2017-Stegmann-The receptor kinase FER is a RAL.pdf

REPORTPLANT SIGNALINGThe receptor kinase FER is aRALF-regulated scaffold controllingplant immune signalingMartin Stegmann,1Jacqueline Monaghan,1*Elwira Smakowska-Luzan,2Hanna Rovenich,1 Anita Lehner,3Nicholas Holton,1Youssef Belkhadir,2Cyril Zipfel1In plants,perception of invading pathogens involves cell-surface immune receptor kinases.Here,we report that the Arabidopsis SITE-1 PROTEASE(S1P)cleaves endogenous RAPIDALKALINIZATION FACTOR(RALF)propeptides to inhibit plant immunity.This inhibition ismediated by the malectin-like receptor kinase FERONIA(FER),which otherwise facilitatesthe ligand-induced complex formation of the immune receptor kinases EF-TU RECEPTOR(EFR)and FLAGELLIN-SENSING 2(FLS2)with their co-receptor BRASSINOSTEROIDINSENSITIVE 1ASSOCIATED KINASE 1(BAK1)to initiate immune signaling.We show thatFER acts as a RALF-regulated scaffold that modulates receptor kinase complex assembly.A similar scaffolding mechanism may underlie FER function in other signaling pathways.Plant immune pattern recognition receptors(PRRs)are often receptor kinases(1).TheArabidopsisthaliana(hereafter,Arabidopsis)receptor kinases FLAGELLIN-SENSING 2(FLS2)and EF-TU RECEPTOR(EFR)bindbacterial flagellin(or the epitope flg22)and EF-Tu(or the epitopes elf18/elf24),respectively,and formligand-induced complexes with their co-receptorBRASSINOSTEROIDINSENSITIVE1ASSOCIATEDKINASE 1(BAK1)(1).To decipher the negative regulation of plantPRR-mediated immune signaling,we screenedfor modifier of bak1-5(mob)mutants that regainimmune responses in the immunodeficient mu-tant background bak1-5(2).Here,we report thecharacterization of MOB6.bak1-5 is impaired in the production of re-active oxygen species(ROS)upon flg22 or elf18treatment(3).We identified the recessive bak1-5mob6 mutant on the basis of restoration of thisresponse(fig.S1A).To map the mob6 locus,wesequenced bulked F2segregants from a back-crossbetweenbak1-5mob6andbak1-5combinedwith phenotypic and genetic analysis of an F2population from a cross between bak1-5 mob6andCol-0seethesupplementarymaterials(SM).WefoundahomozygousmissensemutationwhereSer replaces Pro612(P612S)in At5g19660 encod-ing the subtilase SITE-1 PROTEASE(S1P)/SBT6.1(fig.S1,B and C).Allelism tests and transcomple-mentation assays confirmed that the mob6 phe-notype is caused by a mutation in S1P(fig.S1,Dand E).We thus renamed mob6 as s1p-6.We characterized the effect of mutant S1P al-lelesonimmunesignaling.s1p-3ands1p-6singlemutantsproducedmore ROSinresponse toelf18,flg22,andchitin(fig.S1,DandE;fig.S2A;andfig.S3)and exhibited increased expression of the im-mune marker genes FRK1 and PHI1 upon elf18treatment(fig.S2B).Also,s1p-3ands1p-6mutantsweremoreresistanttothehypovirulentbacteriumPseudomonassyringaepv.tomato(Pto)DC3000coronatine-minus(COR)strain(fig.S2C).Thus,S1P is a negative regulator of immunity.Similartoitshumanortholog,theArabidopsissubtilaseS1Pprocessessubstrateswiththecanon-icalcleavagesiteRxxL/RxLx(whereRisarginine,L is leucine,and x is any amino acid)(4).Theendogenouspeptide RAPID ALKALINIZATIONFACTOR23(RALF23;At3g16570)anestablishedS1P substrate inArabidopsis(5)isa majorhubin a flg22-regulated transcriptional network(6).Consistent with a potential role of RALF23 in im-munity,elf18 treatmentorinoculationwith thetype IIIdeficient strain Pto DC3000 hrcCrap-idly increased the processing of the propeptidePRORALF23(fig.S4,AandB),inanS1P-dependentmanner(fig.S4C).RALF23 treatment led to a dose-dependentinhibitionofelf18-inducedROSproductionmedianinhibitory concentration(IC50)200 nM(Fig.1Aand fig.S5)and resistance to Pto DC3000(Fig.1B).Furthermore,RALF23overexpression(7)inhibitedelf18-triggered ROS production and increasedsusceptibility to Pto DC3000 