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Characterization of defense responses of susceptible and resistant tomato genotypes against bacterial wilt caused by Ralstonia solanacearum, a proteomic approach [Elektronische Ressource] / Diwakar Dahal

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Characterization of defense responses of susceptible and resistant tomato genotypes against bacterial wilt caused by Ralstonia solanacearum, a proteomic approach VonderNaturwissenschaftlichenFakultätderGottfriedWilhelmLeibnizUniversitätHannoverzurErlangungdesGradeseinesDoktors der Naturwissenschaften !Dr.rer.nat.!genehmigteDissertationvonM.Sc. Diwakar Dahal geborenam4.Dezember1974inNepal 2009 Supervisor 1. Prof. Dr. Kerstin Wydra InstituteofPlantDiseasesandPlantProtectionGottfriedWilhelmLeibnizUniversitätHannover,HerrenhäuserStr.230419Hannover,GermanyPresentaddress:CentreforTropicalandSubtropicalAgricultureandForestry(CeTSAF)Georg!AugustUniversitätGöttingen,Buesgenweg137077Göttingen,GermanyTel:0049!(0)511!393909,Fax:0049!(0)511!394556Email:kerstin.wydra@mail.uni!goettingen.deCo!supervisors1.Prof. Dr. Andreas PichInstituteofToxicology(Massspectrometry)MedizinischeHochschule,Carl!Neuberg!Str.130625Hannover,GermanyTel:0049!(0)511!5322808,Fax:0049!(0)511532!2879Email:pich.andreas@mh!hannover.de2.Prof. Dr. Hans-Peter BraunInstituteofPlantGenetic(PlantProteomics)GottfriedWilhelmLeibnizUniversitätHannover,HerrenhäuserStr.230419Hannover,GermanyTel:0049!(0)511!7622674,Fax:0049!(0)511!7623608Email:braun@genetik.uni!hannover.

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Published 01 January 2009
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Characterization of defense responses of susceptible and resistant
tomato genotypes against bacterial wilt caused by Ralstonia
solanacearum, a proteomic approach




VonderNaturwissenschaftlichenFakultät
derGottfriedWilhelmLeibnizUniversitätHannover
zurErlangungdesGradeseines



Doktors der Naturwissenschaften
!Dr.rer.nat.!
genehmigteDissertation
von


M.Sc. Diwakar Dahal
geborenam4.Dezember1974inNepal


2009
Supervisor
1. Prof. Dr. Kerstin Wydra
InstituteofPlantDiseasesandPlantProtection
GottfriedWilhelmLeibnizUniversitätHannover,HerrenhäuserStr.2
30419Hannover,Germany
Presentaddress:
CentreforTropicalandSubtropicalAgricultureandForestry(CeTSAF)
Georg!AugustUniversitätGöttingen,Buesgenweg1
37077Göttingen,Germany
Tel:0049!(0)511!393909,Fax:0049!(0)511!394556
Email:kerstin.wydra@mail.uni!goettingen.de
Co!supervisors
1.Prof. Dr. Andreas Pich
InstituteofToxicology(Massspectrometry)
MedizinischeHochschule,Carl!Neuberg!Str.1
30625Hannover,Germany
Tel:0049!(0)511!5322808,Fax:0049!(0)511532!2879
Email:pich.andreas@mh!hannover.de
2.Prof. Dr. Hans-Peter Braun
InstituteofPlantGenetic(PlantProteomics)
GottfriedWilhelmLeibnizUniversitätHannover,HerrenhäuserStr.2
30419Hannover,Germany
Tel:0049!(0)511!7622674,Fax:0049!(0)511!7623608
Email:braun@genetik.uni!hannover.de

Tag der Promotion: Dezember01,2009

DECLARATION BY CANDIDATE




I, Diwakar Dahal, hereby declare that this thesis, entitled "Characterization of defense
responses of susceptible and resistant tomato genotypes against bacterial wilt caused by
Ralstonia solanacearum, a proteomic approach"isanoriginalworkconductedbymyself
andhasnotbeensubmittedforadegreeinanyotheruniversity.




