植物キチナーゼの構造と抗真菌活性

誌名誌名 Journal of applied glycoscience

ISSNISSN 13447882

巻/号巻/号 573

掲載ページ掲載ページ p. 167-176

発行年月発行年月 2010年7月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

167

J. Appl. Glycosci.， 57， 167-176 (2010) @ 2010 The Japanes巴 Societyof App1ied Glycoscience

Review ~J~~&Q

Structures and Antifungal Activity of Plant Chitinases

(Received January 7， 2010; Accept巴dFebru紅 y23，2010)

Toki Taira1.*

lDepartment of Bioscience and Biotechnology， UniversiりIofthe RyuわIUS

(1， Senbaru， Nishihara-cho， Okinawa 903-0213， Japan)

Abstract: Many chitinases and their genes have been obtained from various plants. To understand structural features of plant chitinases， some classification systems have been proposed by some research groups. How-ever， the complicated multiple classification of plant chitinases frequently confuses researchers. In this article， structures and classification systems of plant chitinases are reviewed， and their issues are discussed. Plant chitinase is considered to protect plants against fungal pathogens by degrading chitin， a major component of the cell walls of many fungi. The antifungal activity of various chitinases derived from various plant sources has been investigated， but a correlation between the structure and the antifungal activity of the proteins has been unclear. To exhibit antifungal activity， a chitinase must bind to fungal cell walls， and then， it should de-grade the chitin in them. Roles of the chitin-binding domain and catalytic domain in antifungal activity of plant chitinases are discussed.

Key words: chitinase， antifungal activity， PR protein， chitin binding， family 19 chitinase

Chitinases (EC 3.2.1.14) cata1yze the hydro1ysis of chi-

tin， which is a s-1，4・linkedhomopo1ymer or oligomer of N-acety1-D-g1ucosarnine (G1cNAc). Many seed p1ants syn-th巴sizevarious chitinases. 1

•2

) However， an endogenous sub-

strate for p1ant chitinases has not yet been found. In the absence of an endogenous substrate， p1ant chitinases may

be invo1ved in白巴 interactionbetween p1ants and mi“

crobes， which produce chitin and chitin-re1ated com-

pounds. It is accepted by many researchers that one of出ephysio1ogica1 ro1es of these chitinases is to protect p1ants against funga1 pathogens by degrading chitin， a 1I悶jorcomponent of the cell wall of many fungiY) There is

strong corre1ative evidenc巴.The 10w constitutive activity

of chitinase found in many p1ants can be dramatica11y in-duced by infection with funga1 pathogens. 1

•2

•5

) The antifun-

ga1 activity of chitinases derived from various p1ants has been investigated in vitro and in vivo. 1.5) However， some chitinases do not show any antifunga1 activity.6.7) P1ant

chitinases are induced not on1y by pathogenesis but a1so by abiotic stress. Severa1 p1ant chitinases are constitutive，

developmenta11y regu1ated， and tissue-and organ-specific. It appe紅 sthat the ro1e of p1ant chitinases does not consist sole1y of defense against pathogen attack. 8-11)

There紅 esevera1 types of chitinase in p1ants. On the basis of their arnino acid sequences， p1ant chitinases have been c1assified into severa1 c1asses. Even in白esame

• Corresponding author (Tel. & Fax. +81-98-895-8802， E-ma幻d枇i1止l上:

tωoke巴y@a勾gr.ルr巧yuk匂y川u必 .jpが).Abbreviations: Ac-AMP， Amaranthus caudatus antimicrobia1 p巴p-tide; BjCHI1， Brassica j目unceachitinase 1; CBM， carbohydrate-binding module; CBP20， 20-kDa chitin-binding protein; FITC， fluor巴sceinisothiocyanate; GBC， gladiolus bulb chitinase; GH， glycoside hydrolase; GlcNAc，件acetyl-D-glucosamine;LysM， ly目

sm巴 motぜ;PDA， potato d巴xtrosewith agar; PLC， pokeweed leaf chitinas巴 PrChi，Pteris ηuわluensis chitinase; PR-proteins， pathogen巴sis-relatedproteins; RSC， rye seed chitinas巴;TBC， tulip bulb chitinase; UDA， Urtica dioica agglutinin; WGA， wheat germ agglutinin.

c1ass， there are various isoforms which have 10w homo1-ogy and/or a different isoe1ectric point. Why do various

structures of chitinase exist in p1ants? Chitinases having different structures shou1d p1ay different ro1es or the sam巴ro1es in different manners. Their biochernica1 characters，

depending on their structures， are expected to re1ate to their physio1ogica1 ro1es.

To address this issue， a corre1ation between the struc-ture of p1ant chitinases and their antifunga1 activity has to

be understood. In this review， first， structures and c1assifi-cation systems of p1ant chitinases ar巴 reviewed，and their issues are discussed. Next， antifunga1 activities of p1ant

chitinases are reviewed， and a corre1ation b巴tweentheir structure and antifunga1 abi1ity is discussed.

Classification of plant chitinases.

According to the CAZy database (http://afmb.cnrs-mrs.

fr/CAZY /)， chitinases are divided into two farnilies， gly-coside hydro1ase (GH) farni1y 18 (GH-18) and 19 (GH-19). GH-18 and GH-19 chitinases differ not on1y in

sequence but a1so in hydro1ytic mechanisms because they operate by retention and inversion， respective1y， of the anomeric configuration.12

) There are both farnilies in p1ant

chitinases. On the other hand， p1ant chitinases have been

c1assified by some research groups on the basis of their arnino acid sequences. First， c1ass 1， II and III chitinases were proposed by Shinshi et al.13

) Collinge et al. pro-posed c1ass IV chitinases that share homo1ogy with c1ass 1 chitinases but are smaller due to some de1etions.1

) Me1ch-

ers et al. proposed a 40-kDa tobacco chitinase homo1o-gous to bacteria1 exo-chitinases as a c1ass V chitinase.14

)

