\PERGAMON Phytochemistry 49 "0888# 850Ð855

9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[PII] S9920!8311 "87#99539!1

A novel elicitin necrotic site revealed by a!cinnamomin sequenceand site!directed mutagenesis

Vale�rie Perez\ Jean!Claude Huet\ Michael O|Donohue0\ Claude Nespoulous\Jean!Claude Pernollet�

Biochimie et Structure des Prote�ines\ INRA\ Domaine de Vilvert\ F!67241 Jouy!en!Josas Cedex\ France

Received 7 July 0887^ revised 2 September 0887

Abstract

Elicitins are 09 kDa proteins secreted by Phytophthora fungi\ that elicit resistance against certain plant pathogens[ Various naturalmolecules\ mutated recombinant elicitins and synthetic peptides were previously shown to di}erentially induce in tobacco leafnecrosis and defence genes\ activities borne by several sites which were identi_ed[ We report a novel necrosis!determining residue atposition 14\ revealed by the comparison of the necrotic activity and sequence of a!cinnamomin with those of other known elicitins[Using a modi_ed recombinant b!cryptogein\ expressed in Pichia pastoris\ we show that the substitution of asparagine 14 by a serineleads to a signi_cant enhancement of the necrotic activity[ Þ 0888 Elsevier Science Ltd[ All rights reserved[

Keywords] Amino acid sequence^ Elicitin^ Necrotic site^ Nicotiana tabacum^ Phytophthora cinnamomi^ Site!directed mutagenesis

0[ Introduction

Fungi of the genus Phytophthora are a major cause ofcrop devastation[ In tobacco most Phytophthora speciesinduce generalized defence responses accompanied byonly limited tissue decomposition[ The active agents aresmall proteins "87 amino acids#\ termed elicitins\ whichare abundantly secreted into the culture medium by mostof the Phytophthora species\ with the exception of Phyto!phthora parasitica var[ nicotianae "Ricci et al[\ 0878^Nespoulous\ Huet\ + Pernollet\ 0881^ Pernollet\aSallantin\ Salle�!Tourne\ + Huet\ 0882^ Kamoun\Young\ Glascock\ + Tyler\ 0882^ Kamoun\ Young\ Fo�r!ster\ Co}ey\ + Tyler\ 0883#[ These elicitor proteins induceremote leaf necrosis in infected tobacco plants and gen!erate defence responses which include a hypersensitive!like reaction\ leading to a systemic acquired resistanceagainst infection by the major tobacco pathogen "i[e[ P[parasitica var[ nicotianae# "Ricci et al[\ 0878# and alsobacterial plant pathogens "Kamoun et al[\ 0882#[ Thesevere\ virulent\ lethal pathogenicity of P[ parasitica var[nicotianae on tobacco appears to be correlated to theabsence of elicitin secretion\ the host does not perceive

0 Current address] Physicochimie et Biotechnologie des Polyme�res\INRA\ Moulin de la Housse\ BP 0928\ F!40576 Reims Cedex 1\ France[

� Corresponding author[ Tel[] ¦22!0!2354!1649^ fax] ¦22!0!2354!1654^ e!mail] pernolleÝjouy[inra[fr[

the fungus and thus fails to initiate defence mechanisms[Transformation of tobacco with a gene encoding b!cryptogein produced an intracellular accumulation ofelicitin in the plant before attack[ This provided thenecessary protein signal not produced by the P[ parasiticavar[ nicotianae and resulted in resistance to the pathogen"Tepfer et al[\ 0887#[

