Analysetranscriptomiquedel’interactiontripartitePseudozymaflocculosa‐Blumeriagraminisf.sp.hordei‐Hordeumvulgare

Mémoire

GowsicaBojarajanRamakrishnan

MaîtriseenbiologievégétaleMaîtreèssciences(M.Sc.)

Québec,Canada

©GowsicaBojarajanRamakrishnan,2016

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RÉSUMÉ

Afind’améliorernospratiquesagricolesdanslecontexted’uneagriculturedurable,

plusieursagentsdeluttebiologique(ALB)ontétédéveloppés,testésetsontmaintenant

utilisésdanslemondepourcombattrelespertesderendementscauséesparlesmaladies.

Blumeriagraminisf.sp.hordei(Bgh)estl’agentpathogèneresponsabledublancdel’orge

etpeutréduire lesrendementsdecetteculture jusqu’à40%.Unchampignonépiphyte,

Pseudozymaflocculosa,aétédécouvertetidentifiéen1987enassociationétroiteavecle

blancdutrèfle.Leschercheursontalorsremarquéquecechampignonexhibaituneforte

activitéantagonistecontreleblancendétruisantlesstructuresdel’agentpathogène.Suite

àd’autrestravaux,ilestapparuquececomportementantagonisteétaitdirigécontretous

lesmembresdesErysiphalesetsemblait liéà lasynthèsed’unglycolipideantifongique

soitlaflocculosine.Toutefois,onn’esttoujourspasparvenusàassocierl’efficacitédel’ALB

avec la productionde ce glycolipide. Ces observations suggèrent qued’autres facteurs

seraient impliqués lorsque les deux protagonistes, l’ALB et le blanc, sont en contact.

L’objectifprincipaldeceprojetétaitdoncdechercherd’autresmécanismesmoléculaires

pouvant expliquer l’interaction P. flocculosa‐blanc et orge, en faisant une analyse

transcriptomiquecomplètedestroisprotagonistesenmêmetemps.

L’interactiontripartiteaétééchantillonnéeàdifférentstempssuivantl’inoculation

deP. flocculosa surdes feuillesd’orgeprésentantdéjàune intensitédeblancd’environ

50%. Les échantillons de feuilles prélevés ont ensuite été utilisés pour l’extraction de

l’ARNquiontétéensuite transformésenADNcpour lapréparationdes librairies.Cinq

répliquats ont été effectués pour chaque temps et le tout a été séquencé à l’aide de

séquençageparsynthèseIlluminaHiSeq.

Lesséquencesobtenues(reads)ontensuiteétéanalyséesàl’aidedulogicielCLC

GenomicsWorkbench.Brièvement,lesséquencesobtenuesontétécartographiéessurles

trois génomes de référence. Suite à la cartographie, les analyses d’expression ont été

conduitesetlesgènesexprimésdefaçondifférentielleontétérecherchés.Cetteétapea

étéconduiteenportantuneattentionparticulièreauxgènescodantpourungroupede

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protéines appelées CSEP pour “candidate secreted effector proteins” qui seraient

possiblementimpliquéesdansl’interactiontripartite.

Parmilesprotéinesexpriméesdefaçondifférentielleenprésencedublancouen

absence de ce dernier, nous avons pu constater que certaines CSEP étaient fortement

expriméesenprésencedublanc.Cesrésultatssontprometteursetnousoffrentunepiste

certainepourl’élucidationdesmécanismesimpliquésdanscetteinteractiontripartite.

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TABLEOFCONTENTS

RÉSUMÉ...............................................................................................................................................iii

TABLEOFCONTENTS.......................................................................................................................v

LISTOFTABLES...............................................................................................................................vii

LISTOFFIGURES...............................................................................................................................ix

ACKNOWLEDGEMENTS...................................................................................................................xi

FOREWARD......................................................................................................................................xiii

CHAPTER1...........................................................................................................................................1

LITERATUREREVIEW......................................................................................................................11.INTRODUCTION........................................................................................................................................12.PSEUDOZYMAFLOCCULOSA..................................................................................................................12.1Biologicalcontrolagents............................................................................................................................12.2Classificationandecology..........................................................................................................................22.3ModeofactionofP.flocculosa.................................................................................................................22.4GeneticsofP.flocculosa...............................................................................................................................32.5UstilagomaydisandPseudozymaflocculosa:Atugofwar.......................................................3

3.Transcriptomicanalysis–ApowerfultoolofNextGenerationSequencing......................44.Powderymildewfungi–Thepathogenofinterest.....................................................................55.Hordeumvulgare–Thehost................................................................................................................66.Effectorbiology–Apathtobeunraveled.......................................................................................77.Hypotheses................................................................................................................................................98.OBJECTIVES..............................................................................................................................................10

CHAPTER2........................................................................................................................................11

MANUSCRIPT....................................................................................................................................11

TranscriptomicanalysisofthetripartiteinteractionPseudozymaflocculosa‐Blumeriagraminisf.sp.hordei‐Hordeumvulgare..............................................................13RÉSUMÉ..........................................................................................................................................................15INTRODUCTION..........................................................................................................................................17MATERIALSANDMETHODS...................................................................................................................19Plantmaterial........................................................................................................................................................19FungalmaterialofPseudozymaflocculosa............................................................................................19InoculationwithPseudozymaflocculosafungus................................................................................19RNAisolation.........................................................................................................................................................19cDNAlibraryconstruction..............................................................................................................................20RNAsequencing....................................................................................................................................................20Mappingreadstoreferencegenome........................................................................................................21Genomeannotation............................................................................................................................................21Identificationofdifferentiallyexpressedgenes................................................................................22

RESULTS........................................................................................................................................................23CharacteristicgrowthofPseudozymaflocculosaincultureconditions................................23

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CalibrationofP.flocculosainresponsetopowderymildewfungus.......................................23TranscriptionaldynamicsofpowderymildewfungusinresponsetoP.flocculosa.......26RNAintegrity,cDNAlibraryvalidationandsequencing...............................................................................26

DifferentialgeneexpressionpatterninP.flocculosa......................................................................31Differentialgeneexpressionpatternofgenesinvolvedinflocculosinproduction.......32P.flocculosaeffectorcandidatesaredifferentiallyexpressedduringinfectionwithB.graminis....................................................................................................................................................................33

Discussion....................................................................................................................................................35ACKNOWLEDGEMENTS............................................................................................................................37REFERENCES................................................................................................................................................37

CHAPTER3........................................................................................................................................41

GENERALCONCLUSIONS...............................................................................................................41

BIBLIOGRAPHY................................................................................................................................45

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LISTOFTABLES

Table1.SamplecollectionpreandpostinoculationofbarleyleaveswithP.flocculosa.................30

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LISTOFFIGURES

Figure1:CharacterizationofPseudozymaflocculosainculturecondition............................................23

Figure2:Developmentofpowderymildewdiseaseovertimeinahealthyplant..............................24

Figure3:AntagonismofPseudozymaflocculosaonbarleypowderymildewcoloniesovertime.25

Figure4:Scanningelectronmicroscopyobservationofbarleypowderymildewfungusbefore

andaftertreatmentwithPseudozymaflocculosa...............................................................................................26

Figure5.TotalRNAisolationfromtheleafsamplesanditsintegritycheckusingbioanalyser...27

Figure6aPrincipleComponentAnalysis(PCA)ofP.flocculosasamplesgrowninvitroandin

vivo.........................................................................................................................................................................................28

Figure6bPrincipleComponentAnalysis(PCA)ofthesamplescollectedatvariousconditions.29

Figure7.Tripartiteinteractionmapping..............................................................................................................30

Figure8.DifferentialgeneexpressionpatternofP.flocculosagenes.......................................................31

Figure9.Differentialgeneexpressionpatternofflocculosinproducinggenes...................................32

Figure10.Differentialgeneexpressionpatternofeffectorcandidatesatdifferenttimepoints..33

Figure11.Differentialgeneexpressionpatternofeffectorcandidates..................................................34

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ACKNOWLEDGEMENTS

Firstandforemost,Iwouldliketothankgodforprovidingmetheopportunityto

cometoCanadaformygraduatestudies.

I like to thank my supervisor Prof. Richard Bélanger for providing me the

opportunitytopursuemygraduatestudiesinhisesteemedlaboratory.Hehasbeenagreat

teacherformeandguidedmetobeabetterscientist.Everyscientificinteractionwithhim

makesmemoredelightfulandenthusiasticaboutscience.Iadmirehispassionforscience,

which has been a great encouragement and inspiration forme duringmy stay in lab.

Duringallmyyearsofhardworkinthelaboratoryhesupportedduringthetoughtime

andappreciatedwhenIhadsuccess.Hisadviceonbothresearchaswellasonmycareer

havebeenpriceless.

I would like to thank Dr. François Belzile for his inputs on my project at lab

meetingsoneveryFridayandbeingapartofmythesiscommittee.Iwouldliketoextend

mythankstoProf.DanielDostalerforbeingacommitteememberofmythesisandforhis

valuablecommentsandsuggestionstoimprovemyproject.

MygratefulthanksgotoCarolineLabbéforallhereffortstoteachandtrainmein

thisproject.AbigthankgoestoHumaforhelpingmeincrucialbioinformaticssteps.

IthankmylabmembersJulien,Rupesh,JulieAnne,Amandine,François,Geneviève,

Bastien,Sarah,Marc‐Olivier,Stéphanie,Marie‐HélèneSamuelandJoanfortheirconstant

supportandencouragement.SpecialthankstoAliyehforbeingaverygoodfriendandfor

sharinglotsoffun‐filledmoments,discussionsandideas.Iamluckyenoughtohavesuch

excellentpeopleascolleagues.

Specialand loadsof thanksgo toallmy familymembers.Wordscannotexpress

how grateful I am to my family. My beloved thanks to my mother Mrs. Pushpam

RamakrishnanandmyfatherMr.Ramakrishnanforallofthesacrificestheyhavemade

throughouttheirlifetoshapeupmeandmycareer.Theirprayersandblessingsforme

were what sustained me so far. I would also like to thank my brother Mr. Arun

Ramakrishnanforhisunconditional loveandcaretowardsme.At theendIwould like

expressappreciationtomybelovedhusbandMr.Girishwaranwhospentlonglonelydays,

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sleeplessnightswithoutmeandstoodbymealwaysasmysupport ineachandevery

momentofmylife.Hislove,care,encouragementandsupportmademetoachievethis

degree.Ihaveaspecialmentiontomydog“Dhivan”wholefthisfootprintsinmyheart

forever.Myfamilyismybackboneandstrength.

