8/2/2019 Jones et.al.1994

1/6

Isolation of the Tomato Cf-9 Gene for Resistance to Cladosporium fulvum by TransposonTaggingAuthor(s): David A. Jones, Colwyn M. Thomas, Kim E. Hammond-Kosack, Peter J. Balint-Kurti, Jonathan D. G. JonesReviewed work(s):Source: Science, New Series, Vol. 266, No. 5186 (Nov. 4, 1994), pp. 789-793Published by: American Association for the Advancement of ScienceStable URL: http://www.jstor.org/stable/2885548 .

Accessed: 12/03/2012 13:34

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of

content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms

of scholarship. For more information about JSTOR, please contact support@jstor.org.

American Association for the Advancement of Science is collaborating with JSTOR to digitize, preserve and

extend access to Science.

http://www.jstor.org/action/showPublisher?publisherCode=aaashttp://www.jstor.org/stable/2885548?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/stable/2885548?origin=JSTOR-pdfhttp://www.jstor.org/action/showPublisher?publisherCode=aaas

8/2/2019 Jones et.al.1994

2/6

M REPORTSdamine and 5-carboxyfluorescein absorb) were fit tothe weighted sum of standard spectrum of a duplexlabeled onlywith donor FD(Xem,490) and the fluores-cence signalof the sample F(Xem'560)xcited at 560nm (where only 5-carboxytetramethylrhodamine b-sorbs)F(Xem,490) =

c * FD(Xem,490) + (ratio)A * F(Xem,560) (2)c and (ratio)A are the fitted weighting factors of thetwo spectral components. The fit was made in therange Xem= 500 to 540 nm (where only Demits)andXem= 570 to 650 nm (where both D and A emit).(ratio)As the acceptor fluorescence signal of theFRET measurement normalized by F(Xem,560)asshown in Eq. 4 (34).

F(Xem,490) -c * FD(Xem,490)(ratio)A= F(Xem,560)

ED(490) SA(490)E S)+A (3)SA(560) 8A(560)(ratio)A is linearly dependent on the efficiency oftransferE; it normalizes the sensitized FRETsignalfor the quantum yield of 5-carboxytetramethylrho-damine, for the concentration of the duplex mole-cule, and for any error in percentage of acceptorlabeling. 8D and _Aare the molarabsorption coeffi-cient of Dand A at the given wavelength. The ratioofabsorption coefficient 8D(490)/SA(560)= 0.43 wasdeterminedfrom the absorbance spectra of the dou-bly labeled molecules and 8A(490)/_A(560) = 0.08was determined from the excitation or absorbancespectrum of a singly 5-carboxytetramethylrho-damine-labeled molecule.S. Arnott, D. W. L. Hukins, S. D. Dover, Biochem.Biophys. Res. Commun. 48,1392 (1972).H. Goldstein,Classical Mechanics (Addison-Wesley,Reading, MA,ed. 2,1980).Sequences of hammerhead riboxymes and sub-strates. S and R represent complementary sets ofoligoribonucleotideswhere eitherRor S carries bothof the dyes. See also reference for RNA duplexes(15).

ConstructA: SAII, RAI1, 5'-GGG CUC UGAUGA GCG CAA GCG AAA CUC C; SA12,RA12, 5'-GGG CCU CUG AUG AGC GCAAGC GAA ACU CC; SA13, RA13, 5'-GGGUCC UCU GAU GAG CGC AAG CGA AACUCC; SA14, RA14, 5'-GGG CUC CUC UGAUGA GCG CAA GCG AAA CUC C; SA15,RA15, 5'-GGG UCU CCU CUG AUG AGCGCA AGC GAA ACU CC; SA16, RA16, 5'-GGG CUC UCC UCU GAU GAG CGC AAGCGA AAC UCC; SA17, RA17, 5'-GGG UCUCUC CUC UGA UGA GCG CAA GCG AAACUC C. B: RB13, 5'-Fl-GGA CCG AAA CCCC-Rh, SB13, 5'-GGG GUdCAGG ACC GCAAGG UCC UCU GAUGAG GUCC; RB14, 5'-Fl-GGA CCG AAA CUC CC-Rh, SB14, 5-GGGAGU dCAG GACCGC AAG GUC CUCUGA UGA GGU CC; RB15, 5'-F1-GGACCGAAA CUG CCC-Rh, SB15, 5'-GGG CAGUdCAGGA CCG CAA GGU CCU CUG AUGAGG UCC; RB17, 5'-F1-GGACCGAAA CUGUGCCC-Rh,SB17, 5'-GGG CAC AGU dCAGGAC CGCAAG GUC CUC UGA UGA GGUCC. C: RC18, SC18, 5'-GGG CCG AAA CUGCCG CAA GGC AGU dCAC CUC C. D:RD20, 5'-FI-GGAGCG UCU GAUGAG GGCCC-Rh, SD20, 5'-GGGCCC GAA ACU GCCGCAAGG CAG UdCA CGCUCC.