To date，出isc1assification system based on the abov巴

出reepapers has been more frequently used than the other (Fig. l(A)). The details of c1asses are described as fo1-10ws. C1ass 1 chitinases consist of an N-t右rrnina1chitin-

binding domain and a GH-19 cata1ytic domain. The chitin -binding domain， b巴10ngingto carbohydrate-binding mod-

168 J. Appl. Glycosci.， Yol. 57， No. 3 (2010)

ule family 18 (CBM-18)， has homology to hevein， an an-

tifungal peptide from rubber latex， and chitin-binding

l巴ctinssuch as wheat germ agglutinin. Class II chitinases

have only a catalytic domain homologous to that of class

1 chitinases. Class rv chitinas巴sshare homology with

class 1 chitinases but紅 esmaller due to four deletion and

C-terminal loop sequences. Both class III and V chitinases

consist of only a GH-18 catalytic domain. Class III and

class V chitinases share the cons巴nsussequence DXDXE，

but otherwise show very low homology to each other and

the molecular mass of cJass V chitinases (about 40 kDa)

is higher than that of class III chitinases (about 30 kDa).

The tertiary structure of hevamine， a class 111 chitinas巴

from rubber latex， had b巴ensolved but that of any cJass V

chitinase has not. However， the tertiary structur巴 of出 巴

catalytic domain of human chitotriosidase， having homol-

ogy to plant class V chitinase， had been solved.15) From

structure-based alignrnent of plant cJass V and class 111

chitinases and human chitotriosidase， it is suggested that

th巴reis a big insertion in cJass V chitinases compared to

cJass III chitinases.16) The cJass V chitinases are homolo-

gous to several chitinases from microbes and animals. The

name“cJass V chitinase" is also used in the cJassification

of fungal chitinase， based on sequence similarity betw巴en

plant and fungal chitinases.17)

Since there are several cJassification systems of plant

chitinases in addition to the above-mentioned cJassifica-

tion system， researchers are frequently confused. Plant

chitinases are members of pathog巴nesis-relatedproteins

(PR-Proteins). Class 1， 11 and IV chitinases belong to

PR-3. Class III and V belong to PR-8 and PR-l1， respec-tiv巴ly.18)N巴uhauset al. 19) had proposed a new cJassifica四

tion system. In this system， nomencJatures of cJass 1， 11，

111 and rv chitinases are almost the same as the above-mentioned classification system， but，“class V chitinase"

indicates a precursor of Urtica dioica (stinging n巴ttle)ag-

glutinin (see Fig. I(B)). The precursor consists of two

hevein domains and a GH-19 chitinas巴ーlikedomain. Ma-

ture UDA protein is composed of only h巴V巴indomains

due to post -translational processing. The GH -19 chitinase-

like domain in the pr巴cursoris unlikely to have any cata

lytic activity， since both catalytic r巴siduesar巴 mutated.

How巴V巴r，expr巴ssion of the catalytic domain in Es-

cherichia coli led

(A) Hevein (i~~~Y~ (CBM 18) Catalytic domain (GH 19)

Class 1

Class 11

Class IV

Class IIL* ・SeeFig. 2

Class 111

Class IIlb

E謹呈

三三量

CataJytic domain (GH 18)

「一一つ=寸 「ー「

DXDXE

5-5 bond

(Class IIb of PR-3)

(Class VII of PR-3)

(Class 111 of PR-8)

Class V 圃 圃 圃 圃E 幽幽昌園田園圃- - f 一 一圃圃園

(8) BjCHI1

(Hev x2+Class 11)

UOA precursor

(Hev x2+Class 11)

Beta vulgarIs Chi (Hev x1/2+Class 11)

22aa 130aa

PrChi-A 白面画面圃r古司画面白歯(LysMx2+Class IIIb)・・1""""'-"圃圃圃E

凶'sdom削 n (ClassI ofPR-II)

(Class V of PR-3)

(C旭S5VI of PR-3)

LysM domain (CBM 50) Class-illb catalytic domain

Fig. 1. Classification of plant chitinases.

(A) Schematic representation of five classes (I-Y) and two sub-classes (IIL and田b)of plant chitinases. Op巴nsquares indicat巴linker regions. Solid lines indicate disulfid巴 bonds.(B) Schematic repl巴sentationof unclassified plant chitinases. BjCHI1， Brassica juncea chitinase-l (AAF02299); UDA precursor， U. dioica aggluti-nin precursor (AAB23919); Beta vulgaris Chi， B. vulgaris chitinas巴

(CAA55883); PrChi-A， Pteris ryukyuensis chitinase-A (BAE98134) GH and CBM indicate glycoside hydrolase and carbohydrate bind ing module， respectively. Blue fonts with brackets indicate classes，

which are based on出巴 classificationsyst巴mproposed by Neuhaus et al.")

not to have gained much popularity. Sinc巴 severalplant

chitinases are not induced by pathogenesis， a cJassification

system should not depend on th巴cJassificationof PR-

proteins. The form巴rclassification system seems to b巴

more acc巴ptableand less confusing for many res巴archers

than the latter. However， the acceptable cJassification system does not

cover some subcJasses of plant chitinases. Th巴reare sev-

eral low mol巴cular weight chitinases which have no

chitin-binding domain and have a GH-19 catalytic domain

with some deletion(s) (Fig. 2). The deletion(s) is/創巴 r巴-

stricted to loop regions and the C-terminal loop r巴gionof

class 11 enzymes.23) There are variations in the number of

deletions and th巴irregions. If these varieties ar巴 reflected

in a cJassification， it will be complicated. Araki and Tori

kata24) have propos巴dthat this low molecular weight sub-

class of class 11 chitinases be designated class IIL

chitinases. This cJassification is simple and acceptable.

