Growing Research Networks on Mycorrhizae for Mutual Benefits

Opinion

Growing

Research

Networks

on

Mycorrhizae

for

Mutual

_Benefits

Olga

Ferlian,

* Arjen

Biere,

Paola

Bonfante,

François

Buscot,

Nico

Eisenhauer,

Ivan

Fernandez,

Bettina

Hause,

Sylvie

Herrmann,

Franziska

Krajinski-Barth,

Ina

C.

Meier,

Maria

J.

Pozo,

Sergio

Rasmann,

Matthias

C.

Rillig,

Mika

T.

Tarkka,

Nicole

M.

van

Dam,

Cameron

Wagg,

and

Ainhoa

Martinez-Medina

*

Researchonmycorrhizalinteractionshastraditionallydevelopedintoseparate disciplinesaddressingdifferentorganizationallevels.Thisseparationhasledto anincompleteunderstandingofmycorrhizalfunctioning.Integrationof mycor-rhiza research at different scales is needed to understand the mechanisms underlying the context dependency of mycorrhizal associations, and to use mycorrhizaeforsolvingenvironmentalissues.Here,weprovidearoadmapfor the integration of mycorrhiza research into a unique framework that spans genestoecosystems.Usingtwokeytopics,weidentifyparallelsinmycorrhiza researchatdifferentorganizationallevels.Basedontwocurrentprojects,we showhow scientific integration creates synergies, and discuss future direc-tions. Only by overcoming disciplinary boundaries, we will achieve a more comprehensiveunderstandingofthefunctioningofmycorrhizalassociations.

TrajectoriesinMycorrhizaResearch

In2000,MillerandKlingstated that‘tosucceed,the mycorrhiza(seeGlossary)research communitymustgobeyondtheirusualdisciplinaryboundariesandintegratetheirworkwith thatofotherresearchers’[1].Althoughsomeadvanceshavebeenmadeinthe18yearssince, mycorrhiza research still largely develops by specializing within different disciplines of biology. During the 19th century, researchinto arbuscular mycorrhiza commenced with the discovery of fungal structures colonizing roots [2,3]. This early research was initially dominatedbythedescriptionsoffungalmorphologicaltraitsandpotentialfungaleffectson plantperformance.Suchearlyresearchstayedattheorganismallevel,revealingmoreabout thefungi,andstudyingeffectsattheindividualhostplantlevel.Duringthesecondhalfofthe 20thcentury,researchonmycorrhizaeandtheirinteractionsdevelopedintotwomaindistinct directions:cellularand,later,subcellularbiologyontheonehand,andecologyontheother hand.

Thisspecializationhasled tomanycrucial discoveriesinmycorrhizalbiology. Focusingon cellularprocesses,ultrastructuralstudiesduringthe1970sledtothefirstdetaileddescriptionof plant–fungalinteractions.Thisincludedarbuscule formation,the identification ofthe peri-arbuscularmembrane,aswellasidentificationoftheinterface(i.e.,thecontactareabetween theinteractionpartners)[4].Ontheecologicalside,therealizationthatunequalbenefitsmaybe providedtodifferentplantspecies[5]ledtoinvestigationsoftheimportanceofmycorrhizaein mediatingplant adaptationand evolutionas well as in structuringplant communities and biogeochemicalcycles[6].Amilestoneherewastheexperimentaldemonstrationofarbuscular mycorrhizal(AM)fungaldiversityeffectsonplantcommunitydiversityandproductivity[7].

Highlights

Mycorrhizaresearch hastraditionally

developedinto distinctdisciplinesat

different organizational levels from

thecellulartoecosystemlevel.

This separation leads to a limited

understandingofmycorrhiza

function-inganditsrolewithinecosystems.

Here,weshowhowthedifferent

dis-ciplinesinmycorrhizaresearch

com-monly address the same general

questionsand how thesequestions

arenestedinthenextorganizational

level.

By integrating different disciplines, these

disciplines are abletocomplementeach

otherandfosterthedevelopmentofa

comprehensive understanding of

mycorrhizalassociations.

Weintroducetwoongoingprojectsas

exampleswheretheintegrationof

dis-ciplines in mycorrhiza research is

alreadycommonpractice.

1

GermanCentreforIntegrative

BiodiversityResearch(iDiv)

