Modeling and experimental determination of infection ... › content › 10.1101 ›...

TITLE1 Modelingandexperimentaldeterminationofinfectionbottleneckandwithin-hostdynamicsofasoil-2 bornebacterialplantpathogen.3 4 AUTHORS5 GaofeiJiang1,2,#,RémiPeyraud2,#,PhilippeRemigi3,AliceGuidot2,WeiDing1,StéphaneGenin2and6 NemoPeeters2*7 8 ORCIDs:9 GJ:0000-0002-5331-739X10 RP:0000-0002-7580-042X11 PR:0000-0001-9023-378812 AG:0000-0001-5282-415713 WD:0000-0001-8536-523314 SG: 0000-0003-2498-240015 NP:0000-0002-1802-076916 17 AUTHORAFFILIATION18 1-LaboratoryofNaturalProductsPesticide,CollegeofPlantProtection,SouthwestUniversity,19 Chongqing,China.20 2-LIPM,UniversitédeToulouse,INRA,CNRS,Castanet-Tolosan,France21 3-NewZealandInstituteforAdvancedStudy,MasseyUniversity,Auckland,NewZealand.22 Tel:(33)561285592;Fax:(33)561285461.23 #Contributedequallytothiswork24 25 CORRESPONDINGAUTHOR26 [email protected] 28 KEYWORDS29 Infectionfoundingpopulation,bottleneck,populationdynamics;modelling;Tomato;Ralstonia30 solanacearumspeciescomplex;bacterialwilt;host-microbeinteraction31 32

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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 29, 2016. . https://doi.org/10.1101/061408doi: bioRxiv preprint

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ABSTRACT(250words)34

Thesoilisknowntobeaverymicrobe-richenvironement.Plantrootsaresurroundedbyacomplex35

microbiotaamongwhich,inwarmclimates,thepathogenicbacteriabelongingtotheRalstoniasp.36

speciescomplex.Weusedacombinationofmathematicalmodellingandexperimentalplant37

infectionmethods,mimickingthenaturalconditions,todefinethekeyparametersdescribingthe38

infection,colonizationandwiltingofthehostplant(“bacterialwilt”disease).Importantly,ourmodel39

takesintoaccountthepossibilityfortheorthologousre-infectionofalreadyinfectedplants.We40

showedthatitisthecaseinourexperimentalsetupandlikelytoalsohappeninnatura.Wewere41

abletomodelandexperimentalymeasuretheplantinfectionbottleneck,underthesenon-forced42

infectionconditions.Wethenquantifiedtowhatextendtheplantnaturalbarrierscanrestrict(upto43

50times)thisbottlenecksize.Wealsomeasuredtheimportanceofthebacterialmainvirulence44

determinants(typeIIIeffectors)toallowinginfection,asthereisareductionofthebottleneckby7045

timeswhenthetypeIIIarsenalisabsent.Wefurthervalidatedthemodelbypredictingastrain’s46

caracteristicsusingonlyafewexperimentallydeterminedparameters.Finallytheanalysisofthe47

globalcolonizationdynamicsallowedanaccurateassessmentoftheinplantabacterialload48

triggeringdisease.49

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SIGNIFICANCESTATEMENT(120words)52

Animalsandplantshaveacomplexcohabitingmicrobiota.Thesemicrobescanbeeitherbeneficialor53

deleterioustothehost.Wehavemodeledandexperimentallymeasuredtheintimateinteraction54

betweenaplanthost(tomato)andapathogenic,soil-associatedbacteriabelongingtotheRalstonia55

sp.speciescomplex.Wewereabletoevaluatehowmanybacteriaareattheoriginoftheinfection56

anddeterminehowtheplantandthebacteriacanimpactonthisbottleneck.Furtherwithin-host57

bacterialdynamicsanalysisallowedtomeasureatwhatinfectionstageaplantwouldbecome58

symptomatic.Sinceourexperimentalconditionallowsnaturalinfection,aswellasre-infectionof59

alreadyinfectedplants,ourglobalinfectionmodelshouldprovefaithfulltotheinnaturaconditions.60

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INTRODUCTION62

Assessileorganisms,plantscan’textractthemselvesfromtheirenvironment.Plant’snatural63

openings(likestomata,hydathodesandrootpermeability)arerequiredforthegazandsolute64

exchangebetweentheorganismanditsnutrient-providingenvironments(air,waterandsoil).Atthe65

sametime,theseenvironmentsarepopulatedbyadiversemicrobiota,representingaconstant66

sourceofplantinoculumofbeneficial,commensalbutalsopathogenicmicrobes.Rootsare67

particularlyexposedassoilisknowntobethemostmicrobe-richenvironment(1).68

Notallpathogenicmicrobespresentinagivenenvironmentwillleadtoasuccessfullplant69

infection.Indeedinfectionandfurthercolonizationisdependentonthepathogen(e.g.virulence70

arsenal),thehost(e.g.developmentalandimmunitytraits)andtheenvironmentalconditions(e.g.71

temperature,hygrometry),aswellasontheinteractionsbetweentheseparameters.These72

constraintsthatsharplylimittheinfectingpopulationsizecreatebottlenecksthatwilldrastically73

influencepopulationdynamicsandtheevolutionofplantpathogens(2).Indeed,thebacterial74

genotypesthatfoundtheinfectingpopulationinthehostplant(i.e.thefounders)willdeterminethe75

geneticmakeupofsubsequentgenerationswithinthehostandalsooffutureinfections.Areduction76

ingeneticvariabilityislikelytocreateafoundereffectwithinthepost-bottleneckpopulation(2).77

Moreover,plantsareinconstantinteractionwiththemicrobiotacommunity,sourceofthepathogen78

populationintheirenvironment.Thisisespeciallytrueforplantroots,inclosecontactwiththe79

abundantsoilmicrobiota,thuslikelytofacere-infections(i.e.homologousmicrobialinfection)or80

superinfection(heterologousmicrobialinfection)(3).81

Infectionbottleneckshavebeenevaluatedbydifferentmeansinseveralhost-microbe82

systems.Themethodologiesusedrangefromtheanalysispost-infectionofafewmarkedquasi-83

species(4,5),severalwild-typeisogenictaggedstrainsorWITS(6),orlargesequencetag-based84

barcodedpopulation(7).Increasingthenumberoftagsisparticularlysuitedforanimalinfection85

systems,asfewerindividualsareneeded.Noneofthesestudies,aimingatquantifyinginfection86

lbottlenecks,actuallyusenaturalinfectionroutesbyexposingtheindividualhoststoaninfectious87