PR-P and PR_Q，25) which are prototypes of cJass 11

chitinases from tobacco， should be grouped under cJass

IIL chitinases du巴 toa deletion. Yamagami et al.26.27) pro-

posed that there is a subcJass of cJass 111 chitinases， bas巴d

on the following findings. Tulip bulb chitinase-l (TBC-l)

and gladiolus bulb chitinase-a (GBC-a) share the consen-

sus sequence DXXDXDXE found in cJass皿 chitinases

but low sequenc巴 homology(below 15%); TBC-l and

GBC-a have no disulfide bonds， but class III chitinas巴s

Antifungal Activity of Plant Chitinases 169

(A) Class I

Class 11

Class IV

1 11 III

匝面ヨー 同ーー一ー園田し一一一一一一一一一一一一一一~ LJ

園圃-・・固.. 置-」ーー一一一一一一一一ーーーーー--' LJ

IV V

一一Lーー」

圃'""'!竺雪

圃 園田

(C/ass VII of PR-3)

PLC-A

Class IIL I scCh;-A 圏直圃 白『 ーーー 圃圃圃圃

(B)

I PR-I'. PR-Q

(Class lIa 0' PR-3)

SgCh;-C 圃

Eご]Ch;Un-b;nd;ng doma;n (CBM 18) _ Ch;l;n-b;nd;珂 doma;n(CBM 5) Calalyt;c doma;n (GH19) 仁二コ Linkerregion _ Loop region

・圃 C-terminalloop

Class 11 (2BAA)

Class IIL homolog (1WVU)

Fig. 2. Yariation of class Ill.， chitinas巴s.

(A) Schematic r巴pr巴sentationof GH-19 chitinases. PLC-A， poke weed leaf chitinase-A (Q7MIQ9); BcChi-A， Bryum coronatum chitinase-A (BAF99002); PR-P and PR-Q， pathogenesis-relat巴dpro-tein P and Q from Nicotiana tabacum (CAA35790 and CAA 35789); SgChi-C， Chitinase-C from Streptomyces griseus (BAA 23739). I-Y indicate loop regions in catalytic domain of class 1 and I1 chitinases. Solid lin巴sindicate disulfide bonds. (B) Ribbon mod-els of barley 26-kDa chitinase (left) and a catalytic domain of Chi-C from S. griseus (right). Gr巴巴nribbons indicat巴 αhelices. Red color indicates the regions which are lost in some plant class I1L chitinases or bacterial GH-19 chitinases. Blue fonts with brackets indicate classes， which ar巴 bas巴don the classification system pro-posed by Neuhaus et al. 191

hav巴 threeconserved disulfide bonds; TBC-1 and GBC-a

do not lys巴 Micrococcusluteus c巴11walls， but hevamine

and pokew巴巴dleaf chitinase-B (PLC-B)， typical class III

chitinases， lyse these cell walls. Furthermore， hydrolysis

of (GlcNAc)5 by TBC-1 and GBC-a yields mainly

(GlcNAc)3 and (GlcNAc)2; however， PLC-B hydrolyzes

(GlcNAc)5 to yi巴ld mainly (GlcNAc)4 and GJcNAc.28)

Therefore， they proposed that chitinases such as TBC-1

and GBC-a should be plac巴din subclass IIIb (Fig. l(A)).

Many homologues to bulb chitinas巴shav巴 beenfound in

major crops such as rice， corn and soybeans， and in a fern

plant.29.3O) In this review， five classes and their two sub-

classes are adopted.

In addition to the five classes and th巴irtwo subclasses，

s巴veralminor chitinas巴shave been rep01ted. Zhao and

Chye31) isolated a B. juncea cDNA巴ncodingBjCHI1， a

chitinase consisting of two hevein-like chitin-binding do-

mains and a GH-19 catalytic domain. By the presence of

two chitin-binding domains， BjCHIl resembles the precur-

sor of UDA but， unlike UDA， BjCHIl retains its chitinase

catalytic domain after post-translational processing (Fig. 1

(B)). Onaga and Taira30) showed that PrChi-A， an antifun-

ig襲撃..瀦jLiquid medium糊 thfungalspores in 96ト-wellplate

FiJterpa問r∞nlainingchitinase on agar plate or well， is punched into agar plale，∞ntaining chitinase

+++ +ー

Fungal hyphae

o V /~司‘ \ +

O ~~ ↑ん川崎 首圏直判 +++‘温 -Os t-lL 0 / い巴圃_ 0/ h圃E園・・・圃・

o 7 =;~~!j~~Phal\ぎ喝./ ln川ionzone 一-、ー Fungalhyphae An agardisk叩山fungalhyphae is placed at

the center of a solid medium in Petri dish

Filterpa問 ← 、山一_.containing chitinase -.ーζ二一ー

Solid medium containing fungal spores in Petri dish

I~

Fungal spores on solid medium in 24-well同ate

Fig. 3. Variation of antifungal assays

For details， see the text

+++ + ー

Fungal colonies (hyphae)

Q@⑧ Fungal colc川田(hyphae)

gal chitinase from leaves of P. ryu勾uensis，consists of

two lysine motif (LysM) domains and a catalytic domain

of class IIIb chitinase (Fig. l(B)). LysM domains 紅 e

found in a vari巴tyof peptidoglycan-and chitin-binding

proteins.32) This is the only report of a plant family-18

chitinas巴 havingan extra LysM domain. If thes巴 minor

sequences紅巳 incorporatedinto the classification system，

many researchers will be confused. Therefor巴 th巴s巴

chitinas巴shaving minor sequ巴ncesshould b巴 leftas non-

classified chitinases until their reported numbers have in-

creased.

Antifungal assay of plant chitinases.

Antifungal activiti巴sof various plant chitinas巴shave

been reported. However， res巴arch巴rspoorly understand

what structures of chitinases have high antifungal activity.

It is difficult to compare antifungal ability of plant

chitinases with different methods and different test fungi.