Halle-Jena-Leipzig,DeutscherPlatz5e,

04103Leipzig,Germany

2

InstituteofBiology,Leipzig

University,DeutscherPlatz5e,04103

Leipzig,Germany

3

NetherlandsInstituteofEcology

(NIOO-KNAW),Droevendaalsesteeg

10,6708PBWageningen,The

Netherlands

4

DepartmentofLifeSciencesand

SystemsBiology,UniversityofTorino,

VialeMattioli25,10125Torino,Italy

5

DepartmentofSoilEcology,

Helmholtz-CentreforEnvironmental

Withmethodologicalandconceptualadvances,thefieldofmycorrhizaresearchhasextended furtherfollowingstudiesonthephysiologyofplants[8]andonecosystemeffects[7,9].Eachof thefieldsaddresses differentscalesandlevelsoforganization.Consequently, theyhave developedindistinctdirectionsintermsofthequestionsaddressed,scope,methods,and, perhaps most importantly, training and specialization of researchers. This division of the researchfieldisillustratedbythefactthattherearetwomaininternationalscientificconferences onmycorrhizalresearch:theInternationalConferenceofMycorrhiza(ICOM),focusingmostly onecologicalresearch onmycorrhiza, andthe International MolecularMycorrhiza Meeting (iMMM),focusingmostlyonmolecularmycorrhizaresearch.Albeiteffectiveinmanyrespects, thishistoricpartitioningintoseparatefieldssometimeshindersacomprehensiveunderstanding ofhowmycorrhizaefunctionandinfluencetheirbioticandabioticenvironment.Forinstance, ourknowledgeoftheimpactofmycorrhizaeonecosystemfunctionsandservicesisrelatively incomplete.Thisisduetothecontext-dependentnatureofthesymbiosis,whichisdrivenby environmentalconditionsaswellasthespeciesidentityoftheplantand/orfungalpartner.The interactioncanexistalongacontinuumofpossibleoutcomesfortheplant,frommutualisticto detrimental[10].Onlybydevelopingnewinvestigationmodelsthatintegratemultiplelevelsof organizationwillwebeabletounderstandhowenvironmentalcontextimpactstherelationships ofplantswiththeirmycorrhizalsymbionts.Thiswillallowustomakerealisticpredictionsofthe contributionofmycorrhizaetothefunctioningofecosystemsandtoagriculturalpractices[11]. Thus,wearguethatmycorrhizaresearchcanreachanewlevelofinsightbyintegratingthe strengths of the increasingly separated research disciplines, both from the cellular to the ecosystemlevelandviceversa.

Here, we highlight how major topics in mycorrhiza research have been independently addressedbydifferentdisciplinesacrossdifferentorganizationallevels, andhowtheycould complementeachother.Withineachtopic,wefocusonfourorganizationallevelsoflife:the cellularandsubcellularlevel(hereaftercalledthecellularlevel),theplantphysiologicallevel, theplantcommunity level,and theecosystemlevel.To stressthe gainsofintegrating researchacrosslevelsoforganization,wepresentanoverviewofthebenefitstheymayprovide toeach other.Moreover,westressthat,among theorganizationallevels, aconsensuson researchpracticesmustbereached.Finally,wegiverecommendationsforfuturedirections towards integrative research networks, which aim at a more complete understanding of mycorrhizalassociationsandtheirfunctioning.Asproofofconcept,weintroducetworecently developedprojectsinwhichsucheffortsarealreadyunderway.Wefocusonthetwomost commonlystudiedmycorrhizaltypes,namelyAMandectomycorrhizae(ECM),whicharealso themostcommonmycorrhizalsymbiosesinnaturalecosystems.Studiesofmycorrhizaeatthe physiologicalandcellularlevelarestronglybiasedtowardsAMcomparedwithECM.However, thisimbalance,whichisreflectedinthepredominanceofAMcomparedwithECMexamplesin ourpaper, does notimpede the validityof thepresented framework, because this can in principlebeappliedtoanytypeofmycorrhizae.

Multi-LevelConnectionsinMycorrhizaResearch

Mycorrhizaresearchspanslower-organizationallevelprocesses[e.g.,inorganicphosphorus (Pi)uptakeandtransportmechanisms,andmodulationofplantimmunity]to

higher-organiza-tionallevelsthataffectplantcommunityandecosystemfunctioning(e.g.,biogeochemical cyclesormultitrophicinteractions).Whilemoststudiesfocusononeortwolevelsof organiza-tion,mycorrhiza-relatedprocessesatonelevel arelikely nested inand,thus,may provide mechanismsunderlyingthefindingsof,thenexthigherlevel(Figure1,KeyFigure).Here,we illustratethepotentialnestingandlinksacrossthedifferentorganizationallevelsinmycorrhiza

Research–UFZ,

Theodor-Lieser-Straße4,06120Halle,Germany

6

LeibnizInstituteofPlant

Biochemistry,Weinberg3,06120

Halle,Germany

7

InstituteofBiology,Leipzig

University,Johannisallee21-23,04103

Leipzig,Germany

8

PlantEcology,Universityof

Goettingen,UntereKarspüle2,37073

Göttingen,Germany

9

DepartmentofSoilMicrobiologyand

SymbioticSystems,Estación

ExperimentaldelZaidín(CSIC),Prof.

