Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20

Structure, Heterologous Expression, and

Properties of Rice (Oryza sativa L.) Family 19
Chitinases

Nam-Hai TRUONG, Seung-Moon PARK, Yoko NISHIZAWA, Takeshi WATANABE,

Takuji SASAKI & Yoshifumi ITOH

To cite this article: Nam-Hai TRUONG, Seung-Moon PARK, Yoko NISHIZAWA, Takeshi
WATANABE, Takuji SASAKI & Yoshifumi ITOH (2003) Structure, Heterologous Expression,
and Properties of Rice (Oryza�sativa�L.) Family 19 Chitinases, Bioscience, Biotechnology, and
Biochemistry, 67:5, 1063-1070, DOI: 10.1271/bbb.67.1063

To link to this article: https://doi.org/10.1271/bbb.67.1063

Published online: 22 May 2014.

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Biosci. Biotechnol. Biochem., 67 (5), 1063–1070, 2003

Structure, Heterologous Expression, and Properties of Rice (Oryza sativa L.)

Family 19 Chitinases
Nam-Hai TRUONG,1,* Seung-Moon PARK,1,** Yoko NISHIZAWA,2 Takeshi WATANABE,3
Takuji SASAKI,2 and Yoshifumi ITOH1,†
1National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
2National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
3Department of Biosystem Science, Graduate School of Science and Technology, Niigata University,

8050 Ikarashi-2, Niigata 950-2102, Japan

Received December 3, 2002; Accepted January 31, 2003

We identiˆed four new family 19 chitinases in Oryza
sativa L. cv. Nipponbare: one class I (OsChia1d), two
class II (OsChia2a and OsChia2b), and one class IV
(OsChia4a). OsChia2a resembled (about 60z identity)
the catalytic domains of class I chitinases, but OsChia2b
was almost identical (95z identity) to that of the class
IV enzyme. OsChia1c, OsChia1cDCBD (a deletion of
OsChia1c lacking a chitin-binding domain, CBD), and
OsChia2b were separately expressed and puriˆed in
Pichia pastoris. OsChia1c inhibited fungal growth sig-
niˆcantly more than OsChia1cDCBD or OsChia2b. The
activities of these enzymes on chitin polymers were simi-
lar, but they acted diŠerently on N-acetylchitooligosac-
charides, (GlcNAC)n. OsChia1c slowly hydrolyzed
(GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and
(GlcNAC)5. In contrast, OsChia2b e‹ciently hydro-
lyzed these oligosaccharides. The high antifungal activi-
ty and low hydrolytic activity of the class I enzyme
towards (GlcNAC)n imply that it participates in the
generation of N-acetylchitooligosaccharide elicitors
from the cell walls of infecting fungi.
Key words: rice (Oryza sativa L); chitinase; N-acetyl-
chitooligosaccharides; antifungal activity;
chitin-binding domain
Plant defense against potential pathogens includes
a hypersensitive response, the production of reactive
oxygen species (oxygen burst), the activation of
pathogenesis-related (PR) genes, structural changes
in the cell wall, and phytoalexin synthesis.1–3)
Elicitors generated by pathogen infection induce
the hypersensitive response, oxygen burst, and PR-
protein as well as phytoalexin synthesis to destroy
plant cells and prevent pathogen growth at the attack
site. The hypersensitive response results in the forma-
tion of signal substances such as ethylene, as well as
salicylic, jasmonic, and nitric acids, which provoke
systemic acquired resistance and transient resistance
throughout the plant to subsequent attack by patho-
gens.1,2) As well as elicitors, these signals activate PR
protein and phytoalexin synthesis. The PR proteins
constitute 11 groups (PR-1 through PR-11).4) Among
these, chitinases (PR-3, PR-4, PR-8, and PR-11) and
b-1,3-glucanase (PR-2) have been more frequently
studied because of their ability to inhibit, particularly
in combination, fungal growth in vitro, through
hydrolytic activities on the chitin and b-1,3-glucan
that are major components of fungal cell walls.5,6)
The participation of these enzymes in plant defense
has been substantiated by the increased resistance
to fungal pathogens of transgenic plants that con-
stitutively express high levels of chitinase and W or
b-1,3-glucanase.7,8) Plant chitinases appear to be
responsible for the formation of elicitors ( N-acetyl-
chitooligosaccharides) from fungal cell walls to acti-
vate their own synthesis and other types of the
defense response.9–12)
Plant chitinases belong to structurally unrelated
families 18 (PR-8 and PR-11) and 19 (PR-3) of
glycosylhydrolases.5,13,14) Family 18 includes classes
III (PR-8) and V (PR-11) enzymes. Family 19 con-
sists of three major classes (I, II, and IV). Class I and
IV enzymes have a diagnostic cysteine-rich chitin-
binding domain (CBD) at the amino terminus,5,14)
and class II enzymes lack this domain (Fig. 1A). The
CBD domain is dispensable for catalytic and antifun-
gal activities but can increase antifungal function.15)
Several enzymes of both families occur in the same
plant, allowing the expression of tissue- or develop-
mental stage-speciˆc chitinases or generation of
chitinases in response to distinct stimuli.5,16,17) Each