COR(Fig.1,C andD)and to the fungus Plectosphaerella cucu-merina(7).Conversely,loss of RALF23(fig.S6A)led to increased elf18-triggered ROS production(Fig.1E)and resistance to Pto DC3000 COR(Fig.1F).RALF23 similarlyinhibitedflg22-triggeredROS production(fig.S7).Furthermore,treatmentwith RALF23 suppressed the heightened elf18-induced ROS production in s1p-6 mutants(fig.S8),which suggests that the negative regulatoryfunction of S1P is executed by the processing ofRESEARCHStegmann et al.,Science 355,287-289(2017)20 January 20171 of 31The Sainsbury Laboratory,Norwich Research Park,Norwich,NR4 7UH,UK.2Gregor Mendel Institute(GMI),AustrianAcademy of Sciences,Vienna Biocenter(VBC),1030 Vienna,Austria.3Protein Technologies Facility,Vienna Biocenter CoreFacilities(VBCF),Vienna,Austria.*Present address:Biology Department,Queens University,Kingston,Ontario K7L 3N6,Canada.Present address:Laboratory ofPhytopathology,Wageningen University,6708 PB Wageningen,Netherlands.Corresponding author.Email:cyril.zipfeltsl.ac.ukFig.1.The endogenous peptide RALF23 nega-tively regulates immunity.(A)ROS productionin Col-0 leaf discs treated with 100 nM elf18,1 mM RALF23,or cotreatment all in 2 mM 2-(N-morpholino)ethanesulfonic acidpotassium hy-droxide(MES-KOH),pH 5.8.Values are meansof total photon counts over 30 min SE,n=16.Letters indicate significance in one-way analysisof variance(ANOVA)(a and b,P 0.001;a to c,P 0.001;b and c,P 0.05).Kinetics are shownin fig.S18A.(B)Colony-forming units(cfu)of PtoDC3000 after syringe inoculation in leaves pre-treated with mock treatment,1 mM elf18,1 mMRALF23,or cotreatment(all in 2 mM MES-KOH,pH 5.8)for 24 hours,determined 2 daysafter inoculation.Values are means SD,n=4(one-way ANOVA;P 0.05).(C and E)ROSproduction after elicitation with 100 nM elf18 orwater.Values are means of total photon countsover 30 min SE.Letters indicate significance inone-way ANOVA(a and b,P 0.001;a to c,P 0.001;b and c,P 0.05).Kinetics are shown infig.S18,B and C.(D and F)Number of PtoDC3000 CORbacteria determined 3 days aftersurface inoculation.Values are means SD,n=4(one-way ANOVA;P 0.05).Similar results wereobtained in three independent experiments.on December 28,2018 http:/science.sciencemag.org/Downloaded from RALF23 or related peptides.For example,thecloselyrelatedRALF33(At4g15800)peptide(figs.S9A and S10A)(8,9)could also inhibit elf18-induced ROS production(figs.S6B;S9,B and C;and S10B).We conclude thatRALF23,aswellasRALF33,negatively regulates immunity.The Arabidopsis genome encodes about 35RALF peptides(8,9).Only 11 RALFs(includingRALF23 and RALF33)display a S1P cleavage site(fig.S10A).Cotreatment with RALF34(contain-ing a predicted S1P cleavage site)inhibitedelf18-induced ROS burst to the same extentas RALF23 and RALF33,whereas RALF32(lack-ing a predicted S1P cleavage site)did not(fig.S10B).This suggests that S1P-cleaved RALFsinhibit immunity.RALF23,RALF33,and the moredivergent RALF32 triggered seedling growth inhi-bition(fig.S10C)in a way similar to RALF1(9).However,RALF32 does not affect elf18-triggeredROS production(fig.S10B).All RALF peptidespreviously tested induced alkalinization(8,10),suggesting that immune inhibition is not a gen-eral property of RALFs and is independent of thealkalinization activity,which is consistent withour bioassays involving RALF peptides being per-formed under buffered conditions(SM).The Arabidopsis malectin-like receptor kinaseFERONIA(FER;At3g51550)was recently identi-fied as a receptor for RALF1(9).RALF1,RALF23,andRALF33arecloselyrelated(fig.S9AandS10A)and have overlapping gene expression patternswith FER(fig.S11)(8,9).fer-2 and fer-4 mutantswereinsensitivetotheinhibitoryeffectofRALF23orRALF33 peptide on elf18-induced ROSproduc-tion(Fig.2A and fig.S12A),which was