HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv . glycines . We have determined the critical amino acid residues important for hypersensitive response [HR] elicitation by random and site-directed mutagenesis of HpaG and its homologXopA . A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR.A total of 52 mutants were obtained, including 51 single missensemutants and 1 double missense mutant . The HR elicitation activitywas abolished in the two missense mutants [HpaG[L50P] and HpaG[L43P/L50P]].Seven single missense mutants showed reduced activity, and theHR elicitation activity of the rest of the mutants was similarto that of wild-type HpaG . Mutational and deletion analysesnarrowed the region essential for elicitor activity to the 23-amino-acidpeptide [H₂N-NQGISEKQLDQLLTQLIMALLQQ-COOH] . A synthetic peptideof this sequence possessed HR elicitor activity at the same concentration as the HpaG protein . This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp . The secondary structure of the peptide is predicted to be an -helix, as is the HrpW region that is homologous to HpaG . The predicted -helix of HpaG is probably critical for the elicitation of theHR in tobacco plants . In addition, mutagenesis of a xopA geneyielded two gain-of-function mutants: XopA[F48L] and XopA[F48L/M52L].These results indicate that the 12 amino acid residues betweenL39 and L50 of HpaG have critical roles in HR elicitation intobacco plants.

 
In many interactions between gram-negative plant-pathogenicbacteria and plants, hrp [for hypersensitive reaction and pathogenicity] genes are required for pathogenicity in the host plant and induction of the hypersensitive response [HR] in nonhost plants [17]. Regions that contain a cluster of hrp genes and other components required for pathogenicity are designated pathogenicity islands [PAIs] [3, 15] . Most hrp genes that encode components of thetype III protein secretion system mediate the translocationof effector proteins, such as Avr [avirulence] proteins, acrossthe bacterial membrane and the walls and plasma membranes ofplant cells [10].

HR is a highly localized plant cell death that occurs when nonhost plants or resistant cultivars of host plants are infiltratedwith the plant pathogen or HR elicitor molecules, such as Avrproteins and harpins . HR is thought be a resistance reactionof plants to microbial pathogens [11] . Harpins are a group of HR elicitors that are secreted by the type III secretion pathwayand elicit HR when infiltrated into the apoplast of leaves ofnonhost plants . Unlike Avr proteins, which must be deliveredinside the cell to exert their functions, harpins can elicitHR when delivered to the intercellular space of plant cells[10] . Since the first known harpin, HrpN, was identified fromErwinia amylovora, many harpins have been reported from Pseudomonas,Ralstonia, and Xanthomonas species [4, 8, 12, 14, 15, 27] . Harpinsare glycine-rich, heat stable, and lack cysteine, but the biochemicalmechanisms of HR elicitation in nonhost plants are unclear.One reason for this is that the amino acid sequences of harpinsdo not share significant homology with other known proteinsor among themselves.

The mode of action of harpins is still controversial . HrpZ of Pseudomonas syringae pv . syringae associates with the walls rather than the membranes of plant cells, and the protein elicitsno response from protoplasts, which lack walls [13] . However, HrpZ of P . syringae pv . phaseolicola binds to lipid bilayers and forms an ion-conducting pore [16] . The N-terminal 109 aminoacids and the C-terminal 216 amino acids of HrpZ are able toelicit HR to a level similar to full-length HrpZ [2] . Kim etal . and Charkowski et al . showed that the HrpW harpins of E.amylovora and P . syringae pv . tomato are composed of two domains—theN-terminal harpin domain and C-terminal Pel [pectate lyase]domain—and proposed that HrpW acts in the cell wall [8, 14].

We previously published the first report of a harpin from Xanthomonas species, HpaG [15] . At 13.4 kDa, HpaG is smaller than otherknown harpins [15] . Four additional Xanthomonas HpaG homologshave been reported . HpaG shows a true harpin-like activity,and Hpa1 of X . oryzae pv . oryzae possesses HR elicitor activityat relatively high concentrations [i.e., >5 µM] . However,XopA of X . campestris pv . vesicatoria does not induce HR [15].To understand the nature of the HR induction by HpaG homologsin nonhost plants, we performed detailed mutational analysisof hpaG, identifying 23 amino acid residues that are essentialand sufficient for the elicitor activity of HpaG . Using site-directedmutagenesis, we determined the amino acid residues that havethe most influence on the elicitor activity . Finally, we obtaineda gain-of-function mutant of XopA.

