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We identiˆed four new family 19 chitinases in Oryza tion of signal substances such as ethylene, as well as
sativa L. cv. Nipponbare: one class I (OsChia1d), two salicylic, jasmonic, and nitric acids, which provoke
class II (OsChia2a and OsChia2b), and one class IV systemic acquired resistance and transient resistance
(OsChia4a). OsChia2a resembled (about 60z identity) throughout the plant to subsequent attack by patho-
the catalytic domains of class I chitinases, but OsChia2b gens.1,2) As well as elicitors, these signals activate PR
was almost identical (95z identity) to that of the class protein and phytoalexin synthesis. The PR proteins
IV enzyme. OsChia1c, OsChia1cDCBD (a deletion of constitute 11 groups (PR-1 through PR-11).4) Among
OsChia1c lacking a chitin-binding domain, CBD), and these, chitinases (PR-3, PR-4, PR-8, and PR-11) and
OsChia2b were separately expressed and puriˆed in b-1,3-glucanase (PR-2) have been more frequently
Pichia pastoris. OsChia1c inhibited fungal growth sig- studied because of their ability to inhibit, particularly
niˆcantly more than OsChia1cDCBD or OsChia2b. The in combination, fungal growth in vitro, through
activities of these enzymes on chitin polymers were simi- hydrolytic activities on the chitin and b-1,3-glucan
lar, but they acted diŠerently on N-acetylchitooligosac- that are major components of fungal cell walls.5,6)
charides, (GlcNAC)n. OsChia1c slowly hydrolyzed The participation of these enzymes in plant defense
(GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and has been substantiated by the increased resistance
(GlcNAC)5. In contrast, OsChia2b e‹ciently hydro- to fungal pathogens of transgenic plants that con-
lyzed these oligosaccharides. The high antifungal activi- stitutively express high levels of chitinase and W or
ty and low hydrolytic activity of the class I enzyme b-1,3-glucanase.7,8) Plant chitinases appear to be
towards (GlcNAC)n imply that it participates in the responsible for the formation of elicitors ( N-acetyl-
generation of N-acetylchitooligosaccharide elicitors chitooligosaccharides) from fungal cell walls to acti-
from the cell walls of infecting fungi. vate their own synthesis and other types of the
defense response.9–12)
Key words: rice (Oryza sativa L); chitinase; N-acetyl- Plant chitinases belong to structurally unrelated
chitooligosaccharides; antifungal activity; families 18 (PR-8 and PR-11) and 19 (PR-3) of
chitin-binding domain glycosylhydrolases.5,13,14) Family 18 includes classes
III (PR-8) and V (PR-11) enzymes. Family 19 con-
Plant defense against potential pathogens includes sists of three major classes (I, II, and IV). Class I and
a hypersensitive response, the production of reactive IV enzymes have a diagnostic cysteine-rich chitin-
oxygen species (oxygen burst), the activation of binding domain (CBD) at the amino terminus,5,14)
pathogenesis-related (PR) genes, structural changes and class II enzymes lack this domain (Fig. 1A). The
in the cell wall, and phytoalexin synthesis.1–3) CBD domain is dispensable for catalytic and antifun-
Elicitors generated by pathogen infection induce gal activities but can increase antifungal function.15)
the hypersensitive response, oxygen burst, and PR- Several enzymes of both families occur in the same
protein as well as phytoalexin synthesis to destroy plant, allowing the expression of tissue- or develop-
plant cells and prevent pathogen growth at the attack mental stage-speciˆc chitinases or generation of
site. The hypersensitive response results in the forma- 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.
were washed with 0.2 ml of the buŠer and lyophi- green shoots (9,212 clones), etiolated shoots (5,103
lized. Bound enzymes were then dissociated from the clones), and panicles (15,128 clones), was low
complexes by boiling in 0.1 ml of sample buŠer23) and (Ã0.02z). In contrast, the frequency of OsChia1d
20-ml portions of samples were analyzed by SDS- EST clones was high in panicles, particularly in ‰ow-
PAGE. Amounts of the bound proteins were meas- ing panicles (0.35z). OsChia2b and OsChia4a clones
ured by comparing densities of protein bands with also occurred in panicle clones, although at a lower
that of known amounts of proteins on the gels. frequency (about 0.04z).
