poorww.4399

ORIGINALPAPERInter-andintracellularcolonizationofArabidopsisrootsbyendophyticactinobacteriaandtheimpactofplanthormonesontheirantimicrobialactivityAnnevanderMeij.
JoostWillemse.
MartinusA.
Schneijderberg.
ReneGeurts.
JosM.
Raaijmakers.
GillesP.
vanWezelReceived:20November2017/Accepted:3January2018/Publishedonline:15January2018TheAuthor(s)2018.
ThisarticleisanopenaccesspublicationAbstractManyactinobacterialiveincloseassoci-ationwitheukaryotessuchasfungi,insects,animalsandplants.
Plant-associatedactinobacteriadisplay(endo)symbiotic,saprophyticorpathogeniclifestyles,andcanmakeupasubstantialpartoftheendophyticcommunity.
Here,wecharacterisedendophyticacti-nobacteriaisolatedfromroottissueofArabidopsisthaliana(Arabidopsis)plantsgrowninsoilfromanaturalecosystem.
ManyoftheseactinobacteriabelongtothefamilyofStreptomycetaceaewithStreptomycesolivochromogenesandStreptomycesclaviferaswellrepresentedspecies.
WhenseedsofArabidopsiswereinoculatedwithsporesofStrepto-mycesstraincoa1,whichshowshighsimilaritytoS.
olivochromogenes,rootswerecolonisedintercellu-larlyand,unexpectedly,alsointracellularly.
Subse-quentexposureofendophyticisolatestoplanthormonestypicallyfoundinrootandshoottissuesofArabidopsisledtoalteredantibioticproductionagainstEscherichiacoliandBacillussubtilis.
Takentogether,ourworkrevealsremarkablecolonizationpatternsofendophyticstreptomyceteswithspecictraitsthatmayallowacompetitiveadvantageinsideroottissue.
KeywordsStreptomycesPlant–microbeinteractionsPlanthormoneCrypticantibioticsElectronmicroscopyIntroductionActinobacteriarepresentadiversephylumcomposedofbothrod-shapedandlamentousbacteriathatcanbefoundinsoil,marineandfreshwaterecosystems(Goodfellow2012).
Thelamentousactinobacteriaareversatileproducersofbioactivenaturalproducts,includingtwo-thirdsofallknownantibioticsaswellasmanyanticancer,antifungalandimmunosuppressiveagents(Barkaetal.
2016;Berdy2005;HopwoodElectronicsupplementarymaterialTheonlineversionofthisarticle(https://doi.
org/10.
1007/s10482-018-1014-z)con-tainssupplementarymaterial,whichisavailabletoauthorizedusers.
AnnevanderMeijandJoostWillemsehavecontributedequallytothiswork.
A.
vanderMeijJ.
WillemseG.
P.
vanWezel(&)MolecularBiotechnology,InstituteofBiology,LeidenUniversity,Sylviusweg72,2333BELeiden,TheNetherlandse-mail:g.
wezel@biology.
leidenuniv.
nlM.
A.
SchneijderbergR.
GeurtsDepartmentofPlantSciences,WageningenUniversity,Wageningen,TheNetherlandsJ.
M.
RaaijmakersDepartmentofMicrobialEcology,NetherlandsInstituteofEcology(NIOO-KNAW),Wageningen,TheNetherlands123AntonievanLeeuwenhoek(2018)111:679–690https://doi.
org/10.
1007/s10482-018-1014-z2007).
Theactinobacteriaarealsomajorproducersofindustriallyrelevantenzymes(Anneetal.
1990;vanDisseletal.
2014).
Hence,theactinobacteriaareofutmostimportanceforhumanhealth,agricultureandbiotechnology.
Theconceptofactinobacteriaasfree-livingbacte-riahasmorerecentlybeenchallengedbystudiespointingtotheirintimaterelationshipswithdiverseeukaryotes(Seipkeetal.
2012;vanderMeijetal.
2017).
Indeed,theyhavebeenfoundinassociationwithvertebrates,invertebrates,fungiandplants.
Actinobacteriaareoftenwelcomegueststotheirhostsduetotheirabilitytoproducechemicallydiversenaturalproducts.
Muchofthechemicaldiversityofsecondarymetabolitesproducedbyactinobacteriahaslikelyevolvedbecauseoftheirinteractionswithother(micro)organismsinhighlydiverseenvironments(Seipkeetal.
2012;vanderMeijetal.
2017).
Itisbecomingincreasinglyclearthatactinobacteriaplayakeyroleinmaintainingplanthealthbycontributingtobioticandabioticstresstolerance(Viaeneetal.
2016).
Forexample,actinobacteriaproducesiderophoresforironacquisitionaswellasantibacterialsandantifun-galstoprotecttheirhostagainstpathogens(Viaeneetal.