IwouldliketoextendmyheartfeltthankstomyfriendSenthilKrishnasamywho

supportedmeandguidedme in lot of things inmy life. I hugely appreciatemy friend

Preyesh,JinaandmylittleangelIshithafortheirkindnessandsupporttowardsme.

IliketothankWajidBhatandAmbreenfortheirkindhelpduringmyinitialdaysin

Canada. I thankRanjan,Hemanta,Prakash,Pallavi,Priyanka,Dinesh,Prenitha,Ramesh

and all Indian buddies inQuebecCity formakingmy stay pleasant andmemorable in

Quebec.

LastbutnotleastIwouldliketodedicatemythesistotwomostinspiringwomen

inmy lifeMrs.NatchiyarShanmugamandMrs.SeeniyammalBojararajan.Theyaremy

grand‐motherswhoalwayswantedmetoachievegreatheightsinmylifeandstoodbeside

me during my hardships. A special mention goes to living legend Mrs. Natchiyar

Shanmugamforherunconditionallove,careandsupport,encouragementtowardsmein

mylife.

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FOREWORD

Thisthesisincludesaliteraturereviewthatoutlinesthecurrentknowledgerelated

totheadvancementinthefieldofbiologicalcontrolagentsinplantprotection.Itdiscusses

theimportanceofeffectorproteinsandrevealstheirroleinfungal‐fungalinteractions,a

big milestone in effector biology. The first part of the first chapter includes the

classification, etiology and genetics of thebiocontrol agentPseudozyma flocculosa.The

secondpartofthefirstchapterrevealstherelationshipbetweenP.flocculosaandUstilago

maydisandthestructuralsimilaritybetweenflocculosinandustilagicacid.Thehypothesis

ofthisthesiswasbasedonthecomparativeanalysisofP.flocculosawithcloselyrelated

organismsthatrevealedfeaturesuniquetoP.flocculosa.

Thesecondchapterofthisthesisispresentedintheformofaresearchmanuscript

where I am the principal author. The manuscript deals with the identification of

mechanismsofactionofP. flocculosadefining itsantagonisticactivityagainstpowdery

mildew fungi using a novel next generation sequencing technique of transcriptomic

analysis.ThedifferentialgeneexpressionanalysisofRNA‐sequencingdatahighlightsthe

roleofeffectorsintheinteractionP.flocculosa‐powderymildewfungi,thefirstsuchreport

forfungal‐fungalinteractions.

Thethirdandfinalchapterofthisthesisconcludesthepresentresultsinabroader

context.

CHAPTER1

LITERATUREREVIEW

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1.Introduction

Plants are a main source of life on earth but their production is constantly

threatenedbypathogenssuchasfungi,oomycetes,bacteria,virusesandnematodes.These

pathogensneedtobecontrolledtomaintainthequalityandabundanceoffood,feed,and

fiberproducedbygrowersaroundtheworld.Cropprotectionisaconstantconcernfor

humancommunitiessincethebeginningofagriculture.Differentapproachesmaybeused

toprevent,mitigateorcontrolplantdiseases.Beyondgoodagronomicandhorticultural

practices,growersoftenrelyheavilyonchemicalpesticides.However,theintensiveusage

ofpesticidescompromisessustainabilityandenvironmentalhealth.Inrecentdecades,the

search for new approaches for crop protection that are both more effective and less

damagingtotheenvironmentandhumanhealthhasledtothedevelopmentofpromising

newtoolsinspiredbybiologicalandecologicalprocesses.Indeed,manymicroorganisms

havethenaturalabilitytoinhibitthegrowthorevenkillotherspeciesinordertoprotect

theirecologicalnicheorhaveasourceofnutrients.Thedevelopmentofnovelalternatives

tocontrolplantdiseases,basedontheexploitationofbeneficialorganisms,hasbeenatthe

forefrontofmanyresearchendeavorsaroundtheworld.Theuseoflivingorganismsto

combat other living organisms presupposes a thorough knowledge of their ecology.

However, many technical and scientific challenges remain to be resolved before a

widespreadcommercialuseofthesebeneficialorganismscanbeenvisioned.

2.PSEUDOZYMAFLOCCULOSA

2.1Biologicalcontrolagents

In agriculture, the microbial flora in the environment of cultivated plants has

becometheobjectofgreatinterest.Withtheadventofsophisticatedmoleculartechniques,

newmicrobialspeciesarediscoveredatanunprecedentedpace,screenedanddescribed

for their beneficial or harmful properties. Exploiting these microorganisms for their

beneficial properties is an approach called "biological control". Biological control is

definedasthereductionofinsects,mites,weedsandplantdiseasesbynaturalenemies

andalsosometimeswiththehelpofanactivehumanrole.Itisacomponentofintegrated

pestmanagementstrategiesandaimstocontrolanyinsectsorplantdiseasesbytheuse

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ofotherspeciesthataretheirnaturalantagonistorthatpromoteplantdefensereactions

(Flint et al., 1998). Biological controlwill try to favor the growth and the dispersal of

natural antagonists in agricultural systems to fight plant diseases, as an ecological or

naturalalternativetofungicides.Mostbiologicalcontrolagents(BCAs)identifiedtodate

havebeencategorizedasexertingtheiractivitythroughthemanifestationofoneormore

of fourmodes of action: competition, parasitism, antibiosis and/or induced resistance

(BélangerandAvisetal.,2001;Whippsetal.,2001).UnderstandingpreciselyhowBCAs

act on their targets will increase their efficacy at reducing various plant disease. For

instance, in cases where parasitism appeared to be the predominant mode of action,

several attempts have been made to increase production of lytic enzymes such as

chitinasesandglucanases(Kubiceketal.,2001;Loritoetal.,2001).Theapproachtargeted

eithertheselectionofBCAstrainswithsuperiorabilitytoproducesuchenzymesorthe

direct cloning and over‐expression of relevant genes conferring greater degrading

properties.Inordertocontroltheseplantdiseases,thebroadspectrumofmechanisms

thatfilamentousfungalpathogensusetocolonizehostplantsneedstobeelucidated.

2.2Classificationandecology

The1980swereparticularlyfertileinthesearchforpotentialBCAsagainstvarious

plantdiseases.ThediscoveryandcharacterizationoftheBCAP.flocculosawaspartofthis

trend. Pseudozymaflocculosa was discovered in 1987 and originally identified as

Sporothrixflocculosa,anascomycetousyeast.Itwasfirstdiscoveredasanepiphyticyeast

onpowderymildew‐infectedcloverleaves(Traquairetal.,1988).Sincethen,ithasbeen

extensivelystudiedforthedevelopmentofanefficientbiofungicidetocontrolpowdery

mildews in many crops. P. flocculosa is an anamorphic fungi which lacks sexual

developmentandwerefoundtobemorphologicallyandphylogeneticallyrelatedtothe

Ustilaginales. The phylogenetic analysis and comparison of diagnostic ribosomal DNA

sequencesmadeittoreclassifyunderUstilaginalesfamily(Begerowetal.,2000).

2.3ModeofactionofP.flocculosa

InitialstudieswithP.flocculosashowedthatitwasneitherastrongcompetitorin

the phyllosphere nor a direct hyperparasite, since no direct contact between the

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biocontrol agent and target powderymildewwas required to observe an antagonistic

activity.PowderymildewcellsexposedtoP.flocculosasufferedrapidplasmolysis,which

ledtotheconclusionthatantibiosiswasitsmodeofaction(Hajlaouietal.,1993).Thiswas

further supported by the discovery of an unusual glycolipid produced byP.flocculosa,

called flocculosin thatexhibitedstrongantifungalactivity (Mimeeetal.,2005).Shortly

thereafter,itwasdiscoveredthatthemoleculewasnearlyidenticaltoustilagicacid,found

in the culture filtrates ofU.maydis in 1951. This finding brought a direct link to the

reclassificationofP.flocculosaamongtheUstilaginales(Begerowetal.,2000).

2.4GeneticsofP.flocculosa

Molecularsignallingbetweenaplantpathogenanditshostplaysafundamental

roleinpathogenesisandintheestablishmentoftheinteraction.Theseinteractionshavea

profoundeffectfordesigningnewstrategiestocombatdiseases.Tounderstandgenesand

theirroleinthebiologyandthegeneticsofanorganism,itisnecessarytounderstandthe

genomesequences.Assuch,sequencingandassemblyoftheP.flocculosagenomebecame

anecessityforunderstandingtheimplicationofflocculosininthebiocontrolactivityofP.

flocculosa.UsingRoche454Titaniumtechnology,525Mbofshotgundataand167Mbof

2.6and4.5kbmate‐pairsequencesforaca.30Xcoverageofthegenomewasgenerated

forgenomesequencingofP. flocculosa.TheP. flocculosa genome is23Mband includes

6877 predicted proteins. The assembly yielded 1583 contigs from which 1187 were

orientedandorderedinto37scaffoldstowhichthreecontigslargerthan2Kbwereadded.

ThemaindifferenceobservedbetweenthegenomestructuresofP.flocculosaandother

Ustilaginaleswerefoundintheproportionofguanineandcytosine(GC)residuesandin

thestructureofgenes.ThegenesidentifiedinP.flocculosacontainedanaverageoffour

timesmoreintronsthanU.maydisbutthenumberoftransposableelementsandsimple

repeatsfoundinP.flocculosagenomeissimilartothatofU.maydis(Lefebvreetal.,2013).

2.5UstilagomaydisandPseudozymaflocculosa:Atugofwar

AlthoughthephylogeneticproximitybetweenU.maydisandP.flocculosahasbeen

demonstratedseveralyearsago,detailsaboutthecontentanddistributionofgenesinP.

flocculosa remainedthesubjectofspeculation. Indeed,ononehand, thegenomeof the

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pathogenU.maydishasbeenavailablesince2006,andhasledtoimportantdiscoveries

andgenerateda lotofuseful information fordifferentresearchaspects (Kämperetal.,

2006).Forinstance,usingtheU.maydiscyp1cDNAasaprobeagainstallknownspeciesof

Pseudozyma, it was possible to show that it hybridized specifically with P. flocculosa

(Marchandetal.,2007),theonlyotherstrainproducingflocculosin.This indicatedthat

cyp1hadtobe involved in flocculosinproduction.Thepresenceofcyp1 inP. flocculosa

raisedtheobviouspossibilityoftheexistenceofaclustersimilartotheonefoundinU.

maydisregulatingtheproductionofflocculosin.Onthebasisofsequencehomologywith

genesfoundinU.maydis,ageneclustercomprising10genesthatwerenecessaryforthe

biosynthesisofflocculosinasidentified(Teichmannetal.,2010).Incontrasttothecluster

ofU.maydis,theflocculosinbiosynthesisclustercontainsanadditionalgeneencodingan

acetyl‐transferaseandislackingagenehomologoustotheα‐hydroxylaseAhd1necessary

forUAhydroxylation.Thefunctionsofthreeacyl/acetyl‐transferasegenes(Fat1,Fat2and

Fat3) including the additional acetyl‐transferase were studied by complementing the

corresponding U. maydis mutants (Teichmann et al., 2010). This showed that the

additionalacetyl‐transferaseisnecessaryforacetylationoftheglucosemoiety,explaining

thedifferencesbetweenthetwomolecules.