Albareda1,18008Granada,Spain

10

InstituteofBiology,Universityof

Neuchâtel,RueEmile-Argand11,

2000Neuchâtel,Switzerland

11

InstituteofBiology,FreieUniversität

Berlin,Altensteinstr.6,14195Berlin,

Germany

12

Berlin-BrandenburgInstituteof

AdvancedBiodiversityResearch

(BBIB),Altensteinstr.6,14195Berlin,

Germany

13

InstituteofBiodiversity,Friedrich

SchillerUniversityJena,Dornburger

Str.159,07743Jena,Germany

14

DepartmentofEvolutionaryBiology

andEnvironmentalStudies,University

ofZurich,Winterthurerstrasse190,

CH-8057Zürich,Switzerland

*Correspondence:

olga.ferlian@idiv.de(O.Ferlian)and

ainhoa_martinez.medina@idiv.de(A. Martinez-Medina).

Glossary

Arbuscule:ahighlybranched

structureproducedbyAMfungi

insidearootcorticalcelloftheir

host.Arbusculesareconsideredto

bethemainsiteofnutrientexchange

betweenthefungalandplant

symbioticpartners.

Cellularlevel:researchthatfocuses

onthestructureandfunctionsof

plantandfungalcellsinvolvedinthe

symbiosis.

Commonmycelialnetworks

(CMNs):abelowgroundnetworkof

mycorrhizalhyphaelinkingrootsof

plantsofthesameordifferent

species.

Defensepriming:aprocessthat

conditionsplantspeciesforthe

enhancedinductionofdefenses,

oftenresultinginenhancedpestand

diseaseresistanceandabioticstress

tolerance.

Discipline:fieldofresearchwitha

specificfocus.Itoftenfocusesona

specificleveloforganization.

Ecosystemfunctioning:physical,

geochemical,andbiologicalactivities

andtheireffectswithinan

ecosystem.Theycanbegrouped

intosizes(orstocks),suchas

nutrientpools,andratesof

processes,andfluxesofmaterial.

Ecosystemlevel:researchthat

focusesonthewholeecosystem

(i.e.,acommunityoforganismsthat

interactwitheachotherandtheir

environment).

Leveloforganization

(organizationallevel):unitwithina

hierarchicalsystemofbiological

structuresandsystems

characterizinglife.Witheachlevel,

organizationalcomplexityincreases

becauseitcomprisestheprevious

level;notethat,inthispaper,we

refertoasystemwithfourlevels

(ecosystem,plantcommunity,plant

physiological,andcellularlevel),

whichmaydifferfromtheclassical

ecologicallevelsoforganization.

Mycorrhiza:asymbioticassociation

betweenasoilfungus(the

mycorrhizalfungus)andaplantroot,

inwhichplantphotosynthatesare

exchangedformineralresources

acquiredbythefungusfromthesoil.

Periarbuscularmembrane:novel

symbiosis-specificmembrane,

derivedfromtheplantand

developedatthemomentoffungal

penetrationandarbuscule

researchbyusingtwoexamplesofkeytopicsinmycorrhizaresearch:Piuptakeand

multi-trophicinteractions.

Example1:EffectofMycorrhizalAssociationsonPlantPhosphorusUptake

TheAMsymbiosis provides aneffective way(the AM pathway)to scavenge Pifrom large

volumesof soilandrapidlydeliver it to rootcorticalcells, thereby bypassingdirect rootPi

uptake.CellularresearchrevealedPitransportersinvolvedinthemycorrhizalphosphateuptake

KeyFigure

Schematic

Overview

of

Research

at

the

Different

Levels

of

Organization

and

the

Processes

Studied

in

Mycorrhizal

Biology

in

the

Context

of

(A)

Inorganic

Phosphorous

(P

)

Uptake

and

(B)

Multitrophic

Interactions

P_i uptake Mul trophic interac ons

Ecosystem level Plant community level Plant physiology level Cellular level PO PO PO

PO soil organic ma erSoil/

Primary producers Herbivores Decom-posers Herbivore a ack Plant stress signals Mycorrhizal signaling Periarbuscular space Plant cell AMF Pi Pi Pi PiH⁺ H⁺ H⁺ ADP ATP + Plant cell wall Plant cell Effectors MTI AMF PRR (B)

(A) Figure1.Eachdrawingdepictsonelevel

oforganizationcoveringthecellular,plant

physiological,plantcommunity,and

eco-systemlevel.Eachlevelwithitsprocesses

isnestedwithinthenexthigherlevel,as

indicatedbythemagnifying circles. (A)