Several r巴sultsof antifungal activity of plant chitinases

using sev巴ralmethods and various fungi are sumrnmized in Table 1. 5-7.14.30，3}-48)

Methods of antifung，α1 assay-S巴veralm巴thodsto de-

tect antifungal activity of chitinas巴shave been developed

(Fig. 3): method 1， hyphal growth inhibition assay in liq-

uid medium using a microtit巴rplate49); method 2， hyphal

extension inhibition assay on a solid medium containing

agar with potato巴xtract-dextroseor malt ext:ract using a

Petri dish5); method 3， hyphal growth inhibition assay on

solid medium using a Petri dish34>; method 4， hyphal growth inhibition assay on solid medium using a 24-well

plate!O) Methods 1 and 2 have b巴enemployed in many

170 J. Appl. Glycosci.， Yol. 57， No. 3 (2010)

(A)

(8)

想思腔(C)

An agar disk with fungal hyphae is F拍cedin the center of a PDA plate

....-Linear line made by hyphae

Linear extension

Several agar disks with fungal hyphae placed in a st団ightline on a PDA plate

Fig.4. Th巴 hyphal巴xt巴nsioninhibition assay and the linear hyphal-ext巴nsioninhibition assay

(A) The hyphal extension inhibition assay of rye se巴dchitinases and its domains against Trichoderma sp. was巴xaminedas de-scrib巴dby Taira et al 則 Thewells contained 10 μL of th巴 follow-ing solutions: 1， blank (sterile water); 2， 90 pmol of RSC-a (basic class 1); 3， 90 pmol of RSC-c (basic class 11); 4， 90 pmol of a cata-lytic domain (derived from RSC-a); 5， 90 pmol of a chitin-binding domain (derived from RSC-a); 6， 90 pmol of出巴 catalyticdomain with 90 pmol of出echitin-binding domain. (B) Th巴 linearhyphae-extension inhibition assay was examined as described by Taira et al剛 Thenumerical values in w巴l1sindicate amollnt of RSC-c (ba-sic class 11) as pmol. (C) Sch巴maticrepresentation of conventional hyphal extension inhibition assay (left) and lin巴arhyphae-extension inhibition assay (right). Reproduc巴d，with permission， from Ref 50).

studies. In the case of method 1， fungal-growth inhibition

was measur巴dby turbidity using a micro-plate reader and

this method is quantitative. In th巴 caseof method 2，

hyphal-extension inhib山 onwas judged by shap巴 and紅白

of inhibition zone around a w巴11or paper disc containing

the chitinase tested (Fig. 4(A)). Method 2 is relatively

easy， but is not quantitative. In this method， how巴ver，

qualitative data such as ext巴ntof hyphal branching and di-

ameter of hypha could be obtained through a microscope.

Usua11y， one study group uses only one of these methods

Therefore， it is difficult to comp氾-ethe sensitivity and ac

curacy of one method with those of another. Based on

common sense， method 2 s巴巴msto be closer to nature

than method 1， b巴ca~se in plant tissue， hyphae of patho-

genic fungi extend into intra-or inter-ce11ular gel like the

gel of an agar plate. Therefore， quantitative assay on an

agar plate is preferabl巴.Taira et al.50) developed a linear

hyphal-ext巴nsion inhibition assay which could obtain

s巴mi-quantitativ巴 dataon a PDA plat巴 (Figs.4(B) and 4

(C)). In addition to this novel assay， a hyphae passing-

through slit assay has also been developed (Fig. 5). Taira

et al.50) proposed that in the case of a conventional hyphal

(A)

(8)

(C)

(D)

当 /¥Hyphaepassed !hrough !he sli!

Fig. 5. The hyphae passing-through slit assay of basic class 1 and basic class 11 chitinas巴s

The hyphae passing-through slit assay was examined as described by Taira et al.50

) WelJs 1 to 6， 30 pmol of RSC-a (basic class 1); W巴l1s7 to 12， 30 pmol of RSC-c (basic class 11); welJ b， st巴rilewater. (A)， (B) and (C)， after 24， 36 and 48 h of incubation， respec tlV巴ly;(D)， schematic repres巴ntationof the hyphae passing-through slit assay. Reproduced， with permission， from Ref. 50)

-ext巴nsioninhibition assay， th巴effectof chitinases on hy-

phal tips is mainly observed. On the other hand， in the

abov巴 novelassays， the effect of chitinases is observed on

not only hyphal tips but also th巴 lateralwa11s of hyphae.

By using these novel assays， n巴wevid巴ncewas obtained

as mentioned later. It is n巴cessaryto choose a method

which suits th巴 aimof a study.

Test fungus forα仰nαザifu仰ngl伊α1assαのIy一Va.rious白ng♂ihav巴

b凶巴巴叩nu凶se吋dfor a叩ntifi白u肌1汀m時l沼galaωss印ay0ぱfplant chi此tina旧aおs巴白s.In vari-

ous fungi test巴d，g巴nusTrichoderma such as T. viride and

T. ressei seems to be more sensitive against chitinase than

anoth巴r.This reason has b巴巴nunknown; however， the rea-

son should b巴 attributedto the structur巴 offungal c巴11

wa11s. It is reasonable that growth inhibition against 00-mycetes by plant chitinas巴 wasnot detected， since th巴lr

ce11 wa11s are composed of ce11ulose rather than chitin.34•51

)

The sensitivity of a fungus may be related to the degr巴巴

of deacetylation in the fungal ce11-wa11 chitins.52) To date，

th巴1・巴 is no 巴vidence of fungus s巴lectivity of plant

chitinase; this means that chitinase A is effective for fun-

gus A but is not巴ffectivefor fungus B， wher巴aschitinase

B is effective for fungus B but is not effectiv巴 forfungus

171

of 0.5μg of tobacco class 1 chitinase a10ne did not cause

interference with the growth of F. solani. However， the combination of 1同 of CBP20 and 0.5μg of the

chitinase inhibited the growth of F. solani in a synergistic

manner.53) These synergistic effects of chitinase and other

defense-related proteins are very interesting in plant de-

fense mechanisms and are veηimportant to produce

pathog巴n-resistanttransgenic plants.

Antifungal Activity of Plant Chitinases

Relationships between antifungal abilities

chitinases and their family and charge.

There are many reports of antifunga1 activity of plant

GH-19 chitinases; however， reports of antifunga1 activity of plant GH-18 chitinases紅 every limited (Table 1). In th巴 sameGH-19 chitinases， reports of antifungal activity

of basic isozym巴紅emore由加 thoseof acidic isozym巴.1

checked antifungal abilities of various plant chitinases

having various structures and isoelectric point (pI)

of plant

A. It is necessary to study the cell-wall structure of sev-

eral fungi to understand the mechanisms of白eantifungal

action of plant chitinases.

