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Isolation and Characterization of an Endopolygalacturonase from Cochliobolus sativus and a Cytological Study of Fungal Penetration of Barley

November 1997 , Volume 87 , Number  11
Pages  1,148 - 1,159

Ronald P. Clay , Carl W. Bergmann , and Melvin S. Fuller

First and second authors: Complex Carbohydrate Research Center, The University of Georgia, Athens; and third author: Department of Botany, The University of Georgia, Athens

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Accepted for publication 11 July 1997.

Endopolygalacturonase (EPG) of Cochliobolus sativus was produced in shake culture and purified by high-performance liquid chromatography. The enzyme had a molecular mass of 34,000 Da, an isoelectric point in the range of 9.0 to 9.5, exhibited endo activity, was nongly-cosylated, and was inhibited by polygalacturonase-inhibiting proteins from bean, pear, and tomato. The amino terminus contained a 14 amino acid region homologous to a region at the N terminus of an EPG of C. carbonum. C. sativus EPG-specific monoclonal antibodies (MAbs) were generated. Western blot analysis confirmed the specificity of the antibodies for the EPG and detected the enzyme in an extract from Hordeum vulgare (cv. Golden Promise) leaf segments infected with C. sativus. Using conventional immunogold and enzyme-gold cytochemical methods, homogalacturonan, esterified pectin, and cellulose were localized in healthy and infected barley leaf epidermis at the electron microscope level. Additionally, the leaf cell wall polysaccharides recognized by purified C. sativus EPG were localized at the electron microscope level, using the purified enzyme as a primary cytochemical reagent, followed by a gold-labeled MAb specific for the enzyme. Loss of polygalacturonic acid in the vicinity of the invading pathogen was visualized cytochemically at the electron microscope level. These observations suggest the involvement of EPG during host penetration by the fungus.

Additional keywords: amino acid sequence, cytochemistry, Edman degradation, electron microscopy, ELISA, light microscopy, mass spectrometry, pectinase.

© 1997 The American Phytopathological Society