N-acetylchitooligosaccharides (1 mM; Seikagaku
Kogyo, Tokyo, Japan) were incubated with 1 mg of Heterologous expression and puriˆcation
enzyme as described above. After the indicated incu- All eŠorts to express active OsChia1c, Os-
bation period, reaction products were resolved and Chia1cDCBD, and OsChia2b in E. coli were unsuc-
detected on silica gel 60 plates (Merck; Darmstadt, cessful. The proteins were produced as inactive and
Germany) as described.21) Antifungal activity was insoluble forms in inclusion bodies even when cul-
assayed by disk-plate diŠusion26) using Trichoderma tures were incubated at 159C, conditions that we
reesei IFO31329 as the test fungus.25) used to produce the barely active class II chitinase in
E. coli.27) Attempts by other groups to express rice
Results family 19 chitinases in E. coli also met with the
formation of inclusion bodies or very low levels of
Family 19 chitinases of O. sativa L cv. Nipponbare synthesized active enzyme.28–30) We accordingly tried
Sequencing the cDNA inserts (Æ1 kb) with to use P. pastoris as the host, as eukaryotic proteins
similarity to known class I chitinase in the EST have often been produced in this organism. When
library (48,526 clones) of the Rice Genome Project cultured in BMMY medium, P. pastoris cells harbor-
(http://rgp.aŠrc.go.jp) discovered four new family ing a recombinant chitinase cDNA produced, ex-
19 chitinases named OsChia1d (DDJB accession num- tracellularly, satisfactory quantities of the corre-
ber AB096139), OsChia2a (AB016497), OsChia2b sponding chitinase. Enzyme formation was maximal
(AB003194), and OsChia4a (AB096140). A com- by 36 h (about 0.2 mg W 50 ml) and remained
parison of their deduced amino acid sequences with unchanged for at least up to 72 h (data not shown).
the sequences of other plant family 19 chitinases The enzymes were puriˆed to apparent homogeneity
showed that OsChia1d and OsChia4a have a CBD after Ni 2+ a‹nity column chromatography, as
at the N-terminal, but OsChia2a and OsChia2b judged by SDS-PAGE (Fig. 3A).
appeared to lack this domain, since a signal peptide
was adjacent to the catalytic domain (Fig. 2). The Chitin-binding a‹nity of CBD and its increase of
catalytic domains of OsChia1d and OsChia2a resem- antifungal activity
bled (about 60z identity) the corresponding regions To conˆrm the binding-a‹nity of rice CBD to
of other class I enzymes. OsChia2b and the catalytic chitin, we bound OsChia1c, OsChia1cDCBD, and
domain of OsChia4a were very similar (95z identity) OsChia2b (10 mg each) to colloidal chitin (1 mg) in
(Fig. 1B) and resembled (60–70z identity) the class 0.2 ml of mixture (see Materials and Methods) and
IV chitinases of other plants rather than the class I measured the amounts of free and bound proteins by
enzymes (º60 identity). In addition, three deletions protein assay and by SDS-PAGE, respectively.
and a cysteine residue located at the carboxyl termi- OsChia1cDCBD and OsChia2b (9.2 and 9.4 mg, re-
nal are features of class IV chitinases.5,14) spectively) were recovered in the supernatant, but
only 0.6 mg of OsChia1c was recovered from the
Frequency of the family 19 EST clones supernatant. Association of OsChia1c with the
The frequency of the OsChia1a, OsChia1b, binding substrate was conˆrmed by SDS-PAGE
OsChia1c, and OsChia2a EST clones in the total EST (Fig. 3B); 9.0, 0.7 and 0.5 mg of OsChia1c,
clones from various tissues, roots (4,079 clones), OsChia1cDCBD and OsChia2b, respectively, were
Structures and Properties of Rice Family 19 Chitinases 1067
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 Fig. 5. Activities of OsChia1c and OsChia2b toward N-Acetyl-
chitooligosaccharides and Reaction Products.
similar Km and Vmax values and thermal stability N-Acetylchitooligosaccharides were incubated with OsChia1c
(Table 2). The catalytic properties of OsChia2b with or OsChia2b (1 mg each) at 379C for 30 min and reaction
the tested substrate were similar, although it was a products at indicated incubation times were analyzed by TLC.21)
little more active than class I enzymes toward glycol The N-acetylchitooligosaccharides used as substrates are: 1,
chitin. However, the activities of the class I and class (GlcNAC)4; 2, (GlcNAC)5; 3, (GlcNAC)6. M, standard N-acetyl-
chitooligosaccharides.