3.Transcriptomicanalysis–ApowerfultoolofNextGenerationSequencing

Sincethestartofgenomicsresearch,genome‐wideexpressionstudieshavebeen

used as a tool to improve our understanding of the involvement of genes in various

biologicalprocesses.Measuringgeneexpressionpatternssimultaneouslyacrossallgenes

inthegenome,i.e.transcriptomics,isauniquelypowerfultechnologytoexplorepotential

novel candidate genes for a particular process. Identifying the full set of transcripts

including large and small RNAs, novel transcripts from unannotated genes, splicing

isoformsandgene‐fusiontranscriptsservesasafoundationforthecomprehensivestudy

of the transcriptome. Whole‐transcriptome analysis is of growing importance in

understandinghowalteredexpressionofgeneticvariantscontributestocomplexplant

diseases. The analysis of genome‐wide differential RNA expression provides greater

insights into biological pathways and molecular mechanisms that regulate cell fate,

developmentanddiseaseprogression(DeVosetal.,2005).Transcriptomicapproaches

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arearevolutionaryfunctionalgenomictoolfordecipheringplant‐pathogeninteractions

inthepathogenomicsera.Thetechniqueisextremelypowerfulasafirststeptoimplicate

novelgenesandpathwaysthatmaybeinvolvedorassociatedwithaparticularcondition.

Transcriptomic data from next generation sequencing technology give us information

about theactivityofgenesthatchangetheirexpressionpattern inresponsetoasignal

originating from the host plant or in the host tissue and may reveal mechanisms of

pathogenesisandbiocontrolactivityasinitiatedbyfungalpathogensandfungalBCAs.

4.Powderymildewfungi–Thepathogenofinterest

Powderymildewsareamongstthemostcommon,widespreadandrecognizableof

allplantdiseases.Theyareaptlynamed,fortheinfectionproducesawhitelawnoffungal

mycelium that covers the plant surface, while chains of aerial conidia give the

characteristicpowderyappearance.Powderymildewscaninfectawiderangeofhosts,

includingover9000dicotyledonousandover650monocotyledonousplantspecies.The

cereals,particularlywheatandbarley,areamongthemostimportantagriculturalcrops

thatsufferfrompowderymildewdiseases.Indeed,intemperateregions,barleypowdery

mildewcancauseyieldlossesofsome5‐20%andoccasionallyasmuchas40%.Taken

collectively,powderymildewscausegreaterlossesintermsofcropyieldthananyother

single“type”ofplantdisease.Thepowderymildewdiseasesarecausedbymanyspecies

ofAscomycetefungi,groupedintoseveralgenera.Theyaretrueobligatebiotrophs,which

meansthatgrowthandreproductionofthesefungidependontheirparasitizinglivinghost

plants.DespitethelackofrobustandreliableDNA‐mediatedtransformationmethodand

mutational analysis, significant progress hasbeenmadeover thepast decade towards

understandingpowderymildew‐hostinteractionsatboththecellularandmolecularlevel

(Hacquardetal.,2013).

Blumeriagraminis f. sp.hordei (E.O.SpeerDC) isapowderymildewfungusthat

infectsbarleyanditcanreducecropyieldasmuchas40%(Wieseetal.,1987). Itwas

earlierincludedinorderErysiphalesbutlateritsmolecularstudiesplacedtheseintoa

new taxonBlumeria. The genome ofB. graminis f. sp.hordei (Bgh) has recently been

sequenced (Spanuetal.,2010). This genome is of size 120Mb and extremely rich in

repetitive elements derived mostly from retrotransposons with 90% transposable

6

elements.Additionally,6540geneswereannotated,fromwhich437encodedcandidate

effectorproteinsand165fornon‐secretedcandidateeffectorproteins.Theabilitytoinfect

tetraploidaswellasdomesticatedhexaploidwheat,wasseentobetheresultofmildew

genomesbeingmosaicsofancienthaplogroupsthatexistedbeforewheatdomestication.

Thishasallowedwheatpowderymildewtomaintaingenetic flexibility,variabilityand

thusagreatpotentialforpathogenvariation.

5.Hordeumvulgare–Thehost

HordeumvulgareL.(barley)istheworld'sfourthmostimportantcerealcropand

animportantmodelforecologicaladaptation.Itwasoneofthefirstdomesticatedcereal

grainsoriginatingintheFertileCrescentover10,000yearsago.Abouttwo‐thirdsofthe

globalbarleycropisusedforanimalfeed,whiletheremainingthirdunderpinsthemalting,

brewing,anddistillingindustries.Althoughthehumandietisnotaprimaryuse,barley

haspotentialhealthbenefits,andisstillthemajorcaloriesourceinseveralpartsofthe

world.Barleyisamemberofthegrassfamily.Itisaself‐pollinating,diploidspecieswith14

chromosomes. Genome of barley was sequenced in 2012 by the International Barley

GenomeSequencingConsortium(IBSC)andalsotheUKBarleySequencingConsortium.

Thegenomeiscomposedofsevenpairsofnuclearchromosomes,withatotalof5000Mbp.

In the IBSC assembly, ~2.6 million sequenced contigs were generated using whole‐

genome shotgun sequencing (WGS). Of these, ~723,000 are assigned to specific

chromosomal positions (Klaus et al., 2012). It is one of the largest diploid genomes

sequencedtodate.

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6.Effectorbiology–Apathtobeunraveled

Interactionsbetweenorganismsarecontrolledbyexchangesof signalsbetween

partners. Plants can be colonized by fungi that have adopted highly diverse lifestyles,

rangingfromsymbiotictonecrotrophic.Fungihaveadopteddiversestrategiestointeract

with host plants and to overcome a complex network of plant defense mechanisms.

Colonizationisgovernedinallsystemsbyhundredsofsecretedfungaleffectormolecules

(Sonahetal.,2016).Theseeffectorssuppressplantdefenseresponsesandmodulateplant

physiology to accommodate fungal invaders and provide themwith nutrients. Fungal

effectorseitherfunctionintheinteractionzonebetweenthefungalhyphaeandhostorare

transferredintoplantcells.

Effectorproteinsaremostlysecretoryproteinsthatalterhostcellstosuppresshost

defense mechanisms and facilitate the interaction by the pathogen so it can derive

nutrientsfromthehost.Effectorswillalsoactivateresistancemechanismsintheresistant

plant genotypes. Candidate secreted effector proteins (CSEPs) are defined as fungal

proteinswithasignalpeptideforsecretion,notrans‐membranedomainsandnosimilarity

withotherobviousproteindomainsarefairlysmall insizeandusuallyspecies‐specific

(Jones andDangl, 2006;Djameietal., 2011). The centrality of effector proteins in the

biologyofplantpathogenicmicrobesisdemonstratedbythepresenceofvastarraysof

effector‐likegenesthatarefoundinpracticallyallpathogengenomes.Thisisparticularly

striking in the genomes of the obligate biotrophic fungi that cause powderymildews

(Spanuetal.,2010; Wickeretal.,2013). In these fungi, many commonly large gene

familiesarereducedtoveryfewmembers,andsomegenesarelostaltogether.Insharp

contrast, the effector‐like gene superfamilies described in cereal powdery mildews

compriseover7%oftheconventionalprotein‐codinggenecapacityofthegenome.From

thepathogens'pointofview,theseeffectorsareessentialtoolstogainentryandswitch

offthehosts'defensemechanisms.Ingeneral,effectorproteinsinterferewithrecognition

of microbes at the surface of cells and intercellular spaces; they can also target the

intracellularimmune‐signalingpathwaysallthewayuptoandincludingtheactivationof

the transcriptionofgenes involved inresistanceandthedefenseresponse. Indeed, the

studyofproteineffectorsisausefulinstrumenttoinvestigateanddefinethemechanisms

8

oftheimmuneresponseitself.Recentdevelopmentsandadvancesincomputationaltools

and in development of various pipelines make it easier to perform genome‐wide

identification of CSEPs (Sonah et al., 2016). This gives us more information on the

distributionandorganizationofCSEPswithinagivenspecies.InU.maydis,426secretory

proteins were identified, 70% of which were with unknown function based on their

homologysearch.Itwasalsofoundthateffectorproteinsexistasclustersof3‐26genes

per cluster. Knockout studies of specific genes or clusters identified about 50 effector

proteins that were involved in pathogenesis (Kamper et al., 2006). More recently the

generation and analysis, including annotation, of the complete genome ofP.flocculosa

with a comparative analysis to the genomes of U.maydis, S.reilianum and U.hordei

highlightedsimilaritiesanddifferenceswithrespecttoeffectorproteins(Lefebvreetal.,

2013). The comparative genomic analysis of phylogenetically closely related species

revealedahigherconservationofvirulentsecretedproteinsinthethreepathogensanda

near complete loss inP. flocculosa.These results highlighted that themain difference

between phytopathogenic Ustilaginales and P. flocculosa could be attributed to a few

specificeffectorproteins,thusconfirmingtheimportantroleofsuchproteinsindefining

plant‐pathogeninteractions.Focusingonpotentialeffectorsrevealedthat,incomparison

toU.maydis,thegenomeofP.flocculosahasnearlythesamenumberofpredictedsecreted

proteins.Foritspart,P.flocculosapossesses200specificCSEPswherenoorthologsare

foundincloselyrelatedspecies.P.flocculosaalsopossesstwoNPP1containingproteins,

absentinallpathogenicUstilaginales.Understandingtheroleofeffectorproteinswilllead

ustonewpathsofdefiningthepotentialfactorsinvolvedinthebiocontrolpropertiesofP.

flocculosa.(Lefebvreetal.,2013).

9

7.Hypotheses

Extensive studies have characterized many infection‐responsive genes in the

pathogenandhostplant,separately.Tounderstandtheplant‐pathogeninteractionand

pathogen‐BCAinteractioncomprehensively,itisvaluabletomonitorthegeneexpression

profileofalltheinteractingorganismssimultaneouslyinthesameinfectedplanttissue.