22. T. Tuschl and F. Eckstein, Proc. Natl. Acad. Sci.U.S.A. 90, 6991 (1993).23. H.-Y.Mei,T. W. Kaaret,T. C. Bruice,Proc. Nati.Acad.Sci. U.S.A.86, 9727 (1989).24. F. U. Gast,K.M.A.Amiri, . J. Hagerman,iochem-

istry 33, 1788 (1994).25. E. Westhof, P. Dumas, D. Moras, J. Mol. Biol. 184,119 (1985).26. K. J. Hertel et al., Nucleic Acids Res. 20, 3252(1992).27. T. F6rster, Fluoreszenz Organischer Verbindungen(Vandenhoeck & Ruprecht, Gottingen, 1951); J. R.Lakowicz,Principlesof Fluorescence Spectroscopy(Plenum Press, New York, 1983).28. C. Massire,C. Gaspin, E.Westhof, J. Mol. Graphics12, 201 (1994).29. T. Tuschl, M. M. P. Ng, W. Pieken, Benseler, F.Eckstein, Biochemistry32, 11658 (1993).30. P. Theisen et al., TetrahedronLett.33, 5033 (1992).31. H. Aurup,T. Tuschl, F. Benseler, J. Ludwig,F. Eck-stein, Nucleic Acids Res. 22, 20-24 (1994).

32. K. J. Hertel, D. Herschlag, 0. C. Uhlenbeck, Bio-chemistry 33, 3374 (1994).33. A. C. Jeffries, R. H. Symons, NucleicAcids Res. 17,1371 (1989).34. R. N. Clegg, Methods Enzymol. 211, 353 (1992).35. We thank R. Clegg and G. Vamosi, Gottingen, fordiscussions and advice, U. Kutzke,G. Heim, and A.Zechel, Gottingen, for technical assistance, A.Heaton, B. Patel, and C. S. Vortler,Gottingen, forcritical reading of the manuscript. Supported by theDeutsche Forschungsgemeinschaft and the Fondsder Chemischen Industrie (F.E.) and an EEC grant(E.W.).Coordinates of the P atoms have been de-posited at the Brookhaven Protein Data Bank.9 August 1994; accepted 11 October 1994

Isolation of the Tomato Cf-9 Gene for Resistanceto Cladosporium ulvumby TransposonTaggingDavid A. Jones, Colwyn M. Thomas, Kim E. Hammond-Kosack,Peter J. Balint-Kurti,* onathan D. G. Jonest

The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporiumfulvum that carry the avirulence gene Avr9. The Cf-9 gene was isolated by transposontagging with the maize transposable element Dissociation. The DNA sequence of Cf-9encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predictedprotein shows homology to the receptor domain of several receptor-like protein kinasesin Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants, and to othermembers of the leucine-rich repeat family of proteins. This structure is consistent withthatof a receptor that could bind Avr9 peptide and activate plant defense.

Plants can defend hemselvesgainstn-fection by viruses,bacteria,fungi, nema-todes,insects,and even other plants.Plantdefensesare often activatedby specificin-teractionbetweenthe productof a diseaseresistance R) gene in the plant and theproduct f a correspondingvirulenceAvr)genein the pathogen 1). Withouteitherofthesegenes,plantdefenses renot activatedandinfectionbythe pathogen s permitted.To understandow specificplant defense sregulated,t is necessaryo leamthe natureof the R and Avr gene products, he waythey interact,and the chain of eventsthatresults.In the interactionbetweentomato(Ly-copersiconsculentum)nd the leaf mouldfungusCladosporiumulvum, he avirulencegeneAvr9has beencharacterized2). Avr9specifiesa 28-amino acidsecretedpeptidethat elicitsa necroticresponsewheninject-ed into tomato plants carrying he Cf-9resistance ene.We have nowisolatedCf-9by transposonagging,usinga maizeActi-vator-Dissociationlement (Ac-Ds)-basedsystem o targeta specificgene from oma-Sainsbury Laboratory,John Innes Centre, Norwich Re-search Park,Colney Lane, Norwich, NR4 7UH, UK.*Present address: Laboratoryof Cellular and Develop-mental Biology, National Instituteof Diabetes and Diges-tive and Kidney Diseases, National Institutes of Health,Bethesda, MD20892, USA.tTo whom correspondence should be addressed.

to. To tagCf-9,we useda transgenicoma-to line (3) carrying Ds element ocated3centimorgans(4) from the Cf-9 locus,which had previouslybeen mapped o theshortarmofchromosome (5). To activatethis Ds element,we used a geneticallyun-linkedstabilizedAc (sAc), itself incapableof transposition 6). Appropriatecrossesandselectionswerecarried ut (Fig. 1A) toproduceplantsheterozygousorDs andsAcandhomozygousorCf-9.Theseplantswerecrossed o plants ackingCf-9 but homozy-gous oranAvr9 ransgene7, 8) (Fig.1A).The progenyof this cross,which werehet-erozygousor Cf-9 and Avr9,becamene-croticanddied shortlyafterseed germina-tion, but those mutantfor Cf-9 survived(Fig. IB).Approximately160,000 progenyweregerminated(Fig. iB) and 118 survivorswererecovered.Of these,65 arosebyclonalpropagation f 10 independentmutations(8). The remaining 3 arose ndependently,givinga totalof 63 independentmutations.Of these, 21 were variegated or necrosis(Fig. IC) andcarriedboth Ds and sAc,33were stable and carriedDs, and 9 werestablebut did not carryDs. In addition othe 21 variegatedmutations hat were in-ferredto carryDs insertions n Cf-9, 16morewere identifiedamong he stablemu-tantsbyactivationwithsAc,which suggestsa totalof at least37 independentDs inser-

