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

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.