Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol159.Issue1.Oct2000:

Pepper gene encoding a basic class II chitinase is inducible by

pathogen and ethephon

Jeum Kyu Hong, Ho Won Jung, Young Jin Kim

1

_{, Byung Kook Hwang *}

Molecular Plant Pathology Laboratory,Department of Agricultural Biology,College of Natural Resources,Korea Uni6ersity,

Seoul136-701,South Korea

Received 3 November 1999; received in revised form 16 May 2000; accepted 6 June 2000

Abstract

A chitinase cDNA clone (designated CAChi2) was isolated from the cDNA library of pepper leaves infected withXanthomonas campestrispv. 6_{esicatoria. The 1004-bp full-length CAChi2 cDNA encodes a basic chitinase with an N-terminal 24 amino acid} signal peptide followed by a catalytic region. An analysis of its sequence indicates that CAChi2 is a class II chitinase, because it does not have chitin-binding domain and C-terminal extension sequences. The deduced amino acid sequence of CAChi2 has a high level of identity with class II chitinases from potato, tomato, tobacco and petunia. Southern analysis demonstrated that the CAChi2 chitinase is encoded by a single or two copy genes in the pepper genome. Following X.campestris pv. 6_{esicatoria or}

Phytophthora capsiciinfection, the CAChi2 chitinase mRNA was more highly expressed in the incompatible interaction, compared to expression in the compatible interaction. Treatment with ethylene-releasing ethephon resulted in a strong accumulation of the transcripts in the leaves. In contrast,DL-b-amino-n-butyric acid, salicylic acid and methyl jasmonate were not effective in inducing CAChi2 transcripts in pepper leaves. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Capsicum annuum; Chitinase cDNA; mRNA expression; Pathogen; Abiotic elicitors

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1. Introduction

Chitinase is one of the most important enzymes acting as components of pathogenesis-related mechanisms in plants. Since its natural substrate, chitin (a b-1,4-linked biopolymer of N -acetylglu-cosamine), is a major structural component of fungal cell walls [1], the induction in plants of chitinases with antifungal activity in vitro may also act as defenses against fungal pathogens [2,3]. The level of chitinase activity is dramatically

in-duced by wounding or infection of plants with fungal, bacterial, or viral pathogens [4 – 6]. Chiti-nase activity is also induced in plants by exoge-nously applied chemicals such as mercuric chloride [3,7]. Purified plant chitinases have antifungal ac-tivity against some fungi in vitro [3,8], which can act synergistically with plant b-1,3-glucanases to inhibit fungal growth [9,10]. In recent studies, such antifungal activity of chitinases makes this protein an attractive candidate for the production of transgenic crop plants with increased expression of chitinase [11].

Induction of chitinase mRNAs by pathogen in-fection was detected in a variety of plants. The chitinase mRNAs encoding 26-, 27- and 30-kDa chitinases, respectively, were strongly induced in tomato plants by inoculation with Cladosporium ful6_{um [12]. Although the major putative role of} chitinase is to defend against fungal pathogens,

Nucleotide sequence data have been deposited in the EMBL/

GenBank™ database under accession number AF091235 (CAChi2). * Corresponding author. Tel.: +82-2-32903061; fax: + 82-2-9251970.

E-mail address:bkhwang@mail.korea.ac.kr (B.K. Hwang).

1_{Present address: Boyce Thomson Institute, Cornell University,}

Tower Road, Ithaca, NY 14850, USA.

chitinases were also induced by infections with X.

campestris pv. 6_{esicatoria [13], Ralstonia (Pseu} -domonas) solanacearum [14] and TMV [15].

In addition to a pathogen attack, exogenous application of chemicals and wounding have been found to activate or enhance biochemical defenses against potential pathogen attack. Class I tobacco chitinase CHN50 mRNA was shown to be in-duced by pathogen infection, salicylic acid, ethyl-ene and wounding [16 – 19]. Acidic class III cucumber chitinase mRNA was strongly expressed by treatment with salicylic acid and 2,6-dichloroi-sonicotinic acid (INA) [20]. Treatment with ben-zothiadiazole induced the accumulation of acidic and basic class III tobacco chitinase mRNAs [21]. Class V chitinase mRNA homologous to bacterial exochitinases was found to be induced by TMV infection, ethephon, wounding and UV irradiation [22]. More recently, analysis of the chitinase gene structure including the promoter region suggests a close relationship between unique sequences in promoter region and expression of chitinase gene [19,23].