DiwakarDahal
GottfriedWilhelmLeibnizUniversitätHannover
December2009,Hannover












IZusammenfassung
ZUSAMMENFASSUNG

Bakterielle Welke, verursacht durch Ralstonia solanacearum, ist eine der verheerendsten
bakteriellen Erkrankungen an Kulturpflanzen in den Tropen und Subtropen. Der Anbau
resistenterSortenbleibtdiepraktikabelsteMaßnahme,welchebeiEingliederungimRahmen
eines integrierten Pflanzenschutzkonzeptes, eine Bekämpfung in einigen Regionen
ermöglicht.Dennoch,dieResistenzistnichtstabilundderMechanismusderResistenzauf
molekularer Ebene weitestgehend ungeklärt. Daher wurden zur Aufklärung von möglichen
Resistenzmechanismen in diesen Arbeiten molekulare und biochemische Methoden.
verwendet. Wir analysierten die Protein!Profile von anfälligen und resistenten Genotypen
vonSolanum lycopersicumbeiBefallmitR. solanacearum.
ZunächstwurdedasProteomdesgesamtenmittlerenStängelabschnittsuntersucht,indemin
vorangehendenArbeitenderArbeitsgruppeResistenzmechanismengegenBakterielleWelke
lokalisiert worden sind Nur die anfälligen Pflanzen reagierten auf die Inokulation des
PathogensmitunterschiedlicherRegulationderdetektierbarenProteine,welchenFunktionen
inanderPathogenabwehr,StressantwortundimMetabolismuszugeschriebenwurden.Die
SensitivitätderMethodewurdedurchAnalyseeinerSubfraktiondesGewebes,desZellwand!
Proteoms des mittleren Stängelabschnitts, weiter erhöht. Ebenso konnten konstitutive,
genotypische Unterschiede zwischen zwei S. lycopersicum Linien, die sich im Grad der
Resistenz gegen R. solanacearum unterscheiden, erfolgreich identifiziert werden
(Primärstoffwechsel! , Abwehr! und Stress!induzierte Proteine ). Unterschiede in der
Proteinregulierung wurden in S. lycopersicum Genotypen auch nach Inokulation mit dem
Pathogen festgestellt: In dem anfälligen Genotyp waren nach Induktion durch Infektion
Pathogenese!assoziierte (PR)!Proteine stärker exprimiert, wohingegen bei der resistenten
LinieProteasenundSignalproteineauftraten.WeiterhinkonnteeineverminderteExpression
vonStress!induzierten!undantioxidativwirksamenProteinenbeidenresistentenGenotypen
ermitteltwerden,währendbeideranfälligenLinieProteinedesZellwandstoffwechselsinder
Interaktion mit dem Pathogen herunterreguliert waren. Proteine des Primär! und
Energiestoffwechsels zeigten unterschiedliche Expressionsstärken in beiden Genotypen.
IIZusammenfassung
Schließlich wurde das Proteom des Xylemsaftes, eine weitere wichtige Schnittstelle der
Pflanze!Pathogeninteraktion,erstmalsindiesemZusammenhanganalysiert.Diesesbeinhaltet
einumfangreichesNetzvon208ProteinenundermöglichteinenÜberblickderFunktionen
des Xylemsaftes in einer Gefäßpflanze. Der Vergleich der Xylem!Proteome von gesunden
Pflanzen zweier unterschiedlich resistenter Genotypen zeigte einen höheren Prozentsatz an
Proteasen, Peroxidasen und anderen an der Verteidigung beteiligten Proteinen in den
resistentenPflanzenauf,wohingegenbeianfälligenPflanzenderAnteilvonProteinenhöher
war,welcheinSignalwegeundTranskriptionsfaktoreninvolviertsind.
Zusammengefasst zeigt die vorliegende Arbeit konstitutive Unterschiede zwischen
resistentenundanfälligenS. lycopersicumGenotypenaufProteom!EbeneimXylemsaftund
in den Zellwänden des Stängels. Weiterhin wurden pathogen!induzierte Differenzen in der
Proteinexpression sowohliam mittleren Stängelabschnitt als auch auf Zellwandebene
festgestellt. Die Ergebnisse liefern einen wichtigen Beitrag für das Verständnis der
Resistenzmechanismen der Tomate gegen bakterielle Welke und können in
Züchtungsprogrammenverwendetwerden.
Schlüsselwörter:
Bakterielle Welke von Tomaten / Proteomics und die Massenspektrometrie / Ralstonia
solanacearum /SekretorischProteine/Stängels,ZellwandundXylemsaftesproteome









