ORIGINAL ARTICLE

Sung Chul Lee Æ Byung Kook Hwang

Identification of the pepper SAR8.2 gene as a molecular markerfor pathogen infection, abiotic elicitors and environmental stressesin Capsicum annuum

Received: 23 May 2002 /Accepted: 12 July 2002 / Published online: 1 October 2002� Springer-Verlag 2002

Abstract Pepper (Capsicum annuum L.) SAR8.2 genes,designated CASAR82A, B and C, which are induced byall the biotic and abiotic stresses, were isolated from apepper cDNA library constructed with the mRNAsfrom pepper plants infected with Xanthomonas campes-tris pv. vesicatoria. The pepper CASAR82A, B and Cgene products, which are very similar to each other inamino acid sequences, have 43–50% homology withthose of tobacco SAR8.2 genes. The CASAR8.2 geneswere not constitutively expressed in any of the organs ofhealthy pepper plants. In contrast, the CASAR82A genewas locally or systemically induced in pepper plants in-fected by X. campestris pv. vesicatoria, Colletotrichumcoccodes or Phytophthora capsici. Strong induction ofthe CASAR82A gene also was found in pepper leavestreated with ethylene, methyl jasmonate, indole-3-aceticacid, abscisic acid, salicylic acid, benzothiadiazole, DL-b-n-amino butyric acid or hydrogen peroxide. Interest-ingly, the transcription of the CASAR82A gene wasrapidly triggered by high salinity, drought or low-tem-perature stresses, but not by mechanical wounding. Insitu hybridization results revealed that the CASAR82AmRNAs were localized in phloem and epidermal cells ofpepper leaf and stem tissues infected by C. coccodes andP. capsici, or treated with salicylic acid. These resultsthus suggest that pepper SAR8.2 genes may be valuableas a molecular marker for the detection of variouspathogen infections, abiotic elicitors and environmentalstresses.

Keywords Abiotic elicitor Æ Capsicum annuum ÆEnvironmental stress Æ SAR8.2 gene Æ Systemicacquired resistance

Abbreviations ABA: abscisic acid Æ BABA: DL-b-amino-n-butyric acid Æ BTH: benzothiadiazole Æ DIG:digoxigenin Æ HR: hypersensitive response Æ IAA:indole-3-acetic acid Æ PR: pathogenesis-related Æ SA:salicylic acid Æ SAR: systemic acquired resistance

Introduction

Plants, like animals, are continuously exposed topathogen attack. However, most plants normally with-stand invasion by potential pathogens because theypossess effective defense mechanisms that render themresistant to pathogen attacks (Collinge and Slusarenko1987). Some of the defense mechanisms are establishedin the plant prior to the arrival of pathogens, whereasothers are inducible upon the perception of pathogenicsignals and can protect the plant not only at the site ofinfection, but also systemically throughout the plant.

The defense response of plants involves specific rec-ognition of the avirulent pathogen mediated by aviru-lence genes. One form of this defense response is therestriction of pathogen growth in plant tissues immedi-ately adjacent to the infection site. Infected plant cellsthen rapidly undergo programmed cell death, which istermed the hypersensitive response (HR). Localized ac-quired resistance occurs within a narrow zone aroundthe HR lesion, and is characterized by a strong stimu-lation of defense responses (Dorey et al. 1998).

Systemic acquired resistance (SAR) is a broad-spec-trum resistance that can be triggered by a predisposinginfection with necrotizing pathogens (Ryals et al. 1996)and by treatment with certain chemicals such as salicylicacid (SA; Malamy and Klessig 1992). Pathogen-inducedSAR has been extensively studied in tobacco (Ward et al.1991), cucumber (Rasmussen et al. 1991) andArabidopsis(Ukness et al. 1993) in which inoculation with necrotizing

Planta (2003) 216: 387–396DOI 10.1007/s00425-002-0875-5

Nucleotide sequence data of CASAR82A, CASAR82B and CA-SAR82C have been deposited in the EMBL/GenBank databaseunder accession numbers AF112868, AF313765, AF313766,respectively.

S.C. Lee Æ B.K. Hwang (&)Laboratory of Molecular Plant Pathology,College of Life and Environmental Sciences,Korea University, Seoul 136-701, KoreaE-mail: bkhwang@korea.ac.krFax: +82-2-9251970

pathogens leads to both local and systemic increases in SAlevels. Accumulation of SA is suggested to be critical forthe induction of the signalling pathway capable of devel-oping SAR (Malamy and Klessig 1992). Furthermore,SAR is associated with the coordinate expression of a setof so-called SAR genes (Ward et al. 1991). These SARgenes include genes encoding pathogenesis-related (PR)proteins. The PR proteins include enzymes that not onlymodify the cell wall (chitinase and b-1,3-glucanase;Schroder et al. 1992), but also have antimicrobial activity(thaumatin group;Woloshuk et al. 1991).Another groupsof defense-related proteins are thionins (Bohlmann andApel 1987; Bohlmann 1994), defensins (Broekaert et al.1995) andSAR8.2 (Ward et al. 1991). SAR8.2 comprises asmall gene family that is induced by all of the resistance-inducing stimuli. SAR8.2 is induced to a lesser extent butfaster in tobacco than the other SAR-related genes (Wardet al. 1991). In tobacco andArabidopsis, extracellular PR-1 is the main protein induced during SAR, whereas incucumbers, extracellular PR-1 is weakly expressed but achitinase (PR-8) is highly expressed (Kessmann et al.1994). In Arabidopsis, induction of SAR by Fusariumoxysporum and turnip crinkle virus infections, as well astreatment with 2,6-dichloroisonicotinic acid and SA,correlated with the systemic induction of several SARgenes (Ukness et al. 1993).

