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 sh ake culture and purified by high-performance liquid chromatography. Th e enzyme had a molecular mass of 34,000 Da, an isoelectric point in th e range of 9.0 to 9.5, exhibited endo activity, was nonglycosylated, a nd was inhibited by polygalacturonase-inhibiting proteins from bean, p ear, and tomato. The amino terminus contained a 14 amino acid region h omologous to a region at the N terminus of an EPG of C. carbonum. C. s ativus EPG-specific monoclonal antibodies (MAbs) were generated. Weste rn blot analysis confirmed the specificity of the antibodies for the E PG and detected the enzyme in an extract from Hordeum vulgare (cv. Gol den Promise) leaf segments infected with C. sativus. Using conventiona l immunogold and enzyme-gold cytochemical methods, homogalacturonan, e sterified 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 purifie d enzyme as a primary cytochemical reagent, followed by a gold-labeled MAb specific for the enzyme. Loss of polygalacturonic acid in the vic inity of the invading pathogen was visualized cytochemically at the el ectron microscope level. These observations suggest the involvement of EPG during host penetration by the fungus.