† To whom correspondence should be addressed. Tel: ＋81-298-38-8075; Fax: ＋81-298-38-7996; E-mail: yosifumi＠nfri.aŠrc.go.jp
* Present address: Institute of Biotechnology, National Centre for Natural Science and Technology, Nghia do, Tu Liem, Hanoi, Viet-
nam
** Present address: Basic Science Research Institute, Chonbuk National University, Chonju 561-756, Korea
1064 N.-H. TRUONG et al.

isoform may have distinct catalytic properties in

Fig. 1. Schematic Structures (A) and Structural Relationship (B)
of Rice Family 19 Chitinases.
Amino acid sequences of the enzymes were aligned to identify
relevant structures and were used to create the tree using Clustal
W software (www.ddbj.nig.ac.jp). Sequences are taken from:
OsChia1a (accession number D16221), OsChia1b (D16222),
OsChia1c (D16223), OsChia1d (AB096139), OsChia2a
(AB016497), OsChia2b (AB003194), OsChia4a (AB096140),
and ChiC (family 19 chitinase of Streptomyces griseus;
AB009289). A: CatD, catalytic domain; CBD, chitin-binding
domain; SP, signal peptide; L, linker sequence. Regions are de-
ˆned as in Fig. 2. B: Numbers at branches are bootstrap esti-
mates and bar indicates substitution Wresidue.
terms of elicitor formation, antifungal spectrum, or
synergistic eŠect with other chitinases or b-1,3-
glucanses on antifungal action.5) Like many other
plants, Oryza sativa L. cv. Nipponbare ( japonica
ssp.) possesses several family 19 chitinases as predict-
ed by Southern blotting.18,19) Three class I genes
(Cht-1, Cht-2, and Cht-3) have been identiˆed in this
cultivar (Table 1).18) To avoid confusion with the
chitinases of other rice cultivars and other plants, we
refer to Cht-1, Cht-2, and Cht-3 as OsChia1a,
OsChia1b, and OsChia1c, respectively, according
to the recommended nomenclature of plant
chitinases.20) These enzymes appear to be expressed at
diŠerent sites and distinctly regulated,11,18,19) perhaps
playing particular roles in the defense mechanism. To
further understand the physiological roles of rice
chitinases, a complete set of the relevant genes must
be identiˆed and the properties of the corresponding
products should be understood. We have identiˆed
and characterized two family 18 chitinase (OsChib3a
and OsChib3b) cDNAs21) in the EST library
(about 50,000 clones) of Nipponbare that had
been constructed by the Rice Genome Project
(http://rgp.dna.aŠrc.go.jp).
We describe here the structures of four novel fami-
ly 19 chitinases (one class I, two class II, and one
class IV) isolated from Nipponbare and the proper-
ties of OsChia1c (a representative of class I),
OsChia1cDCBD (a class I-type catalytic domain),
and OsChia2b (a class IV-type catalytic domain) that
are expressed in P. pastoris.
Materials and Methods
Strains and media. We used Escherichia coli DH5a
(Bethesda Research Laboratory, Bethesda, MA,
USA) as the host in plasmid construction, as well as
E. coli BL21 (DE3) (Novagen; Madison, WI, USA)

