NOVEL OSMOTICALLY INDUCED ANTIFUNGAL CHITINASES AND BACTERIAL EXPRESSION OF AN ACTIVE RECOMBINANT ISOFORM

NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predom inant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class I I acidic chitinase with similarity to PR-Q. The four predominant chiti nases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyse s, two of these were identified as class I chitinase isoforms, one sim ilar to the N. tomentosiformis(H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14: 357-368) protein (T1) and the other ho mologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] P lant Mot Biol 16: 1-10) protein (T2). The other two proteins (T3 and T 4) were determined to be novel chitinases that have sequence similarit y with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and T richoderma longibrachiatum hyphal growth in vitro, although the isofor ms containing a chitin-binding domain were somewhat more active. Condi tions were established for the successful expression of soluble and ac tive bacterial recombinant T2. Expression of soluble recombinant T2 wa s achieved when isopropyl beta-D-thiogalactopyranoside induction occur red at 18 degrees C but not at 25 or 37 degrees C. The purified recomb inant protein exhibited antifungal activity comparable to a class I ch itinase purified from NaCl-adapted tobacco cells.