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THE MYCOTA

A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied

Research

Biology of the Fungal Cell

Third Edition

Dirk Hoffmeister

Markus Gressler

Volume Editors VIII

TheMycota

I Growth,DifferentiationandSexuality 1steditioned.byJ.G.H.WesselsandF.Meinhardt 2ndeditioned.byU.KuesandR.Fischer 3rdeditioned.byJ.Wendland

II GeneticsandBiotechnology 1stand2ndeditioned.byU.Ku ¨ ck

III BiochemistryandMolecularBiology 1stand2ndeditioned.byR.BramblandG.Marzluf 3rdeditioned.byD.Hoffmeister

IV EnvironmentalandMicrobialRelationships 1steditioned.byD.WicklowandB.So ¨ derstro ¨ m 2ndeditioned.byC.P.KubicekandI.S.Druzhinina 3rdeditioned.byI.S.DruzhininaandC.P.Kubicek

V PlantRelationships

1steditioned.byG.CarrollandP.Tudzynski 2ndeditioned.byH.B.Deising

VI HumanandAnimalRelationships 1steditioned.byD.H.HowardandJ.D.Miller 2ndeditioned.byA.A.BrakhageandP.F.Zipfel

VII SystematicsandEvolution 1steditioned.byD.J.McLaughlin,E.G.McLaughlin,andP.A.Lemke{ 2ndeditioned.byD.J.McLaughlinandJ.W.Spatafora

VIII BiologyoftheFungalCell 1stand2ndeditioned.byR.J.HowardandN.A.R.Gow 3rdeditioned.byD.HoffmeisterandM.Gressler

IX FungalAssociations

1stand2ndeditioned.byB.Hock

X IndustrialApplications

1steditioned.byH.D.Osiewacz 2ndeditioned.byM.Hofrichter

XI AgriculturalApplications 1stand2ndeditioned.byF.Kempken

XII HumanFungalPathogens

1steditioned.byJ.E.DomerandG.S.Kobayashi 2ndeditioned.byO.Kurzai

XIII FungalGenomics

1steditioned.byA.J.P.Brown 2ndeditioned.byM.Nowrousian

XIV EvolutionofFungiandFungal-LikeOrganisms Ed.byS.Po ¨ ggelerandJ.Wo ¨ stemeyer

XV PhysiologyandGenetics:SelectedBasicandAppliedAspects 1steditioned.byT.AnkeandD.Weber 2ndeditioned.byT.AnkeandA.Schuffler

TheMycota

AComprehensiveTreatiseonFungias

ExperimentalSystemsforBasicandApplied Research

BiologyoftheFungalCell 3rdEdition

VolumeEditors: DirkHoffmeisterandMarkusGressler

SeriesEditor

ProfessorDr.Dr.h.c.mult.KarlEsser

AllgemeineBotanik Ruhr-Universitat 44780Bochum,Germany

Tel.:+49(234)32-22211

Fax.:+49(234)32-14211

e-mail:Karl.Esser@rub.de

VolumeEditors

ProfessorDr.DirkHoffmeister

Friedrich-Schiller-Universitat

PharmazeutischeMikrobiologie WinzerlaerStrasse2 07745Jena,Germany

Tel.:+49(3641)949851

Fax:+49(3641)949852

e-mail:dirk.hoffmeister@leibniz-hki.de

Dr.MarkusGressler

Friedrich-Schiller-Universitat

PharmazeutischeMikrobiologie WinzerlaerStrasse2 07745Jena,Germany

Tel.:+49(3641)949855

e-mail:Markus.Gressler@leibniz-hki.de

ISBN978-3-030-05446-5ISBN978-3-030-05448-9(eBook) https://doi.org/10.1007/978-3-030-05448-9

LibraryofCongressControlNumber:2019933208

# SpringerNatureSwitzerlandAG2001,2007,2019

Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthe materialisconcerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation, broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionorinformation storageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilarmethodology nowknownorhereafterdeveloped.

Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublication doesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevant protectivelawsandregulationsandthereforefreeforgeneraluse.

Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbook arebelievedtobetrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsorthe editorsgiveawarranty,expressorimplied,withrespecttothematerialcontainedhereinorforanyerrors oromissionsthatmayhavebeenmade.Thepublisherremainsneutralwithregardtojurisdictionalclaims inpublishedmapsandinstitutionalaffiliations.

ThisSpringerimprintispublishedbytheregisteredcompanySpringerNatureSwitzerlandAG Theregisteredcompanyaddressis:Gewerbestrasse11,6330Cham,Switzerland

KarlEsser (born1924)isretiredProfessorofGeneralBotany andDirectoroftheBotanicalGardenattheRuhrUniversitatBochum(Germany).Hisscientificwork focusedonbasicresearchinclassicalandmolecular geneticsinrelationtopracticalapplication.Hisstudieswerecarriedoutmostlyonfungi.Togetherwith hiscollaboratorshewasthefirsttodetectplasmidsin higherfungi.Thishasledtotheintegrationoffungal geneticsinbiotechnology.Hisscientificworkwas distinguishedbymanynationalandinternational honors,especiallythreehonorarydoctoraldegrees.

MarkusGressler (born1984)gainedaPh.D.inbiochemistryatthe FriedrichSchillerUniversityandtheLeibnizInstitute forNaturalProductResearchandInfectionBiology—HansKno ¨ llInstitute(HKI)inJena(Germany) in2015.From2015till2017hewasapostdoctoral fellowatInstitutPasteur,Paris,beforehepursuedhis researchinterestsin2017attheFriedrichSchiller University,Jena,addressingthebiosynthesisand theecologicalaspectsofpolyketidesandnonribosomalpeptidesinfungi.

DirkHoffmeister (born1972)isamycologistandFullProfessorfor PharmaceuticalMicrobiologyattheFriedrichSchiller UniversityJena(Germany).Heisalsoaffiliatedwith theLeibnizInstituteforNaturalProductResearchand InfectionBiology—HansKno ¨ llInstitute(HKI).Particularlyemphasizingthebasidiomycetes,researchinhis groupisfocusedonthebiochemicalandgeneticbasis underlyingfungalnaturalproductbiosyntheses.

SeriesPreface

Mycology,thestudyoffungi,originatedasasubdisciplineofbotanyandwasa descriptivediscipline,largelyneglectedasanexperimentalscienceuntiltheearly yearsofthiscentury.AseminalpaperbyBlakesleein1904providedevidencefor self-incompatibility,termed“heterothallism,”andstimulatedinterestinstudies relatedtothecontrolofsexualreproductioninfungibymating-type specificities.Soontofollowwasthedemonstrationthatsexuallyreproducing fungiexhibitMendelianinheritanceandthatitwaspossibletoconductformal geneticanalysiswithfungi.ThenamesBurgeff,KniepandLindegrenareall associatedwiththisearlyperiodoffungalgeneticsresearch.

ThesestudiesandthediscoveryofpenicillinbyFleming,whosharedaNobel Prizein1945,providedfurtherimpetusforexperimentalresearchwithfungi. Thus,beganaperiodofinterestinmutationinductionandanalysisofmutants forbiochemicaltraits.Suchfundamentalresearch,conductedlargelywithNeurosporacrassa,ledtotheonegene:oneenzymehypothesisandtoasecondNobel PrizeforfungalresearchawardedtoBeadleandTatumin1958.Fundamental researchinbiochemicalgeneticswasextendedtootherfungi,especiallyto Saccharomycescerevisiae,andbythemid-1960sfungalsystemsweremuch favoredforstudiesineukaryoticmolecularbiologyandweresoonableto competewithbacterialsystemsinthemoleculararena.