SCIENCE * VOL. 266 * 4 NOVEMBER1994 789

8/2/2019 Jones et.al.1994

3/6

tions into Cf-9. Of these, 28 have beenmapped to the same 3-kb region of thetomatogenome (Fig.2). All stablemutantstested weresusceptible o race 5 of C. ful-vum,which indicates oncordance etweenthe loss of response o the Avr9transgeneand loss of resistance o a raceof the funguscarrying Avr9. The correlation betweenmultiple independent mutations of Cf-9andmultiple ndependentDs insertionsn a

defined region, togetherwith the sAc-de-pendent nstability f these mutations,pro-vides strong evidence that the Cf-9 genehas been tagged.Mutant 18 (Fig. 2), carryinga singlestable Ds, was used to isolate Cf-9 by plas-mid rescue (9). Analysis of the flankinggenomicsequence uggestedhat Cf-9 con-tains an uninterruptedpen reading rame(ORF) encoding a protein of 863 amino

acids.Five complementaryDNA (cDNA)clones with sequences dentical o the Cf-9genomic sequencewere identified from aCf9 cDNA library10). The predictedpro-tein (Fig. 3) containsseven structural o-mains.The NH2-terminaldomain A (23amino acids) is consistent with a signalpeptide (11). Domain B, the presumedNH2-terminus f the matureprotein,con-tains severalcysteines.Domain C consistsof 28 imperfect opiesof a 24-amino acidleucine-richrepeat (LRR) that has beenshownin many organismso be importantin protein-proteinnteraction.Domain D(28 amino acids)has no conspicuous ea-tures.Domain E (18 amino acids) is veryacidic,with 10 negativelycharged esiduesandno positivelycharged esidues.The hy-drophobicdomain F (37 amino acids) isconsistentwith a transmembrane omain.The COOH-terminal omainG (21 aminoacids) is very basic, with eight positivelychargedresiduesand only two negativelycharged residues. The COOH-terminusconcludeswith the residuesKKRY 12). Inanimalsandyeast,the COOH-terminatingmotif KKXX whereX is any amino acid)(12) functionsas a signal for retrievalofA s nxzV U ^ \YZ F . -Z AS S 23Ba iPHLZPE:D ;Q--S-] : SQ~-Z Y, -AL_ 0'KNY_TN?:: YYZ. 57

ywP D I S Y P R T tc S !_ _ S r;.1_L ;,3 91V; AL D L R LA `.GK F.Fr. 111

T , KRL C FF ^S 136FiEFs: .s:-7.LDS L:SES S ? r rSX 160I1HLSKLhVLRIhCDvY:LS.V-YF- EL- 188LS(31I;QLRELN-LESN0 S 17 I S 211

__ SHL' TTLL S G- ELGEi IL. 2 34VFHL S iL QS L HL S VMPPQ L T VIF P K 2 60W$;r CASLLMTLYVr S VINI A DR I P K 284PSHLN_SL LYXMGR -NL^PIPK P 3 08L WN=N IFLHIL N1HIi E5P I SIH{ 3 3 1FTIFECLKRLL- N :FDG LEF 354LS0 1NTQLERLD LSSN SLTG nIPPSt". 37 8C GLQNLECLYLSSENt ?.-LP CIPSW 4 02XFS5C5LPSDEL3LFI~E;iF- 426

KSKTLSAVTLKQMXLKG 7.cNC 448LT-ZjNQKNL1-LL LLS Gs%7R*- SA 472I-NLK T7L. DLGSLN NLET1rPQC-V 497N~~YI~SHLDLS~CNSGt.N, 521F :S'7uG-N LRVI SLHi0.G-KLTGxVPF. c 545MINCKYLT LDL- GL FPNW 569LGYIFQL7K1LSL.RSNKLH:GPIKSS;N 595TOb7>FMGLQL L DLS SN-G.F SZNLPE ts. 620

'-NLQ-(9ID5S `PEYISDPY 644YYIx'L-'^I ST.KGQ` YDsvp: 666

L"ZSNIXEE1N0KNFEGHIPS7I 687I LVG L. T LNU41hNrLEG H I P AS 711FQ,r VLLO QL -L S SSNXI S _G P P 735LASLTFL_VL-'lu NIT LV I P K 759D Ku- ^S YQS-1- 3 F P K. _ 787E ED-Q1V'TPAELTDQEEiEED 805F a2 :SWQ

8/2/2019 Jones et.al.1994

4/6

REPORTSmembrane-bound roteins from the Golgiapparatus o the endoplasmic reticulum(13). Domains E, F, and G are consistentwith a transmembrane omain flanked bycharged anchoring domains-positivelychargedon the cytoplasmicide and nega-tively chargedon the extracytoplasmicide(14). Twenty-twoN-glycosylation ites aredistributedamong domains B, C, and D.The Cf-9 protein herefore ppears o be anextracytoplasmic lycoproteinanchored oa cell membrane,with the majorityof theextracytoplasmicomainmadeupof LRRs.Cf-9 was introgressednto tomatofromL. pimpinellifolium.f-9probe1 (Fig.2) washybridizedo DNA gel blots of genomicDNA from the tomato cultivar Money-maker line CfO), roma near-isogenic inecarryingCf-9 (line Cf9), and fromL. pen-nellii Fig. 4A). There are at least 11 majorhybridizing andsin Cf9, severalof themunique,whichsuggestshattheyare on theintrogressed egmentof DNA that carriesCf-9.This has been confirmed ythe cross-ing ofCf9 with L. pennel1iiand examinationof the segregationof Cf-9-homologousbandsamong he F2progeny.Threebands,including he 6.7 kb Bgl II Cf-9 band (9),

