Analysis of Tomato spotted wilt virus effector-triggered immunity

D. de Ronde

Research output: Thesisinternal PhD, WU

Abstract

ResistanceinCapsicumagainsttheTomatospottedwiltvirus(TSWV),typespeciesof the Tospovirusgenuswithinthe Bunyaviridaefamily,employsthe singledominant resistancegeneTsw.Thisresistance hasmeanwhilebeenbrokenbyresistance breaking (RB) TSWV isolates and is causing increasing problems in many different (Capsicumcultivating)countries.Theresearchdescribedhereaimedtoidentify andcharacterise theviralproteintriggeringTswresistanceandprovidefurther insightintothemechanismofTsw-mediatedresistance.Knowledgegainedfrom thegeneticandphenotypiccharacterisationofTsw-resistancebreakingisolateswas usedtodevelopdiagnosticmarkersfordetectionofTsw-breakingpathotypesin fieldcultivations.

TheNSsRNAsilencingsuppressor(RSS)proteinwasidentifiedastheavirulence determinant ofTsw-mediatedresistance(Chapter2).WhiletheNSsproteinfrom theTSWVresistanceinducer(RI)isolatewasactiveasRNAsilencingsuppressorand avirulencedeterminant,theNSsproteinfromtwodifferentTSWVRBisolateslacked bothfunctionsasevidencedfromtransientassays.Surprisingly,thecorresponding resistancebreakingvirusisolatesstillexhibitedRNAisuppressoractivity. Noneof the other viral proteins were able to aid in the transient recovery of RSS activity. Electrophoreticmobilityshift assays(EMSAs)usingplantextractscontaining transientlyexpressedNSsproteinsshowedashift ofsiRNAswithNSsRI,indicative forbinding,butnotwithNSsRB.InagreementwiththelocalleafRSSassaysusinga virusinfection,plantextractsofvirusinfectedleaveswereabletoshiftthesiRNAs, showing recovery of the RSS activityduring virus infection.

 

 

The linkage of RNAi suppression and avirulence in NSs was further investigated bymutationalanalysis(Chapter3).AlargesetofNSsmutantswasgeneratedusing alaninesubstitutions ofauthenticTSWVNSsaminoacidsandwastestedfortheir abilitytotriggerTsw-mediatedHRandabilitytosuppressRNAi.Theseassaysshowed thatthe N-terminaldomainofNSscarried mostimportantresiduesinvolvedwith bothactivities. However,singlemutationscouldbeintroducedthatdisruptedone function,whilemaintainingtheotheroneandviceversaindicatingthatRSSactivity andavirulencewerenotfunctionally linked.SwappingofdomainsbetweenNSsRI andNSsRB  notonlyconfirmedtheimportanceoftheN-terminaldomainbutalso thespecificitywithintheTSWVspecies,sincedomainswapsbetweenNSsRIandNSs fromGRSV,arelatedbutdistinct Tospovirus,couldnottransfertheAvrphenotype toGRSV.MutationofaGW/WG-motifintheNterminalregionofNSsRI  leadtoa lossofbothfunctionsandindicatedthatthismotif, knowntobeinvolvedinAGO1 interactionof other viral RSS, was of biological relevance for TSWV NSs.

Theputativeinteraction ofAGO1andNSswasinvestigatedbyusingdifferent approaches to co-immunoprecipitate (Co-IP) on transiently co-expressed tagged- AGO1and(His-)NSs(Chapter4).Initialindicationsforsuchinteraction were obtained,howeverfurthersupportforthisputativeinteraction willhavetocome fromcomplementaryexperiments,e.g. Yeast-2-hybrid (Y2H), FRET-FLIM or BiFC.

Severaladditional TSWVisolateswereanalysedthatbesidestheknownresistance inducing-and resistance breaking-phenotype showed a temperature-dependent phenotype(Chapter5).IsolatesclassifiedtothistypeexhibitedanRIphenotypeat standardgreenhouseconditions (~22°C)whileatelevatedtemperatures(≥28°C), butstillbelowtemperaturesthatinactivatedtheR-geneproduct(≥31°C),wereable tobreaktheresistance.Viruschallengingassaysatvariousconditionsindicatedthat inductionofTswresistanceatalower temperaturebythesesocalledtemperature dependentresistancebreakingisolates(TempRB)involveddenovosynthesisofthe avirulenceprotein,i.e.NSs,andthat proteinfoldingmight play arole. NSsproteins clonedandexpressedfromthisadditional newsetofTSWVresistanceinducing, resistancebreakingandtemperature dependentresistancebreakingisolates revealedvariableresultsregardless oftheircorrespondingvirusphenotype,when tested for their abilitytoinduceTsw-mediated HR andsuppress RNAi at normal greenhouseconditions(22°C).However,similarassaystoanalysetheiractivity attheelevatedtemperature(28°C)failedwhenusingAgrobacteriummediated transientassays.Sofar,themechanismoftemperature dependencyhasnotbeen clarified yetandneedsfurtherinvestigation.Usingtheinformationobtained,a diagnostictoolwasdevelopedtoscreenforthepotential presenceofresistance breakingisolatesofTSWVusingreversetranscription-polymerasechainreaction amplification(RT-PCR).Aprimersetwasdesignedtargetinganimportantcodon ataaposition79andshowedtobeabletodistinguishRB-isolatesfromRI-isolates. However,afewRB-isolatesstillescapedfromdetection indicatingthelimitedand conditionaluse of this tool.

In  summary,  NSs  has  been  identified  as  Avr-determinant  of  Tsw-mediated resistance,butthisfunctionisnottightlylinkedtoitsRNAisuppressor-activity.  Preliminarydataindicateaputativeinteraction betweenAGO1andNSs.Besides the typicalRIandRBphenotypes,athirdphenotypicclassofTSWVisolates has beenidentified thatexhibitsatemperaturedependencyontriggeringTsw- mediatedresistance andpossiblyinvolvesanalteredproteinfoldingofNSs.A diagnostic toolhasbeendevelopedtodetectresistancebreakingisolatesinthe fieldbasedonRT-PCR,butthistoolstillallowsforescapesofRBisolates.Theresults onNSsarediscussedinlightofitsroleaseffectorwithinthe‘Zig-zag-model’of planthostdefenceresponses.Finally,TSWVNSsisbriefly discussedandcompared totheanimal-infecting(NSs)paralogsoftheBunyaviridaefamily,alsoinlightof functional andstructuralhomologiesbetweenthesensorsofinnateimmunityin plant(R-genes)and animal (NLRs/TLRs) cell systems.

 

 

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Vlak, Just, Promotor
  • Kormelink, Richard, Co-promotor
Award date8 Nov 2013
Place of PublicationS.l.
Print ISBNs9789461737212
Publication statusPublished - 2013

Keywords

  • tomato spotted wilt virus
  • plant viruses
  • disease resistance
  • immunity
  • virulence
  • gene mapping
  • genetic markers
  • genetic analysis
  • capsicum annuum
  • sweet peppers

Fingerprint Dive into the research topics of 'Analysis of Tomato spotted wilt virus effector-triggered immunity'. Together they form a unique fingerprint.

Cite this