Defense responses induced locally and systemicallyinclude a broad spectrum of proteins such as PR proteinsand products of resistance genes (Bent 1996). PR geneexpression is regulated in a spatio-temporal manner byvarious plant signals (Lee et al. 2000a; Orozco-Cardenaset al. 2001). Among these signals, SA, systemin, ethyleneand jasmonate have been documented to play a crucialrole in the establishment of SAR (Sticker et al. 1997). SA isan important signalling factor in the induction of plantdisease resistance (Ryals et al. 1996). In an increasingnumber of plant species, elevated levels of SA have beenassociated with resistance of plants to the invadingpathogen (Metraux et al. 1990;Ukness et al. 1993).A largebody of evidence suggests that this systemic increase in SAlevels is important for the induction of SAR (Ryals et al.1996). Ethylene has been suggested to act as a signal in-volved in SAR (Raz and Fluhr 1992). Van Loon (1977)demonstrated that in tobacco, pinpricking leaves withethephon induces the accumulation of PR proteins andenhances resistance to tobacco mosaic virus. This exper-iment has been interpreted to indicate that ethylene isinvolved in PR gene expression and the induction of SAR.

Ethylene is produced when cells undergo necrosisresulting from pathogen infection (Mauch et al. 1992).The application of ethylene was found to induce theaccumulation of defense-related proteins, such as phe-nylalanine ammonia-lyase (PAL) and vacuolar hydro-lases (Mauch et al. 1992). Jasmonate and its methyl esterare natural compounds in plants. They affect physio-logical processes and ethylene biosynthesis when appliedat low concentrations (Sembdner and Parthier 1993). Anadditional function of jasmonates has been attributed tothe induction of defense-related proteins in plants

(Rickauer et al. 1997). It has been postulated thatjasmonates might constitute lipid-derived messengers inthe signal transduction chain preceding the activation ofdefense gene expression (Farmer et al. 1998; Watanabeand Sakai 1998).

Plants are exposed to numerous environmentalstresses, including dehydration, high salt and low tem-perature. Water deficiency, which is one of the mostdetrimental environmental stresses in plants, leads tolarge physiological changes such as membrane modifi-cation, osmolyte biosynthesis and transcriptionalregulation of various genes (Shinozaki and Yamaguchi-Shinozaki 1997). A number of genes have been describedthat respond to drought and salinity stress in plants(Seki et al. 2001). Abscisic acid (ABA) modulates growthand development of plants, particularly during seedformation and during environmental stresses, includingloss of water (Shinozaki and Yamaguchi-Shinozaki1997). Treatment with ABA hardens plants against en-vironmental stresses and results in expression of ABA-regulated genes, so that their products might be involvedin recovery from the stress (Chandler and Robertson1994). Cold acclimation via changes in the expression ofsome genes has previously been suggested (Guy 1990).Cold-regulated genes that play a role in protecting cellsfrom low temperature have been isolated and charac-terized from different plants (Thomashow 1990).

Using a pepper cDNA library and differential displaytechniques, we isolated a cDNA clone encoding SAR8.2from pepper leaves infected by the avirulent strain Bv5-4a of X. campestris pv. vesicatoria. More recently, it hasbeen demonstrated that various PR protein genes in-duced by X. campestris pv. vesicatoria are also strikinglyinduced by other pathogens or various elicitors (Jungand Hwang 2000). To better understand pepper SAR8.2gene functions, we examined the expression of CA-SAR82A in response to pathogens, abiotic elicitors andenvironmental stresses. Based on the results of the pre-sent study, we propose that the SAR8.2 gene functionsas a molecular marker for pathogen infection, abioticelicitors and environmental stresses in pepper plants.

Materials and methods

Plant materials

Pepper (Capsicum annuum L. cv. Hanbyul) was used in this study.The seeds were sown in a plastic tray (55 cm · 35 cm · 15 cm)containing a steam-sterilized soil mix (peat moss, perlite, andvermiculite, 5:3:2, by vol.), sand and loam soil (1:1:1, by vol.). Sixpepper seedlings at the two-leaf stage were transplanted to a plasticpot (5 cm · 15 cm · 10 cm) containing the above-described soilmix. Pepper plants were raised in a growth room at 27±1�C withapprox. 80 lmol photons m–2 s–1 (white fluorescent lamps) for 16 ha day.