II enzymes distinctly diŠered towards N-acetyl-
chitooligosaccharides, (GlcNAC)n. OsChia1c pro-
duced mostly (GlcNAC)3 from (GlcNAC)6 and small all tested chitooligosaccharides to predominantly
amounts of (GlcNAC)2 and (GlcNAC)4 and was yield (GlcNAC)2. To identify their cleavage modes on
minimally active towards (GlcNAC)4 and (GlcNAC)5 the N-acetylchitooligosaccharides we analyzed the
(Fig. 5). In contrast, OsChia2b actively hydrolyzed products of (GlcNAC)5 and (GlcNAC)6 as a function
1068 N.-H. TRUONG et al.
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 cultivars (Table 1). Like the Nipponbare class I
towards (GlcNAC)5 was very weak, yielding very enzyme, synthesis of these enzymes is induced by
small amounts of (GlcNAC)2 and (GlcNAC)3 over elicitors.28,31,32) Vacuole OsChia1b is constitutively
20 min (Fig. 6A). OsChia1c hydrolyzed (GlcNAC)6 expressed at relatively high levels in roots but not in
more actively and generated (GlcNAC)3 as the major leaves, and it is not induced by elicitors.18) OsChia1d,
product as well as very small amounts of (GlcNAC)2 as well as its counterpart (100z identity)
and (GlcNAC)4. These products were not further OsChia1;175 of indica cv. IR24,30) is rather distantly
hydrolyzed after a longer incubation or by larger related to other class I enzymes (Fig. 1B). Consistent
amounts of the enzyme. OsChia1c1cDCBD similarly with the high-level of OsChia1;175 expression in
cleaved (GlcNAC)5 and (GlcNAC)6 as OsChia1c ‰oral organs,30) the EST clones of OsChia1d account-
(data not shown). In contrast, OsCha2b hydrolyzed ed for 0.35z of the total number of clones obtained
(GlcNAC)5 and (GlcNAC)6 at comparable rates and from ‰owering panicles.
both chitooligosaccharides were completely hydro- Class II enzymes (OsChia2a and OsChia2b) are
lyzed within 10 min into (GlcNAC)2 and (GlcNAC)3 also diŠerently regulated. OsChia2a is the probable
(Fig. 6B). (GlcNAC)4, which had been formed from counterpart of RCHT2 in the indica cv. Cheon-
(GlcNAC)6 during the ˆrst 5 min, was degraded into gcheongbyeo, as they share 95z sequence identity.
two molecules of (GlcNAC)2 during further incuba- Rcht2 is formed in response to glycol chitin or fungal
tion. The product (GlcNAC)3, but not (GlcNAC)2, elicitors.10) Another class II enzyme (OsChia2b) with
was slowly hydrolyzed into GlcNAc and (GlcNAC)2 a class IV-type catalytic domain (Fig. 1A) has a
by further incubation. The relative activities of sequence that is identical to that of the partially
OsChia2b to (GlcNAC)5 and (GlcNAC)6 were deduced Chi-4, which has been identiˆed as a husk
roughly 20- and 5-fold those of OsChia1c. protein expressed under high temperatures that
induces seed dormancy.33) The antifungal property of
Discussion the class II enzyme (Fig. 4) implies that it helps to
protect rice seeds from fungal infection together with
These and other studies revealed that O. sativa L. molilactones (phytoalexins), which are also formed
cv. Nipponbare has at least 7 family 19 chitinases under the same conditions.33)
(Fig. 1A and Table 1). The formation of these en- OsChia4a, is a class IV chitinase that has similarity
zymes appears to be regulated by diŠerent mechan- (about 70z identity) to both maize ChiA and
isms. OsChia1a and OsChia1c (84z identity each Arabidopsis ChIV of class IV chitinases.34,35) This
other) are expressed at low levels in roots and leaves notion is in accordance with the hypothesis that the
and are induced to high levels by ethylene, salicylic class I and IV genes diverged before the separation of
acid, and N-acetylchitooligosaccharide elicitors.11,19) dicots and monocots.14) Class II genes might have
CH16, RCH10, and RC24 are the probable counter- arisen from the relevant class I genes by the deletion
parts of OsChia1a (94z identity) and CH6 (91z of CBD14) or perhaps class I genes were created by the
identity) is likely the OsChia1c counterpart in other transposition of a CBD sequence into a class II
Structures and Properties of Rice Family 19 Chitinases 1069
gene.36,37) The high sequence conservation (95z was supported in part by a grant from the Program
identity) between the catalytic domains of OsChia4a for Promotion of Basic Research Activities for In-
(class IV) and OsChia2b (class II) provides an exam- novative Biosciences. N. H. T. was a fellow support-
ple of the direct and recent diversion of their genes ed by the UNU-Kirin fellowship program.