Synergisticそffectof chitinases with s-l，3・.glucanaseor other d，φnse-related proteins-Except for Tricho-

derma species， many fungi are not sensitive to several

plant chitinases. However， many fungi are sensitive to a

combination of chitinase and s-lふgluca回 se，which de-grades s-l，3-g1ucan in the fungal cell walls.14

，34，40) 30-印 a

Ribosome-inactivating protein from barley seeds exhibits antifunga1 activity by inhibiting protein synthesis in target cells by specifically modifying 28S rRNA戸)Leah et al. 37)

showed that inhibition of funga1 growth by 26-kDa

chitinase or RIP30 alone is slight， but is greatly enhanced by mixing the two proteins. One microgram of 20北Da

chitin-binding protein (CBP20) from tobacco leaves inhib-

ited growth of T. viride strongly by itself; however， it did

that of Fusarium solani slightly. In like manner， addition

Antifungal activities of various chitinases using severa1 fungi and diffi巴rentmethods. Table 1.

Referenc巴

Bean (Phaseolus vulgaris) Bean chitinase Basic Bぽl巴y(Hordeum vulgare) 28 kDa AFP Basic Barley (H. vulgare) 28 kDa AFP Basic P巴a(Pisum sativum) Chl Basic Pea (P. sativum) Chl Basic Pea (P. sativum) Chl Basic Barley (H. vulgare) Chitinase T Basic Barley (H. vulgare) Chitinase C Basic Arabidopsis thaliana A. thalimωchitinase Basic Barley (H. vulgare) CHI 26 Basic B紅 l巴Y(H. vulgare) CHI 26 Basic Bean (P. vulgaris) Bean chitinase CH5B Basic Maize (Zea mays) Chit A Basic Maize (Z mのほ ChitA Basic Maize (Z mays) Chit A Basic Tobacco (N. tabacum) 32 kDa Chi-1 Basic Tobacco (N. tabacum) Chi-II PR-3a Acidic Tobacco (N. tabacum) Tob (chitnase A) Basic Tobacco (N. tabacum) Chi-V Basic Tobacco (N. tabacum) Chi-V Basic Tobacco (N. tabacum) Chi-V Basic Tobacco (N. tabacum) Tl Basic Tobacco (N. tabacum) T2 Basic Grape (Vitis labruscana) CBC Basic Grape (V. labruscana) CBC Basic Rye (8ecale cereale) RSC-a Basic Ry巴 (8.cereale) RSC-a Basic Ry巴(8.cereale) RSC-a Basic Rye (8. cereαle) RSC-c Basic Ry巴 (8.cereale) RSC-c Basic Rye (8. cereale) RSC-c Basic B. juncea BjCHIl Acidic Leucaena leucoceph呪la KB2 Chitinase Basic Rice (0ηza sativa) OsChialc Acidic Ric巴 (0.sativa) OsChia2b Basic Pineapple (Ananas comosus) PLChi-A Acidic Pineapple (A. comosus) PLChi-B Basic Pineapple (A. comosus) PLChi-C Acidic Gazyumaru (Ficus microcarpa) GLxChi目A Acidic Gazyumaru (F. microcarpa) GLxChi-B Basic Gazyumaru (F. microcarpa) GLxChi-C Basic Wheat (Triticum aestivum) Cht2 Acidic Wheat (T. aestivum) Cht2 Acidic Wheat (T. aestivum) Cht2 Acidic P.ηuわluensis PrChi-A Acidic

Schlumbaum et al.') Roberts and SelitrennikoffJ3

)

Roberts and Seli佐ennikoff")Mauch et al.34

)

Mauch et al.'4) Mauch et al. '4) J acobsen et al .35) Jacobsen et al.35

)

Verburg and Huyn'6) Leah et al.'η

Leah et al.17)

Broglie et al .拘Huynh et al.39

)

Huynh et al.39)

Huynh et al.39)

S巴la-Buurlageet al.40)

Sela-Buurlage et al.40)

1s巴liet al.41)

Me1chers et al.14)

Me1chers et al .叫M巴1cherset al. 14)

Yun et al.相

Yun et al:2)

Salzman et al.4l)

Salzman et al.4l)

Taira et al.叫

Taira et al.叫

Taira et al .44) Taira et al.叫

Taira et al.叫

Taira et al.判)

Fung et al :') Kaom巴ket al:6

)

Truong et alア)Truong et α1.47

)

Taira et al") Taira et al ..)

Taira et al .. ) Taira et al. 7)

Taira et al.7)

Taira et al.7)

Singh et al :') Singh et al. 48)

Singh et al :') Onaga and Taira'O)

Method

222333222112222442444222222222222222222222222

Test fungus

Trichoderma viride T. reesei Alternaria alternaria T. viride F. solani Penicillium digiωtum T. viride T. viride T. reesei T. reesei F. sporotrichiodes

Rhizoctonia salani A. solani F. 0砂 sporumT. viride

F. solani F. solani T. viride T. viride A. radicina F. solani T. longibrachiatum F.。巧IsporumGuignardia bidwellii BotηItiS cinerea

F. o.巧IsporumR. solani Trichoderma sp. F. 0.巧IsporumR. solani Trichoderma sp T. viride F. 0.砂 sporumT. reesei T. reesei T. viride T. viride T. viride T. viride T. viride T. viride P estalotia theae R. solani

Fusarium sp・

T. viride

Amount*

0.04-2.6 0.2-2.0 2.5-25 0.32-2.6 ND**(2.6) ND*ホ(2.6)0.5-5.0

5 1か 4.00.05-1.5 0.5-1.5 4.0-16 1.0-10 2.0-10 1か 102.5-5.0

ND**(50) 1.8-18 2.0

5.0一10ND**(100)

40 50

50一10050-100

1.0 1.0 1.0 0.8 0.8 0.8

20キ**

1か 2.02.0-10 5.0-10

NDホキ(1.3)

1.7 2.0***

ND**(1.7) 1.6 1.4

100-300 100-300 100-300 4.2

Class

LU

皿

+

IHEI---HIHHIwwwIEIVVVIII-IIIEHEm--Em--EIHEEE品

C

F

2

L

今ん

Charge Chitinase n釘neSource

*Shown as μg per w巴IIor pap巴rdisc.キ*Notdetectable at th巴 amountus巴din the parentheses. * * * Show巴dvery low extent of antifungal ac-tivity at the amount us吋.