Pathogens and inoculation procedures

The two strains Ds1 and Bv5-4a of Xanthomonas campestris pv.vesicatoria used in this study were virulent and avirulent to the

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pepper cultivar Hanbyul, respectively (Lee and Hwang 1996).Pepper plants at the six-leaf stage were inoculated by vacuum-in-filtrating bacterial suspensions (108 cfu ml–1) into the abaxial sideof fully expanded leaves. To examine whether or not the CA-SAR82A gene is systemically induced in upper, non-inoculatedleaves, pepper plants at the four-leaf stage were inoculated with X.campestris pv. vesicatoria on the lower first leaves. The inoculated,first leaves and the non-inoculated upper, second leaves weresampled separately at 1 and 2 days after inoculation. The inocu-lated pepper plants were incubated in a growth room, as describedpreviously (Lee and Hwang 1996).

The two isolates S197 and CBS 178.26 of Phytophthora capsici,which were virulent and avirulent to pepper cultivar Hanbyul, re-spectively (Kim and Hwang 1992), were grown on oatmeal agarplates for 10 days. After sporulation under fluorescent lamps for48 h at 28�C, a zoospore suspension was prepared from the cul-tured plates, as previously described (Kim and Hwang 1992). Asmall quantity of cotton soaked in zoospore suspension (105

zoospores ml–1) was placed on the bottom region of each pepperstem. The inoculated pepper plants were incubated in a growthroom as described previously (Kim and Hwang 1992).

The isolate 2-25 of Colletotrichum coccodes was grown onoatmeal agar plates for 5–7 days at 28 �C under fluorescent lamps.A conidial suspension (105 conidia ml–1) was prepared for inocu-lation of the pepper leaves, as previously described by Hong andHwang (1998). Pepper plants at the four- and eight-leaf stages wereinoculated with the conidial suspension using a foliar spray meth-od. The inoculated pepper plants were incubated in a moistchamber for 36 h in the dark at 28 �C and then returned to thegrowth room.

Application of plant hormones, abiotic elicitorsand environmental stresses

Pepper plants at the four-leaf stage were enclosed in stoppered500-ml glass bottles and ethylene was injected to yield a final con-centration of 5 ll per liter. Pepper plants alsowere removed from soiland their roots were soaked in ABA (100 lM) andNaCl (10, 10, 100and 200 mM).Methyl jasmonate (0.1, 1, 10, 50, and 100 lM), indole-3-acetic acid (IAA; 100 lM), SA (0.01, 0.1, 1, 2, and 5 mM),benzothiadiazole CGA245704 (BTH; 0.01, 0.1, 1, 2, and 10 lM),DL-b-amino-n-butyric acid (BABA; 0.1, 1, 5, 10, and 20 mM) andhydrogen peroxide (100 lM) supplemented with 0.01% Tween 20were sprayed onto pepper plants at the six-leaf stage. Pepper plantsalso were treated by vacuum-infiltrating 100 lM IAA into the ab-axial side of fully expanded leaves. Pepper plants treated withmethyljasmonatewere incubated in a vinyl bag.Control plantswere sprayedwith water. For the low-temperature stress, the plants were trans-ferred to an incubator in which the temperature was set at 4 �C. Forthe drought-stress treatment, watering the plants was withheld untilthe plants were visibly wilted. The treated pepper plants were incu-bated in a growth room at 27±1 �C with approx. 80 lmol photonsm–2 s–1 (white fluorescent lamps) for 16 h a day.

Differential display

To search for the differentially expressed cDNAs, total RNA fromeither the healthy control or X. campestris pv. vesicatoria-infectedpepper leaf tissues was used for reverse transcription in a 20-llreaction volume. The cycling parameters for PCR reactions were:94 �C for 30 s, 40 �C for 2 min, 72 �C for 30 s for 40 cycles, fol-lowed by 72 �C for 5 min using arbitrary primer and poly-dTprimer provided by the manufacturer (GenHunter). The PCRproducts were visualized on 6% denaturing polyacrylamide gelsfollowing the methods of Liang and Pardee (1992) and Liang et al.(1993). RNA map kits from GenHunter Corporation (Brookline,Mass., USA) were used for differential display PCR following themanufacturer’s instructions. After developing the X-ray films,cDNA bands of interest, which code for mRNAs induced only inthe leaves infected by X. campestris pv. vesicatoria, were purified

from the polyacrylamide gels. One of these cDNAs, designatedC19U1, was cloned into the TA cloning system from Invitrogen(San Diego, Calif., USA) to use as a probe for screening forCASAR82 cDNAs from the pepper cDNA library.

Screening for CASAR82 cDNA

A cDNA library in kZAPII (Stratagene) was constructed frompoly(A+) RNA of pepper plants infected with the X. campestris pv.vesicatoria strain Bv5-4a (Kim and Hwang 2000). The peppercDNA library was screened for CASAR82 cDNA with a C19U1cDNA probe according to the manufacturer’s protocol. Two cyclesof plaque purification were carried out to obtain single independentplaques. After isolation of single plaques, the cDNA inserts wereexcised from pBluescript SK (–) phagemids containing cDNA in-serts according to the method of Sambrook et al. (1989).