ThePcycleofan ecosystemandthe

contribution of plant communities (top

offigure).Zoomingintothese

contribu-tions,mycorrhizalinteractionswithinthe

plantcommunityandtheirdiverse

myce-lialnetworksbecomevisible.Plant

phy-siologicalresponsestomycorrhizalfungi,

suchasalteredplantgrowthandaltered

susceptibilitytoantagonists,driveplant

communityresponses.Atthebase,

sub-cellularandcellularprocessesofPi

trans-portattheinterfaceofthefungusandthe

plantrootcelldetermineplant

physiolo-gicalresponses.(B)Organismal

interac-tionsatthewholeecosystemlevel(top

figure). Zooming into the interactions

among mycorrhizal plants, multitrophic

interactions via signaling pathways

(inducedbyherbivoreattackand

subse-quent parasitoid recruitment) become

apparentaswellassignalingofherbivore

attack within the plant community via

common mycelial networks. The

pro-cesses by which the plant allocates

defensecompoundsto itsleaves

(fos-teredbytheassociationwith

mycorrhi-zae),which preventtheherbivorefrom

feeding,become apparent atthe next

level.Atthebase,mycorrhizalfungi

mod-ulateplantimmunitytoestablishthe

sym-biosis, leading to a defense signaling

cascadethatcanenhanceplantimmunity

againstherbivores.Abbreviations:AMF,

arbuscular mycorrhizal fungus; MTI,

microbial-associated molecular pattern

(MAMP)-triggered immunity; PRR:

pathway.Someofthesetransportersshowconstitutive transcriptlevelsinnon-mycorrhizal roots[12],whileothersaremostlyexpressedinarbuscule-containingcells[13] (Figure1A). TheseplantPitransportersarelocatedintheperiarbuscularmembraneandtakeupphosphate

ionsfromtheperiarbuscular space,theapoplastic compartmentbetweentheplant periar-buscular membrane and fungal plasma membrane in arbuscule-containing cells. Recent molecularstudiesdemonstratedthatspecificH+-ATPaseproteinsintheperiarbuscular mem-branearerequiredtogeneratetheprotongradientnecessaryfortheactionofPitransporters

[14].Theseresultsarerelevantfor understandingtheregulationofPiacquisitioninthe AM

symbiosis.However,withoutaddressingthephysiologicallevel,thisinformationisnotsufficient fordeterminingtherelevanceoftheAMpathwaywithrespecttothedirectPiuptakepathway.

ThesameappliestoassessingitsnetcontributiontoplantPiuptakeand,thus,plantnutrition

andfitness.

PlantphysiologicalresearchrevealedthattheAMpathwayhasamajorroleinPiuptakeand,

consequently,inplantgrowth(Figure1A).Experimentswithsingleplantsandplant communi-tiesshowedthatAMfungicontributeupto90%ofplantPlevels[15–17].However,colonization bydifferentAMfungalspeciescanresultindifferentfitnessresponsesoftheplant[18].Similarly, colonizationbythesameAMfungalspeciesdoesnotnecessarilyresultinthesamefitness responseindifferentplantspecies.Thisindicatesthatthereisconsiderablefunctionaldiversity amongplant–AMfungalsymbioses.Indeed,AMassociationshavealsobeenshowntovaryin theirphysiologicalcharacteristics[19].Still,themajorforcesdrivingthisfunctionaldiversityare widelyunknown.Combiningacellularapproachwithphysiologicalexperimentsisapowerful approach to illuminatemycorrhizal functions, whileproviding a genetic explanationfor the functionalvariationobserved.Suchinformationcanevenbeimplementedinthedevelopment ofbiomarkersfortheassessmentofmycorrhizalfunctionaldiversityandfortheselectionof efficient crop–fungus combinations for increasing agricultural productivity. Moreover, the symbiotic efficiency in termsof plant Pi uptake is further influenced by fungalcommunity

composition[7].TopredictthenetimpactofmycorrhizaeonPiuptake,itisessentialtocombine

physiologicalstudieswithstudiesatthecommunitylevel,whereplantsarepartofcomplex species interaction networks. To achieve this, it is crucial that techniques, such as high-throughputsequencing,transcriptomics,andmetabolomics,areusedinthefield.

Studiesattheplantcommunitylevelshowedthatboththeproductivityandcompositionof plant communities arealtered by mycorrhizalassociations. Mycorrhizae indirectly causea redistributionofsoilresourcesamongplants(Pi)byenhancingtheresourceacquisitionand

competitiveabilitiesofsomeplantsoverothers [20].Furthermore,agreaterrichnessofAM fungalspecieshasbeenshownnotonlytoincreasenetprimaryproductivitythroughPisupply,

butalsotomaintainamorediverseplantcommunityonwhichotherorganismsmaydepend[9]. Directly, Pi allocation among plants is also changed through common mycelial networks

(CMNs)[16] (Figure1A). However,our knowledgeofPitransport(and nutrientsingeneral)

amongplantsandcarbontransferbetweenplantsandmycorrhizalfungiinCMNsislimited[21]. Assessingthepotentialcausesandeffectsoftheseresourcefluxesoncommunityfunctioning requiresanunderstandingofthecellularandphysiologicalprocessesdrivingthistransfer,and thefactorsregulatingtheseprocesses.Moreover,abioticfactors,suchassoilnutrient avail-ability,aswellasmicro-andmacroclimate,canaffectthenetimpactofmycorrhizaeonplantPi

uptakeand distribution in the community.The globalimpact of such factorscan only be assessedinecologicalsettings.