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microbiota.Anotherlayerofcomplexitycomesfromthedynamicsofbacterialpopulationswithinthe88

host.Aglobal,quantitativeanalysisoftheseriesofeventsoccurringfrominfectiontocolonization89

andideallytodisseminationiscurrentlylackingformosthost-microbialpathogenssystems(3).The90

exactassesmentofalltheseparametersandtheirinteractionsshouldallowtoidentifythekey91

parametersofaninfectionandunderstandtheirinfluenceonpopulationstructureofpathogenic92

agents(8).Furthermorethebotteleckpredictionandexperimentaldeterminationwasperformed93

undernon-forced,naturalinfectioncondition,includingthepossibilityofsubsequentre-infection.94

Here,wequantifiedspecificandkeyparametersofaplantpathogenicinteractionusinga95

diseasemodelingapproachandthen,predictandexperimentalmeasuredthesizeoftheinfection96

bottleneck(ornumberoffounders)ofthepathogen.Thisworkwasconductedonthemodel97

bacterialsoil-borneplantpathogenRalstoniasp.tounderstandtheglobalparametersofplant98

infectionandcolonizationbyasoil-bornepathogen(9).Thisbacteriuminfectitshoststhroughthe99

rootsandfurthermoreitispossibletocompleteitsfulllifecycleinareasonableamountoftime:100

fromasoilinoculationmimickinganaturalinfectiontocompletewiltinganddecayofthehostplant101

(tomato)takeslessthan2weeks.Inthiswork,wereliedonthissimpleinfectionprocedurefollowed102

bydiseasescoringandbacterialloadmeasurementstomodelthewholelifecycleofthepathogen.103

Toourknowledge,ourstudyisthefirstthatprovidesaglobalmodelingofthewithin-hostpopulation104

dynamicsforaplantbacterialpathogen.Furthermorethebottleneckpredictionandexperimental105

determinationwasperformedundernon-forced,naturalinfectioncondition,includingthepossibility106

ofsubsequentre-infection.107

RESULTS108

Infectionprocessmodelingrevealsmultiplefactorsshapingtheinfectionbottlenecksize.109

ThelifecycleofpathogenicRalstoniasp.inhostplantcanbedividedinfivesubsequentsteps(Fig.1):110

1)rootinvasion,2)passagethroughtherootcortex,3)proliferationwithinthexylemvessels,4)a111

phenotypicswitch,inducingexopolysaccharide(EPS)production,and5)wiltingofaerialorgans112


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leadingtothekillingoftheplantandallowingpathogentransmissiontothesoil.Inordertoidentify113

howthosestepsinfluencetheinfectionfounders(F)uponaprolongedexposureoftheplanttothe114

pathogen(allowingsusequentre-infectionevents(3)),wedesignedamathematicalmodelthat115

reproducesthebacterialpopulation(B)dynamicsuponthese5steps(SeeSIAppendix,MethodS1,116

forthedetailsofthismodel).Weinvestigatedtheimpactofthemodelparametersontheinfection117

bottlenecksizeofRalstoniapseudosolanacearumstrainGMI1000.Fiveoftheseparametersleadtoa118

reductionoftheamountoffounderswhentheirvalueincreased.Theseparametersare:i)theplant-119

pathogenassociationconstant(K,Fig.2A),ii)thereductionofthemaximumentryflowovertime(z,120

Fig.2C),iii)thegrowthrateinxylem(µ,Fig.2D),iv)thequorumsensingthreshold(Q,Fig.2E),andv)121

thesteepnessofplantwiltingresponsetoEPSconcentration(α,Fig.2F).122

Overall,thenumberoffoundersisdeterminedbyatrade-offbetweenbacterialentryflow123

intotheroots(controlledbyK,Vfmaxandz)andthewiltingkinetics(controlledbyµ,Qandα).ForK124

andzthedecreaseofthenumberoffoundersisduetoareductionofaneffectiveentryflowofcells.125

Ontheotherhandincreasingparametersµ,Qandα,leadstoadelayedtransmissionphaseandthus126

reducingtheamountofbacteriaenteringfromsubsequentre-infections.However,themaximum127

numberofsuccessfulfounderscandramaticallyincreasethefinalfounderflowvalue(Vfmax,Fig.2B).128

Interestingly,thislastparameterrepresentsthestrengthoftheplantbottlenecks,i.e.theplant129

barrierandtheimmunesystemclearance.Hence,weidentifiedthatthestrengthofthesetwo130

blottleneckscanrepresentthehighestsourcesoffounders’variabilityevenifthewithin-host131

populationdynamicsalsoplaysarole.132

133

ExperimentalquantificationofkeyparametersofthetomatoinfectionbyRalstonia134

pseudosolanacearumstrainGMI1000135

Wesoughttoexperimentallydeterminethevaluesofthekeyparametersmentionedaboveinorder136

toquantitativelypredictthesizeoftheinfectionbottleneck.Wehypothesizedthatplantscouldbe137


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re-infectedbythesoil-inoculatedpathogen.Totestthisintomato,weperformednon-simultaneous138

inoculationswithtwoequallyvirulentstrains(SIAppendix,Fig.S1A)ofR.pseudosolanacearum,the139

wildtypestrainGMI1000andthegentamicin-resistantderivativeGRS540(SIAppendix,Fig.S2A).140

Tomatoplantsweresoil-inoculatedwithGMI1000andre-infectedwithGRS540at1,8,23and48141

hoursafterthefirstinoculation(hourspost-infectionorhpi).CheckthepresenceofGRS540inthe142

bacterialloadat5dpishowedthat60%oftheplantshadbeenre-infectedwhenthesecond143

inoculationoccurredat23hpi.Onlyfewre-infectionsweredetectedat48hpi.Hence,thecapacityof144

subsequentinfectiondecreasedovertimeatarateof2±0.1%h-1(Fig.3A).145

Next,themedianinfectiondose(MID)ofR.pseudosolanacearumwasquantifiedtoevaluate146

theinfectivityofR.pseudosolanacearumintomato(Fig.3B)usingthemethodofReedandMuench147