DNA sequencing and analyses

The CASAR82A cDNA clones screened were sequenced on anABI 310 DNA sequencer (Applied Biosystems) using a Thermo-cycle sequenase kit (Amersham) with either T3 or T7 primer. Thesequence analysis was carried out using the PC/Gene softwaresystem and BLAST network services (Altschul et al. 1990).Searches in the GenBank databases were performed using theBLAST world-wide web Server at the National Center for Bio-technology Information. The amino acid alignments were manuallyadjusted to compare the cDNA clones of CASAR82A, CA-SAR82B, and CASAR82C with those of other organisms.

RNA preparation and gel-blot analyses

Total RNA was prepared from the tissues of pepper leaves, stems,roots, flowers, and fruits by the guanidine isothiocyanate method(Chomczynski and Sacchi 1987). The RNA was quantified spec-trophotometrically, normalized to equal concentrations, and veri-fied by gel electrophoresis. Equal quantities of RNA were separatedon 1.2% formaldehyde–agarose gels in the presence of ethidiumbromide and transferred to nylon membranes (Hybond N+;Amersham). Equal loading of samples was verified under UV il-lumination after the gel was run. The CASAR82A cDNA was la-beled with 32P-labeled dCTP using the random prime kit(Boehringer Mannheim). Prehybridization and hybridization wereperformed at 65 �C in 5% (w/v) dextran sulfate, 0.25 M disodiumphosphate (pH 7.2), 7% (w/v) sodium dodecyl sulfate (SDS), and1 mM EDTA. The membranes were washed twice with 2· SSC,0.1% SDS for 10 min each at room temperature and then threetimes with 0.1· SSC, 0.1% SDS for 5 min each at 65 �C. Thehybridized blots were exposed to X-ray film.

In situ RNA localization

In situ localization of the transcripts of the CASAR82A gene wasconducted, as described by Lee et al. (2000c). The samples(5·5 mm2) from pepper leaves and stems inoculated with C. coc-codes and P. capsici or treated with SA (5 mM) at the four-leafstage, respectively, were fixed in 1· PBS (phosphate-buffered saline:30 mM sodium phosphate and 130 mM sodium chloride), con-taining 4% paraformaldehyde and 1 ll ml–1 Triton X-100 byvacuum infiltration and shaking for 2 h. Fixed leaf samples werewashed with 1· PBS buffer, dehydrated in a graded ethanol andxylene series, and embedded in paraplast.

Tissue sections (10 lm in thickness) were cut from the diseasedor SA-treated sites and attached to poly-L-lysine-coated microscopeslides. After removal of the paraplast with xylene and then rehy-dration through an ethanol series, the sections were treated with1% bovine serum albumin (BSA) in 10 mM Tris–HCl (pH 8.0),proteinase K (5 mg ml–1) in 50 mM EDTA, 100 mM Tris–HCl

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(pH 8.0), for 30 min at 37 �C and 0.25% acetic anhydride in100 mM triethanolamine (pH 8.0), for 10 min at room tempera-ture. The EcoRI/XhoI insert carrying the CASAR82A cDNA wasdigoxigenin (DIG)-labeled with a DIG High prime kit (BoehringerMannheim). Sections were prehybridized and hybridized with theDIG-labelled probe CASAR82A in 50% formamide, containing4· SSC, 0.5% blocking reagent, 150 lg ml–1 tRNA for 18 h at42 �C. After hybridization, the sections were rinsed twice with 50%formamide and 4· SSC, twice with 4· SSC and then with DEPC-sterile water at 42 �C. The hybridization signals were detected withdiluted nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) at room temperature followingthe procedures described by the suppliers (Boehringer Mannheim).

To demonstrate the specificity of the hybridization, the level ofnon-specific hybridization of the CASAR82A probe was evaluatedby omitting the CASAR82A probe from the standard protocol. Thespecificity of the CASAR82A probe was also examined on unin-fected, healthy pepper leaf tissue.

Results

Isolation and sequence analysis of CASAR82 cDNAs

Total RNA isolated from X. campestris pv. vesicatoria-infected and non-infected pepper leaves was subjected tomRNA differential display analysis. The three cDNAclones of the putative pepper SAR8.2 gene, designatedCASAR82A, B, and C (Capsicum annuum SAR8.2 A, Band C), were isolated from the pepper cDNA libraryfrom HR lesions of leaves infected with an avirulentstrain, Bv5-4a, of X. campestris pv. vesicatoria. TheCASAR82A, B and C genes contain 580, 626 and 450 bpwith a putative open reading frame (ORF) of 86 aminoacids (data not shown). Sequence analyses of CA-SAR82A, B and C revealed that the ORF encoded aputative protein of 86 amino acids, with a eukaryoticsecretion signal sequence of 25 amino acids. The aminoacid sequences of the CASAR82A, B and C cDNAs weresimilar to those of the proteins encoded by tobaccoSAR8.2 genes (Altschul et al. 1990). A significant se-quence similarity was found in CASAR82A, B and C.The amino acid sequence homology search revealed thatthe CASAR82A, B and C gene products have 43–50%homology with the amino acid sequences of TOB-SAR82A, TOBSAR82B, TOBSAR82C, TOBSAR82Dand TOBSAR82E (Fig. 1). A characteristic feature ofthe putative pepper SAR8.2 gene is the presence of acysteine-rich domain (six cysteines) at the C-terminus.