The complete sequences of 01 elicitins are known "Ricciet al[\ 0878^ Huet + Pernollet\ 0878\ 0882^ Nespoulous etal[\ 0881^ Huet\ Nespoulous\ + Pernollet\ 0881^ Huet\Mansion\ + Pernollet\ 0882^ Huet\ Salle�!Tourne\ +Pernollet\ 0883^ Huet\ Le Caer\ Nespoulous\ + Pernollet\0884#\ consist of 87 amino acids\ and show ×56)sequence identity[ In b!cryptogein\ three conserved dis!ul_de bridges maintain a 2!D structure which comprisessix a!helices and a peculiar motif\ whose sequence ishighly conserved among the elicitin family\ composed ofa short antiparallel two!stranded b!sheet and an V loop"Boissy et al[\ 0885^ Fefeu\ Bouaziz\ Huet\ Pernollet\ +Guittet\ 0886#[ Elicitins display a variable ability toinduce necrosis and independently trigger defenceresponses\ with b!"basic# elicitins producing morenecrosis than a!"acidic# elicitins "Nespoulous et al[\ 0881^Pernollet et al[\ 0882^ Huet et al[\ 0884#[ Possible necrotic!determining residues can be identi_ed at positions 1\ 02\61 and 83[ The nature of the side!chain of residue 02 ishighly correlated to necrotic potential\ as demonstratedby the results of a mutagenesis study of this position in a

V[ Perez et al[:Phytochemistry 49 "0888# 850Ð855851

synthetic b!cryptogein gene "O|Donohue et al[\ 0884#[Other sites responsible for the residual necrosis inductionof the a!elicitins have been determined together withthose responsible for inducing plant defences using aseries of synthetic peptides and tobacco plants trans!formed with a reporter gene controlled by defence genepromoters "Perez\ Huet\ Nespoulous\ + Pernollet\ 0886#[

The comparison of necrosis induction by various elic!itin isoforms has previously shown that the acidic andbasic isoforms of cinnamomin secreted by Phytophthoracinnamomi are the most necrogenic in their respectiveclasses "Pernollet et al[\ 0882#[ In this paper\ we reportthe complete sequence of a!cinnamomin and reveal anovel key residue by comparison to the b!cinnamominsequence "Huet + Pernollet\ 0878# and those of otherelicitins[ Site!directed mutagenesis of the synthetic b!cryptogein gene expressed in Pichia pastoris "O|Donohueet al[\ 0885# provide further evidence for the prominentrole of this residue in necrosis induction[

1[ Results

1[0[ Amino acid sequencing of a!cinnamomin

The N!terminal sequence was determined with 3!vinyl!pyridine reduced and alkylated a!cinnamomin[ Over!lapping peptides "Fig[ 0# were obtained either after diges!tion with CNBr or cleavage of the performic acid oxidizedprotein with trypsin[ With an initial yield of 69) and arepetitive sequencing yield of 82)\ the N!terminal endwas sequenced up to Ala 27 and was found to be over!lapping with the CNBr peptide B0[ The tryptic peptideOx0 allowed the alignment of the peptides B0\ B1 andB2[ The latter overlapped with the C!terminal trypticpeptide Ox1[ The good agreement between the IS!MSmeasured Mr "09\195[429[8# and that calculated fromthe amino acid sequence assuming three disul_de bridgesas in other elicitins "09\195[6# validates the sequence ofa!cinnamomin[ It comprises 87 amino acid residues andis devoid of side chain modi_cation\ like other Phyto!phthora elicitins and in contrast to Pythium elicitin!likeproteins "Huet et al[\ 0884#[ The far UV circular dich!

Fig[ 0[ Alignment of a!cinnamomin peptides[ N!ter is N!terminus of reduced and alkylated a!cinnamomin\ B0\ B1 and B2 are CNBr!cleaved peptides\Ox0 and Ox1 are tryptic peptides obtained after digestion of the oxidized protein with performic acid[ Outlined residues indicate those di}erent fromb!cinnamomin[

roism "CD# spectrum of a!cinnamomin "not shown# wasquite comparable to those of natural "Nespoulous et al[\0881^ Huet et al[\ 0881# and recombinant "O|Donohue etal[\ 0885# elicitins[

