Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

Relation of glycosidases to bean hypocotyl growth

The enzymes β-glucosidase, α-glucosidase, β-galactosidase, α-galactosidase, and β-xylosidase were detected in Phaseolus vulgaris L. var. Red Kidney bean hypocotyl tissue throughout the first 13 days of development with p-nitrophenyl glycosides as substrates. Activities of all enzymes except β-glucosidase declined as a function of increasing tissue age. In contrast, β-glucosidase activity increased rapidly 3 days after imbibition to a maximal activity at 5 days and then subsided to one-third the maximum by day 7. This activity peak immediately preceded the logarithmic phase of hypocotyl growth. This enzyme is strongly associated with cell walls during extraction, suggesting that it is wall-bound in situ. Various polysaccharide substrates were used to evaluate the specificity of this enzyme.     

The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl

Extracellular components released from mycelia of the α and β races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible α race and compatible β race. Elicitor activity, however, was greater in the α race fractions. Elicitor purified from α race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function.     

Production and Properties of Xylan-Degrading Enzymes from Cellulomonas uda

Xylan degradation and production of β-xylanase and β-xylosidase activities were studied in cultures of Cellulomonas uda grown on purified xylan from birchwood. β-Xylanase activity was found to be associated with the cells, although in various degrees. The formation of β-xylanase activity was induced by xylotriose and repressed by xylose. β-Xylosidase activity was cell bound. Both constitutive and inducible β-xylosidase activities were suggested. β-Xylanase and β-xylosidase activities were inhibited competitively by xylose. β-Xylanase activity had a pronounced optimum pH of 5.8, whereas the optimum pH of β-xylosidase activity ranged from 5.4 to 6.1. The major products of xylan degradation by a crude preparation of β-xylanase activity, in decreasing order of amount, were xylobiose, xylotriose, xylose, and small amounts of xylotetraose. This pattern suggests that β-xylanase activity secreted by C. uda is of the endosplitting type. Supernatants of cultures grown on cellulose showed not only β-glucanase but also β-xylanase activity. The latter could be attributed to an endo-1,4-β-glucanase activity which had a low β-xylanase activity.     

The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an α-galactosidase, an α-mannosidase, a β-N-acetyl-glucosaminidase, a β-glucosidase, and two β-galactosidases. The pH optimum of each of these enzymes was determined. The pH optima, together with inhibition studies, suggest that each observed glycosidase activity represents a separate enzyme. Three of these enzymes, β-glucosidase, α-galactosidase, and one of the β-galactosidases, have been shown to be associated with the cell surface. The enzyme activities associated with the cell surface were shown to possess the ability to degrade to a limited extent isolated sycamore cell walls. It was found that the activities of β-glucosidase and of one of the β-galactosidases increase as the cells go through a period of growth and decrease as cell growth ceases.     

The Role of beta-Galactosidases in the Modification of Cell Wall Components during Muskmelon Fruit Ripening

The changes in activities of soluble β-galactosidase and two forms of wall-bound β-galactosidases extracted with NaCl and EDTA were investigated throughout the development of muskmelon (Cucumis melo L. cv Prince) fruits. DEAE-cellulose ion-exchange chromatography of soluble β-galactosidase revealed the presence of two isoforms. Soluble isoform I was detected in all stages throughout the fruit development, whereas soluble isoform II appeared around 34 d after anthesis when fruit ripening initiated. Both NaCl- and EDTA-released β-galactosidase activities also increased as ripening proceeded. The soluble and wall-bound forms behaved differently upon ion-exchange chromatography. Enzymological properties such as optimum pH, optimum temperature, K_m values for p-nitrophenyl β-d-galactopyranoside, and inhibition by metal ions were nearly similar in all forms. Molecular sizes of pectic polymers and hemicelluloses extracted from fruit mesocarp cell walls were shifted from larger to smaller polymers during ripening, as determined by gel filtration profiles. NaCl-released β-galactosidase from cell walls of ripe fruits had the ability to degrade in vitro the pectin extracted from preripe fruit cell walls to smaller sizes of pectin similar to those that were observed in ripe cell walls in situ. Both soluble isoform I and II were able to degrade in vitro the 5% KOH-extractable hemicellulose from preripe fruit cell walls to sizes of molecules similar to those that were observed in ripe cell walls in situ. Soluble isoform I and the NaCl-released form from ripe fruits were able to modify in vitro 24% KOH-extractable hemicellulose from preripe cell walls to sizes of molecules similar to those that were observed in ripe fruits in situ.     

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.     

Evidence for Plasmid Linkage of Raffinose Utilization and Associated α-Galactosidase and Sucrose Hydrolase Activity in Pediococcus pentosaceus

The ability to ferment the trisaccharide raffinose was linked with the presence of plasmid DNA in three strains of Pediococcus pentosaceus. Parental strains showed associated inducible α-galactosidase and sucrose hydrolase activities when grown in α-galactosides and sucrose, respectively. Derivative strains of PPE1.0, PPE2.0, and PPE5.0, which had lost 30-, 28-, and 23-megadalton plasmids, respectively, had no α-galactosidase or sucrose hydrolase activity.     