IIISummary
SUMMARY

Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating bacterial
diseases in the tropics and subtropics. Use of resistant cultivars remains the most useful
individual control measure which, after incorporation in a framework of integrated disease
management, provides good control in some regions. However, the resistance is rather
unstableandthemechanismofresistanceatthemolecularlevelislargelyunclear.Therefore,
weinitiatedinvestigationsonthemolecularlevelofresistancemechanismbyanalyzingthe
protein profiles that are specific to susceptible and resistant tomato genotypes against
bacterialwiltcausedbyR. solanacearum.
The proteome was examined first from the whole mid!stem, where resistance mechanisms
hadpreviouslybeenreportedafterrootinoculation.Onlythesusceptibleplantsrespondedto
pathogen challenge by differentially regulating their proteins, which were identified as
pathogenesisaswellasstressrelatedandmetabolicproteins.Thesensitivityoftheanalysis
wasfurtherincreasedbystudyingthecellwallproteomefrommid!stems,andsuccessfully
revealed genotypic differences primarily metabolic, defence and stress related proteins
between the two genotypes. Similarly, plants of both genotypes showed the differential
regulation of proteins in response to pathogen inoculation. PR proteins in susceptible and
proteaseaswellasasignalingproteinsinresistantplantswereupregulatedwhereasstress
related proteins as well as an antioxidant in resistant and cell wall metabolic proteins in
susceptible genotypes showed down regulation during the interaction. Proteins of primary
and energy metabolism also displayed differential regulation in both genotypes. Finally,
xylemsap,anotherkeysiteforplant!pathogeninteraction,wereanalyzedwhichforthefirst
time demonstrated as many as 208 proteins. They included large networks of proteins
providinganoverviewofthexylemsapfunctionsinavascularplant.Thecomparisonofthe
xylemproteomeofhealthyplantsoftwogenotypesalsodisclosedthehigherpercentageof
protease,peroxidaseandotherdefenserelatedproteinsinresistantplants,whilesusceptible
plantscontainedmainlysignalingandtranscriptionrelatedproteins.
IVSummary
In conclusion, the present study provided constitutive differences in tomato genotypes of
variable degrees of resistance on proteome level in the xylem sap and stem cell walls.
Additionally, pathogen!induced differences in whole stem as well as in stem cell wall
proteome present an important contribution to understanding of bacterial wilt resistance in
tomato. The results give valuable information for future breeding programmes and genetic
improvementoftomatobacterialwiltresistance.
Key words:
Bacterial wilt of tomato / Proteomics and mass spectrometry / Ralstonia solanacearum
/Secretoryprotein/Stem,cellwallandxylemsapproteome