cosegregatewith Cf-9, whereasotherswerelinked either distally or proximally (4).Therefore,Cf-9 appearso be a member f asmall, clusteredmultigene amily,which isconsistentwith the geneticcomplexitypro-posed for this locus (5) and observed orresistance oci in other plant species (15,16). The disease-sensitiveCfOand L. pen-neUii ines show no more than five or sixmajor hybridizingbands. Differences incopy number could be a consequence ofgene duplication ndunequal rossing-over,which is consistent with proposedmodelsfor the evolutionof complexresistance oci(1, 15).Cf-9 probe2 (Fig. 2) washybridizedoRNAgel blots of mRNAfrom ines CfO ndCf9 (Fig.4B). A singleband of about3 kb,a size consistent with the size of the Cf-9cDNA clones,was observed n both lines.The similarhybridizationntensity,broad-ness of the bands, and a slight but consis-tentlyreducedmobility n Cf9 suggest im-ilar familiesof transcriptswith only minordifferencesn size bothwithinand betweenthe two lines.Cf-9 was found to be homologoustomanymembersof the LRRsuperfamily f

proteins 17). Two classesof plantproteinscarryingLRRswere the most homologousto Cf-9:(i) thereceptor-like roteinkinases(RLPKs)RLK5,TMK1,and a kinase-defec-tive homolog TMKL1 from Arabidopsis(18); and (ii) antifungalpolygalacturonase-inhibiting proteins (PGIPs) from severalplants 19) (Table1). Although he homol-ogy detected n these proteins s due mainlyto the LRRs, here is also homologywithdomainsB and D outside he LRRsof Cf-9(Fig. 5).The 24-amino acid LRRsof these ex-tracellular roteinsdiffer romthe predom-inantly23-aminoacidLRRs f intracellularproteins 4) by the insertionof a glycine nthe consensus sequence LXXLXXLXXLX-LXXNXLXXIPXXwhereX is any aminoacid) (12), to producethe consensus se-quence LXXLXXLXXLXLXXNXLXGXIP-XX in plants,or by the insertionof a leu-cine or alanine to producethe consensussequenceL/A)LXXLXXLXXLXLXXNXLX-XIPXX in other species (Table 1). Apartfrom some N-glycosylationites, there arefew conserved minoacids nterstitialo theLRR backbone, except amongthe PGIPs(4). Complete equencealignments repos-siblefor the PGIPs,because he numberofLRRs,the location of severalN-glycosyla-tion sites,and some of the sequence hat isinterstitialo the LRRs s conserved, ut thisis not so for Cf-9 and the RLPKs.Clearly,Cf-9shares nevolutionaryelationwith thePGIPsand RLPKs ut has diverged onsid-erablywith respectto (i) the numberandinterstitialsequences of its LRRs, (ii) itsattachment o a cell membranescomparedwith thePGIPs'ackofattachment, nd(iii)its lack of a cytoplasmic erine-threonineproteinkinasedomainascomparedwith theRLPKs,whichsuggestshatit mayalso havedivergedunctionally.Recently,the ArabidopsisPS2gene for

Fig. 4. DNA and RNAgel blots probed with Cf-9. (A) Genomic A BDNAs (3 ,ug) from the tomato cultivars Moneymaker (0), the 9 o p 0 9Moneymaker near-isogenic line Cf9 (9) carrying Cf-9, and L. kbpennellii (P)were digested with Bgl II,electrophoretically sep- 12arated on a vertical 1%agarose gel, and capillary-blotted onto 8a Hybond N filter.The filterwas hybridizedwith the [32P]dCTP- jlabeled probe 1 (Fig. 2) and washed with high stringency at _ wi6EP 3 kb650C in 0.2 x standard saline citrate and 0.1% SDS. (B) _ ^ 5Polyadenylated RNAs (10 ,g) from mature unchallenged 4leaves of the tomato cuftivars Moneymaker (0) and Cf9 (9)were electrophoretically separated on a 1.4% agarose gel and _ 3capillary-blotted onto a Hybond N filter. The filter was probedwith 2P]dCTP-labeled probe 2 (Fig. 2) and washed with high . . ....stringency at 650C in 0.1 x saline sodium phosphate EDTA g 2and 0.1% SDS.

Table 1. Comparison of the extracytoplasmic LRRs of Cf-9, PGIPs, andseveral membrane-spanning LRRproteins (12). The consensus LRR s shownfor each protein, with frequent amino acid substitutions indicated below(dashes indicate any amino acid). Abbreviations: GP, platelet glycoprotein;LH, eutenizinghormone; CG, chorogonadotropin; MA,membrane-anchored

transmembrane proteins with only a very short cytoplasmic domain; S, se-creted; TM, transmembrane proteins with substantial cytoplasmic domains;7M, seven membrane-spanning domains; PGs, polygalacturonases; vWf,vonWillebrand actor; ser-thr PK, serine-threonine protein kinase; IL-1R, interleu-kin-1 receptor; and ABP, actin-binding protein.Protein LRR consensus Repeat Membrane Signaling Referencenumber association Liadmechanism

Cf-9 L--L--L--LDLSSNNL-G-IPS- 28 MA Avr9? Unknown This workF N FPGIPs L--L--L--L-LS-N-L-G-IP-- 10 S Fungal PGs - (19)RLK5 L--L--L--L-L--N-LSG-IP-- 21 TM Unknown Ser-thrPK (18)TMK1 L--L- -L- -L-L--N-L-G-IP-- 13 TM Unknown Ser-thr PK (18)TMKL1 L--L--L-SL-L--N-LSG-LP-- 7 TM Unknown Unknown (18)Toll (Drosophila) LF-H--NL--L-L--N-L--LP-- 15 TM Activated IL-1R-ike (25)spatzleproteinGP lba LL--LP-L--L-LS-N-LTTLP-G 7 TM Activated vWf ABP binding (26)(mammal)LH-CG receptor AF--L-----L-IS---L--LP-- 12 7M LH,CG G-protein- (27)(mammal) L I coupledSCIENCE * VOL.266 * 4 NOVEMBER994 791