172 J. Appl. Glycosci.， Vol. 57， No. 3 (2010)

Class I B， sterile water as blank

1-1， RSC-a (class 1， p/=9.7)

1-2， PLChi-B (class 1， p/=7司9)

ト3，GlxChi-B (class 1， p/=9.3)

ト4，CrChi-B (class 1， p/=7.1)

ト5，PLChi-C (class 1， p/=4.6)

Class 11 & 11しB， sterile water as blank

11-1， RSC-c (class 11， p/>10)

11-2， GLxChトC1(class 11， p/>10)

11-3， GLxChi-C2 (class 11， p/>10)

11・4，GLxChi-C3 (class 11， p/>10)

iト5，PLC-A (class IIL， p/=3.7)

Class 111， IIIb & V

B， sterile water as blank

111-1， PLC-B (class 111， p/=9.1)

111-2， TBC-1 (class IlIb， p/=5.5)

111・3，PLChi-A (class 111， p/=4.4)

111-4， GLxChi鮒A(class 111， p/=4.0)

111-5， CrChトA(class V， p/=5.6)

Fig. 6. Antifungal activities of various plant chitinases having various pI.

The antifungal activity of various plant chitinas巴sagainst T. vi ride was examined as described by Taira et al.5O

) Each welJ con-tained 100 pmol of plant chitinase. RSC， rye se巴dchitinas巴;PLChi， pineapple leaf chitinase; GlxChi， gazyumaru latex chitinase; CrChi， Cycas revoluta chitinase; PLC， pokeweed leaf chitinas巴;TBC， tulip bulb chitinase.

(Fig. 6). Basic class 1 and II chitinases巴xhibitstrong anti-

fungal activities. A basic class III chitinase has a low

level of antifungal activity. Acidic chitinases had no or a

very low level of antifungal activity. These results indiω

cate the charge of plant chitinases is very important for

their antifungal activities. In addition， the antifungal ac-

tiviti巴sof GH -19 chitinases are higher than those of

GH 18. Taira et al:4) showed that basic class II chitinase

bound to the fungal cell引 lallcolumn， packed with ce11-

wa11 fraction prepared from mycelia of Trichoderma sp.，

under pH 6.0 and low ionic strength conditions. The bind-

ing ability of basic class II chitinase was reduced by ele-

vating pH or ionic strength. Many antimicrobial peptides

have high basicity， and basicity of these p巴ptideswould

be sufficient for antifungal activity. An antifungal activity

of Ac-AMP (Amaranthus caud，αtus antimicrobial p巴ptide)，

a hevein-like chitin-binding peptide， is reduc巴dat high巴r

ionic strength.5臼斗) These f白indingssuggest that basicity of

plant chitinase contributes to the affinity of th巴 prot巴into

t出h巴 fungal c 巴11 蜘屯surf;白'ace.Th 巴 smface of microbial ce11s has

a negative charge due to negatively charged phospholipid

headgroups. Basic chitinases might bind to the fungal ce11

-surface by ionic interaction. It seems to be fundamental

manner of natural immunity to distinguish between the

self and nonself by features of their surfaces.

P. ryuわ'uensischitinase時A (PrChi-A) is the only plant

GH-18 chitinase having chitin-binding domains.30) PrChi帥

A exhibits antifungal activity， whereas， a mutant without

two chitin例 bindingdomains does not. Tobacco class V

chitinase exhibits antifungal activity against T. viride and

A. radicina .14) The antifungal activities of tobacco class V

chitinase and PrChi叩 A were clearly lower than those of

basic class 1 and II chitinases from rye seed日 (Ohnuma

and Onaga， unpublished data). Chi19F， a GH-19 chitinase

having a chitin-binding domain， from S. coelicolor巴xhib-

ited antifungal activity， whereas GH-18 chitinases from

th巴samestrain did not， in spite of the chitinases having a

chitin-binding domain.55) Thes巴 findingssugg巴stthat GH-

19 chitinases are mor巴 adaptiveto disrupt the fungal hy-

phae than GH-18. In addition， there may be adaptive chitin働bindingdomains and norトadaptivechitin“binding

domains to bind onto the fungal c巴11-walls.

Roles of chitill-bindillg domaill ill an死向ngalactivity

of class 1 chitillases.