Theexperimentalachievementsinresearchonthegeneticsandmolecular biologyoffungihavebenefitedmoregenerallystudiesintherelatedfieldsof fungalbiochemistry,plantpathology,medicalmycology,andsystematics.Today, thereismuchinterestinthegeneticmanipulationoffungiforappliedresearch. ThiscurrentinterestinbiotechnicalgeneticshasbeenaugmentedbythedevelopmentofDNA-mediatedtransformationsystemsinfungiandbyanunderstanding ofgeneexpressionandregulationatthemolecularlevel.Appliedresearchinitiativesinvolvingfungiextendbroadlytoareasofinterestnotonlytoindustrybutto agriculturalandenvironmentalsciencesaswell.

Itisthisburgeoninginterestinfungiasexperimentalsystemsforappliedas wellasbasicresearchthathaspromptedpublicationofthisseriesofbooksunder thetitleTheMycota.Thistitleknowinglyrelegatesfungiintoaseparaterealm, distinctfromthatofeitherplants,animals,orprotozoa.Forconsistencythroughoutthisseriesofvolumes,thenamesadoptedformajorgroupsoffungi(representativegenerainparentheses)areasfollows:

Pseudomycota

Division:Oomycota(Achlya,Phytophthora,Pythium)

Division:Hyphochytriomycota

Eumycota

Division:Chytridiomycota(Allomyces)

Division:Zygomycota(Mucor,Phycomyces,Blakeslea)

Division:Dikaryomycota

Subdivision:Ascomycotina

Class:Saccharomycetes(Saccharomyces,Schizosaccharomyces)

Class:Ascomycetes(Neurospora,Podospora,Aspergillus)

Subdivision:Basidiomycotina

Class:Heterobasidiomycetes(Ustilago,Tremella)

Class:Homobasidiomycetes(Schizophyllum,Coprinus)

WehavemadethedecisiontoexcludefromTheMycotatheslimemoldswhich, althoughtheyhavetraditionalandstrongtiestomycology,trulyrepresent nonfungalformsinsofarastheyingestnutrientsbyphagocytosis,lackacell wallduringtheassimilativephase,andclearlyshowaffinitieswithcertainprotozoantaxa.

Theseriesthroughoutwilladdressthreebasicquestions:whatarethefungi, whatdotheydo,andwhatistheirrelevancetohumanaffairs?Suchafocusedand comprehensivetreatmentofthefungiislongoverdueintheopinionofthe editors.

Avolumedevotedtosystematicswouldordinarilyhavebeenthefirstto appearinthisseries.However,thescopeofsuchavolume,coupledwiththe needtogiveseriousandsustainedconsiderationtoanyreclassificationofmajor fungalgroups,hasdelayedearlypublication.Wewish,however,toprovidea preambleonthenatureoffungi,toacquaintreaderswhoareunfamiliarwith fungiwithcertaincharacteristicsthatarerepresentativeoftheseorganismsand whichmakethemattractivesubjectsforexperimentation.

Thefungirepresentaheterogeneousassemblageofeukaryoticmicroorganisms.Fungalmetabolismischaracteristicallyheterotrophicorassimilativefor organiccarbonandsomenonelementalsourceofnitrogen.Fungalcellscharacteristicallyimbibeorabsorb,ratherthaningest,nutrientsandtheyhaverigidcell walls.Thevastmajorityoffungiarehaploidorganismsreproducingeither sexuallyorasexuallythroughspores.Thesporeformsanddetailsontheir methodofproductionhavebeenusedtodelineatemostfungaltaxa.Although thereisamultitudeofsporeforms,fungalsporesarebasicallyonlyoftwotypes: (1)asexualsporesareformedfollowingmitosis(mitospores)andculminate vegetativegrowth,and(2)sexualsporesareformedfollowingmeiosis(meiospores)andareborneinoruponspecializedgenerativestructures,thelatter frequentlyclusteredinafruitbody.Thevegetativeformsoffungiareeither unicellular,yeastsareanexample,orhyphal;thelattermaybebranchedto formanextensivemycelium.

Regardlessofthesedetails,itistheaccessibilityofspores,especiallythedirect recoveryofmeiosporescoupledwithextendedvegetativehaploidy,thathave madefungiespeciallyattractiveasobjectsforexperimentalresearch.

Theabilityoffungi,especiallythesaprobicfungi,toabsorbandgrowonrather simpleanddefinedsubstratesandtoconvertthesesubstances,notonlyinto essentialmetabolitesbutintoimportantsecondarymetabolites,isalsonoteworthy. Themetaboliccapacitiesoffungihaveattractedmuchinterestinnaturalproducts

chemistryandintheproductionofantibioticsandotherbioactivecompounds. Fungi,especiallyyeasts,areimportantinfermentationprocesses.Otherfungiare importantintheproductionofenzymes,citricacid,andotherorganiccompounds aswellasinthefermentationoffoods.

Fungihaveinvadedeveryconceivableecologicalniche.Saprobicforms abound,especiallyinthedecayoforganicdebris.Pathogenicformsexistwith bothplantandanimalhosts.Fungievengrowonotherfungi.Theyarefoundin aquaticaswellassoilenvironments,andtheirsporesmaypollutetheair.Some areedible;othersarepoisonous.Manyarevariouslyassociatedwithplantsas copartnersintheformationoflichensandmycorrhizae,assymbioticendophytes orasovertpathogens.Associationwithanimalsystemsvaries;examplesinclude thepredaceousfungithattrapnematodes,themicrofungithatgrowinthe anaerobicenvironmentoftherumen,themanyinsectassociatedfungi,andthe medicallyimportantpathogensafflictinghumans.Yes,fungiareubiquitousand important.Therearemanyfungi,conservativeestimatesareintheorderof 100,000species,andtherearemanywaystostudythem,fromdescriptive accountsoforganismsfoundinnaturetolaboratoryexperimentationatthe cellularandmolecularlevel.Allsuchstudiesexpandourknowledgeoffungi andoffungalprocessesandimproveourabilitytoutilizeandtocontrolfungifor thebenefitofhumankind.

Wehaveinvitedleadingresearchspecialistsinthefieldofmycologyto contributetothisseries.Weareespeciallyindebtedandgratefulfortheinitiative andleadershipshownbytheVolumeEditorsinselectingtopicsandassembling theexperts.Wehaveallbeenabitambitiousinproducingthesevolumesona timelybasisandthereinliesthepossibilityofmistakesandoversightsinthisfirst edition.Weencouragethereadershiptodrawourattentiontoanyerror,omission,orinconsistencyinthisseriesinorderthatimprovementscanbemadein anysubsequentedition.

Finally,wewishtoacknowledgethewillingnessofSpringer-Verlagtohost thisproject,whichisenvisionedtorequiremorethan5yearsofeffortandthe publicationofatleastninevolumes.