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present studyare listed in Table 1 . Escherichia coli cells were cultivated at 37°C in Luria broth [LB; USB] or on LB agar plates . X. campestris pv . vesicatoria strains were grown at 28°C inLB or on YDC [1% yeast extract, 2% calcium carbonate, 2% D-glucose] agar plates . Antibiotics were used in E . coli cultures at 100 µg/ml for ampicillin and 34 µg/ml for chloramphenicol.

 
DNA manipulations. Standard methods were used for DNA cloning, restriction mapping,and gel electrophoresis [23] . The vector DNA was treated withappropriate restriction enzymes, and extraction of DNA fragmentsfrom gels was carried out by using the QIAEX II gel extractionkit as described by the manufacturer [Qiagen, Valencia, Calif.].All other standard molecular biological methods were carriedout as described by Sambrook et al . [23] . The oligonucleotidesused for the mutagenesis of hpaG and xopA are listed in Tables2 and 3 . All oligonucleotides were designed by using the PrimerSelectprogram [DNASTAR] to minimize secondary structure and dimerformation and were chemically synthesized by CoreBioSystem [Seoul,Korea].

 
Random mutagenesis. Random mutagenesis of hpaG was performed by using the error-pronePCR [25] and hydroxylamine mutagenesis [18] methods, with modifications. For the error-prone PCR, pTJ1 was used as a template, and the primers hpaGfrw [5'-GCGGCCATATGAATTCTTTGA-3'] and hpaGrev [5'-GGATCCTTACTGCATCGATC-3']were used . The PCR products were cleaned, digested with NdeIand BamHI, separated by agarose-gel electrophoresis, purifiedfrom the gel, and fused between the NdeI and BamHI sites ofthe plasmid pET14b [Novagen, Madison, Wis.] . For the hydroxylaminemutagenesis, 10 µg of pJ14 DNA were incubated in a reactionmixture containing 0.5 M hydroxylamine and 5 mM EDTA in 0.1 M potassium phosphate [pH 6.0] at 50°C for 4, 8, or 12 hor at 37°C for 12 or 24 h . After the treatment, the plasmidswere diluted in TE buffer [10 mM Tris-HCl [pH 8.0], 1 mM EDTA]and dialyzed overnight to remove the hydroxylamine . The dialyzedplasmid DNA was precipitated with ethanol and transformed intoE . coli BL21[DE3].

Deletion mutagenesis. Deletion derivatives of HpaG—HpaGN67, HpaGC66, HpaGC75, HpaGC77, HpaGC79, HpaGC81, and HpaGC83—were constructedby PCR with pTJ1 as a template . The upstream T7 promoter-specificprimer was complementary to the template DNA upstream of thehpaG insert and included a unique NdeI site . The downstreamprimers were complementary to an internal region of hpaG andincluded a translational stop codon and a unique BamHI site.The downstream primers designed for each deletion mutant are listed in Table 2 . Each 50-µl PCR contained 10 mM Tris-HCl[pH 8.3], 1.5 mM MgCl₂, 50 mM KCl, 20 ng of pTJ1 DNA, 200 µMconcentrations of deoxynucleoside triphosphates, 2 µM concentrations of primers, and 1.2 U of Taq polymerase [TaKaRa Shuzo Co . Shiga, Japan] . The reaction mixtures were heated for 2 min at 94°C and then amplified over 10 cycles of 1 minat 94°C, 1 min at 45°C, and 1 min at 72°C, followedby 20 cycles of 1 min at 94°C, 1 min at 50°C, and 1min at 72°C . The PCR products were purified with phenoland chloroform extraction and the DNA was precipitated withethanol . After digestion with NdeI and BamHI, the PCR productswere fused between the corresponding sites in the pET14b vector.