8/2/2019 Jones et.al.1994

5/6

resistance o Pseudomonasyringae nd thetobacco N gene for resistance o tobaccomosaic virus have been reported o carryLRRs(20). However, these LRRs are notwellconserved, requite variablen length,and lack the conservedglycine of plantextracytoplasmic RRs.N issuggested o beacytoplasmic roteinand RPS2mayalsobecytoplasmic.N and RPS2 aremore homol-ogousto one another than to Cf-9. Con-ceivably, here are two distinctclassesof Rgenes with LRRs; those like Cf-9, whichhave extracellularigands,and those likeNandpossiblyRPS2,which have intracellularligands.The LRRdomainsof a number f recep-tors have been shown to bind proteinli-gands 21) (Table1).Thepresence f LRRsin Cf-9 is therefore onsistentwiththe ideathat Cf-9 acts as a receptor or a specificprotein ligand,most likely the Avr9 pep-tide. However, the LRR region of Cf-9seemsexcessively argefor bindingof the28-amino acidAvr9 peptide to be its solefunction. An Avr9-bindingdomain mayoccupy only a small portionof the LRRregion,with the remainingLRRsprovidingstructure o the protein.Alternatively, helargeLRRdomaincouldreflect nteractionwith other Cf-9 moleculesor with anotherplant protein,perhapsone bindingAvr9.TheLRRmotif s areceptormodule hathas been combined with severaldifferentcytoplasmicsignalingmechanisms(Table

1). Unlike these receptors,Cf-9 does nothave any obviouscytoplasmic ignalingca-pacity.However,Cf-9 could generatea cy-toplasmic ignal by interactionbetween itstransmembraneomainor cytoplasmic ailand a cytoplasmic signaling mechanism.Structurally, f-9 resembles he membrane-boundreceptordomainof RLPKs ut lacksthe proteinkinasedomain.Conversely,hetomatoPtogenefor resistance o Pseudomo-nas syringae v. tomatoencodes a proteinkinase (16) resembling the membrane-boundkinasedomainof RLPKs utlackingthe receptordomain. Cf-9 and Pto mightrepresent omponentsof receptorand sig-naling mechanisms hat are analogoustoT cell activation by CD4, which has anextemal receptor domain, a transmem-branedomain,and a 38-amino acid cyto-plasmic domain (22). This cytoplasmicdomain interacts with a tyrosine proteinkinase, p561ck, that is attached to themembraneby an NH2-terminalmyristoyl-ation site (23), as postulatedfor the Ptokinase (16). Altematively, the cytoplas-mic tail of Cf-9 mayhave no interactivefunction at all but maybe simplyan an-choring domain required or attachmentof an extracytoplasmic protein to themembrane.Membrane ttachmentmaybenecessary o enable Cf-9 to interactwithanother membrane-bound xtracytoplas-mic componentthat is attached to a cy-toplasmicsignalingmechanism.

Additional genes, Rcr-l and Rcr-2,areneeded for full Cf-9 function (24) andmight encode proteins hat cooperatewithCf-9 in the production f a defense activa-tion signal.The next challenge s to discov-er the natureof the proteins hat cooperatewith Cf-9 to activate plantdefenses n re-sponse to Avr9.REFERENCESAND NOTES

1. T. Pryor and J. Ellis, Adv. Plant Pathol. 10, 281(1993); N. T. Keen, Annu. Rev. Genet. 24, 447(1992).2. G. F. J. M. Van Den Ackerveken, J. A. van Kan, P. J.G. M. De Wit, Plant J. 2, 359 (1992).3. C. M. T. Rommens et al., Plant Mol. Biol. 20, 61(1992).4. D. A. Jones, C. M. Thomas, K. E. Hammond-Ko-sack, P. J. Balint-Kurti, . D. G. Jones, unpublisheddata.5. D. A. Jones, M. J. Dickinson, P. J. Balint-Kurti,M.Dixon, J. D. G. Jones, Mol. Plant-Microbe Interact.6,348 (1993).6. S. R. Scofield, K. Harison, S. J. Nurrish,J. D. G.Jones, Plant Cell 4, 573 (1992).7. K. E. Hammond-Kosack, K.Harrison,J. D. G. Jones,Proc. Natl. Acad. Sci. U.S.A. 91, 10445 (1994).8. Two tagging experiments were done. Inexperimentone, two female tagging parents were increased to19 by propagation of cuttings. Fruits from crossesdone on up to 10 fruitingtrusses per plant wereharvested, and seed from each fruit was kept andsown separately to enable detection of clonally rep-resented mutation events. In experiment two, 140tagging parents were used. Fruits romcrosses doneon up to fourfruiting russes per plantwere harvest-ed, and seed from each fruit truss was kept andsown separately as above.9. To isolate Cf-9, genomic DNA from mutant 18 wascut with Bgl II,which does not cut within the Dselement, then recircularizedwith T4 DNA ligase andused to transform Escherichia coli by electropora-tion. The Ds element contained an origin of DNAreplication and a chloramphenicol resistance gene,both functional in E. coIl (3).The plasmid recovered,pM18B, carried 6.7 kb of tomato DNA, including2066 base pairs (bp) of the 5' end of an ORF trun-cated at the 3' end by the Bgl II ite used forplasmidrescue and interruptedby the Ds element 93 bp 3' tothe start codon. The remainder of the gene wasrecovered by plasmid rescue with the use of Xba I,which cuts within the Ds element but still allowedrescue of one end of the Ds and its flanking tomatoDNA, up to an Xba Isite about 14.5 kb 3' of the Bgl11 ite. The plasmid recovered, pM18X, carried anadditional522 bp of ORF 3' of the Bgl II ite. The tworegions of tomato DNA flanking the Ds in pM18Bwere subcloned in both orientations as Bam HI-BglIIfragments, with the use of Bam HI sites just insideeither end of the Ds, into the Bam HIsite of a pBlue-script polylinker.Deletion derivativeswere created byrestriction endonuclease digestion and religationwith the use of sites within the pBluescript polylinkerand the cloned DNA. These were sequenced withpBluescript or Ds primers. Sequencing over the re-striction sites used for making deletions, over se-quence gaps, and over the 3' end of the ORF con-tained inpM18X was completed with specific prim-ers. The region spanning the site of the Ds insertionwas polymerase chain reaction (PCR) amplifiedfrom Cf-9 by means of flanking primers-one 5' ofthe Xho Isite and the other 3' of the Sst Isite. ThePCR product was digested with Xho I and Sst I andcloned into pBluescript to generate pCf9XS, whichcontained the insert used as probe 1 (Fig.2 and Fig.4A). DNAsequencing confirmed the inferredtargetsequence.10. RNAwas prepared (6) from the cotyledons and firstleaves of 400 14-day-old, glasshouse-grown Cf9seedlings that had been injected with a concentra-tion of crude Avr9 peptide that was one-quarter ofthe minimum concentration required to induce ne-