In several types of plant chitinases， class 1 chitinase is

of interest due to the role of the chitin-binding domain in

antifungal activity. An antifungal activity of class 1 and II

chitinases had been demonstrated by using some bioas-

says. Basic chitinase C (class II) derived from barley seed

inhibited the growth of the fungus T. viride as we11 as ba-

sic chitinase T (class 1) did. CHN A (basic class 1

chitinase derived from tobacco) and its mutant， ~C CHN

(CHN A without a chitin同 bindingdomain)， were capable

of inhibiting the growth of工 viride，although CHN A

was about five times more effective than ~C CHN.56) Tob

chitinase (tobacco basic class 1) and Tob~H chitinase

(Tob chitinase without the chitin-binding domain) have

been found capable of inhibiting the growth of T. viride，

although Tob chitinase was more active; the inhibition

zone exceeded 1.5 mm at 4.5μg of enzyme per well，

whereas 18 /lg of Tob~H chitinase was necessary to ob-

tain the same effect.41) Taira et al.50

) showed that RSC附昌

(basic class 1 chitinase from rye seeds) and RSC-c (basic

class II chitinase with 92% sequence similarity to the

catalytic domain of RSC-a) exhibit巴dantifungal activity to

a similar extent by using a conventional hyphal略目tension

inhibition assay. However， the actions of RSC糊 aand -c on

hyphae were shown to be different by using a novel bio-

assay. The assay， a hyphae passingωthrough slit assay，

show巴dt出ha瓜tt治h巴 hyphalextension was more peαTS討ist匂er羽1t1砂y

inl制1吋ib説it犯巴dby RSC白 atめhant白ha祇taf仔f巴ct旬巴dby RSC但 cand t治h巴

hyphae affected by RSC.ι.蜘蜘句-公句

(Fig. 5). These reports indicate that the chitin-binding do-

main of class 1 chitinase contributes to the antifungal abil-

ity of the chitinase. How does the domain contribute to

the antifungal action? There is only one study on the an欄

tifungal ability of the chitin四 bindingdomain obtained by

limited hydrolysis of class 1 chitinase.50) The chitin-

binding domain of basic class 1 chitinase from rye seeds

had no or a very low level of antifungal activity， whereas the catalytic domain of this chitinase exhibits antifungal

activity as we11 as basic class II chitinase (Fig. 4(A)). In

addition， the antifungal activity of a mixture of the chitin-

binding domain and the catalytic domain was similar in

extent to that of the catalytic domain alone. These results

su

Antifungal Activity of Plant Chitinas巴s 173

cell-surface. Th巴targ巴tshould be a chitin.

During apical growth in filamentous fungi， chitin and

s-glucan fibers ar巴 synth巴sizedsimultaneously in the tip

of th巴 growinghypha. In the mature fungal cell walls， at

a distant part白・omthe hyphal tip， the polysaccharides are

cross-linked to form mixed chitin-glucan fibers and may

be overlaid by other polysaccharides and protein layers.

These findings support the following idea. At the hyphal

tip， the exposed nascent chitin chains ar巴 onlyaccessible

to hydrolysis by chitinase， whereas the chitin lay巴rin the

mature cell walls is inaccessible to degradation by the en-

zyme.') Benhamou et al.57) measured th巴 densityof cell-

wall chitin of R. solani at various times after巴xposureto

basic bean c1ass 1 chitinase by using WGA-ovomucoid

gold complex. By 30 min after exposure to th巴chitinas巴，

the density of cell-wall chitin in hyphal tips fell to 51%

of that of non-treatment cell-walls， whereas that in septa

and lateral walls r巴mainedat 65 and 83%， r巴spectively.

After 60 min， the densiti巴sof cell-wall chitin in hyphal

tips， septa and lateral walls were 32， 58 and 64%， resp巴c-

tively. After 360 min， the densities of cell-wall chitin in

hyphal tips， septa and lateral walls fell to b巴low5%

These observations indicate that chitin in the hyphal tip is

certainly sensitive to basic c1ass 1 chitinas巴 however，the

chitinase could degrade the chitin in septa and lateral

walls as well as the chitin in hyphal tips. Taira et al.44)

showed that FITC-labeled RSC-a bound to the hyphal

tips， lateral walls and septa but FlTC-labeled RSC-c

bound only to the hyphal tips (Fig. 7). Furthermore，

RSC-a had a greater affinity for the cell walls than

RSC-c. RSC-a liberat巴da larger amount of reducing sugar

from the cell walls than RSC-c did. Th巴seresults sug-

g巴stedthat basic c1ass 1 chitinase binds to the lateral walls

and septa， consisting of th巴 maturecell walls， and de-

grad巴smature chitin fiber， while basic c1ass 11 binds only

to the hyphal tips follow巴dby d巴gradationof only nascent

chitin (see Fig. 9(B))

Taira et al刊 showedthat the antifungal activity of ry巴

seed basic c1ass 1 chitinas巴 athigh salt concentrations is

(E)

〆 .....

歯圃・・・・・圃園圃・・・・・圃Fig. 7. A FITC-chitinase binding study.

A FITC-chitinas巴 bindingstudy was done as described by Taira et al叫 (A)ーの)and (E)一(H)were exposed to FITC-RSC-a and FITC-RSC-c， r巴sp巴ctively.(A)， (C)， (E) and (G)紅巳 theoptical im-ages and (B)， (D)， (F) and (H) are the resp巴ctivecorresponding fluorescent images. Bar = 50μm. Reproduced， with p巴nl'USSIOn，from Ref. 44).

slightly stronger than that under low ionic strength condi-

tions. At high salt concentrations， the c1ass 1 chitinase had

affinity to the fungal cell-walls， wher巴asthe catalytic do-

main of th巴 chitinasedid not. Acidic c1ass 1 chitinase

from pineappl巴 leavesinhibited hyphal growth of T. viride

at high salt concentrations， whereas the chitinase did not

exhibit antifungal activity und巴rlow ionic strength condi-

tions.6) The chitin-binding ability of basic c1ass 1 chitinase

from gazyumaru latex was enhanced by increasing th巴 salt

concentration.7) The antifungal activity of rye seed c1ass 1

chitinase mutant W23A (Trp23 to Ala in 由巳 chitin-

binding domain)， which is impむr巴din chitin-binding ac-

tlvlty， was w巴ak巴nedwith increasing salt concentl'ations

in the culture medium (Fig. 8).58) The binding of the

carbohydrate-binding modul巴 (CBM17)from Clostridium cellulmノoranscellulase 5A to cellopentaose and cellohexa-

ose was illcr巴asedby the presence of NaCl and the p紅ー

ticipation of tryptophan residues in ligand binding was in-

dicat巴d.59)In the case of Bacillus circulans chitinase A1，

replacem巴ntof Trp687 (in th巴 chitin-binding domain)

with phenylalanine significantly reduced chitin-binding ac-

tivity at low巴rsalt concen位ationsbut allowed s位ongbind-

ing to chitin at high salt conc巴ntrations.60)Thes巴 findings

indicate that the chitin-binding domain binds to a chitin

by hydrophobic interaction through hydrophobic arnino

acid residues， since tryptophan is the most hydrophobic

amino acid and high salt concentrations should increase

Fil!. 8. Effects of ionic strenσth on the antifunl!al activitv of basic 0"...... .........L.L ...，~....... '-'""-~'-"'"'''' "'...........0.... ....，，， ......... ....................0........ .......，...... '''J

class 1 chitinas巴mutants.63)

The samples to be tested were plac巴dinto the wells in 10 mL of distill巴dwat巴rwith 50 pmol of purified chitinases. 1， bla叫((dis-tilled water); 2， RSC-a; 3， recombinant RSC-a; 4， E126Q; 5，

W23A; 6， RSC-c. (A) and (B) contain culture medium with 0 and 0.1 M NaCl， r巴spectively.Reproduced， with perrnission， from Ref 63).