via either deletion or insertion mechanisms within
rice. References
The class IV catalytic domains have three deletions
(Fig. 1A) that correspond to the loops between a- 1) Ebel, J., and Mith äoter, A., Early events in the elicita-
helices C and D, F and G, and G and H.38) These tion of plant defence. Planta, 206, 335–348 (1998).
loops do not appear to signiˆcantly contribute to 2) Lamb, C. J., Ryals, J. A., Ward, E. R., and Dixon,
R. A., Emerging strategies for enhancing crop
stability and activity towards chitin polymers
resistance to microbial pathogens. Bio W Technology,
(Table 2). However, the hydrolytic activities of 10, 1436–1445 (1992).
OsChia2b towards N-acetylchitooligosaccharides 3) Jig, C., Smith-Becker, J., and Keen, N. T., Genetics
were signiˆcantly distinct from those of OsChia1c of plant-pathogen interaction. Cur. Open.
and OsChia1cDCBD. As proposed by X-ray crystal- Biotechnol., 9, 202–207 (1998).
lography, molecular dynamics simulations, and 4) van Loon, L. C., Pierpoint, W. S., Boller, T., and
kinetic studies,38–40) barley class II chitinase with the Conejero, V., Recommendations for naming plant
class I-type catalytic domain would bind to a sub- pathogenesis-related proteins. Plant Mol. Biol. Rep.,
strate at subsites -3 through +3 (or A through F) 12, 245–264 (1994).
and cleave the substrate between -1 and +1 sites by 5) Collinge, D. B., Kragh, K. M., Mikkelsen, J. D.,
a single-displacement inverting mechanism.39,41) Nielsen, K. K., Rasmussen, U., and Vad, K., Plant
chitinases. Plant J., 3, 31–40 (1993).
OsChia1c appears to share the similar binding-
6) Simmons, C. R., The physiology and molecular
subsite structure with the barley enzyme.39) Accord- biology of plant 1,3-b-D-glucanases and 1, 3;1,4-b-D-
ing to this binding model, OsChia2b may lack the glucanases. Crit. Rev. Plant Sic., 13, 325–387 (1994).
loop between a-helices F and G that contains the 7) Cornelissen, B. J. C., and Melchers, L. S., Strategies
substrate-binding residue K188 of sites -3 and -2. for control of fungal diseases with transgenic plants.
The protein also has a substitution of T69 at the Plant Physiol., 101, 709–712 (1993).
site +2 to G. Regardless of these altered binding- 8) Jack, G., G äornhardt, B., Mundy, J., Logemann, J.,
subsite structures, OsChia2b e‹ciently hydrolyzes Pinsdorf, E., Leah, R., Schell, J., and Maas, C.,
(GlcNAC)4 and (GlcNAC)5, while OsChia1c, in Enhanced quantitative resistance against fungal dis-
which the proposed binding residues are perfectly ease by combinatorial expression of diŠerent barley
antifungal proteins in transgenic tobacco. Plant J., 8,
conserved, does so very poorly (Figs. 6A and B). At
97–109 (1995).
this stage the structures responsible for the diŠerent
9) Inui, H., Yamaguchi, Y., Ishigami, Y., Kawaguchi,
hydrolytic activities are unknown. The activities of S., Yamada, T., Ihara, H., and Hirano, S., Three
plant chitinases towards N-acetylchitooligosaccha- extracellular chitinases in suspension-cultured rice
rides are quite important with respect to their roles cells elicited by N-acetylchitooligosaccharides. Biosci.
in elicitor formation (see below). The P. pastoris Biotechnol. Biochem., 60, 1956–1961 (1996).
expression system allows mutant enzymes to be pro- 10) Kim, C. Y., Gal, S. W., Choe, M. S., Jeong, S. Y.,
duced that have a speciˆc amino acid substitution(s) Lee, S. I., Cheong, Y. H., Lee, S. H., Choi, Y. J.,
at a position(s) of interest for elucidation of its role in Han, C., Kang, K. Y., and Cho, M. J., A new class II
substrate binding and catalysis. chitinase, Rcht2, whose induction by fungal elicitor is
The antifungal properties of OsChia1c and abolished by protein phosphate 1 and 2A inhibitor.