By using two-dimensional SDS polyacrylamide gel electrophoresis (PAGE), we have demonstrated previously that some PR proteins not detectable in healthy pepper plants were induced to accumulate by infection with Phytophthora capsici or X.

campestris pv. 6_esicatoria [24,25]. In further stud-ies, we reported differential induction and accu-mulation of chitinase isoforms in pepper plants inoculated with these pathogens or treated with mercuric chloride [2,13]. The roles of individual isoforms of chitinases were also evaluated in the compatible and incompatible interactions. More recently, we isolated and purified the chitinases and other chitin-binding proteins from pepper stems treated with mercuric chloride [3]. The purified chitinases contained high ratios of cys-teine and glycine at the N-terminal chitin-binding domain and showed antifungal activity against some plant pathogenic fungi.

We have started to investigate the molecular expression of the chitinase gene family in pepper plants. In this paper, we report the isolation, sequencing, and expression analysis of CAChi2 cDNA clone encoding a basic class II chitinase in pepper plants. The gene was induced in pepper plants by infection withX.campestris pv.6esicato

-ria or P. capsici, treatment with ethephon and benzothiadiazole.

2. Materials and methods

2.1. Plant, pathogens, and elicitors

Pepper (Capsicum annuum L.) cultivar Hanbyul was used in this study. Pepper seeds were sown in plastic trays (55×35×15 cm) containing steril-ized soil, sand and compost (1:1:1, v/v/v). Seedlings at the two-leaf stage were transplanted to plastic pots (5×15×10 cm) containing the same soil mixture. Pepper plants were raised in a growth room under 16-h day illumination at 279

2°C.

Virulent (Ds1) and avirulent (Bv5-4a) strains of

X. campestris pv. 6_esicatoria were used in this study. Pepper plants were inoculated by vacuum-infiltrating the bacterial suspension into the abax-ial side of the fully expanded leaves with an atomizer. Virulent (S197) and avirulent (CBS178.26) isolates of P. capsici were grown on oatmeal agar plates for 10 days. To induce sporu-lation, the plates were placed under fluorescent light at 28°C for 3 days. The mycelia and sporan-gia were incubated in sterile water for 1 h at 4°C and then 1 h at room temperature to release zoospores. A small quantity of sterile cotton soaked in a zoospore suspension (105 _zoospores

ml−1_{) of the two isolates was placed on the stems}

of pepper plants at the six-leaf stage wounded by making a 1-cm longitudinal slit.

Entire plants were sprayed with 10 mM ethep-hon (Amchem Products), 25mM methyl jasmonate (Aldrich), 1000 mg ml−1

DL-b-amino-n-butyric

acid (BABA) (Sigma), 20 mg ml−1

benzothiadia-zole (BTH) (Novartis), or 5 mM salicylic acid (Sigma). Pepper plants treated with ethephon and methyl jasmonate were covered with vinyl bags. At various intervals after inoculation and elicitor treatment, pepper leaves or stems were harvested from the inoculated plants for RNA isolation.

2.2. Construction of cDNA library

A leaf cDNA library was constructed using poly (A)+ _{mRNA from pepper (cv. Hanbyul) leaves}

infected with avirulent strain Bv5-4a ofX.campes

-trispv.6_{esicatoria. Poly (A)}+_{mRNA was isolated}

La Jolla, CA) and packaged in vitro into phage particles following the manufacturer’s instructions (Amersham).

2.3. Preparation of the chitinase-coding region pchit II

The coding region, named pchit II, of the class II pepper chitinase from pepper genomic DNA was amplified by polymerase chain reaction (PCR) using two primers that introduced restriction sites at the ends of the amplification products. The two primers, named CLASS2N and CLASS2C, were synthesized using a DNA synthesizer. The 5% and 3% primers were CLASS2N 5%-AACAACCC(A/ T)GATTTAGT(A/G)GC-3% _{and CLASS2C 5}%

-GTTCCTTT(A/G)GTTGTTACAGT-3%, which

included amino acid sequence alignments of the highly conserved regions of different plant chiti-nases, respectively (Fig. 2). Amplification of the chitinase-coding region pchit II by PCR was per-formed in a DNA thermal cycler (Perkin Elmer). The amplification reaction mixture of 20 ml con-sisted of 100 ng pepper genomic DNA, 10 pmol of the CLASS2N primer, 10 pmol of the CLASS2C primer, 3.0 mM NTP, and Taq DNA polymerase in 10× PCR buffer. The DNA-denaturation step was set at 94°C for 5 min, the primer annealing step at 44°C for 45 s, and the primer extension step at 72°C for 2 min. The reaction was allowed to run for 40 cycles. The PCR-amplified DNAs were fractionated on a 1% low melting agarose gel (FMC Bioproducts, ME). Desired fragments were purified from the gel, cloned into pT7Blue vector and sequenced. The probe pchit II (the CLASS2N-CLASS2C probe) for screening pepper chitinase cDNAs was prepared from the recombinant plas-mid by digesting with BamHI and HindIII.