2016).
Actinobacteriacanmakeupasubstantialpartoftherootendophyticcommunityacrosstheplantkingdom,whichislargelydeterminedbyanincreasedrelativeabundanceofthefamilyofStreptomycetaceae(Bul-garellietal.
2012;Edwardsetal.
2015;Lundbergetal.
2012).
Thecompositionofrhizosphericandrootendophyticbacterialcommunitiesisstronglyinu-encedbysoiltype(Bulgarellietal.
2012)aswellasbytheplantgenotype(Perez-Jaramilloetal.
2017).
Therefore,partofthemicrobiomecompositionoftherhizosphereandendosphereofArabidopsisthalianagrownundercontrolledconditionsindiffer-entnaturalsoilsisconservedwithspecicbacterialtaxaincludingmembersoftheactinobacteria(Bul-garellietal.
2012).
Despitetheiroccurrenceintheendosphere,verylittleisknownonhowmostactinobacteriacolonisetheendosphereandwheretheyreside.
AnexceptionisStreptomycesscabies,thecausalagentofpotatoscab,whichhasbeenstudiedindetail(Loriaetal.
2006;Jourdanetal.
2016;Bignelletal.
2010;Bukhalidetal.
1998).
Colonizationofrootandshoottissuebyactinobac-teriaisdependent,atleastinpart,onchemicalcuesfromtheplantaswasshownforenrichmentofactinobacteriabytheplanthormonesalicylicacid(SA)(Lebeisetal.
2015).
SAplaysaroleinavarietyofphysiologicalandbiochemicalprocessesandisimportantasanendogenoussignalmediatinglocalandsystemicplantdefenseresponsesagainstpathogensandabioticstressfactors(Rivas-SanVicenteandPlasencia2011).
SAisdetectableinArabidopsisleavesandrootsatconcentrationsupto1lg/gfreshweight(vandeMorteletal.
2012).
Hence,endophyticactinobacteriaaremostlikelyexposedandresponsivetoSAandotherplanthormonessuchasjasmonicacidandauxin(Halimetal.
2006;Zhao2010).
InthisstudyweisolatedendophyticactinobacteriafromArabidopsisroots,grownundercontrolledconditionsaswellasfromplantsgrowninanecologicalsetting.
Colonizationpatternsweremoni-toredandvisualisedbyconfocalaswellaselectronmicroscopyforStreptomycesstraincoa1,whichadaptedfromasoildwellingtoanendophyticlifestyle,onaxencially-grownA.
thaliana.
Finally,weinvestigatedhowspecicplanthormonesaffecttheantimicrobialactivityofendophyticStreptomycesisolatedfromwildArabidopsisprovidingarststeptowardsevaluatingtheconceptofplant-mediated'antibioticproductionondemand'.
ResultsanddiscussionIsolationofendophyticactinobacteriaToconrmthepresenceofactinobacteriaintheendosphere,sterileA.
thalianaCol-0plantsweregrowninapottingsoil:sandmixturefor2weeksundercontrolledconditions,followedbyharvestingrootsandshoot,surface-sterilizationandhomogenizationoftheroottissueandplatingontovariousmediaselectiveforactinobacteria(Zhuetal.
2014b).
Tenmorpholog-icallydistinctactinobacterialisolateswereobtainedfortheCol-0plants.
AsimilarisolationapproachwasadoptedforA.
thalianaecotypemossel(Msl)obtainedfromanaturalecosystem(Mossel,Veluwe,theNetherlands).
NexttoplatingontoselectivemediafortheisolationofactinobacterialendophytesfromMslplants,totalDNAwasextractedineightreplicatesfromsamplesobtainedfromthesoil,therhizosphere,therootendosphereandtoothpicksinsertedinthesoilwereutilisedaswoodcompartment(Bulgarellietal.
2012).
TheseDNAsampleswerethenanalyzedby680AntonievanLeeuwenhoek(2018)111:679–69012316SrDNA-ampliconsequencing.
Basedontotalsequencereadstheresultsofthelatteranalysisshowedthatactinobacteriarepresentedonaverage22%ofthetotalendophyticpopulation(Fig.
1a).
Amongtheactinobacterialoperationaltaxonomicunits(OTUs),theStreptomycetaceaeandMicromonosporaceaewereoverrepresented.
Additionally,10%thereadsoftheendophyticpopulationwasrepresentedbyonlytwoOTUs(137and48),belongingtothefamilyofStreptomycetaceae(Fig.
2b).
Thisenrichmentwasnotasmuchobservedforthesoil,therhizosphereandbacteriaassociatedwithatoothpick,suggestingaspecies-specicselectionamongtherootendophyticactinobacteriaofA.
thaliana.