Regulation of CASAR82A gene expression by pathogens

RNA gel blot analysis was performed with the CA-SAR82A cDNA probe to ascertain the existence ofCASAR82A mRNA in leaf, stem, root, flower and fruittissues of pepper plants. As a result, the CASAR82Atranscripts were found not to be expressed in any of theorgans of healthy pepper plants (data not shown).

Pepper leaves infected with the virulent strain Ds1 ofX. campestris pv. vesicatoria did not develop typicalsymptoms at 30 h after inoculation, but chlorotic and

necrotic areas had developed after 6 days. In contrast,pepper leaves infected with the avirulent strain Bv5-4ashowed very distinct HRs at 18 h after inoculation (Leeand Hwang 1996). Infection by either the virulent Ds1strain or avirulent Bv5-4a strain stimulated an increasein the abundance of CASAR82A mRNA (Fig. 2a). Inthe compatible interaction, CASAR82A mRNA wasstrongly induced and accumulated at 18–30 h after in-oculation. In the incompatible interaction, CASAR82AmRNA began to accumulate at 12 h after inoculation,followed by a maximum in transcript levels at 30 h.

At the two-leaf stage, lesions coalesced on pepperleaves at 4 days after inoculation with C. coccodes.However, lesions on pepper leaves inoculated at the eight-leaf stage were expressed as light-brown or gray flecks(Hong and Hwang 1998). At the four-leaf stage, thetranscripts began to accumulate in the infected leaves at12 h after inoculation, reaching a relatively high level at48 h and the transcript level declined at 72 h (Fig. 2b). Atthe eight-leaf stage, the accumulation of CASAR82Atranscripts was pronounced at 12 h after inoculation.The transcript abundance remained at high levelsuntil 36 h during the incompatible interaction. A slightdecrease in themRNA level occurred in the infected leavesthereafter.

In the compatible interactions with virulent isolateS197 of P. capsici, the plants had blighted foliage, leafdefoliation and rapidly growing stem lesions. In the in-compatible interactions with avirulent isolate CBS178.26,the plants had superficial brownish-purple speckles thatdeveloped slowly on the stem at 5 days after inoculation.

Fig. 1 Comparison of amino acid sequences of pepper (Capsicumannuum) CASAR82A, CASAR82B and CASAR82C cDNAs withtobacco (Nicotiana tabacum) SAR8.2 genes (TOBSAR82A, Gen-Bank accession no. g292610; TOBSAR82B, GenBank accession no.M97359; TOBSAR82C, GenBank accession no. M97360; TOB-SAR82D, GenBank accession no. M97361; TOBSAR82E, Gen-Bank accession no; M97362) (Ward et al. 1991). Black boxesSequence identity of marked amino acids. Dashes Gaps introducedfor alignment of homologous regions. Arrowhead Putative secretionsignal peptide. Asterisk (*) C-terminal cysteine-rich domain

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A remarkable expression of CASAR82A was found inpepper stem tissues at 1 day after inoculation in both thecompatible and incompatible interactions (Fig. 2c). In thecompatible interactions, the CASAR82A mRNA abun-dance remained at high levels until 3 days after inocula-tion, whereas the transcripts completely disappeared at4 days when the infected pepper plants were almost dead.In the incompatible interactions, however, levels of thetranscripts were extremely high in pepper stem tissues at4 days after inoculation.

We examined whether or not transcripts of CA-SAR82A were systemically expressed in pepper plantsimmunized at the four-leaf stage with virulent or avir-ulent strains of X. campestris pv. vesicatoria (Fig. 2d).CASAR82A transcripts were detected in systemic, up-per, second leaves in both compatible and incompatibleinteractions. In the incompatible interactions, CA-SAR82A transcripts were more strongly induced in thesystemic, upper, second leaves, compared to those of thecompatible interactions.

Regulation of CASAR82A gene expressionby abiotic elicitors

To assess the involvement of abiotic elicitors in CA-SAR82A gene induction, abiotic elicitors such as SA,BTH, and BABA were sprayed onto pepper plants(Fig. 3). No CASAR82A transcripts were detectable inthe healthy, control pepper plants. Several signal mole-cules, in addition to SA, have been shown to be involvedin the induction of the genes that participate in the plantdefense against pathogens (Enyedi et al. 1992). The threeabiotic elicitors SA, BTH and BABA strongly inducedthe expression of CASAR82A in pepper leaves at dif-ferent concentrations and during the 24 h after treat-ment (Fig. 3a–c). In general, treatment with BTH was

Fig. 3 Time courses of accumulation of CASAR82A mRNA inpepper leaves treated with the abiotic elicitors salicylic acid (a),benzothiadiazole (b), and DL-b-amino-n-butyric acid (c) at variousconcentrations. Total RNA (30 lg) from each sample was loadedin each lane. The EcoR1/Xho1 fragment of the putative pepperCASAR82A cDNA insert in pBluescript SK(–) was labeled andused as a probe. A duplicate gel was stained with ethidium bromideas a control for RNA loading. C Control