2.4. Screening of cDNA library

To isolate full-length cDNA clones of pepper chitinases, the cDNA library in lZAP II was screened with the chitinase-coding region pchit II as a probe. The probe pchit II was labeled with digoxygenin (DIG) using a random primer label-ing kit (Boehrlabel-inger Mannheim). About 50 000 plaques of the cDNA library were transferred onto duplicate nylon membranes (Hybond N+_,

Amer-sham). The blots were hybridized for 18 h at 65°C with the DIG-labeled probe. Hybridization was

performed in a solution containing 5× SSC, 0.1%

N-lauroylsarcosine, 0.02% SDS and 1% blocking reagent. Blots were washed twice at room temper-ature for 10 min in 2× SSC and 0.1% SDS, and then twice at 65°C for 5 min in 0.1× SSC and 0.1% SDS. The membranes were exposed to X-ray film for autoradiography.

2.5. Sequencing of cDNA and comparati6_e analysis

DNA sequences of the pchit II and putative pepper chitinase cDNA CAChi2 were determined by the dideoxynucleotide chain termination method using Sequenase Version 2.0 (USB, Cleve-land, OH). The deletion mutants of the CAChi2 cDNA clone were generated using Erase-A-Base kit (Promega, Madison, WI). DNA sequence data were assembled and analyzed using PC/Gene soft-ware and the BLAST and ORF finder network services at the National Center for Biotechnology Information [26]. DNA sequences were further

analyzed using DNASTAR (DNASTAR,

Madison, WI).

2.6. Genomic DNA isolation and Southern blotting

Genomic DNA was extracted from pepper leaves by the modified method of Kim et al. [27]. Fresh leaf tissue (2 g) was ground with liquid nitrogen in a mortar. Genomic DNA was ex-tracted in an urea extraction buffer (42% urea, 3.125 mM NaCl, 50 mM Tris – HCl, pH 8.0, 20 mM EDTA, pH 8.0). The genomic DNA (10 mg) extracted was digested with EcoRI, BamHI, and

HindIII, and fractionated by electrophoresis on a 0.8% agarose gel. The genomic DNA was trans-ferred onto a nylon membrane by vacuum blot-ting. Hybridization was performed using the

32_{P-labeled CAChi2 cDNA as a probe [28].}

2.7. RNA isolation and Northern blotting

(1:100:2, v/v/v) was added to the homogenates. After centrifugation, the aqueous phase was mixed with 1 vol. isopropanol, and then placed at −20°C for at least 1 h to precipitate RNA. The pellet was dissolved in TNE buffer (50 mM Tris – HCl, pH 7.5, 10 mM EDTA, 0.5% SDS) and again precipitated by adjusting the solution to 2 M LiCl. After centrifugation, the RNA pellet was washed with 80% ethanol, dried and dissolved in DEPC-treated water. For Northern blot analysis, total RNA (20 mg) was separated on a denaturing 1.5% formaldehyde agarose gel. The RNA was transferred to Hybond N+ _membrane

(Amer-sham), followed by UV cross-linking. Hybridiza-tion was performed overnight at 50°C in a solution containing 7% SDS, 50% formamide, 5× SSC, 2% blocking reagent, 50 mM sodium phos-phate (pH 7.0), and 0.1% N-lauroylsarcosine. Membranes were washed twice with 2× SSC and 0.1% SDS at room temperature for 10 min, and then twice with 0.1× SSC and 0.1% SDS at 65°C for 5 min. Membranes were exposed to X-ray films.

3. Results

3.1. Isolation of a cDNA clone encoding a basic class II chitinase

Two primers, N-terminus CLASS2N and C-ter-minus CLASS2C, were designed and synthesized from the conserved regions of the class II chitinase sequences previously reported in many plant spe-cies. The polymerase chain reaction (PCR) proce-dures were performed to amplify the coding region of the class II pepper chitinase from pepper genomic DNA using the two primers synthesized. After the PCR amplification, a distinct DNA frag-ment was visualized on an agarose gel. The size of the DNA fragment amplified was similar to the size expected, approximately 310 bp, which corre-sponds to the coding region of the class II chiti-nases between the two primers. The fragment was cloned into pT7Blue cloning vector and se-quenced. The amplified fragment, named pchit II, of the class II pepper chitinase was identified to be 308 bp (data not shown). The pchit II sequence showed 79, 76, and 71% identities to potato, tomato and tobacco class II chitinase, respectively.