NumbersontotalsequencereadsandOTUsarelistedinTableS1.
IsolationofactinobacteriafromtheendosphereofA.
thalianaecotypeMslresultedin35morphologi-callydifferentisolates.
The34-465regionofthe16SRNAgeneofallisolateswassequencedandtheclosesthitsontheEzBioCloudwereconrmedusingtheMLSA-basedphylogenypublishedrecently(Labedaetal.
2017).
LinkingtheOTUsdetectedby16SrDNAampliconsequencingtoasingleStrepto-mycesspecieswasnotalwayspossibleduetoarelativelylowresolution(Girardetal.
2013;Labedaetal.
2017).
Nevertheless,thetwoisolatesthatwereanalysedbothbelongedtothemostabundantOTUsStreptomycesclaviferandStreptomycesolivochromo-genes(Fig.
2a).
Outofthe35isolates,vewerecloselyrelatedtoS.
claviferandfourtoS.
olivochro-mogenes.
Supportiveevidencecomesfromseveralindependentstudies,reportingS.
olivochromogenesfromtheendosphereofChinesecabbageroots,potatotubers,medicinalplantsandpurplehenbit(SinghandGaur2016;Leeetal.
2008;Doumbouetal.
1998;Kimetal.
2012).
Additionally,strainssimilartoS.
claviferwerepreviouslyisolatedfromsugarcane,lucerneplantsandwheat(Kruasuwanetal.
2017;Francoetal.
2017;MiskandFranco2011).
Subsequentphenotypingoftheseendophyticacti-nobacterialisolatesbyhighresolutionimagingbyscanningelectronmicroscopy(SEM)revealedspinysporesthataretypicalofS.
clavifer(Goodfellow2012),supportingthe16SrDNA-basedtaxonomicclassicationoftheisolatesasS.
clavifer(Fig.
2b).
Incontrast,S.
olivochromogenespoorlydevelopssporu-latingmyceliumaccordingtoBergey'smanual,whichisshownaswellforanisolateclassiedasS.
olivochromogenes(Fig.
2c).
However,impairedsporulationwasencounteredfrequently,aphenotypethatwasdependentonthegrowthmedia,whichmakesthisfeaturenon-specic(seenextparagraph).
TheFig.
1Relativeabundanceoftheactinobacteriaintheendo-phyticcompartmentofA.
thalianaMsl.
aAmpliconsequencingdatashowthatactinobacteriarepresent22%ofthetotalendophyticcompartment(EC),whileinsoilandrhizosphere(RS)communitiesandthoseinassociationwithatoothpick(TP)insertedinthesoil,theytakeuproughly2%ofthetotalbacterialcommunity.
bTheenrichmentofactinobacteriaispredomi-natelydrivenbyStreptomycetaceaeOTU48and137.
OTU48and137makeup10%ofthetotalECandalmost50%ofalltheactinobacteriapresentintheEC.
TheseOTUsarenotasmuchenrichedinthesamplesderivedfromnon-endophyticoriginsAntonievanLeeuwenhoek(2018)111:679–690681123endophyticisolatesofA.
thalianaecotypeMslshoweddiversecolonymorphologies(forexamplesseeFig.
3).
Remarkably,20outof35isolatesfailedtodeveloponR5agarplates,suggestingtheyhadeitherlostkeysporulationgenesorlackedtheabilitytodevelopintheabsenceofspecicnutrientsortraceelements.
Forexample,R5agarisknowntolacksufcientironandcopper,whichisamajorreasonwhybldmutantscannotsporulateonthismedia(Keijseretal.
2000;Lambertetal.
2014).
Addition-ally,severalendophytesshowedthepropensitytoopenuptheircolony,withtheirvegetativemyceliumfacingupward.
ThisfeatureiswellexempliedbyStreptomycessp.
MOS31andMOS18(Fig.
3h,c).
MOS18istaxonomicallyrelatedtoS.
claviferandMOS31istheonlyendophyticisolatecloselyrelatedtoStreptomycesbobili,a'neighbour'ofS.
olivochro-mogenes(Labedaetal.
2017).
Tostudythemorpho-logicalcharacteristicsofMOS31athigherresolutionSEMwasapplied,whichrevealedathicksheetofhyphaethatturnedawayfromtheinsideofthecolony(Fig.
4a).
Additionally,weobservedhyphaeextend-ingfromthesesheets,awayfromthevegetativemycelium(Fig.
4b).
EndophyticcolonizationofArabidopsisbyStreptomycesWethenwantedtoknowifandwheretheisolatesentertherootendosphereofA.
thalianatogetmoreinsightintotheyetelusiveendophyticbiologyofStrepto-myces.
ThereforeweinoculatedsporesofStrepto-mycesstraincoa1,whichwasrecruitedbysterileA.
thalianaCol-0plantsgrowninapottingsoil:sandmixture,ontosterilisedseedsofA.
thalianaCol-0.