1[1[ Sequence homology of elicitins

Figure 1 illustrates the alignment of the a!cinnamominsequence with those of mature elicitins secreted by Phyto!phthora species and the two recombinant proteins usedin this study[ The natural sequences are ordered fromtop to bottom according to increasing necrotic power totobacco detached leaves "Pernollet et al[\ 0882#[ Boxesindicate residues involved in elicitin biological activities\thick lines correspond to the sites determined by sequencecomparison and site!directed mutagenesis\ thin linesthose identi_ed by synthetic peptides "Perez et al[\ 0886#[The strict homology between both cinnamomins "73[6)#is comparable to the sequence homologies of other elicitinisoforms belonging to a same species "i[e[ 81) for P[drechsleri elicitins "Huet et al[\ 0881# and 76) for P[megasperma megasperma elicitins "Huet + Pernollet\0882#[ The di}erences between a! and b!cinnamomins areessentially those which are commonly found between a!and b!elicitins[ Six heterologous residues were located atpositions 1\ 02\ 17\ 61\ 82 and 83\ which belong to the00 sites already identi_ed to be involved in the elicitinbiological activities[ Comparison of cinnamomins to theother 00 natural Phytophthora elicitin sequences showedcomplete homology and no deletion was necessary foralignment[ The match between this set of elicitins was56[2) and showed large consensus regions\ emphasizedin Fig[ 1 by a solid line which separates acidic from basicelicitins[ The a!cinnamomin sequence exhibited only onenovel feature\ distinguishing cinnamomins from otherelicitins[ This di}erence\ indicated by bold circles in Fig[1\ corresponds to the replacement at position 14 of anAsn by a Ser[

1[2[ Characterization of recombinant mutated elicitins

In order to con_rm the peculiar role of the residue 14\we decided to change b!cryptogein Asn 14 into Ser to test

V[ Perez et al[:Phytochemistry 49 "0888# 850Ð855 852

Fig[ 1[ Sequence comparison of natural and recombinant Phytophthoraelicitins[ Cacto\ Phytophthora cactorum elicitin "Huet et al[\ 0882#^ Cap\capsicein\ Phytophthora capsici elicitin "Ricci et al[\ 0878#^ Cina and b\P[ cinnamomi acidic and basic elicitins\ respectively "this work and Huet+ Pernollet\ 0878#^ Cryb\ b!cryptogein\ Phytophthora cryptogea basicelicitin "Ricci et al[\ 0878#^ Drea and b\ Phytophthora drechsleri acidicand basic elicitins\ respectively "Huet et al[\ 0881#^ Inf\ Phytophthorainfestans elicitin "Huet et al[\ 0883#^ MgMa and b\ Phytophthora mega!sperma megasperma acidic and basic elicitins\ respectively "Huet +Pernollet\ 0882#^ Para\ parasiticein\ P[ parasitica var parasitica elicitin"Nespoulous et al[\ 0881#^ rCry\ recombinant b!cryptogein "O|Donohueet al[\ 0885#^ N14S\ rCry mutated at position 14 "this work#[ The naturalsequences are ordered from top to bottom according to increasingnecrotic power to tobacco detached leaves "Pernollet et al[\ 0882#[Circles indicate the residues typical of P[ cinnamomi elicitins[ Boldcharacters show the residues of the recombinant elicitins which havebeen modi_ed compared to b!cryptogein[ The regions of consensus areemphasized with a continuous line which separates acidic from basicelicitins[ Boxes indicate residues involved in elicitin biological activities\thick lines correspond to the sites determined by sequence comparisonand site!directed mutagenesis\ thin lines those identi_ed by syntheticpeptides and activation of defence genes[

the in~uence of this sole mutation[ The production oflarge quantities of highly pure recombinant natural"rCry# or mutated "N14S# b!cryptogeins was achievedusing the P[ pastoris expression system as described by"O|Donohue et al[\ 0885#[ The N!termini of these mol!ecules exhibit some minor sequence di}erences withrespect to wild!type b!cryptogein\ since they weredesigned to optimize the signal peptide removal "i[e[ Thr0 is replaced by Gly and Ala 1 by Thr\ and an additionalArg constitutes the N!terminal amino!acid# as reportedin Fig[ 1[ Both the N!terminal sequences and the Mr ofthe puri_ed recombinant proteins were veri_ed prior tobiological assays] rCry exhibited a measured Mr of09\360[220[1 "calculated 09\360[0# and N14S a Mr of09\333[320[9 "calculated 09\333[9#[