The cDNA library, which was constructed from mRNAs in pepper leaves inoculated with the avir-ulent strain Bv5-4a of X. campestris pv. 6esicato

-ria, was screened with the PCR-derived clone pchit

II. Among approximately 50 000 plaques

screened, more than 150 plaques showed positive hybridizations with the pchit II probe at high stringency. Following plaque purification and in vivo excision of the pBluescript SK (−) recombi-nant plasmid from the lZAP II, the sizes of 15 clones were determined by agarose gel elec-trophoresis and Southern blot analysis (data not shown). The two cDNA clones of ca. 1.5 and 0.4 kb, which hybridized to the pchit II probe, were sequenced. However, the 1.5-kb cDNA clone pchit II 1-6 was confirmed to encode a putative basic chitinase of pepper in the sequence comparison by the BLAST program. The putative basic chitinase cDNA pchit II 1-6 was designated CAChi2.

3.2. Nucleotide and deduced amino acid sequences of CAChi2 cDNA

The complete nucleotide and deduced amino acid sequences of the CAChi2 cDNA encoding a putative basic chitinase of pepper revealed that the CAChi2 cDNA contains 1004 bp with a 759-nu-cleotide open reading frame (data not shown). The N-terminal 24 amino acids exhibit the characteris-tics of a signal peptide with a highly hydrophobic core and characteristic amino acid composition near the cleavage site [31]. The CAChi2 does not have cysteine-rich domains and a C-terminal ex-tension signal sequence, which is necessary for vacuolar targeting of mature proteins [32].

compared to the conserved regions in potato and tomato chitinases [12,33,34]. The high levels of sequence identity, from 69 to 86%, were found between the pepper chitinase CAChi2 and each of class II chitinases of potato, tomato, tobacco, and petunia. In contrast, a much lower identity was found between the pepper chitinase and other classes of plant chitinases (data not shown).

A phylogenetic analysis of the 15 deduced chiti-nase amino acid sequences in Genbank was used to

compare the relationship of the chitinase encoded by pepper CAChi2 to other chitinases. As shown in Fig. 2, well-separated branches of plant chitinases can be identified in the phylogenetic tree. The deduced proteins of the CAChi2 chitinase cDNA clone falls into a class II chitinase group. Chitinases of each class are clustered together. Class II chiti-nase group including CAChi2 has a high amino acid sequence homology to class I chitinases, but sequence dissimilarities to class IV chitinases.

Fig. 2. Phylogenic analysis of the aligned amino acid sequences of pepper chitinase CAChi2 cDNA and 15 other plant chitinase genes. The other chitinases are indicated by the names of the plants of origin and their GenBank/EMBL accession numbers. The relative homologies between chitinase sequences are proportional to the horizontal length of branches.

3.3. Genomic organization of pepper chitinase gene corresponding to CAChi2 cDNA

Genomic DNA was digested with the restriction enzymes EcoRI, BamHI, and HindIII, respec-tively, for which there are no internal restriction sites in the sequence of CAChi2 cDNA. The com-pletely digested DNA was subjected to Southern blot hybridization with the EcoRI –XhoI insert from the isolated chitinase cDNA clone CAChi2 as a probe (Fig. 3). The CAChi2 cDNA hybridized to one EcoRI band, one BamHI band and two

HindIII bands. All of these band sizes are long enough to represent intact genes, suggesting that the CAChi2 chitinase in pepper genome is encoded by single or two copy genes.

3.4. Expression of CAChi2 mRNA upon infection with pathogens and treatment with abiotic elicitors

RNA gel blot analysis was performed to moni-tor the expression of the chitinase CAChi2 mRNA upon infection with eitherX. campestrispv.6_esica

-toria or P. capsici. No transcripts homologous to CAChi2 cDNA were detected in the healthy pep-per leaves. In the compatible interactions of X.

campestris pv. 6esicatoria with pepper, accumula-tion of CAChi2 mRNA was considerably induced in the leaves by 30 h after inoculation (Fig. 4). In the incompatible interaction, the CAChi2 mRNA

was first detected 12 h after inoculation, followed by a maximum in transcript levels at 30 h. P.

capsici-infected pepper stems also showed different accumulation of CAChi2 transcripts in the com-patible and incomcom-patible interactions (Fig. 5). In-duction of CAChi2 appeared in both interactions 1 day after inoculation. In the compatible

interac-Fig. 3. Genomic DNA gel blot analysis of the putative CAChi2 cDNA of pepper plants. Genomic DNA was di-gested with EcoRI, BamHI, and HindIII, fractionated on 0.8% (w/v) agarose gel, and blotted onto nylon membrane (Hybond N+_{). The membrane was hybridized with a CAChi2}