Noteworthy,16SrDNAanalysisclusterscoa1intheS.
olivochromogenesbranchbasedonphylogeneticrelationshipswithinthefamilyofStreptomycetaceae(Labedaetal.
2017).
AttachmentofthesporestotheseedswasconrmedbySEM(Fig.
S1).
Seven-day-oldseedlingsgrownfromthetreatedseedswerestainedwithpropidiumiodideandsubjectedtoconfocaluorescencemicroscopy.
Theresultsshowedthattheendophytehadcolonisedbothleavesandroots(Fig.
5).
Straincoa1attachedtothelateralrootsindensepellets,whereascolonizationoftheleavesinvolvedhyphalgrowthovertheleafsurface.
ThecolonisedArabidopsisrootswerethenxedforsectioningandhighresolutionimagingwithtransmis-sionelectronmicroscopy(TEM).
Regionsofinterestwereidentiedbyobtaining1-lmsectionsthatwereFig.
2CharacterizationofStreptomycesendophytesandtheirtaxonomicdistribution.
aSeveralisolatesshowhighestsimi-laritywithStreptomycesclaviferorStreptomycesolivochromo-genesonthebasisof16SrDNAanalysis.
Themajorityoftheisolatesshowedawidevariationinclosestspeciesassigned,indicatingadiverseendophyticcompartment.
'Unclassied'meansthattheseactinobacteriacouldnotbeclassiedatthespecieslevelbasedonthe16SrDNAsequence.
bScanningelectronmicrographofStreptomycessp.
MOS18,whichproducesspinyspores.
Scalebar3lM.
cScanningelectronmicrographofStreptomycessp.
MOS38showingpoorsporu-lation.
Scalebar5lM.
StrainsweregrownonSFMmediafor6days682AntonievanLeeuwenhoek(2018)111:679–690123stainedwithtoluidinetovisualisethebacteria(Fig.
6a).
Theresultsshowedthatcoa1notonlycolonisedtherootsurfacebutalsotheinternalroottissueand,remarkably,theintracellularspace(Fig.
6b,c).
Noplantcelldefectswereobservedintheimagedsamples.
Strikingly,thereisnoplantFig.
3Streptomycesendophytesdisplayawiderangeofmorphologies.
StrainsshownabovearegrownonSFMagarplatesfor6days.
S.
coelicolorM145andS.
griseusDSM40236areshownasreferencestrains.
aS.
coelicolorM145,bS.
griseusDSM40236andtheendophyticstreptomycetesMOS18(c),MOS38(d),MOS14(e),MOS32(f),MOS25(g),MOS31(h)andMOS35(i).
Scalebar2mmFig.
4ScanningelectronmicrographofStreptomycessp.
MOS31.
ImagesofmycelialsheetsasisseeninFig.
3H.
aThicklayersofvegetativemyceliummadeupofhyphaeandextracellularmatrixturnawayfromtheinnerpartofthecolony.
Scalebar100lM.
bHyphaeextendingfromthemycelialsheets,therebygrowingawayfromthevegetativemycelium.
Scalebar30lM.
Streptomycessp.
MOS31wasgrownonSFMagarplatesfor6daysAntonievanLeeuwenhoek(2018)111:679–690683123cellularmembraneseparatingStreptomycesfromtheintracellularspace.
PreviousstudiesshowedcolonizationoftherootsurfaceofArabidopsisbyactinobacteria,andlettuce,turniprapeandcarrotbyStreptomyces(Bulgarellietal.
2012;Bonaldietal.
2015;Kortemaaetal.
1994).
Inaddition,endophyticcolonizationofgerminatingwheatseedhasbeenreportedbyreintroductionofaFig.
5ColonizationofArabidopsisbyStreptomycesendophytecoa1.
aConfocalmicrographofacolonisedlateralroot.
Thesampleisstainedwithpropidiumiodide,resultinginreduorescenceofbothbacterialandplantcells.
Coa1attachestotherootasadensepellet.
Scalebar50lM.
bConfocalmicrographoftheborderoftheleaf.
Singlehyphaearegrowingovertheleafsurface(arrowheads).
Scalebar15lMFig.
6SectionsofArabidopsisrootsinvadedbyStreptomycescoa1.
aToluidinestainedsectionofanArabidopsisrootinvadedbyStreptomycescoa1.
Coa1enterstherootviatheepidermis(EP)cellsandcolonisestherootinbetweenthecortexcells(CO)andEPcells.
Scalebar10lM.
Boxedpartoftheimageisshownasmagnicationontheright.
b,cTransmissionelectronmicrographsofArabidopsisrootsinvadedbycao1.
Coa1colonisestherootintracellularly.