Fig[ 2[ Comparison of necrosis induction on tobacco detached leavesby elicitins[ Cryb "Ð E Ð#\ b!cryptogein\ Phytophthora cryptogea basicelicitin^ Cina "Ð Ž Ð# and Cinb "Ð R Ð#\ acidic and basic Phytophthoracinnamomi elicitins\ respectively^ rCry "Ð × Ð#\ recombinant b!cryp!togein and N14S "Ð ( Ð#\ recombinant mutated b!cryptogein[ The ver!tical ordinate indicates the mean proportion of necrotized areameasured on _ve detached leaves\ standard deviations are indicatedwhen di}erent from zero[ Detached leaves that were kept in nutrientsolution at room temperature in the dark for 37 h[

1[3[ Necrotic properties on tobacco leaves

Figure 2 shows the doseÐresponse curves obtained witha! and b!cinnamomins\ b!cryptogein and the recom!binant proteins[ The endpoint responses "9 and 099)necrotized area# were identical from one experiment toanother\ whereas the transition responses were subject tosome variability "standard deviations indicated in Fig[2#[ Cinb and Cryb were found to be the most necroticmolecules\ whereas Cina was the less active one[ Thesevalues are in good agreement with the previous obser!vations "Pernollet et al[\ 0882#[ The recombinant proteinrCry was found to induce necroses in a range close to thatof Cina\ while the mutated N14S recombinant elicitin wasnearly as necrotic as Cinb[

2[ Discussion

Compared to the other Phytophthora elicitins\ a!cin!namomin exhibits comparable size\ amino acid compo!sition\ absence of side!chain modi_cation and globalconformation\ as revealed by circular dichroism[ Thehigher necrosis induction of a! and b!cinnamomins\ com!pared to acidic and basic elicitins\ respectively "Pernolletet al[\ 0882#\ appears to be correlated with the presence ofserine instead of asparagine at position 14[ This potential\novel necrotic!determining residue was directly inves!tigated by site directed mutagenesis of a recombinant b!cryptogein produced by the P[ pastoris system[

The determination of the necrotic potential of rCRY

V[ Perez et al[:Phytochemistry 49 "0888# 850Ð855853

)

)CMYK Page 853

revealed that the presence of an extra Arg at the N!terminus of the b!cryptogein recombinant protein sig!ni_cantly reduces its necrotic power\ compared to thatof the wild!type Cryb\ to almost the level of Cina[Examination of the 2!D structure of b!cryptogein "Boissyet al[\ 0885^ Fefeu et al[\ 0886# shows that seven of the02 residues "including Arg 9# involved in the biologicalactivities are grouped in two clusters at the borders of adepression centered around Tyr 76[ One of these sitesinvolves the active residues 69 and 61 "Perez et al[\ 0886#\which are located close to the extra Arg 9 "Fig[ 3a# whosepositive charge considerably alters the electric potentialof this region\ which in Cryb is principally due to Asp 61[This modi_cation probably explains the loss of necroticactivity associated with this extra arginine\ but the pres!ence of a threonine at position 1 instead of alanine mightalso contribute to the necrotic activity reduction "Perezet al[\ 0886#[

The replacement of Asn 14 by Ser in rCRY\ thus imit!ating the natural cinnamomin sequence context\ sig!ni_cantly restored the necrotic activity of this protein toa level close to that of b!cryptogein and b!cinnamomin[Fig[ 3 shows that the residue 14 is located in a solvent!exposed position on the second a!helix close to Ala 11and Ser 17\ amino acids previously shown to be of import!ance for biological activity "Perez et al[\ 0886#[ Our pre!sent data indicate a crucial\ necrotic!determining role forthese three exposed residues[ Interestingly\ these residuesare located on the same face of the elicitin as residue 02\a key amino acid for necrosis induction "O|Donohue etal[\ 0884^ Perez et al[\ 0886#[ It is therefore very likely