Fig. 4. Northern blot analysis of chitinase (CAChi2) mRNA accumulation in pepper leaf tissue during compatible and incompatible interactions after infection by virulent strain Ds1 and avirulent strain Bv5-4a ofXanthomonas campestris

pv. 6esicatoria, respectively. (A) Northern blot analysis of CAChi2 mRNA at various times after inoculation. (B) Den-sitometric comparison of CAChi2 mRNA levels between compatible and incompatible interactions. As a control, each blot was hybridized with a 25S rRNA probe fromCapsicum annuum. The Northern blot analysis was repeated three times with similar results. Vertical bars represent S.E.

monate, benzothiadiazole and salicylic acid re-sulted in little accumulation of the transcripts in the leaves (Fig. 6A). Time course induction of CAChi2 transcripts by ethephon treatment is shown in Fig. 6B. The transcript level began to increase at 18 h after treatment with ethephon, with a maximum at 24 h. After application of ethephon, a chemical releasing gaseous ethylene, we examined whether or not hydrochloric acid (HCl) and phosphonic acid (H3PO3) as breakdown

products of ethephon when applied to pepper plants induced transcripts of CAChi2. There was no induction of the transcript accumulation in pepper leaves by treatment with the two chemicals (data not shown). When treated with methyl

jas-Fig. 5. Northern blot analysis of chitinase (CAChi2) mRNA accumulation in pepper stem tissue during compatible and incompatible interactions after infection by virulent isolate S197 and avirulent isolate CBS178.26 ofPhytophthora capsici, respectively. (A) Northern blot analysis of CAChi2 mRNA at various times after inoculation. ‘W’ indicates total RNA extracted from the wounded stem tissues 1 day after wound-ing. (B) Densitometric comparison of CAChi2 mRNA levels between compatible and incompatible interactions. As a con-trol, each blot was hybridized with a 25SrRNA probe from

Capsicum annuum. The Northern blot analysis was repeated three times with similar results. Vertical bars represent S.E.

tions, CAChi2 slightly increased 2 days after inoc-ulation and then diminished after 3 days after inoculation when pepper plants began to die due to severe P. capsici infection. In the incompatible interaction, however, drastic increase of CAChi2 mRNA occurred 2 – 3 days after inoculation. After 4 days, the transcripts decreased to the level of 1 day after inoculation. There was no expression of CAChi2 mRNA in the wounded stems.

jas-Fig. 6. (A) Expression pattern ofCAChi2 mRNAs in pepper leaves treated with ethephon, methyl jasmonate,

DL-b-amino-n-butyric acid, benzothiadiazole, and salicylic acid. (B) Time course of induction of CAChi2 mRNA in pepper leaves treated with 10 mM ethephon. As a control, each blot was hybridized with a 25S rRNA probe from C. annuum. The Northern blot analysis was repeated three times with similar results.

CAChi2 belongs to the class II chitinase. The chitin-binding domain is known to be a major characteristic of class I chitinase [38]. Class I and II chitinases have leader sequences and catalytic regions as common features, whereas class II chiti-nase do not have a cysteine-rich chitin-binding domain, a hinge region, and a C-terminal exten-sion sequence present in the class I chitinase [39]. Comparison of N-terminal amino acid sequences of basic pepper chitinases showed that the basic CAChi2 chitinase is not identical with the basic chitinases b1 (32 kDa, pI 9.0) and b2 (22 kDa, pI 9.1) previously purified from pepper plants [3], indicating the presence of another chitinase iso-form in pepper plants. Cluster analysis of the deduced peptide of CAChi2 cDNA with the align-ments of chitinases from 23 known chitinases also confirmed that CAChi2 can be classified into the class II chitinases (Fig. 2). With a tobacco class I chitinase CHN50, it has been suggested that some C-terminal extension sequences are necessary for targeting the protein to the vacuole [32,40] and that sequence changes in the vacuolar targeting peptide allow a gradual transition from vacuolar retention to secretion [40]. C-Terminal extension sequences were not found in the pepper CAChi2 chitinase, as in the petunia class II chitinase which has been found to be extracellularly located [35]. The calculated isoelectric point (pI) of CAChi2 is 9.39 that is not typical in the class II chitinases known to be usually more acidic proteins [38]. Although the CAChi2 chitinase gene may encode a basic chitinase, its localization seems to be extra-cellular, because of absence of a C-terminal exten-sion signal. These suggestions are well supported by the findings of Kombrink et al. [41], which showed that potato basic chitinase was targeted to the extracellular space. Our recent observation that the activity of a basic chitinase isoform with pI 9.8, i.e. similar to that of CAChi2 chitinase, was induced in the intercellular washing fluid of X.

campestris pv. 6esicatoria-infected leaves [13] sup-ports the possible extracellular localization of CAChi2 chitinase in pepper leaves.