Fig. 5. Sequence alignments be- NH2-terminal homologytween Cf-9 and homologous pro- A_ B-teins. Alignments for the NH - C mcvtvP TFLCQLALS9 LS*PN1pa A-SLLQFKNMFT 452 ~~~~~~~~~~TPtZI2VrS LLLVV1 ?L PAS P SLS VR IP K:: 39t --S DI}(domains A, B, and part of C) and PPGIP NELKFSTFLSLTLLFSSVLNPALS DLN7DDKKV --- L-QTKKAP-G 43TR1 MG_EARFLHwVTFFFV, .LH.CHCGT SLSGsSSD- -VXK -LL>G-K:XS S 1QG 4 6COOH-termini domain D and part T'lla KRDTFrLLSFTYLLLSLS KASDGD -SAM-SLKKSL-- 39of Cl of Cf-9 and the PGIPs/1olTMKlb C-terminlal of TMIXa ............ GE:CD}P---RKS->L-Lt-ASSF-- 335of C) of Cf-9 and the PGIPs (19), T~bT3a. EDRK-LLAS-/v'RLKS5 )MYCTI1LLLCLSSTY'_ P ? S ",,,%-DATLR> - ,G.L,S PAOSL- - 42represented by tomato (T) andpear (P)PGIPsand the RLPKs(18). Cf-9 NPFASDYCYORTYlVIQSYPRTL- S---S--WDVTCE 87TMK1has two regions of homology PPGIP N - PYVA 2- - -W-D---Y-WVKCR 63with the NH2-terminus f Cf-9, TMKL N- SESLLLS-S--W= VP----VCQi-WRGvKWV- 71TNKla N - - PPF-----SFGW--SDP-D--FPCK-WT H VT-- 61whichis consistentwith an NH2- Tlb D - --- YPPRLAES--W-KGN--D--PCTNWIGACSN 361terminalduplication.Thefirst, locat- RLK5 -------s-s--w- SDND PCK-WLGVSCDA 63ed at the NH2-terminus, is desig- LRRI _3 LRR2 62 ~~~~~~~Cf9 TT-GQVIAtL-,LC S QLQGK7H S jEL enr.L S _L_KpRO I --F\QGS 1 iSK13 6natedTMK1a, nd the second, lo- TPGIP KTNRTNA_TVF QA i I-PAAVC-DLPY ELEEFH- VaGT-IPPA 110cated between two blocks of L s PIP TTNRINSLTIFAGQVSGQIPA--LVGDLPYLETLEFHK-PNLGP- IQPA 114cato between twvo blvosof LRR8 -1l -yXDQSLSyNs7LLSQPAS 1and preceded by a short stretch of Tla GI! 106VTRIQ SGLQGT1SP3- - LPX,AELERLEL WGP - VP- 3 106TMlllb GEalj_- VI SLEKELr;TGTI SPE - -F-AI TKCL QRI; LG- - IN=GM- IPQE 4 a5hydrophobic residues, is designat- RLKR TSN-VS V:LSSF5LVG PFPS- - ILCNLPS LHSLS LY- ;IZ-LSA 108ed TMK1 . The site of signal pep- COOH-terminal homologytide cleavage (indicatedby a gap) is LRRn- - LRRn -W DCsimilarthroughout. Cysteines con- Cf-9 LESLDLSSN.KSGCEiPQlALTFL5V1NLSENN~LVGLCZPK-3--XKQF--D 763served at the NH 2-terminus but not T Ip P 1SLDLi.`HKT'1GSLPPYLF-PLN-EQFFL,V'-GQ>G!- -T-1- -Q 305at the COOH-terminusof Cf-9 are T3L VKSLLSS.FE-L 2VEGG E-LESLUUiING-PDFGESK-FuxE 273TEla LESLS3LPR:;NSPTGP'vPA-S; r S;LESLKWvK-',-N;2lSlwv7PVPss&XS S D-- 302highlighted ngreen. The conserved T9Xlb --QR I L$:N .BNLTGNIFQEL--TT.LPiN-LKT LDVSSNNKLIFGKVPGFRSN--V- -- 433LRR1 s truncated, begins with V or RLK5 L-N-YLDLSSNQFSGEI-PLrELQNL-KLN-VNLSYNNILSGK:FIPPLYANK-I--Y 595I ratherthan L,and lacks the char-actenstic N as compared with the TPGIP SFiYSYns- HN-K-anGd-GP 327other LRRs. The LRR backbone, PPGIP SFDEYSYF-HN-RCLCGAPLPSCK 330TNIri SFE----- GN;S P C-L-GSS? ..... :M and SN doma-ns 295LXXLXXLXXLXLXXNXLXGXIPXX TIala SVDLDKD--SNSFCL-SSP ............. N-term-na_ of T1Mih 318TK1lb VVWNN----GN- PDIGKDK. .. ..SIG, T and PS domains 447(12), nwhichL s oftenreplacedby RLK h HD----FIGN-PGILCVDLDGLCRKITRSKN. .. TM and PS domtains 621I, F, or V, and I by L, F, or V, ishighlighted in blue. Othersequence identities are highlightedin red. N-glycosylationsites are underlinedand those conserved in LRR2and LRRnare highlightedin purple.Abbreviations:Tm, transmembrane:PK, protein kinase; S/G, serine-glycine--rich.792 SCIENCE * VOL.266 * 4 NOVEMBER994