TheresearchonP.flocculosamademanyassumptionsaboutthegeneticbasisrelatedto

itsmolecularbiology.TheclosephylogeneticlinkbetweenP.flocculosaandU.maydishas

offeredunexpectedopportunitiestodefinethefactorsinherenttospecificlifestylesthat

characterize fungi.More specifically, it allowed the study of genetic determinants that

conferred the phytopathogenic nature of some Ustilaginales. On the other hand, the

biocontrolprocessappearstobemediatedbyaninteractioninvolvingnutrientsproduced

bytheplant,harvestedbythephytopathogenandexploitedbyP.flocculosa.Itwouldthus

constituteatripartiteinteraction.Ourhypothesesareasfollows:

1. P.flocculosawillonlydeveloponpowderymildewcolonies/sporespresentonaliving

plant;itwillnotantagonizepowderymildewsporesseparatedfromtheirhost

2. ThedevelopmentofP.flocculosaisstoppedassoonasthepowderymildewsporesare

ruptured thus interrupting the flow of nutrients from the plant. This shows that

Pseudozyma flocculosa issomehowdependentontheplantandtakingthenutrients

fromtheplantthroughthepowderymildewfungus.

Thesehypothesesleadtothefollowingquestions:

1. WhatarethefactorsthatcouldberesponsibleforthiskindofinteractionbetweenP.

flocculosaandB.graminis?

2. HowdoesP.flocculosarecognizeB.graminisasahost?

3. What are the genes involved in this process, i.e., that are activated or suppressed

duringthisprocess?

10

8.Objectives

Basedontheavailableliterature,fewstudieshavebeencarriedouttodetermine

thevirulencefactorsofaBCAagainstanotherfungus,mainlyinthecontextofatripartite

interaction. Our major objective is to understand the biocontrol mechanisms of

PseudozymaflocculosaagainstB.graminis.

Thepresentworkaims tounravel thegeneticdeterminantsassociatedwith the

processofsuppressionofBghandtobetterunderstandthemodeofactionofP.flocculosa

to improve its effectiveness as a biological control agent. In short, the objectives are

designed to reveal the most interesting properties associated with the biology of the

biocontrolagent.

Thespecificobjectivesare:

1) To optimize the methodology of the sampling time and technique of

Pseudozymaflocculosaincultureconditionsandinbiologicalcontrolcondition

2) Toacquireafundamentalunderstandingofthegeneticprinciplesthatregulate

thebiocontrolactivityofP.flocculosaagainstB.graminis.

3) Toidentifythegene(s)responsibleforthetripartiteinteractionP.flocculosa‐

Bgh–Hordeumvulgare

CHAPTER2

MANUSCRIPT

13

Transcriptomic analysis of the tripartite interaction Pseudozyma flocculosa‐

Blumeriagraminisf.sp.hordei‐Hordeumvulgare

ABSTRACT

Blumeria graminisf. sp.hordei(Bgh) is a powdery mildew fungus that infects

barleyandcanreducecropyieldbyasmuchas40%.TheepiphyticfungusPseudozyma

flocculosa,isoftenfoundincloseassociationwithpowderymildewofcloverleavesand

exhibitsastrongantagonisticactivitybyrapidlydestroyingtheinvasivestructuresofthe

pathogen. The objective of this work was to understand the molecular mechanisms

dictatingtheinteractionbetweenabiologicalcontrolagents(BCA)P.flocculosaandaplant

pathogen(Bgh).Inthepresentstudy,tounderstandgeneexpressiondynamicsduringa

host‐pathogen‐BCA interaction, a complete RNA‐seq transcriptome profiling was

performedonthetripartiteinteraction.Thetranscriptomeprofilingstrategywasusedto

understand the genetic determinants of the interaction involving P. flocculosa, the

pathogenBgh,andthehostplant,barley.OurresultsdeterminedthesubtlechangesinP.

flocculosa gene expression under in vitro and biocontrol conditions. The analysis of

differentially expressed genes (DEGs) as performed with an initial emphasis on 200

unique candidate secretory effector proteins (CSEPs) ofP. flocculosa in an attempt to

determinetheirroleininfluencingitsinteractionwithbarleypowderymildew.Over30

CSEPswereupregulatedduringP.flocculosainteractionwithBgh,includingpf02826that

hadanear1000‐foldchange.TheseresultssuggeststronglythatCSEPsareinvolvedinthe

biocontrolactivityofP.flocculosa,andrepresent,toourknowledge,thefirstsuchreport

forafungal‐fungalinteraction.

15

RÉSUMÉ

Blumeriagraminisf. sp.hordei(Bgh)est l’agentpathogènecausant leblancde l’orge. Il

peutàluiseul,causerdespertesallantjusqu’à40%danscetteculture.Unchampignon

épiphyte,Pseudozymaflocculosa,aétédécouvertetidentifiéen1987enassociationétroite

avecleblancdutrèfle.Leschercheursontalorsremarquéquecechampignonexprimait

une forte activité antagoniste contre le blanc en détruisant les structures de l’agent

pathogène.L’objectifdecetravailétaitdecomprendrelesmécanismesmoléculairessous‐

jacents de l’interaction de lutte biologique entre P. flocculosa et l’agent pathogène B.

graminisf.sp.hordei.Pourcefaire,uneanalysetranscriptomiquecomplèteparséquençage

descDNA(RNA‐Seq)destroisprotagonistesdel’interactiontripartiteaétéeffectuée.Une

analysedesgènesexprimésdefaçondifférentielleaétéeffectuéeavecuneattentiontoute

particulièreàlaclassedesprotéinescandidateseffectricesouplussimplement,CSEPs.Il

enestressortique30CSEPs,sur les200étudiées,présentaientdegrandesdifférences

d’expressiondelapartdeP.flocculosalorsqu’ilsetrouvaitenabsenceouenprésencede

B.graminis.Parexemple,laprotéineCSEPpf02826,avaituneexpressionprèsde1000fois

supérieurelorsqueleP.flocculosaétaitenprésencedeblanc.Cerésultatmetenévidence

quelesCSEPssontimpliquéesdansl’activitédeluttebiologiquedeP.flocculosa.Demême,

pourunepremière fois, nousmettonsen lumière l’implicationdeprotéines effectrices

dansuneinteractionchampignon‐champignon.

17

INTRODUCTION

Barley isoneof themostwidelygrowncrops intheworld.Thebarleypowdery

mildew fungus, Blumeria graminis f. sp. hordei (E.O. Speer DC) (Bgh) is an obligate

biotrophicpathogendefinedasaseriousbarleydiseaseworldwidewhereitcanreduce

yield by as much as 40%. This plant pathogen is able to overcome host defense

mechanismsandsubsequentimmunitymanifestedduringformationoftheintracellular

feedingstructureofthefungus,thehaustorium.

Because of their ectotrophic growth, powdery mildews are readily exposed to

naturalenemiesandafewfungalspecieshavebeentestedfortheirpotentialasbiocontrol

agents(Kiss2003).Amongthem,Pseudozymaflocculosa(Traquair,ShawandJarvis)hasa

significantantagonisticactivityagainstpowderymildews(Avisetal.,andPaultizetal.,

2001). Pseudozymaflocculosa was discovered in 1987 and originally identified as

Sporothrixflocculosa,anascomycetousyeast(Traquairetal.,1988)andlaterreclassified

asabasidiomyceterelatedtotheanamorphsoftheUstilaginales(Begerowetal.,2000).

Whileaneffectiveantagonistofpowderymildews,itsspecificactivitytowardthis

particulargroupofplantpathogensappearstobealotmoreintricateandcomplexthan

whatwashypothesizedintheliterature.EarlierstudieswithP.flocculosashowedthatit

wasneitherastrongcompetitorinthephyllospherenoradirecthyperparasite,sinceno

directcontactbetweenthebiocontrolagentandtargetpowderymildewwasrequiredto

observeanantagonisticactivity.PowderymildewcellsexposedtoP.flocculosasuffered

rapidplasmolysis,whichledtotheconclusionthatantibiosiswasitsmodeofaction.This

wasfurthersupportedbythediscoveryofanunusualglycolipidproducedbyP.flocculosa,

calledflocculosinthatexhibitedstrongantifungalactivity(Mimeeetal.,2005).

The structure of flocculosin is highly similar to the structure of ustilagic acid

producedbytheplantpathogenUstilagomaydis.Thisfindingbroughtadirectlinktothe

reclassificationofP. flocculosawith theUstilaginales.With theavailabilityofU.maydis

genome in 2006 (Kämper et al., 2006) and P. flocculosa genome, the gene clusters

responsibleforthesynthesisofustilagicacidandflocculosinwereidentified(Teichmann

etal.,2010).Morerecently,in2013,acomparativeanalysisofP.flocculosatothegenomes

18

of U.maydis, Sporisoriumreilianum and Ustilagohordei highlighted similarities and

differences with respect to effector proteins (Lefebvre et al., 2013). The comparative

genomic analysis of phylogenetically‐related species revealed a higher conservation of

virulentsecretedproteinsinthethreepathogensandanearcompletelossinP.flocculosa.

TheseresultshighlightedthatthemaindifferencebetweenphytopathogenicUstilaginales

andP.flocculosacouldbeattributedtoafewspecificeffectorproteins,thusconfirmingthe

importantroleofsuchproteinsindefiningplant‐pathogeninteractions.Thefocusingon

potentialeffectorsrevealedthat,incomparisontoU.maydis,thegenomeofP.flocculosa

has nearly the same number of predicted secreted proteins. For its part,P. flocculosa

possesses200specificCSEPswherenoorthologsarefoundincloselyrelatedspecies.

Thecentralityofeffectorproteins inthebiologyofplantpathogenicmicrobesis

demonstrated by the presence of vast arrays of effector‐like genes that are found in

practically all pathogen genomes. This is particularly striking in the genomes of the

obligate biotrophic fungi that cause powdery mildews (Spanuetal.,2010;

Wickeretal.,2013).On theotherhand, Lefebvreetal. (2013) identified someunusual

genesunique toP. flocculosa that couldaccount for theelusiveproperties linked to its

biocontrolactivity.P.flocculosaalsopossessestwoNPP1containingproteins,absentinall

pathogenicUstilaginales.Theseobservationsledustothehypothesisthatfeaturesunique

ofP.flocculosawouldberesponsibleforitsspecificitytowardsbarleypowderymildew.

