A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana

JournalofPlantPhysiology169 (2012) 1017–1022

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JournalofPlantPhysiology

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AnovelSec14phospholipidtransferproteinfromNicotianabenthamianaisup-regulatedinresponsetoRalstoniasolanacearuminfection,pathogenassociatedmolecularpatternsandeffectormoleculesandinvolvedinplantimmunity

AkinoriKibaa, ,MasahitoNakanoa,PatrickVincent-Popeb,HirotakaTakahashic,TatsuyaSawasakic,YaetaEndoc,KouheiOhnishid,HirofumiYoshiokae,YasufumiHikichia

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LaboratoryofPlantPathologyandBiotechnology,FacultyofAgriculture,KochiUniversity,Nankoku783-8502,Japan

DepartmentofCellandDevelopmentalBiology,LinebergerComprehensiveCancerResearchCenter,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC27599-7090,USAc

EhimeUniversity,Cell-FreeScienceandTechnologyResearchCenter,DivisionofProteomedicalSciences,Cell-FreeScienceandTechnologyResearchCenter,Japand

ResearchInstituteofMolecularGenetics,KochiUniversity,Nankoku783-8502,Japane

LaboratoryofDefenseinPlant–PathogenInteractions,GraduateSchoolofBioagriculturalSciences,NagoyaUniversity,Chikusa-ku,Nagoya464-8601,Japan

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Articlehistory:

Received17January2012

Receivedinrevisedform5April2012Accepted5April2012

Keywords:NbSEC14

NicotianabenthamianaPlantimmunity

Ralstoniasolanacearum

Sec14phospholipidtransferprotein

Toelucidatethemolecularmechanismsofplantimmuneresponses,weisolatedgeneswhoseexpressionwasregulatedbyinoculationwithRalstoniasolanacearum.Here,wereportthecharacterizationofNico-tianabenthamianabelongingtotheSEC14-genesuperfamilydesignatedasNicotianabenthamianaSEC14(NbSEC14).NbSEC14rescuedgrowthdefectsandimpairedinvertasesecretionassociatedwiththeyeastsec14ptemperature-sensitivemutant,whilerecombinantNbSec14proteinhadphospholipidstransferactivity.NbSEC14expressionwasup-regulatedinN.benthamianaleavesafterinoculationwithviru-lentoravirulentR.solanacearum.ExpressionofNbSEC14wasinducedbytreatmentwithchitin, g22,andbyAgrobacterium-mediatedtransientexpressionofINF1elicitin,AvrAfromR.solanacearum,andco-expressionofthecapsidproteinfromTobaccomildgreenmosaicviruswithitscognateresistanceL1protein.NbSEC14-silencedplantsshowedacceleratedgrowthofboththevirulentandavirulentR.solanacearumaswellasaccelerationofdiseasedevelopment.ThisstudymayprovideusefulinformationforthefurtheranalysisofthefunctionofplantSec14proteinhomologsintheregulationofplantimmuneresponses.

© 2012 Elsevier GmbH. All rights reserved.

Introduction

Ralstoniasolanacearumisadevastatingsoil-bornepathogenwithaglobaldistributionandawidehostrange.Intomatoes,resistancetoR.solanacearumiscontrolledbyseveralloci(Thoquetetal.,1996),whereasinArabidopsisthaliana,itismonogenicandconferredbytheRRS1-Rgenethatencodesanovelresistanceprotein(Lahaye,2002).Recently,PopP2,thecognateavirulence

Abbreviations:ETI,effector-triggeredimmunity;HAI,hourafterin ltra-tion;HR,hypersensitiveresponse;NbSEC14,NicotianabenthamianaSEC14;SFHs,Sec14homologues;PAMPs,pathogenassociatedmolecularpatterns;PATL,patelinproteins;PC,phosphatidylcholine;PCR,polymerasechainreaction;PI,phos-phatidylinositide;PITP,phosphatidylinositol/phosphatidylcholinetransferprotein;PIPs,phosphoinositides;PTI,PAMPStriggeredimmunity;qRT-PCR,quantitativereversetranscription-polymerasechainreaction;Sec14p,Sec14protein;SSHs,soy-beansec14homologues.

Correspondingauthor.Tel.:+81888645196;fax:+81888645196.E-mailaddresses:akiba@kochi-u.ac.jp,akiba@cc.kochi-u.ac.jp(A.Kiba).

proteinforRRS1-R,wasidenti edandshowntointeractwiththeRRS1-Rprotein(Deslandesetal.,2003).AlthoughtherehavebeenextensiveeffortstoidentifyresistancegenestoanalyzeR.solanacearum–plantinteractions,littleisknownaboutthemolec-ulareventsinplantsduringtheestablishmentofresistanceorsusceptibilitytoR.solanacearum.Recently,weemployeddiffer-entialdisplaytoisolatefragmentsfromgenesthatareregulatedintobaccoplantsinoculatedwithR.solanacearum.Weidenti edR.solanacearum-responsivegenes(RsRGs)involvedinsignaltrans-duction,defense/stressresponses,andcellularmetabolism,aswellasgeneswithunknownfunctions(Kibaetal.,2007).Wehaveusedthevirus-inducedgenesilencing(VIGS)methodtoscreenforgenesinvolvedinR.solanacearum–plantinteractionsusingNicotianaben-thamianaandthepotatovirusX(PVX)vectorsystem(Maimboetal.,2010).Here,wedescribetheidenti cationandcharacteriza-tionoftheRsRG920gene,whichencodesaproteinwithaputativeSec14motif(NbSEC14),whichbelongstothelargeyetuncharac-terizedSec14protein(Sec14p)superfamily.Sec14pwasoriginallyisolatedfromSaccharomycescerevisiaeandfunctionsasaninterface

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