Researchatthe ecosystemlevel revealedthatmycorrhizal associationshave a keyrole in shapingecosystemstructureandfunctionbyalteringthedistributionofPamongspecies,how

development;alsoknownasthe

perifungalmembrane.

Plantcommunitylevel:research

thatfocusesoninteractionswithina

plantcommunity(i.e.,all

co-occurringplantsinagiventimeand

space).

Plantphysiologicallevel:research

thatfocusesontheinternal(chemical

orphysical)functioningofplantsand

theirpartsthataffectplantfunctions,

itisrecycledbetweenabove–belowgroundecosystemcompartments,andultimatelyretained withinanecosystem[7,22](Figure1A). Alltheseprocessesmaydiffer betweenecosystem typesandareaffectedbydifferentabioticfactors,suchas thosementionedabove.Thisis typicallyreferredtoas‘contextdependency’.However,withoutknowledgeofthephysiological andplant community perspectiveon processesof resource distribution among plants via CMNs,itishardtopredicthowchangesintheprocessesareaffectedbydifferentcontextsand toidentifythedriversofthechanges.

Example2:EffectsofMycorrhizalAssociationsonMultitrophicInteractions

Cellular biological research on multitrophic interactions with mycorrhizae has yielded two important insights.First,following theexchange of signalsduring the presymbioticphase, plantsinitiallyrecognizemycorrhizalfungitoacertainextentaspotentialinvaders,triggeringa mild immune response [23]. This immune response resembles the microbial-associated molecularpattern(MAMP)-triggeredimmunity(MTI)mountedafterpathogenrecognition.Such plantresponsescansubsequentlybecounteractedbythesecretionoffungaleffector mol-ecules[24,25]and/orbytheplantperceptionofMYCfactors[26].Asaresultofthismolecular dialogbetweenbothpartners,thelevelsofseveraldefense-relatedphytohormonesandother metabolitescan bealtered in mycorrhizalroots, and even shoots[27,28]. Second,recent studiessuggestthat,asaconsequenceofthismodulationofplantimmunitybymycorrhizal fungi,mycorrhizalplantscanenter intoastateofsensitization oftheimmune system.This defense-primedstateallowsplantstorespondfasterand/orstrongerwhentheyare chal-lenged by subsequent herbivore and pathogen attack [28–30] (Figure 1B). The pathway regulatedbytheplanthormonejasmonicacid(JA)hasacrucialroleinplantdefensepriming [28,31].Althoughmorerobustplantdefenseisusuallyassociatedwithbetterperformancein timesofstress,boostinginduceddefenseresponsesdoesnotalwaysprovideanadvantageto theplant[32].Theincorporationofaphysiologicalapproachisessentialto understandthe effectsonwhole-plantperformance.Forinstance,fundamentalquestionsonthecontribution ofmycorrhizal-boosteddefenses toplant resistanceagainstantagonists, orontheirrole in herbivore-inducedchangesincarbonassimilationandpartitioningofassimilates,canonlybe addressed byintegrating the mycorrhizalimpact on plant defensesignaling networks into whole-plantmodels.

Plantphysiological researchrevealedthat both AMand ECMfungiinducechanges inthe immune system of a plant that impact the interaction of the plant with other community membersatdifferenttrophiclevels[33](Figure1B).Giventhatmycorrhizalfungiprimeplants forJA-signaleddefenses,itwasspeculatedthatmycorrhizalcolonizationwouldpredominantly negatively affect leaf-chewing herbivores and necrotrophic pathogens. Both attackers are mostlyresponsivetoJA-mediateddefenses[34].Atthesametime,mycorrhizalcolonization wouldbenefitpiercingandsap-suckingherbivores,andbiotrophicpathogens,whicharemore responsivetosalicylic(SA)-mediateddefenses.However,studiesshowedhighvariationinthe effect of mycorrhizal colonization on plant–herbivore interactions [35]. This suggests that additionalfactorsare involvedindetermining the finaloutcome ofmycorrhiza–plant–insect interactions[36].Moreaccurate predictionsat thewhole-plantlevelwouldrequirea better integrationofcellularandphysiologicalstudies,providinginsightintothemechanismsinvolved withstudiesoftheecologicalfunctionoftraitsthatareinfluencedbymycorrhizae.Furthermore, thiscombinationofphysiologicalandcellbiologicalinformationhasthepotentialtoprovidethe foundationfortheapplicationofmycorrhizaeinpestmanagementstrategies.Besidesdirectly affectingplantantagonists,mycorrhizaemightinfluenceplant–herbivoreinteractionsby affect-ingtheattractionofnaturalenemiesofinsectherbivores[37].Additionally,theycanalterthe competitiveabilityofplantswithinthecommunity[38].Finally,plantscanexchange

