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Induction and purification of chitinase in Brassica napus L. ssp. oleifera infected with Phoma lingam

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Abstract

A pathogen-induced chitinase (EC 3.2.1.14) was isolated from cotyledons of oilseed rape (Brassica napus cv. Bienvenu) 8 d after inoculation with Phoma lingam. The purified chitinase has a molecular weight of 30 kDa, and an isoelectric point of approx. 9.1. A partial amino-acid sequence obtained after tryptic digestion of the protein shows high sequence similarity to basic chitinases from bean, tobacco, potato, Arabidopsis, barley and rice, as well as to acidic chitinases from tobacco and petunia. A close serological relationship was found between the chitinase isoenzyme and an isoenzyme from sugar-beet (Beta vulgaris L.). When resistant and susceptible cultivars were inoculated with P. lingam there was a significant difference in the increase in chitinase activity during the early stage after inoculation. The resistant cultivars showed a rapid increase in chitinase activity, in contrast to susceptible cultivars where an increase in activity was delayed until 24 h after infection. By measuring the chitinase activity from the mycelium of P. lingam, it was concluded that the increase in chitinase activity found in infected plants was of plant origin. The chitinase activity was found to be restricted to the site of pathogen attack and was not systemically induced in other parts of the plant.

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Abbreviations

CHIT:

chitinase

FPLC:

fast protein liquid chromatography

HPLC:

high-performance liquid chromatography

RP:

reverse phase

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Plant Biology Section, Environmental Science and Technology Department, Risø National Laboratory, DK-4000, Roskilde, Denmark

    Ulla Rasmussen & Henriette Giese

  2. Biotechnological Research Division, Danisco A/S, DK-1001, Copenhagen, Denmark

    Jørn Dalgaard Mikkelsen

Authors
  1. Ulla Rasmussen
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  2. Henriette Giese
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  3. Jørn Dalgaard Mikkelsen
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Additional information

We wish to thank H.E. Larsen for expert technical assistance and Dr. I. Svendsen, Carlsberg Laboratory for doing the amino-acid sequencing. Dr. D.B. Collinge is acknowledged for critical reading of the manuscript. U. Rasmussen was supported by a grant from the Center for Plant Biotechnology.

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Rasmussen, U., Giese, H. & Mikkelsen, J.D. Induction and purification of chitinase in Brassica napus L. ssp. oleifera infected with Phoma lingam . Planta 187, 328–334 (1992). https://doi.org/10.1007/BF00195656

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  • DOI: https://doi.org/10.1007/BF00195656

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