(10).TheMIDcorrespondstothebacterialinoculumsthattriggerdiseasefor,atleast50%,ofthe148

inoculatedplants(SIAppendix,Fig.S3).TheMIDgraduallydeclineduntil10dpitoanasymptotic149

valueof5.9·106cells(Fig.3B).ToensurethattheMIDisafeatureofthesoil-rootinterface,we150

performedasimilarassaybutusingsteminjectiontobypasstheroots(SIAppendix,Fig.S4).Here,151

theMIDstemwasonly4.1103bacteriaat5dpi(SIAppendix,Fig.S4D).152

Inordertoevaluatebacterialgrowthrateinthestem,thebacterialloadininfectedplants153

wasquantifiedatdifferenttimesfollowingrootinfection(Fig.4A).Fittingdatapointsbetweendays2154

and5yieldedanexponentialgrowthrateof0.196h-1±0.039(R2=0.42)(SIAppendix,Fig.5B).The155

weaknessofthecorrelationcoefficientsresultsfromtheintrinsicvariabilityofthetimingofthefirst156

infectionassociatedwithsoilinoculationsprocedures.Inordertoaddressthis,thesameexperiment157

wasrepeatedusingasteminoculationprocedure.Wefoundagrowthrateat0.233±0.012h-1(R2=158

0.928±0.024)(Fig.4B),whichisintherangeoftheinvitrogrowthrateinminimalmedium:0.253h-1159

±0.034(R2=0.97±0.02)(SIAppendix,Fig.S5A).160

Duringtheaboveexperiment(Fig.4A),thediseaseindex(DI)ofeachplantwasalsorecorded.161

ThisDIscoringusesa0to4scalerepresentingeachaquartileoftotalplantleafwilting;from25%162


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leaveswilted(DI=1),to100%,orcompletewilting(DI=4).Symptomaticplants(DI>0)havea163

bacterialloadrangingfrom108cfu·g(FW)-1to1011cfu·g(FW)

-1(Fig.5A),withthenotableexceptionofa164

fewdried-outplantsthathadreachedDI=4,severaldaysbeforebacterialloadassessment(SI165

AppendixFig.S6,S7).Thedatasetwasusedtoidentifytheinplantabacterialthresholdtriggering166

plantsymptoms.Weusedatwoclassclassifier,predictingwiltingonset(DI>0)ornotifthebacterial167

loadexceedathreshold(q)andcomparedthepredictorperformanceinexperimentalvaluefor168

variousthresholdlevels.TheMatthewscorrelationcoefficient(MCC)(11),whichisameasureofthe169

qualityofapredictor,wasusedtofindthebestthresholdvalue.AmaximumMCCvalueof0.896was170

obtained(SIAppendix,Fig.S8)forabacterialthresholdof6.107±0.97.107cfu·g(FW)-1.171

Wefurtherexploitedthislargedatasetofmorethan500individualinfectedplantsscored172

forbacterialloadandsymptomstoextrapolatethewiltingtimeresponsetotheEPSproductionin173

tomatoplants.Forthispurpose,allwiltingkineticsofdiseasedindividualsweresynchronisedtothe174

firstdaywhenwiltingsymptomswereobserved(Fig.5B).Thesteepness,α,ofthefittedwiltingtime175

responsecurvewas:0.612± 0.012.Then,wedeterminedusingtheequation4theinplantaEPS176

concentrationfromtheknownfluxofEPSproduction,VEPS,ofR.pseudosolanacearum(Peyraudetal.177

submitted)andthemeasuredbacterialloadandgrowthrate.ThisEPSconcentrationovertimewas178

usedtoassignthescallingfactor,β=2.31,toobtainthewiltingdoseresponsecurvetotheEPS179

concentration.180

Finally,wedeterminedthereductionoftheproliferation,µ-,duetothecollapseofthehost181

tissuesatlateinfection.Frommeasureddataofadvancedwiltingsymptomsandbacterialloadwe182

fittedavalueof0.25h-1.183

184

PredictionandexperimentalmeasurementoftheinfectionbottlenecksizeforRalstonia185

pseudosolanacearumstrainGMI1000186


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Theaboveexperimentallydefinedparameters,namelyK=5.9·106,z=0.02,µ=0.23,187

q=6.107,α=2.31,β=0.612andµ-=0.25,wereusedasinputinthemathematicalmodelinorderto188

predictthesizeoftheinfectionbottleneckforR.pseudosolanacearumstrain.Firstweestimatedthe189

maximumeffectiveentryflowoffounders,Vfmax,fromfittingthemodelpredictiontoexperimental190

wiltingonsetdata(extractedfromFig.4Adata).WefoundVfmaxtobeintherangeof5to22cells·h-1.191

Themodelpredictsthattheactualnumberoffoundersat7dpishouldbeintherangeof90to476192

foraninnoculumat5.107cell·ml-1. 193

Wethenexperimentallydeterminedthesizeoftheinfectionbottleneck,i.e.thenumberof194

founders(Ni)intomatousingasimplestatisticalmethodbasedontheco-infectionofGMI1000195

mixedwithminutefractions(from1%tolessthan0,1%)ofthegentamycin-markedstrainGRS540.196

Importantly,bothstrainswereshowntobeofequalfitnessinplanta(SIAppendix,Fig.S1).The197

probabilityofaninfectedplanttoalsocontainthemarkedstrainisdirectelycorrelatedwiththe198

proportionofthemarkedstrainintheoriginalinoculum(p<1%,seeMaterialsandmethods)andthe199

size(Ni)oftheinfectionbottleneck.Setsof32plantswereinoculatedwithagivenGMI1000/GRS540200

ratio(p).TheresultingproportionofplantsinfectedbyGRS540wasthenusedtogenerateasingle201

estimationofNithroughaprobabilisticanalysis(seeM&MandSIAppendix,Fig.S9).Afterextensive,202