VTable of contents
TABLE OF CONTENTS

DECLARATION BY CANDIDATE ...................................................................................... I
ZUSAMMENFASSUNG ....................................................................................................... II
SUMMARY ........................................................................................................................... IV
TABLE OF CONTENTS ..................................................................................................... VI
ACKNOWLEDGEMENT ...................................................................................................... X
ABBREVIATIONS ............................................................................................................... XI
LIST OF TABLES ............................................................................................................. XVI
LIST OF FIGURES .......................................................................................................... XVII
GENERAL INTRODUCTION .............................................................................................. 1
BACTERIALWILTANDITSCAUSATIVEAGENT.........................................................................1
DISEASEMANAGEMENTANDRESISTANCEMECHANISM...........................................................3
PROTEOMICS...........................................................................................................................5
GELELECTROPHORESISANDMASSSPECTROMETRY................................................................6
RESEARCHFRAMEWORK.........................................................................................................7
CHAPTER 1: PATHOGENESIS AND STRESS RELATED, AS WELL AS
METABOLIC PROTEINS ARE REGULATED IN TOMATO STEMS INFECTED
WITH RALSTONIA SOLANACEARUM. ........................................................................... 10
1.1INTRODUCTION...............................................................................................................11
1.2MATERIALSANDMETHODS.............................................................................................14
1.2.1Plantmaterialandbacterialstrain...........................................................................14
1.2.2Plantgrowthconditionsandinoculation.................................................................15
1.2.3Bacterialquantification...........................................................................................15
1.2.4Monitoringandevaluationofdiseasesymptoms....................................................15
VITable of contents
1.2.5Stemproteomeanalysis..........................................................................................16
1.2.5.1Proteinextractionandsamplepreparation.......................................................16
1.2.5.2Two!dimensionalgelelectrophoresis..............................................................17
1.2.5.3Proteinstaining,gelscanningandimageanalysis...........................................18
1.2.5.4Massspectrometricanalysisanddatainterpretation.......................................18
1.3RESULTS.........................................................................................................................19
1.3.1Symptomdevelopmentandbacterialpopulationsinstems....................................19
1.3.2Analysisofthestemproteome................................................................................20
1.3.3 Characterization of tomato proteins induced after inoculation with R.
solanacearum...................................................................................................................22
1.4DISCUSSION.....................................................................................................................26
1.4.1Proteinsinvolvedinplantdefence..........................................................................28
1.4.2Proteinsinvolvedinplantstress.............................................................................29
1.4.3Proteinsinvolvedincarbohydratemetabolism.......................................................29
1.4.4Proteinsinvolvedinenergyproduction..................................................................30
1.5CONCLUSION...................................................................................................................30
CHAPTER 2: ANALYSIS OF CELL WALL PROTEINS REGULATED IN STEM OF
SUSCEPTIBLE AND RESISTANT TOMATO GENOTYPES AFTER
INOCULATION WITH RALSTONIA SOLANACEARUM, A PROTEOMIC
APPROACH .......................................................................................................................... 32
2.1INTRODUCTION................................................................................................................33
2.2MATERIALSANDMETHODS............................................................................................34
2.2.1Plantmaterialandinoculumpreparation................................................................34
2.2.2Plantgrowthconditionsandinoculation.................................................................35
2.2.3Bacterialquantification...........................................................................................35
2.2.4Diseasesymptomsevaluation.................................................................................35
2.2.5Proteinextractionfromcellwallsoftomatostems................................................35
2.2.6Proteinseparationwith2!Dand3!DSDS!PAGE..................................................36
2.2.7Proteinstaining,gelscanningandimageanalysis..................................................37
VIITable of contents
2.2.8Trypticdigestion.....................................................................................................37
2.2.9 Matrix assisted laser desorption and ionization!time of flight tandem mass
spectrometry(MALDI!TOFMS/MS).............................................................................38
2.2.10Electrosprayinonization(ESI)iontrapMS..........................................................38
2.3RESULTS.........................................................................................................................39
2.3.1Symptomdevelopmentandbacterialpopulationsinstems....................................39
2.3.2Cellwallproteinanalysis........................................................................................40
2.3.2.1Proteinregulationinresistantgenotype...........................................................42
2.3.2.2Proteinregulationinsusceptiblegenotype......................................................45
2.3.2.3Proteinvariationingenotypiccomparison......................................................48
2.3.3ResolutionofcellwallproteinsatbasicpIrange...................................................51
2.3.4Predictionofsecretionsignals................................................................................52
2.4DISCUSSION.....................................................................................................................54
2.4.1Expressionofplantdefensemechanisms...............................................................54
2.4.2Changeincellwallmetabolism..............................................................................57
2.4.3Metabolicactivitiesalteration.................................................................................58
2.4.3.1Variationinprimarymetabolism.....................................................................58
2.4.3.2Suppressionofenergymetabolism..................................................................59
2.4.4Variationinotherproteins......................................................................................60
2.4.5Constitutivedifferencesintomatogenotypes.........................................................61
2.4.6Natureofcellwallproteins.....................................................................................62
2.4.7Resolutionofbasicproteins....................................................................................63
2.5CONCLUSION...................................................................................................................63
CHAPTER 3: HIGH-THROUGHPUT EXPRESSION PROFILING OF XYLEM SAP
PROTEOME OF SUSCEPTIBLE AND RESISTANT TOMATO GENOTYPES
REVEALED NETWORKS OF METABOLIC, DEFENSE AS WELL AS CELL WALL
RELATED AND SIGNALLING PROTEINS.................................................................... 64
3.1INTRODUCTION................................................................................................................65
3.2MATERIALSANDMETHODS............................................................................................67
VIII