Inordertodefineandunderstandthespectrumofgenesinvolvedinthebiocontrol

properties of P. flocculosa, transcriptomic analysis could represent a powerful tool to

identify thespecific factorsdefining the interactionbetweenP. flocculosaandpowdery

mildew.Thetechniqueisessentialasafirststeptoimplicatenovelgenesandpathways

thatmaybeinvolvedorassociatedwithaparticularcondition.Transcriptomicdatafrom

nextgenerationsequencingtechnologygivesusinformationabouttheactivityofgenes

thatchangetheirexpressionpatterninresponsetoasignaloriginatingfromthehostplant

orinthehosttissueandmayrevealmechanismsofpathogenesisandbiocontrolactivity

asinitiatedbyfungalpathogensandfungalBCAs.

ThisstudyaimedtoidentifythedifferentialgeneexpressionpatterninP.flocculosa

in response to barley powdery mildew (Bgh) by transcriptome analysis. The main

19

objectiveoftheworkwastouncovergenesresponsibleforthebiocontrolactivityofP.

flocculosaand specificity towardsbarleypowderymildewwith a specific emphasis on

thosecodingforeffectorproteins.

MATERIALSANDMETHODS

Plantmaterial

Barley (Hordeumvulgare)plants cv.Fosterdisplayinghighsusceptibility toBgh

wereusedforthisstudy.Barleyseedsweresowninthegreenhouse.Threeweekslater,

theseedlingswereexposedtonaturalinfectionwithBgh.Thefunguswasallowedtogrow

onthesurfaceofleavesundermoistconditionsfor4‐5daysuntilitcovered30‐40%ofthe

leafsurface.

Fungalmaterial

Pseudozymaflocculosawasgrowninyeastmaltpeptonedextrosebroth(YMPDB)

andharvestedatfourdifferenttimepoints:2h,8h,18hand30hspanningthedifferent

growthphasesof the fungus(Hammamietal.,2011).This fungalmaterialwasusedas

controlforP.flocculosagrowninbiologicalcontrolcondition.

InoculationwithPseudozymaflocculosa

From a 3‐day‐old culture, sporidia titer was adjusted to 1 x 107 cfu/ml and

inoculatedontheplantshighlyinfectedwithBgh.Waterfromthesporidiasolutionwas

lefttoevaporatefor20minutesandtheplantswerecoveredwithplasticbagstomaintain

ahighhumiditylevel.Barleymildewleafsampleswerecollectedat12h,24hand36hpost

inoculation.Watersprayswereusedascontrol.

RNAisolation

TotalRNAwasisolatedfromcontrol,powderymildewinfected,andP.flocculosa

growingonpowderymildewofbarleyinfectedleaves,andflaskculturesofP.flocculosaat

differenttimepointsusingtrizolfollowedbyRNeasyminikitfromQiagen.Concentration

and purity of the extractedRNAswere subsequentlymeasured byNanodrop and also

evaluatedbygelelectrophoresison0.8%agarosegelsat130Vandstainedwithethidium

20

bromide.TheRNAwaschecked for integrityonaBioanalyzer2100algorithm(Agilent

Technologies)beforemakingthecDNAlibraries.

cDNAlibraryconstruction

Library constructionwasdone for all the sampleswith the Illumina®TruSeq®

RNASamplePreparationKitv2.ThiskitwasusedtoconvertthemRNAintotalRNAinto

a library of template molecules suitable for subsequent cluster generation and DNA

sequencing.Thefirststepinlibrarypreparationinvolvespurifyingthepoly‐Acontaining

mRNAmoleculesusingpoly‐Toligo‐attachedmagneticbeads.Followingpurification,the

mRNAisfragmentedintosmallpiecesusingdivalentcationsunderelevatedtemperature.

ThecleavedRNAfragmentsarecopiedintofirststrandcDNAusingreversetranscriptase

and random primers. This is followed by second strand cDNA synthesis using DNA

polymeraseI.ThesecDNAfragmentsthengothroughanendrepairprocess,theaddition

ofasingle‘A’base,andthenligationoftheadapters.Theproductsarethenpurifiedand

enrichedbyPCR to create the final cDNA library. The cDNAwas checked for integrity

beforeperformingthesequencingprocessonaBioanalyzer2100(AgilentTechnololgies).

RNAsequencing

Replicationsandrandomizationareessentialcomponentsofawell‐plannedand

properlyanalyzedRNA‐seqdesign.Inourstudyweusedfivereplicationsforeachsample

atdifferenttimepointsandbeforelibrarypreparationsallthesampleswererandomized

todifferentgroupsandeachgroupcontainedsixindividuallibraries.RNAsequencingwas

performed on 48 cDNA library samples. We performed multiplexing and each cDNA

library was labeled or barcoded with sample specific sequences that allow multiple

samplestobeincludedinthesamesequencingreactionwhilemaintaininghighfidelity

sample identities downstream. Six librarieswere pooled togetherwhilemaintaining a

properratioofRNAquantityforeachlibraryandthensequencedinonelaneofaflowcell

usingIlluminaHiSeq2000sequencingtechnology.Twosequencingrunswereperformed

attheMcGillUniversityandGénomeQuébecInnovationCentre(McGillUniversity,Montréal,

Canada).

21

Mappingreadstothereferencegenome

Raw sequences in FASTQ format obtained from the sequencing platform were

analyzedusingCLCGenomicsWorkbenchv8.0.1(CLCbio,Aarhus,Denmark).Low‐quality

bases (Q< 15)were trimmed frombothendsof the sequences and theadapterswere

trimmedand theprocessed readswereused for further analysis. The sequenceswere

mappedtotherespectivereferencegenomesofP.flocculosa,Bghandbarleyusingaseries

ofprograms, includingBowtie2 forshort‐readmappingandTopHatv1.3.3 fordefining

exon–intron junctions. Theupdated genome sequences and annotations ofBgh canbe

found in http://www.blugen.org/. The complete genome sequence of P. flocculosa,

assembled into1,281scaffolds,wasused forprotein identificationbasedonhomology

withU.maydissequences(Lefebvreetal.,2013).Thewhole‐genomeshotgunsequencing

informationwasobtained fromGenBankunder theaccessionnumberAOUS00000000.

The recent genome sequence of barley was obtained from

http://plants.ensembl.org/Hordeum_vulgare. The principal component analysis (PCA)was

performedbyCLCGenomicsWorkbenchv8.0.1aftermappingasameasureofqualitycontrol

tocheckwhethertheoverallvariabilityofthesamplesreflecttheirgrouping.

Genomeannotation

Gene functions were predicted using InterproScan v4.8 (database v38.0)

(Quevillon et al., 2005).Annotationof CAZymeswasperformedusing thedbCANWeb

server (Yin et al., 2012). A search for genes involved in the biosynthesis of secondary

metaboliteswasperformedusingJCVISecondaryMetaboliteUniqueRegionsFinderWeb

server (SMURF) (Khaldi et al., 2010). Annotation of secreted proteins and CSEPswas

accomplished according to the method described by Mueller et al. (2008). Secreted

proteinswereselectedbaseduponSignalPv3.0(Bendtsenetal.,2004)D‐valueandDmax

cutoffs,TargetPv1.1(Emanuelssonetal.,2000)predictedlocationofproteins,TMHMM

v2.0(Kroghetal.,2001)predictedthenumberoftransmembranedomainsandposition

accordingtocleavagesite,andfinally,correlationtoLocDBorPotLocDBProtCompv9.0

(http://www.softberry.com) databases. Based on InterproScan‐assigned domains,

proteinslackingenzymaticfunctionswereclassifiedascandidateeffectors(CSEP).

22

Identificationofdifferentiallyexpressedgenes

The Illumina HiSeq reads were normalized and the expression level of each

transcript was expressed as the number of reads per transcript kilobase per million

fragmentsmapped(RPKM)value,whichwascalculatedbasedonthenumberofmapped

reads. A fold change >4 and FDR‐corrected p‐value <0.05was used as a parameter to

detectdifferentiallyexpressedgenesatdifferentconditionsineachlibrarybasedonRPKM

values. A gene ontology (GO) term was assigned to each transcript based on the GO

annotationsforbiologicalprocessineachofthereferencegenome.TheGOannotationsof

P.flocculosacanbefoundinfromGenBankundertheaccessionnumberAOUS00000000.

23

RESULTS

CharacteristicgrowthofPseudozymaflocculosaincultureconditions.

Pseudozyma flocculosa growth was analyzed in YMPD culture conditions and

characterized morphologically. The different growth stages of P. flocculosa showing

germination, mycelial growth and sporidia formationweremonitored at various time

points (Figure 1). Time 0 corresponds to the biological status of P. flocculosa in a

subculture fromanactivelygrowing,72h‐old liquidculture.At thisstage, the fungus is

present only in the formof ovoid spores called sporidia. After 4 h, in a freshmedium

containingthenecessarynutrientsfortheirdevelopment,sporidiagerminateanddevelop

hyphae.At8h,thecultureexpandsinmycelialgrowth,withnoresidualsporidia.After12

h,mycelialfilamentsstartedproducingnewsporidia,aprocessthatcontinuedoverthe

next 24 h. After 18h, the presence of needle‐like structures was observed, a clear

indication of flocculosin production by the fungus. After 36 h, only sporidia could be

observed.Basedontheseobservations,thesporephase(2h),exponentialgrowthphase

(8h)andflocculosinproductionphase(18h)wereconsideredasdistinctivegrowthphases

ofP.flocculosaandselectedfortranscriptomicstudies.

Figure1:CharacterizationofPseudozymaflocculosainculturecondition.GrowthanddevelopmentalphasesofP.flocculosaculturedinYMPDmediumover72h.

CalibrationofP.flocculosainresponsetopowderymildewfungus

Barleyplantscv.Fostergrowninagreenhouseshowedclearsignsofinfectionafter

two weeks (Figure 2). For their part, powdery mildew colonies inoculated with P.

24

flocculosawerecoveredbytheBCAasearlyas12hafterinoculation(Figure3).Overtime,

thepowderymildewfungalstructuresappearedtobeembeddedinamycelialnetwork

thatlookedlikea"spiderweb"(24h),whichledtotheirprogressiveandcompletecollapse

at36h.

Electron microscopy results clearly highlighted the destruction of powdery mildew

conidiabyP.flocculosaovertime(Figure4).TheseresultsclearlyshowedthatP.flocculosa

progressivelyinvadedthecoloniesofpowderymildewleadingtotheircollapseovertime.

Figure2:Developmentofpowderymildewdiseaseonbarleyplants.(a)Twoweek‐oldHordeumvulgareplantscv.Fostergrowningreenhouse(b)plantsexposedtonaturalinfectionwithBlumeriagraminisf.sp.hordei.

25

A B

Figure3:AntagonismofPseudozymaflocculosaonbarleypowderymildewcoloniesovertime.(A)Barleyleafsamplessprayedwithwaterasacontrol;(B)barleyleafsamplesinoculatedwithbiocontrolagentP.flocculosa.