ThebacteriumcanbefoundinbetweentheCO-andEPcells,andinbetweentheendodermis(ED)andCOcells(imageB).
Inaddition,inEPcellsintracellulargrowthofStreptomycescoa1wasobserved(imageC).
Scalebar2lM684AntonievanLeeuwenhoek(2018)111:679–690123GFPexpressingStreptomycesstrain(Francoetal.
2017).
Tothebestofourknowledge,ourresultsshowforthersttimethepresenceofastreptomycetewithinanArabidopsisrootcell.
Furthermore,thehyphaeofcoa1appearedlesselectrondenseintheendospherethanontheplantsurface,whichmayreectphysio-logicaldifferencesbetweenlifeinsideandoutsidetheplant.
Stilltherearemajorgapsinourunderstandingoftheendophyticbiologyofstreptomycetes.
Forexample,wedidnotndreproductivestructuresorsporeswithintheendosphere.
Itthereforeremainstobeseenwhetherendophyteshaveacompletelifecycleinsideplantsoronlyremaininthevegetativegrowthphase,althoughwehavetotakeintoaccountthata7daytimeframemightnotbesufcienttoformsporeswithinaplant.
Ithasalsobeensuggestedthattheendophyticlifestyleofactinobacteriamayincludetheformationofsmallprotoplast-likecellsthatlackacellwall(Ramijanetal.
2016).
Theseso-calledL-formsmayexplainhowtherelativelylargemycelialacti-nobacteriacanstillmigrateandproliferateinsideplanttissue.
TofurtherunraveltheendophyticbiologyofStreptomyceswewillfocusfutureexperimentsontheidenticationofmajorgeneticormorphologicaltraitsassociatedwiththeendophyticlifestyle.
ResponseofendophytestophytohormonesActinobacteriaplayanimportantroleinantibiosisandasprobioticstotheplantduetotheproductionofadiversearrayofbioactivemolecules(Viaeneetal.
2016).
Weassessedtheantimicrobialactivityoftheactinobacterialendophytesinoverlayassays,usingBacillussubtilis(Gram-positive)andEscherichiacoli(Gram-negative)asthetarget.
Byapplyingagardiffusionassayswiththestreptomycetesgrownonminimalmediumagarwithmannitolandglycerolweshowedthat11%oftheisolatesproducedoneormorecompoundsthatinhibitedgrowthofE.
colicells,while14%inhibitedgrowthofB.
subtilis.
Generally,thevastmajorityofactinomycetesisolatedfromsoilsamplesareabletoinhibitB.
subtilisgrowthunderroutinelaboratoryconditions,whichdiffersfromwhatwendforourcollectionofendophytes(Zhuetal.
2014b).
Whilethesmallnumbersmakeitdifculttoapplystatistics,wecannotruleoutthatthereducedantibioticactivityagainstGram-positivebacteriaandinpartic-ularFirmicutes,maybetypicalofendophyticactinobacteria.
Genomesequencingrevealedthatactinobacteriahavealotofbiosyntheticgeneclustersfornaturalproductsthatarepoorlyexpressed(KolterandvanWezel2016;Nettetal.
2009).
Thisoffersavastreservoirofpotentiallyimportantbioactivemolecules,includingantibiotics.
Toallowscreeningofthesecompounds,strategiesarerequiredtoelicittheirexpression(RutledgeandChallis2015;vanWezeletal.
2009;Wuetal.
2015;Zhuetal.
2014a).
Weassessedthepotentialofphytohormonesaselicitors,therebymimickingthechemicalenvironmentoftherhizosphereandendosphere.
Forthis,weexposedtheendophytestotheplanthormonessalicylicacid(SA),indoleaceticacid(IAA,knownasauxin)orjasmonicacid(JA).
Thestrainsweregrownonminimalmedia,withorwithout0.
01,0.
001or0.
0001%(w/v)ofeitherplanthormoneasconcentrationsofSAcanbeashighas0.
01%(w/w).
Square12912cmagarplateswereinoculatedwithspotsfromsporestocksoftheendophytes.
Interestingly,thepercentageofstrainsexhibitingantibioticactivityroughlydoubled,with20%ofthestrainsinhibitingE.
colicells,and29%inhibitinggrowthofB.
subtilis(TableS1).
ThiseffectcouldmostlybeattributedtoIAA,whichhadamoresignicantelicitingeffectontheendophytesthanSAorJA(TableS2).
AnexampleofelicitedantimicrobialproductionbyIAAispresentedinFig.
S2.
Addition-ally,weobservedincreasedactivityagainsttheindicatorstrainsaswell,indicatingeithermoreproductionofthesameantibioticorproductionofadifferent(setof)antibiotic(s)(SeeFig.
S3).
