Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-β-Glucanases and Chitinases

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M(r)] 35 kilodaltons [kD], isoelectric point [pI] approximately 6.4) showed 1,3-beta-glucanase activity, while the other one (M(r)26 kD, pI approximately 6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-beta-glucanases isolated from other plant species, one additional 1,3-beta-glucanase (M(r)33 kD) and three additional chitinases (M(r) 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-beta-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.