Site-directed mutagenesis with megaprimer PCR. Site-directed mutagenesis of hpaG was performed by using thePCR-mediated megaprimer method [5] . In the first PCR amplification, the template DNA, pHpaGMT, was constructed as follows . The 0.4-kb NdeI-BamHI fragment from pTJ1 was fused between the corresponding sites in pBSN, generating pBHPAG . To delete the T7 promoter sequence region, pBHPAG was digested with SmaI and BssHII, blunt ended with the Klenow fragment [TaKaRa Shuzo Co.], and religated, generating pHpaGMT . In the first PCR, pHpaGMT was used as the template DNA, and the M13-20 primer and individual mutagenicprimers were used [Table 3] . The PCR conditions used were the same as in the deletion mutagenesis method described above.The PCR products were isolated from 1.2% agarose gels, purifiedby using the QIAEX II gel extraction kit, and resuspended indistilled water for use in subsequent PCRs as megaprimers . Inthe second PCR amplification, pTJ1 DNA was used as the template;the T7 promoter primer and the megaprimer of the gel elutionproduct of first PCR were used as primers . The reaction conditionsused were the same as in the first PCRs . The second PCR productswere purified with phenol and chloroform extraction, followedby precipitation with ethanol . After digestion with NdeI andBamHI, the digested DNA was fused between the correspondingsites in pET14b . Site-directed mutagenesis of xopA was performedas described above . Mutants with single amino acid substitutionsare denoted as the one-letter notation of the original aminoacid and its position in the HpaG amino acid sequence, followedby the substituted amino acid.

DNA sequencing and data analysis. Mutagenized hpaG and xopA DNA fragments in pET14b were sequencedto confirm the presence of the appropriate mutation . For DNAsequencing, plasmid DNAs containing the hpaG and xopA mutantclones were purified by using the QIAprep Spin Miniprep Kit[Qiagen] . The T7 promoter primer and the T7 terminator primer [5'-CTAGTTATTGCTCAGCGGT-3'] were used in sequencing reactions.The reactions were carried out by using the ABI Prism BigDyeterminator cycle sequencing kit [version 2.0; Perkin-Elmer Corp.,Norwalk, Conn.] on an ABI 3700 DNA Analyzer [Applied Biosystems,Foster City, Calif.] at the National Instrumentation Centerfor Environmental Management, Seoul, Korea . DNA sequence datawere analyzed by using the SEQMAN and MEGALIGN software [DNASTAR]and GENETYX-WIN software [Software Development, Tokyo, Japan].

Overexpression and purification of HpaG and HpaG mutant proteins. The site-directed and deletion mutant clones were introducedinto the E . coli strain BL21[DE3][pLysS] for protein overexpression. Strains harboring each mutant clone were grown overnight inLB containing ampicillin and chloramphenicol, and the overnightcultures were diluted 100-fold in LB and grown at 37°C withagitation . At an optical density of 0.8 at 600 nm, IPTG [isopropyl-ß-D-thiogalactopyranoside] was added to a final concentration of 1 mM, the cultures were grown at 37°C for 2 h with agitation, and the cells werethen harvested by centrifugation . The cells were concentrated100-fold by resuspending the pellet in 20 mM Tris-HCl [pH 8.0],sonicated, and boiled for 10 min . After centrifugation, theprotein in the partially purified samples was quantitated byusing the Bradford method, with bovine serum albumin as thestandard [7], and the protein samples were used for the primaryanalysis of HR elicitor activity on tobacco leaves . The proteinswere also visualized by sodium dodecyl sulfate [SDS]-polyacrylamidegel electrophoresis, followed by staining with Coomassie brilliantblue R.

N-terminal His-tagged proteins were used when more highly purified proteins were required . E . coli BL21[DE3][pLysS] cells carrying the hpaG or xopA mutants fused into the pET14b vector were grownin LB broth, and the His-tagged proteins were expressed after the addition of IPTG . Cells were harvested and lysed by sonication in 0.5 ml of lysis buffer [10 mM imidazole, 20 mM Tris-HCl [pH 8.0]] . After centrifugation at 10,000 x g for 20 min at roomtemperature to pellet the cellular debris, the supernatant wasloaded onto a Ni-NTA spin column [Qiagen], binding His-taggedprotein . The Ni-NTA matrix was centrifuged at 1,000 x g for2 min at room temperature, and the matrix was then washed twotimes with washing buffer [20 mM imidazole, 20 mM Tris-HCl [pH8.0]] to remove unbound protein . His-tagged protein was elutedby stepwise addition of 0.1 ml of elution buffer 1 [0.5 M imidazole,20 mM Tris-HCl [pH 8.0]] and 0.1 ml of elution buffer 2 [1 Mimidazole, 20 mM Tris-HCl [pH 8.0]] . The eluted protein wasdialyzed with 20 mM Tris-HCl [pH 8.0] to remove the imidazole,and the concentration of the purified protein was measured bythe Bradford method with bovine serum albumin as the standard[7].

