PLANT PHYSIOLOGY , Vol 111, Issue 4 1219-1225, Copyright © 1996 by American Society of Plant Biologists
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WHOLE PLANT, ENVIRONMENTAL, AND STRESS PHYSIOLOGY |
Novel Osmotically Induced Antifungal Chitinases and Bacterial Expression of an Active Recombinant Isoform
D. J. Yun, M. P. D'Urzo, L. Abad, S. Takeda, R. Salzman, Z. Chen, H. Lee, P. M. Hasegawa and R. A. Bressan
Center for Plant Environmental Stress Physiology, 1165 Horticulture Building, Purdue University, West Lafayette, Indiana 47907-1165
NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells
accumulate and secrete several antifungal chitinases. The predominant
protein secreted to the culture medium was a 29-kD peptide that, based on
internal amino acid sequence, was determined to be a class II acidic
chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2,
T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells
were purified. Based on N-terminal sequence analyses, two of these were
identified as class I chitinase isoforms, one similar 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 homologous to the N.
sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16: 1-10)
protein (T2). The other two proteins (T3 and T4) were determined to be
novel chitinases that have sequence similarity with class I chitinases, but
each lacks a chitin-binding domain. All four chitinases inhibited Fusarium
oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth
in vitro, although the isoforms containing a chitin-binding domain were
somewhat more active. Conditions were established for the successful
expression of soluble and active bacterial recombinant T2. Expression of
soluble recombinant T2 was achieved when isopropyl
[beta]-D-thiogalactopyranoside induction occurred at 18[deg]C but not at 25
or 37[deg]C. The purified recombinant protein exhibited antifungal activity
comparable to a class I chitinase purified from NaCl-adapted tobacco cells.