OsChia2b (Fig. 4) support the notion that they par- Plant Mol. Biol., 37, 523–534 (1998).
11) Nishizawa, Y., Kawakami, A., Habi, T., He, D. Y.,
ticipate in defense against fungal pathogens. Shibuya, N., and Minami, E., Regulation of the
OsChia1c has high antifungal activity, but low activi- chitinase gene expression in suspension-cultured rice
ty towards (GlcNAC)n which is less than hexameric cells by N-acetylchitooligosaccharides: diŠerences in
(Figs. 6A and B), and which can elicit chitinase the signal transudation pathways leading to the acti-
synthesis,9,11,19) thus making it (and its isoform vation of elicitor-responsive genes. Plant Mol. Biol.,
OsChia1a) a likely generator of N-acetylchitooligosac- 39, 907–914 (1999).
charide elicitors from the cell walls of infecting fungi. 12) Minami, E., Kuchitsu, K., He, D.-Y., Kouchi, H.,
OsChia2b and OsChia4b may not signiˆcantly contri- Midoh, N., Ohtsuki, Y., and Shibuya, N., Two novel
genes rapidly and transiently activated in suspension-
bute to elicitor formation. Rather they would degra-
cultured rice cells by treatment with N-acetylchito-
de elicitors due to their powerful hydrolytic activity
heptaose, a biotic elicitor for phytoalexin production.
on N-acetylchitooligosaccharides (Figs. 6A and B). Plant Cell Physiol., 37, 563–567 (1996).
13) Henrissat, B., and Bairoch., A., New families in the
Acknowledgments classiˆcation of glycosyl hydrolases based on amino
acid sequence similarity. Biochem. J., 293, 781–788
We thank RGP for proving EST clones. This study (1993).
1070 N.-H. TRUONG et al.
14) Hamel, F., Boivin, R., Tremblay, C., and Bellemare, Plant Mol. Biol., 30, 387–401 (1996).
G., Structure and evolutionary relationships of 29) Pan, C.-H., Rhim, S.-L., and Kim, S.-I., Expression
‰owering plants. J. Mol. Evol., 44, 614–624 (1997). of two cDNAs encoding class I chitinase of rice in
15) Iseli, B., Boller, T., and Neuhaus, J.-M., The N- Escherichia coli. Biosci. Biotechnol. Biochem., 60,
terminal cysteine-rich domain of tobacco class I 1346–1348 (1996).
chitinase is essential for chitin binding but not for 30) Takakura, Y., Ito, T., Saito, H., Inoue, T., Komari,
catalytic or antifungal activity. Plant Physiol., 103, T., and Kuwata, S., Flower-predominant expression
221–226 (1993). of a gene encoding a novel class I chitinase in rice
16) Sela-Buurlage, M. B., Ponstein, A. S., Bres- (Oryza sativa L.). Plant Mol. Biol., 42, 883–897
Vloemans, S. A., Melchers, L. S., van den Elzen, P. (2000).
J. M., and Cornelissen, B. J. C., Only speciˆc 31) Kim, Y. K., Baek, J. M., Park, H. Y., Choi, Y. D.,
tobacco ( Nicotiana tabacum) chitinases and b-1,3- and Kim, S. I., Isolation and characterization of
glucanase exhibit antifungal activity. Plant Physiol., cDNA clones encoding class I chitinase in suspension
101, 857–863 (1993). cultures of rice cells. Biosci. Biotechnol. Biochem.,
17) Busam, G., Kassemeyer, H.-H., and Matern, U., 58, 1164–1166 (1994).
DiŠerential expression of chitinases in Vitis Vinifera 32) Zu, Q., and Lamb, C. J., Isolation and characteriza-
L. responding to systemic acquired resistance activa- tion of a rice gene encoding a basic chitinase. Mol.
tors or fungal challenge. Plant Physiol., 115, Gen. Genet., 226, 289–296 (1991).
1029–1038 (1997). 33) Nakazaki, T., Tomimoto, Y., Ikehashi, H.,
18) Nishizawa, Y., Kishimoto, N., Saito, A., and Hibi, Kowyama, Y., Yano, M., Yamamoto, K., and
T., Sequence variation, diŠerential expression and Sasaki, T., A novel chitinase in rice (Oryza sativa L.)
chromosome location of rice chitinase genes. Mol. detected from husk proteins and its gene locus. Breed-
Gen. Genet., 241, 1–10 (1993). ing Sci., 47, 363–369 (1997).