Bochum,GermanyKARL ESSER Auburn,AL,USAPAUL A.LEMKE April1994 SeriesEditors

VolumePreface

Sincetheturnofthetwenty-firstcentury,anexponentialamountoffungal genomeshasbeensequencedandanalyzedandarenowavailableonlinewith openaccess.However,itquicklyturnedoutthatthemassivecollectionofin silico-generateddataisnotsufficientforbiotechnologicaldownstreamapplicationprocessesorforanin-depthunderstandingofinterspeciescross-talks:

Thecurrentthirdeditionof TheMycotaVIII highlightsaspectsoffundamentalfungalcellbiology.Theacademicviewonthefieldoffungalcellgrowth—long timedenouncedasatraditionalbutoutrangedfieldoffungalbiology—has undergoneadrasticchangewithinthelast20years.Thearisingneedforfungal productsnecessitatestheoptimizationoflarge-scalefermentationtechniques whichinturnrequiresadetailedknowledgeinfungaldevelopment,celldivision, cellwallproduction,andintracellularsignaltransduction:Infoodandpharmaceuticalindustries,theproductionofmedicallyimportantfungalmetabolitesor foodcontaminants,suchastheaflatoxins,isstrictlydependentonthedevelopmentalstageoftheproducingfungi.Inaddition,someimmune-modulating drugsarebasedonlargefungalcellwallpolysaccharidesbutduetotheirstickiness,theirproductionishighlyenergy-wastingandcomplicated.However,phenotypicswitchesfromfilamentoustoyeast-likegrowthformseasenotonlythe fermentationprocessbutalsothesubsequentdownstreamprocessingofthe fungalmaterial.Insum,extendedknowledgeinfungalcellbiologyenables defined,precisecultivationconditionsandthereforehighlyadvancesthewhite biotechnologyinpharmaceuticalandfoodindustry.

Fromtheecologicalpointofview,fungiareindispensablesymbiontsofmost ofhigherplantrootssupportingeachother’sgrowth.Thetightfungus–plant interplaynotonlyrequiresashiftofthefungalcellularshape,butalsoanadapted metabolisminresponsetodefinedextracellularsignals.Thediscoveryoffungal signaltransductionpathwayseasestheunderstandingofthefine-tunedmetabolic cross-flowbetweenbothsymbiontsandisthereforeofincreasedinterestin agriculture.

Theeditorsarepleasedtoworkwithanenthusiasticgroupofinternational expertsonthefieldoffungalcellbiologywhocollectedandcombinedthecurrent knowledge,wrotethearticles,anddepictedmainfindingsinillustratedfigures.The closecollaborationwiththeeditorsenabledaholisticviewonfungalcelldivision anddevelopmentcoveringthetwomostimportantdivisionsoffungi,ascomycetes andbasidiomycetes.Thevolumehighlightsfurtheraspectsofcell–cellconnections, cellshapeswitches,polarizedgrowth,andproteintransportwithinthecells.Asa specialfeature,thecurrentvolumespansthedividebetweenfungalcellgrowthand signaltransmission/transductionandshowsitsinterconnectivity.

TheeditorsthankStevenD.Harris,Ph.D.,whoinitiatedthisvolume.Wealso cordiallythankDr.AndreaSchlitzbergerofSpringerNatureforthediscussion andcoordinationofthiscurrentvolumeof TheMycota.Theeditorsaregrateful tobenefitfromtheprofoundexperienceofSeniorEditor,ProfessorEmeritusKarl Esser,andthankhimforhiskindandencouragingadvice.

Jena,Germany November2018

MarkusGressler DirkHoffmeister

FungalCellGrowth

TheWoroninBody:AFungalOrganelleRegulatingMulticellularity

MARUYAMA,KATSUHIKO KITAMOTO SeptumFormationandCytokinesisinAscomyceteFungi

SEILER,YVONNE HEILIG

TheCytoskeletonandPolarityMarkersDuringPolarizedGrowth

YUANWEI ZHANG,HECHUN JIANG,LING LU

ListofContributors

REINHARD FISCHER

DepartmentofMicrobiology,InstituteforAppliedBiosciences,Karlsruhe InstituteofTechnology(KIT),Karlsruhe,Germany

KAP-HOON HAN

DepartmentofPharmaceuticalEngineering,WoosukUniversity,Wanju, RepublicofKorea

YVONNE HEILIG

InstituteforBiologyII–MolecularPlantPhysiology,Albert-LudwigsUniversity Freiburg,Freiburg,Germany

HECHUN JIANG

JiangsuKeyLaboratoryforMicrobesandFunctionalGenomics,Jiangsu EngineeringandTechnologyResearchCenterforMicrobiology,CollegeofLife Sciences,NanjingNormalUniversity,Nanjing,China

MIN-JU KIM

SchoolofFoodScienceandBiotechnology,InstituteofAgriculturalScienceand Technology,KyungpookNationalUniversity,Daegu,RepublicofKorea

KATSUHIKO KITAMOTO

DepartmentofBiotechnology,TheUniversityofTokyo,Tokyo,Japan

MI-KYUNG LEE

BiologicalResourceCenter(BRC),KoreaResearchInstituteofBioscienceand Biotechnology(KRIBB),Jeongeup-si,RepublicofKorea

ROGER R.LEW

DepartmentofBiology,YorkUniversity,Toronto,Canada

LING LU

JiangsuKeyLaboratoryforMicrobesandFunctionalGenomics,Jiangsu EngineeringandTechnologyResearchCenterforMicrobiology,CollegeofLife Sciences,NanjingNormalUniversity,Nanjing,China

FRANCIS MARTIN

Laboratoired’excellenceARBRE,CentreINRAdeNancy-Lorraine,UMRINRA/ Universite ´ deLorraine1136‘InteractionsArbres/Microorganismes’, Champenoux,France

JUN-ICHI MARUYAMA

DepartmentofBiotechnology,TheUniversityofTokyo,Tokyo,Japan

HEE-SOO PARK

SchoolofFoodScienceandBiotechnology,InstituteofAgriculturalScienceand Technology,KyungpookNationalUniversity,Daegu,RepublicofKorea

CLE ´ MENT PELLEGRIN

Laboratoired’excellenceARBRE,CentreINRAdeNancy-Lorraine,UMRINRA/ Universite ´ deLorraine1136‘InteractionsArbres/Microorganismes’, Champenoux,France

STEPHAN SEILER

InstituteforBiologyII–MolecularPlantPhysiology,Albert-LudwigsUniversity Freiburg,Freiburg,Germany

FreiburgInstituteforAdvancedStudies(FRIAS),Albert-LudwigsUniversity Freiburg,Freiburg,Germany

NORIO TAKESHITA

FacultyofLifeandEnvironmentalSciences,UniversityofTsukuba,Tsukuba, Japan

CLAIRE VENEAULT-FOURREY

Laboratoired’excellenceARBRE,CentreINRAdeNancy-Lorraine,UMRINRA/ Universite ´ deLorraine1136‘InteractionsArbres/Microorganismes’, Champenoux,France

JAE-HYUK YU

DepartmentofBacteriology,UniversityofWisconsin,Madison,WI,USA DepartmentofSystemsBiotechnology,KonkukUniversity,Seoul,Republicof Korea

YUANWEI ZHANG

JiangsuKeyLaboratoryforMicrobesandFunctionalGenomics,Jiangsu EngineeringandTechnologyResearchCenterforMicrobiology,CollegeofLife Sciences,NanjingNormalUniversity,Nanjing,China

FungalCellGrowth

JUN-ICHI MARUYAMA1,KATSUHIKO KITAMOTO1

CONTENTS

I.MulticellularityofFilamentousFungi ......3

II.TheWoroninBody,anOrganelleSpecificto Pezizomycotina .............................4

