presentations and meeting abstracts€¦ · deciphering the role of fungal secondary metabolites...

Microbial and Plant Systems Modulated by Secondary Metabolites Meeting July 24-26, 2017 Walnut Creek, California

Speaker Presentations

and Meeting Abstracts

All information current as of July 24, 2017

JGI Contact: Stephanie Canon

DOE Joint Genome Institute

[email protected]

The Joint Genome Institute is a user facility of

the Department of Energy Office of Science

DOE Joint Genome Institute: www.jgi.doe.gov

DOE Office of Science: science.energy.gov

TableofContents

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TableofContentsAgenda................................................................................................................................................................4Monday,July24................................................................................................................................................................4Tuesday,July25...............................................................................................................................................................4Wednesday,July26........................................................................................................................................................6

SpeakerPresentations..................................................................................................................................7Convergenceofmodelsystemsandthenaturalworld..................................................................................7Plant-associatedmicrobiomesandtheeffectsondiseaseoutcomes.......................................................7Understandingtheroleoftherootmicrobiomeandplanthealth.............................................................8Specializedmetabolisminrootnodulecommunities.....................................................................................8Mappingthecolonizationofasyntheticmicrobialcommunityinoculumfromsugarcanemicrobiomeinmaizeandsoybeanplants.......................................................................................................8

Root-associatedmicrobiomeofaditerpene-deficientmaizemutant......................................................9Understandingtheroleofspecializedmetabolicnetworksmediatingbioticinteractionsinmaizethroughintegratedmultidisciplinaryapproaches....................................................................9

Dissectionofmetabolicandphenotypictraitsinmajorcrops..................................................................10Metabolomicsactivityscreening...........................................................................................................................10Decipheringtheroleoffungalsecondarymetaboliteswithinanaerobicmicrobialcommunities..............................................................................................................................................................11

Metabolomics-guidedisolationofsignificantbiologicalplant,soil,andmicrobialsecondarymetabolites..........................................................................................................................................11

Harnessingnaturetohelpfarmerssustainablyfeedtheplanet..............................................................12Miningtheplantmicrobiomefornovelagriculturalpestcontrolsolutions......................................13Strategiestodesigncustommicrobesformultiplemarketsusingafoundrymodel.....................13OptimizingconditionsthatinduceorrepresstoxinproductionusingPhenotypeMicroArrays...............................................................................................................................................................13

Plantgrowthregulatorsandplantgrowth-promotingmicrobesforcropenhancement............14Omicsapproachestodeorphanizefungalnaturalproducts.....................................................................14Membranetraffickingatthehost-pathogeninterface.................................................................................14Comparativephylogenomicsidentifiesfungalsecondarymetabolitesalliedwithvirulencetoplanthosts........................................................................................................................................15

Discoveryandengineeringofnovelnaturalproductsviasyntheticbiology......................................15Harnessingthechemistryandbiologyofplantmetabolism.....................................................................16Metagenomicandsyntheticbiologyapproachesfornaturalproductdiscovery..............................16Genomeminingfornewherbicides......................................................................................................................17Harnessingsyntheticbiologyfortheproductionoffineandspecialtychemicals..........................17RedesigningthegeneticsofsecondarymetabolisminStreptomyces.....................................................17DiscoveryofnovelsignalingsecondarymetabolitesfromClostridium................................................18Interkingdomsignaling:aPopuluscasestudy.................................................................................................18

PosterPresentations...................................................................................................................................20Streptomyces'dynamicroleintherootofArabidopsisthaliana.............................................................20Seedsofantagonism:AnABCtransporteranditsadjacenttranscriptionfactorinFusariumverticillioidesarerequiredforpyrrocidineBtolerance.....................................................20

Exploringthesoybeanmicrobiomeusingcomplementaryspatialmetabolomicstechniques..................................................................................................................................................................21

Enzymatictransformationofthesiderophorepyochelinthroughimagingmassspectrometryofbacteria-fungiinteraction..................................................................................................22

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Metabolicengineeringaprobioticyeasttoincreaseastaxanthinproduction..................................22EffluxtransporterscontributetovirulenceandhostcompatibilityofPseudomonassyringaeB728a.........................................................................................................................................................23

Volatileterpenesecondarymetabolisminswitchgrassrootsinthebiotic/abioticenvironment..............................................................................................................................................................23

Aprotein-proteininteractionnetworkcenteredonc-di-GMPsignalingintheplantgrowthpromotingrhizobacteriaP.fluorescens.........................................................................................24

Nanoscaleclusteringofenzymesinafungalsesquiterpenebiosyntheticpathway.......................25Droughtstressresultsinacompartment-specificrestructuringofrice-root-associatedmicrobiomes..............................................................................................................................................................25

TherhizosphereofthebeachgrassAmmophilabreviligulataasamodelforplant-microbiomeinteractions......................................................................................................................................26

HydroxycinnamicacidamidesareexportedbyaMATEtransporterinArabidopsis.....................26Mappingthecolonizationofasyntheticmicrobialcommunityinoculumfromsugarcanemicrobiomeinmaizeandsoybeanplants....................................................................................................27

Decipheringtheroleoffungalsecondarymetaboliteswithinanaerobicmicrobialcommunities..............................................................................................................................................................28

Root-associatedmicrobiomeofaditerpene-deficientmaizemutant...................................................29Genomesequenceofanabundance-drivenmicrobiomesyntheticcommunitywithbeneficialeffectsonplantdevelopment........................................................................................................29

Community-basedculturecollectionasastrategyfortargetingbeneficialplant-associatedbacteriafromthesugarcanemicrobiome..............................................................................30

Metabolomics-guidedisolationofsignificantbiologicalplant,soil,andmicrobialsecondarymetabolites..........................................................................................................................................31

Evolutionanddiversityofabiosyntheticgeneclusterforproductionofavinylglyine................31Changesinrootexudationandmicrobiomerecruitmentbymaizeinresponsetophosphatelimitation..............................................................................................................................................32

Microbialvariationamonghighandlowmethaneemittingricecultivars.........................................33UseofLC-MS/MSinthecharacterizationofproteinbioactivesfromBacillusspores...................33Strigolactoneimpactsonsoybeanrhizospheremicrobialcommunityassembly............................33IdentificationofunknownsecondarymetabolitesbyhybridNMR/MSapproach:applicationtostudyingthefloweringtimeinArabidopsisthaliana..................................................34

Examiningthesecondarymetaboliteactivityinthelichencommunity..............................................35ChangesintherootmetabolomeofcitrusplantsinfectedwithCandidatusLiberibacterasiaticus.......................................................................................................................................................................35

Assessingmicrobialcommunitycontributiontoplantabioticstresstolerance:acasestudyinserpentinesoils......................................................................................................................................36

MetabolomicstounderstandanddetectC.Liberibacterasiaticusinfection......................................36Chemistryoftheplantexudationandsubstrateutilizationpreferencesofsoilmicroorganismsunderlyingrhizospheremicrobiomeassemblyinannualandperennialgrasses.....................................................................................................................................................37

Broad-host-rangeexpressionrevealsnativeandhostregulatoryelementsinfluencingheterologousantibioticproductioninGram-negativebacteria..........................................................38

TomatoRhiz'OMICS....................................................................................................................................................38Functionalgenomics-guideddiscoveryofcrypticmetabolitesinvolvedinpathogenicplant-microbeinteractions.................................................................................................................................39

ChemicaldiversitygenerationusingRiPPpathways...................................................................................39ImpactofHLBonthemetallomeandmetabolomeofCitrus1H.............................................................40Investigatingagenome-to-phenotypepipelineformodelgrasses........................................................40Establishingagenome-to-phenotypepipelineformodelgrasses..........................................................41

TableofContents

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CharacterizationofthemicroviridinbiosyntheticgeneclusterfromChryseobacterium.............42SurveyofthebiosyntheticpotentialofthePopulusmicrobiome............................................................42EngineeringthebiocatalyticselectivityofiridoidproductioninSaccharomycescerevisiae.....................................................................................................................................................................43

Genomeminingoffungalnaturalproducts......................................................................................................43

JGIMicrobialandPlantSystemsModulatedbySecondaryMetabolites

2017Conference

AgendaJuly24to26,2017

WalnutCreekMarriott,WalnutCreek,CA

AllfunctionswillbeheldattheWalnutCreekMarriott.

Monday,July244to5PM OpeningKeynotePresentation Speaker:JoHandelsman,WisconsinInstituteforDiscovery

Convergenceofmodelsystemsandthenaturalworld

5to6PM RapidFireTalks Postersessionattendees

6to9PM OpeningReceptionandPosterSession Host:YasuoYoshikuni

Tuesday,July259to9:10AM Welcome&Introduction AxelVisel

9:10to9:40AM JGISyntheticBiology YasuoYoshikuni

9:40to10:10AM JGIMetabolomics TrentNorthen

10:10to10:30AM Break

10:30to11AM CarolineRoper,UniversityofCalifornia,Riverside Plant-associatedmicrobiomesandtheeffectsondiseaseoutcomes

11to11:30AM RonniedeJonge,UtrechtUniversity,Utrecht Understandingtheroleoftherootmicrobiomeandplanthealth

11:30tonoon MatthewTraxler,UniversityofCalifornia,Berkeley Specializedmetabolisminrootnodulecommunities

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Noonto12:15PM NataliaDamasceno,CenterofMolecularBiologyandGeneticEngineering Mappingthecolonizationofasyntheticmicrobialcommunityinoculumfrom

sugarcanemicrobiomeinmaizeandsoybeanplants(shorttalk)

12:15to12:30PM KatherineMurphy,UniversityofCalifornia,Davis Root-associatedmicrobiomeofaditerpene-deficientmaizemutant(shorttalk)

12:30to1:30PM WorkingLunch

1:30to2PM AlisaHuffaker,UniversityofCalifornia,SanDiego Understandingtheroleofspecializedmetabolicnetworksmediatingbiotic

interactionsinmaizethroughintegratedmultidisciplinaryapproaches

2to2:30PM JieLuo,HuazhongAgriculteralUniversity Dissectionofmetabolicandphenotypictraitsinmajorcrops

2:30to3PM GarySiuzdak,TheScrippsResearchInstitute Metabolomicsactivityscreening

3to3:15PM CandiceSwift,UniversityofCalifornia,SantaBarbara Decipheringtheroleoffungalsecondarymetaboliteswithinanaerobicmicrobial

communities(shorttalk)

3:15to3PM HeinoHeyman,PacificNorthwestNationalLab Metabolomics-guidedisolationofsignificantbiologicalplant,soil,andmicrobial

secondarymetabolites(shorttalk)

3to3:30PM Break

4to4:20PM VictoriaKnight-Connoni,IndigoAgriculture Harnessingnaturetohelpfarmerssustainablyfeedtheplanet

4:20to4:40PM JillPaulik,AgBiome Miningtheplantmicrobiomefornovelagriculturalpestcontrolsolutions

4:40to5PM JohanKers,GinkgoBioworks Strategiestodesigncustommicrobesformultiplemarketsusingafoundry

model

5to5:20PM BarryBochner,Biolog OptimizingconditionsthatinduceorrepresstoxinproductionusingPhenotype

MicroArrays

5:20to5:40PM MarciSurpin,ValentBiosciences Plantgrowthregulatorsandplantgrowth-promotingmicrobesforcrop

enhancement

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Wednesday,July269to9:30AM NancyKeller,UniversityofWisconsin,Madison Omicsapproachestodeorphanizefungalnaturalproducts

9:30to10AM SophienKamoun,TheSainburyLaboratory Membranetraffickingatthehost-pathogeninterface

10to10:30AM GillianTurgeon,CornellUniversity Comparativephylogenomicsidentifiesfungalsecondarymetabolitesalliedwith

virulencetoplanthosts

10:30to11AM Break

11to11:30AM HuiminZhao,UniversityofIllinois,Urbana-Champaign Discoveryandengineeringofnovelnaturalproductsviasyntheticbiology

11:30AMtonoon SarahO’Connor,JohnInnesCentre Harnessingthechemistryandbiologyofplantmetabolism

Noonto1PM WorkingLunch

1to1:30PM DavidMead,VarigenBiosciences Metagenomicandsyntheticbiologyapproachesfornaturalproductdiscovery

1:30to2PM YiTang,UniversityofCalifornia,LosAngeles Genomeminingfornewherbicides

2to2:30PM ErikoTakano,TheUniversityofManchester Harnessingsyntheticbiologyfortheproductionoffineandspecialtychemicals

2:30to3PM Break

3to3:30PM MichaelSmanski,UniversityofMinnesota RedesigningthegeneticsofsecondarymetabolisminStreptomyces

3to4PM WenjunZhang,LawrenceBerkeleyNationalLaboratory DiscoveryofnovelsignalingsecondarymetabolitesfromClostridium

4to5PM ClosingKeynotePresentation Speaker:GeraldTuskan,OakRidgeNationalLab

Interkingdomsignaling:aPopuluscasestudy

5PM EndofSecondaryMetaboliteConference

Note:AMandPMrefreshmentswillbeservedafterthemeetingbegins,whileworkisbeingperformed.

Attendanceisrequiredduringthesetimes.

SpeakerPresentations

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SpeakerPresentationsConvergenceofmodelsystemsandthenaturalworld

JoHandelsman([email protected])

WisconsinInstituteforDiscovery,Madison,WI,USA.

Secondarymetaboliteshaveprovedcriticaltomicrobialcommunication,competition,andbehaviorin

binaryinteractionsinthelaboratory.Therolesofthesecompoundsinmorecomplexmicrobial

interactions—particularlyinmultispeciescommunities—remainsmurky.Thechallengeinstudyingthese

interactionsinnaturalsystems,suchastherhizosphereandsoil,liesinthecomplexityofthesemicrobial

communities,manyofwhichcontainthousandsofspeciesandaresubjecttocontinualandoften

unpredictablechanges.

Themonumentalgrowthofbiologyoverthelastcenturyispredicatedonseveralmodelorganismsthat

haveprovidedpowerfultoolsforunmaskingthesecretsofmolecular,cellular,andorganismal

processes.Likewise,modelsthatsimplifystudyofmicrobialcommunitiescouldcatapultmicrobial

ecologytoanewlevelofunderstanding.Toaddresstheneedforsuchamodel,wedesigneda

communitycontainingthreemembersisolatedfromtherhizospheresoffield-grownsoybeanplants.

Communitymemberswerechosenfromamongagroupof“Bacilluscereushitchhikers,”whicharebacteriathatco-isolatewithB.cereusfromsoybeanroots.Fromalargecollectionofhitchhikerswe

chosetwoisolates,PseudomonaskoreensisandFlavobacteriumjohnsoniae,whichwhencombinedwith

B.cereusrepresentthethreemajorphylaoftherhizosphere.

Thismodelcommunityrevealedinteractionsthatwerenotevidentinbinaryinteractions.The

communitymembersproduceseveralsecondarymetabolitesthataffectothermembersdifferently

whencommunitymembersarepresentindifferentcombinations.Moreover,thecommunityforms

robustbiofilmsthatarenotobservedwithanyofthethreeindividuallyorinpairs.Genomesequences

andgeneticanalysishavethepotentialtorevealtherulesthatgovernestablishmentandrobustnessof

thismodelcommunity.

Plant-associatedmicrobiomesandtheeffectsondiseaseoutcomes

CarolineRoper([email protected])

UniversityofCalifornia,Riverside,CA,USA.

Pierce’sdisease(PD)ofgrapevine,causedbythexylem-limitedbacteriumXylellafastidiosa(Xf),isamajorthreattothegrapevineindustry.InvineyardsthatareunderhighPDpressure,thereare

interestingexamplesofvinesexhibitingeithernosymptomsorverymildPDsymptoms(disease-

escaped).Thesedifferencesarelikelynotattributedtothegeneticsoftheplantbecauseallvinesina

vineyardareclonal.Wehypothesizethatthemicroorganismsinhabitingthexyleminthesedisease-

escapedvinesareinhibitorytoXfandsubsequentlyreducediseaseseverity,duetotheirshared

ecologicalniche.WehavecharacterizedthemicrobialcommunitiesresidinginPD-infectedvinesand

comparedthemtodisease-escapedvinesandidentifiedasubsetofbeneficialorganismsthatare

antagonistictoXf.Weenvisionharnessingthesemicrobestoconstructabeneficialsynthetic

phytobiomethatcanbedeployedintograpevinesduringthenurserypropagationprocess.Wearealso


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currentlyassessingthesecondarymetaboliteprofilesofendophyticgrapevinemicrobesandare

developingsomeofthesediscoveriesintoPDmitigationtools.

Understandingtheroleoftherootmicrobiomeandplanthealth

RonniedeJonge([email protected])

UtrechtUniversityInstituteofEnvironmentalBiology,Utrecht,Netherlands.

Theinteractionbetweengenotype-dependentrootexudationandtherhizospheremicrobiome

compositionandactivitywillbediscussed.

Specializedmetabolisminrootnodulecommunities

MatthewTraxler([email protected])

UniversityofCalifornia,Berkeley,CA,USA.