T0h

T12h

T24h

T36h

T0h

T12h

T24h

T36h

26

Figure 4: Scanning electronmicroscopy observations of barley powderymildewfungus(A)beforeand(B)aftertreatmentwithPseudozymaflocculosa.

TranscriptionaldynamicsofpowderymildewfungusinresponsetoP.flocculosa

RNAintegrity,cDNAlibraryvalidationandsequencing

Theleafsamplescollectedatvarioustimepointspreandpost‐inoculationwithP.

flocculosaarepresentedinTable1.Qualityofthesampleswasassessedwithabioanalyser

andfoundtobeveryhigh(Figure5).Intotal,3200millionreads,each100nucleotides

long,weregenerated,withapproximately200millionreadsfromeachlane.Intotal,2%

ofthereadsalignedtorRNAandwereremovedpriortomappingtoreferencegenomes.

The principal component analysis (PCA) performed on P. flocculosa samples

revealedadistinctdifferencebetweentheinvitroandbiocontrolconditions(Figure6a).

Inthesamemanner,therewasaclearclusteringofthedifferenttreatmentsalongwiththe

replicationsassociatedwitheachinteraction(Figure6b).

A

B

27

TheprocessedreadswerethenalignedtotheP.flocculosareferencegenome.The

remainingreadsweremappedagainstthegenomeofB.graminisandtheprocessrepeated

against thebarleyreferencegenome(Figure6).At time0h,before inoculationwithP.

flocculosa,thereadsmappedinequalproportionsbetweenB.graminisandbarley.At12

h,thereadsthatmappedtotheP.flocculosagenomerepresentedaround1%andthiswas

accompaniedbyadecreaseandincreaseintherespectivereadsofB.graminisandbarley.

Thesametrendwasobservedat24hforthepathogenandtheplant,whileP.flocculosa

readsmorethandoubled.Interestingly,thedifferencesbetweenB.graminisandbarley

amplifiedat36hbutreadsassociatedwithP.flocculosareceded(Figure7).

Figure5.TotalRNA isolation frombarley leaf samplesand integritycheckusingbioanalyser. Total RNA isolation was performed on barley leaf samples collected atvarioustimepointsusingtrizolfollowedbyRNeasyMiniKitfromQiagen.TheintegrityoftheRNAwasmeasuredwithanAgilentbioanalyzer.

28S 18S

28

Figure6a.PrincipalComponentAnalysis(PCA)ofP. flocculosasamplesgrown invitroandinvivo.PrincipalcomponentanalysisgeneratedfromthegeneexpressiondataofP.flocculosainartificialculturemediaincomparisonwithbiologicalcontrolconditions.Eachcolordotintheplotrepresentsthesamplesandtheirreplicatescollectedatdifferenttimepoints.

29

Figure6b.PrincipalComponentAnalysis(PCA)ofbarleyleafsamplescollectedatvariousconditions.Principalcomponentanalysisgenerated fromthegeneexpressiondataofthesamplesofbarley,theinteractionBlumeriagraminisf.sp.hordei‐barleyandP.flocculosa‐B.graminis‐barleycollectedunderdifferentconditions.Eachcolordot in theplotrepresentsthesamplesandtheirreplicatescollectedatdifferenttimepoints.

30

Table 1. Sample collection pre and post‐inoculation of barley leaves withPseudozymaflocculosaandBgh.Theleafsampleswerecollectedatfourdifferenttimepointsunderthreedifferentconditions.Ateachtimepoint3‐6biologicalreplicateswerecollectedforstatisticalsignificance.

Figure7.Tripartiteinteractionmapping.Distributionofthetotalreads(rRNAreadsremoved)ontothereferencegenomeofHordeumvulgare,Blumeriagraminisf.sp.hordeiandPseudozymaflocculosabeforetheinoculationwiththebiocontrolagent(sterilewater

Samples Time0 Time12hours

Time24hours

Time36hours

Barley(Greenleaf) A1,A2,A3 A4,A5,A6 A7,A8,A9 A10,A11,A12

Barley+Blumeriagraminis+Water

B1,B2,B3 B4,B5,B6 B7,B8,B9 B10,B11,B12

Barley+B.graminis+P.flocculosa

N/A C1,C2,C3,C4,C5,C6

C7,C8,C9,C10,C11,C12

C13,C14,C15,C16,C17,C18

31

usedascontrol)andhourspost‐inoculation(hpi)withthebiocontrolagentP.flocculosaat12,24and36hours.

DifferentialgeneexpressionpatterninP.flocculosa

Differentially expressed genes (DEGs)were detected between in vitro grownP.

flocculosaandP.flocculosagrowingonBgh.Geneswithafalsediscoveryrate(FDR‐BH)

less than 0.05 and fold change greater than 4 were considered to be differentially

expressed. Our analysis revealed that 1948 genes, 1459 genes, and 1699 genes were

differentiallyexpressedandupregulatedinP.flocculosainbiocontrolconditionsat12,24

and36h,respectively(Figure8a).At thesametime,1541,1105and1409geneswere

differentiallyexpressedanddownregulatedinP.flocculosainbiocontrolconditionsat12,

24and36h,respectively(Figure8b).

A B

Figure 8. Differential gene expression pattern of Pseudozyma flocculosa genes.DifferentialgeneexpressionpatternofP.flocculosagenesinbiologicalcontrolconditionsagainst Blumeria graminis f.sp. hordei in comparison with in vitro cultures. The Venndiagramindicatesthenumberofgenesthataredifferentiallyexpressedateachspecifictime point. Intersection region in the Venn diagram indicates the number of genes

32

expressed in common between the time points. A. Differentially upregulated genes B.Differentiallydownregulatedgenes.Differentialgeneexpressionpatternofgenesinvolvedinflocculosinproduction

Flocculosinisacellobioselipidwithantifungalproperties,andinitiallythoughtto

be involved in thebiocontrol activityofP. flocculosa. Theexpression level of11genes

involvedinflocculosinproductionbyP.flocculosa isshowninFigure9.Comparedtoin

vitroconditions,nogenesoftheflocculosinclusterappeartobehighlyexpressedonBgh,

althoughalowerbutconsistentlevelofexpressionisseenforallgenes.

Figure 9. Differential gene expression pattern of flocculosin producing genes.Differential gene expression pattern of genes found in the flocculosin cluster betweenbiological control conditions against Blumeria graminis f.sp. hordei (red) and in vitro

33

cultures (blue).Theexpression levelof the11genes isexpressed inRPKM(ReadsPerKilobaseperMillionmappedreads).

P.flocculosaeffectorcandidatesaredifferentiallyexpressedduringinfectionwith

B.graminisf.sp.hordei

Effectorproteinsarekeyfactorsinestablishingtheinteractionbetweenaplantand

apathogenbutlittleisknownoftheirrolebetweentwointeractingfungi.Here,wepresent

resultsderived from theanalysisof the200uniqueCSEPs inP. flocculosa reportedby

Lefebvreetal.(2013).Amongtheeffectorgenes,33,39and41CSEPswerefoundtobe

specificallyupregulatedat12,24and36h,respectively(Figure10).Ofthisgroup,pf02826

hadthehighestfoldchangereachingnearly1000.Intotal,27CSEPshadmorethanasix‐

fold change, suggesting that CSEPs played an important role in the interaction of P.

flocculosawithBgh(Figure11).

Figure10.Upregulationofeffectorcandidatesatdifferenttimepoints.Upregulationof candidate secreted effector proteins (CSEPs) in Pseudozyma flocculosa in biological

34

controlconditionagainstBlumeriagraminisf.sp.hordeiatdifferenttimepointsof12,24and36hourspostinoculationwithP.flocculosa.

Figure 11. Differential gene expression pattern of effector candidates. Differential gene expression pattern of candidate secreted effector proteins (CSEPs) in Pseudozyma flocculosa between biological control condition against Blumeria graminis f.sp. hordei (BCA) and in vitro cultures (flask) condition.

35

Discussion

Transcriptomicanalysisofthemolecularinteractionsbetweenaplant(Hordeum

vulgare) – a pathogen (Blumeria graminis) – a biological control agent (Pseudozyma

flocculosa)showed thedifferentiallyexpressedgenes involved in thebiological control

activityofP.flocculosa.Thisatlasofdifferentiallyexpressedgenesrevealedtheroleand

importance of candidate secreted effector proteins (CSEPs) in the antagonism of P.

flocculosatowardapowderymildewfungus.Theseresultsarepartofacomprehensive

effortaimedatunderstandingthemolecularcrosstalkinthetripartiteinteractionbetween

aplant,pathogenandbiocontrolagent.

Pseudozymaflocculosaisaneffectiveantagonistofpowderymildewsbutitsspecific

activitytowardthisparticulargroupofplantpathogensisintricateandcomplex.Itcannot

parasitizeplantsbut is a powerful antagonist of powderymildews (Jarvisetal., 1989;

HajlaouiandBélanger,1993;Clement‐Mathieuetal.,2008). Inearlierclassification,all

smutfungiwereecologicallycharacterizedbytheirabilitytoinfectplantsandshareda

similarlifecyclewithayeast‐likehaploidphaseandaparasiticdikaryophase,culminating

in the production of numerouspowderyblack teliospores, hence, their commonname

(Begerow et al., 2006). However, a number of anamorphic fungi lacking sexual

development,initiallyplacedindeuteromycetoustaxa,werefoundtobemorphologically

andphylogeneticallyrelatedtotheUstilaginales.Inordertointegratetheseanamorphs

intothegeneralphylogeneticsystemofUstilaginomycetes,Begerowetal.(2000)analyzed

and compared diagnostic ribosomal DNA sequences of teleomorphic and anamorphic

speciesofUstilaginomycetes.TheiranalysesconfirmedthatspeciesofPseudozymaand

Ustilaginalesparasitizinggrasses formamonophyleticgroup.Pseudozyma species thus

representthesoleknownmembersoftheUstilaginalesthatcannotparasitizeplants.

