Some Pathogenesis-Related Proteins Are Chitosanases with Lytic Activity Against Fungal Spores. Jean Grenier. Départment de Phytologie, Faculté des Sciences de l’Agriculture et de l’Alimentation, Université Laval, Québec, Canada G1K 7P4. Alain Asselin. Départment de Phytologie, Faculté des Sciences de l’Agriculture et de l’Alimentation, Université Laval, Québec, Canada G1K 7P4.. MPMI 3:401-407. Accepted 23 July 1990. Copyright 1990 The American Phytopathological Society.

Proteins from intercellular fluids (IFs) of nonstressed and stressed barley leaves, cucumber cotyledons, and tomato leaves were analyzed for chitinase and chitosanase activity in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Glycol chitin incorporated in the gel matrix was used as the substrate for detection of chitinases, while glycol chitosan was the substrate of chitosanases. Chitinases with molecular masses between 25 and 35 kDa were stimulated in stressed barley and cucumber. Chitosanases with molecular masses between 10 and 24 kDa were stimulated in stressed barley, cucumber, and tomato. Chitosanase activities in IF extracts of stressed tissue were also analyzed after two-dimensional gel electrophoresis involving separation in native gels at pH 8.9 (Davis system) and 4.3 (Reisfeld system) in the first dimension and SDS gels in the second dimension. In stressed barley leaves, four acidic (three at 22 kDa, one at 19 kDa) and two basic (one at 22 kDa, one at 19 kDa) chitosanases were detected. Four acidic chitosanases (one at 10 kDa, one at 12 kDa, and two at 14 kDa) were also observed in stressed cucumber cotyledons, while one basic chitosanase (24 kDa) was found in stressed tomato. Chitosanases were also evaluated for their capacity to hydrolyze fungal spores of pathogens. Spore suspensions were embedded in polyacrylamide gels, and lysis was observed visually by transparency through the opaque spore suspension. In one-dimensional SDS gels, lysis of Fusarium oxysporum f. sp. radicis-lycopersici was observed with stressed barley and tomato IF extracts. In tomato, the activity lysing spores corresponded to the major basic chitosanase at 24 kDa. In barley, the spore lytic activity migrated exactly as did basic chitosanase 6 (19 kDa) after analysis in a two-dimensional gel system. Verticillium albo-atrum and Ophiostoma ulmi spores were also lysed by the same chitosanases. By using these new gel assays, up to six chitosanase activities of low molecular mass (below 24 kDa), distinct from chitinase activities, can be detected in IF extracts of stressed tissue.