The Southern blot analysis of the pepper chiti-nase gene indicated that the basic chitichiti-nase CAChi2 is encoded by one or two genes in pepper plants (Fig. 3). The fragment restricted with

EcoRI or BamHI was hybridized as a single band, whereas the fragment restricted with HindIII was hybridized as double bands. These bands may monate, DL-b-amino-n-butyric acid,

benzothiadia-zole or salicylic acid, the CAChi2 transcript did not accumulate in the pepper leaves during the incubation for 48 h.

4. Discussion

In this study, the pepper basic chitinase CAChi2 cDNA was isolated from the cDNA library of pepper leaves infected with X. campestris pv.6_esi

-catoria, using a probe amplified from pepper ge-nomic DNA as a template by PCR. The PCR product was amplified by the two primers, which were degenerated based on the conserved regions of class II chitinase amino acid sequences of differ-ent plant species previously reported [12,35,36].

reflect either two chitinase gene copies differently located in the pepper chromosomes or a chitinase gene that has an internal cleavage site for HindIII in the intron region.

Interestingly, the CAChi2 chitinase mRNA in-creased more markedly in response to the aviru-lent strain than to the viruaviru-lent strain of X.

campestrispv.6_{esicatoria. Strong expression of the} CAChi2 chitinase mRNA in the incompatible in-fection of X. campestris pv. 6esicatoria may result in a high accumulation of basic chitinase activity in the intercellular space of pepper leaves, as re-cently demonstrated by Lee and Hwang [13]. In the incompatible interaction, the mRNA expres-sion of CAChi2 chitinase greatly increased at 18 – 24 h, when a hypersensitive response, like rapid and localized tissue collapse, appeared against the avirulent strain infection. The great accumulation of the CAChi2 chitinase mRNA at 24 – 30 h after appearance of hypersensitive response may be sig-nificant in defense of pepper plants against X.

campestris pv. 6_{esicatoria infection. In contrast,} mRNA induction of CAChi2 chitinase gene was relatively low until 30 h after inoculation with the virulent strain. At this time, no symptoms ap-peared on the pepper leaves. Chlorotic and suscep-tible lesions began to occur 3 days after inoculation with the virulent strain. P. capsici in-fection in pepper stems greatly induced the

accu-mulation of the CAChi2 mRNA in the

incompatible interaction rather than in the com-patible interaction (Fig. 5), which suggested that the CAChi2 chitinase gene may be required for the expression of defense against P. capsici infection. Application of ethephon (2-chloroethylphospho-nic acid) induced a strong expression of CAChi2 mRNA in pepper leaves, whereas methyl jas-monate, BTH and salicylic acid induced little ex-pression. Enhancement of the CAChi2 mRNA expression in pepper leaves upon treatment with ethylene-releasing ethephon suggests that expres-sion of CAChi2 gene may be controlled by an ethylene-dependent signal transduction mecha-nism, as described recently for the PR-1 gene expression in pepper plants [42]. Recently, Tornero et al. [43] have demonstrated that ethep-hon, but not each of the breakdown products phosphonic acid and hydrochloric acid, induced PR-1 genes in tomato. Ethylene is thought to be the natural mediator of PR protein accumulation [44].

Taken together, it seems likely that in pepper defense- or pathogenesis-related plant responses, induction of CAChi2 mRNA is intrinsically asso-ciated with ethylene biosynthesis. However, fur-ther detailed studies will be necessary to precisely elucidate the role of ethylene in the induction of CAChi2 gene expression in pepper plants.

References

[1] S. Bartnicki-Garcia, Cell wall chemistry, morphogenesis and taxonomy of fungi, Annu. Rev. Microbiol. 73 (1968) 487 – 500.

[2] Y.J. Kim, B.K. Hwang, Differential accumulation of b-1,3-glucanase and chitinase isoforms in pepper stems infected by compatible and incompatible isolates ofPhy

-tophthora capsici, Physiol. Mol. Plant Pathol. 45 (1994) 195 – 209.

[3] Y.J. Kim, B.K. Hwang, Purification, N-terminal amino acid sequencing and antifungal activity of chitinases from pepper stems treated with mercuric chloride, Phys-iol. Mol. Plant Pathol. 48 (1996) 417 – 432.

[4] M. Legrand, S. Kauffmann, P. Geoffroy, B. Fritig, Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chiti-nases, Proc. Natl. Acad. Sci. USA 84 (1987) 6750 – 6754.

