoli. The insert from the partial pea cDNA was used to probe a genomic library derived from pea leaves of the same cultivar. One of the genomic clones, pPIG4-3, contained the complete coding sequence for a mature -1,3-glucanase protein. The predicted amino acid sequence of the pea -1,3-glucanase has 78% identity to bean -1,3-glucanase, 62% and 60% to two tobacco -1,3-glucanases, 57% to soybean -1,3-glucanase, 51% to barley -1,3-glucanase, and 48% to barley -1,3-1,4-glucanase. Genomic Southern analysis indicates that the pea genome contains only one -1,3-glucanase gene corresponding to the probe used in this study. Accumulation of -1,3-glucanase mRNA homologous with the pPIG312 probe was detected in pea pods within 4 to 8 h after challenge with F. solani f. sp. phaseoli, f. sp. pisi, a compatible strain, or the elicitor, chitosan. In the incompatible reaction, mRNA accumulation remained high for 48h, whereas it rapidly decreased in the compatible reaction. After fungal inoculation of whole pea seedlings, the enhanced mRNA accumulation occurred mainly in the basal region (lower stem and root). This -1,3-glucanase glucanase mRNA was constitutively expressed in the roots of pea seedlings. The sustained levels of -glucanase mRNA expression induced by the incompatible pathogen in the resistance response suggests that the enzyme contributes to the pea plant's general defense.     

Localization of Fungal Components in the Pea-Fusarium Interaction Detected Immunochemically with Anti-chitosan and Anti-fungal Cell Wall Antisera

Antisera specific for purified cell walls of Fusarium solani f. sp. pisi and phaseoli and of shrimp shell chitosan were utilized as immunochemical probes to determine the location of fungal components in the pea-Fusarium interaction.     

Characterization of a 20 kDa DNase elicitor from Fusarium solani f. sp. phaseoli and its expression at the onset of induced resistance in Pisum sativum

DNase released from Fusarium solani f. sp. phaseoli (Fsph DNase) has previously been reported to induce pathogenesis-related (PR) genes, phytoalexin accumulation and disease resistance against subsequent challenge with the true pea pathogen, Fusarium solani f. sp. pisi (Fspi). This report is a further analysis of DNase production with probes specific for both the gene and protein. N-terminal analysis of the ≈20 kDa Fsph DNase protein facilitated both the development of anti-Fsph DNase antiserum and the cloning of the Fsph DNase gene. Utilizing the anti-Fsph DNase antiserum to prepare an affinity column, we demonstrated that the retention and recovery of the DNase activity was associated with this protein. Fsph DNase protein was detectable by Western analysis in both the fungi and plant cytoplasm within 6–8 h following inoculation of the pea endocarp surface. Partially purified DNase detected via catalytic activity began accumulating within pea tissue at 3 h post-inoculation. Enhanced fragmentation of pea DNA occurred within 5 h following treatment of pods with Fsph DNase or inoculations with the two fungi. DNA cleavage within the nuclei of endocarp pea cells was detectable via a TUNEL assay at 3 h post-inoculation. As a result of these findings, we propose that the entrance of Fsph DNase into the pea cell and the signalling of plant defence responses is temporally associated with the damage of host DNA.     

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.     

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.     

Production of Chitinases and β-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO₃. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Surface ��-1,3-Glucan Facilitates Fungal Stealth Infection by Interfering with Innate Immunity in Plants

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant''s defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants'' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.     

Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M_r 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (M_r 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

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.     

Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani

Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin.     

Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of Bacillus strains and decreasing the amount of Fusarium strains. Quantitative PCR confirmed that the populations of the soil-borne fungi Fusarium oxysporum and Trichoderma spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi.     

Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption

The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic β-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, β-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther β-1,3-glucanase is serologically unrelated to the basic β-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as β-1,3-glucanase are specific to each process.     

An improved test for evaluating the pathogenicity of isolates of Fusarium solani f.sp. pisi on pea

An improved in vitro test is described for determining the pathogenicity of Fusarium solani f.sp. pisi isolates on pea. This technique involves the use of polypropylene fibre Milcap plugs to suspend peas in boiling tubes containing spore suspensions in 0.1% water agar. Results were available after 14 days of incubation at 25°C. Four levels of pathogenicity were detected on pea cultivars Little Marvel and Dark Skinned Perfection using a total of eight isolates and strains of F. solani f.sp. pisi.