Polysaccharide-degrading enzymes of Botrytis allii and Sclerotium cepivorum. Enzyme production in culture and the effect of the enzyme on isolated onion cell walls

Mankarios, A. T.; Friend, J.

doi:10.1016/0048-4059(80)90010-7

Cited by 54 publications

(17 citation statements)

References 18 publications

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“…In the primary cell wall, the cellulose-xyloglucan framework is embedded in a matrix of pectic polysaccharides, which thereby control the access of hydrolytic enzymes to the cellulose and hemicellulose substrates (1,7). Thus, pectinases are often the first cell wall degrading enzymes produced by plant pathogens when cultured on purified plant cell walls or during infection (8,9). Due to the structural complexity of the pectin matrix a synergistic or sequential action of several different pectinolytic enzymes is required for efficient breakdown (5).…”

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confidence: 99%

Molecular Cloning and Characterization of a Rhamnogalacturonan Acetylesterase from Aspergillus aculeatus

Kauppinen

Stephan

Kofod

et al. 1995

Journal of Biological Chemistry

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A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH 2 -terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. aculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.Pectic polysaccharides are located predominantly in the middle lamella and primary cell wall of dicotyledonous plants (1). The main backbone in pectins can be divided into linear homogalacturonan (smooth) regions of up to 200 residues of (1,4)-linked ␣-D-galacturonic acid (GalUA) 1 and highly branched rhamnogalacturonan (hairy) regions consisting of repeatingIn general, about half of the Rha residues in the hairy regions are substituted with neutral oligosaccharides such as arabinans, galactans, and arabinogalactans. Most pectic substances are furthermore esterified with acetyl or methyl groups at some of the GalUA residues in the backbone (1, 4).Many saprophytic and plant pathogenic fungi and bacteria possess an array of extracellular enzymes involved in the degradation of plant cell wall polymers (5, 6). In the primary cell wall, the cellulose-xyloglucan framework is embedded in a matrix of pectic polysaccharides, which thereby control the access of hydrolytic enzymes to the cellulose and hemicellulose substrates (1, 7). Thus, pectinases are often the first cell wall degrading enzymes produced by plant pathogens when cultured on purified plant cell walls or during infection (8, 9). Due to the structural complexity of the pectin matrix a synergistic or sequential action of several different pectinolytic enzymes is required for efficient breakdown (5). For example, the hydrolysis of smooth regions of pectin by polygalacturonases is highly dependent upon demethylation of the homogalacturonan backbone by pectin methylesterase (5). Similarly, the degradation of rhamnogalacturonan by rhamnogalacturonases (RGases) depends on the removal of the acetyl esters from the substrate (10, 11). Thus, the presence of a rhamnogala...

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confidence: 99%

Molecular Cloning and Characterization of a Rhamnogalacturonan Acetylesterase from Aspergillus aculeatus

Kauppinen

Stephan

Kofod

et al. 1995

Journal of Biological Chemistry

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“…Among the different cell wall-degrading enzymes known to be produced by pathogens, most attention has been focused on those that depolymerize pectin, a major component of the primary plant cell wall and middle lamella (29). Pectin-degrading enzymes are frequently the first cell wall-degrading enzymes produced by plant fungal pathogens in infected tissues (6,21,28). Depolymerization of pectin would not only provide a carbon source for fungal growth and development (13) but also expose other cell wall components to degradation, causing further cell wall breakdown (3,23).…”

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Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris

1995

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Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectindegrading enzymes are thought to be important for pathogenesis. Antibodies prepared against a pectininducible pectate lyase (pectate lyase A [PLA]) produced by a phytopathogenic fungus, Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection. The gene (pelA) and its cDNA were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F. solani f. sp. pisi with the pelA cDNA as the probe. A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced. The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F. solani pisi culture grown in the presence of glucose as the sole carbon source. The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F. solani f. sp. pisi but no significant homology with other pectinolytic enzymes. The first 16 amino acid residues at the N terminus appeared to be a signal peptide. The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa. PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography. Purified PLB showed optimal lyase activity at pH 10.0. A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion. Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other. The 5 flanking regions of both pelA and pelB were translationally fused to the ␤-glucuronidase gene and introduced into F. solani f. sp. pisi, and ␤-glucuronidase activities of the transformants were measured. Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression. Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F. solani f. sp. pisi infects pea epicotyl.

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“…It is well known that the fungal metabolite oxalic acid is produced by S. cepivorum in culture and in infected onion plants and plays a pivotal role in the progress of onion infection (Stone and Armentrout 1985). Additionally, S. cepivorum secretes a group of extracellular enzymes during invasion of onion tissues that degrade the cell wall, including polygalacturonase and pectin methylesterase (Abd El-Razik et al 1974), cellulases, arabanases and xylanases (Mankarios and Friend 1980). Conversely, many fungal metabolites have been used as a potential opportunity to control plant pathogens.…”

Section: Discussionmentioning

confidence: 99%

Efficiency assessment of the antifungal metabolites from Sclerotium cepivorum against onion white rot disease

et al. 2015

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The inhibitory effects of secondary metabolites of Sclerotium cepivorum against itself were studied both in vitro and in vivo. The highest inhibition of fungal growth and a 98.6 % reduction in the number of sclerotia was obtained with 50 % culture filtrate. No sclerotial germination was observed on media amended with culture filtrates at 10, 25 and 50 %. Methanol extract of mycelia and sclerotia, and ethyl acetate extract of culture filtrate both exhibited over 90 % inhibition of S. cepivorum growth at 1200 ppm. A chloroform extract of culture filtrate resulted in 71.1 % inhibition at 1200 ppm. All extracts completely suppressed the formation and germination of sclerotia at 400, 800 and 1200 ppm. In greenhouse experiments, no disease was observed with the ethyl acetate extract when onion seedlings were dipped in the extract for 4 or 8 h. The GC-MS analysis revealed 28 compounds in the methanol extract. The methanol extract contained mainly hexadecanoic acid methyl ester (31.16 %), whereas 5-hydroxymethylfurfural, HMF, (36.40 %) was the main constituent of the ethyl acetate extract. These results suggest that the secondary metabolites of S. cepivorum are potential and promising antifungal agents for the control of onion white rot. These results can be also applied to reduce the incidence and severity of other diseases in different crops.

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Polysaccharide-degrading enzymes of Botrytis allii and Sclerotium cepivorum. Enzyme production in culture and the effect of the enzyme on isolated onion cell walls

Cited by 54 publications

References 18 publications

Molecular Cloning and Characterization of a Rhamnogalacturonan Acetylesterase from Aspergillus aculeatus

Molecular Cloning and Characterization of a Rhamnogalacturonan Acetylesterase from Aspergillus aculeatus

Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris

Efficiency assessment of the antifungal metabolites from Sclerotium cepivorum against onion white rot disease

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