Afterelicitation,comparativemetabolomicsand/orgeno-micsonsamplesobtainedfromproducingandnon-producingconditionscanbeusedtoidentifythecompoundandgeneclusterofinterest,respectively(Gubbensetal.
2014;Nguyenetal.
2013).
Althoughtheconceptofelicitingantimicrobialproductionbyactinobacteriaisalreadywell-estab-lished,thisexperimentalsetupwasconnectedtothebioticinteractionsbetweenplantandstreptomycete,aimedtomimicthechemicalenvironmentoftheplant.
Plant-endophyteinteractionshavelikelyplayedakeyroleintheevolutionofthechemicaldiversityofactinomycete-derivednaturalproductsandsignalsthatcontroltheproductionoftheseantimicrobialsarelikelytiedtothebioticinteractions.
Thisideaisfurtherexploredbythe''cryforhelp''hypothesis,whichadditionallystatesthatactinobacteriaencountertrade-offsbetweenthecostsofproducingcomplexnaturalAntonievanLeeuwenhoek(2018)111:679–690685123productsandtheirbenets,andmaythereforeproducethesemoleculesspecicallyinresponsetoecologicaldemands(vanderMeijetal.
2017).
Tobroadenourunderstandingofthisconceptitwouldbeofinteresttoknowwhichtype(s)ofcompoundsareproducedinresponsetophytohormoneexposure.
Identicationoftheantimicrobialcompoundsproducedinresponsetoexposuretophytohormonesandthecorrespondingmetabolicnetworkswillgivebetterinsightsintothebacterialresponsestotheplant's''cryforhelp''.
Inaddition,theconcentration-dependenteffectofphy-tohormonesmaybeofgreatimportanceaslocalphytohormoneconcentrationsinaplantvary.
Inordertoharnessplant-actinomyceteinteractionsaselicitorsforantimicrobialproduction,furtherstudiesshouldfocusontheseconcentration-dependenteffects,aswellassynergismorantagonismofdifferentphythor-moneswhenusedaselicitor.
ConclusionInthisstudy,weshowtherecruitmentofStreptomycesendophytesbyA.
thalianaCol-0andA.
thalianaMsl.
OurpilotstudyshowsthatisolatesfallingwithinthegroupsofS.
olivochromogenesandS.
claviferareoverrepresentedintheendosphere,suggestingthattheseendophyticstreptomycetesmayhavespeciccharacteristicsthatallowthemtoadapttolifeinsidetheplant.
Thisneedstobestudiedinmoredetail,andmayalsobeextendedtothestudyofotheractinobac-terialgenerafoundintheendosphere.
Additionally,weprovidearststeptowardstheproofofconceptforthe'cryforhelp'hypothesis,wherebyplanthormones,inparticularIAA,haveastimulatingeffectonantibioticproductionbyendophyticactinobacteria.
Thesebaselineexperimentshighlighttheimportanceofexploringandexploitingplant-actinomyceteinter-actionsaselicitorsfor'antibioticproductionondemand'.
MaterialsandmethodsBacteria,plantsandgrowthconditionsStreptomycescoelicolorA3(2)M145wasobtainedfromtheJohnInnesCentrestraincollectioninNorwich,UK,andStreptomycesgriseusDSM40236fromtheDSMZculturecollection(Braunschweig,Germany).
S.
coelicolorM145andS.
griseusDSM40236weregrownonSFMfor6daysat30°C,unlessindicateddifferently.
AsterileA.
thalianaecotypeColumbia(Col-0)wasusedfortherecruit-mentofendophyticactinobacteriaunderlabcondi-tions.
Plantsweregrownonamixtureof9:1substratesoilandsand(HollandPotgrond)at21°C,a16hphotoperiod,and70%relativehumidity.
After2weeksofgrowth,theplantswereharvested.
SoilfortheeldexperimentwithA.
thalianaecotypeMossel(Msl)wascollectedinMay2016attheMosselareaatthe'HogeVeluwe'intheNetherlands(coor-dinates:N52°03035.
500E5°45006.
400),fromfourdiffer-entspotswithinaradiusof100m.
Iftherewasanyvegetationpresent,thetop5–10cmsoilwasremoved.
Thesoilwashomogenisedandalllargepartssuchasdeadrootsandstoneswereremoved.
Thesoilwaskeptinacoldroomat4°Cuntiluse.
Seedsweresterilisedin49dilutedhouseholdbleachfor10min,washedseventimeswithsterileMQwater,ashortrinsewith70%ethanolandtransferredtoplateswithawetlterpaper,placedat4°Cfor48handthenmovedtoa21°Cincubatorinthedark.
Mosselsoilwasplacedinatraywith393cmpotsandwatered.
Toremovetheendogenousseedpopulation,thetraywasplacedinthegreenhousefor2days.