[5] N. Majeau, J. Trudel, A. Asselin, Diversity of cucumber chitinase isoforms and characterization of one seed basic chitinase with lysozyme activity, Plant Sci. 68 (1990) 9 – 16.

[6] D. Roby, K. Broglie, R. Cressman, P. Biddle, I. Chet, R. Broglie, Activation of a bean chitinase promoter in transgenic tobacco plants by phytopathogenic fungi, Plant Cell 2 (1990) 999 – 1007.

[7] W. Nasser, M. De Tapia, G. Burkard, Maize pathogene-sis-related proteins: characterization and cellular distri-bution of b-1,3-glucanases and chitinases induced by brome mosaic virus infection or mercuric chloride treat-ment, Physiol. Mol. Plant Pathol. 36 (1990) 1 – 14. [8] A. Schlumbaum, F. Mauch, U. Vo¨geli, T. Boller, Plant

chitinases are potent inhibitors of fungal growth, Nature 324 (1986) 365 – 367.

[9] F. Mauch, L.A. Staehelin, Functional implications of the subcellular localization of ethylene-induced chitinase and b-1,3-glucanase in bean leaves, Plant Cell 1 (1989) 447 – 457.

[10] Y.J. Kim, B.K. Hwang, Isolation of a basic 34 kilodal-tonb-1,3-glucanase with inhibitory activity againstPhy

-tophthora capsicifrom pepper stems, Physiol. Mol. Plant Pathol. 50 (1997) 103 – 115.

[11] K. Broglie, I. Chet, M. Hollyday, R. Cressman, P. Biddle, S. Knowlton, C.J. Mauvais, R. Broglie, Trans-genic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani, Science 254 (1991) 1194 – 1197.

-infected tomato, Plant Mol. Biol. 22 (1993) 1017 – 1029.

[13] Y.K. Lee, B.K. Hwang, Differential induction and accu-mulation of b-1,3-glucanase and chitinase isoforms in the intercellular space and leaf tissues of pepper by

Xanthomonas campestris pv. 6_{esicatoria infection, J.} Phytopathol. 144 (1996) 79 – 87.

[14] L. Godiard, F. Raguch, D. Froissard, J.J. Leguay, J. Grosset, Y. Chartier, Y. Meyer, Y. Marco, Analysis of the synthesis of several pathogenesis-related proteins in tobacco leaves infiltrated with water and with compat-ible and incompatible isolates of Pseudomonas solanacearum, Mol. Plant-Microbe Interact. 3 (1990) 207 – 213.

[15] K. Lawton, E. Ward, G. Payne, M. Moyer, J. Ryals, Acidic and basic class III chitinase mRNAs accumula-tion in response to TMV infecaccumula-tion of tobacco, Plant Mol. Biol. 19 (1992) 735 – 743.

[16] J. Grosset, I. Marty, Y. Chartier, Y. Meyer, mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible, Plant Mol. Biol. 15 (1990) 485 – 496. [17] Y. Fukuda, M. Ohme, H. Shinshi, Gene structure and

expression of a tobacco endochitinase gene in suspen-sion-cultured tobacco cells, Plant Mol. Biol. 16 (1991) 1 – 10.

[18] M. van Buuren, J.M. Neuhaus, H. Shinshi, J. Ryals, F. Meins, The structure and regulation of homeologous tobacco endochitinase genes of Nicotiana syl6_estrisand

N.tomentosiformisorigin, Mol. Gen. Genet. 232 (1992) 460 – 469.

[19] Y. Fukuda, H. Shinshi, Characterization of a novel

cis-acting element that is responsive to a fungal elicitor in the promoter of a tobacco class I chitinase gene, Plant Mol. Biol. 24 (1994) 485 – 496.

[20] K.A. Lawton, J. Beck, S. Potter, E. Ward, J. Ryals, Regulation of cucumber class III chitinase gene expres-sion, Mol. Plant-Microbe Interact. 7 (1994) 48 – 57. [21] L. Friedrich, K. Lawton, W. Ruess, P. Masner, N.

Specker, M.G. Rella, B. Meier, S. Dincher, T. Staub, S. Uknes, J. Me´traux, H. Kessmann, J. Ryals, A benzoth-iadiazole derivative induces systemic acquired resistance in tobacco, Plant J. 10 (1996) 62 – 70.

[22] L.S. Melchers, M. Apotheker-deGroot, J.A. van der Knaap, A.S. Ponstein, M.B. Sela-Buurlage, J.F. Bio, B.J.C. Cornelissen, P.J.M. van den Elzen, H.J.M. Linthorst, A new class of tobacco chitinases ho-mologous to bacterial exochitinases displays antifungal activity, Plant J. 5 (1994) 469 – 480.