III.MolecularBasisofWoroninBody Structure ....................................6

IV.WoroninBodyBiogenesisfrom Peroxisomes ................................7

V.SeptalTetheringoftheWoroninbody ......7

VI.ProteinsAssociatedwiththeWoronin Body ........................................10

VII.ConclusionsandPerspectives ...............11 References. .................................12

I.MulticellularityofFilamentous Fungi

Filamentousfungiformstraighthyphaevia polarizedtubularextensionofthehyphaltip andconsistofanetworkofstraightprimary hyphaewiththeformationofbranches.Hyphae arecompartmentalizedintodistinctcellsbythe formationofaseptum,whichisnotfrequently observedinearly-divergingfungi,suchas membersofthephyla Mucoromycota and Chytridiomycota,butisregularlyfoundinlaterdivergingfungibelongingtothephyla Ascomycota and Basidiomycota (Jedd 2011).Theseptumisproposedtoincreasethemechanical integrityofhyphaeandtodividethemycelium intosectionsofdistinctgrowthstates,suchas mitoticandnon-mitoticcells(FiddyandTrinci 1976;Momanyetal. 2002;Nayaketal. 2010;

Edgerton-MorganandOakley 2012).However, theseptumdoesnotcompletelyseparate hyphaeduetothepresenceofaseptalpore, whichisaperforatedstructurethatallowsthe exchangeofthecytoplasmicconstituents, includingorganelles,betweenadjacenthyphal cells(Lew 2005;Teyetal. 2005;Ngetal. 2009; Bleichrodtetal. 2015).Suchcell-to-cellconnectivityresemblesgapjunctionsinanimalcells andplasmodesmatainplantcells.

Cell-to-cellconnectivitythroughtheseptal poreisassociatedwiththecatastrophicriskof cytoplasmiclossbycellsadjacenttoindividuallydamagedhyphae.Thisriskwasdemonstratedbythephysicaldamageofhyphaewith arazorblade(TrinciandCollinge 1974)or pulselaser(Lichiusetal. 2012),treatment withcellwall-destabilizingreagents(Bowman etal. 2002),andexposuretohypotonicshock (Fig. 1a)(JeddandChua 2000;Maruyamaetal. 2005).Celldeathisinducedbyagingandheterokaryonincompatibility(FleißnerandGlass 2007).Despitesuchdamage,thecytoplasmof cellsimmediatelyadjacenttothewoundedcell istypicallyretained(Fig. 1b)(JeddandChua 2000;Maruyamaetal. 2005).Theprotected cellstheninitiateregrowthofthemyceliumby producinganewhyphaltip(JeddandChua 2000;Maruyamaetal. 2006;Maruyamaand Kitamoto 2007;Lichiusetal. 2012).Theseprocessesrepresentaninherentdefensesystemfor promotingsurvivalbypreventingthesimultaneouslossofcytoplasmfrommultiplecells uponhyphalwounding.Hence,theseptalpore isanimportantsubcellularstructureformaintainingthemulticellularityoffilamentous fungi.

1 DepartmentofBiotechnology,TheUniversityofTokyo, Tokyo,Japan;e-mail: amarujun@mail.ecc.u-tokyo.ac.jp BiologyoftheFungalCell,3rd Edition

Fig.1 Hyphalwoundingandthepreventionofexcessivecytoplasmicloss.(a)Hyphaltipburstingupon hypotonicshock.Hyphaltipsatthemarginofan A. oryzae colonygrownonagarmediumwereobservedby differentialinterferencecontrast(DIC)microscopy beforeandafterfloodinghyphaewithwater.Bar: 50 mm.(b)Preventionoftheexcessivelossofcytoplasm

II.TheWoroninBody,anOrganelle

Specificto Pezizomycotina

Filamentousfungalspeciesthatformsepta haveevolvedtopossessspecialized membrane-boundorganelleslocatedaround theseptum;the Pezizomycotina (Ascomycota) containstructurescalledthe Woroninbody

uponhyphalwounding.Thecytoplasmislabeledby expressingEGFP.Anarrowheadandarrowindicatea bursthyphaltipandtheadjacentseptum,respectively. Notethatthecell(2nd)adjacenttothelysedcell(1st) retainsitscytoplasmicconstituents,asdeterminedby DICandfluorescencemicrocopy.Bar:10 mm

(MarkhamandCollinge 1987),and Agaricomycotina (Basidiomycota)haveanendoplasmic reticulum(ER)-relatedseptalporecap(Mu ¨ ller etal. 1998).Woroninbodieswerefirstidentifiedashighlyrefractiveparticleslocatednear theseptuminthefilamentousascomycete Ascoboluspulcherrimus bytheRussianmycologist MichaelStepanovitchWoronin(Woronin 1864).TheorganellewaslaternamedbyBuller

A B

Fig.2 MorphologyoftheWoroninbody.(a)TransmissionelectronmicroscopicobservationofWoronin bodies(arrows)neartheseptumin A.oryzae.Bar: 500nm.(b)Differentialinterferencecontrast(DIC)

(1933)inrecognitionofWoronin’sdiscovery andisrestrictedto Pezizomycotina species (MarkhamandCollinge 1987).TheWoronin bodyhastwomorphologicallydistinctsubclasses;itpredominantlyappearsbytransmissionelectronmicroscopyasaspherical electron-densestructureneartheseptum (Fig. 2a),althoughinasmallnumberofspecies,suchas Neurosporacrassa,Woroninbodiesformhexagonalcrystallinestructuresthat

imageofahexagonalWoroninbodylocalizedtothe cellcortexin N.crassa.Woroninbodiesareindicated byarrows.Bar:1 mm

areoccasionallyvisiblebylightmicroscopy (Fig. 2b)(Markham 1994).Bybothelectron andlightmicroscopy,Woroninbodieswere observedtoplugtheseptalporeuponhyphal wounding(MarkhamandCollinge 1987). Therefore,itwaslongsuspectedthattheprimaryfunctionofWoroninbodieswasthepreventionofexcessivecytoplasmiclossfrom cellsadjacenttodamagedorlysedcells (Fig. 3a).

Fig.3 Woroninbodyfunction.(a)SchematicrepresentationoftheseptalpluggingfunctionofWoroninbodies.(b)ConfocalimagesofWoroninbodies(red, arrowheads)andsepta(green)before(left)andafter

(right)hyphalwounding.Woroninbodiesandsepta werelabeledbyDsRed2andEGFP,respectively(Maruyamaetal. 2005).Bar:2 mm

Hyphal wounding After hyphal wounding
body y

III.MolecularBasisofWoroninBody Structure

AlthoughWoroninbodieswerediscovered approximately150yearsago,thecomposition ofWoroninbodieslongremainedunclear.Proteasedigestionofultrathinsectionssuggested thatWoroninbodiescontainedproteinaceous materials(McKeen 1971;MasonandCrosse 1975).Woroninbodiesfrom N.crassa were firstpurifiedin2000throughdifferentialand densitygradientcentrifugation,allowingfor theidentificationof Hex1 asa majorstructural protein (JeddandChua 2000;Tenneyetal. 2000).GenesencodingHex1areconservedin Pezizomycotina species(JeddandChua 2000; Asiegbuetal. 2004;Curachetal. 2004;Soundararajanetal. 2004;Maruyamaetal. 2005; BeckandEbel 2013).Deletionofthe hex1 generesultsindefectiveWoroninbodyformationandseverecytoplasmicbleedingupon hyphalwounding(JeddandChua 2000;Maruyamaetal. 2005;BeckandEbel 2013).When theapicalneighboringcellisselectivelyruptured,thesubapicalcellsreinitiatehyphal growthbyproducinganewbranch,butdeletionofthe hex1 geneseverelyreducesor completelylosestheactivityofgrowthreinitiation(TegelaarandWo ¨ sten 2017).