Afterweeding,thesterileseedlingsontheplatesweretransplantedtothetraywithMosselsoilandafter7daystheplantsincludingthesoilwereplantedintototheMosseleld.
After6weeksofgrowthintheeld,plantswereharvestedusingasmallshovel3–4cmaroundthebaseoftheplant.
IsolationofendophytesA.
thalianaCol-0wassurfacesterilisedbywashingtheplantthreetimesin70%EtOH,afterwhichtheplantmaterialwascrushedinMQ.
SterilizationwasconrmedbyaddingasterilisedplantontoLBagar,afterwhichnobacterialgrowthwasobserved.
RoottissueofA.
thalianaMslwascleaned,sonicatedandgroundwithmortarandpestlein1mLphosphatebuffer(perlitre:6.
33gofNaH2PO4H2O,10.
96gofNa2HPO42H2Oand200lLSilwetL-77).
Boththecrushedplantmaterial(Col-0)andsonicatedplantmaterial(Msl)werespreadontothesurfaceofarangeofselectiveisolationmediaincludinghumicacidagar(HA)(HayakawaandNonomura1987),glucose686AntonievanLeeuwenhoek(2018)111:679–690123caseinagar(GCA)(Zhang1985),soyourmannitolmedium(SFM)orminimalmedium(MM)(Kieseretal.
2000).
Initialselectionwasdoneonmediasupplementedwiththeantifungalagentnystatin(50lg/mL)andtheantibacterialagentnalidixicacid(10lg/mL).
Plateswereincubatedat30°Cfor4–25days.
PlantharvestingandDNAisolationofthemicrobialcommunityForA.
thalianaMslweappliedtheharvestingprotocolasdescribedbefore(Lundbergetal.
2012).
Inshort,rootsincludingrhizosphericsoilwerecollectedina50mLtubecontaining25mLofphosphatebuffer,andvortexedfor15s.
Replacingthebufferandvortexingwasrepeateduntilthebufferstayedclear.
Rootsweretransferredtoa15mLtube,sonicated(5burstsof30swith30sbreaks),vortexed,washedwithphosphatebufferanddriedonlterpaper.
Then,therootswereeithergroundforbacterialisolation,orashfrozenandstoredat-80°CforlaterDNAisolation.
Fourindividualplantswerepooledintoonesample.
UntreatedtoothpickswereinsertedintotheMslsoilataminimaldepthof4cmandwereutilisedaswoodcompartment.
DNAwasisolatedfromsoilusingtheMoBioPowerSoilkitandECandtoothpicksampleswiththeMPBioFastDNAspinkit.
QualityandquantityoftheDNAwascheckedbynanodropandgelelectrophoresis.
Around400ngwassentfor16srDNAsequencingatBeijingGenomicsInstitute(BGI).
AmpliconsequencingUsingprimers515F(50-GTGYCAGCMGCCGCGG-TAA-30)and806R(50-GGACTACNVGGGTWTC-TAAT-30),theV4regionwassequencedatBGIontheHiSeq2500sequencingplatform(Illumina).
RawdatafromBGIwasprocessedusingapreviouslyreportedcustomimplementation(Perez-Jaramilloetal.
2017)ofQIIME(Caporasoetal.
2010)withminormodi-cations(describedbySchneijderbergetal.
,inprep).
Inshort,readswerequalitylteredandlteredforchimerasusingChimeraSlayer.
Usinga97%identitythreshold,denovoOTUsweredetermined,whichweretaxonomicallyassignedusingtheRDPclassier2.
10(Coleetal.
2014)withtheGreenGenesdatabase28(DeSantisetal.
2006).
OTUsrelatedtomitochondialandchloroplastsequenceswereremoved,asweretheOTUsthatdidnothave25readsinatleast5samples(''raretaxa'').
ToobtainrelativeabundanceoftheOTUs,thenumberofreadsfromasingleOTUpersamplewasdividedbythetotalnumberofreadsofthatsampleafterlteringforraretaxa.
Analysisofactinobacteriabasedon16SrRNAsequencesThe16SrRNAgenesoftheactinobacteriawereampliedbyPCRfromliquid-grownmyceliausingprimersF1(50-GCGTGCTTAACACATGCAAG-30)andR1(50-CGTATTACCGCGGCTGCTG-30),whichcorrespondtontpositions15-34and465-483ofthe16SrRNAlocusofS.
coelicolor(vanWezeletal.
1991),respectively.
PCRswereconductedasdescribed(Colsonetal.
2007)andsequencedusingoligonucleotideF1.
SequencingwasdoneatBase-ClearinLeiden,theNetherlands.
16SrRNAgeneanalysiswasperformedusingwebbasedidentifytoolonEzBioCloud(https://www.
ezbiocloud.
net/identify).