8/2/2019 Jones et.al.1994

6/6

REPORTScrosis within 24 hours. Eighty seedlings were har-vested 0, 3, 6, 12, and 24 hours after injection. Anequal mass of total RNA taken at each time pointwas pooled, and polyadenylated [poly(A)+]RNA asprepared by oligo(dT)cellulose chromatography. AnAmersham cDNA cloning kit was used to prepareand clone cDNA as described by the manufacturer.Eco RI adapters were ligated to double-strandedcDNA and subsequently ligated to Eco RI-digestedarms of the phage vector Xgt10. The phage waspackaged and used to infect E coli C600 cells. Ap-proximately 5 x 105 recombinant plaques weretransferred o Hybond N+ membranes and screenedwith probe 1 (9). FiveCf-9 cDNA clones were recov-ered, and the full-length nserts were cloned as BamHI fragments into pUC119 and sequenced withprimersderived fromthe genomic sequence of Cf-9and withprimers lanking he polylinker loning site ofpUC119. The full-length nsert of one of these cloneswas used as probe 2 (Fig.2 and Fig. 4B).11. G. von Heijne,Nucleic Acids Res. 14, 4683 (1986).12. Single-letter abbreviations for the amino acid resi-dues are as follows: A, Ala;C, Cys; D, Asp; E, Glu;F,Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N,Asn; P, Pro;Q, Gln; R, Arg; S, Ser; T, Thr;V, Val;W,Trp;and Y, Tyr.13. F. M.Townsley and H. R. B. Pelham, Eur.J. Cell Biol.64, 221 (1994).14. G. von Heijne and Y. Gavel, Eur.J. Biochem. 174,671 (1988).

15. T. Pryor,Trends Genet. 3, 157 (1987); M. A. Sudu-pak, J. L. Bennetzen, S. H. Hulbert,Genetics 133,119 (1993).16. G. B. Martin t al., Science 262, 1432 (1993).17. The Brookhaven Protein Data Bank, SWISS-PROT,Protein IdentificationResource, and GenPept pro-tein databases were searched withthe BlastP algo-rithmof S. F. Altschul, G. Warren, W. Miller,E. W.Myers, and D. J. Lipman [J. Mol. Biol. 215, 403(1990)].18. J. C. Walker,Plant J. 3, 451 (1993); C. Chang et al.,Plant Cell4,1263 (1992); C. Valon, J. Smalle, H. M.Goodman, J. Giraudat, Plant Mol. Biol. 23, 415(1993).19. H. U. Stotz, J. J. A. Contos, A. L. T. Powell, A. B.Bennett, J. M. Labavitch, Plant Mol. Biol. 25, 607(1994); H. U. Stotz et al., Plant Physiol. 102, 133(1993); P. Toubart et al., Plant J. 2, 367 (1992).AntirrhinumFIL2 M.Steinmayr, P. Motte, H. Som-mer, H. Saedler, Z. Schwarz-Sommer, ibid. 5, 459(1994)] also showed homology to Cf-9 but is includ-ed among the PGIPs because of its extensive ho-mology withthem.20. A. F. Bent et al., Science 265,1856 (1994); M. Min-drinos, F. Katagiri,G. Yu, F. M. Ausubel, Cell 78,1089 (1994); S. Whithamet al., ibid., p. 1101.21. T. Braun, P. R. Schofield, R. Sprengel, EMBOJ. 10,1885 (1991); J. Ware etal., J. Clin.Invest. 92, 1213(1993); N. Suzuki et al., Proc. Natl. Acad. Sci. U.S.A.87, 8711 (1990); J. Field et al., Science 247, 464(1990).22. B. Tourvieille,S. D. Gorman, E. H. Field,T. Hunka-piller,J. R. Parnes, Science 234, 610 (1986).23. A.Veillette,M. A. Bookman. E.M.Horak,J. B. Bolen,Cell 55, 301 (1988).24. K. E. Hammond-Kosack, D. A. Jones, J. D. G.Jones, Plant Cell 6, 361 (1994).25. C. Hashimoto, K. L. Hudson, K. V. Anderson, Cell52, 269 (1988); S. Roth, Curr.Biol. 4, 755 (1994).26. R. K.Andrews and J. E. B. Fox, J. Biol. Chem. 267,18605 (1992); J. A. Lopez et al., Proc. Natl. Acad.Sci. U.S.A. 84, 5615 (1987).27. H. Loosfelt et al., Science 245, 525 (1989); K. C.McFarlandet al., ibid., p. 494.28. We thank C. Rommens and J. Hille or making avail-able the Ds stock used; S. Perkins,J. Darby,and M.Shailer for plantcare; P. Bovill orassistance inseedprocessing; J. Maxon-Smith for producing some ofthe test cross seed under contract, and A. Cavill orcarrying out some of the DNA sequencing undercontract. Supported by AFRCPMBgrants 522, 523,and PG83/570; by an EECBRIDGEgrant;and by agrant to the Sainsbury Laboratory rom The GatsbyCharitableoundation.7 September1994;accepted4 October1994