60independentinoculationsofsetsof32plants,themedianofNiwasestimatedtobe458cells(SI203

AppendixFig.S10).WeobservedahighvariabilityofNiwithatwotimesstandarddeviationof2734204

cells.ThisvariabilitycouldreflectthetruebiologicalvariabilityofNi,howeveritmayalsoarisefrom205

thestochasticsamplingofthemarkedstraininherenttoourexperimentalsetup.Thus,inorderto206

evaluatethecontributionofastochasticsamplingofthemarkedstrainonNivariation,wesimulated207

arandomsamplingbasedonthesameparametersastheexperimentalsetup(numberofplants,208

concentrationofmarkedstrainininnoculum).ForatrueNivalueof458thestochasticityanalysis209

gaveamedianNiof454cells(SIAppendixFig.S10),withatwotimesstandarddeviationof507cells.210

Hence,weevaluatedthatthestochasticityofindividualsamplingduetoourexperimentalsetup211

couldrepresentupto18%(507/2734)ofthevariabilityobservedintheexperimentalNiestimation.212


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PlantphysicalbarriersandpathogentypeIIIeffectorsaremajordriversoftheinfectionbottleneck214

size.215

Weusedawounded-rootinoculationproceduretoestimatethecontributionofplantnatural216

rootbarrierstotheinfectionbottlenecksize.At4dpi,allwounded-rootplantshadsymptoms,217

indicatinganacceleratedinfectionandcolonization.Weusedthesamemethodologyofmixed218

inoculationstodeterminetheimpactofwoudingontheinfectionbottlenecksize(NWT-wounding).We219

wereabletoshowthatNWT-woudingdramaticallyincreasedtoamedianof24215cells(Fig.7,blue220

circles),significantlylargerthanNWT(medianof458cells)aspreviouslydefined(MannWhitneytest,221

P-value<0.0001).AnalysisofvariationinmodelparameterswhichfitssuchhighNivaluesand222

earliestsymptomsonset(4days),revealsthatahighentryflow,Vfmax,around1065cells·h-1likely223

occurredinadditiontothereductionofthedelaytime,d,tocrossrootcortex.224

Wethentreatedtomatoplantswithanhrpmutant(R.pseudosolanacearumstrainGMI1694,225

hrcVmutant)anditsisogenicgentamycin-markedstrain(GRS743,SIAppendixFig.S1B),toevaluate226

theimpactofacompletelossofbacterialtypeIIIsecretionontheinfectionbottlenecksize.Tomato227

plantswereharvestedat10dpiinordertomaximizethecolonizationofplantsbythehrpdefective228

R.pseudosolanacearumstrain.Here,theinfectionbottlenecksizeofthehrpmutant,Nhrp,was229

estimatedatamedianof6cells(andmeanof7±4cells,Fig.7,greensquares),significantlylower230

thanNWT(MannWhitneytest,P-value=0.0014).231

232

Usingthemodeltoinferlife-historytraitsofabacterialmutant233

Theresultsobtainedsofarindicatedthatourmodelisabletocorrectlypredictthesizeofthe234

infectionbottleneckfromafewkeyparametersthatweredeterminedexperimentally.Wewondered235

ifthemodelcouldbeusedwiththeoppositegoal,i.e.inferringastrain’scharacteristicsfroma236

measurednumberoffounders.InR.pseudosolanacearum,thetypeIIIsecretionsystem(orTTSS)is237


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requiredforbacteriatoenterxylemvessels(12).AmutantintheTTSSapparatus(hrpmutant)is238

unabletokillplants,butcansustainalimitedpopulationsizesinxylemvessels(13).239

Wethereforecombinedthewoundingprocedureandtheinoculationwiththehrpstrains.Samples240

wereharvestedtoestimatethenumberoffoundersofahrpmutantbywounding(Nhrp_wounding)at7241

dpi.Thefounderamountincreasedtoameanof62±40cellsandamedianof53cells.Themeanof242

Nhrp_woundingwasabout9timeshigherthanNhrp(P-value=0.019)but482timeslowerthanNWT_wounding243

(P-value=0.0006).244

Weusedthesedatatoinferthestrengthoftheimmunesystemclearanceontheentering245

bacterialcellsandtheinplantagrowthrateforGMI1694.Theentryflowoffounders,Vfmax(hrp_wounding),246

wasestimatedtobearound3cells·h-1.Thus,ifconsideringthattheentryflowinwoundedrootsof247

thehrpmutantissimilartothewt(1065cells·h-1)andthatthesuccesfullfounderflowcorresponds248

tothesurvivorstotheimmunesystemresponse,wefoundthatthestrengthoftheimmunesystem249

clearancewas99.7%oftheenteredcells.Comparisonbetweenthefoundingnumberofthewt,458,250

andthehrpmutantstrain,around6,leadstoasimilarvalue,98.7%.Then,weestimatedthe251

maximalgrowthrateofGMI1694byconsideringthatnosymptomsoccursandthusbacterialload252

remainsbelowthequorumsensingtreshold,q,forbothintactandwoundedrootconditions.We253

foundthatthemaximalgrowthrateinplantashouldbeintherangeof0.9to0.11h-1.We254

experimentallytestedthispredictionbymeasuringthegrowthrateoftheGMI1694straininstem255

xylemusingthesteminjectionprocedure(SIAppendix,Fig.S11)andfoundagrowthrateof0.112±256

0.005h-1,withR2=0.873±0.06.Hence,theclearanceofthehrpmutantbacteriabytheplant257

immunesysteminthestemwasevaluatedtobeatamaximumof52%.258

259


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DISCUSSION260

Ourobjectiveinthisworkwas(i)tomodeltheplantinfectionprocessandthewithin-hostbacterial261

populationdynamics,(ii)predictandexperimentalmeasuretheinfectionbottlenecksizefor262