Table 1. Family 19 Chitinases of Nipponbare and Other Cultivars

Nipponbare Other cultivarsa Expression Ref.

regulated by)
(Tissue W
Class I
OsChia1a (Cht-1)b RCH10 (94z), RC24 (94z) Leaf W
elicitors 11, 18, 19, 28, 31, 32)
CH16 (94z)
OsChia1b (Cht-2) Root vacuole W constitutive 18)
OsChia1c (Cht-3) CH6 (91z) Leaf Welicitors 11, 18, 31)
OsChia1d OsChia1;175 (100z) Pestle W
tissue-speciˆcity 30)
Class II
OsChia2a RCHT2 (95z) Leaf W
elicitors 10)
OsChia2b (Chi-4) Husk Wtissue-speciˆcity, high temperature 33)
Class IV
OsChia4a tissue-speciˆcity
Floral organ W
a Amino acid sequence similarities to the corresponding Nipponbare counterparts are shown in parentheses as percent identities. The origins of enzymes are:
CH6 and CH16, japonica cv. Nakdongbyeo; RCH10 and RC24, indica cv. IR36; OsChia1;175, indica cv. IR24; RCHT2, indica cv. Cheongcheongbyeo.
b Alternative names of the Nipponbare family 19 chitinases are indicated in parentheses.
 Structures and Properties of Rice Family 19 Chitinases
and Pichia pastoris G115 (Invitrogen; San Diego,
CA, USA) for the production of recombinant
chitinases. Escherichia coli was grown in Luria-
Bertani (LB) medium (1z tryptone, 0.5z yeast
extract, and 1z NaCl) with ampicillin (100 mg W ml)
when necessary. Pichia pastoris was cultured in YPD
medium (1z yeast extract, 2z peptone, and 2z dex-
trose) for general growth, on MM agar (1.34z yeast
nitrogen base, 4×10－5z biotin, 1z glucose, and
1.5z agar) for selection of His＋ transformants, and
in BMMY medium (1z yeast extract, 2z peptone,
100 mM potassium phosphate (pH 6.0), 1.34z yeast
nitrogen base, 4×10－5z biotin, and 1z methanol)
for the production of recombinant chitinase, as
recommended by the manufacturer (Invitrogen).
Construction of cDNA library and nucleotide
sequencing. We extracted and puriˆed mRNA from
various O. sativa L. cv. Nipponbare tissues as
described.22) The corresponding cDNAs were then
synthesized using a SuperScript kit (Bethesda
Research Laboratory, Bethesda, MD, USA), ligated
to the Sal I and Not I sites of plasmid pBluescript II
SK＋ (Stratagene; La Jolla, CA, USA), and used to
transform E. coli NM522 (Stratagene). Nucleotides
were sequenced as described22) or using an ABI377
DNA sequencer with Big-dye primer and Big-Dye ter-
minator sequencing kits (Applied Biosystems, Foster
City, CA, USA). Amino acid and DNA sequences
were analyzed using the Clustal W and Blast pro-
grams at the DDJB (http://www.ddbj.nig.ac.jp).
Plasmid and strain construction. We attempted
to express class I chitinase OsChia1c, truncated
OsChia1c without CBD (OsChia1cDCBD), and
OsCha2b in E. coli. The corresponding cDNA
regions were ampliˆed by PCR using the primers, P1
(5?-ATGGCCATGGAGCAGTGCGGCAGCCAG-
3?) and P2 (5?-ACTCGAGGGAAGGCGGGTAG-
GGCCTCTGGT-3?) (for OsChia1c), P3 (5?-TTGG-
CCATGCCGACCCGCCCTCCAGC-3?) and P2
(for OsChia1cDCBD), and P4 (5?-GCCATGGCAG-
CCGGCGTGTCTGTGGAGA-3?) and P5 (5?- ACT-
CGAGACAGTAGAGGTTCCCCCCGGG-3?) (for
OsChia2b). After conˆrming the nucleotides by
sequencing, the ampliˆed fragments were cloned
between the Nco I and Xho I sites of plasmid
pET-22b (＋) (Novagen) as Msc I (or Nco I) and
Xho I fragments to produce pET-OsChia1c,
pET-OsChia1cDCBD and pET-OsChia2b with the
chitinase sequences fused in-frame between the pelB
leader sequence and the His-tag sequence of the plas-
mids. Because these enzymes were expressed in E.
coli BL21 (DE3) as the inactive form in inclusion
bodies, we tried to express the chitinases using a
Pichia expression kit (Invitrogen; San Diego, CA,
USA). The chitinase genes in the recombinant
