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

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.