R.pseudosolanacearumstrainGMI1000ontomatoandfinally,(iii)tounderstandthefactorsthat263

participatetothebacterialinfectionbottleneck.Forthispurposeweusedanatural,soil-drenching,264

plantinfectionbioassays.Plantswerescoredfordiseaseappearance(bacterialwilt)andbacterial265

load.Thelowestlevelofbacterialsoilsaturationtoobtainreliablewilting(>50%)inourconditions266

(12days),isasymptotictoavalueof6.106cfu·ml-1ofinoculum(seeFig.3B).Inourexperimental267

setupthisequatestoa4.6106cfu·g-1DryWeightsoilinoculum,comparabletothe105-107cfu·g-1Dry268

Weightofsoildetectedintherhizosphereofdiseasedtomatoplantsinthefield(14).Wealso269

showedthatthereisapotentialforsubsequentre-infection,overatimeframeof24-48h(seeFig.270

3A).Thesuccessofthesefurtherre-infectionsislikelytobedependentonthedensityofthelocal271

inoculumswhichcandeclineothertime(15)andontheinteractionbetweenthehostimmune272

responseandthebacterialvirulencearsenal.Indeed,theloweringofthere-infectionprobabilitywith273

time(seeFig.3A)couldbecorrelatedwiththeonsetofplantimmuneresponses(13).274

Weshowed(Fig.5A)thatasymptomaticplantsaredisplayingdifferentlevelsofbacterialcolonization275

whereasdiseasedplantsarealwayswellcolonized(>108cfu/gFW).Wefurtherrefinedthis276

relationshipbyanalyzingthelargedatasetofindividualplantsforwhichwehaveobtainedboththe277

diseasescoreandthebacterialcount.Wewereabletodeterminetheinplantabacterialload278

(6.107Cfu/gFW)thatwouldbethebestpredictoroflaterwilting(seeSIAppendix,Fig.S8).As279

Ralstoniasp.plantpathogenicstrainsarerestrictedtothexylemvesselsofplants,representing280

probablylessthana1/10ofthevolume/weightofastem,wecouldconsiderourinplanta281

experimentalvalidationasbeinginthesamerangeasthethreshold(107-108cfu/ml)determinedto282

triggerEPSproductioninvitro(16).283


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Inordertounderstandanddeterminealltheimportantparametersofthelifecycleofpathogenic284

Ralstoniasp.,webuiltamodelthatcapturesthegloballifecycleofthebacteriumfrominfectionto285

disseminationintheenvironmentaftercompletewiltingofthehostplant.Weexperimentally286

estimatedthesizeoftheinfectionbottleneck(i.e.thenumberoffounders)byaprobabilistic287

approachevaluatingthepresence/absenceofamarkedstraininthecolonizedplantsattheearliest288

stagepossible.Thisexperimentallydeterminednumberoffounders(medianNi=458cells)was289

obtainedaftertheanalysisofalargenumberofindividualplantsandisinthesamerangeastheone290

predictedfromthemodel(Ni=90to476).Toourknowledge,itisthefirstdeterminationofthehost-291

bacterialpathogeninfectionbottleneckthattakesintoaccountre-infectioninanexperimental292

systemmimickingnaturalinfection.293

Thequantificationofthisbottleneckimposedbytheplantonthebacterialpopulationpresentinthe294

soilisrequiredtoevaluatethepossibilityofa“foundereffect”.Thislatterisdefinedasabottleneck295

toonarrowtoallowthegeneticdiversityoftheinfectingpopulationtomirrorthegeneticdiversityof296

thesoilresidentpopulation,leadingtoapotentialgeneticdrift.TheRalstoniasolanacearumSpecies297

Complex(RSSC)hasawidespecies-to-continentgeographicdistribution(17).Somestudiesshowthat298

diversityanalysisofsingleisolatesfromdifferentinfectedplantscanyieldupto42-51haplotypes299

mainlyfrom2RSSCspecies(R.solanacearumandR.pseudosolanacearumformerlydefinedas300

phylotypesIIandI(17))overanarrowgeographicaldistribution(18,19).Sofarnostudieshave301

exploredtheRSSCpopulationdiversityinthesoilofplantedfields,thusitisnotpossibletoestablish302

whetherthesizeoftheinfectionbottleneckasdeterminedinthisworkfortheGMI1000strainwould303

perseresultinageneticdriftornot.Inaddition,inthisstudy,theinfectionbottleneckwasmeasured304

usingaclonalpopulation(individualsofthesameclone,GMI1000andisogenicmarkedstrain).305

However,inalocalnaturalpopulation,differentbacterialgenotypescouldalsocontributetothe306

infectionbottlenecksize(i.e.presenceofstrainswithdifferentvirulencearsenals,different307

metaboliccapacities).Studyingthecontributionofdifferentbacterialgenotypestotheinfection308

bottlenecksizewouldbeaninterestingquestionforfuturestudies.Aninterestingcorollaryof309


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bottleneckrestrictionduringinfectionisrelatedtokinselection.Indeedonecouldimaginethatthe310

restrictionimposedbythebottleneckwillprevent(orreduce)theinfectionbynon-kinbacteria.This311

wouldlimitthesuper-infectionbyopportunisticmicrobesorre-infectionbykin-cheaters,generating312

ahighlyrelatedinfectingpopulation(3,20).Studyingtheabilityofdifferentbacterialgenotypesto313

alterbottlenecksizesinisolationorwithindiverse(multi-strainormulti-species)communities314

will/wouldbeaninterestingquestionforfuturestudies.315

Inthisstudywealsoevaluatedtheeffectofplantandbacterialparametersonthesizeofthe316

infectionbottleneck.Asexpected,thebottlenecksizeincreasedsignificantly(>50times)after317

mechanicallywoundingtheroots.Wealsoshowedthatthisbottleneckwasdramaticallyreduced318