Plant assays. For HR tests, tobacco [Nicotiana tabacum cv . Samsun NN] plantswere inoculated with HpaG, HpaG derivatives, XopA, or XopA derivativesin 20 mM Tris-HCl [pH 8.0], and the responses of the plantswere observed for 12 to 24 h after inoculation.

Immunodetection of HpaG mutants. Purified HpaG mutant proteins were separated by SDS-polyacrylamidegel electrophoresis [on a 15% acrylamide gel] and then transferredto Hybond-P membrane [Amersham Pharmacia Biotech, Buckinghamshire,United Kingdom] by electroblotting at 25 V for 60 min in transferbuffer [48 mM Tris, 39 mM glycine, 0.037% [wt/vol] SDS, 20%[vol/vol] methanol [pH 8.3]] . For immunoblot detection, a rabbitpolyclonal anti-HpaG antibody was used as the primary antibodyand alkaline-phosphatase-conjugated goat anti-rabbit immunoglobulinG [Pierce Biotechnology, Rockford, Ill.] was used as the secondaryantibody . Positive signals were detected by using One-Step NBT/BCIPsolutions [Pierce].

Peptide synthesis. The HpaG peptide [H₂N-NQGISEKQLDQLLTQLIMALLQQ-COOH] and theHpaG[L50P] peptide [H₂N-NQGISEKQLDQLLTQLIMAPLQQ-COOH] were synthesizedby A&PEP [Chungnam, Korea].

Protein secondary structure prediction. The secondary structures of HpaG, HpaG derivatives, XopA, andXopA derivatives were predicted by using the protein structureprediction server HNN Secondary Structure Prediction Methodat Network Protein Sequence @nalysis [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html] [9].

 
Deletion analysis of hpaG. To determine the regions in HpaG that are critical for the inductionof HR in nonhost plants, we constructed seven truncated HpaGderivatives and tested their ability to induce HR in tobaccoplants . HpaGC66, HpaGC75, HpaGC77, HpaGC79, and HpaGC81 hadelicitor activity equivalent to that of the wild-type HpaG, whereas HpaGN67 and HpaGC83 failed to induce HR even at concentrationsgreater than 10 µM [Fig. 1] . These results indicate thatthe N-terminal 52 amino acids of HpaG are sufficient for elicitoractivity and that the C-terminal 75 amino acid residues arenot essential for elicitor activity.

 
Random and site-directed mutagenesis of the hpaG gene. To determine the amino acid residues of HpaG that have criticalroles in HR elicitor activity, we used site-directed mutagenesisto generate 46 HpaG derivatives with single amino acid substitutionsand one mutant, HpaG[L43P/L50P], in which two amino acids werealtered [Fig . 2] . In addition, three mutants, HpaG[I120T], HpaG[L121P],and HpaG[A126V], were constructed by using error-prone PCR methods,and two mutants, HpaG[A126T] and HpaG[S131L], were generatedby using hydroxylamine mutagenesis [Fig . 2] . Among the 52 missensemutants, the HR elicitor activity of 43 mutants was the sameas that of wild-type HpaG, but 7 mutants produced less HR activitythan the wild-type [data not shown and Fig . 2] . Two mutant proteins,HpaG[Q45A] and HpaG[L50A], elicited HR on tobacco leaves at1 µM but failed to induce HR at 0.5 µM [Fig. 3].HpaG[L39A], HpaG[L39P], and HpaG[L46A] elicited HR on tobaccoleaves at concentrations greater than 2.5 µM, and HpaG[L42D]and HpaG[L43P] elicited HR at concentrations greater than 5µM [Fig . 3].

 
To confirm that the reduced elicitor activity of the mutantproteins was not due to protein instability, all of the expressedproteins were detected by using immunoblots . As shown in Fig.4, each HpaG derivative was successfully expressed, exhibitedwild-type heat stability, and cross-reacted with polyclonalanti-HpaG antibodies . This indicated that the loss of activityof the mutants was not due to instability of the proteins.