comple-mented in a fer-4/FERGREEN FLUORESCENTPROTEIN(fer-4/FER-GFP)line(Fig.2A).Fur-thermore,RALF23 overexpression in fer-2 had noeffectonelf18-inducedROSproduction(fig.S12B).The genetic dependence of FER in RALF23-,RALF33-,and RALF32-triggered growth inhi-bition(fig.S10C)suggested that FER may bindadditional RALF peptides.Biotinylated RALF23 bound to recombinantFER ectodomain(ectoFER)but not the unrelatedEFR receptor ectodomain(ectoEFR)(Fig.2B).Similarly,ectoFER expressed and purified frominsect cells bound to biotin-RALF23 but not biotin-elf24(Fig.2C),with dissociation constant(Kd)values 300 nM and 600 to 900 nM,as re-vealed by microscale thermophoresis and iso-thermal titration calorimetry,respectively(Fig.2Dand fig.S13),which is consistent with valuesreported for other ligand-receptor kinase pairs(11,12)and the IC50for RALF23-mediated inhi-bition of elf18-triggered ROS production(fig.S5).Thus,in addition to RALF1(9),FER is also a re-ceptor for RALF23.FER is enriched in detergent-resistant mem-branes after flg22 treatment(13).The fer-2 andfer-4 mutants were hyposensitive to elf18,flg22,and chitin(Figs.2A and 3A and figs.S12 andS14)and were more susceptible to Pto DC3000COR(Fig.3B),indicating that FER can alsopositively regulate immunity.FER weakly asso-ciates with both FLS2 and BAK1,with the latterbeing strongly enhanced upon flg22 treatment(Fig.3C).Flg22-induced FLS2-BAK1 complex for-mation was reduced in fer-4 and restored in fer-4/FER-GFP(Fig.3D).Cotreatment with RALF23reduced ligand-induced FLS2/EFR-BAK1 com-plex formation(Fig.3,E and F).RALF23 over-expression had a similar effect on flg22-inducedStegmann et al.,Science 355,287-289(2017)20 January 20172 of 3Fig.2.RALF23-mediatedinhibitionofimmunityisFERdependent.(A)ROS production after treatmentwith 100 nM elf18 alone or cotreated with 1 mM RALF23(all in 2 mM MES-KOH,pH 5.8).Values are meansof total photon counts over 40 min SE,n=16.Letters indicate significance in one-way ANOVA(a and b,P 0.001;a to c,P 0.05;b and c,P 0.01).Kinetics are shown in fig.S18D.(B)In vitrobinding assay with maltose-binding protein(MBP)ectoFER or MBP-ectoEFR purified from E.coli.Pull-down assay for immunoprecipitation(IP)was done with neutravidin beads;Western blots(WB)wereprobed with antibody against MBP(a-MBP).(C)In vitro binding assay with ectoFERV5-6xHis(ectoFERtagged with V5 antibody epitope and hexahistidine)produced in insect cells.Pull-down was performedwith streptavidin beads;Western blots were probed with antibody against V5(a-V5).(D)Quantitativebinding analysis using synthetic RALF23 peptide and ectoFERV5-6xHis using microscale thermopho-resis.Similar results were obtained in three independent experiments,except for(C),where the assayswere performed twice with identical results.Fig.3.FER is a RALF-regulated scaffold forimmunereceptorcomplexes.(A)ROS productionafter elicitation with 100 nM elf18 or water.Valuesare means of total photon counts over 30 min SE,n=8.Letters indicate significance in one-wayANOVA(a and b,P 0.05;b and c,P 0.05;b tod,P 0.05;a to c,P 0.001;a to d,P 0.001).Kinetics are shown in fig.S18E.(B)Number ofPto DC3000 CORbacteria 3 days after surfaceinoculation.Values are means SD,n=4(one-way ANOVA;P 0.05).