Thistalkwilldescribeoureffortstoexplorespecializedmetabolisminmicrobialcommunitiesthat

inhabitrootnodulesoflegumeplants.Wehopetodeveloptherootnoduleasamodelsystemfor

studyinganecologicallyrelevantmicrobialcommunity.Thiswillincludemetabolomicandmetagenomic

characterizationoffavabeanrootnodules.

Mappingthecolonizationofasyntheticmicrobialcommunityinoculumfromsugarcanemicrobiomeinmaizeandsoybeanplants

NatáliaDamasceno([email protected])

CenterforMolecularBiologyandGeneticEngineering,UNICAMP,Campinas,Brazil,andDepartmentof

GeneticsandEvolution,UNICAMP,Campinas,Brazil.

Plantsliveinassociationwithahighlycomplexcommunityofbacteriaandfungigroups.Recent

advancesinmicrobialstudieshaveshownthathostgenotypesinfluencethediversity,structure,and

compositionofplantmicrobiomes.However,thegeneticandmolecularmechanismsinvolvedinplant-

microbecommunicationthatareresponsibleforestablishingthemicrobialcommunityinplanttissues

remainunknown.Unravelingthetraitsinvolvedinmicrobe-hostinteractionduringmicrobiome

assemblageisanimperativesteptowardsbuildingbiotechnologicaltoolstotransfermicrobesandtheir

beneficialfunctionstoeconomicallyrelevantplants.Inthiswork,wesoughttoexplorethegeneticand

molecularmechanismsinvolvedincross-speciesmicrobialcompatibilitybystudyingthecolonizationof

twoimportantcropplantsfromdistinctphysiologicalgroups,maizeandsoybean.Ourmainstrategy

involvestransferringabacterialcommunitytoanewC4plantandtoaC3planttoevaluateifthe

bacterialmembersofthiscommunityarecapableofcolonizingbothplantswithcontrasting

physiologicalprofiles.First,wedesignedasyntheticconsortiumofmicroorganismscomposedofthe

mostabundantbacterialgroupsfromsugarcane,aC4bioenergycrop.Secondly,weestablisheda

platformforinoculationassaysusingmaize,anotherC4crop,asaplantmodel.Maizeseedsare

germinatedandplantedinsterileconditionsandareinoculatedwiththesyntheticcommunity.Withthis

platform,wecanevaluatetheeffectofthepresenceofthebacterialcommunityinplantgrowthand

alsotestdifferentnutrientsolutionconcentrationsorstressconditions.Thepresenceofthesynthetic

communityimprovesplantgrowthevenunderlownutrientavailability.Wealsotestedwhetherthis


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communitywouldbeabletocolonizetheplanttissues.Byusingculture-independenttechniquesto

profileinoculatedandnon-inoculatedplants,weshowthatonlyapartofthesyntheticcommunity

robustlycolonizesthemaizeplants.Thisdataindicatesthatevenamongphylogeneticallycloseplants

theremightexisttraitsunderplayinghostcolonizationbyspecificmicrobialgroups.Additionally,plants

inoculatedwiththesyntheticmicrobialcommunityhadtheirtotalfreshanddrybiomassonaverageup

tothreetimesincreasedwhencomparedwithuninoculatedplants.Wesoughttoinvestigateifthe

syntheticcommunityisalsoabletocolonizesoybeanplantswithasimilarpatternandabeneficialplant

response.DualRNA-seqofplantandmicrobialcommunitywillrevealgenesandpathwaysinvolvedin

theestablishmentofthemicrobialcommunityanditsbeneficialfunctions.

Root-associatedmicrobiomeofaditerpene-deficientmaizemutant

KatherineMurphy([email protected])

DepartmentofPlantBiology,UniversityofCalifornia,Davis,CA,USA.

Plantsdeployspecializedmetabolitestocommunicatewithotherorganismsandcopewith

environmentalchallenges.Thisincludesinteractionswithmicrobialcommunities,inwhichplants

exchangesugarsforavailablenutrientsaswellasprotectionagainstenvironmentalstressors.However,

themolecularmechanismsbywhichaplantrecruitsitsparticularmicrobialcommunityandtheroleof

specializedmetabolitesinthiscommunicationarepoorlyunderstood.Here,wereportthatmaizeroot

diterpenes,agroupofspecializedmetaboliteswithversatilefunctionsinstressresilience,influence

rhizospherebacterialcommunities.Inadditiontothepreviouslydescribedkauralexinmetaboliteswith

keyrolesinmaizepathogenandenvironmentalstressresistance,werecentlyelucidatedanovelgroup

ofmaize-specializedditerpenes,termeddolabralexins,whichalsoshowactivityagainstbioticand

abioticstress.Distinctfromthegibberellinbiosynthesispathway,bothkauralexinsanddolabralexinsare

synthesizedviathecopalyldiphosphatesynthaseZmAn2beforebranchingintoseparatepathways.The

an2(antherear2)maizemutantisdeficientinformingbothkauralexinanddolabralexinmetabolites,

andexhibitsenhancedstresssusceptibility.Using16SrRNAsequencing,wedeterminedthebacterial

communitycompositionsofthean2mutantcomparedtoitswildtypesibling.Underwell-watered

conditions,distinctbacterialcommunitiesanddiversitieswereobservedbetweenmutantandwildtype

plants,whereasthemicrobiomecompositionsbecameindistinguishableunderdroughtconditions.

Thesefindingssuggestthatditerpenesplayanimportantroleinshapingtherhizospheremicrobiome,

whilealternatemechanismsmaybedominantunderdroughtstress.

Understandingtheroleofspecializedmetabolicnetworksmediatingbioticinteractionsinmaizethroughintegratedmultidisciplinaryapproaches

AlisaHuffaker([email protected])

UniversityofCalifornia,SanDiego,CA,USA.

Allplantsbiosynthesizecomplexblendsofspecializedmetabolitesthatenableandshapecommunity

interactionswithotherorganisms.Plantedonover140,000squaremilesofarableUSfarmlandand

geneticallytractable,maize(Zeamays)isanexcellentPoaceousmodelforstudyingeffectsofchemical

diversityonbioticinteractions.Focusingonrootandcrowntissuesofmaize,weusemetabolomic

approachestoidentifymicrobe-elicitedmetaboliteproduction.Torapidlyuncoverunderlyingregulatory

genesresponsible,classicalforwardgeneticsusingbiparentalmappingandgenome-wideassociation

studies(GWAS)readilyrevealmetabolite-basedquantitativetraitloci(mQTL)andultimatelyregulatory


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genes.InsightsfrommQTLapproachesinmaizeenabledtheidentificationofunexpectedcandidate

genesinvolvedinthebiosynthesisofdiversechemicalclasses.Invitrobiochemicalapproachescoupled

withgenesynthesisspeedstheconfirmationoftopcandidategenes.Toexamineentirepathway

deletionsinvivo,CRISPR/Cas9mutagenesisenablesthecreationofknockoutsinhistoricallychallenging

duplicatedgenes.Asanexample,usingCRISPR/Cas9wecreatedframe-shiftmutationsinterpene

synthases(Tps)6/11toremovethedominantfungal-elicitedsesquiterpeneacidsinmaize.Zmtps6/11

mutantsdisplaysignificantsusceptibilitytoattackbybothfungi(Fusariumgraminearum)andbacteria

(Pantoeastewartii).Thedevelopmentofmorecomprehensivebiosyntheticpathwaymutantlibrariesin

maizewillenablesystematicanalysesofmetaboliteeffectsonbioticinteractionsinfieldenvironments.

Dissectionofmetabolicandphenotypictraitsinmajorcrops

JieLuo([email protected])

HuazhongAgriculteralUniversity,Wuhan,China.

Applicationofanewlydevelopedwidely-targetedmetabolomicsstrategysimultaneouslydetected

hundredsofbothprimaryandsecondarymetabolitesinriceanddisclosedanumberofsubspecies-

specificmetabolitesthatmayreflect,aswellasaffect,thesubspeciesdifferentiationofrice.Distinctand

overlappedaccumulationwasobservedandcomplexgeneticregulationofmetabolismwasrevealedin

twodifferenttissuesbysubsequentmetabolicQTL(mQTL)mappingandmetabolicgenome-wide

associationstudy(mGWAS).Hundredsoflociwithhighresolutionandlargeeffectswereuncovered.

Interactivegene/metaboliteidentification/annotationwasfacilitatedforbothfunctionalgenomicsand

metabolomics.Dataminingrevealedalargenumberofcandidategenesunderlyingmetabolitesthatare

ofphysiologicalandagronomicalimportance,andalsocanbeappliedtothebulkidentificationoftailing

enzymescontributingmosttometabolicdiversity.Similarapproacheswerealsoappliedtothe

understandingofthemaizekernelmetabolome.Furthermore,comparativemGWASbetweenriceand

maizeresultedingreatlyincreasedpowerandresolutioninbothspecies.Inaddition,parallelmetabolic

andphenotypicGWASidentifiednewcandidategenesresponsibleforbothmetabolicandmorphologic

traitssuchasgrainwidthandleafsenescence,revealingdirectlinkagebetweenthemetabotypeandthe

phenotype.Ourstudynotonlyrevealsnovelbiochemicalandgeneticinsightsofimportantaspectsof

plantandhumansuchasdevelopment,stressresistance,andnutrition/health-promoting,butalso

providesavastamountofhigh-qualitydataforfurtherunderstandingplantmetabolomesandwhich

mayhelpbridgethegapbetweenthegenomeandphenome.Thestrategyisapowerfultoolforlarge-

scalegeneidentification,pathwayelucidation,andknowledge-basedcropgeneticimprovement.

Metabolomicsactivityscreening

GarySiuzdak([email protected])

TheScrippsResearchInstitute,La Jolla, CA, USA.

Systems-wideanalysishasbeendesignedandimplementedintoourcloud-basedmetabolomicplatform

(XCMSOnline.scripps.edu)toguidelarge-scalemulti-omicexperiments.Thisautonomousapproach

superimposesmetabolomicdatadirectlyontometabolicpathways.Thesedataarethenintegratedwith

transcriptomicandproteomicdata.Todate,theutilityofthisplatformhasbeendemonstratedin

thousandsofstudies.Theapproachfacilitatesbiomarkerdiscovery,mechanisticdataanalysis,and

metabolomicsactivityscreening(MAS).


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Decipheringtheroleoffungalsecondarymetaboliteswithinanaerobicmicrobialcommunities

CandiceSwift([email protected])

UniversityofCalifornia,SantaBarbara,CA,USA.

Anaerobicfungithriveincompetitivemicrobialenvironmentssuchasthedigestivetractsofmanylarge

herbivoresdespitebeingvastlyoutnumberedbyothermicroorganisms.Inadditiontosecreting

powerfulbiomass-degradingenzymes,theseuniquenon-modelorganismsalsopossessaricharrayof

biosyntheticmachineryforproducingsecondarymetabolites,whosefunctionsremaintobe

determined.Wehypothesizethatsecondarymetabolitesfromgutfungiplayimportantrolesin

anaerobiccommunitiesviamicrobialdefense,communication,orstresstolerance.Here,wehave

combinedcomplementaryapproachesingenomics,transcriptomics,massspectrometry,andNMRto

probethefunctionsofsecondarymetabolitesinthecomplexanaerobicdigestiveenvironmentof

herbivores.First,weuncoveredaplethoraofgeneclustersencodingbiosyntheticenzymesfor

secondarymetabolitesfromdiversechemicalclassesbyminingthegenomesandtranscriptomesof

threeanaerobicfungifromtheprimitivephylumNeocallimastogomycota.Keysecondarymetabolite

clustersincludecanonicalpolyketidesynthases(PKS)andnonribosomalpeptidesynthetases(NRPS).

Severaloftheclustersareexpressedunderstandardlaboratorygrowthconditions,whereasothersare

silent.RNAsequencingallowsustotestenvironmentalconditionsthatactivatethebiosyntheticgene

clustersthathavebeenidentifiedinthefungalgenomes.Theenvironmentalconditionswearetesting

includeheatshock,oxidativestress,pH,andnutrientavailability.Inadditiontoabioticfactors,weare

alsoco-cultivatingtheanaerobicfungiwithbacteriaandarchaeatoscreeninteractionsthatmay

enhancetranscriptionoftheclusters.Finally,weareworkingwithJGIandEMSLtoemployanalytical

techniqueslikemassspectrometryandNMRthatallowustodetectanddeterminethestructuresofkey

secondarymetabolites.Todate,wehavedetected~100likelysecondarymetabolitesandputatively

identifiedanantioxidantpolyketide,baumin,whichisalsoproducedbyadistantlyrelatedfungusfrom

thephylumBasidiomycota.Massspectrometryfacilitateshighthroughput,rapidscreeningof

metaboliteswithinacomplexmixtureandyieldsachemicalfingerprintofeachmoleculethatcanbe

comparedwithreferencedatabasesfordereplicationwithknownnaturalproducts,whileNMRpermits

detailedstructuralcharacterizationofisolatedcompounds.Thesecondarymetabolismofanaerobic

fungirepresentsacompletelyuntappedreservoirofbiosyntheticpotential,whichcouldbedrawnupon

fornoveltherapeutics,newchemicalbuildingblocks,andenzymesforbioengineeringnaturalproducts.

Ourintegratedapproachallowsustostudysecondarymetabolismatalllevels,fromDNAtoRNAto

metabolites,thusmaximizingdiscoveryofnovelmetabolitesandunmaskingtheirnativefunctions.

Metabolomics-guidedisolationofsignificantbiologicalplant,soil,andmicrobialsecondarymetabolites

HeinoHeyman([email protected])

EarthandBiologicalSciencesDirectorate,PacificNorthwestNationalLaboratory,Richland,WA,USA.

Oneofthegrandchallengesfacingthemetabolomicscommunityistheidentificationofunknown

metabolites,withunknownsecondarymetabolitescontributingthemosttothischallenge.Thelarge

chemicalheterogeneityofthemetabolomehasmadeidentificationandannotationofbiologically

significantmetabolitesoneofthemostimportantbottlenecksinuntargetedmetabolomicsanalyses.

Themassspectrometry-basedapproachesusedformetabolomicsandlipidomicsanalysesarecapableof


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detectingthousandsofmoleculesinasinglerun,butcomprehensiveannotationoftheassociated

metabolitesislimitedtospectralreferencelibrarymatchingand/ordirectcomparisontoanalytical

standards.Currenttandemmassspectrallibrariesaresmallcomparedtothelargenumberof

metabolitesfoundinthebiosphere.Incaseswheremetabolitesofinterestcanbematchedtoreference

compounds,MScangiveunequivocalidentification,butforunambiguousidentificationofpartiallyor

completelyunknownmolecules,NMRspectroscopyisindispensable.Inthediverseapplications

environmentwithinwhichtheEnvironmentalMolecularSciencesLaboratoryoperates,withamultitude

ofdifferentorganismsandbiologicalsystemsunderinvestigationviaUserProjects,thisbottleneckisa

commonproblemandthusleavesasignificantpartoftheinterpretationofthemetabolomicsresults

ambiguous.BymakinguseofcomplementaryspectraldatafrombothMSandNMR,wearereducingthe

timeforstructuralelucidationofmetabolitesbyincorporatingcandidaterejectionandsubstructural

conformation.InthistalkIwilldescribethecapabilitiesofanewpipeline,MetaboliteIdentificationand

CharacterizationPipeline(MICP),�currentlybeingdevelopedatEMSLtoboosttheidentificationof

unknownmetabolitesandIwillpresentselectedapplicationstofungalandsoilstudieswherewe

developedaspecificdirectedfractionationandisolationroadmapwhichwasusedtopurify,isolate,

identify,andcharacterizethefungalandsoilunknownmetabolitesthatareofbiologicalimportance.

Thenewlyidentifiedmetaboliteswillbeaddedtoourever-growingPacificNorthwestNational

Laboratorymetabolitedatabase,whichservesasarepositoryforfuturevalidatedmetabolomicsdata.

ThenovelmetabolitesidentifiedbyourMICPwillbeusedtoimproveourunderstandingoftheroles

thatbioticandabiotictransformationshaveonplantsandsoilmicroorganismswithenvironmental

changes.

Harnessingnaturetohelpfarmerssustainablyfeedtheplanet

VictoriaKnight-Connoni([email protected])

IndigoAgriculture,Charlestown,MA,USA.

Plantsrelyonavastarrayofnatural,beneficialmicrobestosupporttheirhealthandproductivity.The

communityofmicrobeslivinginandaroundaplant—itsmicrobiome—worksinharmonywiththeplant

toprovidelife-sustainingbenefitsthroughouttheplant’slifecycle.Themicrobiomehelpstheplant

absorbnutrientsinthesoilandbolstersitsresiliencetoenvironmentalstresses,inmanycasesmediated

bythesecondarymetabolitesindividualmicrobesproduce.Microbeshaveevolvedinconjunctionwith

plantsovermillionsofyears,inmanycasestooptimizetheirhealthandmaximizetheirproductivity.

Developmentofmicrobialproductstoachievelong-termagriculturalsustainabilityisagrowingindustry.