Pseudozyma flocculosa was grown in artificial media for morphological

characterization at different time points. Obligate biotrophs likeB.graminis present a

numberofchallengessincetheycannotbeculturedoutsidetheirhost;theirlifecycleis

closelytiedwiththeinfectionprocessandthehost’sresponse.P.flocculosaonlygrowsas

anepiphyteontheleafsurface,butwillratherdevelopextensivelywheninpresenceof

powderymildewcolonies.Withthesechallengesinhand,itwasdifficulttooptimizethe

36

timepoints for thegrowthofP. flocculosa inbiological control conditions.Preliminary

experimentsandsmall‐scalesequencing(MiSeq)wereperformedtooptimizeabioassay

soitcouldbeusedfortranscriptomicanalysis.Basedontheresultsoftheseexperiments

andthegrowthofP.flocculosaininvitroconditions,thefunguswasinoculatedonplants

and the samples were collected 12, 24 and 36 hours post‐inoculation (hpi) with P.

flocculosa.Themicroscopyresultsshoweddistinctdifferencesbetweentheleafsamples

inoculatedwithwaterandP.flocculosa.DestructionofconidiaofBghbyP.flocculosawas

clearlyvisibleasearlyas12hpiandcompleteat36hpi inelectronmicroscopy.These

resultsjustifiedourtimepointschosenforsamplecollectionfortranscriptomicanalysis.

Earlier studies aboutP. flocculosa showed that flocculosin is an activemolecule

involvedinthemodeofactionofP.flocculosa(Mimeeetal.,2005).Followingthediscovery

of flocculosin and its structural similarity with ustilagic acid, a cluster of 11 genes

responsible for flocculosin production was identified (Teichmann et al., 2010). These

resultshighlightthattheproductionofunusualglycolipidsbytworelatedyetdisparate

organismsistheresultofanintricateandwell‐conservedenzymaticprocessexclusiveto

thetwostudiedfungi.Fromabiologicalorevolutionarypointofview,onehastoassume

thatconservationofthisgeneclusterservesadistinctpurpose,eventhoughevidenceto

that effect is still lacking. Inour transcriptomic analysis the expression level of the11

geneswithin thegenecluster responsible for flocculosinproductionwashighwhenP.

flocculosawascultured inartificialmedia incomparisonwiththebiocontrolcondition.

These results proved that P. flocculosa relies on mechanisms other than flocculosin

productiontoantagonizebarleypowderymildewaspreviouslysuggestedbyMarchandet

al.,(2007).

Followingthereleaseofthefirstfungalgenomesandthedevelopmentofreliable

bioinformaticstoolstopredictsecretionsignalsinproteinsequences,emphasishasbeen

placedon the studyof effectorproteins asdeterminantsofpathogenicity (Tortoetal.,

2003).Inavarietyofplantpathogens,includingsmutfungi,powderymildews,rusts,and

oomycetes, effectors have been found to affect virulence, suppress plant defense

responses,dictatehostspecificity,and/ortomaintainabiotrophicinteraction.Basedon

themethoddescribedbyMuelleretal.(2008),547secretedproteinswereidentifiedinP.

37

flocculosa. Among them, 345 could not be assigned an enzymatic function and were

thereforeconsideredasCSEPs.Ofthose,200werefoundtobeuniquetoP.flocculosa.In

our differential gene expression analysis, we closely followed the 200 CSEPs that are

uniquetoP. flocculosa.OurresultsshowedthatmanyofthemareupregulatedwhenP.

flocculosaantagonizespowderymildewstructures.Oneofthemhadnearlya1000‐fold

change,whichwouldclassifyitasagoodcandidatetoinvestigatefurtherforfunctional

studies.Takentogether,theseresultsstronglysuggestthateffectorproteinsplayavery

importantroleintheinteractionofP.flocculosawithbarleypowderymildew.

In conclusion this is the first study of transcriptomic analysis of a tripartite

interaction plant‐pathogen‐biocontrol agent. RNA‐seq results highlighted the global

changesinthegeneexpressionpatterninP.flocculosainresponsetothepathogenandthe

host. Inparticular,DGEpatternanalysis gaveus the atlasof effectorproteinspossibly

involvedintheinteractionbetweenP.flocculosaandBgh.Thisinvestigationoftheroleof

effectorproteinsinsuchinteractionsopensnewopportunitiestowardunderstandingthe

potentialfactorsinvolvedinbiologicalcontrol.Toourknowledge,thisisthefirstreport

linkingeffectorproteinsinfungal‐fungalinteractions.

ACKNOWLEDGEMENTS

WethankInstitutdeBiologieIntegrativeetdesSystems(IBIS)atLavalUniversity

for their technical assistance. This work was supported by grants from the Natural

Sciences and Engineering Research Council of Canada, Centre SÈVE and the Canada

ResearchChairsProgramtoR.R.B

REFERENCES

Avis,T.J.andBélanger,R.R.(2001).Specificityandmodeofactionoftheantifungalfattyacid cis‐9‐heptadecenoic acid produced by Pseudozyma flocculosa. Applied andEnvironmentalMicrobiology,67:956‐60.

Begerow, D., Bauer, R. and Boekhout, T. (2000). Phylogenetic placementsustilaginomycetous anamorphs as deduced from nuclear LSU rDNA sequences.MycologicalResearch,104:53‐60.

Begerow, D., Stoll, M. and Bauer, R. (2006). A phylogenetic hypothesis ofUstilaginomycotinabasedonmultiplegeneanalysesandmorphologicaldata.Mycologia,98:906‐91.

38

Bélanger,R.R., Labbé, C. and Jarvis,W.R. (1994). Commercial‐scale control of rosepowderymildewwithafungalantagonist.PlantDisease,78:420–424.

Bendtsen,J.D.,Nielsen,H.,Heijne,G.V.andBrunak,S.(2004).ImprovedPredictionofSignalPeptides:SignalP3.0.JournalofMolecularBiology,340:783‐795.

Clément‐Mathieu, G., Chain, F.,Marchand, G. and Bélanger, R.R. (2008). Leaf andpowdery mildew colonization by glycolipid‐producing Pseudozyma species. FungalEcology,1:69‐77.

Emanuelsson,O.,Nielsen,H.,Brunak,S.andHeijne,G.V.(2000).Predictingsubcellularlocalization of proteins based on their N‐terminal amino acid sequence. Journal ofMolecularBiology,300:1005‐1016.

Hajlaoui,M.R. andBélanger,R.R. (1993). Antagonism of the yeast‐like phyllophanefungusSporothrix flocculosaagainstErysiphegraminisvartritici.BiocontrolScienceandTechnology,3:427‐434.

Hammami,W.,Castro,C.Q.,Remus‐Borel,W.,Labbé,C.andBélanger,R.R. (2011).EcologicalbasisoftheinteractionbetweenPseudozymaflocculosaandpowderymildewfungi.AppliedandEnvironmentalMicrobiology,77:926‐933.

Jarvis,W.R., Shaw, L.A. and Traquair, J.A. (1989). Factors affecting antagonism ofcucumber powderymildew by Stephanoascus flocculosus and S. rugulosus.MycologicalResearch,92:162‐165.

Kämper, J.,Kahmann,R.,Bölker,M.,Ma,L.J.,Brefort,T.,Saville,B.J.etal. (2006).InsightsfromthegenomeofthebiotrophicfungalplantpathogenUstilagomaydis.Nature,444:97‐101.

Khaldi, N., Seifuddin, F.T., Turner, G., Haft, D., Nierman,W.C.,Wolfe, K.H. andFedorova, N.D. (2010). SMURF: Genomic mapping of fungal secondary metaboliteclusters.FungalGeneticsandBiology,47:736‐741.

Kiss,L.(2003)Areviewoffungalantagonistsofpowderymildewsandtheirpotentialasbiocontrolagents.PestManagementScience,59:475–483.

Krogh, A., Larsson, B., Heijne, G.V. and Sonnhammer, E.L. (2001). PredictingtransmembraneproteintopologywithahiddenMarkovmodel:applicationtocompletegenomes.FungalGeneticsandBiology,305:567‐580.

Lefebvre,F., Joly, D.L., Labbé, C., Teichmann,B., Linning, R., Belzile, F. et al. (Bélanger, R.R.) (2013). The transition from a phytopathogenic smut ancestor to ananamorphic biocontrol agent deciphered by comparative whole‐genome analysis. ThePlantCell,25:1946‐1959.

Marchand,G.,Clément‐Mathieu,G.,Neveu,B.andBélanger,R.R.(2007).Enhancingbiologicalcontrolefficacyofyeaststocontrolfungaldiseasesthroughbiotechnology.InZ.K. Punja, S.H. De Boer, and H. Sanfaçon (Eds.), Biotechnology and plant diseasemanagement(p.518‐531).CABI,Wallingford,UK.

39

Mimee,B., Labbé, C., Pelletier,R. andBélanger,R.R. (2005). Antifungal activity offlocculosin,anovelglycolipidisolatedfromPseudozymaflocculosa.AntimicrobialAgentsandChemotherapy,49:1597‐1599.

Mueller,O.,Kahmann,R.,Aguilar,G.,Trejo‐Aguilar,B.,Wu,A. anddeVries,R.P.(2008).ThesecretomeofthemaizepathogenUstilagomaydis.FungalGeneticsandBiology,45(Suppl.1),S63‐S70.

Paulitz,T.C.andBélanger,R.R.(2001).Biologicalcontrolingreenhousesystems.AnnualReviewofPhytopathology,39:103‐133.

Quevillon,E.,Silventoinen,V.,Pillai,S.,Harte,N.,Mulder,N.,Apweiler,R.andLopez,R.(2005).InterProScan:proteindomainsidentifier.NucleicAcidsResearch,33(Suppl.2),W116‐W120.

Spanu,P.D.,Abbott,J.C.,Amselem,J.,Burgis,T.A.,Soanes,D.M.,Stüber,K.etal.(2010).Genomeexpansionandgene loss inpowderymildewfungireveal tradeoffs inextremeparasitism.Science,330:1543‐1546.

Teichmann,B.,Liu,L.,Schink,K.O.andBölker,M.(2010).Activationoftheustilagicacidbiosynthesisgenecluster inUstilagomaydisby theC2H2zinc finger transcriptionfactorRua1.AppliedandEnvironmentalMicrobiology,76:2633‐2640.

Torto,T.A.,Li,S.,Styer,A.,Huitema,E.,Testa,A.,Gow,N.A.etal.(2003).ESTminingand functional expressionassays identifyextracellulareffectorproteins from theplantpathogenPhytophthora.GenomeResearch,13:1675‐1685.

Traquair,J.A.,Shaw,L.A.andJarvis,W.R.(1988).NewspeciesofStephanoascuswithSporothrixanamorphs.CanadianJournalofBotany,66:926‐933.

Wicker,T.,Oberhaensli,S.,Parlange,F.,Buchmann,J.P.,Shatalina,M.,Roffler,S.,Ben‐David,R.,Doležel, J., Simková,H., Schulze‐Lefert, P., Spanu, P.D.,Bruggmann,R.,Amselem,J.,Quesneville,H.,vanThemaat,E.V.L.,Paape,T.,Shimizu,K.K.andKeller,B.(2013).Thewheatpowderymildewgenomeshowstheuniqueevolutionofanobligatebiotroph.NatureGenetics,45:1092–1096.