Fig[ 3[ Location of key residues in the 2!D structure of b!cryptogein[Protein Data Bank identi_cation of b!cryptogein coordinates] 0BEO"Boissy et al[\ 0885#[ Substantial aminoacids\ numbered according toFig[ 1\ are shown in space!_lling model\ the rest of the molecule asstrand[ The second a!helix is indicated by a1[ "a# The concave faceshowing the crevace centered around Tyr76\ the additional N!terminalArg residue "carbon atoms colored in cyan# and the active residues 1\69 and 61 "carbon atoms colored in magenta#[ "b# The opposite convexface showing the residues 02\ 11\ 14 "carbon atoms colored in yellow#and 17[

that residues 11\ 14 and 17 are involved in a molecularinteraction which also involves residue 02[

The optimization of elicitin!induced resistance inplants expressing an elicitin!encoding gene would necessi!tate the simultaneous augmentation of the elicitorpotency of the transfected gene product and the negationof its high necrotic power[ However\ to attain this attract!ive biotechnological goal\ all of the amino acids involvedin both elicitin activities need to be accurately identi_ed[These present results\ along with previously publisheddata on the necrotic!determining residues of elicitins\ goa long way to meeting this requirement and the devel!opment of tailored!made elicitin genes for use in tobaccoplant transformation can now be envisaged[ These mol!ecules will have an increased ability to elicit resistanceagainst Phytophthora while lacking undesirable sidee}ects such as necrosis development[ Despite the numer!ous studies which have provided an experimental descrip!tion of many of the cellular events that arise from elicitinactivity\ the comprehensive molecular basis of the hostspeci_city of Phytophthora is not currently known andthe identi_cation of the biological functions of elicitinsstill remains to be established "Grant\ Ebert\ + Gayler\0885#[ Our present results also contribute to the under!standing of this novel family of proteins which comprisesnumerous related members distinct from authenticelicitins whose precise modes of action are still unknown"Baillieul\ Fritig\ + Kau}mann\ 0885^ Kamoun\Lindqvist\ + Govers\ 0886#[

On a more general point of view\ these results con_rmseveral reports on structureÐactivity relationships studiesof other fungal elicitors that have also proven that singlechanges in the amino acid sequence may signi_cantlyalter their biological activity\ as shown for a peptideoriginating from a 31 kDa glycoprotein elicitor from P[megasperma megasperma "Nu�rnberger et al[\ 0883# andthe AVR8 peptide of Cladosporium fulvum "Kooman!Gersmann\ Voselgang\ Hoogendijk\ + De Wit\ 0886#[This supports the idea that fungal phytopathogens arebeing recognized by the plant in a receptor!like manner[

3[ Experimental

3[0[ Protein puri_cation

Natural proteins "a! and b!cinnamomins and b!cryp!togein# were puri_ed according to the general elicitinprocedure using the fungi isolates as described in "Per!nollet et al[\ 0882#[ Recombinant proteins secreted by P[pastoris were puri_ed as described by O|Donohue et al[\0885[ The elicitin proteins were lyophilized and storedat −19>C before use[ Their purity was checked usinganalytical RPLC\ N!terminal sequencing and mass spec!trometry[ Both the N!terminal sequences and the Mrwere veri_ed before biological assays[

V[ Perez et al[:Phytochemistry 49 "0888# 850Ð855 854

3[1[ Protein sequencing

Fifty mg of a!cinnamomin were reduced with 1!mer!captoethanol and alkylated with 3!vinyl!pyridine accord!ing to Henschen\ 0875[ The reduced and alkylated proteinwas submitted to N!terminal Edman degradation andto peptide cleavage with CNBr "Huet et al[\ 0881#[ Inaddition\ 49 mg of a!cinnamomin\ previously oxidizedwith performic acid\ were digested with sequencing grademodi_ed trypsin "EC 2[3[10[3# obtained from Promegaaccording to "Huet et al[\ 0882#[ Automated Edmandegradation was performed using either an Applied Bio!systems 364A sequencer and its on!line phenyl!thiohydantoin amino acid analyzer model 019A or aPerkin Elmer Procise 383!509A sequencer with reagentsand methods of the manufacturers[