IndigoAgriculture,Inc.(www.indigoag.com)isfocusedonproductsthatusemicrobialendophytes,and

hasdevelopedalargeandsophisticatedpipelinetodiscovermicrobesthatenhanceplantperformance

infieldtrialsandfarmerfields.Indigoreleaseditsfirstproductin2016,IndigoCotton™,througha

collaborationwithTexasA&MledbyDr.GregSword.IndigoCotton™demonstratedanaverageyield

improvementof11%intargetfields,demonstratingtheeffectivenessofourdiscoveryanddevelopment

model.Toensureproductsafetyandefficacy,wehavefolloweddifferentmetabolitesproducedbythe

productmicrobesaswellastheplantsandseedsgeneratedbytheintroductionofthesemicrobes.


13

Miningtheplantmicrobiomefornovelagriculturalpestcontrolsolutions

JillPaulik([email protected])

AgBiome,ResearchTrianglePark,NC,USA.

AtAgBiome,wearefocusedondeliveringinnovativesolutionsforsomeofthegreatestchallengesin

agricultureandcropprotection.Wedothisbyexploringandscreeningourlargeanddiversemicrobial

collectionviaaproprietaryGENESIS™technologyplatform.Withinthisplatform,~40,000individual

microbialisolateshavebeencompletelysequencedatthewholegenomelevelandarecontinuously

screenedforrelevantgenesequencesandbiologicalactivityagainstmajorinsectandnematodepests,

plantdiseases,andweeds.Leveragingtheknowledgegainedbycouplingempiricalscreeningvia

validatedscreenswithgenomecomparisonsfacilitatesthediscoveryofthemosteffectivestrainsfor

productdevelopment.Ourfirstproductisbeinglaunchedthisyear.Itisauniquebiologicalfungicide

thatishighlyeffectiveagainstarangeofsoil-borneandfoliarfungaldiseases.

Strategiestodesigncustommicrobesformultiplemarketsusingafoundrymodel

JohanKers([email protected])

GinkgoBioworks,Boston,MA,USA.

Thereisanemergingdemandforsourcingnaturalproductsusingengineeredmicrobes.Whilerecent

advancesinsyntheticbiologyandmetabolicengineeringprovidefeasibleapproachestoengineering

suchorganisms,commercialsuccessfordevelopingthese“cultured”ingredientspresentsspecific

challenges.Unlikebiofuels,whereeffortscanbefocusedononeparticularmoleculegiventhe

enormousmarketsize,culturedingredientsrequiredevelopingdifferentorganismlinesinarapidand

low-costfashion.Thisrequiresascalablesolutionforbiomanufacturingoforganisms,whichisprovided

byourstate-of-the-artfoundrythatcontinuestoinnovateandgrow.Iwilldescribehoworganism

developmentatGinkgoleveragesourfoundrytoacceleratethedesign/build/testusingspecific

examples.Inparticular,Iwillhighlightthevalueofcombiningcomputer-aidedengineeringsoftware

tools,lowcostgenesynthesis,andhighresolution-accuratemassLCMStodevelopengineered

microbes.

OptimizingconditionsthatinduceorrepresstoxinproductionusingPhenotypeMicroArrays

BarryBochner([email protected])

Biolog,Hayward,CA,USA.

PhenotypeMicroArray(PM)technologyallowsabiologisttosimultaneouslyculturemicrobialcellsin

nearlytwothousandmicroscalecultureconditions.Itcanthereforebeusedtoquicklyandefficiently

discovercultureconditionsthatinduceorsuppressproductionofsecondarymetabolitesincluding

toxins.Oneapproach,takenbyGardiner,Kazan,andMannerstodiscoverinducersoftrichothecene

toxinbyFusariumgraminearum,wastouseaGFP-toxingeneconstructandlookforwellswithgreen

fluorescence.Asecondapproach,takenbySinghtodiscoverinducersofanti-fungalsecondary

metabolites,employedscaled-downLCMSanalysisoftheculturefluidfromthemicroplatewells.Our

approachwastodevelopageneralcolorimetriccell-basedtoxicityassaythatcouldbeusedtoscreenfor

conditionsthateitherinduceorsuppressproductionoftoxinsorothertoxicsecondarymetabolites.We

firstdemonstratedtheapproachbymeasuringtheeffectofculturesupernatantfluidsfromClostridium


14

difficileonCHOcells.WethenwentontodemonstratethatthisapproachusingCHOcellsorother

sensitivecellscanworkforawiderangeoftoxin-producingbacteria.

Plantgrowthregulatorsandplantgrowth-promotingmicrobesforcropenhancement

MarciSurpin([email protected])

ValentBiosciences,WalnutCreek,CA,USA.

ValentBioSciences,LLC,develops,manufactures,andmarketsbiorationalproductsgloballythathelp

addressgrowerneedsinasustainablemanner,safeguardcropspostharvest,providevectorcontrol

solutionsthatprotecthumanhealth,maintaingreenforests,anddelivermeasurablevaluetoour

customersandstakeholders.Biorationalproductsaretypicallyderivedfromnaturalorbiologicalorigins

andincludebiologicalpesticidesaswellasproductsforcropstressmanagement,enhancedplant

physiologybenefits,androotgrowthmanagement.Theyarecharacterizedasbeinghighlyspecificin

theiractivitywhiledeliveringdistincteconomic,health,and/orenvironmentalbenefits.Thisis

accomplishedviatheBiorationalCropEnhancement,BiorationalCropProtection,andBiorational

Rhizosphereplatforms.Thistalkwillgiveabroadoverviewofourdiscoverypipelinewithanemphasis

ontherolesofsecondarymetabolitesintheCropEnhancementandRhizosphereplatforms.

Omicsapproachestodeorphanizefungalnaturalproducts

NancyKeller([email protected])

UniversityofWisconsin,Madison,WI,USA.

Filamentousfungiarerenownfortheproductionofadiversearrayofsecondarymetabolites(SMs).

Thesenaturalproductsarevaluedfortheirbioactivepropertiesstemmingfromtheirfunctionsin

microbialbiology,includingprotectionfromabioticandbioticstressandestablishmentofasecure

niche.Thistalkwillpresentmethodswehavetakentoaddressthechallengesinactivatingand

connectingchemicalproduct(s)toSMbiosyntheticgeneclusters,includingcreationof“fungalartificial

chromosome”libraries,creationofinducibleheterologousclusters,andtranscriptomics/metabolomics

assessmentofinter-microbialwars.

Membranetraffickingatthehost-pathogeninterface

SophienKamoun([email protected])

TheSainsburyLaboratory,NorwichResearchPark,Norwich,UK.

Manyplantpathogenicandsymbioticmicrobesproducespecializedstructuresthatinvadehostcellsbut

remainenvelopedbyhost-derivedmembranes.Themechanismsunderlyingthebiogenesisand

functionsofsuchhost–microbeinterfacesarepoorlyunderstood,buttheseinterfacesarethoughtto

mediatemetaboliteandmacromoleculetraffickingenablinginter-organismalcommunication.TheIrish

potatofaminepathogenPhytophthorainfestansisanoomyceteeukaryoticmicrobethatinfects

solanaceousplants.P.infestansformshaustoria,whicharehyphalextensionsthatinvaginatethehost

cellmembrane.Ahost-derivedmembrane,calledtheextrahaustorialmembrane(EHM),separates

haustoriafromtheplantcellandconstitutesthehaustorialinterface.SomeP.infestansstrainsinfectthemodelplantNicotianabenthamianaanddevelophaustoriaininfectedleafcells.Wehaveexploitedthe


15

N.benthamianaexperimentalsystemtoperformfast-forwardcellbiologyofthehaustorialinterface.

Thisrevealeddynamicchangesinhostmembranecompartmentformationandreroutingin

haustoriatedplantcells.Inparticular,wediscoveredthatselectiveautophagyandothertrafficking

pathwaysaredivertedtothepathogeninterface.OurworkingmodelisthatP.infestans-secretedproteins(effectors)co-opthostmembranetraffickingtopromoteplantcolonization.

Comparativephylogenomicsidentifiesfungalsecondarymetabolitesalliedwithvirulencetoplanthosts

GillianTurgeon([email protected])

CornellUniversity,Ithaca,NY,USA.

Filamentousfungiproducechemicallydiversesecondarymetabolites(SMs)thathavepositiveand

negativeeffectsonotherorganismsandareimplicatedinvirulencetohostsanddevelopmentof

structuresassociatedwithreproduction.SMsalsocombatnutritionalandenvironmentalstressesin

nichesituations,makingthemcentraltofungalsurvivalandproliferation.SMsareattheveryheartof

theinformationnetworksthatplayoutwithinasingleorganism,andbetweencommunitiesof

interactingorganisms.Sequencingofthegenomesofhighlyaggressiveplantpathogensinthegenus

CochliobolusandinthecloselyrelatedgenusSetosphaeriahasprovidedaclearerpictureoftheplethoraofSMgeneclustersencodingunknownSMs.Ourlabemploysacombinationofgeneticandcomparative

phylogenomicmethodstoexplorefunctionofSMstomakeinitialfunctionalpredictions(guiltby

association).ComparativeanalyseshaverevealedthatthesuitesofSM-encodinggenesfromallCochliobolusspeciesareastoundinglydiversebetweenspeciesbutremarkablyconservedamong

isolatesofthesamespecies,exceptforlifestyle-definingexamplesthatgenerallymaptounique

genomicregions.Thispatternisalsofoundwhencomparisonsaremadebetweenthecloselyrelated

CochliobolusandSetosphaeriagenera.Functionalanalysisofseveralofthesestrain-uniqueSMsreveals

astrongcorrelationwitharoleinvirulencefornecrotrophsand,surprisingly,alsoforhemibiotrophs.

Discoveryandengineeringofnovelnaturalproductsviasyntheticbiology

HuiminZhao([email protected])

UniversityofIllinoisatUrbana-Champaign,Champaign,IL,USA.

Microorganismsareamajorsourceofnewtherapeuticagents.Mygrouphasbeendevelopingnew

genomics-driven,syntheticbiology-enabledstrategiestodiscoverandproducenovelnaturalproducts

fromsequencedgenomesandmetagenomes.Onestrategyistorefactortargetcrypticgeneclustersin

heterologoushosts.Asproofofconcept,weusedthisstrategytoawakenthesilentpolyketide

spectinabilinpathwayfromStreptomycesspectabilisinStreptomyceslividansandactivateacrypticpathwaycontainingapolyketidesynthase–non-ribosomalpeptidesynthetasefromStreptomycesgrieseusinStreptomyceslividans,whichledtothediscoveryoftwonoveltetramicacidnaturalproducts

thathaveneverbeenreportedinliterature.Toincreasethethroughput,weareestablishingafully

integratedroboticsystemtoautomateallthestepsingeneclusterrefactoringandproductdetection.

Anotherstrategyistoactivatethetargetcrypticgeneclustersintheirnativehostsbyknocking-instrong

promotersupstreamofthetargetcrypticgeneclustersusingaCRISPR/Cas9system.Wesuccessfully

activatedmorethan10crypticgeneclustersfromfivedifferentStreptomycesanduncoveredanumber

ofnovelnaturalproducts.


16

Harnessingthechemistryandbiologyofplantmetabolism

SarahO’Connor([email protected])

JohnInnesCentre,Norwich,UK.

Plants,whichmakethousandsofcomplexnaturalproducts,areoutstandingchemists.Throughthe

concertedactionofenzymesthatareassembledintometabolicpathways,naturecreateschemical

complexityfromsimplestartingmaterials.Inthistalk,Iwillhighlightsomeoftheunusualenzymatic

transformationsthatplantsusetomakecomplex,bioactivenaturalproducts,andwillalsodiscuss

methodsbywhichthesepathwayscanbeharnessedformetabolicengineering.Thefocusisonthe

biosynthesisofthemonoterpenescallediridoids,andthealkaloidsderivedfromiridoids,knownasthe

monoterpeneindolealkaloids.Thediscovery,functionalcharacterization,andmechanisticstudyof

enzymesinvolvedinthebiosynthesisoftheseimportantcompoundsinseveralmedicinalplantspecies

willbediscussed.

Metagenomicandsyntheticbiologyapproachesfornaturalproductdiscovery

DavidMead([email protected])

VarigenBiosciences,Madison,WI,USA.

Soilmicroorganismscontainvastreservoirsofbioactivenaturalproducts;however,themajorityofthem

arerecalcitranttocultivationinthelab.Inthisstudyalarge-insertsoilmetagenomicclonelibrary

(~110kband19,200clones)wasconstructedfromanagriculturalsoil(CullarsRotation,Auburn,AL)

usingabroadhostrangeshuttleBACvector,pSmartBAC-S.Thisinsertsizeiscapableofharboringmany

intactsecondarymetabolitebiosyntheticpathwayssuchasTypeIPKSpathwaysthatareusually>40kb.

Identificationofbiosyntheticgeneclusterswasconductedusingmultiplemethods,includingDNA

hybridization,PCR,andnext-generationsequencing.Inthefirsttwomethodswetargetedaconserved

domainofTypeIpolyketidesynthases(PKS)andidentifiedclonesbymacroarrayhybridizationorPCR,

resultingin12and110pathway-containingclones,respectively.Inaddition,weusedastrategyinwhich

plates,rows,andcolumnswereseparatelypooled,andbar-codedDNAsequencesfromeachpoolwere

subjectedtoIlluminaHiSeqsequencing.Contigswereassembledfromeachpoolandscreenedfor

secondarymetabolitegeneclustersusingantiSMASH3.0.Weidentified593clonesthatcontainedaPKS

and/ornon-ribosomalpeptidesynthetasepathwayamong1,516totalbiosyntheticpathwaysidentified.

Theclonedpathwaysareverydivergentfromknownpathways,withthe%G+Ccontentvaryingfrom

34%to79%andthenearestBLASThitofketo-synthasedomainsrangingfrom19%to95%aminoacid

identity.BiosyntheticclustersidentifiedviaPCRwereasubsetoftheclonesidentifiedvianext-gen

sequencing,whichwerebothnumericallymoreabundantandrepresentativeofnovelpathwayshighly

divergentfromknownpathways.139identifiedpathway-containingBACcloneswithlimitedhomology

toknownPKSpathwaysweretransformedintoStreptomycescoelicolorM1154andscreenedforthe

synthesisofantibacterialcompoundsbyvariousbioassaysagainstbacterialandfungalhuman

pathogensincludingmethicillin-resistantStaphylococcusaureus(MRSA)andCandidaalbicans.ClonesexpressingantimicrobialactivitywerefurthercharacterizedbyLC/MSanalysis.Theseresultsindicate

thathighlynovelbiosyntheticclusterscanbeclonedintactfromcomplexmetagenomesand

heterologouslyexpressedtoproducesecondarymetabolites,therebyexpandingouravailableresources

ornaturalproductdiscovery.Improvedmethodsforcapturing,cloning,andoverexpressingnatural

productpathwaysarebeingdevelopedtofurtheracceleratethediscoveryofnovelbeneficialmolecules.


17

Genomeminingfornewherbicides

YiTang([email protected])

UniversityofCalifornia,LosAngeles,CA,USA.

Duetorapidemergenceofweedsthatareresistanttocommercialherbicidessuchasglyphosateand

glufosinate,theneedfornewherbicideswithanovelmodeofactionismoreurgentthanever.Herewe

describerecentdiscoveriesinourlabs(TangandJacobsen).Onediscoveryisanaturalproductthat

inhibitsdihydroxyaciddehydrogenase(DHAD),along-sought-aftertargetforherbicidedevelopment.

Thecompoundwasminedfromsequencedfungalgenomesusingatarget-basedapproach.We

developedascalableplatformforproducingthecompoundinyeastandshowedthatitcanpotently

inhibitplantDHAD(Ki~300nM)andcaneffectivelyshutdownplantgrowthinplanta.Wealsofounda

resistanceenzymethatcanbeusedasatransgeneforgeneratingtransgeniccropplants.

Harnessingsyntheticbiologyfortheproductionoffineandspecialtychemicals

ErikoTakano([email protected])

TheUniversityofManchester,Manchester,England.

Manymicrobialgenomesencodethemachinerytoproducediversebioactivemoleculesthatcanbe

usedinhealthcare,agriculture,andfood.Itsnaturalmodularitymakesthismachineryaparticularly

attractivetargetforsyntheticbiology.There-engineeringofthebiosyntheticcapacityofmicrobes

requiresthedevelopmentofawiderangeofexperimentalandcomputationaltools.Theseinclude

orthogonaltranscriptionalcontrolcircuitsandbacterialmicrocompartments,computationaltoolsfor

thedetectionandanalysisofsecondarymetabolitebiosynthesisgeneclustersthatenrichourlibraryof

partsandbuildingblocksforpathwayengineering,andhigh-resolutionmassspectrometryanalysisfor

thedebuggingoftheengineeredsystems.