(C to F)Coimmunopre-cipitation of(C)FER-GFP from fer-4/FER-GFP;(D)FLS2 from Col-0,fer-4,or fer-4/FER-GFP;(E)FLS2-GFP from Col-0/FLS2-GFP;or(F)EFR-GFP fromefr/EFR-GFPseedlings treated with or without theindicated peptides(100 nM flg22,100 nM efl18,1 mM RALF23,or water)for 10 min.Western blotswere probed with antibodies a-GFP,a-BAK1,anda-FLS2.CBB,Coomassiebrilliantblue.Similarresultswere obtained in three independent experiments.RESEARCH|REPORTon December 28,2018 http:/science.sciencemag.org/Downloaded from FLS2-BAK1 complex formation(fig.S15).RALF23or the loss of FER did not affect accumulation ofFLS2,EFR,or BAK1(Fig.3,D to F,and fig.S15).Thus,ligand-induced complex formation betweenFLS2/EFR and their co-receptor BAK1 is pro-moted by FER and inhibited by RALF23.Our data suggest that FER acts as a scaffoldto regulate immune receptorcomplex formation.FER may reside in plasma membrane micro-domains as part of preformed signaling“plat-forms,”together with receptors and co-receptors.Whether FER-mediated regulation intersects withother regulators of FLS2/EFR-BAK1 complex for-mation,such as BIR2 and IOS1(14,15),will bean interesting topic for future investigation.Two-thirds of RALF proteins lack a predictedS1P cleavage site and are devoid of a propeptideregion(fig.S10A).Treatment with one of these,RALF17,induced ROS production and acted ad-ditively to elf18(fig.S16,A and B).RALF17 pre-treatment was also sufficient to induce resistanceto Pto DC3000(fig.S16C).As RALF17-inducedROS production is dependent on FER(fig.S16B),the activity is not caused by a possible contam-ination with synthetic peptides(e.g.,flg22 or elf18)used in our laboratory.PRORALF23 is processed by S1P within min-utes of elicitor perception(fig.S4),which sug-gests a negative-feedback mechanism to inhibitthe scaffolding function of FER and to dampenimmune signaling.Fungal pathogens secrete pep-tides with homology to RALF23(16,17);thesefungal RALF orthologs may suppress immunityby inhibiting the formation of active receptorcomplexes.FER has emerged as a regulator of manybiological processes,ranging from fertilizationto inhibition of cell elongation and growth(18).Many of these processes involve receptor kinasesand co-receptors(19).This suggests that FERmay have a similar scaffolding function withinother receptor kinase complexes.Different,butoverlapping,expression patterns of FER and RALFgenes(fig.S11)(9,20)suggest that a variety ofFER-RALF modules may regulate diverse recep-tor kinase complexes during growth,development,or environmental sensing.REFERENCES AND NOTES1.D.Couto,C.Zipfel,Nat.Rev.Immunol.16,537552(2016).2.J.Monaghan et al.,Cell Host Microbe 16,605615(2014).3.B.Schwessinger et al.,PLOS Genet.7,e1002046(2011).4.C.Rautengarten et al.,PLOS Comput.Biol.1,e40(2005).5.R.Srivastava,J.-X.Liu,H.Guo,Y.Yin,S.H.Howell,Plant J.59,930939(2009).6.R.Niebergall,thesis(University of East Anglia,2012);https:/ueaeprints.uea.ac.uk/42367/.7.A.Dobn et al.,PLOS Pathog.11,e1004800(2015).8.A.Morato do Canto et al.,Plant Physiol.Biochem.75,4554(2014).9.M.Haruta,G.Sabat,K.Stecker,B.B.Minkoff,M.R.Sussman,Science 343,408411(2014).10.G.Pearce,Y.Yamaguchi,G.Munske,C.A.Ryan,Peptides 31,19731977(2010).11.J.Wang et al.,Nature 525,265268(2015).12.J.Santiago et al.,eLife 5,e15075(2016).13.N.F.Keinath et al.,J.Biol.Chem.285,3914039149(2010).14.T.Halter et al.,Curr.Biol.24,134143(2014).15.Y.-H.Yeh et al.,Plant Cell 28,17011721(2016).16.S.Masachis et al.,Nat.Microbiol.1,16043(2016).17.E.Thynne et al.,Mol.Plant Pathol.(2016).18.C.Li,H.M.Wu,A.Y.Cheung,Plant Physiol.171,23792392(2016).19.X.Ma,G.Xu,P.He,L.Shan,Trends Plant Sci.21,10171033(2016).20.E.Murphy,I.De Smet,Trends Plant Sci.19,664671(2014).ACKNOWLEDGMENTSThis research was funded by the Gatsby Charitable Foundation(C.Z.),the European Research Council(grant“PHOSPHinnATE”to C.Z.),the Austrian Academy of Science through theGregor Mendel Institute(Y.B.),the Deutsche Forschungsgemeinschaft(Fellowship STE 2448/1 to M.S.),the European Molecular BiologyOrganization and the U.K.Biotechnology and Biological SciencesResearch Council(Fellowships ALTF 459-2011 and BB/M013499 toJ.M.),and the Erasmus Mundus program(H.R.).We thankL.Stransfeld,the John Innes Centre horticultural service and TheSainsbury Laboratory tissue culture service for technicalassistance,and all