defense-relatedinformationwithotherplantsinthecommunityviaCMNs,asshowninlaboratorystudies [39].Therefore,including thecommunity perspectiveinreal-worldscenariosis essentialto predictthenetimpactofmycorrhizaeonplant–herbivoreinteractionsatthewhole-plantlevel. Theeffectsofmycorrhizationonplantphysiologytrickleuptoinfluencemicrobial,animal,and plantcommunitystructureandecosystemfunctioning[40].Besidesmodifyingaboveground plantproductivity,mycorrhizalassociationshavebeenshowntoinfluencethediversityofplant species as well as that of higher trophic levels, such as herbivores and parasitoids [41]. Moreover,CMNsconnectingmycorrhizalplantswithinacommunityactnotonlyasconduits forcarbonandmineralnutrients,butalsoasanundergroundmessagingsystem(‘signaling highway’)[42].Thisallowsneighboringplantstoactivatedefensesbeforetheyareattacked themselves[43,44](Figure1B).Althoughthisphenomenonhasbeenexclusivelystudiedin laboratorysettings,CMNshavethepotentialtodeterminetheoutcomeofmultitrophic inter-actionsbeyondtheindividualplantlevel,byconferringinformationaboutherbivorepresence withinacommunity.Consequently,theymayalsochangepredatorandparasitoidrecruitment atthecommunitylevel.Still,thecostandbenefitsofinterplantsignaling,andtheirevolutionary consequences,remainmostlyunknown.AnalyzingtheimpactofCMNsinplant-and insect-relatedtraitsatthephysiologicallevelandthemainmechanismsofinterplantsignalingatthe cellularlevelwill helptoaddressthesequestions.Typically,innature,theoutcome ofsuch interactionsisnotexclusivelydeterminedbytheinteractionpartnersperse.A multitudeof environmentalfactorsthatcanonlybemeasuredattheecosystemlevel(underfieldconditions) maystrengthenorweakenspeciesinteractions.

Mycorrhizal fungi alter plant community performance and higher-trophic level community structure, thereby impacting element and energy cycling of the whole ecosystem (Figure1B).Forexample,bychangingthechemicalcompositionofleavesandroots, mycor-rhizaewill likelyaffectthedecompositionofplant litter[45],whichservesasthemainbasal resourceinthebelowgroundfoodweb.Changesinbasalresourcesaffectthetrophicstructure andmultitrophicinteractionsofthefoodweb,andaddtoplantcommunityeffectsonnutrient cyclingofthewholeecosystem.Patternsinmultitrophicinteractionsobservedatthe ecosys-temlevelwillremaincorrelativeifnotintegratedwithmoremechanisticstudiesattheplant physiologicalandcommunitylevels. Thisisalso importantto applymycorrhiza researchto agricultureand/orcropproduction.Forinstance,theapplicationoffertilizersandpesticidesis subject to strong political and industrial pressures. Understanding the mechanisms and processes involved in AM function could underpin the development of novel crop plant– mycorrhizalassociationsforoptimalnutrientuptakeefficiencyand,simultaneously,forhigher resistanceagainstantagonists.

OpportunitiesandChallengesRelated totheIntegration ofDisciplinesin MycorrhizaResearch

To create a conceptual framework that transcends individual disciplines in mycorrhiza research,researchersneedtolearnhowtoappreciatethereciprocalbenefitsthatthedifferent disciplinesoffer. Forexample,molecular toolsof modelorganisms canprovide meansfor identifyinggeneswithimportantrolesinmycorrhizalecology.Thisisessentialforour under-standingofthedistributionandfunctionofmycorrhizaeinspaceandtime,andforpredicting howmycorrhizaerespondtochangesintheirenvironment.However,mycorrhizaecologists interested inusing genomics toolsare hampered by the low numberof fungal and plant genomescurrentlyavailable. Nevertheless,the rapidlyincreasing numberofplant genome sequencesbecomingavailablewillallow thesetoolstobeappliedmoreoftento ecological studies.Inaddition,noveltechniquesforgeneticengineering,suchasCRISPR/Cas9,willallow

for the elucidation of the function and ecological relevance of single or multiple genes in mycorrhizalfunctioninnon-modelorganisms.Theapplicationofthesegenetictools,combined withknowledgeatthe cellularlevel,will becrucial forbetterunderstanding thefunctionof mycorrhizaeintheecosystem.Bycontrast,cellularbiologistsaremakingimportantadvances inunderstandingthemolecularandcellularprocessesinvolvedinmycorrhizalestablishment andfunctioning.Nonetheless,thefunctionsofthousandsofgenesidentifiedby high-through-putgenomicsandtranscriptomicsremainpoorlydeciphered.Ecologistscanprovideguidance astothespecificcontextsandsituationsinwhichtoexaminethefunctionalecologyof‘omics patterns.Thiscanonly berealizediflaboratoryapproachesandmethodsare usedinfield settings.