Interaction of a Protein Phosphatase with anArabidopsis Serine-Threonine Receptor KinaseJulie M. Stone, Margaret A. Collinge, Robert D. Smith,Mark A. Horn, John C. Walker*

A protein phosphatase was cloned that interacts with a serine-threonine receptor-likekinase, RLK5,from Arabidopsis thaliana. The phosphatase, designated KAPP (kinase-associated protein phosphatase), is composed of three domains: an amino-terminalsignal anchor, a kinase interaction (KI)domain, and a type 2C protein phosphatasecatalytic region. Association of RLK5with the KIdomain is dependent on phosphorylationof RLK5and can be abolished by dephosphorylation. KAPPmay function as a signalingcomponent in a pathway involving RLK5.

Many signal transductionpathwaysin-volved in the controlof cell proliferationand differentiationoriginate with trans-membranereceptorscontaining cytoplas-mic protein kinase domains. Althoughmuchof the researchdone has focusedonreceptortyrosinekinases (RTKs) (1), re-ceptorserine-threoninekinaseshave beenidentifiedaswell. These includethe trans-forminggrowth actorM andactivinrecep-tor superfamily 2) and all known recep-tor-likekinases fromhigherplants (3, 4).We report here the identification of aprotein phosphatase that interacts withthe phosphorylated form of a putativeplant receptor serine-threonine kinase.This interaction is reminiscent of themechanismsby which RTKsactivate cel-lularsignalingevents. The signaling pro-cess of RTKs includes recognition of apolypeptide igand, dimerization f the re-ceptor, and autophosphorylationof ty-rosineresidues n the cytoplasmicportionof the molecule.Thesephosphorylatedy-rosines with their flanking amino acidsserve as high-affinitybindingsites forcel-lularproteinscontaining Src homology2(SH2) domains (5). Therefore,activationof RTKs eadsto the formationof proteincomplexes at the plasmamembrane hatare capableof transmitting ignalsto thenext molecule in the signal cascade.The RLK5 enefromArabidopsishalianaencodesa proteinwithfeatures haracteris-tic of the polypeptidegrowth actorrecep-tor kinases:a largeNH2-terminal xtracel-lulardomain,a single transmembraneo-main,andaCOOH-terminal roteinkinasecatalytic domain (3). The protein kinasedomainofRLK5,whenexpressed sa fusionproteinin Escherichiaoli,autophosphoryl-ates exclusivelyon serine and threonineresidues 6).J. M. Stone, Department of Biochemistry, UniversityofMissouri-Columbia,Columbia, MO 6521 1, USA.M. A. Collinge, R. D. Smith, M. A. Horn, J. C. Walker,Division of Biological Sciences, Universityof Missouri-Columbia, Columbia, MO 6521 1, USA.*To whom correspondence should be addressed.

To identify omponents f a signal rans-ductionpathway nvolvingRLK5, nterac-tion cloning(7, 8) wasused.An ArabidopsiscomplementaryDNA (cDNA) expressionlibrarywasscreened orproteins hat inter-act withtheproteinkinasecatalyticdomainof RLK5(RLK5CAT).The probe used inthisfilter-binding ssaywasaglutathione-S-transferaseGST)-RLK5CATfusion pro-tein labeledwith 32P at a proteinkinaseArecognition ite at thejunctionof the fusion(8). A positive clone was purified,sub-cloned,andsequenced 9). The cDNA in-sertencodes a 239-amino aciddomainre-ferred o as the KIdomain.Sequencecom-parisonhasnot revealedanystronghomol-ogies with previouslyreportedsequences(10).The possibility that interaction be-tween the KI domain and RLK5 is phos-phorylation-dependentwasexploredby invitrobindingstudies.Analyses of protein-protein interaction on membranefilters(11) demonstrated hat the KI domain iscapableof bindingto RLK5CAT,which isautophosphorylatedn E. coli (6). Treat-ment with a type 1 serine-threoninepro-tein phosphatase,ZmPP1(12), abolishedthe interaction, whereas phosphatasetreatment n the presenceof okadaicacid,an inhibitor of type 1 protein phospha-tases, did not interfere with the interac-tion. Furthermore,he KIdomaindoes notbind a mutant form of RLK5 hat is inca-pable of autophosphorylation(Fig. 1).These resultsshow that associationof theKIdomainwith RLK5requiresphosphor-ylation. Furthermore,he KIdomain doesnot indiscriminately bind phosphopro-teins, including32P-GST,or an autophos-phorylatedreceptor-likekinase from Zeamays(13).Full-lengthcDNA clones were identi-fied by screeningof an ArabidopsisDNAlibrarywith a nucleic acid probe corre-sponding to the KI domain. The full-length cDNA encodes a 582-amino acidprotein with a predictedmolecularweightof 65 kD (Fig. 2A). The NH2-terminus

SCIENCE * VOL. 266 * 4 NOVEMBER1994 793