3[2[ Protein characterization

Mr were determined by micro ion spray mass spec!trometry "Sciex API099\ Perkin!Elmer# using a positivemode from 299 to 2999 a[m[u[ with 9[4 a[m[u[ steps anda 0 ms dwell!time[ The ion spray needle voltage was 5999V and the ori_ce plate voltage 39 V\ at a ~ow rate of 9[1ml=min−0[ Mass spectrometry data were analyzed with thePerkin Elmer Sciex Bio!Multi!View 0[1 software[ Theaverage Mr were calculated from the sequence using thePerkin Elmer Sciex Peptide Map 1[1 software[ Circulardichroism spectra were recorded using a Mark V Jobin!Yvon "Longjumeau\ France# dichrograph\ as previouslyreported "O|Donohue et al[\ 0885#[

3[3[ Recombinant protein production and site directedmutagenesis

The production of recombinant elicitins was performedin P[ pastoris according to "O|Donohue et al[\ 0885#[The position 14 was mutated from Asn to Ser using aTransformerTM mutagenesis kit "Clontech#\ based on theunique site elimination system "Deng + Nickolo}\ 0881#as previously described "O|Donohue et al[\ 0884#[ Themutagenic primer sequence "with the mutant serine antic!odon TCG in italic# was] 4?p AGA GCA CTG GCTGAA AGA CGC GTC 2?[ The selection primer wasdesigned to direct the substitution of an A~ II recognitionsite by a Nde I site^ the sequence of this primer "with theNde I site in italic# was] 4?p TCA CAC CGC ATA TGGTGC ACT C 2?[ Both proteins di}er from the wild!typeb!cryptogein at their N!terminus "RGT instead of TA#[

3[4[ Tests for necrotic activities on tobacco leaves

Puri_ed natural and recombinant proteins were testedfrom approximately 59!day old greenhouse grown tob!acco plants "Nicotiana tabacum cv Xanthi XHFD7# bypetiole inoculation on detached leaves[ Inoculations of

09 ml were applied to still expanding leaves with elicitinamounts of 09 ng\ 099 ng\ 499 ng\ 0 mg and 09 mg dilutedin pure water[ Water was also inoculated as a negativecontrol[ All solution concentrations were spectro!photometrically checked by UV absorbance at 166 nmwith the proper extinction coe.cient "Nespoulous + Per!nollet\ 0883# before they were inoculated[ Once the elicitinsolution drop had been taken up by the petiole\ usuallywithin 29 s\ the petiole was dipped into a nutrient solutionadapted from "Baudet et al[\ 0875#[ Treated leaves wereincubated at room temp in the dark to allow the necrosesto develop[ Five replicates of each treatment wereincluded in each experiment and all elicitin moleculeswere assayed simultaneously to reduce variability[ Symp!toms reached their maximum extent after 1 days[ Therelative necrotic index of an isoform was de_ned as thesum of the normalized necrotic area\ equal to 0 whena leaf was totally necrotized and zero for no necrosis\obtained with doses of 09 ng\ 099 ng\ 0 and 09 mg[

Acknowledgements

The authors are grateful to Christian Ouali for hisskillful technical assistance[ This study was supported inpart by a grant from the Association pour la Recherchesur le Cancer "ARC#[

References

Baillieul\ F[\ Fritig\ B[\ + Kau}mann\ S[ "0885#[ Molecular PlantÐMicrobe Interactions\ 8\ 103[

Baudet\ J[\ Huet\ J[ C[\ Jolivet\ E[\ Lesaint\ C[\ Mosse�\ J[\ + Pernollet\J[ !C[ "0875#[ Physiologia Plantarum\ 57\ 597[

Boissy\ G[\ de la Fortelle\ E[\ Kahn\ R[\ Huet\ J[ !C[\ Bricogne\ G[\Pernollet\ J[ !C[\ + Brunie\ S[ "0885#[ Structure\ 3\ 0318[

Deng\ W[ P[\ + Nickolo}\ J[ A[ "0881#[ Analytical Biochemistry\ 199\70[

Fefeu\ S[\ Bouaziz\ S[\ Huet\ J[ !C[\ Pernollet\ J[ !C[\ + Guittet\ E["0886#[ Protein Science\ 5\ 1168[

Grant\ B[ R[\ Ebert\ D[\ + Gayler\ K[ R[ "0885#[ Australian PlantPathology\ 14\ 037[