plasmids were PCR-ampliˆed using the primers, P6
1065
(5?-ATTACGTAGAGCAGTGCGGCAGCCAGG-
C-3?) and P7 (5?-TGCGGCCGCTCAGCGGTGGC-
AGCAGCCAACT-3?) (for OsChia1c), P8 (5?-ATT-
ACGTACCGACCCCGCCCTCCAGC-3?) and P7
(for OsChia1cDCBD), and P9 (5?-ATTACGTAG-
CAGCCGGCGTGTCTGTGGAG-3?) and P7 (for
OsChia2b), and cloned as Sna BI and Not I fragments
into plasmid pPIC9 between the corresponding
restriction sites. Sequencing the DNA conˆrmed
that the resultant plasmids pPIC-OsChia1c, pPIC-
OsChia1cDCBD, and pPIC-OsChia2b carried the
correct chitinase cDNA tagged with histidine codons
and that it was fused in-frame downstream of the a-
factor signal peptide. The resultant plasmids were
linearized with the restriction endonuclease Bgl II,
then used to transform P. pastoris G115 (His－).
The His＋ transformants, P. pastoris OsChia1c-1,
OsChia1cDCBD-1, and OsChia2b-1, were selected
on MM agar. We conˆrmed that the corresponding
enzyme was produced by the recombinant strains by
measuring chitinase activities in BMMY culture
medium.
Expression and puriˆcation of chitinases. We incu-
bated P. pastoris cells harboring a recombinant
chitinase cDNA in BMMY medium (50 ml) at 309 C
for 36 h to fully express the chitinase as described.21)
The chitinases were puriˆed by passage through a
Ni2＋-a‹nity column (His-Trap column, Amersham
Biotech; Buckinghamshire, UK) according to Park
et al.21) Puriˆed enzymes were thoroughly dialyzed
against 20 mM phosphate buŠer (pH 7.2) containing
1 mM EDTA. The induction and purity of the enzyme
were analyzed by SDS-polyacrylamide gel elec-
trophoresis (SDS-PAGE).23)
Assays of enzyme, chitin-binding, and antifungal
activities. Chitinase activity was assayed in 0.5 ml of
0.1 M citrate-phosphate buŠer (pH 4.8 or pH 5.2)
containing 0.2z glycol chitin (or 0.2z colloidal
chitin) and enzyme (0.05 ml) according to the modi-
ˆed procedures of Schales.24) One unit was deˆned as
the amount of enzyme that is required to produce
1 mmol of reducing sugar (as N-acetylglucosamine)
per min. Km and Vmax values were calculated from
double reciprocal plots (1 W v versus 1 W
s). To examine
heatstability, enzymes were incubated in 20 mM phos-
phate buŠer (pH 7.2) at various temperatures for
10 min and remaining activities were measured. For
binding assays, puriˆed chitinase (10 mg) was incu-
bated with 1 mg of colloidal or regenerated chitin as
binding substrates in the above citrate-phosphate
buŠer (0.2 ml) for 1 h on ice with occasional mix-
ing.25) Thereafter, free enzymes were separated from
enzyme-substrate complexes by centrifugation at
15,000×g for 20 min at 09C and measured using
protein assay kit (Bio-Rad Laboratories, Hercules,
CA, USA). Enzyme-substrate complexes in pellets
1066
Fig. 2. Amino-terminal Sequences of Family 19 Chitinases.
Amino acid sequences were aligned as in Fig. 1. N-terminal ends of catabolic enzyme domains correspond to that of mature barley
class II enzyme.38)
were washed with 0.2 ml of the buŠer and lyophi-
lized. Bound enzymes were then dissociated from the
complexes by boiling in 0.1 ml of sample buŠer23) and
20-ml portions of samples were analyzed by SDS-
PAGE. Amounts of the bound proteins were meas-
ured by comparing densities of protein bands with
that of known amounts of proteins on the gels.
N-acetylchitooligosaccharides (1 mM; Seikagaku
Kogyo, Tokyo, Japan) were incubated with 1 mg of
enzyme as described above. After the indicated incu-
bation period, reaction products were resolved and
detected on silica gel 60 plates (Merck; Darmstadt,
Germany) as described.21) Antifungal activity was