CharacterizationofHex1proteinrevealed thatitspontaneouslyself-assemblestoforma solidcore,therebyprovidingmechanicalresistanceagainsttheprotoplasmicstreamingpressurethatisgenerateduponhyphalwounding (JeddandChua 2000;Yuanetal. 2003).The crystalstructureofHex1consistsofthreeintermolecularcontactsthatpromoteself-assembly (Yuanetal. 2003).Interestingly,thestructural propertiesofHex1resemblethoseofeukaryotic translationinitiationfactor5A(eIF5A)proteins (Kimetal. 1998;Peatetal. 1998),suggestingthat the hex1 geneevolvedfrom eIF-5A bygene duplicationintheancestorof Pezizomycotina. PhosphorylationofHex1alsocontributestothe formationofthemultimericcoreoftheWoroninbody(Tenneyetal. 2000;Juvvadietal. 2007).

The N.crassahex1 geneencodesasingle translationalproduct(JeddandChua 2000), althoughalternativesplicingofthe hex1 gene

wasreportedinseveralspecies,including Magnaporthegrisea, Aspergillusoryzae,and Aspergillusfumigatus (Soundararajanetal. 2004; Maruyamaetal. 2005;Becketal. 2013).Both splicedandnon-splicedtranscriptsyieldtwo polypeptidesthatsedimentinhigh-density proteinfractions(Maruyamaetal. 2005),suggestingthatthesepeptidesparticipateinthe self-assemblyandformationoftheWoronin bodycorematrix.Moreover,itwasdemonstratedthatapoly-histidinemotifencoded withinanalternativelysplicedregiontargets Hex1totheseptalpore(Becketal. 2013).

Althoughthehexagonalcrystalstructureof the N.crassa Woroninbodyiseasilyobserved bylightmicroscopy,thefusionofHex1witha fluorescentproteinenablestheseptalplugging activityofWoroninbodiestobevisualizedin most Pezizomycotina species(Fig. 3b)(Maruyamaetal. 2005;BeckandEbel 2013).Using thisapproach,Bleichrodtetal.(2012)reported thattheWoroninbodyreversiblyplugsthe septalporeduringnormalgrowth,acharacteristicthatcontraststheconventionalviewofthis organellefunctioninginwoundhealing.The analysisofgeneexpressionactivityforindividualhyphaerevealedthatalthoughwild-type cellsexhibitedheterogeneousactivity,cells lackingWoroninbodiesthroughdeletionof hex1 (Dhex1 strain)displayedmoreuniform geneexpressionactivity(Bleichrodtetal. 2012).Thus,itwasproposedthatWoronin bodiescontributetothegenerationofhyphal populationswithdifferentcellularactivitiesby occasionallypreventingcell-to-cellconnectivityviathepluggingoftheseptalpore.The generationofsuchcolonialheterogeneity undernormalgrowthconditionsmayprotect cellsagainstenvironmentalstresses,asevidencedbythesensitivityofthe A.oryzae Dhex1 straintoheatstress(Bleichrodtetal. 2012).Thus,itappearsthatWoroninbodies haveagatekeeperroleinregulatingcell-to-cell channelsbysimplepluggingofseptalpore, similartothatobserveduponhyphalwounding (JeddandPieuchot 2012).

Inadditiontoseptalplugging,Hex1hasotherphysiologicalimpactsatthecellularlevel.Forexample,deletionofthe hex1 generesultsindefectiveconidiation

6J.MaruyamaandK.Kitamoto

(asexualsporeformation)(Yuanetal. 2003;Sonetal. 2013),impairedgrowthundernitrogenstarvation (Soundararajanetal. 2004),andincreasedsensitivity tocellwallandmembrane-destabilizingagents(Beck etal. 2013).Inplantpathogenicfungi,Hex1isalso requiredforefficientpathogenesisandviralRNAaccumulation(Soundararajanetal. 2004;Sonetal. 2013). Theselinesofevidencesuggestadditionalphysiological functionsofWoroninbodies,whichneedfurtherinvestigationoftheactionmechanisms.

IV.WoroninBodyBiogenesisfrom Peroxisomes

Electronmicroscopicstudiesexaminingthe subcellularoriginofWoroninbodiesdemonstratedtheinclusionoftheseorganelleswithin microbodies (Wergin 1973;Camp 1977).This findingwassupportedbythespecificbinding ofantibodiesagainstmicrobody-specificsignal peptidestoWoroninbodies(Kelleretal. 1991). Arelationshipbetweenperoxisomesandthe Woroninbodywasclearlyindicatedbythe findingthattheC-terminusofHex1protein containsperoxisomaltargetingsignalsequence 1(PTS1)(JeddandChua 2000).Inaddition, time-lapseimaginganalysisrevealedthatWoroninbodiesbudfromperoxisomes(Teyetal. 2005).

Woroninbodyformationoccursatthe hyphalapexthroughaprocessinvolvingapicallybiasedexpressionofthe hex1 gene(Tey etal. 2005).Woroninbodybiogenesisrequires peroxinsthatmediatetheimportofperoxisomalmatrixandmembraneproteins (Fig. 4a)(Managadzeetal. 2007;Liuetal. 2008;Lietal. 2014 ).Specifically,Hex1associateswithPex26,aperoxinthatisenrichedin Woroninbody-containingperoxisomesand recruitstheAAAATPasesPex1andPex6to theperoxisomalmembraneforreceptorrecycling(Liuetal. 2011).Therecruitmentof Pex26leadstotheformationofaparallelactivationloopforPex5recyclingandHex1 import,allowingfortheefficientbiogenesis ofWoroninbodies.Fam1wasoriginallyidentifiedin Colletotrichumorbiculare asa Pezizomycotina-specificorthologofPex22,which functionsintherecyclingofPTSreceptors fromperoxisomestothecytosol(Fig. 4a).

Fam1isspecificallylocalizedonthemembrane ofWoroninbodies,raisingthepossibilitythat recyclingofPTSreceptoroccursinWoronin bodies(Kuboetal. 2015).

Inaddition,thebuddingofWoroninbodies fromperoxisomesappearstorequiredynaminrelatedproteins(Wurtzetal. 2008).TheperoxisomeproliferatorproteinPex11isneededfor thedifferentiationofWoroninbodiesfromperoxisomes(Fig. 4b)andalsoaffectstheseptal pluggingfunctionoftheseorganelles(Escan ˜ o etal. 2009).Thegeneticscreeningof N.crassa mutantsdefectiveinWoroninbodybiogenesis identifiedseveralperoxinsandtheWoronin bodysortingcomplex(WSC)proteinthat werecriticalforthisprocess(Liuetal. 2008).