Henschen\ A[ "0875#[ In] Wittmann!Liebold\ Salnikow + Erdmann"Eds[#\ Advanced methods in protein microsequence analysis[ Berlin]Springer!Verlag\ p[ 133[

Huet\ J[ !C[\ + Pernollet\ J[ !C[ "0878#[ FEBS Letters\ 146\ 291[Huet\ J[ !C[\ + Pernollet\ J[ !C[ "0882#[ Phytochemistry\ 22\ 686[Huet\ J[ !C[\ Le Caer\ J[ !P[\ Nespoulous\ C[\ + Pernollet\ J[ !C[ "0884#[

Molecular PlantÐMicrobe Interactions\ 7\ 291[Huet\ J[ !C[\ Mansion\ M[\ + Pernollet\ J[ !C[ "0882#[ Phytochemistry\

23\ 0150[Huet\ J[ !C[\ Nespoulous\ C[\ + Pernollet\ J[ !C[ "0881#[ Phytochemistry\

20\ 0360[Huet\ J[ !C[\ Salle�!Tourne\ M[\ + Pernollet\ J[ !C[ "0883#[ Molecular

PlantÐMicrobe Interactions\ 6\ 291[Kamoun\ S[\ Lindqvist\ H[\ + Govers\ F[ "0886#[ Molecular PlantÐ

Microbe Interactions\ 09\ 0917[Kamoun\ S[\ Young\ M[\ Fo�rster\ H[\ Co}ey\ M[ D[\ + Tyler\ B[ M[

"0883#[ Applied and Environmental Microbiology\ 59\ 0482[Kamoun\ S[\ Young\ M[\ Glascock\ C[ B[\ + Tyler\ B[ M[ "0882#[

Molecular PlantÐMicrobe Interactions\ 5\ 04[

V[ Perez et al[:Phytochemistry 49 "0888# 850Ð855855

Kooman!Gersmann\ M[\ Voselgang\ R[\ Hoogendijk\ E[ C[ M[\ + DeWit\ P[ J[ G[ M[ "0886#[ Molecular PlantÐMicrobe Interactions\ 09\710[

Nespoulous\ C[\ + Pernollet\ J[ !C[ "0883#[ International Journal ofPeptide and Protein Research\ 32\ 043[

Nespoulous\ C[\ Huet\ J[ !C[\ + Pernollet\ J[ !C[ "0881#[ Planta\ 075\440[

Nu�rnberger\ T[\ Nennstiel\ D[\ Jabs\ T[\ Sacks\ W[ R[\ Hahlbrock\ K[\+ Scheel\ D[ "0883#[ Cell\ 67\ 338[

O|Donohue\ M[ J[\ Boissy\ G[\ Huet\ J[ !C[\ Nespoulous\ C[\ Brunie\S[\ + Pernollet\ J[ !C[ "0885#[ Protein Expression and Puri_cation\ 7\143[

O|Donohue\ M[ J[\ Gousseau\ H[\ Huet\ J[ !C[\ Sallantin\ M[\ Tepfer\D[ A[\ + Pernollet\ J[ !C[ "0884#[ Plant Molecular Biology\ 16\ 466[

Perez\ V[\ Huet\ J[ !C[\ Nespoulous\ C[\ + Pernollet\ J[ !C[ "0886#[Molecular PlantÐMicrobe Interactions\ 09\ 649[

Pernollet\ J[ !C[\ Sallantin\ M[\ Salle�!Tourne\ M[\ + Huet\ J[ !C[ "0882#[Physiological and Molecular Plant Pathology\ 31\ 42[

Ricci\ P[\ Bonnet\ P[\ Huet\ J[ !C[\ Sallantin\ M[\ Beauvais!Cante\ F[\Bruneteau\ M[\ Billard\ G[\ + Pernollet\ J[ !C[ "0878#[ EuropeanJournal of Biochemistry\ 072\ 444[

Tepfer\ D[\ Boutteaux\ C[\ Vigon\ C[\ Aymes\ S[\ Perez\ V[\ O|Donohue\M[ J[\ Huet\ J[ !C[\ + Pernollet\ J[ !C[ "0887#[ Molecular PlantÐMicrobe Interactions\ 00\ 53[