Fig. 2a–d Time courses of accumulation of CASAR82A mRNA inpepper tissues during the compatible and incompatible interactionof pepper with Xanthomonas campestris pv. vesicatoria, Colletotri-chum coccodes or Phytophthora capsici. a Northern blot analysis ofCASAR82A mRNA in pepper leaves at various time intervals afterinoculation with virulent strain Ds1 and avirulent strain Bv5-4a ofX. campestris pv. vesicatoria at the six-leaf stage. H Healthy leaf. bNorthern blot analysis of CASAR82A mRNA in pepper leavesinoculated with strain 2-25 of C. coccodes at the four-leaf and eight-leaf stages. H Healthy leaf. c Northern blot analysis of CASAR82AmRNA in pepper stems at various time intervals after inoculationwith virulent isolate S197 and avirulent isolate CBS178.26 of P.capsici. H Healthy stem, W wounded stem. d Northern blotanalysis of CASAR82A mRNA in local (L) (first) and systemic (S)(second) leaves at 1 and 2 days after inoculation of leaves withvirulent strain Ds1 and avirulent strain Bv5-4a of X. campestris pv.vesicatoria at the four-leaf stage. Total RNA (30 lg) from thesamples at various time intervals after inoculation was loaded ineach lane. The EcoR1/Xho1 fragment of the putative pepperCASAR82A cDNA insert in pBluescript SK(–) was labeled andused as a probe. A duplicate gel was stained with ethidium bromideas a control for RNA loading

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more effective than that with SA or BABA in activatingthe CASAR82A gene in pepper leaf tissues.

Regulation of CASAR82A gene expression by planthormones and hydrogen peroxide

Expression of CASAR82A was examined in pepper leaftissues at 18 h after treatment with ethylene and methyljasmonate at various concentrations. Ethylene andmethyl jasmonate induced the accumulation of CA-SAR82A mRNA in a dose- and time-dependent manner(Fig. 4a, b). In particular, methyl jasmonate stronglyactivated the CASAR82A gene even at lower concen-trations and at an earlier time after treatment. TheCASAR82A mRNA began to be induced in pepperleaves 1 h after treatment with IAA, followed by a highlevel at 24 h (Fig. 4c). As shown in Fig. 4d, CASAR82Awas strongly induced in leaf and stem tissues by treat-ment with ABA. The CASAR82A mRNA started toaccumulate at 1 h after treatment and remained at a

constant levels until 24 h after treatment. To assess theinvolvement of active oxygen species in CASAR82A geneinduction, hydrogen peroxide was applied to pepperplants (Fig. 5). TheCASAR82AmRNAwas induced andaccumulated at considerable levels in pepper leaves at18–24 h after treatment with hydrogen peroxide.

Regulation of CASAR82A gene expression by highsalinity, drought and low temperature

To examine the effects of high salinity, drought and coldstresses on the CASAR82A gene induction, pepperplants were subjected to NaCl, dehydration and low-temperature treatments (Fig. 6). Northern blot analysisrevealed that CASAR82A was not expressed in all themock-treated pepper tissues. However, treatment withNaCl, dehydration and low temperature strongly in-duced the transcription of CASAR82A in leaf and stemtissues of pepper. In particular, high-salinity stress in-duced by NaCl treatment was effective in activatingCASAR82A in pepper plants (Fig. 6a). The transcriptsof CASAR82A gradually increased in the leaf tissue, asthe NaCl concentration became higher. In all the treatedstem tissues, the CASAR82A transcripts remained athigh levels, irrespective of the NaCl concentration andtimes. As shown in Fig. 6b, the expression of CA-SAR82A was distinctly induced in both leaf and stemtissues at 2–6 h after dehydration, but gradually de-clined thereafter. The effect of drought on the CA-SAR82A gene remained stronger in the stem tissue thanin the leaf tissue. As shown in Fig. 6c, cold stress causedstrong induction of the CASAR82A transcripts in leafand stem tissues, and the transcript abundance remainedconstant throughout the exposure of pepper plants tolow temperatures.

In situ localization of CASAR82A mRNAin leaf and stem tissues infectedby pathogens or treated with SA

To examine the subcellular localization of expression ofCASAR82A in response to pathogen infection and SAtreatment, in situ hybridization was performed using the

Fig. 4 Time courses of accumulation of CASAR82A mRNA inpepper leaves treated with the plant hormones ethylene (a), methyljasmonate (b), indole-3-acetic acid (c), and abscisic acid (d). TotalRNA (30 lg) from each sample was loaded in each lane. TheEcoR1/Xho1 fragment of the putative pepper CASAR82A cDNAinsert in pBluescript SK(–) was labeled and used as a probe. Aduplicate gel was stained with ethidium bromide as a control forRNA loading. C Control

Fig. 5 Expression of CASAR82A mRNA in pepper leaves atdifferent time intervals after treatment with 10 mM hydrogenperoxide. Total RNA (30 lg) from each sample was loaded in eachlane. The EcoR1/Xho1 fragment of the putative pepper CA-SAR82A cDNA insert in pBluescript SK(–) was labeled and used asa probe. A duplicate gel was stained with ethidium bromide as acontrol for RNA loading. C Control