InthistalkIwillexplorethepossibilitiescreatedbytheapplicationofthedesign/build/test/learncycle

ofsyntheticbiologytotheengineeringofmicrobialmetabolismfortheproductionofhigh-value

chemicals,asimplementedinthehigh-throughputplatformoftheBBSRC/EPSRC-fundedManchester

SyntheticBiologyResearchCentre,SYNBIOCHEM.Iwillalsodiscussthegrowingtoolboxoftechniques

andapproaches,illustratedwithconcreteapplicationcasestudies.

RedesigningthegeneticsofsecondarymetabolisminStreptomyces

MichaelSmanski([email protected])

UniversityofMinnesota,Minneapolis,MN,USA. GeneticmanipulationofnaturalproductgeneclustersinStreptomycesiscomplicatedbytheirlargesize,

complexorganization,andarelativelylimitedtoolboxforgeneticengineering.Weareleveragingrecent

advancesinDNAsynthesisandassemblytechniquestore-buildnaturalproductgeneclustersfroma

collectionofcharacterizedgeneticelements.WedemonstratetheapplicationsofourDNAassembly

pipelinetoward(1)generatingchemicaldiversityand(2)improvingyieldusingadesignedbiosynthetic

pathwayfortheneuroprotectivenaturalproductserofendicacid.


18

DiscoveryofnovelsignalingsecondarymetabolitesfromClostridium

WenjunZhang([email protected])

LawrenceBerkeleyNationalLaboratory,Berkeley,CA,USA.

Medicinallyactivenaturalproductshavefunctionalgrouparraysandscaffoldarchitecturesthatoffer

advancedplatformsforthedevelopmentofsuccessfulnewdrugs.Thestudyofbiosyntheticroutesto

naturalproductswillfacilitatetheproductionoftargetmoleculesincludingcommercialdrugsandkey

startingmaterialsforchemicalderivatizationandsemisynthesis.Ourlabisinterestedinstudyingthe

biosynthesisofvariouspharmaceuticallyimportantnaturalproducts,includingbutnotlimitedto

antimycin-typedepsipeptides,themanumycinfamily,andpyrroloindolealkaloids.Wearealso

developinggeneralplatformsofinsitunaturalproductslabelingforvariousapplications.Small-molecule

secondarymetabolites(SSMs)areoftenemployedbymicrobestoaccessinformationaboutboththeir

intracellularphysiologicalstatusandtheirextracellularenvironment,andtheyareoftencriticalin

controllingcomplexprocessesincludingmorphologicaldifferentiation,multicellularity,biofilm

formation,secondarymetaboliteproduction,andvirulence.Inordertoblockthebacterial

communicationleadingtopathogenicity,itisimportanttounveiltheidentityandfunctionofthehidden

SSMsinpathogenicbacteria.WeareinterestedinunveilinghiddenSSMsfrommycobacteria,followed

bymodeofactionstudies.ThecharacterizationofenzymaticmachineryforthebiosynthesisofSSMs

andtheirfunctionalnetworkwillpromotethedevelopmentofnewanti-bacterialtreatments.

Additionally,wearealsointerestedinuncoveringtheidentityandfunctionofhiddensignalingSSMsin

Clostridia,withthegoalofimprovingABEfermentationperformance.Bacterialvolatilesrepresenta

sourcefornewbiofuelcompoundsinadditiontothetraditionalbioethanolandplant-oil-derived

biodiesel.Therelevantvolatilecompoundsthathavebeenidentifiedincludevariousshort-tomedium-

chainalkanes,alkenes,alcohols,andisoprenoids,whichhavegreatpotentialtoreplaceorsupplement

petroleum-derivedchemicalsandfuels.However,littleisknownabouttheenzymaticlogicforthe

biosynthesisofmanyofthevolatileorganiccompounds(VOCs)producedbyvariousbacterialcultures.

Inordertodevelopmoresustainableandeconomicallyfeasiblebiochemicalsandbiofuels,itis

importanttofullycharacterizetheenzymaticmechanismsofbiosynthesisandsecretionofthese

hydrocarbonsaswellastoengineertheirheterologousproductionwithincreasedyieldandefficiencyin

preferredhosts.

Interkingdomsignaling:aPopuluscasestudy

GeraldTuskan([email protected])

OakRidgeNationalLaboratory,OakRidge,TN,USA.

Experimentalevidencepointingtointer-kingdomsignalingbetweenPopulusanditsendophyticcommunitywillbepresentedspecificallyrelatedtoLaccariacolonization,smallsecretedprotein

signaling,andquorumsensing.ThesedatainitiallyemergedfromtheJGI-based,sequenced,assembled

andannotatedPopulusgenome,whereover35archaeal,bacterial,andfungalgenomesweredetected

andassembled.Sincethenover500bacterialendophyticand35fungalassociateshavebeen

sequenced,assembled,andannotatedfromPopulus.Asaresultoftheseefforts,Populuslectinreceptor-likekinase(RLK)hasbeenidentifiedthatcontrolLaccariacolonization.ThePopulustransgeneshavebeentransformedintoArabidopsis,whichresultedintheformationofaHartig-net,thefirstreport

ofamycorrhizalassociationinArabidopsis.ThePtRLKgeneinducesmetabolicchangesinArabidopsisthatmimicfungalchallengebutinthepresenceofLaccariatheseresponsessubside.FromthePopulus


19

genomeassemblyover200smallsecretedproteinshavebeenidentifiedandcharacterized.Severalof

theseareactivelytakenupbyfungalassociatesandarethensubsequentlylocalizedtothenucleusby

Laccaria.ThepresenceofthePopulus-secretedproteinsintheLaccarianucleicauseschangesinLaccariahyphalbranching.FromthePopulus-basedendophyticbacterialcollection,severalgenera,i.e.,Rhizobium,Rahnella,AlbidiferaxandPseudomonas,werefoundtocontainquorum-sensinggenesthat

respondtoPopulusleafmacerate.Theplantsignalisactivelytransportedandismostlikelyadipeptide,

resemblingaD-Ala-D-Leudimer.ItappearsthatPopulushasmetabolicsignalsthatattractfavorable

bacteriaviaco-optionoftheirquorumsensingmachinery.Strategiesforleveragingthisinformation

indicatewemaybeabletointentionallyandspecificallymanagethePopulusmicrobiomeinan

environmentallyrelevantmanner.Inter-kingdomsignalingbetweenaplanthostanditsmicrobiome,

throughexchangeofmetabolitesandproteins,appearstobepervasive.

PosterPresentations

20

PosterPresentationsStreptomyces'dynamicroleintherootofArabidopsisthaliana

Sloan,SarahStuartC([email protected])1;Grant,DavidL

1;Massey,JacobC;Lebeis,SarahL

1

1TheUniversityofTennessee,Knoxville,TN,USA.

WhileconsistentpresenceofStreptomyceswithinrootsofanumberofplantspeciessuggestsspecific

selection,mechanismsthatfacilitateinternalrootcolonizationareuncharacterized.Hereweseekto

teaseapartplantfrommicrobe-derivedmechanisms,andmorespecificallyrevealStreptomyces’abilitytopositivelyandnegativelyinfluenceco-occurringmicrobes.Wehypothesizethatselectsoil

StreptomycesareinfluentialmembersoftheArabidopsisthalianarootcommunity,capableof

harnessingtheirmetabolicpotentialtojoinandsculpttherootmicrobiome.Toaddressthis,weexplore

thepotentialofselectStreptomycesisolatesasdriversofrootcommunityestablishment,employing

antimicrobialmetabolicstoselfishlytargetpotentialmicrobialcompetitors.Weutilizedthemodel

organismArabidopsisthalianatoinvestigateisolate-specificinfluencesonplantphenotype.Plant-Streptomycesexperimentsrevealisolate-dependentphenotypes,withalteredrootstructureandvarying

colonizationpotential.Microbe-microbeexperimentshaveshownStreptomyces’abilitytoinhibitgrowthofselectsoilmicrobes,includingseveralBacilliisolates.Preliminaryexperimentsexploringcolonization

ofseedlingsinoculatedwithStreptomycesandanaturalmixedcommunitydifferinbiomass,plant

growth,androotstructures.Sequencingofthe16Sgenewillfurtherelucidatecommunitycompatibility

andstructuraldynamics,providingevidenceofshiftsinmicrobialrelativeabundances.Obtainingfull

genomesequencesoftheseStreptomycesisolatesallowstargetedinvestigationofpotentialchemical

mechanismsdrivinginteractionsandcontinuetoinformexperimentdesignandinterpretationof

findings.Initialexperimentsinvestigatingtheroleofbacterially-derivedmelanins,phenazine,andindole

products,theirinfluenceonplantrootcolonization,andabilitytoaffectmicrobialcolonizersprovide

evidenceofdifferentialproduction,potentialmicrobialgrowthinfluence,andconsistentplantroot

interactionphenotypes.TwooffourStreptomycesisolatesuniquelycapableofproducingmelanin

consistentlyout-competeothermicrobesforcolonizationwithintheroot.Wearecurrentlyconducting

targetedexperimentstounderstandthepotentialroleofthismetaboliteincolonization.Additionally,

preliminaryinvestigationoftheindolederivativeauxinsuggestsauxin-independentrootphenotypes,

indicatingapotentialroleforisolate-specificnon-indolemetabolitesinrootgrowthpromotion.

Together,thepreliminaryandanticipatedfindingssuggestmicrobeandhostrelationshipdynamics

responsibleformicrobialcommunitymodification.Thesefindingsfurtherourcurrentunderstandingof

Streptomyces’activitiesintherootofArabidopsisthaliana.

Seedsofantagonism:AnABCtransporteranditsadjacenttranscriptionfactorinFusariumverticillioidesarerequiredforpyrrocidineBtolerance

Gao,MingluZ([email protected])1;Gu,Zi

2;Glenn,AnthonyE

3;Gold,ScottE

3

1DepartmentofPlantPathology,TheUniversityofGeorgia,Athens,GA,USA.

2InstituteofBioinformatics,

TheUniversityofGeorgia,Athens,GA,USA.3ToxicologyandMycotoxinResearchUnit,UnitedStates

DepartmentofAgriculture,AgriculturalResearchService,Athens,GA,USA.

Theprevalentseed-bornemaizeendophyte,Fusariumverticillioides,isthecausalagentofseverekernelrotdisease.Itsproductionoffumonisinmycotoxinisaworldwidefoodsafetyconcern,ascontaminated

PosterPresentations

21

cornisassociatedwithhumanandanimaltoxicosis.Anothermaizeendophyte,Sarocladiumzeae,cohabitstheecologicalnicheofmaizeseedswithF.verticillioides.Withinmaizekernels,F.verticillioidesisprimarilyconfinedtothepedicel,whileS.zeaeisobservedinembryos.Invitrocompetitionassays

haveindicatedS.zeaecaninhibitthegrowthofF.verticillioides.Alactam-containingantibioticproduced

byS.zeae,namedpyrrocidineB,isassociatedwiththeantagonism.LC-MSanalysisofliquidcultures

indicatedthatF.verticillioidescandefenditselfbydegradingpyrrocidineB,whentheconcentrationdoesn’tsignificantlyimpedeitsgrowth.Toexploretheantagonisticmechanismonthesideof

F.verticillioides,RNA-seqexperimentswereconductedbychallengingtheF.verticillioidesliquidculturewithpyrrocidineBatsubinhibitoryconcentrationstoinducetranscriptomicchanges.Tengeneswith

dramaticinductioninpyrrocidineBtreatmentwereselectedastargetstogenerategenedeletion

mutants.PhenotypicanalysesrevealedthatdeletionofanABCtransportergene(FVEG_11089)orits

adjacenttranscriptionfactor(FVEG_11090)elevatedsensitivityofF.verticillioidestopyrrocidineB.QuantitativePCRshowed13,530-foldinductionofABCtransportergeneinresponsetopyrrocidineB

exposure,andtheinductionwasdependentontheadjacenttranscriptionfactor.Hence,wetheorize

thatFVEG_11089functionsinpyrrocidineBresistancebyextrudingtheantibioticoutofthecell,and

FVEG_11090positivelyregulatesinductionofFVEG_11089toahighlevelinresponsetopyrrocidineB

exposure.Theexplorationoftheantifungalresistancemechanismaddressesthecompetitive

relationshipsofthetwomaizeseedendophytescolonizingthesameecologicalniche.

Exploringthesoybeanmicrobiomeusingcomplementaryspatialmetabolomicstechniques

Anderton,ChristopherR([email protected])1;Veličković,Dušan

1;StopkamSylwia

2;

Agtuca,Beverly3;Chu,Rosalie

1;Koppenaal,David

1;Vertes,Akos

2;Stacey,Gary

3;Paša-Tolić,Ljiljana

1

1EnvironmentalMolecularSciencesLaboratory,EarthandBiologicalSciencesDirectorate,Pacific

NorthwestNationalLaboratory,Richland,WA,USA.2DepartmentofChemistry,TheGeorgeWashington

University,Washington,DC,USA.3DivisionsofPlantSciencesandBiochemistry,C.S.BondLifeSciences

Center,UniversityofMissouri,Columbia,MO,USA.

Inanefforttoattainmoresustainableagriculturalpractices,thereisagreatinterestinunderstanding

metabolicprocesseswithinplantsystemsknowntoacquirenitrogenthroughbiologicalnitrogen

fixation.Thesymbioticassociationbetweennitrogen-fixingsoilbacteria(Rhizobiaceae)andplantsofthefamilyLeguminosaeareonesuchsystemofinterest.Thissymbiosisgeneratesspecializedorgans,called

rootnodules,whererhizobiareduceN2intobioavailableproductsaccessibletothehostplant,andin

exchangetheplantprovidesacarbonsourcetothebacteriatoensure(amongotherthings)sufficient

energyfornitrogenfixation.However,littleisknownaboutthearrayofmetabolitesinvolved,andtheir

spatialdistribution,whichinfluencetherhizobia-legumeassociation.Sincethesebiologicalprocesses

areinherentlythree-dimensionalphenomena,wherelocalizedmetabolismcanexistwithindifferent

anatomicalcompartmentsoftherootnodule,wedescribetheuseofbothlaserablationelectrospray

ionization(LAESI)andmatrix-assistedlaserdesorption/ionization(MALDI)massspectrometry(MS)

methodstospatiallyobservethearrayofmetabolitesthatinfluencetherhizobia-legumeassociationof

Bradyrhizobiumjaponicumandsoybean(GlycinemaxWilliams82).Wewereabletoidentifyandmapa

numberofmoleculesinvolvedinbiologicalnitrogenfixation,suchashemeB,riboflavin,andadenine.

Wecouldalsovisualizethedistributionsofsecondarymetabolites(e.g.,flavonoids,saponins,and

glucosides)thatmodulatedthissymbiosis.TandemMS,pre-massanalysisionmobilityseparation,and

highmassresolutionandmassaccuracymeasurementsoftheisotopicenvelopepermittedustomove

beyondprovidingputativeidentificationsbasedonaccuratemeasuredmassalone,andprovidedhigher

confidenceintheidentityandlocalizationofmetabolites.Wefurtherappliedthisinformationto

PosterPresentations

22

elucidatetheactivemetabolicpathwayswithinthesoybeanrootnodule,whichaffordedabetterview

ofactualmetabolismthanobtainedfromproteomicsandtranscriptomicsalone.

Enzymatictransformationofthesiderophorepyochelinthroughimagingmassspectrometryofbacteria-fungiinteraction

Ying-Ning,Ho([email protected])1;Chi-Ting,Hsieh

1;Yu-Liang,Yang

1

1AgriculturalBiotechnologyResearchCenter,AcademiaSinica,Taiwan.

Manyspeciesofplant-associatedbacteriasecretenaturalproductsthatinhibitthedevelopmentor

growthofplantpathogens.Inturn,pathogensmaydevelopresistancetoantagonisticmolecules.

However,littleisknownaboutenzymatictransformationsofsecretedmetabolitesthatoccurduring

interactionsbetweenbacteriaandfungi.Burkholderiacenocepaciastrain869T2,anendophyticbacterium,showedapromisingantagonisticeffectagainstPhellinusnoxius,apathogenthatcausesbrownrootrotdisease.Tounderstandthefunctionalbacterial-fungalinteractioncomprehensively,itis

agoodstrategytomonitormetabolitesecretionandgeneexpressionprofilesofbothinteracting

organismssimultaneously.PyochelinisonesiderophoreofPseudomonasaeruginosaandBurkhoderiasp.thatisabletoinduceplantISR(inductionofsystemicresistance)andhasbeenidentifiedasan

antifungalantibiotic.ThroughIMSandRNA-seq,wefoundthatinsteadofinhibitingthegeneexpression

ofpyochelininstrain869T2,P.noxiuscouldmodifypyochelin(m/z325)tom/z383,whileP.noxiusfacedtoBurkholderiacenocepaciastrain869T2.Wethenisolatedpyochelinandpyrrolnitrin,an

antifungalcompoundfrom869T2,andfoundthattheinhibitedzonecausedbypyrrolnitrindecreased

overtime.However,thecombinationofpyochelinandpyrrolnitrinhasshownalongerinhibitoryeffect

againstP.noxius.ThisresultmightexplainwhyP.noxiustriedtomodifythestructureofpyochelin.We

alsofound869T2couldtransformm/z393tom/z409andinhibitsomemetabolitessecretedfromP.noxiusinresponse.Thoroughunderstandingofthisbacterial/fungalinteractionwouldfurtherthedevelopmentofbiologicalcontrolstrategies,andmayleadtothediscoveryofamethodtocontrol

brownrootrotdisease.

