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Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi

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Abstract

Chitinase (EC 3.2.1.14.) activity increased in pea (Pisum sativum L.) leaves inoculated with both virulent and avirulent isolates of Ascochyta pisi Lib. Three basic chitinase isoenzymes were purified: two, A1 and A2, separated by high performance liquid chroma tography, had a relative molecular mass (Mr) of approx. 34 000, with an isoelectric point (pI) of 8.5, and one, B, had an Mr of approx. 25 000, with a pI of 9.0. Elevated levels of chitinase isoenzymes were found in the leaves: thus 150 h after inoculation, there was a ninefold increase for isoenzymes A1 and A2 combined, with a threefold increase for isoenzyme B in susceptible plants, and a sevenfold increase for isoenzymes A1 and A2 combined, with a twofold increase for isoenzyme B in hypersensitive-reacting plants. The N-terminal 15–23 amino-acid residues of the three chitinase isoenzymes were sequenced, and considerable sequence similarity was found to chitinase sequences from tobacco, potato and bean, as well as to hevein and wheat-germ agglutinin. Purified chitinase protein cross-reacted with specific antiserum raised against a chitinase isoenzyme from sugar beet (Beta vulgaris L.). Immunoblots prepared using leaf proteins isolated at different time points following inoculation revealed the accumulation of polypeptides corresponding to at least two of the chitinase isoenzymes.

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Abbreviations

FPLC:

fast protein liquid chromatography

HPLC:

high-performance liquid chromatography

Mr :

relative molecular mass

pI:

isoelectric point

RH:

relative humidity

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Section for Plant Pathology, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark

    Knud Vad & David B. Collinge

  2. Biotechnology Research Division, Danisco A/S, Langebrogade 1, DK-1001, Copenhagen K, Denmark

    Jørn D. Mikkelsen

Authors
  1. Knud Vad
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  2. Jørn D. Mikkelsen
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  3. David B. Collinge
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Additional information

We wish to thank E. Bollerup and T. Kufa (Pajbjergfonden, Odder, Denmark) for permission to quote unpublished data and for supplying plant and fungal material, K.G. Welinder (Dept. of Biochemical Genetics, University of Copenhagen, Denmark) for peptide sequencing, and S. Dreboldt (Danisco A/S, Copenhagen, Denmark), U. Jagd and C. Boesgaard Jensen for technical assistance. We are grateful to P.L. Gregersen, K.M. Kragh, V. Smedegaard-Petersen and H. Thordal-Christensen (Dept. of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark) for their constructive criticism during this work, and to L. Rossen (The Genetic Engineering Group, Lyngby, Denmark) and A.J. Slusarenko (Institut für Pflanzenbiologie, Zürich, Switzerland) for critically reading drafts of the manuscript. This study is supported by Centre for Plant Biotechnology and Statens Jordbrugsog Veterinærvidenskabelige Forskningsråd.

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Vad, K., Mikkelsen, J.D. & Collinge, D.B. Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi . Planta 184, 24–29 (1991). https://doi.org/10.1007/BF00208231

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  • DOI: https://doi.org/10.1007/BF00208231

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