(>70times),whenplantswereinfectedwithahrpmutant,devoidofanytypeIIIeffectors,and319

knowntoproducesymptomlessbutcolonizedplants(13).ThislaterresultshowsthattypeIII320

effectorsarecollectivelyrequiredtoestablishthefirstinfectingfront,likepreviouslyshownfor321

specifictypeIIIeffectorsinaninvitroinfectionmodel(21,22).Wecanhypothesizethatoneofthe322

prevalentroleofthesevirulencedeterminantswouldbetosuppressplantimmunity,butwecan’t323

ruleouttheirroleinnutrientmobilization,evenattheseearlyinfectionstages.Finally,wetestedthe324

predictionpowerofourglobalinfectionmodelbytestingthepredictedinplantagrowthrateofthe325

hrpmutantstrainwheninoculatedonwoundedplants.Astheexperimentallymeasuredgrowthrate326

fitswiththepredictedmaximalgrowthrate,ourstartinghypothesisofsameentryflow(inwounded-327

rootplants)butgreaterclearanceofthehrpmutantbacteriabytheplantimmunesystemholdstrue.328

Severalimportantquestionsarestillunresolvedregardingtheparameterscontrollingthesizeofthe329

infectionbottleneck(2)duringtheinfectionoftomatoplantsbyRalstoniasp.Inparticularitwould330

beinterestingtoevaluatevariationintheinfectionbottlenecksizewiththeplantrootstatus,bothin331

termofrootgrowth(youngvsoldplants)andintermsofrootdevelopmentalplasticity(e.g.in332

differenttypeofsoils).Atasmallerscaleeven,onecouldimaginethattherecouldbespecific333

bottelnecksforthebacteriatocrossseveraldistinctcelllayerswithintherootorgan(12),likeitwas334

shownfortheprogressionoftheanthraxbacteriaindifferentmurinetissues(4).Anotherlevelof335


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complexitywouldbetounderstandthecontributionofthemicrobiometothisbottleneck.336

Specifically,itwouldbeinterestingtoevaluatewhethertheprotectionofplantsfrombacterialwilt337

bytrophiccompetition(15)couldpartlybeexplainedbyan“infection-sitecrowding”thusinducinga338

likelyreductionintheinfectionbottlenecksize.339

340

341

342


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MATERIALANDMETHODS343

Infectionmodel344

Wedesignedamathematicalmodeloftheinfectionkineticstodescribethepopulation345

dynamicsofR.pseudosolanacearumandthefoundernumberwithinthexylem.Themodelis346

composedof3compartments(c),thesoil(s),therootcortex(r),andthexylem(x).Thevolume347

dimentionofthemodelwassetinml,sinceitcorrespondstotheconcentrationofcellsusingsoil348

drenchingmethods,andthebacterialloadmeasureding(freshweight)wichcanbeassumedtobe349

intherangeof1mlvolume.ThevariablesaretheconcentrationofthebacterialcellsBcwithinthe3350

compartments(incell·ml-1),thefoudernumber,F,intherootcortex,Fr,andxylem,Fx,(incell·ml-1),351

theEPSconcentrationinxylem,EPSx,(inmmol·ml-1ofN-acetylglucosamine),thewiltinglevelofthe352

plant,W,(inDI).BandFcanbeinthemagnitudeoffewcellsintissues,thusweusedentiervaluefor353

thesevariables.Theequations,describedinthesupplementaryinformation,aresolvednumerically354

withatimestepof1hour.Thetransmissiontimewasdefinedasthetimewhenthewiltingsymptom355

reachesDI>3.5.Whennowiltinglevelabove3.5isreachedafter30days,thistimepointistakenfor356

theevaluationoftheinfectionbottleneck.357

Bacterialstrains,plantmaterialandcultureconditions358

TheR.pseudosolanacearumstrainsusedinthisworkwere:GMI1000,thewild-typestrain;GRS540,a359

derivativestrainfromGMI1000withagentamicincassetteinsertion(23);GMI1694,atypeIII360

secretionsystemmutant(hrcVmutant)withspectinomycincassetteinsertion(24);GRS743,a361

derivativestrainfromGMI1694withtheintegrationofthegentamicincassetteofGRS540(this362

study).Thegentamicincassettedoesnotalterthepathogenfitness,aswaspreviouslyshownby363

steminjection(23)andSIAppendix,Fig.S1.R.pseudosolanacearumstrainswereroutinelygrownat364

28°CincompleteBGmedium(25).Antibioticswereusedatthefollowingfinalconcentrations:10365

μg·L-1gentamicinand40μg·L-1spectinomycin.Solanumlycopersicumvar.SuperMarmandewas366

cultivatedingreenhouse.Squaretrays(30×30×4.5cm)wereusedtogrow16tomatoseedlingsand367


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keptinthegreenhouseunderthefollowingconditions:75%humidity,12hlight28°C,and12h368

darkness27°C.Plantswereinoculatedafter4weeksofgrowthindedicatedquarantinefacilities.369

Determinationofthemedianinfectiondose370

Wild-typeR.pseudosolanacearumGMI1000wasusedtodeterminatethemedianinfectiondose371

(MID)intomato.Groupsof16tomatoplantsinonetrayweresoil-drenchinginoculatedwith5.108,372

5.107,5.106,5.105or5.104cfu·ml-1in500mlofbacterialsuspension.Forsteminjection,10µlof373

bacterialsuspensionswasdirectlyinjectedintostemwithamicrosyringe(Hamilton,Reno,NV,374

U.S.A.)atthelevelofthecotyledon.Theinjectioninoculumsweredilutedto5.107,5.106,5.105,5.104375

and5.103cfu·ml-1ofR.pseudosolanacearum.Symptomappearancewasscoreddailyforeachplant.376

Diseaseindex(DI)wasusedtodescribetheobservedwilting:0fornowilting;1for25%ofleaves377

wilted;2for50%;3for75%and4forcompletewilting.Symptomonset(DI>0)wasdefinedasan378

infectionsuccessofR.pseudosolanacearumintomato.TheMIDofR.pseudosolanacearumintomato379

wasusedatthe50%endpointmethodofReedandMuench(10). First,wecalculatedtheReedand380

Muenchindex(RMI)forthe50%endpointfromthesedilutions.TheobtainedRMIwasthen381

correctedbythedilutionfactor.382

𝑅𝑀𝐼 = % !"#$%&$' !" !"#$%"&' !"##$%!&'$() !"#$% !"% ! !"%% !"#$%&$' !" !"#$%"&' !"##$%!&'$() !"#$% !"% !(% !"#$%&$' !" !"#$%"&' !"##$%!&'$() !"#$% !"%).383