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pepper leaf and stem tissues (Fig. 7). In situ hybridiza-tion of sections with a DIG-labeled CASAR82A cDNAprobe resulted in no labeling of uninfected, healthypepper leaf midrib and stem tissues (Fig. 7a, d). At 24 hafter inoculation with C. coccodes, the CASAR82AmRNA was intensely localized in the epidermis, endo-dermis and phloem cells of leaf midrib tissue, but xylemvessels were almost free of labeling (Fig. 7b). The la-beling intensity at the subcellular sites of CASAR82Aexpression in the infected leaf tissues was similar to thatobserved for corresponding tissues treated with SA(Fig. 7c). In the early stage of Phytophthora develop-ment at 24 h after inoculation with P. capsici, a low levelof CASAR82A transcripts was found, confined tophloem cells of stem tissue, but xylem cells remainedunlabeled (Fig. 7e). However, treatment with SAstrongly induced the accumulation of CASAR82Atranscripts around the phloem area of vascular bundlesas well as the epidermal cells of stem tissues (Fig. 7f).

Discussion

The identification of PR genes induced in plants uponpathogen infection is an important strategy for under-standing defense mechanisms in plants. We have isolatedsome PR genes from pepper plants using the differential

display strategy (David et al. 1998; Lee et al. 2000a). Somepepper SAR8.2 genes, designated CASAR82A, B and C,were isolated from a pepper cDNA library constructedwith the mRNAs from the pepper plants infected byX. campestris pv. vesicatoria. The putative pepper CA-SAR82A, B and C were found to be homologous to theSAR8.2 genes isolated previously from tobacco (Wardet al. 1991). The putative proteins encoded byCASAR82A, B and C cDNAs showed all the character-istic features of SAR8.2, including 86 amino acids, whichshare significant homology with the five tobacco SAR8.2genes: TOBSAR82A, TOBSAR82B, TOBSAR82C,TOBSAR82D, and TOBSAR82E (Ward et al. 1991). Theprimary structure of the entire protein encoded byCASAR82A, including an N-terminal signal peptidesequence and an SAR8.2 sequence, resembles thosereported for tobacco SAR8.2 precursors. The SAR8.2homologous region in the CASAR82A polypeptide ispreceded by a stretch of 25 amino acids characteristic of atransmembrane leader sequence (von Heijne 1985). ThisN-terminal domain is highly hydrophobic and its aminoacid constituents closely resemble those predicted forsignal peptides (von Heijne 1985, 1986).

Participation of the SAR8.2 gene in the pathogenesisand defense programs was demonstrated earlier in to-bacco tissues infected by pathogens (Ward et al. 1991).Our results of northern blot analysis revealed that theCASAR82A transcripts accumulated rapidly andstrongly in the incompatible interactions of pepperleaves with X. campestris pv. vesicatoria, which suggeststhat the CASAR82A gene may be involved in the ex-pression of the defense response to bacterial infection inpepper leaves. However, the CASAR82A gene seemedalso to be required for disease development by thebacterial pathogen. As shown in Fig. 2b, CASAR82Awas differentially expressed in pepper leaves infected byC. coccodes, as pepper plants aged: CASAR82A mRNAsaccumulated at lower levels when plants were infected atthe four-leaf stage than at the eight-leaf stage. This maybe because the symptoms on pepper leaves infected by C.coccodes were different between the four- and eight-leafstages. Pepper plants at the four-leaf stage were sus-ceptible, whereas pepper plants at the eight-leaf stagebecame increasingly resistant. Accordingly, the strongexpression of CASAR82A in pepper leaves at latergrowth stages may be very important in triggering theresistance response of pepper to the anthracnose. Theaccumulation of CASAR82A transcripts also occurredin pepper stems in response to infection by P. capsici. Inthe stem tissues infected by P. capsici, the transcriptswere strongly induced in the compatible and incompat-ible interactions. The expression patterns of the CA-SAR82A gene were quite similar in the compatible andincompatible interactions. The systemic induction ofCASAR82A was found in upper leaves after inoculationwith X. campestris pv. vesicatoria on the lower leaves. Aprevious report suggests that expression of the SARgenes coincides with the maintenance of SAR in tobacco(Ward et al. 1991). The induction of CASAR82A in

Fig. 6 Time courses of accumulation of CASAR82A mRNA inleaves and stems of pepper plants after treatment with sodiumchloride (a), drought (b), and low temperature (c). Total RNA(30 lg) from each sample was loaded in each lane. The EcoR1/Xho1 fragment of the putative pepper CASAR82A cDNA insert inpBluescript SK(–) was labeled and used as a probe. A duplicate gelwas stained with ethidium bromide as a control for RNA loading.C Control

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systemic, upper leaves indicates that this gene serves as amolecular marker for the onset of systemically inducedresistance in pepper plants.

To determine whether or not accumulation of CA-SAR82A mRNA was induced by abiotic elicitors andplant hormones, we treated pepper leaves with SA,BABA, BTH, ethylene, methyl jasmonate, IAA andABA. Interestingly, expression of the CASAR82AmRNA could be induced in pepper leaves by treatmentwith a variety of abiotic elicitors and plant hormones(Figs. 3, 4). These Northern blot data revealed that allbiotic and abiotic stresses tested, except mechanicalwounding, triggered the transcription of CASAR82A inpepper tissues, which suggest that CASAR82A mayfunction as an indicator in pepper plants exposed toexternal stresses.