WSCisa Pezizomycotina-specificproteinthat recruitstheHex1assemblytothematrixsideof theperoxisomalmembraneandfacilitatesbuddingoftheWoroninbody(Fig. 4b)(Liuetal. 2008).WSCbelongstothePMP22(peroxisome membraneprotein)/MPV17(myeloproliferativeleukemiavirus17)genefamilyandisproposedtohaveevolvedtopossessnew functionalproperties,includingself-assembly andHex1binding,fromancestralPMP22 (Jedd 2011).

ApsB,acomponentofthemicrotubuleorganizingcenter(MTOC)thathasfunctional peroxisomaltargetingsignalsequence 2(PTS2),interactswithHex1(Zekertetal. 2010).Inaddition,TmpL,atransmembrane proteininvolvedinredox-relatedsignaltransduction,islocalizedtoWoroninbodies(Kim etal. 2009).However,itisunclearhowApsB andTmpLareinvolvedinWoroninbodybiogenesisandfunction.

V.SeptalTetheringoftheWoronin body

Inmost Pezizomycotina species,Woroninbodiesaretypicallytetheredtotheseptumbya filamentatadistanceof100–200nm(Momany etal. 2002;Maruyamaetal. 2005).Thetethering structurewasshowntohaveelasticproperties, asdemonstratedbytherapidreturnofthe Woroninbodiestotheseptumafterphysical separationbylasertrapping(Bernsetal.

Fig.4 Woroninbodybiogenesisfromperoxisomes.(a) Schematicdiagramsoftheimportmachineriesofperoxisomalmatrixandmembraneproteins.Peroxins labeledinbluewerereportedtobeinvolvedin

1992).Moreover,Woroninbodiesrapidly insertintotheseptalporeuponhyphalwoundingandalsoreversiblyplugtheseptalpore duringnormalgrowth(Bleichrodtetal. 2012). Theseprocessesrequiretheproperpositioning oftheWoroninbodyandsufficientflexibilityof thetetheringlinker.

Incontrast,theWoroninbodiesofafew membersofthegenera Neurospora and Sordaria areassociatedwiththecortexinadelocalizedpattern(Plamann 2009).In N.crassa, newlysynthesizedHex1proteinsareimported intoperoxisomesintheapicalcompartment (Teyetal. 2005),andthenewlyformedWoroninbodiesaretheninheritedintosubapical

Woroninbodyfunction. PTS1 peroxisomaltargeting signal1, mPTS membraneperoxisometargetingsignal. (b)ModelofWoroninbodybiogenesisfrom peroxisomes

compartmentsviaassociationwiththecellcortex(Teyetal. 2005;Liuetal. 2008).Duringa screeningformutantsthataccumulateWoroninbodiesintheapicalcompartment,Ngetal. (2009)identifiedthe leashin locus,whichis comprisedoftheadjacentgenes lah-1 and lah2.TheN-terminalregionofLAH-1bindsto Woroninbodiesviathemembraneprotein WSC,whereastheC-terminalregionmediates theassociationofWoroninbodieswiththecell cortex(Fig. 5a)(Ngetal. 2009).Incontrast, LAH-2,whichlocalizestothehyphaltipand septum,isnotinvolvedinWoroninbodyfunction(Fig. 5a).In N.crassa cellsexpressingan LAH-1/LAH2fusionconstruct,Woroninbodies

Fig.5 SubcellularlocalizationofWoroninbodiesand tetheringproteins.(a)DelocalizedpatternofcortexassociatedWoroninbodiesviaLAH-1protein.(b)Sep-

accumulateatbothsidesoftheseptum,which onlyhaspartialabilitytopreventtheexcessive lossofcytoplasmuponhyphalinjury(Ngetal. 2009).Thisfindingindicatesanadditional requirementofthetetheredWoroninbodies for septalporeplugging. Otherspecies,suchas A.fumigatus and A. oryzae,withtetheredWoroninbodieshave large LAHproteins consistingofasinglepolypeptideofover5000aminoacids(Becketal. 2013;Hanetal. 2014).LAHisrequiredforthe tetheringofWoroninbodiestotheseptumand isinvolvedintheWoroninbodyfunctionof preventingtheexcessivelossofcytoplasmbut toalesserextentthanthemajorWoroninbody proteinHex1(Hanetal. 2014).Thisproperty maybeexplainedbythefactthatuntethered Woroninbodiesareabletoplugtheseptalpore, butnotasquicklyastetheredones.

taltetheringofWoroninbodiesviaLAHprotein.(c) IllustrationofthegenerationoftwoLAHproteinsfrom theancestralLAHprotein

LargeLAHproteincanbefunctionally dividedintoconservedN-andC-terminal regionsandanon-conservedcentralregion (Becketal. 2013;Hanetal. 2014):TheNterminalregionassociateswithWoroninbodiesinaWSC-dependentmanner,andtheCterminalregioncontainingatransmembrane spanningregionmediateslocalizationofLAH totheseptum(Fig. 5b)(Becketal. 2013;Han etal. 2014;Leonhardtetal. 2017).Atruncated LAHproteinconsistingofonlytheN-andCterminalregionsretainstheabilitytotether Woroninbodiestotheseptum;however,the Woroninbodiesarelocatedcloser(~50nm) totheseptumthanthoseinwild-typecells (Hanetal. 2014).Thisdifferenceisroughly consistentwiththe70-nmlengthoftheapproximately2700-amino-acidcentralregionof LAH,aswasestimatedbasedonthelength

Septum
Septal pore
Woronin body
LAH-2
Gene splitting
Cell cortex association in LAH-1
New promoter for LAH-2 expression
Septum
Septal pore
Woronin body
LAH WSC

10J.MaruyamaandK.Kitamoto

(1 mm)ofthe4-mDaproteintitin(Naveetal. 1989).TheelasticityoftheWoroninbody tetherthatwaspreviouslydemonstratedby laser-captureexperiments(Bernsetal. 1992) isconferredbythecentralnon-conserved regionofLAH(Hanetal. 2014).ThenonconservedcentralregioninLAHispredicted tobedisordered(Hanetal. 2014)andpresumablyfunctionsasamolecularspring,similarly tothemuscleproteintitin,whichexhibits molecularspring-likeelasticityviaitsintrinsicallydisorderedregion(Lietal. 2001).Moreover,incellsexpressingLAHproteinlacking thecentralregion,tetheredWoroninbodiesdo notplugtheseptalpore,evenafterhyphal wounding(Hanetal. 2014).Collectively,efficientseptalpluggingrequiresnotonlythat Woroninbodiesaretetheredtotheseptum butalsothatthetethermusthavesufficient elasticitytoallowfortherelativelyunrestricted movementofWoroninbodies.Theoccasional observationthatWoroninbodiesfromtherupturedcellsideplugtheseptalporesuggestsan activemechanismofWoroninbodymovement (Steinbergetal. 2017a).Surprisingly,ATP depletionbytherespirationinhibitorcarbonyl cyanide m-chlorophenylhydrazineinducesthe translocationofWoroninbodiesintotheseptal porewithoutcellwounding.Thissuggeststhat ATPisrequiredtopreventtheseptalplugging byWoroninbodies,leadingtothespeculation thatATPmaybindtothetetheringprotein LAHforpreventingaconformationalchange andcontractionoftheprotein(Steinbergetal. 2017b).

