Cell Surface Interactions between Bean Leaf Cells and Colletotrichum lindemuthianum: Cytochemical Aspects of Pectin Breakdown and Fungal Endopolygalacturonase Accumulation

After a brief period of biotrophic growth, the anthracnose fungus Colletotrichum lindemuthianum (Sacc. et Mgn.) Bri et Cav. develops extensively in bean leaf cells, causing severe wall alterations and death of the host protoplast. Aplysia gonad lectin, a polygalacturonic acid-binding agglutinin, was complexed to gold and used to study the extent of pectin breakdown during the necrotrophic phase of the infection process. In view of its specific binding properties for the endopolygalacturonase produced by C. lindemuthianum, a polygalacturonase-inhibiting protein isolated from bean cell walls was successfully tagged with gold particles and used for localizing the sites of enzyme accumulation in infected host tissues. The basal level of endopolygalacturonase produced by C. lindemuthianum grown in culture was found to increase severalfold when the fungus developed in host plant tissues. The enzyme was able to diffuse freely in the host cell wall, causing drastic degradation of the pectic material of primary walls and middle lamella matrices. The enzymatic alteration of plant cell walls was accompanied by the release of pectic fragments and by the accumulation of pectic molecules at specific sites, such as intercellular spaces and aggregated cytoplasm of infected host cells. The occurrence of pectic molecules at those sites where fungal growth is likely to be restricted is discussed in relation to their origin and their implication in the plant's defense system.     

Purification and characterization of two isozymes of polygalacturonase from Botrytis cinerea. Effect of calcium ions on polygalacturonase activity

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl₂, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.     

Biotic elicitors of defense reactions in lodgepole pine

Elevated levels of defensive chemicals (monoterpenes) were detected in lodgepole pine (Pinus contorta var. latifolia) phloem surrounding sites inoculated with living mountain pine beetles (Dendroctonus ponderosae) a blue-staining fungus (Ceratocystis clavigerum), a pectic fragment from tomato leaves (PIIF) and a fungal cell wall fragment (chitosan). Chitosan elicited the greatest production of monoterpenes at the lowest concentrations, and also elicited greater responses in large, fast-growing trees. Chitosan may prove to be a useful material for assaying the resistance of conifers to lethal bark beetle attacks. The results suggest a common recognition-defense mechanism in higher plants.     

Enzymatic Deconstruction of Backbone Structures of the Ramified Regions in Pectins

The pectic enzymes are a diverse group of enzymes that collectively degrade pectin, a mixture of highly heterogeneous and branched polysaccharides rich in d-galacturonic acids forming a major component of the primary cell wall of plants. This review covers key enzymes that function to deconstruct the “ramified region” of pectin. The enzymes include glycoside hydrolases and polysaccharide lyases that degrade complex pectic domains consisting of rhamnogalacturonans, xylogalacturonans, and other heterogeneous polymers. The chemical nature of the pectic substrates for the enzymes is presented. The biochemical properties of the enzymes, the mechanisms of enzyme actions, and related structures and functions, are described. Applications of these enzymes in fruit juice processing and in the production of bioactive compounds, as well as their technological relevance to the deconstruction of cell wall structures for biomass conversion are discussed.     

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches’ broom disease in cacao

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.     

The life style of Aureobasidium pullulans and the multiple forms of its polygalacturonase

The life style of Aureobasidium pullulans on pectin medium and its production of extracellular polygalacturonases are closely related. Polygalacturonases with random action pattern (EC 3.2.1.15) were formed in the first phases of cultivation, whereas exopolygalacturonases (EC 3.2.1.67) with terminal action pattern on pectin were produced during the whole growth of this yeast-like fungus. The production and inactivation of individual enzyme forms during cultivation were strongly dependent on the pH value of the pectin medium. Various kinds of stress can support the prolongation of the phase of endo-acting enzyme production, as well as the increase of their activity.     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999     

Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus,specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases. Correspondence to: A. G. J. Voragen     

Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.     

Polygalacturonase activity and location in arbuscular mycorrhizal roots of Allium porrum L.