Metabolicengineeringaprobioticyeasttoincreaseastaxanthinproduction

Lin,Yu-JuLuLu([email protected])1,2;Chang,Jui-Jen

3;Lin,Hao-Yeh

1;Thia,Caroline

1;Kao,Yi-Ying

1;

Huang,Chieh-Chen2;Li Wen-Hsiung

1;2(correspondingauthor)

1BiodiversityResearchCenter,AcademiaSinica,Nankang,Taiwan.

2DepartmentofLifeSciences,

NationalChungHsingUniversity,Taiwan.3DepartmentofMedicalResearch,ChinaMedicalUniversity

Hospital,ChinaMedicalUniversity,Taiwan.

Inthisstudy,anastaxanthin-biosynthesisKluyveromycesmarxianusstrainSm23wasfirstconstructed,

whichcouldproduce31µg/gDCWastaxanthin.Then,genomeintegrationofthekeyastaxanthin-

biosynthesisgenesHpchybandbktwasdonetoincreasegenecopynumberandastaxanthinyield.Four

improvedstrainswereobtainedandtheyieldofastaxanthinandthetotalyieldofcarotenoidsinastrain

increasedwiththecopynumbersofHpchybandbkt.Toimprovetheyieldfurther,thegeneHpchyb

fromHaematococcuspluvialiswasmodifiedbysite-directedmutagenesistoincreasetheenzyme

efficiencyand/ortopreventtheheterologousproteindegradationbyubiquitination.Usingrepeated-

integrationapproachofbktandthemutatedHpchybintoSm23,theS3-2strainwasobtainedand

showntoproducethe3S,3’S-astaxanthinat9972µg/gDCWina5Lfermentor.

PosterPresentations

23

EffluxtransporterscontributetovirulenceandhostcompatibilityofPseudomonassyringaeB728a

Helmann,TylerC([email protected])1;Lindow,Steven

1

DepartmentofPlantandMicrobialBiology,UniversityofCalifornia,Berkeley,CA,USA.

PlantpathogenssuchasPseudomonassyringaeencounterdiverseplant-producedmetabolitesduring

colonizationofboththeapoplastandsurfacesofplants.Successfulinfectionorepiphyticcolonization

requirestolerancetosuchchemicalsbyeitherdetoxificationoractiveeffluxfromthecell.Pseudomonassyringaepv.syringaeB728aharborsapproximately120multidrugresistancetransporters,representing

severalproteinfamilies.Themajorityofthesegenesareuncharacterized.Sincedifferentplantspecies

producechemicallydiverseantimicrobialcompounds(phytoalexins),onemajorobjectiveistoaddress

theextenttowhichdifferenteffluxpumpsarerequiredindifferenthostplantsandtheextentof

redundancyinthistrait.MexB(ahomologofAcrBinE.coli)isamultidrugresistanceeffluxtransporter

intheResistance-Nodulation-Divisionproteinfamily.Ithaspreviouslybeenshowntocontributeto

tolerancetomanytoxicantsinvitro.Inthebeanapoplast,aMexBmutantgrowstoalowertotal

populationthanwildtype,butthisdifferenceisonlyvisibleafteratleastfourdays.Thisisapproximately

thetimerequiredforbeantoproducephytoalexinsinacompatibleinteraction,andthereforeMexBis

likelyamajorpumpresponsibleforsurvival.P.syringaeB728awasoriginallyisolatedfromcommon

bean(Phaseolusvulgaris),andisalsopathogeniconNicotianabenthamiana.Interestingly,itappearstobeabletogrowintheapoplastofmanyadditionalplanthosts,includingothermembersofthe

FabaceaeaswellashostsintheSolanaceaeandAsteraceae.WhilethemutantstrainlackingMexBis

lessvirulentthanwildtypeinbeanleaves,itgrowsaswellasthewildtypestraininseveralotherplant

species,suggestingthattheseadditionalhostplantsdonotproduceantimicrobialcompoundsthatare

removedbytheMexBtransporter.Abar-codedTnSeqapproachisbeingusedtodeterminethe

contributionofeachofthetransporterssingly,andincombinationwithMexB,tothefitnessofP.syringaeinavarietyofhostplants.InfectedleaftissueisbeinginterrogatedbyLC-MS/MStodetermine

theextenttowhichplantspeciesdifferintheircomplementoftoxicmetabolitesthatmustbeovercome

bypotentialpathogens,andthustheirneedforeffluxpumps.

Volatileterpenesecondarymetabolisminswitchgrassrootsinthebiotic/abioticenvironment

Tholl,DorotheaBC([email protected])1;Muchlinski,Andrew

1;Atwood,Claudia

1;Sawyer,Hannah

2;Pelot,

Kyle3;Zerbe,Philipp

3

1DepartmentofBiologicalSciences,VirginiaTech,Blacksburg,VA,USA.

2VirginiaStateUniversity,

Petersburg,VA,USA.3DepartmentofPlantBiology,UniversityofCalifornia,Davis,CA,USA.

Asaprairiegrass,switchgrass(Panicumvirgatum)producesanextensivefibrousrootsystem,whichis

exposedtoadynamicbioticandabioticbelowgroundenvironment.Switchgrassrootsarerichinvolatile

terpenoidsecondarymetabolites,particularlythemonoterpenoids(-)-borneolanditsderivative(-)-

camphor.Weinvestigatethemultiplefunctionsofthesecompoundsinbeneficialorantagonistic

interactionswithsoil-borneorganisms.Whileitisknownthatvolatileterpenoidsaffectcolonizationby

epiphyticbacteriainabovegroundtissues,theroleofthesecompoundsininteractionwithmicrobesin

therhizosphereandendosphereofrootsislargelyunknown.Wehypothesizethatroot-produced

terpenevolatilesfunctionashost-specificchemo-selectivefactorsthataffectrootmicrobialcommunity

compositionasgrowthinhibitorsorCsources.ToassessthespecificuseofterpenoidsasCsources,

culture-basedmethodsareappliedtoidentifyterpenoid-metabolizingmicrobesintheswitchgrass

PosterPresentations

24

endosphereandrhizosphere.Tofurtherdeterminetheeffectofvolatileterpenoidsontheroot

microbialcommunityinvivo,wearecharacterizingtheterpenesynthase(TPS)genefamilyof

switchgrass.WithintheTPS-bsubfamily,wehaveidentifiedPvTPS04asaleaf-androot-expressed

terpenesynthasethatforms(-)-borneolasamajorproduct.RNAi-basedapproachesareinprogressto

reducetheproductionofborneolandcamphoranddeterminemodificationsoftherootmicrobiome

associatedwithchangesintherootchemicalenvironment.Inanattempttoassesstheeffectofabiotic

(drought)stressonterpenoiddynamicsinswitchgrassroots,wefoundanunexpectedaccumulationof

sesquiterpenoidsandditerpenoidsindicatingchangesintherootchemicalenvironmentspecificto

droughtstressepisodes.Theroleofthesechemicalmodificationsinroot-microbialassociationsand

stressprotectionneedstobefurtheraddressed.

Aprotein-proteininteractionnetworkcenteredonc-di-GMPsignalingintheplantgrowthpromotingrhizobacteriaP.fluorescens

Noirot,Marie-FrancoiseS([email protected])1;Larsen,Peter

1;Malato,Grace

1;Wilton,Rose

1;Noirot,

Philippe1

1ArgonneNationalLaboratory,Lemont,IL,USA.

Plantgrowthpromoting(PGP)rhizobacteriaachievetheirbeneficialeffectsthroughdirectandindirect

interactionswithplantroots.WhilePGPactivitiesareextensivelydescribedataphysiologicallevel,the

underlyingmechanismsbywhichbacteriacolonizeplantroots,sensenutrientcomposition,andmake

compoundsavailabletotheplantremainpoorlycharacterized.Toaddressthisknowledgegap,we

decipheredakeyprotein-proteininteractionnetworkinvolvingafamilyofproteinsknowntoregulate

bacterialsensingandsignalingduringcolonizationofplantrootsbyPseudomonasfluorescens.

PseudomonadsareabundantsoilbacteriawithcharacterizedPGPeffects,includingtheproductionof

auxins(planthormones),siderophores(ironuptake)andsecondarymetabolitesfacilitatingphosphate

solubilization.AlthoughsuccessfulrootcolonizationbyfluorescentPseudomonadsisacriticalinitialstep

inPGP,othermechanismsarealsoinvolved.Inmanybacterialspecies,thechemicalmessengerc-di

GMPhasemergedasakeyplayerinthecontrolofmanycellularprocessesinvolvedindevelopmental

transitionandcellfate,productionofexopolysaccharidesandothermetabolites,andcontrolof

virulence.Inbacteria,c-di-GMPhomeostasisinvolvesthebalanceactivitiesofdiguanylatecyclases

(DGCs)andphosphodiesterases(PDEs).Theseenzymesarecharacterizedbythepresenceofcatalytic

domainsdisplayingsignaturemotifs(GGDEF/EAL/HP-GYP)oftenlinkedwithadditionaldomainsfor

signalinginputsandoutputs.Uponbindingc-di-GMP,theseproteinsexertaregulatoryactionat

transcriptional,post-transcriptionalorpost-transductionallevels.

Thisstudycombinesidentificationofproteincomplexesinvolvedinc-di-GMPregulatorypathwayswith

asystematicCRISPRi-basedfunctionalanalysis.Ahigh-qualitybinaryprotein-interactionmapcentered

onc-di-GMPsignalinginP.fluorescensSBW25wasbuiltfromveryhighconfidenceinteractions.

Targetedon10c-di-GMPbindingproteinsinvolvedinbacteria-rootassociations,ourprotein-protein

interactionnetworkiscomposedof94proteinsconnectedby130interactionsclusteredinconnected

functionalmodules,revealingconnectionsbetweenthec-di-GMPsignalingandvariouscellular

processes.Itexhibitedaremarkablestructuralorganizationthatrevealedfunctionalassociations

connectingc-di-GMPbindingproteinstoparticularmetabolicpathwaysandcellularmachineries

relevantwithplant-rootinteractions,suchascellsignaling,transcriptionalregulations,celladhesion,

andtransportofvariousnutrients.Thisnetworkisofhighbiologicalsignificanceandprovidedawealth

PosterPresentations

25

ofhypothesesforfurtherdecipheringoftherelationshipsbetweenc-di-GMPsignalingandplant

(Aspen)-rootcolonizationbyP.fluorescens.

Nanoscaleclusteringofenzymesinafungalsesquiterpenebiosyntheticpathway

Kistler,H.Corby([email protected])1;Boenisch,MarikeJ

1;Broz,Karen

1;Blum,Ailisa

2

1USDAARSCerealDiseaseLaboratory,St.Paul,MN,USA.

2UniversityofQueensland,St.Lucia,Brisbane,

Queensland,Australia.

Themevalonatepathwayleadstothesynthesisoffarnesyldiphosphate,whichservesasaprecursorfor

bothprimaryandsecondaryterpenoidmetabolites.Sincethesedifferentproductsdrawuponthesame

startingmaterials,howdocellsapportionthesupplyofsharedmolecularprecursorstoprimaryand

secondarymetabolicpathways?Cellularcompartmentalizationmayservetosequesterpathwaysandto

channelmetabolitesforparticularpurposes.Trichothecenesareconditionallyexpressedsesquiterpene

mycotoxinsproducedbythefungusFusariumgraminearum.Uponinductionoftrichothecenesynthesis,

enzymesofthetrichothecenepathwayaswellasupstreammevalonatepathwayenzymesco-localizeto

highlyremodeledendomembranestructurescalledorganizedsmoothendoplasmicreticulum(OSER).

Basedonsuper-resolutionfluorescencestructuredilluminationmicroscopy(SIM)andfluorescence

resonanceenergytransfer(FRET),itcanbeinferredthattheseenzymesformamulti-proteincomplex.

NanoscalelocalizationofproteinsdemonstratesthatintegralERmembraneproteinsfromboth

pathwaysarebroughttogetheralongwithcytoplasmicenzymescapturedwithinthethree-dimensional

cellulararchitectureofOSER.Toidentifyadditionalproteinsassociatedwiththesestructures,

fluorescence-activatedcellsorting(FACS)hasbeenusedtoenrichforfluorescentlylabeledOSERfor

proteomicanalysis.Proteomicsrevealedadditionalproteinsinvolvedinthetrichothecenebiosynthetic

pathwayaswellasanumberofconservedERproteins.Effortscurrentlyareunderwaytodestabilizethe

trichothecenebiosyntheticcomplexandtomislocalizecomponentproteinstotesthowclusteringof

enzymesandancillaryproteinsinfluencetrichothecenesynthesisandotherterpenoidpathwayswithin

thecell.

Droughtstressresultsinacompartment-specificrestructuringofrice-root-associatedmicrobiomes

SantosMedellin,ChristianM([email protected])1;Edwards,Joseph

1;Liechty,Zachary

1;Nguyen,

Bao1;Sundaresan,Venkatesan

1,2

1PlantBiologyDepartment,UniversityofCalifornia,Davis,CA,USA.

2PlantSciencesDepartment,

UniversityofCalifornia,Davis,CA,USA.

Plantrootssupportcomplexmicrobialcommunitiesthatcaninfluenceplantgrowth,nutrition,and

health.Whileextensivecharacterizationsofthecompositionandspatialcompartmentalizationofthese

communitieshavebeenperformedindifferentplantspecies,thereisrelativelylittleknownaboutthe

impactofabioticstressesontherootmicrobiota.Here,wehaveusedriceasamodeltoexplorethe

responsesofrootmicrobiomestodroughtstress.Usingfourdistinctgenotypes,growninsoilsfrom

threedifferentfields,wetrackedthedrought-inducedchangesinmicrobialcompositioninthe

rhizosphere(thesoilimmediatelysurroundingtheroot),theendosphere(therootinterior),and

unplantedsoils.Droughtsignificantlyalteredtheoverallbacterialandfungalcompositionofallthree

communities,withtheendosphereandrhizospherecompartmentsshowingthegreatestdivergence

PosterPresentations

26

fromwell-wateredcontrols.Theoverallresponseofthebacterialmicrobiotatodroughtstresswas

taxonomicallyconsistentacrosssoilsandcultivars,andwasprimarilydrivenbyanenrichmentof

multipleActinobacteriaandChloroflexi,aswellasadepletionofseveralAcidobacteriaandDeltaproteobacteria.Whiletherewassomeoverlapinthechangesobservedintherhizosphereand

endospherecommunities,severaldrought-responsivetaxawerecompartment-specific,apatternlikely

arisingfrompreexistingcompositionaldifferences,aswellasplant-mediatedprocessesaffecting

individualcompartments.Theseresultsrevealthatdroughtstress,inadditiontoitswellcharacterized

effectsonplantphysiology,alsoresultsinrestructuringofrootmicrobialcommunities,andsuggestthe

possibilitythatconstituentsofthealteredplantmicrobiotamightcontributetoplantsurvivalunder

extremeenvironmentalconditions.

TherhizosphereofthebeachgrassAmmophilabreviligulataasamodelforplant-microbiomeinteractions

Izquierdo,JavierA([email protected])1;Zaslow,ShariJ

1;Potter,SavannahJ

1;Hsieh,

Brandon1;Pimentel,Joshua

1;Wanees,AbanoubE

1;Normile,TylerG

1

1DepartmentofBiology,HofstraUniversity,Hempstead,NY,USA.

TheAmericanbeachgrassAmmophilabreviligulataisasalt-lovingplantconsideredtobeanimportant

sanddunearchitectofbarrierislandsandothercoastalenvironmentsinthemid-AtlanticandNortheast.

Avarietyofinterestingadaptationsallowittocolonizeandtrapsandinordertopromotetheformation

ofdunesandofferprotectiontothesecoastalecosystems.Myresearchgroupisinterestedin

understandingtherolethattheroot-associatedmicrobiomesplayinhealthybeachgrassgrowth.We

havecharacterizedthemicrobiomesassociatedwiththesoilsandrootsofbeachgrasssamplescollected

alongthesouthshoreofLongIslandunderavarietyofbeachgrassgrowthandhealthconditions.Weare

learningthatthemicrobialcommunitiesassociatedwithbeachgrassareremarkablydiverseand

extremelywellstructuredacrosssoilmicroenvironments.Moreimportantly,wehavealsoidentifieda

varietyofpatternsinmicrobialcommunitycompositionthatareassociatedwiththehealthoftheplant.

Inaddition,wehavealsobeenabletoisolateandcharacterizeplant-growth-promotingmicrobesthat

couldplayakeyroleinhealthybeachgrassrootgrowth.Genomesequencingofkeyisolateshasallowed

ustoidentifypathwaysthatmayplayaroleintheirmolecularsignalingexchangeswithbeachgrass

throughtheproductionofindoleaceticacid,siderophores,terpenes,andothersecondarymetabolites.