Initiatingandsuccessfullymaintainingcrossdisciplinaryresearchcanbenotonlyrewarding,but alsochallenging.Onespecificchallengeisthedifferenceinconceptsandterminologiesinthe differentdisciplines.Mycorrhizaresearchersmaymeandifferentthingswhenconsideringresults as‘beingproven’[46],whichisoftenequivalenttothe‘gainofmechanisticunderstanding’.For manyecologists,theterm‘mechanism’definesaprocessorinteractionthattakesplaceatthe organizationallevelbeneaththeleveloftheobservationinastudy.Thismeansthatthistermis relativetothelevelofobservation.Forcellularbiologistsandphysiologists,itcommonlydefines somethingthattakesplaceatthecellularlevel(i.e.,itisanabsoluteterm).Furtherchallengesfor crossdisciplinaryresearcharerelatedtothedesignofcollaborativeprojects.Forinstance,what constitutesa‘control’and‘replicate’cansignificantlydifferforecologicalandcellularstudieson mycorrhizafunctioning.Inparticular,ecosystem-levelstudiesinthefieldlackanon-mycorrhizal control,whereasthisisnotthecaseinlaboratorysettings,wheresoilscanbesterilized.Infield experimentalstudies,replicationisoftenbasedonanexperimentalunitthatcanbeanindividual,a species,oraplantcommunity.However,forcellularstudiesconductedinlaboratorysettings,the wholeexperimentcanbereplicatedseveraltimestovalidatetheresults.Inotherwords,thelackof statisticalpowerwithinoneexperimentiscounterbalancedbytheconsistencyofresultsacross experiments.Suchastrategyisnotpossibleinecologicalsettings,sincevariabilityacrossyears, seasons,orweekscanonlybecounterbalancedbyarobustexperimentaldesignandhigh replication. Therefore,amainchallenge ofcombiningmultiplemycorrhiza researchfieldsresidesin integratingtheecologicalcontextdependencyfoundinnaturalsettingswithmechanistic discov-eriesobtainedinartificiallaboratoryconditions.

ConcludingRemarksandFuturePerspectives

Keytoachievingtheintegrationofmycorrhizaresearchacrossdifferentorganizationallevelsis todesignandexecutecommonprojectswhereresearchquestionsareaddressedatdifferent levelsatthesametime(seeOutstandingQuestions).Afruitfulapproachistolinklaboratoryand fieldexperimentsthroughcommonquestions,whichbuildoneachother.Inthelaboratory, importantprocessescanbestudiedbymanipulatingspecificbioticandabioticparameters precisely.Thesefindingsgeneratehypothesesontheirecologicalrole,whichshouldbetested infieldsettings.Here,thetestorganismsaresubjecttodifferenttreatmentswithinthecontextof amultitudeofbioticandabioticinteractions.Inturn,basedonpatternsthatarefoundin real-worldecosystems,single processesandmechanisms can bedisentangledusingtargeted laboratoryexperiments.Giventhatreal-worldecosystemscompriseamultitudeofinteracting andinterwoven processes,it remainschallenging to select single processesto betested independentlyinthelaboratory.Overall,bothapproachescancreatepositivefeedbackloops thatfueleachother,promotinghypothesis-drivenresearchatdifferentlevels.Anothercourseis to incorporate laboratory approaches and methods in field experimental settings for the integrationof differentorganizationallevels. However,themainchallengeisthe application of highly sensitive analytical techniques designed for in vitro laboratory environments.

OutstandingQuestions

Dofield-samplingconditionsallowthe

useofhighlysensitiveanalytical

tech-niquesas doneininvitrolaboratory

environmentstoidentifycellular

mech-anismsinmodelsystems?

Isitpossibletoidentifysinglepotentially

relevantprocessesfromgeneral

pat-ternsinreal-worldecosystemsto

inde-pendentlytestanddisentanglethemin

targetedlaboratoryexperiments?

Howcanwecreateacommon

practi-calbaseintermsofproblemdetection,

hypothesisdevelopment,andanalysis

for building a full crossdisciplinary

framework?

What is the common denominator

regarding concepts, approaches,

anddefinitionsinexperimentalstudies

thatcanlinkalldisciplinesintoa

com-pleteintegrativeresearch?

How can a ‘genes-to-ecosystem’

