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Isolation of Fungal Cell Wall Degrading Proteins from Barley (Hordeum vulgare L.) Leaves Infected with Rhynchosporium secalis

October 2002 , Volume 15 , Number  10
Pages  1,031 - 1,039

Reza Zareie , 1 Dara L. Melanson , 2 and Peter J. Murphy 3

1Department of Applied and Molecular Ecology, Waite Campus, Adelaide University, Glen Osmond, South Australia 5064, Australia; 2South Australian Research and Development Institute, Plant Research Centre, GPO Box 357, Adelaide, South Australia, 5001, Australia; 3Department of Plant Science, Waite Campus, Adelaide University, Glen Osmond, South Australia 5064, Australia

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Accepted 7 June 2002.

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa β-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.

Additional keywords: antifungal proteins , apoplast , PR proteins .

© 2002 The American Phytopathological Society