Theidentifyserviceprovidesprovensimi-larity-basedsearchesagainstquality-controlleddata-basesof16SrRNAsequences.
Thetop-hitinformationforeachIdentifyJobwascheckedagainstStrepto-mycesfocusedMLSAbasedphylogenetictreepub-lishedelsewhere(Labedaetal.
2017).
MicroscopyLightmicroscopyStereomicroscopywasdoneusingaZeissLumarV12microscopeequippedwithaAxioCamMRc,andconfocalmicroscopyusingaZeissObserverMicro-scope.
Forconfocalmicroscopy,sporesoftheendo-phytewereaddedtotheseeds.
SeedswereputonMurashigeandSkoogmediumwith1%sucroseand0.
8%agarandkeptinthedarkat4°for48h.
Afterthecoldshock,seedswereincubatedintheclimateroomfor1week(seebacteria,plantsandgrowthcondi-tions)afterwhichtheywereimaged.
Sampleswerestainedwithpropidiumiodide1:1000(1lg/mL).
Sampleswereexcitedwithlaserlightatawavelength535nmtodetectthepropidiumiodide.
AntonievanLeeuwenhoek(2018)111:679–690687123ElectronmicroscopyMorphologicalstudiesonsinglecoloniesofendo-phytesbySEMwereperformedusingaJEOLJSM6700Fscanningelectronmicroscope.
ForStrep-tomyceteonlysamples,piecesofagarwithbiomassfrom6-day-oldcoloniesgrownonSFMwerecutandxedwith1.
5%glutaraldehyde(1h).
Subsequently,samplesweredehydrated(70%acetone15min,80%acetone15min,90%acetone15min,100%acetone15minandcriticalpointdried(BaltecCPD-030).
Hereafterthesampleswerecoatedwithgoldusingagoldsputtercoater,anddirectlyimagedusingaJEOLJSM6700F.
ForSEMofArabidopissamples,imagingtimingwasincreasedfour-foldtooptimisexationanddehydration,andthe70%acetonestepwasdoneovernight.
Transmissionelectronmicroscopy(TEM)fortheanalysisofcross-sectionsofArabidopsisrootsandStreptomyceswasperformedwithaJEOL1010trans-missionelectronmicroscopeasdescribedpreviously(Pietteetal.
2005).
Samplesweregrowninthesamewayasforthelightmicroscopy,andxedwith1.
5%glutaraldehydefor4h.
Post-xationwasperformedwith1%Osmiumtetroxidefor4h.
Initialdehydrationwith70%ethanolwasdoneovernight.
Hereafterusingahighmagni-cation(9150)stereomicroscope(MZ16AF)rootsectionscontainingStreptomycesgrowthwereselected,followedbydehydrationin1hsteps(80,90,100%ethanol,100%propyleneoxide,50/50propyleneoxide/EPON,100%EPON).
SubsequentlysampleswereembeddedinEPONandpolymerisedfor2daysat60°C.
Ultrathinsectionswerecutonanultramicrotome(ReichertUltracutE),collectedoncoppergridsandexaminedusingaJeol1010trans-missionelectronmicroscopeat70kV.
AntimicrobialactivityassaysAntimicrobialactivityoftheendophyteswastestedagainstB.
subtilis168oraderivativeofE.
coliAS19-RrmA(LiuandDouthwaite2002).
IndicatorbacteriawereculturedinLBbrothandincubatedat37°Covernight.
Antimicrobialassayswereconductedusingthedouble-layeragarmethod.
Briey,actinobacteriawereinoculatedonminimalmediumagarplatescontainingbothmannitolandglycerol(1%w/v)asnon-repressingcarbonsources,sincenotallStreptomycesgrowequallywelloneithermannitolorglycerol.
Theagarplatesweresupplementedwitheither(±)-jasmonicacid(Caymanchemicalcompany,cas:88-30-0),3-indoleaceticacid(Sigma-Aldrich,cas:87-51-4)orsalicyclicacid(AlfaAesar,cas:69-72-7).
Theendophytesweretypicallyincubatedfor5daysat30°C,followingwhichtheywereoverlaidwithLBsoftagar(0.
6%w/vagar)containing300lLofoneoftheindicatorstrains(OD0.
4–0.
6),andthenincubatedovernightat37°C.
Thefollowingday,antibacterialactivitywasdeterminedbymeasuringtheinhibitionzones(mm)oftheindicatorstrainsur-roundingthecolonies.
AcknowledgementsThisworkwassupportedbyaGrants14218and14221fromtheNetherlandsOrganizationforScienticresearch(NWO)toJRandGPvW,respectively.
ConictofinterestTheauthorsdeclarenoconictofinterests.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunre-stricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCom-monslicense,andindicateifchangesweremade.
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