ThemolecularoriginofLAHremainselusive,but thisproteinfirstappearedinthecladeof Pezizomycotina speciestogetherwithWoroninbodies(Jedd 2011).Theancestral lah geneencodesasingle,large polypeptideandwassubsequentlysplitintotwo genes, lah1 and lah2,inthe Neurospora -Sordaria clade.The lah1 geneacquiredanearlytermination sequenceandanewcortexassociationdomainatthe C-terminusandanewpromotercontrolledexpressionofthedownstream lah2 gene(Fig. 5c).Thus,as aresultofthegenesplittingofthe lah gene,the localizationoftheWoroninbodywasshiftedfrom septaltetheringtothecortex. N.crassa and Sordaria fimicola exhibitextensivecytoplasmicstreaming throughseptalpores(Lew 2005;Teyetal. 2005;Ng etal. 2009 )andhaveunusuallyrapidgrowthrates (>1 mm/s)(Ryanetal. 1943).Asthetetheringof

Woroninbodiestotheseptummayhavereduced theexchangeofcytoplasmbetweenadjacentcells,a delocalizedpatternofWoroninbodieswithcortex associationmayhavebeenselectedtosupportthe rapidgrowthofthesespecies.

VI.ProteinsAssociatedwiththe WoroninBody

ThroughthepurificationofWoroninbodyassociatedproteinsandbioinformatics approachesfordetectingproteinsthatcontain intrinsicallydisorderedregions,17 septalporeassociated(SPA)proteins wereidentifiedin N. crassa (Laietal. 2012).Intrinsicallydisordered proteinsrangefrompartiallytocompletely unstructuredandlackingglobularfolds;however,theseproteinsarecapableoffoldingupon bindingtotargetmolecules(WrightandDyson 2009).Thenumberofreportsrelatedtointrinsicallydisorderedproteinshasincreasedsignificantlyinrecentyears,andmanybiological functionsoftheseproteinshavebeenrevealed (OldfieldandDunker 2014).Theloss-of-functionofseveralSPAproteinsleadstoexcessive septation,septalporedegeneration,anduncontrolledWoroninbodyactivation(Laietal. 2012).Spa10proteinisrequiredforthelocalizationofLAHC-terminalregionatthemature septalpore,consequentlystabletetheringof Woroninbodiestotheseptum(Leonhardt etal. 2017).Thesefindingssuggestthatthe septalporeisacomplexsubcellularsitefor theassemblyofunstructuredproteins,which contributetodiversestatesofcell-to-cellconnectivity.

The Pezizomycotina-specificproteinSO, whichwasoriginallyfoundin N.crassa and wasnamedbasedonthe“soft”appearanceof themutantcolony,isimportantforhyphal fusionandsexualreproduction(Fleißneretal. 2005;Enghetal. 2007).SOproteinconsistsof approximately1200aminoacidsandcontainsa singleWWdomain.Pro40,a Sordariamacrospora SOhomolog,functionsasascaffoldby associatingwithproteinkinasesinvolvedincell wallintegrity(Teichertetal. 2014).SOprotein isdispersedthroughoutthecytoplasmunder normalgrowthconditionsbutaccumulatesat

Fig.6 AccumulationofSOproteinattheseptalpore. SOlocalizationwasvisualizedbyexpressingasEGFP fusionprotein.(Left)Uponhyphalwounding,SOaccumulatesattheseptalporetogetherwiththeWoronin

theseptalporeadjacenttowoundedcells (Fig. 6),aswasreportedin N.crassa and A. oryzae (FleißnerandGlass 2007;Maruyama etal. 2010). S.macrospora Pro40proteincolocalizeswithWoroninbodies(Enghetal. 2007).

Deletionofthe so genedelaysseptalplugging andreducesthenumberofhyphaethatprevent excessivecytoplasmiclossin N.crassa and A. oryzae (FleißnerandGlass 2007;Maruyama etal. 2010).Takentogether,thesefindingsindicatethatSOproteinhasseptalpluggingactivity byaccumulatingattheseptalpore,similarto thefunctionofWoroninbodies.Additionally, SOproteinaccumulatesattheseptalporein aginghyphae(FleißnerandGlass 2007)and undervariousstressconditions,including low/hightemperature,highacidity/alkalinity, andnitrogen/carbondepletion(Fig. 6)(Maruyamaetal. 2010).Inresponsetopulselaser treatment,whichphysicallystressescellswithoutcausinghyphalwounding,SOrapidlyaccumulatesattheseptalporenearesttothe stressedhyphalarea(Maruyamaetal. 2010).

body.(Right)SOalsoaccumulatesattheseptalpore understressedconditions.Asteriskindicatestheseptum,andthearrowheadindicatestheaccumulationof SOproteinattheseptalpore.Bars:5 mm

Thus,SOproteinmayregulatecell-to-cellconnectivityviatheseptalporeinastressdependentmanner.

Astudyof Aspergillusnidulans revealedthatmitosis interruptscell-to-cellconnectivitythroughtheseptal pore(Shenetal. 2014).ThemitoticNIMAkinasewas foundtobelocalizedtotheseptumduringinterphase andtocontributetokeepingtheseptalporeopenbutis translocatedintothenucleustoinitiatemitosis,resultinginseptalclosure.Notably,however,themitotic regulationofcell-to-cellconnectivityisindependent ofWoroninbodiesandSOprotein.Identificationof theNIMAkinasesubstrate(s)mayprovideinsight intothemolecularmechanismsunderlyingmitotic interruptionofcell-to-cellconnectivity.

VII.ConclusionsandPerspectives

DespitethediscoveryofWoroninbodiesover 150yearsago,ourunderstandingofthese uniquefungalorganelleswasexclusivelybased onprimitivemicroscopicstudiesconductedin

12J.MaruyamaandK.Kitamoto

thetwentiethcentury.However,inthepast20 years,molecularstructureandfunctionofWoroninbodieshavebeenlargelyelucidated.Genomicchangessuchasgeneduplicationand splittinggeneratedandmodifiedtheWoronin bodytoallowforspecificmulticellularorganizationofindividual Pezizomycotina species. AlthoughtheprimaryfunctionofWoroninbodieshadlongbeenthoughttobewoundhealing, recentmolecularstudieshaverevealednewphysiologicalfunctionsoftheseorganelles.Todate, however,themolecularmachineriescontrolling andmediatingseptalclosureandcellrepair remainmostlyunknown.Furtherinvestigations tofunctionallylinkWoroninbodiesandrelated moleculesinthevicinityoftheseptumwilllead toamorecomprehensiveunderstandingofthe mechanismsregulatingcell-to-cellconnectivity andfungalmulticellularity.