approachinmycorrhizaresearchbe

applied for solving environmental

issues,suchasnutritionofagrowing

Furthermore,it requires the collaboration ofresearchers from both fieldsto overcomethe limitationsofindividualdisciplines.Toachievethisgoal,itisessential,althoughchallenging,to fostercollaborationsincommonprojectsatalllevels;fromproblemdetectionandhypothesis developmenttoexperimental(orobservational)setup,analysis,andinterpretationoffindings [46].Therealizationofjointworkshopsandconferencesmaylayfoundationsforsuch enter-prises.Similarly,integrativeMSandPhDcourseswilltrainthenextgenerationofmycorrhizal scientiststoapplybothecologicalandmolecularapproachesintheirresearchprojects. Initialattemptstomakemycorrhizaresearchcrossdisciplinaryareunderwayinthe PhytOak-meterproject.Here,clonaloaks(QuercusroburL.),whichwereoriginallydevelopedforhighly standardizedmolecularmeasurements,arenowtransplantedintofieldexperimentsoverwide environmentalgradients(Box1). Anotherexampleis the tree diversity experiment,MyDiv, whereplotsconsistoftreecommunitiesthatarecharacterizedbydifferenttypesof mycorrh-ization(namelyECMandAM).MyDivintegratespartoftheclonaloaksfromthePhytOakmeter project,andfurtherimplementsomics’approaches(i.e.,metabolomics)inthefield(Box2). Theseareonlytwoexampleprojectsamongseveralthatareintheprocessofovercomingthe specifictechnicalandscientificboundariesofthesingledisciplines.Suchsynergisticprojects canultimatelyadvancethecomprehensiveunderstandingofmycorrhizalassociationsandtheir functioning that spansfrom genes to ecosystems and howthis can contribute to solving

Box1.TrophinOak-PhytOakmeter:ACloneLaboratoryandFieldExperimentalPlatform

Treeperformancedependsontheinterplaybetweenresource-demandingbeneficialanddetrimentalbiotrophicinteractionsandtheowndevelopmentalprogram.

Thisprogramisnotonlycontrolledbyinternalprocesses,butalsoinfluencedbyexternalfactors[47].TrophinOakandPhytOakmeteraretwocomplementary

experimentalsystemsthatcanbeusedtounravelsuchinterplayatthelaboratoryandfieldlevel,respectively.Theybothuseacloneofcommonoak(Quercusrobur

L.;DF159;FigureIA)becauseofitsendogenousrhythmicgrowth(ERG)characterizedbyalternatinggrowthflushesinshootandrootsparalleledbyresource

allocationshiftsbetweenbelow-andabovegroundparts[48].APetridishsystemwithsterilesoilandrootedmicrocuttingsofDF159wasestablishedtostudythe

impactoftheECMfungusPilodermacroceumontheinterplaybetweenERGandbiotrophicinteractionswithabove-andbelowgroundinteractors(FigureIB)[49].

OaktranscriptomicprofilingusingRNAseqwaspromptedbyestablishingthelargespecificreferencelibraryOakcontigDF159.1[50].ThePhytOakmeterprojectisan

extensionofTrophinOak,releasingclonalDF159oaksasphytometersinthefieldalongEuropeanclimatic,land-useintensity,andplantdiversitygradients(FigureIC).

Such‘PhytOakmeters’wereintegratedinthetreediversityexperimentMyDiv[51](seeBox2inthemaintext).DataonPhytOakmeterdevelopmentand

transcriptomicswillcomplementtheanalysesofphysiologicalandcellularresponseanalysesinrelationtothediversityofAMandECMtreeneighborsmanipulated

inMyDiv.TheuseofclonaloakphytometersallowstheoptimalevaluationandcomparisonofdifferentecosystemfunctionsinMyDiv.Alsoseehttp://www.ufz.de/

trophinoak-phytoakmeter.

(A) (B) (C)

FigureI.MainElementsandStepsintheTrophinOak-PhytOakmeterExperimentalPlatform.(A)InvitropropagationoftheOakDF159,(B)mycorrhization

withPilodermacroceuminmicrocosmsintheTrophinOakproject,and(C)outplantingofthePhytOakmeterstoafieldsite.Adaptedfrom[49](A,B).Photographby

fundamental issues, such as sustainable food production while preserving our planet’s biodiversity.

Acknowledgments

AllauthorsacknowledgefundingfromtheGermanCentreforIntegrativeBiodiversityResearch(iDiv)Halle-Jena-Leipzig

fundedbytheGermanResearchFoundation(DFG;FZT118).A.M.M.,A.B.,M.P.,S.R.,andN.M.v.D.acknowledge

stimulatingdiscussionswithinCOSTActionFA1405andfundingfromEUMC-ITNMiRA(grantno.765290).I.C.M.

acknowledgesfinancialsupportfromtheDFG(grantno.ME4156/2-1)andVolkswagenFoundation[grantno.

11-76251-99-34/13(ZN2928)].M.C.R.acknowledgesfundingfromtheEuropeanResearchCouncil(ERC,AdvancedGrant‘Gradual

Change’)andBMBFfundingfortheproject‘BridginginBiodiversityScience(BIBS)’.M.P.acknowledgessupportfrom

MINECO(grantno.AGL2015-64990-C2-1-R).I.F.acknowledgessupportfromiDivFlexpoolProgram(grantno.RA-185/

17).O.F.andN.E.acknowledgesupportfromtheERC(EuropeanUnion’sHorizon2020researchandinnovationprogram,

grantno.677232).S.R.acknowledgestheSwissScienceFoundation(grantno.159869).F.B.,S.H.,andM.T.

acknowl-edgeDFGforfinancialsupport(GrantsBU941/20-1andTA290/4-1).AlltheauthorsacknowledgesupportfromtheiDiv

OpenSciencePublicationFund.

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