(A) Basic class I

Hydrophobic

interaction

(8) Basic class 11

「一一一 Hyphaltip ↓，..J:l品 N陶'as討ce附n川tc凶h削it，伽"川n

cOllld be d~gr，“dedl高'Yclω'ass 1 &: 11 chilI附"“s皿""，

Lateral wall Maturechitin町bers

co1l1d be degrnded h.l' cltlss 1 cltitimlse

Fig. 9. Difference in binding manner between basic cJass 1 and cJass 11 chitinases.

For details， se巴thet巴xt.

174 J. Appl. Gかcosci.，Vol. 57， No. 3 (2010)

hydrophobic interaction. Basic c1ass 1 chitinase could bind

to the fungal cell-walls both by hydrophobic interaction

through the chitin-binding domain and by ionic interaction

through the catalytic domain (Fig. 9(A)).

Contribution of chitin -hydrolytic activity of plant

chitinasesωtheir antifungal activi，砂.The mutant protein of白e26-kDa chitinase (c1ass 11) of

barley that does not possess chitinolytic activity had 15%

of the antifungal activity of the wild type of chitinase.61)

The mutant protein of c1ass 1 chitinase from chestnut

seeds that had no chitinolytic activity caused as much

morphological change of T. viride hyphae as the wild

type of chitinase.62) By only observation through a light

microscope， researchers proposed that catalysis of c1ass 1

chitinase is not necessary for antifunga1 activity. The mu-

tation of Trp72 to Ala in RSC-c caus巴ddecreases in both

chitin-hydrolytic activity and antifungal activity.63) The

mutant of RSC-c， possessing no chitinolytic activity， made

a ve巧rnarrow inhibition zone on an antifungal assay un-

der high ionic strength conditions.44) Ohnuma et al.58

)

showed that the mutant of RSC-a， having no chitin-

hydrolytic activity， did not exhibit antifungal activity un-der either low or high ionic strength conditions (see

Fig. 8， well 4). A chitin-hydrolytic inactive mutant of

PrChi-A did not inhibit hyphal extension of T. viride .30) It

is true出at some chitinase-mutants having no chitin-

hydrolytic activity exhibit slight antifungal activity; how-

ever， there is no doubt that the major antifungal activity

of chitinases is based on their chitinolytic activity (Fig. 8).

Conclusion.

Chitinase is considered to protect plants against fungal

pathogens by degrading chitin， a major component of the

cell walls of many fungi. To have antifungal activity， first， a chitinase must bind to fungal cell walls， and sec-ond， it must degrade the chitin in it. Many studies support

that basic c1ass 1 chitinas巴 hasthe highest antifungal ac-

tivity among plant chitinases due to its strong binding

ability to fungal cell-walls and chitin-degrading activity.

Following is a summ紅 Yof antifungal action of basic c1ass 1 chitinase (Fig. 9): First， basic c1ass 1 chitinase bound to

hyphal tips and lateral walls and septa， consisting of ma-

ture cell walls， by mainly ionic interaction of the catalytic

domain and by hydrophobic interaction of chitirトbinding

domain， and second， degraded mature chitin fibers as well as nascent chitin by its hydrolytic action. On the other

hand， basic c1ass 11 chitinase bound only to the hyphal tip by mainly ionic interaction by itself， followed by degrada-

tion of only nascent chitin. As a result， the basic c1ass 1

chitinase more effectively inhibited fungal growth than

basic c1ass 11 chitinase did. However， several questions still remain. Why is GH-19

chitinase more effective to inhibit fungal growth than

GH-18 chitinases? In spite of the chitin-binding module，

what is the difference betwe巴nan adaptive chitin-binding

domain and a non-adaptive chitin-binding domain in its

ability to bind to the fungal cell-walls? It is necess紅 Yto

study the cell-wall structure of several fungi to understand

the antifungal action of plant chitinases. To c1arify the re-lationship betw巴enthe structure of plant chitinases and

their antifungal action in detail， it is also nec回 S紅 Yto de-

velop more quantitative assays on solid medium.

1 greatly appreciate the kind gift from of several chitinas巴sDr. Takeshi Yamagami (Kyushu University). This work was supported in p訂 tby Grant-in-Aid for Young Scientists (B) and a grant from the Takeda Science Foundation.

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植物キチナーゼの構造と抗真菌活性

平良東紀1

1琉球大学農学部亜熱帯生物資源科学科

(903-0213沖縄県西原町千原 l番地)

さまざまな植物から多くのキチナーゼおよびその遺伝

子が単離されている.その構造特性を理解するために，

いくつかの研究グループによって複数のクラス分類が提

案されている.しかしながら 複雑で複数あるクラス分

類は本分野の多くの研究者に混乱を引き起こしている.

本稿では，植物キチナーゼの構造とそのクラス分類につ

いてレビューして，その問題点について議論する.一方，

植物キチナーゼは病原性真菌のおもな細胞壁構成多糖で

あるキチンを分解することによって，その感染を抑制す

る生体防御タンパク質と考えられている.さまざまな植

物キチナーゼの抗真菌活性が調べられているが，その構

造と抗真菌活性の相関については明らかにされていない.

キチナーゼが抗真菌活性を発揮するには，まず細胞壁に

結合し，次にそこにあるキチンを分解しなくてはならな

い.本稿では植物キチナーゼのキチン結合ドメインと触

媒ドメインの抗真菌活性における役割について議論する.