Polygalacturonase activity and location were analysed in leek roots (Allium porrum L.) colonized by Glomus versiforme (Karst.) Berch, an arbuscular mycorrhizal (AM) fungus. Polygalacturonase activity in mycorrhizal roots did not differ quantitatively from that found in nonmycorrhizal roots on all of the four harvesting dates. Fractionation of mycorrhizal root extracts by ion-exchange chromatography showed that expression of polygalacturonase was specific to the mutualistic association. Immunofluorescence and immunogold experiments were carried out to locate the polygalacturonase in mycorrhizal roots using a polyclonal antibody raised against a Fusarium moniliforme endopolygalacturonase. Immunolabelling was observed all over the arbuscules (intracellular fungal structures) but particularly at the interface between the arbuscule and the plant membrane. Since pectins are located in this area, we suggest that polygalacturonase produced during the symbiosis could play a role in plant pectin degradation.     

Cloning and Sequence Analysis of the Endopolygalacturonase Gene from the Pitch Canker Fungus, Fusarium circinatum

The fungus Fusarium circinatum causes pitch canker disease on mature pine trees and root rot and damping-off of pine seedlings. Endopolygalacturonases (endoPGs) play a major role during penetration of plants by fungi. Digestion of the pectic polysaccharides in the plant primary cell walls is one of the earliest functions of endoPGs during infection. The research objective was to clone and characterize the gene encoding endopolygalacturonase in F. circinatum. A 970-bp DNA fragment was cloned by using degenerate PCR amplification from F. circinatum DNA. Sequence data for this fragment were used to design specific primers for use in genome walking to amplify and sequence the remaining portion of the F. circinatum endoPG gene (Fcpg). The amino acid sequence predicted from this gene showed 90% and 87% similarity to Fusarium oxysporum and Fusarium moniliforme endoPGs, respectively. Received: 10 August 2000 / Accepted: 30 October 2000     

Purification and Properties of Endo-polygalacturonase from Aspergillus japonicus

An endo-polygalacturonase from culture extracts of Aspergillus japonicus was purified about 34-fold by ammonium sulfate fractionation, SE-Sephadex column chromatography and gel filtration. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis. Using gel filtration a molecular weight of 35,500 was estimated for the enzyme. The enzyme rapidly reduced the viscosity of pectic acid and released reducing groups in a random manner, yielding a mixture of mono-, di- and trigalacturonic acids as end products. The pH optimum of the enzyme for viscosity-reducing activity was 4.5 with pectin and pectic acid as substrates, and that for releasing reducing groups was also 4.5 with various pectic substances. The purified enzyme was able to macerate various kinds of plant tissues by itself.     

Three polygalacturonases constitutively synthesized by Aspergillus alliaceus

Of three molecular forms of polygalacturonases synthesized by Aspergillus alliaceus on glucose media, two were exopolygalacturonases (exoPG₁ and exoPG₂) and one was an endopolygalacturonase (endoPG). Low-methoxylated beet pectin was the preferred substrate for the endoPG and exoPG₂ whereas pectic acid was the optimal substrate for exoPG₁. The highest activities of endoPG, exoPG₁ and exoPG₂ were at pH 5.5, 3.5, and 6.0 and at 35, 45 to 50 and 35°C, respectively. Disks of potato-tuber tissue were macerated by endoPG, but not by exoPG₁ or exoPG₂.The authors are with the Institute of Microbiology, Belarus Academy of Sciences, Zhodinskaya 2, 220141, Minsk, Belarus;     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

Pectic enzyme production and morphogenesis inHelminthosporium atypicum

The production of pectic enzymes byHelminthosporium atypicum and its morphogenesis on different media were studied. It was observed that the fungus produces pectic enzyme (macerating enzyme) adaptively. Increasing concentrations of glucose had an inhibitory effect on enzyme production. Glucose promotes profuse growth and early sporulation whereas presence of pectin slows down the growth and delays sporulation. Delay in sporulation is the effect of presence of pectin and not of the low pH of the medium. It is also suggested that in the case ofH. atypicum low pH of the medium does not allow the fungus to utilize a carbon source as efficiently as at higher pH.     