[23] Y. Fukuda, Interaction of tobacco nuclear protein with an elicitor-responsive element in the promoter of a basic class I chitinase gene, Plant Mol. Biol. 34 (1997) 81 – 87.

[24] B.K. Hwang, J.Y. Yoon, W.D. Ibenthal, R. Heitefuss, Soluble proteins, esterases and superoxide dismutase in stem tissue of pepper plants in relation to age-related resistance to Phytophthora capsici, J. Phytopathol. 132 (1991) 129 – 138.

[25] Y.K. Lee, J.Y. Kim, B.K. Hwang, Bacterial multiplica-tions and electrophoretic patterns of soluble proteins in compatible and incompatible interactions of pepper leaves withXanthomonas campestrispv.6_esicatoria, Ko-rean J. Plant Pathol. 40 (1994) 305 – 313.

[26] J.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403 – 410.

[27] W.T. Kim, A. Campbell, T. Moriguchi, H.C. Yi, S.F. Yang, Auxin induces three genes encoding 1-aminocy-clopropane-1-carboxylate synthase in mung bean hypocotyls, J. Plant Physiol. 150 (1997) 77 – 84. [28] Sambrook, J., Fritsch, E.F., Maniatis, R. Molecular

Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [29] P. Chomczynski, N. Sacchi, Single-step method of RNA

isolation by acid guanidium thiocyanate-phenol-chloro-form extraction, Anal. Biochem. 162 (1987) 156 – 159. [30] J. Logemann, J. Shell, L. Willmitzer, Improved method

for the isolation of RNA from plant tissues, Anal. Biochem. 163 (1987) 16 – 20.

[31] G. von Heijne, Patterns of amino acids near signal-se-quence cleavage site, Eur. J. Biochem. 133 (1983) 17 – 21.

[32] J.M. Neuhaus, L. Sticher, F. Meins, T. Boller, A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole, Proc. Natl. Acad. Sci. USA 88 (1991) 10362 – 10366.

[33] R. Bu¨chter, A. Stro¨mberg, E. Schmelzer, E. Kombrink, Primary structure and expression of acidic (class II) chitinase in potato, Plant Mol. Biol. 35 (1997) 749 – 761.

[34] R.D. Chen, L.X. Yu, A.F. Greer, H. Cheriti, Z. Tabaeizadeh, Isolation of an osmotic stress- and ab-scisic acid-induced gene encoding an acidic endochiti-nase from Lycopersicon chilense, Mol. Gen. Genet. 245 (1994) 195 – 202.

[35] H.J.M. Linthorst, L.C. van Loon, C.M.A. van Rossum, A. Mayer, J.F. Bol, J.S.C. van Roekel, E.J.S. Meulen-hoff, B.J.C. Cornelissen, Analysis of acidic and basic chitinases from tobacco and petunia and their constitu-tive expression in transgenic tobacco, Mol. Plant-Mi-crobe Interact. 3 (1990) 252 – 258.

[36] G. Payne, P. Ahl, M. Moyer, A. Harper, J. Beck, F. Meins, J. Ryals, Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco, Proc. Natl. Acad. Sci. USA 87 (1990) 98 – 102.

[37] N.V. Raikhel, H.I. Lee, W.F. Broekart, Structure and function of chitin-binding proteins, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44 (1993) 591 – 615.

[38] L.S. Graham, M.B. Sticklen, Plant chitinases, Can. J. Bot. 72 (1994) 1057 – 1083.

[39] D.B. Collinge, K.M. Kragh, J.D. Mikkelsen, K.K. Nielsen, U. Rasmussen, K. Vad, Plant chitinases, Plant J. 3 (1993) 31 – 40.

[40] J.M. Neuhaus, M. Pietrzak, T. Boller, Mutation analy-sis of the C-terminal vacuolar targeting peptide of to-bacco chitinase: low specificity of the sorting system, and gradual transition between intracellular retention and secretion into the extracellular space, Plant J. 5 (1994) 45 – 54.

[42] Y.J. Kim, B.K. Hwang, Pepper gene encoding a basic pathogenesis-related 1 protein is pathogen and ethylene inducible, Physiol. Plant. 108 (2000) 51 – 60.

[43] P. Tornero, J. Gadea, V. Conejero, P. Vera, Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for pathogenesis-related gene

during the hypersensitive response and development, Mol. Plant-Microbe Interact. 10 (1997) 624 – 634. [44] Y. Eyal, Y. Meller, S. Lev-Yadun, R. Flur, A basic type

PR-1 promoter directs ethylene responsiveness, vascular and abscission zone-specific expression, Plant J. 4 (1993) 225 – 234.