OurgoalistousethebeachgrassAmmophilabreviligulataasamodelsystemtostudyplant-microbe

interactions,layingthegroundworkfortheutilizationofspecificisolatesandmicrobiomeconfigurations

topromotethehealthofbeachgrassandotherplantsfacingsimilarenvironmentalchallenges.

HydroxycinnamicacidamidesareexportedbyaMATEtransporterinArabidopsis

Rosahl,SabineL([email protected])1,2;Dobritzsch,Melanie

1,2;Gorzolka,Karin

1;Matern,Andreas

1;

Eschen-Lippold,Lennart1

1LeibnizInstituteofPlantBiochemistry,Halle,Germany.

2InterdisciplinaryCenterforCropPlant

Research,Halle,Germany.

ArabidopsisthalianaisabletopreventcolonizationbyPhytophthorainfestans,thecausalagentoflateblightdiseaseofpotato.Thisnonhostresistancedependsonmultilayereddefenseresponses.To

addresstheroleofplantsurface-localizedsecondarymetabolitesforpathogendefense,untargeted

PosterPresentations

27

metaboliteprofilingwasperformed.Inadditiontoindoliccompounds,thehydroxycinnamicacidamide

coumaroylagmatinewasamongthesecretedcompounds.MicroarrayanalysesrevealedP.infestans-activatedandhighlyco-expressedgenes,encodinganagmatinecoumaroyltransferase(ACT)anda

MATEtransporter.InleavesofP.infestans-inoculatedACTknockoutmutants,nocoumaroylagmatine

wasdetectable,suggestingthatbiosynthesiswasimpaired.InMATEmutants,coumaroylagmatine

accumulatedintracellularly,butnotextracellularly,indicatingthattheMATEtransporterisrequiredfor

theexportofcoumaroylagmatine.

InSolanumtuberosum,coumaroylagmatineaccumulatesinresponsetoP.infestansinfectioninleaves,butnotextracellularly,suggestingthatpotatoisnotabletosecretecoumaroylagmatineefficiently.

ExpressionofbothAtACTandAtMATEintransgenicpotatoplantsleadstohighlevelsofextracellular

agmatineandputrescineconjugates.ThissuggeststhattheMATEtransporterisspecificforaroleof

AtMATEfortheexportofaspecificsubsetofhydroxycinnamicacidamides.

Mappingthecolonizationofasyntheticmicrobialcommunityinoculumfromsugarcanemicrobiomeinmaizeandsoybeanplants

Damasceno,NatáliaB([email protected])1,2;deSouza,RafaelSoaresCorrea

1,2;Armanhi,Jaderson

SilveiraLeite1,2;Arruda,Paulo

1,2

1CenterforMolecularBiologyandGeneticEngineering,UNICAMP,Campinas,Brazil.

2Departmentof

GeneticsandEvolution,UNICAMP,Campinas,Brazil.

Plantsliveinassociationwithahighlycomplexcommunityofbacteriaandfungigroups.Recent

advancesinmicrobialstudieshaveshownthathostgenotypesinfluencethediversity,structure,and

compositionofplantmicrobiomes.However,thegeneticandmolecularmechanismsinvolvedinplant-

microbecommunicationthatareresponsibleforestablishingthemicrobialcommunityinplanttissues

remainunknown.Unravelingthetraitsinvolvedinmicrobe-hostinteractionduringmicrobiome

assemblageisanimperativesteptowardsbuildingbiotechnologicaltoolstotransfermicrobesandtheir

beneficialfunctionstoeconomicallyrelevantplants.Inthiswork,wesoughttoexplorethegeneticand

molecularmechanismsinvolvedincross-speciesmicrobialcompatibilitybystudyingthecolonizationof

twoimportantcropplantsfromdistinctphysiologicalgroups,maizeandsoybean.Ourmainstrategy

involvestransferringabacterialcommunitytoanewC4plantandtoaC3planttoevaluateifthe

bacterialmembersofthiscommunityarecapableofcolonizingbothplantswithcontrasting

physiologicalprofiles.First,wedesignedasyntheticconsortiumofmicroorganismscomposedofthe

mostabundantbacterialgroupsfromsugarcane,aC4bioenergycrop.Secondly,weestablisheda

platformforinoculationassaysusingmaize,anotherC4crop,asaplantmodel.Maizeseedsare

germinatedandplantedinsterileconditionsandareinoculatedwiththesyntheticcommunity.Withthis

platform,wecanevaluatetheeffectofthepresenceofthebacterialcommunityinplantgrowthand

alsotestdifferentnutrientsolutionconcentrationsorstressconditions.Thepresenceofthesynthetic

communityimprovesplantgrowthevenunderlownutrientavailability.Wealsotestedwhetherthis

communitywouldbeabletocolonizetheplanttissues.Byusingculture-independenttechniquesto

profileinoculatedandnon-inoculatedplants,weshowthatonlyapartofthesyntheticcommunity

robustlycolonizesthemaizeplants.Thisdataindicatesthatevenamongphylogeneticallycloseplants

theremightexisttraitsunderplayinghostcolonizationbyspecificmicrobialgroups.Additionally,plants

inoculatedwiththesyntheticmicrobialcommunityhadtheirtotalfreshanddrybiomassonaverageup

tothreetimesincreasedwhencomparedwithuninoculatedplants.Wesoughttoinvestigateifthe

syntheticcommunityisalsoabletocolonizesoybeanplantswithasimilarpatternandabeneficialplant

PosterPresentations

28

response.DualRNA-seqofplantandmicrobialcommunitywillrevealgenesandpathwaysinvolvedin

theestablishmentofthemicrobialcommunityanditsbeneficialfunctions.

Decipheringtheroleoffungalsecondarymetaboliteswithinanaerobicmicrobialcommunities

Swift,CandiceL([email protected])1;Louie,Katherine

2;Bowen,Benjamin

2;Bingol,Kerem

3;

Heyman,Heino3;Northen,Trent

2;O'Malley,MichelleA

1

1UniversityofCalifornia,SantaBarbara,CA,USA.

2JointGenomeInstitute,WalnutCreek,CA,USA.

3EnvironmentalMolecularScienceLaboratory,Richland,WA,USA.

Anaerobicfungithriveincompetitivemicrobialenvironmentssuchasthedigestivetractsofmanylarge

herbivoresdespitebeingvastlyoutnumberedbyothermicroorganisms.Inadditiontosecreting

powerfulbiomass-degradingenzymes,theseuniquenon-modelorganismsalsopossessaricharrayof

biosyntheticmachineryforproducingsecondarymetabolites,whosefunctionsremaintobe

determined.Wehypothesizethatsecondarymetabolitesfromgutfungiplayimportantrolesin

anaerobiccommunitiesviamicrobialdefense,communication,orstresstolerance.Here,wehave

combinedcomplementaryapproachesingenomics,transcriptomics,massspectrometry,andNMRto

probethefunctionsofsecondarymetabolitesinthecomplexanaerobicdigestiveenvironmentof

herbivores.First,weuncoveredaplethoraofgeneclustersencodingbiosyntheticenzymesfor

secondarymetabolitesfromdiversechemicalclassesbyminingthegenomesandtranscriptomesof

threeanaerobicfungifromtheprimitivephylumNeocallimastogomycota.Keysecondarymetabolite

clustersincludecanonicalpolyketidesynthases(PKS)andnonribosomalpeptidesynthetases(NRPS).

Severaloftheclustersareexpressedunderstandardlaboratorygrowthconditions,whereasothersare

silent.RNAsequencingallowsustotestenvironmentalconditionsthatactivatethebiosyntheticgene

clustersthathavebeenidentifiedinthefungalgenomes.Theenvironmentalconditionswearetesting

includeheatshock,oxidativestress,pH,andnutrientavailability.Inadditiontoabioticfactors,weare

alsoco-cultivatingtheanaerobicfungiwithbacteriaandarchaeatoscreeninteractionsthatmay

enhancetranscriptionoftheclusters.Finally,weareworkingwithJGIandEMSLtoemployanalytical

techniqueslikemassspectrometryandNMRthatallowustodetectanddeterminethestructuresofkey

secondarymetabolites.Todate,wehavedetected~100likelysecondarymetabolitesandputatively

identifiedanantioxidantpolyketide,baumin,whichisalsoproducedbyadistantlyrelatedfungusfrom

thephylumBasidiomycota.Massspectrometryfacilitateshighthroughput,rapidscreeningof

metaboliteswithinacomplexmixtureandyieldsachemicalfingerprintofeachmoleculethatcanbe

comparedwithreferencedatabasesfordereplicationwithknownnaturalproducts,whileNMRpermits

detailedstructuralcharacterizationofisolatedcompounds.Thesecondarymetabolismofanaerobic

fungirepresentsacompletelyuntappedreservoirofbiosyntheticpotential,whichcouldbedrawnupon

fornoveltherapeutics,newchemicalbuildingblocks,andenzymesforbioengineeringnaturalproducts.

Ourintegratedapproachallowsustostudysecondarymetabolismatalllevels,fromDNAtoRNAto

metabolites,thusmaximizingdiscoveryofnovelmetabolitesandunmaskingtheirnativefunctions.

PosterPresentations

29

Root-associatedmicrobiomeofaditerpene-deficientmaizemutant

Murphy,KatherineM([email protected])1;Edwards,Joseph

1;Mafu,Sibongile

1;Zerbe,Philipp

1

1DepartmentofPlantBiology,UniversityofCalifornia,Davis,CA,USA.

Plantsdeployspecializedmetabolitestocommunicatewithotherorganismsandcopewith

environmentalchallenges.Thisincludesinteractionswithmicrobialcommunities,inwhichplants

exchangesugarsforavailablenutrientsaswellasprotectionagainstenvironmentalstressors.However,

themolecularmechanismsbywhichaplantrecruitsitsparticularmicrobialcommunityandtheroleof

specializedmetabolitesinthiscommunicationarepoorlyunderstood.Here,wereportthatmaizeroot

diterpenes,agroupofspecializedmetaboliteswithversatilefunctionsinstressresilience,influence

rhizospherebacterialcommunities.Inadditiontothepreviouslydescribedkauralexinmetaboliteswith

keyrolesinmaizepathogenandenvironmentalstressresistance,werecentlyelucidatedanovelgroup

ofmaize-specializedditerpenes,termeddolabralexins,whichalsoshowactivityagainstbioticand

abioticstress.Distinctfromthegibberellinbiosynthesispathway,bothkauralexinsanddolabralexinsare

synthesizedviathecopalyldiphosphatesynthaseZmAn2beforebranchingintoseparatepathways.The

an2(antherear2)maizemutantisdeficientinformingbothkauralexinanddolabralexinmetabolites,

andexhibitsenhancedstresssusceptibility.Using16SrRNAsequencing,wedeterminedthebacterial

communitycompositionsofthean2mutantcomparedtoitswildtypesibling.Underwell-watered

conditions,distinctbacterialcommunitiesanddiversitieswereobservedbetweenmutantandwildtype

plants,whereasthemicrobiomecompositionsbecameindistinguishableunderdroughtconditions.

Thesefindingssuggestthatditerpenesplayanimportantroleinshapingtherhizospheremicrobiome,

whilealternatemechanismsmaybedominantunderdroughtstress.

Genomesequenceofanabundance-drivenmicrobiomesyntheticcommunitywithbeneficialeffectsonplantdevelopment

deSouza,RafaelSoaresCorrea([email protected])1;Armanhi,Jaderson

1;DamascenoNatália

1;

Imperial,Juan2,3;Arruda,Paulo

1

1InstituteofBiology,UNICAMP,Campinas,Brazil.

2CentrodeBiotecnologíayGenómicadePlantas,

UniversidadPolitécnicadeMadrid,Madrid,Spain.3ConsejoSuperiordeInvestigacionesCientíficas,

Madrid,Spain.

Thesugarcane-associatedmicrobialcommunityhaslongbeenexploredforitscapabilityofsupporting

plantdevelopmentunderdiverseconditions.Thesesurveyshavemostlyfocusedonspecificbacterial

groupssuchnitrogen-fixingbacteriabyapproachesbasedonisolationondefinedculturemediaand

inoculationofasinglebacteria.However,thesoleuseofmethodsbasedoncultivationisstronglybiased

andmaynotreflecttherealcompositionofthebacterialcommunityintheplant.Furthermore,the

inoculationofasinglebacteriadoesnotrepresentthehighlycomplexdynamicofaplantmicrobiome.

Asaresult,welackfundamentalinformationregardingthemicrobialassemblageanditsfunctionalrole

inassociationwiththesugarcaneplant.Thegoalofthisworkistoexplorethegenetictraitsinthe

untappedmicrobialcommunitythatareresponsibleforplantdevelopment.Ourgrouphasadopteda

strategythatconcomitantlyuseculture-dependentandculture-independenttechniquestotarget

dominantmicrobialgroupsfromthesugarcanemicrobiome.Byusingculture-independenttechniques,

wefoundacoremicrobiomecomposedoflessthan20%ofthetotalmicrobialrichnessandthatsums

uptoover90%ofthetotalmicrobialrelativeabundanceinroots,stalks,andleavesofsugarcane.The

coremicrobiomeismostlyformedofbacterialandfungalgroupswhosefunctionsinassociationwith

PosterPresentations

30

plantshaveneverexplored.Unliketraditionalapproachesthatinvestigatethebeneficialeffectsofthe

microbiotabyselectingmicrobialcandidatessolelybasedontaxonomicalidentity,wedesigneda

syntheticbacterialcommunitybychoosingnaturallydominantgroupsinthesugarcanemicrobiome,

mostlypoorlyexploredintermsofassociationwiththesugarcaneplant.Bacterialcandidateswere

derivedfromacollectionofmicroorganismsfromover5000isolatedcommunitiesfromrootsandstalks

ofsugarcane.Ourresultsshowsthatthesyntheticcommunityrobustlycolonizedplants,stimulatedthe

rootdevelopment,andtripledplantbiomass.Thecommunityprofileshowsthateachmicrobeinthe

syntheticcommunitydisplaysapatternofcolonizationthatdoesnotcorrelatewithphylogenetic

relationship.Wehavesequencedthegenomeofthesyntheticcommunitymemberstoevaluate

potentialgenesandpathwaysconservedamongtherobustcolonizers.

Community-basedculturecollectionasastrategyfortargetingbeneficialplant-associatedbacteriafromthesugarcanemicrobiome

Armanhi,JadersonSilveiraLeite([email protected])1;deSouza,Rafael

1;Damasceno,Natália

1;de

Araújo,Laura1;Imperial,Juan

2,3;Arruda,Paulo

1

1InstituteofBiology,UniversityofCampinas,Campinas,Brazil.

2CentrodeBiotecnologíayGenómicade

Plantas,UPM,Madrid,Spain.3ConsejoSuperiordeInvestigacionesCientíficas,Madrid,Spain.

Recentadvancesinmicrobiomestudieshavelinkedmicrobialdiversitytobiologicalfunctionsassociated

withplantdevelopmentandbiotechnologicalprocesses.Thesoil-plantecosystemharborsavast

microbialdiversitythatvariesaccordingtothesoiltype,plantspecies,organs,andgenotypes,andmost

bacterialgroupsaredifficulttocultivate.Exploringthemicrobialcommunitymembersrequiresmethods

forisolation,annotation,andcross-referencingwithcommunityassemblagedatatotargetorganismsof

interestsuchasbeneficialplant-associated(BPA)microorganisms.However,microbialisolation

traditionallyrequiresseveralroundsofpickingandstreakingtoobtainpurecolonies.Thesemethodsare

time-consumingandcostly,andmayresultinlossesofrelevantbiologicalinformation.Asanalternative

methodformicrobiomeassessment,werecentlyintroducedtheconceptofcommunity-basedculture

collections(CBC).Thisapproachwasusedforisolating,identifying,andinvestigatingnovelBPAbacteria

fromthesugarcanemicrobiome.Coloniesfromprimaryplatingsofthesugarcanerootandstalk

microbiotawerestoredregardlessofwhethertheywereformedbysingleormultiplemicroorganisms.A

multiplexampliconsequencingstrategyoffull-lengthmicrobial16SrRNAgeneswasdevelopedusingthe

PacBioplatform.Thecross-referencingoftheCBC(culture-dependentapproach)withthesugarcane

microbiomeprofile(culture-independentapproach)revealedthattheCBCrecovered399unique

bacteriarepresenting15.9%oftherhizospherecoremicrobiomeandupto65.3%oftheendophytic

coremicrobiomesofthesugarcanestalks.Asyntheticcommunitycomprisedofhighlyabundant

bacteriafromtherootandstalksugarcanecoremicrobiomeswasinoculatedinmaizeandstimulated

rootgrowthandincreasedplantbiomassby3.4-fold.Thenaturalmicrobiotaprofilewasdramatically

changedininoculatedplantsbothindiversityandabundance.Thebacteriaofsyntheticinoculum

displacedthenaturalmicrobiotaandrobustlycolonizedmaizeplants,summingupto53.9%ofthetotal

microbialabundanceinroots.TheCBCmethodcanbeusedtorecoverlargerfractionsofmicrobiota

fromanyenvironmentpreservingtheirputativeinteractions,asitovercomessignificantlimitationsof

currentmicrobiomeresearchrelatedtomicrobiotaculturecollection.Furthermore,theconcomitant

useofculture-dependentandculture-independenttechniquesallowedtargetinghighlyabundantand

beneficialbacterialgroupsfromthesugarcanemicrobiomethathaveyetbeenpoorlyexplored.