assayed by disk-plate diŠusion26) using Trichoderma
reesei IFO31329 as the test fungus.25)
Results
Family 19 chitinases of O. sativa L cv. Nipponbare
Sequencing the cDNA inserts (Æ1 kb) with
similarity to known class I chitinase in the EST
library (48,526 clones) of the Rice Genome Project
(http://rgp.aŠrc.go.jp) discovered four new family
19 chitinases named OsChia1d (DDJB accession num-
ber AB096139), OsChia2a (AB016497), OsChia2b
(AB003194), and OsChia4a (AB096140). A com-
parison of their deduced amino acid sequences with
the sequences of other plant family 19 chitinases
showed that OsChia1d and OsChia4a have a CBD
at the N-terminal, but OsChia2a and OsChia2b
appeared to lack this domain, since a signal peptide
was adjacent to the catalytic domain (Fig. 2). The
catalytic domains of OsChia1d and OsChia2a resem-
bled (about 60z identity) the corresponding regions
of other class I enzymes. OsChia2b and the catalytic
domain of OsChia4a were very similar (95z identity)
(Fig. 1B) and resembled (60–70z identity) the class
IV chitinases of other plants rather than the class I
enzymes (º60 identity). In addition, three deletions
and a cysteine residue located at the carboxyl termi-
nal are features of class IV chitinases.5,14)
Frequency of the family 19 EST clones
The frequency of the OsChia1a, OsChia1b,
OsChia1c, and OsChia2a EST clones in the total EST
clones from various tissues, roots (4,079 clones),
N.-H. TRUONG et al.
green shoots (9,212 clones), etiolated shoots (5,103
clones), and panicles (15,128 clones), was low
(Ã0.02z). In contrast, the frequency of OsChia1d
EST clones was high in panicles, particularly in ‰ow-
ing panicles (0.35z). OsChia2b and OsChia4a clones
also occurred in panicle clones, although at a lower
frequency (about 0.04z).
Heterologous expression and puriˆcation
All eŠorts to express active OsChia1c, Os-
Chia1cDCBD, and OsChia2b in E. coli were unsuc-
cessful. The proteins were produced as inactive and
insoluble forms in inclusion bodies even when cul-
tures were incubated at 159C, conditions that we
used to produce the barely active class II chitinase in
E. coli.27) Attempts by other groups to express rice
family 19 chitinases in E. coli also met with the
formation of inclusion bodies or very low levels of
synthesized active enzyme.28–30) We accordingly tried
to use P. pastoris as the host, as eukaryotic proteins
have often been produced in this organism. When
cultured in BMMY medium, P. pastoris cells harbor-
ing a recombinant chitinase cDNA produced, ex-
tracellularly, satisfactory quantities of the corre-
sponding chitinase. Enzyme formation was maximal
by 36 h (about 0.2 mg W 50 ml) and remained
unchanged for at least up to 72 h (data not shown).
The enzymes were puriˆed to apparent homogeneity
after Ni 2＋ a‹nity column chromatography, as
judged by SDS-PAGE (Fig. 3A).
Chitin-binding a‹nity of CBD and its increase of
antifungal activity
To conˆrm the binding-a‹nity of rice CBD to
chitin, we bound OsChia1c, OsChia1cDCBD, and
OsChia2b (10 mg each) to colloidal chitin (1 mg) in
0.2 ml of mixture (see Materials and Methods) and
measured the amounts of free and bound proteins by
protein assay and by SDS-PAGE, respectively.
OsChia1cDCBD and OsChia2b (9.2 and 9.4 mg, re-
spectively) were recovered in the supernatant, but
only 0.6 mg of OsChia1c was recovered from the
supernatant. Association of OsChia1c with the
binding substrate was conˆrmed by SDS-PAGE
(Fig. 3B); 9.0, 0.7 and 0.5 mg of OsChia1c,
OsChia1cDCBD and OsChia2b, respectively, were
 Structures and Properties of Rice Family 19 Chitinases 1067

