VIEW ARTICLE | DOI: 10.1094/MPMI-2-315
Electrophoretic Forms of Chitinase Activity in Xanthi-nc Tobacco, Healthy and Infected with Tobacco Mosaic Virus. Jean Trudel. Départment de Phytologie, Faculté des Sciences de 1'Agriculture et de 1'Alimentation, Université Laval, Québec, Canada, G1k 7P4.. Patrice Audy, and Alain Asselin. Départment de Phytologie, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Québec, Canada, G1k 7P4.. MPMI 2:315-324. Accepted 28 June 1989. Copyright 1989 The American Phytopathological Society.
Additional keywords: chitinolysis, hydrolases, Nicotiana, two-dimensional polyacrylamide gel.
Tobacco leaf homogenates and intercellular fluid extracts were analyzed in native polyacrylamide gel systems for detection of chitinase activity. They were compared using clarified homogenates from several tobacco organs. In gels specific for acidic proteins, one major and five minor activities were found in diseased tissue, while only the major activity could be detected at a low level in healthy leaf tissue. Up to eight activities, including the six activities from infected leaf tissue, could be revealed in flower parts and apical leaves from healthy tobacco plants. In gels specific for basic proteins, two major and five minor activities were observed both in infected leaf tissue and in healthy flower parts. All tissue extracts contained one of the major basic chitinases. In both gel systems, the precise identification of pathogenesis-related (PR) proteins with chitinase activity was precluded because bands in one-dimensional native gels often contained more than one protein. Thus, chitinase activity was also studied in sodium dodecyl sulfate denaturing gels. Estimated molecular masses were 25-26 kDa and 30 kDa. The most prominent activity was the 30-kDa protein in all tissues, except for some flower parts. This activity was highly stimulated in infected tissue. The activities at 25-26 kDa were also stimulated in infected tissue and present in healthy flower parts. The major acidic chitinase stimulated in infected tissue hydrolyzed 4-methylumbelliferylchitotriose but not 4-methylumbelliferylchitobiose. The two major basic chitinases stimulated in infected tissue hydrolyzed the chitotriose derivative, but the chitobiose substrate was only hydrolyzed by one of the enzymes. Both basic chitinases had lysozyme activity. A two-dimensional gel system was devised to allow identification of individual proteins with chitinase activity. Four acidic proteins could be renatured in such gels, and three proteins corresponded to PR-P, PR-Q, and PR-b6c. Chitinase activity of PR-b6c was much higher than the three other activities combined. Four basic chitinases could be identified. One corresponded to PR-b13 and the others to unidentified proteins. Up to 13 (6 acidic, 7 basic) activities could be detected in Xanthinc leaf tissue infected with tobacco mosaic virus.