Ethylene application led to the expression of pepperSAR genes in pepper plants (Jung and Hwang 2000).This is consistent with the findings of Terras et al. (1998)that ethylene exposure induced resistance to Alternariabrassicicola in radishes. In our earlier studies, it wasdemonstrated that infection by the avirulent X. cam-pestris pv. vesicatoria triggered not only the accumula-tion of PR-1 mRNA, but also, concomitantly, thebiosynthesis of ethylene in pepper leaves (Kim and

Hwang 2000). In pepper–pathogen interactions, there-fore, it seems likely that the rate of ethylene biosynthesisinduced by pathogen infection is directly proportional tothat of PR protein accumulation. CASAR82Atranscripts were rapidly accumulated in pepper leavestreated with ethylene. The PR proteins induced by eth-ylene were induced in plants by methyl jasmonate as well(Buchter et al. 1997). In our present study, CASAR82Atranscripts were also rapidly accumulated in pepperleaves by treatment with methyl jasmonate. These resultssuggest that ethylene and methyl jasmonate may besignal molecules capable of activating the SAR8.2 genein pepper leaves. Application of methyl jasmonate in-duced the accumulation of CASAR82A mRNA at alltreatment concentrations. These observations aresupported by the recent suggestions of Steiner andSchonbeck (1997) that abiotic elicitors induce PRproteins in plants, irrespective of concentration.

As shown in Figs. 4 and 6, CASAR82A transcriptsaccumulated in the leaf and stem tissues followingtreatment with ABA, high salt, drought and low tem-perature. A number of plant genes have been found torespond to drought, salt and cold stresses at the tran-scriptional level (Shinozaki and Yamaguchi-Shinozaki1996; Bray 1997). The functions of some gene productshave been demonstrated to play a significant role inprotecting cells from water deficiency (Bray 1997). Oneof the major signals operating during drought stress isknown to be the plant hormone ABA, which is involvedin many other abiotic stresses (Bray 1997). Most of thegenes that respond to drought, salt and cold stresses arealso induced by treatment with ABA (Shinozaki andYamaguchi-Shinozaki 1996), indicating that the CA-SAR82A gene responds to these environmental stresses.In the case of drought treatment (Fig. 6), CASAR82Atranscripts strongly accumulated in the early stages of

Fig. 7a–f In situ localization of CASAR82A mRNAs in pepperleaf midrib and stem tissues infected by Colletotrichum coccodesand infected by Phytophthora capsici, respectively, or treated withSA. a Non-inoculated leaf midrib tissues. b Leaf midrib tissue at24 h after inoculation with C. coccodes. c Leaf midrib tissue at 18 hafter treatment with SA. d Non-inoculated stem tissues. e Stemtissue at 24 h after inoculation with virulent isolate S197 ofP. capsici. f Stem tissue at 18 h after treatment with SA. Thinsections were hybridized with DIG-labelled pepper CASAR82AcDNA. C Cortex, Co collenchyma cell, E epidermal cell, PMpalisade mesophyll, SM spongy mesophyll, V vascular bundle.Scale bar = 100 lm

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drought, but later decreased very rapidly in the desic-cated leaves. The disappearance of transcripts in leaftissues at 18 h after drought may result from a severedeterioration of the treated tissues.

In situ localization of CASAR82A transcripts inpepper leaf tissues infected with C. coccodes andP. capsici or treated with SA revealed that the CA-SAR82A transcripts accumulated in phloem and epi-dermal cells (Fig. 7), indicating that the SAR8.2 geneproducts have specific functions in the infected host cellsassociated with SA, which is known to play a key role inboth SAR signaling and disease resistance (Ryals et al.1996). The specific accumulation of chitinase, b-1,3-glucanase and thionin have been demonstrated inphloem and epidermal cells of plants (Wubben et al.1996; Lee et al. 2000b). Because phloem is required forthe transport of nutrients, pathogens may exert allpossible efforts to possess the phloem. Therefore,SAR8.2 protein may act as a preformed defense barrierto the vulnerable phloem. An alternative or additionalrole of high, constitutive CASAR82A levels in phloemcells could be the generation of signal molecules inresponse to invading pathogens, thereby activating otherdefense responses in the surrounding tissues.

In summary, the CASAR82A transcripts were inducedin pepper plants by all the tested biotic and abioticstresses, except mechanical wounding. These results sug-gest that theCASAR82A functions as amolecular markergene for various biotic and abiotic stresses. Accordingly,the CASAR82A gene product could be used as a molec-ular marker to perceive pathogen infection, abioticelicitors and environmental stresses in pepper plants.

Acknowledgement This research was supported by a grant(CG1224) from the Crop Functional Genomics Center of the 21stCentury, Frontier Research Program funded by the Ministry ofScience and Technology of Republic of Korea. The authors thankDr. Daniel D. Holte for critically reading the manuscript.

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