Fig. 4. Inhibition of Hyphal Growth of T. reesei by Chitinases.

Growth inhibition was assayed as described.25) Trichoderma
reesei conidia (about 103) were inoculated onto paper disks (T)
placed at the center of plates and incubated for 24 h at 259C.
Enzymes were applied in various amounts onto disks placed at
edges of extending hyphae and plates were incubated for a fur-
ther 24 h. Amounts of the enzymes applied: 1, 0 mg; 2, 1 mg; 3,
2 mg; 4, 5 mg; 5, 10 mg.
Fig. 3. SDS-Polyacrylamide Gel Electrophoresis of Puriˆed
Chitinases (A) and CBD-Dependent Binding to Colloidal Chitin
(B).
A: Puriˆed chitinases (1.5–2 mg) were resolved on 12.5z SDS- Table 2. Properties of OsChia1c, OsChia1cDCBD, and Os-
polyacrylamide gels along with molecular standards (M) and Chia2b Expressed in P. pastoris
stained with Coomassie brilliant blue. B: Chitinases were incu-
bated with colloidal chitin and bound enzymes were analyzed by Properties Colloidal Glycol Optimum Thermal
chitin chitin stabilitya
SDS-PAGE. 1, OsChia1c; 2, OsChia1cDCBD; 3, OsChia2b.
Enzymes Km Vmax Km Vmax pH temp. 9C
(9C)

measured in the complexes. The binding-a‹nity of OsChia1c 0.9 2.1 0.4 52.0 4.8 65 68
OsChia1cDCBD 1.2 1.9 0.2 82.6 4.8 65 68
these proteins to regenerated chitin was essentially
OsChia2b 1.9 2.3 0.4 116.3 5.2 65 68
the same as that to colloidal chitin (data not shown).
Tobacco and bacterial CBD have been shown to Km (mg W ml) and Vmax ( mmol reducing sugars generated W
mg protein W
min)
values are averages of two measurements.
increase antifungal activity about three- and 10-fold, a Enzymes were incubated at various temperatures for 10 min and

respectively.15,25) We examined the eŠects of rice CBD remaining activities were measured. Temperatures that caused 50z
on antifungal function and whether or not class I and reduction of enzyme activity are presented.
class IV type catalytic domains have similar antifun-
gal activities. Inhibition assays of the hyphal growth
of T. reesei with puriˆed OsChia1c, OsChia1cDCBD,
and OsChia2b showed that OsChia1c inhibited
fungal growth more than its deletion derivative
(Fig. 4). The growth inhibition caused by 10 mg of
OsChia1cDCBD roughly corresponded to the action
of 2 mg of OsChia1c. OsChia2b had inhibitory activi-
ty similar to that of OsChia1cDCBD. These results
showed that rice CBD can stimulate the antifungal
activity of the catalytic domain by about 5-fold and
that both class I and class IV catalytic domains have
similar antifungal properties.