Susceptibility to Enzymatic Degradation of Cell Walls From Bean Plants Resistant and Susceptible to Rhizoctonia solani Kuhn

Enzymes in culture filtrates of Rhizoctonia solani Kuhn grown using 4-day old or 20-day old bean (Phaseolus vulgaris L.) hypocotyl cell walls as a carbon source degraded xylan, galactan, galactomannan, araban, polygalacturonic acid, and carboxymethylcellulose. Extracts of lesions from R. solani infected plants, but not healthy plants, contained similar enzymatic activities. These enzyme sources readily solubilized cell wall constituents containing arabinose, galactose, and glucose from 4-day old, but not from 20-day old, bean cell walls. Analysis of cell walls prepared from infected plants revealed that the alterations in cell wall composition in the diseased host were limited largely to the immediate lesion areas and occurred during the early phases of pathogenesis. The cell walls of young susceptible bean seedlings could be degraded by R. solani enzymes, but the cell walls of older plants which are resistant to this pathogen were not susceptible to enzymatic destruction by the same enzyme preparation.     

Host-Pathogen Interactions: VII. Plant Pathogens Secrete Proteins which Inhibit Enzymes of the Host Capable of Attacking the Pathogen

The results presented demonstrate that microbial pathogens of plants have the ability to secrete proteins which effectively inhibit an enzyme synthesized by the host; an enzyme whose substrate is a constituent of the cell wall of the pathogen. The system in which this was discovered is the anthracnose-causing fungal pathogen (Colletotrichum lindemuthianum) and its host, the French bean (Phaseolus vulgaris). An endo-β-1, 3-glucanase present in the bean leaves is specifically inhibited by a protein secreted by C. lindemuthianum. The cell walls of C. lindemuthianum are shown to be composed largely of a 1, 3-glucan.     

Host-Pathogen Interactions : XVII. HYDROLYSIS OF BIOLOGICALLY ACTIVE FUNGAL GLUCANS BY ENZYMES ISOLATED FROM SOYBEAN CELLS

The ability of β-glucosylase I, a soybean cell wall β-glucosyl hydrolase, to degrade elicitors of phytoalexin accumulation was studied. Extensive β-glucosylase I treatment of the glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae results in hydrolysis of 77% of the glucosidic bonds of the elicitor and destruction of 94% of its activity. Soybean cell walls contain some additional factor, probably one or more additional enzymes, which can assist β-glucosylase I in hydrolyzing the glucan elicitor. This was demonstrated by the more rapid hydrolysis of the glucan elicitor by a mixture of soybean cell wall enzymes (containing β-glucosylase I). In a single treatment, the mixture of cell wall enzymes hydrolyzed 91% of the glucosidic bonds and destroyed 85% of the activity of the elicitor. The enzymes from soybean cell walls will also hydrolyze elicitor-active oligoglucosides prepared from the mycelial walls of Phytophthora megasperma var. sojae. The active oligoglucosides are more susceptible than the glucan elicitor to hydrolysis by these enzymes. The mixture of cell wall enzymes or β-glucosylase I, by itself, hydrolyzes more than 96% of the glucosidic bonds and destroys more than 99% of the activity of the oligoglucoside elicitor. Two possible advantages for the existence of these enzymes in the walls of soybean cells are discussed.     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

A Lichen Lectin Specifically Binds to the α-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae

A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with α-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of β-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.Key Words: arginase, cell wall, evernia prunastri, lectin ligand, phycobiont, urease     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Glycosidases in Cell Wall-degrading Extracts of Ripening Tomato Fruits

Enzyme preparations were obtained from cell wall debris of tomato (Lycopersicon esculentum L. cv. Tropic) fruits at various stages of ripeness and were assayed for glycosidase and polysaccharidase activities. In addition to polygalacturonase (mol wt 40,000), ripening fruits contain β-galactosidase (mol wt 63,000) and β-1, 3-glucanase (mol wt 12,000). The β-glycosidases, unlike polygalacturonase, are active in extracts of green fruits. Placental tissue shows very low polygalacturonase but increasing β-galactosidase and β-1, 3-glucanase activities as ripening proceeds. A large change in the susceptibility of the walls to hydrolase action occurs before the stage in which the greatest polygalacturonase activity occurs. The possibility that the β-glycosidases contribute to the wall modifications that lead to fruit softening is discussed.     

Regulation of alpha-amylase activity in bean stem tissues

α-Amylase activity was assayed in 1-centimeter sections taken from bean (Phaseolus vulgaris var. Kentucky Wonder) hypocotyls and epicotyls at measured distances from the cotyledons. The activity was low throughout the hypocotyl for the first 7 days. An increase was first observed with etiolated hypocotyls in the basal region, becoming higher in the more central regions by 14 to 17 days. By 21 days the activity was highest in the upper region, but had decreased in the lower regions. A comparable pattern was observed for the epicotyl from etiolated seedlings, the activity increasing first in the region closest to the cotyledons. These increases were associated with loss of cells from the pith in the hypocotyl and epicotyl of both dark- and light-grown plants. Since the changes were observed in tissues virtually devoid of starch, it is hypothesized that the control mechanism is related to the cellular disassembly associated with the mobilization of materials released during senescence rather than to a regulation by the enzyme's substrate or products.     

An Antifungal Exo-α-1,3-Glucanase (AGN13.1) from the Biocontrol Fungus Trichoderma harzianum

Trichoderma harzianum secretes α-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type α-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.