Induction of chitinases and of translatable mRNA for these enzymes in melon plants infected with Collectotrichum lagenarium

Abstract

In melon plants infected with Colletotrichum langenarium, there is a strong increase in the activity of chitinase, an enzyme with a potential defensive function against pathogens. In order to investigate the molecular mechanisms involved in the regulation of chitinase gene expression, antisera have been raised against two purified chitinases (I and II) from infected melon plants. Changes induced by infection in the rate of synthesis of chitinases were determined using direct immunoprecipitation of enzymes labelled in vivo with [ 35S]methionine. A large but transient increase in the rate of chitinase synthesis occurred 5 days after inoculation. The in vitro synthesis of chitinases was then studied in healthy and infected melon plants. Poly(A)RNA was fractionated by sucrose gradient density centrifugation, and translated in a rabbit reticulocyte lysate. The transition products were then separated by fast protein liquid chromatography (FPLC) and further analysed by SDS-polyacrylamide gel electrophoresis. The identification of in vitro synthesized chitinases was performed by immunoprecipitation. The obtained results indicated the presence of chitinases in in vitro translation products of mRNA from infected, but not from healthy melon seedlings. It is concluded therefore, that infection of melon seedlings by a pathogen caused an increase in the translatable mRNAs for host chitinases.