PosterPresentations

31

Metabolomics-guidedisolationofsignificantbiologicalplant,soil,andmicrobialsecondarymetabolites

Heyman,HeinoM([email protected])1;Bingol,Kerem

1;Swift,Candice

2;O'Malley,Michelle

2;

Lipton,Mary1;Tfaily,MalakM

1;Metz,ThomasO

1

1EarthandBiologicalSciencesDirectorate,PacificNorthwestNationalLaboratory,Richland,WA,USA.

2DepartmentofChemicalEngineering,UniversityofCalifornia,SantaBarbara,CA,USA.

Oneofthegrandchallengesfacingthemetabolomicscommunityistheidentificationofunknown

metabolites,withunknownsecondarymetabolitescontributingthemosttothischallenge.Thelarge

chemicalheterogeneityofthemetabolomehasmadeidentificationandannotationofbiologically

significantmetabolitesoneofthemostimportantbottlenecksinuntargetedmetabolomicsanalyses.

Themassspectrometry-basedapproachesusedformetabolomicsandlipidomicsanalysesarecapableof

detectingthousandsofmoleculesinasinglerun,butcomprehensiveannotationoftheassociated

metabolitesislimitedtospectralreferencelibrarymatchingand/ordirectcomparisontoanalytical

standards.Currenttandemmassspectrallibrariesaresmallcomparedtothelargenumberof

metabolitesfoundinthebiosphere.Incaseswheremetabolitesofinterestcanbematchedtoreference

compounds,MScangiveunequivocalidentification,butforunambiguousidentificationofpartiallyor

completelyunknownmolecules,NMRspectroscopyisindispensable.Inthediverseapplications

environmentwithinwhichtheEnvironmentalMolecularSciencesLaboratoryoperates,withamultitude

ofdifferentorganismsandbiologicalsystemsunderinvestigationviaUserProjects,thisbottleneckisa

commonproblemandthusleavesasignificantpartoftheinterpretationofthemetabolomicsresults

ambiguous.BymakinguseofcomplementaryspectraldatafrombothMSandNMR,wearereducingthe

timeforstructuralelucidationofmetabolitesbyincorporatingcandidaterejectionandsubstructural

conformation.InthistalkIwilldescribethecapabilitiesofanewpipeline,MetaboliteIdentificationand

CharacterizationPipeline(MICP),�currentlybeingdevelopedatEMSLtoboosttheidentificationof

unknownmetabolitesandIwillpresentselectedapplicationstofungalandsoilstudieswherewe

developedaspecificdirectedfractionationandisolationroadmapwhichwasusedtopurify,isolate,

identify,andcharacterizethefungalandsoilunknownmetabolitesthatareofbiologicalimportance.

Thenewlyidentifiedmetaboliteswillbeaddedtoourever-growingPacificNorthwestNational

Laboratorymetabolitedatabase,whichservesasarepositoryforfuturevalidatedmetabolomicsdata.

ThenovelmetabolitesidentifiedbyourMICPwillbeusedtoimproveourunderstandingoftheroles

thatbioticandabiotictransformationshaveonplantsandsoilmicroorganismswithenvironmental

changes.

Evolutionanddiversityofabiosyntheticgeneclusterforproductionofavinylglyine

Okrent,RachelA([email protected])1;Trippe,Kristin

1;Manning,Viola

1;Davis,Edward

2

1USDAARSForageSeedandCerealResearchUnit,Corvallis,OR,USA.

2DepartmentofBotanyandPlant

Pathology,OregonStateUniversity,Corvallis,OR,USA.

Thebiologicalherbicideandantibiotic4-formylaminooxyvinylglycine(FVG)wasoriginallyidentifiedin

strainsofPseudomonasfluorescensisolatedfromtherhizosphereofwheatandothergrasses.

Biosyntheticenzymes,regulatoryfactors,andtransportersessentialforFVGproductionand

accumulationareencodedbythegvgbiosyntheticgenecluster.Inordertounderstandmoreaboutthe

diversityofFVGproduction,weinvestigatedtheevolutionofthegvgcluster.Wesequencedthe

genomesofmultipleFVG-producersandcombinedthesedatawithminingofsequencedatabases.We

PosterPresentations

32

identified>30isolateswithgvgclusters,includingP.fluorescens,P.syringae,andseveralnon-pseudomonads.Byobservingthepatternofdistributionofthegvgcluster,examiningitsgeneticcontext

indifferentstrains,andcomparinggeneandspeciestrees,weconcludedthatthegvgclusterhasbeen

insertedmultipletimesindifferentlineagesthroughhorizontalgenetransfer.Thefateofgvgclusters

afterinsertionrevealsexamplesofgenelossandgenedecaybutlittlerearrangement.Thefrequencyin

whichthegvgclusterappearsinunrelatedstrainssuggestsausefulfunctionforFVGfordiversebacteria

livinginavarietyofenvironmentalhabitats.

Changesinrootexudationandmicrobiomerecruitmentbymaizeinresponsetophosphatelimitation

Brisson,VanessaL([email protected])1,2,3

;Zhang,Zheyun1,2;Vogel,John

1,2;Northern,Trent

1,2;Gaudin,

Amélie1,3

1LawrenceBerkeleyNationalLaboratory,Berkeley,CA,USA.

2DOEJointGenomeInstitute,WalnutCreek,

CA,USA.3UniversityofCalifornia,Davis,CA,USA.

Beneficialmicrobialcommunitiesareimportantforsupportingplantgrowthandproductivity,

particularlyundernon-idealnutrientconditions.Modulationofrootexudatecompositionisonewayin

whichplantscaninfluencetheirrhizospheremicrobialcommunitiesandpotentiallyrecruitbeneficial

microbesinresponsetodifferentgrowthconditions.Domesticationandbreedingforhighinput

agriculturalproductionmayhaveaffectedtheabilityofmaizetoadapttonutrient-limitedconditions.

Herewepresentaninvestigationintotheresponseofmaizeplantstophosphatelimitationintermsof

exudedmetabolites,rhizospheremicrobiomerecruitment,andplantgrowth.Wefirstperformedan

experimentwithasinglemodernmaizehybridgrownwithandwithoutavailablephosphate(provided

as85mg/LKH2PO4).Phosphatelimitationresultedinsignificantdifferencesinexudedmetabolites,with

approximately10%ofdetectedmetabolitesshowingdifferentialexudationbetweenthetwogrowth

conditions.Phosphatelimitationalsoresultedinashiftinbiomassallocationfromshoottoroot

production,withrootscomprising36%and58%oftotalbiomassforgrowthwithandwithout

phosphate,respectively.Wethenperformedanadditionalexperimentwithanevolutionarypanelof

fourmaizeaccessionsgrownunderthreephosphateconditions(lowphosphate[5mg/LKH2PO4],high

phosphate[85mg/LKH2PO4],andinsolublephosphate[1.5gFePO4.2H2Oper1.5Lpot]).Thepanel

includedoneteosinte(wildrelative),oneancientlandrace,oneinbredparentofmodernmaizehybrids,

andonemodernmaizehybrid.Plantsweregrowninsterilizedsandandinoculatedwithamicrobial

communityderivedfromlowphosphorus(13ppmPi)soilfromunfertilizedwheatplotsatRussellRanch

inDavis,California.Growthwithlowphosphateagainresultedinmorerootbiomasscomparedwith

highphosphateandinsolublephosphate(51%vs.43%and44%oftotalbiomass,respectively).Thetotal

phosphoruscontentofplantleavesindicatedthatforallfouraccessions,plantsgrownwithinsoluble

phosphatewereabletouptakemorephosphatethanthelowphosphatecondition,butlessphosphate

thanthehighphosphatecondition.Overall,rootexudatesfromplantsgrownwithlowphosphatehad

lowerlevelsoftotalorganiccarbon(12ppmTOC/grootdryweight)comparedwithplantsgrownwith

highphosphateorinsolublephosphate(16and15ppmTOC/grootdryweight,respectively).Thistrend

wasapparentintheteosinte,landrace,andinbredaccessions,butwasdifferentforthemodernhybrid,

forwhichtheinsoluble-phosphategrowthconditionproducedthelowestaverageTOCexudation.Initial

plateassaysformicrobialphosphatesolubilizationindicatedifferencesinmicrobiomerecruitment

betweendifferentaccessions,withtheteosintebeingthemosteffectiveatconsistentlyrecruiting

phosphatesolubilizingmicroorganisms.Furtheranalysisofrootexudatemetabolitesandmicrobial

communitycompositionfromthisexperimentareongoing.

PosterPresentations

33

Microbialvariationamonghighandlowmethaneemittingricecultivars

Liechty,ZacharyS([email protected])1;Edwards,Joseph

1;SantosMedellin,Christian

1;Eason,

Shane3;Phillips,Gregory

3;Sundaresan,Venkatesan

1,2

1PlantBiologyDepartment,UniversityofCalifornia,Davis,CA,USA.

2PlantSciencesDepartment,

UniversityofCalifornia,Davis,CA,USA.3CollegeofAgricultureandTechnology,ArkansasState

University,Jonesboro,AR,USA.

Ricecultivationaccountsformorethan15%oftheglobalanthropogenicemissionsofmethane,apotent

greenhousegas,duetomethanogenicarchaeainthesoilthataresupportedbyricerootexudates.The

rateofmethaneemissioninricehasbeenfoundtobedependentonbothgenotypeandlocation.Inthis

study,ahighmethaneemittingcultivar(Sabine),andlowemittingcultivar(CLXL745)weregrownina

fieldsiteinArkansas,andthemicrobiomeofthesoildirectlysurroundingtheroot(rhizosphere)andthe

interioroftheroot(endosphere)weresampledthroughoutthegrowingseason.Surprisingly,the

relativeabundanceofmethanogenicarchaeawashigherinthelateseasonmeasurements,afterthe

periodofpeakmethaneemissions.Therewasastatisticallysignificantincreaseintherelative

abundanceofmethanogensassociatedwiththehighemissioncultivarSabinerelativetothelow

emissioncultivarCLXL745atthisstage.Futureexperimentswillexaminetheroleofphysiology,

structure,androotexudatesindrivingthecultivardifferencesinmethaneemission.

UseofLC-MS/MSinthecharacterizationofproteinbioactivesfromBacillusspores

Hotton,SaraK([email protected])1;Smaldone,Greg

1;Curtis,Damian

1;Tegeler,Tony

1

1BayerUS,CropScience,WestSacramento,CA/USA.

BayerBiologicsisworkingtodeliverintegratedsolutionsforfarmers,includingbiologicalproducts,for

managementofplantpestsanddiseasesaswellasenhancingcropvigorandyieldpotential.Researchat

BayerBiologicsfocusesonscreeningalargemicrobialstraincollectionfornewbioactives.Myresearch

focusesonthecharacterizationofBacillussporeproteinsthatareofinterestasbioactives.ProteinsarefirstisolatedfromsporesandLC-MS/MSmethodsforidentificationandquantitationareusedinprotein

characterization.Successfulrecoveryofsporeproteinsisachievedusingatrichloroaceticacidextraction

protocol.Proteinsarethentrypsin-digestedandanalyzedonanABSciexTripleTOF4600mass

spectrometerforproteinidentificationoranABSciexQTrap4000massspectrometerforprotein

quantitation.Theseanalysesallowforthepreciseandspecifictrackingofunmodifiedsporeproteinsand

areessentialinthecharacterizationofnovelproteinbioactives.

Strigolactoneimpactsonsoybeanrhizospheremicrobialcommunityassembly

Liu,FangNO([email protected])1;Grewal,Parwinder

2;Bernard,ErnestC

1;Lebeis,Sarah

1;Hewezi,

Tarek1;Staton,MegE

1

1UniversityofTennessee,Knoxville,TN,USA.

2UniversityofTexasRioGrandeValley,Edinburg,TX,USA.

Recentresearchincreasinglysuggestsanintimateandmutualisticinteractionbetweenplantsandtheir

associatedmicrobiomes.Therhizosphere,thenarrowregionbetweenplantandsoil,isthemost

dynamicandactiveinterface,withintensivecommunicationbetweenaplantanditsmicrobiome.Inthis

region,ageneralenrichmentofthemicrobialcommunityistriggeredbyplantexudates,whichis

followedbyhost-specificdifferentiationofmicrobiotathatthriveontherhizoplaneandinthe

PosterPresentations

34

endosphere.Agrowingbodyofstudiessuggestsrootexudates,especiallysecondarymetabolites,are

importantcompoundsmediatingplantandmicrobecommunication.Strigolactonesareanovelclassof

carotenoid-derivedphytohormonesthatcontrolmanyaspectsofrootandshootdevelopment.Interms

ofrhizospherecommunication,strigolactoneshavebeendemonstratedtobeinvolvedingerminationof

parasiticplantseeds,inductionofhyphalbranchingofarbuscularmycorrhizalfungi,andpromotionof

nodulation.Wepostulatestrigolactonecouldsimultaneouslyinfluencetherhizospheremicrobial

communityrecruitmentdirectlybyactingasasignalmoleculeafterbeingexudedintosoilorindirectly

throughregulationofrootmorphologicalarchitecture.Toexploretheimpactofstrigolactoneson

soybeanrhizospheremicrobiomerecruitment,threehighlyexpressedgenes(D14,MAXI,andMAXII)

thatparticipateinstrigolactonebiosynthesisanddownstreamperceptionwereidentified.Both

overexpressionandRNAisilenceconstructsofGlycinemaxWilliams82willbegeneratedusinga

transgenichairyrootsystem,andconfirmedbasedonagreenfluorescentprotein(GFP)reporter.After

screening,transgeniclinesandemptyvectorcontrollineswillbegrowninthegreenhouse.Atflowering

stage,rhizospheresoilwillbeharvestedandusedforDNAextraction.16SribosomeRNAamplicon

sequencingtargetedattheV3_V4regionwillbeusedtocharacterizetherhizospheremicrobial

community.Theresultsofthisexperimentwillelucidatewhetherincreasedordecreasedstrigolactone

productionimpactsrhizospheremicrobiomestructureandprovideinitialevidenceonwhichbacteriaare

mostactivelyrespondingtothisparticularrootexudate.

IdentificationofunknownsecondarymetabolitesbyhybridNMR/MSapproach:applicationtostudyingthefloweringtimeinArabidopsisthaliana

Bingol,Kerem([email protected])1;Boiteau,Rene

1;Hoyt,David

1;Nicora,Carrie

1;Kinmonth-

Schultz,Hannah2;Heyman,Heino

1;Washton,Nancy

1;Metz,Thomas

1;Ward,Joy

2

1EarthandBiologicalSciencesDirectorate,PacificNorthwestNationalLaboratory,Richland,WA,USA.

2DepartmentofEcologyandEvolutionaryBiology,UniversityofKansas,Lawrence,KS,USA.

Despiterecentprogressinmetabolomics,alargenumberofsecondarymetabolitesinsoil,microbial,

andplantsystemsarestillunknown,i.e.,theyarenotidentifiedoravailableinanymetabolomics

database.Theseunknownsecondarymetabolitesmustbestructurallycharacterizedandidentifiedinan

efficientandaccuratemanner.Withtheincreasedresolutionofmassspectrometers(MS),the

determinationofaccuratemassesofindividualknownandunknownsecondarymetabolitesisbecoming

increasinglyroutine.Thisinformationallowsonetodeducethemolecularformulaofmetabolitesthat

underlieseachpeakinthecomplexmixturemassspectrum.However,knowledgeofmolecularformulas

doesnoteasilytranslatetotheidentificationofindividualsecondarymetabolitesbecauseofthelarge

degeneracyofthestructuralspacebelongingtoagivenmolecularformula.Withincreasingmass,this

degeneracyincreasesexponentially.Thismakesidentifyingunknownsecondarymetabolitesvery

challengingbyMSalone.Ontheotherhand,NMRspectracandifferentiatesignificantlybetween

isomers.Therefore,integrationofNMRinformationwithMSopensupnewopportunitiestoaddressthe

structureelucidationchallenge.Recently,weproposedahybridNMR/MSmetaboliteidentification

strategy.Thisstrategyfirstidentifiesthechemicalformulasofthemixturecomponentsfromaccurate

massesbyMSandthengeneratesallfeasiblestructuresthatareconsistentwiththesechemical

formulas.Next,NMRspectraofeachmemberofthefeasiblestructuresarepredictedandcompared

withtheexperimentalNMRspectraofthesamemixturetoidentifythemolecularstructuresthatbest

matchtheinformationobtainedfromboththeMSandNMRtechniques.Wedemonstratetheapproach

ontheidentificationofunknownsecondarymetabolitesinArabidopsisthalianainthecontextofdeterminingthemetabolicunderpinningsofshiftsinfloweringtimeinresponsetoatmosphericCO2rise,

PosterPresentations

35

whichwillhavemajorimplicationsonaglobalscale,sinceCO2isexpectedtodisruptcarboncycling

withinecosystems.ThehybridNMR/MSapproachhasbeenroutinelyusedatEnvironmentalMolecular

SciencesLaboratory(EMSL)foridentificationofunknownsoil,microbial,andplantsecondary

metabolites.EMSL’scapabilitiesareavailabletoresearchersworldwidethroughapeer-reviewed

proposalprocess,typicallyatnocost.Formoreinformation,visithttps://www.emsl.pnl.gov/emslweb/.