Catalytic properties
OsChia1c and OsChia1cDCBD hydrolyzed soluble
glycol chitin and insoluble colloidal chitin with
similar Km and Vmax values and thermal stability
(Table 2). The catalytic properties of OsChia2b with
the tested substrate were similar, although it was a
little more active than class I enzymes toward glycol
chitin. However, the activities of the class I and class
II enzymes distinctly diŠered towards N-acetyl-
chitooligosaccharides, (GlcNAC)n. OsChia1c pro-
duced mostly (GlcNAC)3 from (GlcNAC)6 and small
amounts of (GlcNAC)2 and (GlcNAC)4 and was
minimally active towards (GlcNAC)4 and (GlcNAC)5
(Fig. 5). In contrast, OsChia2b actively hydrolyzed
Fig. 5. Activities of OsChia1c and OsChia2b toward N-Acetyl-
chitooligosaccharides and Reaction Products.
N-Acetylchitooligosaccharides were incubated with OsChia1c
or OsChia2b (1 mg each) at 379C for 30 min and reaction
products at indicated incubation times were analyzed by TLC.21)
The N-acetylchitooligosaccharides used as substrates are: 1,
(GlcNAC)4; 2, (GlcNAC)5; 3, (GlcNAC)6. M, standard N-acetyl-
chitooligosaccharides.
all tested chitooligosaccharides to predominantly
yield (GlcNAC)2. To identify their cleavage modes on
the N-acetylchitooligosaccharides we analyzed the
products of (GlcNAC)5 and (GlcNAC)6 as a function
1068
Fig. 6. Course of (GlcNAC)5 and (GlcNAC)6 Hydrolysis by OsChia1c (A) and OsChia2b (B).
Reaction proceeded as described in Fig. 5. After the indicated incubation periods, reaction products were analyzed by TLC.21) M, N-
acetylchitooligosaccharide standards.
of the incubation period. The activity of OsChia1c
towards (GlcNAC)5 was very weak, yielding very
small amounts of (GlcNAC)2 and (GlcNAC)3 over
20 min (Fig. 6A). OsChia1c hydrolyzed (GlcNAC)6
more actively and generated (GlcNAC)3 as the major
product as well as very small amounts of (GlcNAC)2
and (GlcNAC)4. These products were not further
hydrolyzed after a longer incubation or by larger
amounts of the enzyme. OsChia1c1cDCBD similarly
cleaved (GlcNAC)5 and (GlcNAC)6 as OsChia1c
(data not shown). In contrast, OsCha2b hydrolyzed
(GlcNAC)5 and (GlcNAC)6 at comparable rates and
both chitooligosaccharides were completely hydro-
lyzed within 10 min into (GlcNAC)2 and (GlcNAC)3
(Fig. 6B). (GlcNAC)4, which had been formed from
(GlcNAC)6 during the ˆrst 5 min, was degraded into
two molecules of (GlcNAC)2 during further incuba-
tion. The product (GlcNAC)3, but not (GlcNAC)2,
was slowly hydrolyzed into GlcNAc and (GlcNAC)2
by further incubation. The relative activities of
OsChia2b to (GlcNAC)5 and (GlcNAC)6 were
roughly 20- and 5-fold those of OsChia1c.
Discussion
These and other studies revealed that O. sativa L.
cv. Nipponbare has at least 7 family 19 chitinases
(Fig. 1A and Table 1). The formation of these en-
zymes appears to be regulated by diŠerent mechan-
isms. OsChia1a and OsChia1c (84z identity each
other) are expressed at low levels in roots and leaves
and are induced to high levels by ethylene, salicylic
acid, and N-acetylchitooligosaccharide elicitors.11,19)
CH16, RCH10, and RC24 are the probable counter-
parts of OsChia1a (94z identity) and CH6 (91z
identity) is likely the OsChia1c counterpart in other
N.-H. TRUONG et al.
cultivars (Table 1). Like the Nipponbare class I
enzyme, synthesis of these enzymes is induced by
elicitors.28,31,32) Vacuole OsChia1b is constitutively
expressed at relatively high levels in roots but not in
leaves, and it is not induced by elicitors.18) OsChia1d,
as well as its counterpart (100z identity)
OsChia1;175 of indica cv. IR24,30) is rather distantly
related to other class I enzymes (Fig. 1B). Consistent
with the high-level of OsChia1;175 expression in
‰oral organs,30) the EST clones of OsChia1d account-
ed for 0.35z of the total number of clones obtained
from ‰owering panicles.
Class II enzymes (OsChia2a and OsChia2b) are
also diŠerently regulated. OsChia2a is the probable
counterpart of RCHT2 in the indica cv. Cheon-
gcheongbyeo, as they share 95z sequence identity.
Rcht2 is formed in response to glycol chitin or fungal