Soil calcium and plant disease in serpentine ecosystems: a test of the pathogen refuge hypothesis

Ecologists have long sought mechanistic explanations for the patterns of plant distribution and endemism associated with serpentine soils. We conducted the first empirical test of the serpentine pathogen refuge hypothesis, which posits that the low levels of calcium found in serpentine soils provide associated plants with a refuge from attack by pathogens. We measured the range of soil calcium concentrations experienced by 16 wild population of California dwarf flax (Hesperolinon californicum) and experimentally recreated part of this range in the greenhouse by soaking serpentine soils in calcium chloride solutions of varying molarity. When flax plants grown in these soils were inoculated with spores of the rust fungus Melampsora lini we found a significant negative relationship between infection rates and soil calcium concentrations. This result refutes the pathogen refuge hypothesis and suggests that serpentine plants, by virtue of their association with low calcium soils, may be highly vulnerable to attack by pathogens. This interaction between plant nutrition and disease may in part explain demographic patterns associated with serpentine plant populations and suggests scenarios for the evolution of life history traits and the distribution of genetic resistance to infection in serpentine plant communities.     

<Emphasis Type="Italic">Aspergillus flavus</Emphasis> hydrolases: their roles in pathogenesis and substrate utilization

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.     

Pectolytic enzymes in inoculated and uninoculated red clover seedlings

Summary Pectolytic enzymes were studied in inoculated and uninoculated red clover (Trifolium pratense) seedlings grown aseptically in flasks containing distilled water or nitrogen-free salts media. Enzyme activity in root exudates and root extracts depended on the conditions of seedling growth. Tests with sodium polypectate and citrus pectin indicated the presence of two enzymes, specific for pectic acid and pectin respectively. Both enzymes were produced by uninoculated seedlings, and in seedlings inoculated with Rhizobium trifolii, R. leguminosarum or R. lupini, enzyme activity was not correlated with infectivity of the strains. re]19720814     

Purification and Properties of a Pectin trans-Eliminase in Erwin aroideae Formed in the Presence of Nalidixic Acid

A strain of Erwinia aroideae produces a remarkable amount of pectolytic enzyme when the organism was induced by nalidixic acid for the bacteriocin production. This pectolytic enzyme was purified approximately 60-fold from the induced medium by carboxymethyl-cellulose and Sephadex G–75 gel column chromatographies after batchwise treatment with carboxymethyl- and diethylaminoethyl-celluloses. The purified enzyme was almost homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a molecular weight of about 28,000 to 32,000 was determined for this enzyme. The optimum pH of the enzyme activity was about 8.0 to 8.2. The purified enzyme produced reaction products from pectin and methoxylated pectic acid which had a strong absorption at 235 nm indicating a trans-eliminase reaction. Pectin or pectic acid with higher methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectic acid was a substrate. The limit of degradation of pectin and pectic acid with higher methoxyl content (90% esterified) by the enzyme were 6.5% and 43%, respectively. It was concluded that the enzyme is a new endo-pectin trans-eliminase from bacterial origin.     

Arbuscular mycorrhiza reduces susceptibility of tomato to Alternaria solani

Mycorrhiza frequently leads to the control of root pathogens, but appears to have the opposite effect on leaf pathogens. In this study, we studied mycorrhizal effects on the development of early blight in tomato (Solanum lycopersicum) caused by the necrotrophic fungus Alternaria solani. Alternaria-induced necrosis and chlorosis of all leaves were studied in mycorrhizal and non-mycorrhizal plants over time course and at different soil P levels. Mycorrhizal tomato plants had significantly less A. solani symptoms than non-mycorrhizal plants, but neither plant growth nor phosphate uptake was enhanced by mycorrhizas. An increased P supply had no effect on disease severity in non-mycorrhizal plants, but led to a higher disease severity in mycorrhizal plants. This was parallel to a P-supply-induced reduction in mycorrhiza formation. The protective effect of mycorrhizas towards development of A. solani has some parallels to induced systemic resistance, mediated by rhizobacteria: both biocontrol agents are root-associated organisms and both are effective against necrotrophic pathogens. The possible mechanisms involved are discussed.