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SeptumFormationandCytokinesisinAscomyceteFungi

STEPHAN SEILER1,2,YVONNE HEILIG1

CONTENTS

I.Introduction .................................15

II.SpatialCues:MechanismsSpecifyingthe PositionoftheDivisionPlane ...............16

A.PositioningtheCellDivisionPlanein UnicellularYeasts .........................16

B.SeptumPlacementinFilamentous AscomyceteFungi .........................18

C.SignalIntegrationbyAnillin-Type LandmarkProteins ........................19

III.TemporalCue(s):CoordinationofCellCycle ProgressionandSeptationInitiation ........21

A.CoordinationofMitoticExitandInitiation ofSeptationinUnicellularYeasts. ........21

B.TheSeptationInitiationNetworkinthe Pezizomycotina Fungi. ....................23

C.CoordinationofNuclearBehaviorand SeptumFormationin Pezizomycotina Fungi. .....................................24

IV.AssemblyandRegulationoftheContractile ActomyosinRing(CAR) .....................26

A.CARAssemblyinBuddingandFission Yeast. .....................................26

B.CARAssemblyinthe Pezizomycotina .....27

C.CrosstalkBetweentheSINandtheMOR PathwaysforCARRegulation. ............28

V.CellWallBiogenesisandCellDivision ......29

A.CellWallBiogenesis... ....................29

B.SeptumFormationandCellDivisionDuring FungalDevelopment.. ....................30

C.SeptalPore-AssociatedFunctionsin FilamentousAscomyceteFungi... ........32

VI.ConclusionsandPerspectives ...............33 References.. .................................33

1 InstituteforBiologyII–MolecularPlantPhysiology,AlbertLudwigsUniversityFreiburg,Freiburg,Germany;e-mail: Stephan.Seiler@biologie.uni-freiburg.de; 2 FreiburgInstituteforAdvancedStudies(FRIAS),Albert-LudwigsUniversityFreiburg,Freiburg,Germany

I.Introduction

Celldivisionisafundamentalcellularprocess thatisessentialforproliferationofunicellular aswellasmulticellularorganisms.Itreflectsthe finalstageofthecellcycle,duringwhichacellis physicallydividedintotwodaughtercellsthat containafullsetofchromosomesandother cellularorganelles.Cytokinesiscanbedivided intoseveralgeneralstepsthatapplytomost eukaryoticcells(BarrandGruneberg 2007; Eggertetal. 2006):(1)theselectionofthefuture celldivisionplane basedonspatialaswellas temporalcues,(2)the assemblyofacortical actomyosinring(CAR) atthissite,and(3)its constrictioncoupledwithmembraneinvagination.Ingeneral,theformationoftheCAR anditssubsequentconstrictionistightlycoupledtothecellcycletoensurethatcellseparationdoesnotoccurpriortochromosome segregation.(4)Theformationofanextracellularcellwall,theseptum,composedofglucans,chitin,andotherpolysaccharidesinfungi furtherrequirescoordinationofCARconstrictionwithsecretionofcellwallbiosyntheticand remodelingenzymestobuildtheextracellular septum.(5)This primaryseptum iscoveredby additionallayersofcellwallmaterialthatform the secondaryseptum andisfinallydegraded bysecretedhydrolyticenzymesintheunicellularyeastsandduringsexualdevelopmentof filament-formingmoldstoallowdetachmentof thetwocells.

Severaloverviewshaverecentlysummarizedthemechanisticprinciplesunderlyingcell polarizationanddivisioninbuddingyeastand fissionyeast,twounicellularfungithatconsis-

BiologyoftheFungalCell,3rd Edition

TheMycotaVIII

D.Hoffmeister,M.Gressler(Eds.) © SpringerNatureSwitzerlandAG2019

tentlyserveasconceptualframeworkforthe analysisoffungalaswellashighereukaryotic cellbiology(PollardandWu 2010;BiandPark 2012;Weiss 2012;MartinandArkowitz 2014). However,thevastmajorityoffungiforms mycelialcoloniesthatconsistofnetworksof branchedhyphe.Thesecellsgrowbytipgrowth andarecompartmentalizedbyseptumthatpartitioncellularenvironmentswithinthehypha. Incontrasttotheunicellularyeasts,notevery nucleardivisiontriggerstheformationofanew septuminmycelialfungi,andthushyphalcompartmentsaregenerallymultinucleate.Moreover,asmallseptalporeisretainedinhigher fungitoenableintercellularcommunication andtransportofcytoplasmandorganelles betweenadjacenthyphalcompartments.This controlledsegmentationofhyphalunits throughseptalcrosswallsinamulticellular myceliumisthebasisforthemorphological complexityachievedbythefungiduringvegetativegrowth,differentiation,andinfection processesandisthusaprerequisiteforthe evolutionarysuccessofthefungalkingdom (Gull 1978;PringleandTaylor 2002;Blackwell 2011).

Thephylum Ascomycota comprisesthree majorsubphyla: Taphrinomycotina, Saccharomycotina,and Pezizomycotina (McLaughlin etal. 2009;Stajichetal. 2009).Thesubphylum ofthe Pezizomycotina containsover90%ofthe Ascomycota species.Almostallspeciesofthis cladegeneratemultinuclearhyphaethatare compartmentalizedbyseptaandincludethe modelmolds Aspergillusnidulans and Neurosporacrassa.The Saccharomycotina containthe industrialyeastsandparasitic Canididaspec. species,dimorphicfungithatcanswitch betweenyeastandmycelialstates(Sudbery 2011).Mostmembersofthe Saccharomycotina areunicellular,butthisgroupalsoincludesfilamentousforms,suchas Ashbyagossypii.This speciesisverycloselyrelatedto Saccharomyces cerevisiae,andmorethan90%ofthegenesare highlyconservedinthetwofungi(Dietrichetal. 2004;WendlandandWalther 2005),butthetwo specieshavedevelopeddramaticallydifferent growthforms:constitutivemultinucleatetip growthin A.gossypii versusunicellulargrowth in S.cerevisiae.Thethirdsubphylumincludes

the Schizosaccharomycetes andotherearly diverginglineagesandisprimarilyrepresented by Schizosacchomycespombe.Themonophyleticoriginofthissubphylumisstillunder debate,butmostrecentdatasupportthemonophylyofthetaxon(Jamesetal. 2006).Both filamentsandyeastsarefoundinthissubphylum,suggestingthatbothmorphologiesare ancestralin Ascomycota.

Despitetheimportanceofseptumfor hyphae,proliferation,anddifferentiationinfilamentousfungi,ourunderstandingofseptum formationanditsregulationishighlyfragmentary(Harris 2001;SeilerandJusta-Schuch 2010; Mourino-PerezandRiquelme 2013).Inthis review,wewillfocusonrecentprogressthat confirmstheuseofconservedmolecularmodulesduringcelldivisionintheunicellular yeastsandduringseptumformationinthefilamentousfungi.However,itisalsobecoming apparentthatproperplacementandregulated formationandfunctionofseptainthedifferent phylogeneticgroupsof Ascomycota requires significantrewiringandspecies-specificadaptationoftheseconservedmodules(Guetal. 2015).

II.SpatialCues:Mechanisms

SpecifyingthePositionofthe DivisionPlane

A.PositioningtheCellDivisionPlanein UnicellularYeasts

Theregulatorypathwaysthatcontrolthespatial aspectstoplacethefuturecelldivisionplane arepoorlyconservedamongdifferenteukaryoticorganismsdespitethehighimportanceofa tightcoordinationofcytokinesiswithchromosomeandorganellesegregation.Forexample, thetwomodelyeasts S.cerevisiae and S.pombe havedevelopedfundamentallydistinct mechanismstocontrolthespatialaspectsfor placingthefuturecelldivisionplane(Fig. 1a, b).Thebud-siteselectionsystemofbudding yeastreliesoncorticalcuesoftheprevious celldivisioncycleinordertoredirectthedividingnucleustothebudneck,whiletheposition

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“Do you suppose he has heard of that game, Dan?” laughed Walter.

“We’ll soon know,” replied Dan with a smile as he stepped out upon the platform, where the outstretched hand of the harnessmaker grasped his first of all.

“I say, Dan,” demanded Silas, “I hear ye beat ’em all.”

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THE END

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