 
The predicted N-terminal -helix is important for elicitor activity. The amino acid substitutions that affected the HR elicitor activityof HpaG were clustered in the region from L39 to L50 . We thereforeinvestigated the relationship between elicitor activity andpredicted secondary structures in HpaG . Computer-generated predictionsof the HpaG secondary structure showed that the protein hastwo possible -helices and two possible ß-sheet regions[Fig . 2] . The first predicted -helix is formed by the 19 aminoacid residues between S35 and Q53, and the second is formedby the 17 amino acid residues between Q88 and Q104 [Fig . 2].The first predicted ß-sheet is in the five amino acidresidues between S13 and V17, and the second is in the sevenamino acid residues between G119 and L125 [Fig. 2] . Since theterminal 81 amino acid residues of HpaG are not required forelicitor activity, the second predicted -helix and ß-sheetin the C terminus were not taken into consideration for theelicitor activity . The HpaG mutants with mutations in the first putative -helix, including HpaG[L39A], HpaG[L39P], HpaG[L42D],and HpaG[L43P], were affected in the ability to induce HR, asshown in Fig . 5 . However, no null mutant that lacked elicitoractivity was obtained . The mutants described above are predictedto have altered secondary structures, going from the putative -helix to the random coil form in this region . Therefore, changingthis putative -helix into the random coiled form might abolishthe elicitor activity . The HNN secondary structure predictionmethod [available at http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html] was used to predict mutants in which the putative -helix wouldbe affected, revealing two potential mutants, HpaG[L50P] and HpaG[L43P/L50P] . These two mutants were constructed by site-directed mutagenesis . HpaG[L50P] was predicted to have changes in the lower edge of the putative -helix, and the normally most -helical region in the double mutant HpaG[L43P/L50P] was predicted to assume a random coil form [Fig . 5] . Infiltration of HpaG[L50P] and HpaG[L43P/L50P] into tobacco leaves at concentrations of10 µM resulted in no elicitor activity [Fig . 3 and 5].HpaG[F14D], in which the region of the first predicted ß-sheetwas predicted to assume a random coil form, had HR elicitationactivity equivalent to that of wild-type HpaG [Fig. 5] . Thisresult indicates that the 12 amino acid residues between L39and L50 have important roles in HR elicitation in tobacco plantsand that the leucine residue at position 50 is the most criticalfor the elicitor activity.

 
A synthetic peptide comprising 23 amino acid residues of HpaG is sufficient to elicit HR. Based on the analysis of the HpaG mutants produced by deletionand site-directed mutagenesis, we proposed that the putativeN-terminal -helical region, composed of the 19 amino acid residuesfrom S35 to Q53, has an important role in the HR elicitationby HpaG in tobacco plants . We therefore synthesized the HpaGpeptide, composed of the 23 amino acid residues H₂N-NQGISEKQLDQLLTQLIMALLQQ-COOH,and the HpaG[L50P] peptide, composed of 23 amino acid residuesH₂N-NQGISEKQLDQLLTQLIMAPLQQ-COOH, in which the L50 was changedto a proline residue . The peptides were dissolved in 20 mM Tris-HCl[pH 8.0], diluted to various concentrations, and then infiltratedinto tobacco leaves . The HpaG peptide at 0.5 µM elicitedHR on tobacco leaves, but the HpaG[L50P] peptide failed to elicitHR, even at 10 µM [Fig . 3] . These results indicate thatthe HpaG peptide has elicitor activity equivalent to that ofthe wild-type HpaG and that L50 has a critical role in the elicitoractivity of HpaG.

The putative N-terminal -helical region is common in harpins. To determine whether the putative -helical region in the N-terminalregion of HpaG is present in other harpins, we compared theHpaG peptide sequence with the sequences of other harpins . Theamino acid sequence of the HpaG peptide does not have homologywith the sequences of HrpN, HrpZ, or PopA . However, the N-terminalharpin domain of the HrpW proteins from P . syringae pv . tomatoand E . amylovora has some amino acid residues in common withthe HpaG peptide region [Fig . 6A] . However, the HpaG peptideshows no homology with the HrpW proteins of X . axonopodis pv.citri or X . campestris pv . campestris [data not shown] . TheHpaG peptide sequence has 78 and 74% homology with amino acidresidues K72 to M94 of HrpW of P . syringae pv . tomato and residuesQ36 to Q62 of HrpW of E . amylovora, respectively [Fig . 6A].Computer-based secondary structure analysis revealed that thetwo HrpW regions are predicted to have an -helical region similarto the putative -helix of the HpaG peptide [Fig . 6B] . The leucine-rich motif found in the HpaG peptide [LLXXLIXXLL] was identifiedin the corresponding region of two HrpW proteins [Fig . 6B].