19) Nishizawa, Y., and Hibi, T., Rice cDNA gene: cDNA 34) Huynh, Q. K., Hironaka, C. M., Levine, E. B.,
cloning and stress-induced expression. Plant Sci., 76, Smith, C. E., Borgmeyer, J. R., and Shah, D. M.,
211–218 (1991). Antifungal proteins from plants. Puriˆcation,
20) Neuhaus, J.-M., Fruitage, B., Linthorst, H. J. M., molecular cloning, and antifungal properties of
Meins, Jr., F., Mikkelsen, J. D., and Ryals, J., A chitinases from maize seed. J. Biol. Chem., 267,
revised nomenclature for chitinase genes. Plant Mol. 6635–6640 (1992).
Biol. Rep., 14, 102–104 (1996). 35) de A Gerhardt, L. B., Sachetto-Martins, G.,
21) Park, S.-M., Kim, D.-H., Truong, N. H., and Itoh, Contarini, M. G., Sandroni, M., de P Ferreira, R., de
Y., Heterologous expression and characterization of Lima, V. M., Cordeiro, M. C., de Oliveira, D. E.,
class III chitinases from rice (Oryza sativa L.). and Margis-Pinheiro, M., Arabidopsis thaliana class
Enzyme Microb. Technol., 30, 697–702 (2002). IV chitinase is early induced during the interaction
22) Nagasaki, H., Yamamoto, K., Shomura, A., Koga- with Xanthomonas campestris. FEBS Lett., 419,
Ban, Y., Takasuga, A., Yano, M., Minobe, Y., and 69–75 (1997).
Sasaki, T., Rice class III chitinase homologues isolat- 36) Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins,
ed by random cloning of rice cDNAs. DNA Res., 4, Jr., F., Structure of a tobacco endochitinase gene:
379–385 (1997). evidence that diŠerent chitinase genes can arise by
23) Laemmli, U. K., Cleavage of structural proteins transposition of sequences encoding a cysteine-rich
during the assembly of the head of bacteriophage T4. domain. Plant Mol. Biol., 14, 357–368 (1990).
Nature, 227, 680–685 (1970). 37) Ohme-Takagi, M., Meins, Jr., F., and Shinshi, H., A
24) Imoto, T., and Yagishita, K., A simple activity meas- tobacco gene encoding a novel basic class II chitinase:
urement of lysozyme. Agric. Biol. Chem., 35, a putative ancestor of basic class I and acidic class II
1154–1156 (1971). chitinase genes. Mol. Gen. Genet., 259, 511–515
25) Itoh, Y., Kawase, T., Nikaidou, N., Fukada, H., (1998).
Mitsutomi, M., Watanabe, T., and Itoh, Y., 38) Hart, P. J., P‰uger, H. D., Monzingo, A. F., Hollis,
Functional analysis of the chitin-binding domain of a T., and Robertus, J. D., The reˆned crystal structure
family 19 chitinase from Streptomyces griseus of an endochitinase from Hordeum vulgare L. seeds
HUT6037: substrate-binding a‹nity and cis- at 1.8 A resolution. J. Mol. Biol., 248, 402–413
dominant increase of antifungal function. Biosci. (1995).
Biotechnol. Biochem., 66, 1084–1092 (2002). 39) Fukamizo, T., Chitolytic enzymes: catalysis, sub-
26) Roberts, W. K., and SelitrennikoŠ, C. P., Plant and strate binding, and their application. Curr. Protein
bacterial chitinases diŠer in antifungal activity. J. Pept. Sci., 1, 105–124 (2000).
Gen. Microbiol., 134, 169–176 (1988). 40) Honda, Y., and Fukamizo, T., Substrate binding
27) Andersen, M. D., Jensen, A., Robertus, J. D., Leah, subsites of chitinase from barley seeds and lysozyme
R., and Skriver, K., Heterologous expression and from goose egg white. Biochim. Biophys. Acta, 1388,
characterization of wild-type and mutant forms of a 53–65 (1998).
26 kDa endochitinase from barley ( Hordeum vulgare 41) Brameld, K. A., and Goddard III, W. A., The role of
L.). Biochem. J., 322, 815–822 (1997). enzyme distortion in the single displacement mechan-
28) Xu, Y., Zhu, Q., Panbangred, W., Shirasu, K., and ism of family 19 chitinases. Proc. Natl. Acad. Sci.
Lamb, C., Regulation, expression and function of a USA, 95, 4276–4281 (1998).
new basic chitinase gene in rice (Oryza sativa L.).