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 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 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells.
Yun
D J
DJ
Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, Indiana 47905-1165, USA.
D'Urzo
M P
MP
Abad
L
L
Takeda
S
S
Salzman
R
R
Chen
Z
Z
Lee
H
H
Hasegawa
P M
PM
Bressan
R A
RA
D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D002688:Chitinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology* / Q000382:microbiology
D000935:Antifungal Agents; D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D012965:Sodium Chloride; D002688:Chitinases