 
Gain-of-function mutants of XopA. The harpin XopA does not elicit HR in tobacco plants . SinceXopA lacks 16 amino acid residues that correspond to the regionbetween positions 59 to 74 in HpaG, we engineered XopA as achimeric protein to make the protein active . First, we constructeda chimeric protein, XopA-HpaG, by exchanging the C terminusof XopA with the C terminus of HpaG . This was performed by coligatingthe 120-bp NdeI-PvuII fragment of pTXOPA and the 73-bp PvuII-BamHIfragment of pTHG67 between the NdeI and BamHI sites of pET14b.The resulting plasmid, pTXH2, was sequenced to confirm the correctconstruction and then transformed into E . coli strain BL21[DE3].The XopA-HpaGC66 fusion protein contains the XopA domain fromthe start codon to the 41st amino acid residue and the HpaGdomain from the 41st to the 67th amino acid residues . The resultingXopA-HpaG fusion protein does not contain the D40 residue ofHpaG and did not exhibit elicitor activity [Fig . 7].

 
Based on an alignment of the deduced amino acid sequences ofXopA and HpaG, we mutagenized the xopA gene by site-directed mutagenesis . The F and M residues at positions 48 and 52 ofXopA, respectively, differ from the leucine residues in thesepositions in HpaG . Therefore, the codons for residues F48 andM52 of XopA were mutated into leucine codons, both individuallyand in one clone containing both mutations [Fig . 8] . XopA[M52L]was not able to elicit HR, but XopA[F48L] and XopA[F48L/M52L]elicited HR on tobacco leaves at 2.5 and 1 µM, respectively[Fig . 7].

 
To determine whether the inability of XopA to induce HR in tobacco plants is unique to X . campestris pv . vesicatoria strain 82-8 [race 1], we isolated and sequenced xopA genes from strains that represent races 2 and 3, X . campestris pv . vesicatoria strain E-3 [race 2] and LS833 [race 3] . The DNA sequences ofthe isolated xopA genes were identical to that of X . campestris pv . vesicatoria strain 82-8 [race 1] [data not shown].

 
In this study, we investigated the critical amino acid residuesthat determine the HR elicitor activity of HpaG and XopA, byusing random and site-directed mutagenesis . Initially, we foundthat XopA cannot elicit HR in tobacco leaves, even though theamino acid sequence of XopA is very similar to that of HpaG[15] . The major difference between HpaG and XopA is that XopAlacks 16 amino acid residues that correspond to the region betweenpositions 59 and 74 in HpaG [15] . These sixteen amino acid residues, QGQGQGQGGDSGGQGG, are mainly glycine and glutamine residues.Of the 28 glycine residues in the HpaG protein, 9 are in thisregion . A prominent feature of most harpins is a high glycinecontent: 21% in HpaG, 22.6% in HrpN of E . amylovora, 13.2% inHrpZ of P . syringae pv . syringae, 13.9% in HrpW of E . amylovora,and 20.9% in PopA of R . solanacearum [4, 12, 14, 15, 27] . However, XopA has a relatively low content of glycine residues [8% glycine], owing to the lack of the 16-amino-acid region . This observation suggested that the glycine-rich 16-amino-acid region of HpaGhas an important role in the elicitor activity of this protein.However, HpaG deletion analysis showed that the 16 amino acidresidues are dispensable for the elicitor activity and thatthe N-terminal 52 amino acids of HpaG are sufficient to maintainthe elicitor activity and heat stability of the protein . Thisindicates that the inability of XopA to induce HR is not relatedto the lack of the 16 amino acid residues and that the residuesthat are important for the elicitor activity reside outsideof this region . The fact that there are only four glycine residuesin the N-terminal 52-amino-acid region suggests that the glycinerichness of harpins is not important for the elicitor activitybut that it may have other roles.

Site-directed mutagenesis analysis of the N-terminal 52 aminoacid residues showed that amino acid substitutions that affectelicitor activity are located within the 19-amino-acid region SEKQLDQLLTQLIMALLQQ . These 19 amino acids clearly play important roles in inducing HR because the synthetic HpaG peptide, composedof 23 amino acid residues encompassing the 19 amino acid residues, possesses full elicitor activity . The amino acid compositionof the peptide region contains unexpected features . In contrastto the other harpins, which are glycine-rich, the HpaG peptideis rich in glutamine and leucine . The HpaG peptide has onlyone glycine residue [4.3%], but six glutamine [26%] and sixleucine [26%] residues . Since the five HpaG mutants with affectedelicitor activity have substitutions at L39, L42, L43, L46,and L50, and one has a substitution at Q45, the leucine residuesin the 23-amino-acid sequence probably have an important rolein HR elicitation.