𝐿𝑜𝑔𝑎𝑟𝑖𝑡ℎ𝑚 𝑜𝑓 𝑀𝐼𝐷 = logarithm of the dilution above 50% infection + RMI×dilution factor.384

Eachtreatmenthadtwotechnicalrepeatsandthreebiologicalrepeatswith96plantsintotal.385

Monitoringofbacterialcolonizationkineticsandinplantagrowthrate.386

Thesoil-drenchinginoculationwasperformedusingGMI1000at5.107cfu·ml-1.Symptomswere387

recordedbeforeharvestasdescribedabove.Sixteenplantsweresampledateachdpiandthis388

experimentwasrepeatedthreetimeswith48samplesintotal.Forthesteminjectionprocedure,389

eachplantwasinjectedwith10µlbacterialsolutioncontaining5.106cfu·ml-1ofGMI1000.Sampling390

wasconductedstraightawayafterinjection(0hpi),7hpi,24hpiand52hpi.Eachsamplinghad4391

plantsandthisexperimentwasrepeated8timeswith32samplesintotal.The1cmstemfromthe392


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cotyledonsupwardswasharvested,weighed,andhomogenizedbymechanicaldisruptionina393

grinder(RetschFrance,95610EragnysurOise,France).Bacteriawerethenenumeratedbyplating394

serialdilutionsonBGagarplates,usinganeasyspiral(Interscience,SaintNomlaBreteche,France).395

Predictionofbacterialthresholdtoinducesymptom.396

ThefollowingrelationwasusedtopredictwiltingonsetW(W=1)ornowitling(W=0)dependingon397

thebacterialloadb,incfu·ml-1,inplanta.398

W=1ifb>tthenW=0399

Witht,thethresholdabovewhichthewiltingonsetoccurs.Two-classcontingencematrices400

withexperimentalandsimulateddataweregeneratedforchangingvaluesoft.TheMatthews401

CorrelationCoefficient(11)(MCC)wasusedtocomparepredictingcapacitiesoft.TheMCCcan402

rangefrom0(randomassignment)to1(perfectprediction).403

Estimationofthesizeoftheinfectionfoundingpopulation(Ni)404

AmethodwasdevelopedtoquantitativelyestimatethesizeoftheR.pseudosolanacearum405

infectionfoundingpopulationintomato,orNi.Themethodisbasedonthemeasurementofthe406

proportionofplantsinfectedwithagentamycinemarkedstrainwhenthehostwasinoculatedwitha407

mixtureoftwostrains,thewtstrainplusvarioussmall(0.1%to0.01%)proportionofamarkedstrain.408

Thestartingfrequencyofthemarkerstrainintheinoculum,p,wasmeasuredbyserialplating.The409

frequencyofthewtstrainbeing1-p.Risreferredtoastheeventthatatleastonecellofthe410

resistantbacteriaindividualispresentinthehostplant.P(R)istheprobabilityoftheeventR.NRis411

consideredastheeventthatonlythewt(Non-Resistant)strainispresent.P(NR)istheprobabilityof412

theeventNR.Thus,P(R)+P(NR)=1.Basedontheabove:P(NR)=(1-p)NiandP(R)=1-(1-p)Ni.And413

Ni=log(1-P(R))/log(1-p).SIAppendix,Fig.S9highlightsthesigmoidfeatureofN=f(P)foreach414

givenp..Ni,or“infectionbottleneck”canbeestimatedfromthisexpressionthroughinvestigating415

P(R)experimentallyasfollows:Theresistant,markedstrainsareGRS540orGRS743,andtheir416

respectivenon-markedisogenicstrainsareGMI1000andGMI1694(hrpmutant).Thesamplingtime417


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wasat7dpiforrootinoculationofGMI1000andGRS540,4dpiforwoundinginoculationofGMI1000418

andGRS540,10dpiforinoculationofGMI1694andGRS743and7dpiforwoundinginoculationof419

GMI1694andGRS743.Stemsampleswerecollectedasdescribedbeforeandappearanceofthetwo420

strainswasdeterminedbyplatingonBGagarwithandwithoutantibiotics.Thetwoextremeoutput421

valueofNi,i.e.Ni=0andNi=infinity,respectivelyobtainedwhenP(R)=0/32orP(R)=32/32were422

excludedfromtheanalysisastheyartificiallyskewtheestimationofNi.Indeedtheselimitvaluesare423

thetwoasymptotesofthesigmoidcurveofN=f(P)(seeSIAppendixFig.S9)424

425

ACKNOWLEDGEMENTS426

GJwassupportedbyChinaScholarshipCouncil(No.[2013]3009)andTECHNOIIErasmusMundus427

Programme(2015-2016).RPwassupportedbyEMBO(Long-TermFellowshipALTF1627-2011)and428

MarieCurieActions(EMBOCOFUND2010,GA-2010-267146).Thisworkwasperformedwithfunding429

fromtheLABEX“TULIP”(ANR-10-LABX-41).Wealsothankourcolleaguesforusefuldiscussions.The430

fundingbodiesplayednoroleinstudydesign,datacollectionandanalysis,decisiontopublish,or431

preparationofthemanuscript.432

433

AUTHORSCONTRIBUTION434

GJ,RP,PRandNPdesignedtheresearch,GJ,RPperformedresearch,GJ,RP,AG,SGandNPanalyzed435

data,GJ,RP,PR,AG,SG,WDandNPwrotethepaper.Allauthorshavereadandapprovedthe436

manuscriptforpublication.GJandRPcontributedequallytothework.437

438


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439

440

441

REFERENCES442

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14. HoritaM,TsuchiyaK(2005)DiversityofRalstoniasolanacearuminTomatoRhizosphereSoil.470 BacterialWiltDisease,edsPriorP,AllenC,ElphinstoneJ(Allen,C.Prior,P.Hayward,A.C.).471

15. WeiZ,etal.(2015)Trophicnetworkarchitectureofroot-associatedbacterialcommunities472 determinespathogeninvasionandplanthealth.Naturecommunications6:8413.473