elicitors.10) Another class II enzyme (OsChia2b) with
a class IV-type catalytic domain (Fig. 1A) has a
sequence that is identical to that of the partially
deduced Chi-4, which has been identiˆed as a husk
protein expressed under high temperatures that
induces seed dormancy.33) The antifungal property of
the class II enzyme (Fig. 4) implies that it helps to
protect rice seeds from fungal infection together with
molilactones (phytoalexins), which are also formed
under the same conditions.33)
OsChia4a, is a class IV chitinase that has similarity
(about 70z identity) to both maize ChiA and
Arabidopsis ChIV of class IV chitinases.34,35) This
notion is in accordance with the hypothesis that the
class I and IV genes diverged before the separation of
dicots and monocots.14) Class II genes might have
arisen from the relevant class I genes by the deletion
of CBD14) or perhaps class I genes were created by the
transposition of a CBD sequence into a class II
 Structures and Properties of Rice Family 19 Chitinases
gene.36,37) The high sequence conservation (95z
identity) between the catalytic domains of OsChia4a
(class IV) and OsChia2b (class II) provides an exam-
ple of the direct and recent diversion of their genes
via either deletion or insertion mechanisms within
rice.
The class IV catalytic domains have three deletions
(Fig. 1A) that correspond to the loops between a-
helices C and D, F and G, and G and H.38) These
loops do not appear to signiˆcantly contribute to
stability and activity towards chitin polymers
(Table 2). However, the hydrolytic activities of
OsChia2b towards N-acetylchitooligosaccharides
were signiˆcantly distinct from those of OsChia1c
and OsChia1cDCBD. As proposed by X-ray crystal-
lography, molecular dynamics simulations, and
kinetic studies,38–40) barley class II chitinase with the
class I-type catalytic domain would bind to a sub-
strate at subsites －3 through ＋3 (or A through F)
and cleave the substrate between －1 and ＋1 sites by
a single-displacement inverting mechanism.39,41)
OsChia1c appears to share the similar binding-
subsite structure with the barley enzyme.39) Accord-
ing to this binding model, OsChia2b may lack the
loop between a-helices F and G that contains the
substrate-binding residue K188 of sites －3 and －2.
The protein also has a substitution of T69 at the
site ＋2 to G. Regardless of these altered binding-
subsite structures, OsChia2b e‹ciently hydrolyzes
(GlcNAC)4 and (GlcNAC)5, while OsChia1c, in
which the proposed binding residues are perfectly
conserved, does so very poorly (Figs. 6A and B). At
this stage the structures responsible for the diŠerent
hydrolytic activities are unknown. The activities of
plant chitinases towards N-acetylchitooligosaccha-
rides are quite important with respect to their roles
in elicitor formation (see below). The P. pastoris
expression system allows mutant enzymes to be pro-
duced that have a speciˆc amino acid substitution(s)
at a position(s) of interest for elucidation of its role in
substrate binding and catalysis.
The antifungal properties of OsChia1c and
OsChia2b (Fig. 4) support the notion that they par-
ticipate in defense against fungal pathogens.
OsChia1c has high antifungal activity, but low activi-
ty towards (GlcNAC)n which is less than hexameric
(Figs. 6A and B), and which can elicit chitinase
synthesis,9,11,19) thus making it (and its isoform
OsChia1a) a likely generator of N-acetylchitooligosac-
charide elicitors from the cell walls of infecting fungi.
OsChia2b and OsChia4b may not signiˆcantly contri-
bute to elicitor formation. Rather they would degra-
de elicitors due to their powerful hydrolytic activity
on N-acetylchitooligosaccharides (Figs. 6A and B).
Acknowledgments
We thank RGP for proving EST clones. This study
1069
was supported in part by a grant from the Program
for Promotion of Basic Research Activities for In-
novative Biosciences. N. H. T. was a fellow support-
ed by the UNU-Kirin fellowship program.
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