Interestingly, the 19 amino acid residues that affect elicitor activity are predicted to have an -helical structure . Thereappears to be an important correlation between the -helical feature of HpaG and its HR elicitor activity . Alfano et al. noted that HrpZ has nine probable -helices; however, the relationshipbetween the -helices and the HR elicitor activity was not examined[2] . The predicted -helix that is conserved between HpaG andthe HrpW harpins of Pseudomonas and Erwinia species suggeststhat the feature constitutes a key functional domain in harpinelicitor activity . However, there are no clues as to the specificmechanisms of the predicted -helix in HR induction.

Comparing the two Xanthomonas HrpW proteins with the HrpW harpins of Pseudomonas and Erwinia species, the two Xanthomonas HrpWproteins were not reported to be HR elicitors . Therefore, we cannot consider the HrpW proteins of Xanthomonas species as harpins, based on the sequence homology . Comparison of the four HrpW proteins shows that the Xanthomonas HrpW proteins are considerably smaller than the Pseudomonas and Erwinia HrpW harpins . Moreover,the Xanthomonas HrpW proteins have high homology with the pectatelyase domains of the Pseudomonas and Erwinia HrpW harpins butnot with the harpin domains of the HrpW harpins . The two XanthomonasHrpW proteins also have a predicted -helix in the N-terminalregion, but we did not find any significant homology with theHpaG peptide sequence [data not shown].

The characteristics of other peptide elicitors were examinedby way of comparison with those of HpaG . A 13-amino-acid oligopeptide, derived from the Phytophthora megasperma glycoprotein, was shown to be both necessary and sufficient for elicitor activity, as measured by phytoalexin accumulation in parsley [21]; in addition,the AVR9 28-amino-acid oligopeptide of Cladosporium fulvum isable to induce a hypersensitive necrosis in tomato [26] . However,there are no similarities in the amino acid sequences or predictedstructures of these peptide elicitors with those of HpaG . Thisindicates that the importance of the predicted -helix in harpinelicitor activity might be limited to bacterial pathogens.

Since the first report of a harpin, HrpN of E . amylovora, several HrpN homologs have been identified from E . carotovora, E . chrysanthemi,and Pantoea stewartii subsp . stewartii [1, 6, 19], and HrpZ homologs have been identified from P . syringae pv . syringae, P . syringae pv . glycinea, and P . syringae pv . tomato [12, 22].All of the HrpN and HrpZ homologs are true harpins because theycan elicit HR . However, the HpaG homologs from Xanthomonas speciesdiffer in their abilities to elicit HR [15] . Sequence differencesin the critical 23 amino acid residues of the HpaG peptide andthe corresponding regions of other HpaG homologs probably contributeto the differences in HR elicitation ability . Comparison ofHpaG with Hpa1 from X . oryzae pv . oryzae reveals that T44 ofHpaG is changed to a cysteine residue in Hpa1 and that M48 andQ53 of HpaG are both serine residues in the Hpa1 sequence . Likewise,in XopA, L46 and M48 of HpaG are changed to phenylalanine residues,and A49 and L50 of HpaG are changed to serine and methionineresidues [Fig . 8] . Therefore, it is reasonable to propose thatchanges in a few amino acid residues lead to different levelsof elicitor activity . This idea is consistent with the resultsof the mutagenesis of XopA.

In spite of their different abilities to induce HR in nonhost plants, HpaG, Hpa1, and XopA are necessary for full virulencein their respective host plants [15, 20, 28], indicating thatthe HpaG homologs have common roles, which contribute to compatibleinteractions with host plants . The C-terminal region of HpaGis not essential for HR elicitation ability; however, this regionin HpaG homologs may have an unknown role in disease progresswithin the host plant.

 
This study was supported by grant CG1412 from the Crop Functional Genomics Center of the 21st Century Frontier R&D Programof the Ministry of Science and Technology of the Republic ofKorea . J.-G.K., E.J., and J.O . are recipients of graduate fellowshipsfrom the Ministry of Education as part of the Brain Korea 21Project.

 
* Corresponding author . Mailing address: School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea . Phone: 82-2-880-4676 . Fax: 82-2-873-2317 . E-mail: ingyu@snu.ac.kr .

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