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16. CloughSJ,FlavierAB,SchellMA,DennyTP(1997)DifferentialExpressionofVirulenceGenes474 andMotilityinRalstonia(Pseudomonas)solanacearumduringExponentialGrowth.Applied475 andEnvironmentalMicrobiology63(3):844–850.476

17. SafniI,etal.(2014)PolyphasictaxonomicrevisionoftheRalstoniasolanacearumspecies477 complex:proposaltoemendthedescriptionsofRalstoniasolanacearumandRalstoniasyzygii478 andreclassifycurrentR.syzygiistrainsasRalstoniasyzygiisubsp.syzygiisubsp.nov.,R.479 solanacearumphylotypeIVstrainsasRalstoniasyzygiisubsp.indonesiensissubsp.nov.,480 bananablooddiseasebacteriumstrainsasRalstoniasyzygiisubsp.celebesensissubsp.nov.481 andR.solanacearumphylotypeIandIIIstrainsasRalstoniapseudosolanacearumsp.nov.IntJ482 SystEvolMicrobiol64(Pt9):3087–3103.483

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23. GuidotA,etal.(2014)MultihostexperimentalevolutionofthepathogenRalstonia497 solanacearumunveilsgenesinvolvedinadaptationtoplants.MolecularBiologyandEvolution498 31(11):2913–2928.499

24. CunnacS,OcchialiniA,BarberisP,BoucherC,GeninS(2004)Inventoryandfunctionalanalysis500 ofthelargeHrpreguloninRalstoniasolanacearum:identificationofnoveleffectorproteins501 translocatedtoplanthostcellsthroughthetypeIIIsecretionsystem.MolecularMicrobiology502 53(1):115–128.503

25. PlenerL,BoistardP,GonzálezA,BoucherC,GeninS(2012)MetabolicadaptationofRalstonia504 solanacearumduringplantinfection:amethioninebiosynthesiscasestudy.PloSOne505 7(5):e36877.506

507

508


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509 Figure1:ThelifecycleofplantpathogenicRalstoniasp.510

Ralstoniasp.livesinthesoil,penetratesintotheplantthroughtheroots,passtherootcortexand511

thencolonizethexylemvessels.Whenhighbacterialdensitiesarereached,aphenotypicswitchis512

inducedwithproductionofexopolysaccharide(EPS).Thexylemwaterfluxisinterrupted,promoting513

thewiltingsymptoms.Afterplantdeath,thebacteriareturnstothesoilwhereitwillcontinueasa514

saprophyteuntilanewhostisinfected.515

516


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517

Figure2:Sensitivityoftheinfectionbottlenecktodifferentparametersoftheinfectioncycle.518

Simulationswereperformedusingthepopulationdynamicsmodel(seeresults)andbyvaryingthe519

differentvaluesofthemodelparametersK(panelA),Vfmax(panelB),r(panelC),µ(panelD),q(panel520

E),s(panelF),seethelistFig.1andAppendixSIFig.2.Simulationswereperformedfor3521

concentrationsofbacterialsoilinoculum,1.106,1.107and1.108cell·ml-1.522

523


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524

525

Figure3:InfectivityofRalstoniapseudosolanacearumintomato.526

(A)TomatohostiscontinuouslyinfectedbyR.pseudosolanacearumwithin48hours.Plantswerefirst527

inoculatedwiththewildtypeGMI1000strainandsubsequentlyinoculatedwiththemarkedGRS540528

strain(SIFig.1A)at1,8,23and48hoursafterthefirstinoculation.Bacterialloadofthesetwo529

strainswerequantifiedat5dpitoobtainthefractionofGRS540infinalbacterialpopulation.(B)The530

medianinfectiondose(MID)ofR.pseudosolanacearumintomatofollowingsoil-drenching531

inoculation.Five10timesdilutionofR.pseudosolanacearuminoculums,volumeof500mlpertrial,532

werepouredoneachtrayof16plants.Dailyphenotypingwasperformed(SIFig.3)andtheReed-533

MuenchmethodwasadoptedtodeterminetheMIDateachdpi.Thereddottedlinerepresentan534

asymptoticvalueatMID=5.9·106cells.535

536


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537

Figure4:ColonizationdynamicsofRalstoniapseudosolanacearuminplanta.538

(A)Thebacterial loadof individualtomatoplantswasassessedfrom1dpito12dpi.Sixteenplants539

wereanalysedateach time-point (dpi). Thisexperiment is representativeof3biological replicates540

(AppendixSIFig.6).Barrepresentsthemedianofbacterialloadineachgroup.(B)Inplantagrowth541

ofR.solanacearumafterthesteminjectioninoculation.Sampleswereharvestedat0,7,25,52hpi.542

Thirthy-two individualplants from8biological replicateswereanalysed.Aftergrowthcurve fitting,543

theratewasdeterminedat0.233±0.012h-1;R2=0.937±0.024(mean±sd).544

545


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546

Figure5:Bacterialwiltdiseasedynamics.547

ThedatafromFig.4Awasusedforthiscorrelationanalysiswiththreebiologicalrepeats(SIFig.7).548

(A)Correlationsofbacterialloadanddiseaseindex(DI)intomato.Boxplotsrepresentthemedian549

and5-95percentilesineachDIgroup.***indicatesP-value<0.0001,Mann-Whitneytest,ns:P-550

value>0.05.(B)Timeresponsecurveofsymptomonset(wilting)synchronizedtothefirstdayofthe551

symptomappearanceforeachindividualplant.552

553


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554

555

Figure6.ContributionofplantphysicalbarriersandbacteriatypeIIIeffectorstotheinfection556

bottlenecksize.557

WildtypeGMI1000(greydots)andhrpmutantGMI1694(hrp,greensquares)ofR.558

pseudosolanacearumwereharvestedat7dpiand10dpifollowingrootinoculation.GMI1000559

wounding(WT-wounding,bluedots)andGMI1694wounding(hrp_wounding,greenopensquares)560

wereharvestedat4dpiand7dpibywoundingrootinoculation.Eachexperimentalconditioncanbe561

distinguishedfromtheothersasindicatedbytheMann-WhitneytestP-valuesinthetable.562

563 564 565


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