Yin,Y.,Mao,X.,Yang,J.,Chen,X.,Mao,F.andXu,Y.(2012).dbCAN:awebresourceforautomated carbohydrate‐active enzyme annotation. Nucleic Acids Research, 40(W1):W445‐W451.

CHAPTER3

GENERALCONCLUSIONS

43

GENERALCONCLUSIONS

Plant diseases need to be controlled in order to maintain the quality and

abundanceof food, feed,andfiberproducedbygrowersaroundtheworld.Different

approacheshavebeenusedtoprevent,mitigateorcontrolplantdiseases.Beyondgood

agronomic and horticultural practices, growers often rely heavily on chemical

fertilizersandfungicides.Theroadsleadingtothediscoveryofnewtoolsinthefight

againstcroppestsarefilledwithexcitingdiscoveriesandchallenges.Formanyyears,

considerableeffortshavebeendeployedgloballytodevelopmoreeffectivealternatives

to conventional pesticides (Enserink et al., 2013). Consequently, some plant

pathologistshave focused theireffortsondevelopingalternative inputs to synthetic

chemicals for controlling pests and diseases. Among these alternatives are those

referredtoasbiologicalcontrol.Avarietyofbiologicalcontrolmethodsareavailable

forusebutfurtherdevelopmentandeffectiveadoptionarerequiredtoproperlyexploit

biocontrolagents.Inthiscontext,itisimportanttounravelthekeymechanismsofa

biocontrol agent. For this purpose, we need to explore the relationship between a

biocontrolagentanditsinteractionwiththepathogenandtheplant.

SincethediscoveryofPseudozymaflocculosa,continuouseffortshavebeenmade

tobetterunderstanditsgeneticbasisas itrelatestoitsspecificproperties.Overthe

years,hypothesessurroundingitsmodeofactionhaveevolvedandchanged.Fromthe

beginning,theideaofmycoparasitism,similartothatobservedinTrichodermassp.,was

rejectedinfavorofthehypothesispromotingantibiosisastheprimarymodeofaction

(Hajlaoui and Bélanger, 1993). Thereafter, the likelihood of that hypothesis was

reinforced by the discovery of a molecule with antifungal properties, flocculosin,

produced in large quantities by the BCA (Cheng et al., 2003; Mimee et al., 2005).

However, the phylogenetic relationship between P. flocculosa and Ustilago maydis

highlightedthestructuralsimilarityofflocculosinwithustilagicacid(Begerowetal.,

2006;Marchandetal.,2009).Thisfindingforcedthereconsiderationofantibiosisas

themodeofactionofP.flocculosasinceU.maydiswasclearlynotproducingustilagic

acid for the same purpose. The publication of the genome of P. flocculosa was an

importantsteptodeepenourknowledgeaboutthebiologyoftheorganism.Itprovided

44

many tools to validate old and new hypotheses with regards to P. flocculosa could

antagonizebarleypowderymildew.

Inthiswork,thisisthefirsttimethatatranscriptomicanalysisisexploitedto

study the genetic determinants of a tritrophic interaction. Our results have clearly

highlighted that several effector proteins were upregulated in P. flocculosa when

attackingbarleypowderymildew(Bgh),whilegenesrelatedtoflocculosinproduction

are not. To our knowledge, this is the first suggestion that effector proteinswould

dictatetheinteractionbetweentwofungithesamewaytheydobetweenapathogen

andaplant.Thisopensupabrandnewfieldofinvestigationthatcouldleadstomajor

advances in the field of biological control and translational research for practical

applicationinagriculture.

45

BIBLIOGRAPHY

Avis,T.J.andBélanger,R.R.(2001).Specificityandmodeofactionoftheantifungalfattyacid cis‐9‐heptadecenoic acid produced by Pseudozyma flocculosa. Applied andEnvironmentalMicrobiology,67:956‐60.

Begerow, D., Stoll, M. and Bauer, R. (2006). A phylogenetic hypothesis ofUstilaginomycotinabasedonmultiplegeneanalysesandmorphologicaldata.Mycologia,98:906‐91.

Bélanger,R.R.andAvis,T.J.(2002).Ecologicalprocessesandinteractionsoccurringinleaf surface fungi. In S.E. Lindow, E.I. Hecht‐Poinar and V.J. Elliott (Eds.),Phyllospheremicrobiology(p.193‐208).TheAmericanPhytopathologicalSocietyPress.St.Paul,MN:

Cheng,Y.,McNally,D.J.,Labbé,C.,Voyer,N.,Belzile,F.andBélanger,R.R. (2003).Insertionalmutagenesisofafungalbiocontrolagentledtodiscoveryofararecellobioselipidwithantifungalactivity.AppliedandEnvironmentalMicrobiology,69:2595‐2602.

DeVos,M.,VanOosten,V.R.,VanPoecke,R.M.,VanPelt,J.A.,Pozo,M.J.,Mueller,M.J.,Pieterse,C.M.(2005).SignalsignatureandtranscriptomechangesofArabidopsisduringpathogenandinsectattack.Molecularplant‐microbeinteractions,18:923‐937

Djamei,A.,Schipper,K.,Rabe,F.,Ghosh,A.,Vincon,V.,Kahnt,J.,Osorio,S.,Tohge,T.,Fernie,A.R.andFeussner, I. (2011).Metabolicprimingbya secreted fungal effector.Nature,478:395‐398.

Enserink,M.,Hines,P.J.,Vignieri,S.N.,Wigginton,N.S.andYeston,J.S.(2013).Smarterpestcontrol.Thepesticideparadox.Introduction.Science,341:728‐729.

Flint,Maria Louise & Dreistadt, Steve H. (1998) Natural Enemies Handbook: TheIllustratedGuide toBiological Pest Control.University of California Press. ISBN978‐0‐520‐21801‐7.

Hacquard S,KracherB,MaekawaT,Vernaldi S, Schulze‐LefertP,Ver Loren vanThemaatE.V.L.(2013).Mosaicgenomestructureofthebarleypowderymildewpathogenand conservation of transcriptional programs in divergent hosts. Proceedings of theNationalAcademyofSciences,110:E2219‐28.

Hajlaoui,M.R. andBélanger,R.R. (1993). Antagonism of the yeast‐like phyllophanefungusSporothrix flocculosaagainstErysiphegraminisvartritici.BiocontrolScienceandTechnology,3:427‐434.(Voirp.5:HajlaouiandBélanger1993)

Jones,J.D.andDangl,J.L.(2006).Theplantimmunesystem.Nature444:323‐329.

Kämper, J.,Kahmann,R.,Bölker,M.,Ma,L.J.,Brefort,T.,Saville,B.J.etal. (2006).InsightsfromthegenomeofthebiotrophicfungalplantpathogenUstilagomaydis.Nature,444:97‐101.

46

Klaus, P.,andMüller S. (Eds.) (2012). The roots of logistics. A Reader of ClassicalContributions to the History and Conceptual Foundations of the Science of Logistics.Springer‐Verlag,Berlin.

Kubicek,C.P.,Mach,R.L.,Peterbauer,C.K.andLorito,M.(2001).Trichoderma: fromgenestobiocontrol.JournalofPlantPathology,83:11‐23.

Lefebvre,F.,Joly,D.L.,Labbé,C.,Teichmann,B.,Linning,R.,Belzile,F.etal.(Bélanger,R.R.) (2013). The transition from phytopathogenic smut ancestor to an anamorphicbiocontrolagentdecipheredbycomparativewhole‐genomeanalysis.ThePlantCell,25:1946‐1959

Lorito,M.,Scala,F.,Zoina,A.andWoo,S.(2001).EnhancingbiocontroloffungalpestsbyexploitingtheTrichodermagenome.InM.Vurroetal.(Eds.),NATOScienceSeriesI:LifeandBehaviouralSciences:Enhancingbiocontrolagentsandhandlingrisks339:247‐259.

Marchand,G.,Clément‐Mathieu,G.,Neveu,B.andBélanger,R.R.(2007).Enhancingbiologicalcontrolefficacyofyeaststocontrolfungaldiseasesthroughbiotechnology.InZ.K. Punja, S.H. De Boer, and H. Sanfaçon (Eds.), Biotechnology and plant diseasemanagement(p.518‐531).CABI,Wallingford,UK.

Mimee,B., Labbé, C., Pelletier,R. andBélanger,R.R. (2005). Antifungal activity offlocculosin,anovelglycolipidisolatedfromPseudozymaflocculosa.AntimicrobialAgentsandChemotherapy,49:1597‐1599.

Sonah,H.,Deshmukh,R.K. andBélanger,R.R. (2016). Computational Prediction ofEffector Proteins in Fungi: Opportunities and Challenges.Frontiers inPlant Science, 7:article#126,14p.doi:10.3389/fpls.2016.00126

Spanu,P.D.,Abbott,J.C.,Amselem,J.,Burgis,T.A.,Soanes,D.M.,Stüber,K.etal.(2010).Genomeexpansionandgene loss inpowderymildewfungireveal tradeoffs inextremeparasitism.Science,330:1543‐1546.

Teichmann,B.,Liu,L.,Schink,K.O.andBölker,M.(2010).Activationoftheustilagicacidbiosynthesisgenecluster inUstilagomaydisby theC2H2zinc finger transcriptionfactorRua1.AppliedandEnvironmentalMicrobiology,76:2633‐2640.

Traquair,J.A.,Shaw,L.A.andJarvis,W.R.(1988).NewspeciesofStephanoascuswithSporothrixanamorphs.CanadianJournalofBotany,66:926‐933.

Whipps,J.M.(2001).Microbialinteractionsandbiocontrolintherhizosphere.JournalofExperimentalBotany,52(suppl1),487‐511.

Wicker,T.,Oberhaensli,S.,Parlange,F.,Buchmann,J.P.,Shatalina,M.,Roffler,S.,Ben‐David,R.,Doležel, J., Simková,H., Schulze‐Lefert, P., Spanu, P.D.,Bruggmann,R.,Amselem,J.,Quesneville,H.,vanThemaat,E.V.L.,Paape,T.,Shimizu,K.K.andKeller,B.(2013).Thewheatpowderymildewgenomeshowstheuniqueevolutionofanobligatebiotroph.NatureGenetics,45:1092–1096.

47

Wiese,M.V.(1987).Compendiumofwheatdiseases.AmericanPhytopathologicalSociety.pp.124pp.ISBN0‐89054‐076‐4.