Examiningthesecondarymetaboliteactivityinthelichencommunity

Trail,FrancesO([email protected])1,3;Roze,Ludmila

1;Linz,John

2

1DepartmentofPlantBiology,MichiganStateUniversity,EastLansing,MI,USA.

2DepartmentofFood

ScienceandHumanNutrition,MichiganStateUniversity,EastLansing,MI,USA.3DepartmentofPlant,

SoilandMicrobialSciences,MichiganStateUniversity,EastLansing,MI,USA.

Lichensaresomeofthelongest-livingorganismsknown,despitetheirslowgrowth,andtheyveryrarely

appeartodieofdisease.Thelichenizedfungusestablishesthemainlichenthallusinassociationwithan

algaorcyanobacterium.Thisscaffoldbecomesanicheforavarietyofotherfilamentousascomycetes

andbasidiomycetes,yeasts,bacteria,and,occasionally,insects.Lichensareknowntoproducea

plethoraofuniquesecondarycompounds.Theirlongevityandrobustness,despiteacloseassociation

withdiversemicrobes,providesaninterestingstudysystemtoviewtheroleofsecondarymetabolitesin

managingamicrobialcommunity.Weisolatedextractsfrom72lichenspeciesandtestedfortheir

effectsonsporulation,hyphalgrowth,andsecondarymetaboliteproductioninfungalcultures.The

structureofthesecompoundsisunderinvestigation,asistheidentificationofthemicrobialsource.

Interestingly,themostcommonactivitybyfaramongthelichenextractshadtheeffectofarresting

secondarymetaboliteproduction.Thisfindingsuggeststhatlichensattenuatenegativeinteractionswith

theincumbentfungithroughtheirabilitytoregulatesecondarymetabolism.

ChangesintherootmetabolomeofcitrusplantsinfectedwithCandidatusLiberibacterasiaticus

Padhi,EmilyMT([email protected])1;Mishchuk,DaryoO

1;Chin,Elizabeth

1;Godfrey,Kris

2;Slupsky,

CarolynM1,3

1DepartmentofFoodScience&Technology,UniversityofCalifornia,Davis,CA,USA.

2Contained

ResearchFacility,UniversityofCalifornia,Davis,CA,USA.3DepartmentofNutrition,DepartmentofFood

Science&Technology,UniversityofCalifornia,Davis,CA,USA.

Huanglongbing(HLB)isasevere,incurablediseaseaffectingcitrusplantsthatisbelievedtobecausedby

thebacteriumCandidatusLiberibacterasiaticus(CLas).Citrusrootsmaybepreferentiallycolonizedby

CLaspriortoothertissues;however,littleisknownabouttheimpactofCLasinfectiononplant

metabolismintherootsystem.One-year-oldgreenhouse-grownLisbonLemonandWashingtonNavel

orangecitrustreesweregraft-inoculatedwithcitrusbudwoodinfectedwithCLas.Rootswereobtained

fromhealthy(n=17)andinfected(n=17)trees46weekspost-inoculationandanalyzedvia1HNMR

spectroscopytoidentifyandquantifywater-solublerootmetabolites.Mann-WhitneyUtestingand

partialleastsquaresdiscriminantanalysis(PLS-DA)wereusedtodeterminesignificantdifferencesin

metaboliteconcentrationsandtoidentifydistinctmetabolitepatterns.Overall,severalmetabolites

weresignificantlydifferentinrootsobtainedfromplantsinfectedwithCLascomparedtohealthycontrol

plantsanditwaspossibletodetermineinfectionstatusthroughdifferentiatedmetabolomicsprofiles.

ThisstudydemonstratesthatadiscreetmetabolomearisesintherootsofcitrusinfectedwithCLas.

PosterPresentations

36

Assessingmicrobialcommunitycontributiontoplantabioticstresstolerance:acasestudyinserpentinesoils

Igwe,AlexandriaN([email protected])1;Vannette,Rachel

1

1UniversityofCalifornia,Davis,CA,USA.

Metal-contaminatedanddrought-pronesoilshavecharacteristicssimilartothosefoundinserpentine

soil.Becauseofthesesimilaritiesandadaptedplantcommunities,serpentineisanexcellentmodel

systemforstudyingplantandmicrobialadaptationtothesestressors.Serpentinesoilscontainhigh

concentrationsofheavymetals,suchasnickel,cadmium,copper,andlead,butlowconcentrationsof

calciumrelativetomagnesiumandnitrogen.Asafurtherchallengetoplants,serpentinesoilsalsohave

lowwaterholdingcapacity,whichmeansthatmanyplantsmustadapttodroughtstressaswell.Usinga

modelsystemofserpentinesoils,weproposetodefineacoremicrobiomeandmetagenomeassociated

withCalifornianativeplantcongenerswithinthegenusLinanthusgrownonserpentineandnon-serpentinesoil.Weaimtodeterminethefunctionalattributesoftheplantcoremicrobiomeassociated

witheachsoiltypeandtounderstandhowmicrobiomeassemblyisinfluencedbyabioticfactors

associatedwithserpentinesoil.Undergreenhouseandfieldconditions,usingareciprocaltransplant

design,IwilltransplantsterilelygerminatedLinanthusseedlingsfromserpentineandnon-serpentine

soilintoeachsoiltype.IwillcollectmicrobialDNAfromtheplantrhizosphereandendosphereat1,5,

and21daysposttransplant(dpt).Todeterminebiodiversity,allsampleswillhavethe16SrRNAgene

regionandmarkergenesassociatedwithstresstolerancesequenced.Additionally,wholeshotgun

sequencingwillbeconductedonthe21dptfieldsampletoassessthetaxonomiccompositionand

diversityanddeterminethefunctionalroleofmicrobialcommunitiesinserpentinesystems.Therearea

varietyofoutcomestothisexperimentthatwillhelpbothacademicandindustryresearchershighlight

microbesorgenesforfutureresearch.Additionally,resultswillhelpresearchersunderstandcommunity

assemblydynamics,whichwillaidinmicrobiomemanagementforagriculturalproductionand

phytoremediation.Microbiomemanagementisonewaytohelpfarmersoptimizeresourceuseto

ensurethecontinueduseofarableland.

MetabolomicstounderstandanddetectC.Liberibacterasiaticusinfection

Chin,ElizabethL([email protected])1;Mishchuk,Darya

1;Padhi,Emily

1;Slupsky,Carolyn

1,2

1DepartmentofFoodScienceandTechnology,UniversityofCalifornia,Davis,CA,USA.

2Departmentof

Nutrition,UniversityofCalifornia,Davis,CA,USA.

Metabolomicsisananalyticalmethodthatcomprehensivelymeasuresmetabolitestoprovidea

snapshotofthemetabolicstateofanorganism.Metabolitecompositionchangesinresponsetostress,

includinginfectionanddisease.Metabolomicanalysisofcitruscanthereforebeusedasanindirect

methodtodetermineifapathogenispresent,sinceitmeasuresthehostresponse;thisisincontrastto

directmethodsthatrequiredetectingthepathogenitself.Huanglongbing(HLB)isadevastatingcitrus

diseasecausedbythebacterialpathogenCandidatusLiberibacterasiaticus(CLas).EarlydetectionofCLasintreesiscriticaltocontrolthespreadofHLB.However,sinceCLasisnotevenlydistributed

throughoutthetissueoftreesandcanbepresentatverylowlevels,directmethodsoftenfailtodetect

thepathogen.Wehavepreviouslyshownthatmetabolomicscandetectmetabolitechangesinfruit

fromcitrusinfectedwithCLasregardlessofthepresenceofvisiblesymptoms.Here,wereporton

metabolomicanalysisofcitrusrootsandleavesuponCLasexposureandcitrusleavesexposedtothe

insectvectorofCLas,theAsiancitruspsyllid(ACP).Metabolitedifferenceswereobservedbetweenroots

PosterPresentations

37

fromhealthyandinfectedgreenhousetrees.Infectedfieldtreeswerealsodistinguishedfromhealthy

treesbasedontheirmetabolitepattern.SinceACPsarethemainmodeofCLastransmissioninthefield,

theeffectsofACPfeedingintheabsenceofCLaswerealsostudied.ThechangesduetoCLas-freeACP

feedingweredifferentfromchangesmeasuredduringCLasinfection.Together,theseresultssuggesta

roleformetabolomicsforimprovingdetectionofCLas.

Chemistryoftheplantexudationandsubstrateutilizationpreferencesofsoilmicroorganismsunderlyingrhizospheremicrobiomeassemblyinannualandperennialgrasses

Zhalnina,KaterynaV([email protected])1;Schlaefer,SasseJoelle

1;GaoJian

1;Tepper-FobesEden

1;

Louie,KatherineB1;Karaoz,Ulas

1;Loque,Dominique

2;Bowen,BenjaminP

1;Firestone,MaryK

1;Brodie,

EoinL1,3;Northen,TrentR

1

1LawrenceBerkeleyNationalLaboratory,Berkeley,CA,USA.

2JointBioEnergyInstitute,Emeryville,CA,

USA.3UniversityofCalifornia,Berkeley,CA,USA.

Plant-soil-microbialinteractionsregulatetheavailabilityofnutrientsandcarbon(C)transformationin

soil.Plantsexudeadiverserangeofcompoundsintothesoilsurroundingtheirroots;theseexudatesare

thoughttoattractandsupportmicroorganismsthatmayimproveplantnutrientacquisition,drought

tolerance,andresistancetopathogens.Additionally,plant-microbialinteractionscoulddefinethefuture

fateofrootC,andspecificallywhetheritisrespiredtotheatmosphereorstabilizedinsoil.Herewe

usedmass-spectrometry-basedmetabolomicstoidentifykeymetabolitesthatwesuggestcanbe

importantplayersinbidirectionalplant-microbeinteractionsinsoil.Theseincludeexudationpatternsof

twoannualandperennialgrasses,metaboliteexchangebetweenplantandrhizospheremicroorganisms,

andpotentialchemicalmechanismsofrhizospherecommunityassembly.

Takingamulti-scaleapproachincludingfield,greenhouse,andhighlycontrolledlabexperiments,our

goalistodeterminetheprocessesthatunderlieplant-soil-microbialrelationships.Weidentified

exudationpatternsoftheannualgrassAvenabarbataandlinkedchangesinAvenaexudatecomposition

tosubstrateutilizationpreferencesofbacterialisolatesfromtheAvenarhizosphere.Similarly,we

analyzedsuccessionalchangesinexudationprofilesoftheperennialgrassswitchgrass.Currentlyweare

studyingalargecollectionofswitchgrassisolatesandtheexudationprofileofswitchgrasstodefine

metabolicmechanismsunderlyingswitchgrassrhizosphereassembly,includingpossiblebeneficial

effectsofrhizospherebacteriaonplantnutritionanddroughttolerance.

Inourstudy,weproposethatlong-termrelationshipsbetweenplantsandrhizosphereorganismsare

mediatedbydynamicrootexudationandmicrobialsubstrateselectivity.

PosterPresentations

38

Broad-host-rangeexpressionrevealsnativeandhostregulatoryelementsinfluencingheterologousantibioticproductioninGram-negativebacteria

Zhang,JiaJiaR([email protected])1;Tang,Xiaoyu

1,2;Zhang,Michelle

1;Nguyen,Darlene

1;Moore,Bradley

S1,3

1CenterforMarineBiotechnologyandBiomedicine,ScrippsInstitutionofOceanography,Universityof

CaliforniaatSanDiego,LaJolla,CA,USA.2GenomicMedicine,J.CraigVenterInstitute,LaJolla,CA,USA.

3SkaggsSchoolofPharmacyandPharmaceuticalSciences,UniversityofCaliforniaatSanDiego,LaJolla,

CA,USA.

Heterologousexpressionhasbecomeapowerfultoolforstudyingmicrobialbiosyntheticgeneclusters

(BGCs).AlthoughGram-positiveheterologoushostssuchasStreptomyceshavebeendevelopedandoptimizedtosupportdiversesecondarymetabolicreactions,therehasbeencomparativelylessworkon

Gram-negativehosts,someofwhichgrowfasterandareeasiertoworkwith.Here,weextendthe

transformation-associatedrecombinationcloningandheterologousexpressionplatformformicrobial

BGCstoincludeGram-negativehosts.Usingabroad-host-rangeexpressionplatform,wetesttheimplicit

assumptionthatbiosyntheticpathwaysaremoresuccessfullyexpressedinmorecloselyrelated

heterologoushosts.CloningandexpressionoftheviolaceinBGCfromPseudoalteromonasluteoviolacea2ta16revealedrobustproductionintwoproteobacterialhosts,PseudomonasputidaKT2440andAgrobacteriumtumefaciensLBA4404,butverylittleproductionoftheantibioticinvariouslaboratorystrainsofEscherichiacoli,despitetheircloserphylogeneticrelationship.Weidentifiedanon-clustered

LuxR-typequorumsensingreceptorfromP.luteoviolacea2ta16,PviR,thatincreasespathwaytranscriptionandviolaceinproductioninE.coliby~60-foldindependentlyofacyl-homoserinelactone

autoinducers.AlthoughE.coliharborsthemostsimilarhomologofPviRidentifiedfromallhoststested,

overexpressionofvariousE.colitranscriptionfactorsdidnotresultinastatisticallysignificantincreaseinviolaceinproduction,whileoverexpressionoftwoA.tumefaciensPviRhomologssignificantly

increasedproduction.Thus,thisworknotonlyintroducesanewgeneticplatformforheterologous

expressionofmicrobialBGCs,butitalsochallengestheassumptionthathostphylogenyisanaccurate

predictorofhostcompatibility.Wearguefortheuseofadiversesetofheterologoushosts,whichmay

alsoprovideinsightsintobiosyntheticmechanismandbiologicalfunction.

TomatoRhiz'OMICS

Korenblum,Elisa([email protected]);Goldmann,ItaiA;Szymanski,JedrzejJ1;Massalha,

Hassan1;Rogachev,Ilana

1;Meir,Sagit

1;Aharoni,Asaph

1

1WeizmannInstituteofScience,Rehovot,Israel.

Therhizospheremicrobialcommunityaffectsthehostphysiology,andviceversa.However,theintricate

processes,i.e.,environmentalandhostmolecularfactorscombined,thatshapethemicrobiomeofthe

rhizospherearestillgreatlyunknown.Here,twoapproacheswereusedinordertocorrelatethetwo

majorcomponentsofthetomatorhizosphere:thetomatorootandthesoilbacterialdiversity.First,we

assessedthegeneticfactorsthataffecttherhizospherebacterialcompositioninasetof76introgression

lines(ILs)ofSolanumlycopersicumcarryingonlyasinglechromosomesegmentfromthewildspecies

Solanumpennellii(LA0716).Forthattomatopopulation,acoremicrobiomewasdefinedbasedon16S

rRNAampliconsequencing,consistingof154abundantOTUsthatchangedquantitativelyacrossmost

ILs.TestingoftheseOTUs’abundancesforcosegregation,4hostquantitativetraitloci(QTL)show

significantlinkagewithrelativeabundancesofspecificbacterialOTU.TheseQTLaffectbacterialOTUby

PosterPresentations

39

increasingitsabundance,eithercontrollinganindividualOTUorvariousOTUsspanningadiverse

taxonomicrange.Inaddition,inordertounderstandhowtherootexudationinfluencesthetomato

rhizospheremicrobiota,tomatorootswerechallengedwithsoilmicrobialcommunitiesestablished

usingadilution-to-extinctionapproach.Themetabolicpatterns,analyzedbyLC-MSandGC-MS,of

tomatorootsandexudatesaretailoredbysoilmicrobialdiversityandcomposition.Themetabolic

changesofhostplantsinresponsetosoilmicrobialdiversitymightalsoaffecttherhizospheremicrobial

ecology.

Functionalgenomics-guideddiscoveryofcrypticmetabolitesinvolvedinpathogenicplant-microbeinteractions

Chooi,Yit-Heng([email protected])1

1SchoolofMolecularSciences,UniversityofWesternAustralia,Perth,Australia.

Plantpathogenicfungiproduceanarsenalofhydrolyticenzymes,proteinaceouseffectors,and

secondarymetabolitestofacilitatetheirinfectionofhosts.Comparedtoourunderstandingof

proteinaceouseffectors,ourunderstandingoftherolesofsecondarymetabolitesinpathogenicplant-

microbeinteractionsisstillpoor.Ourunderstandingispartiallyhinderedbyconditionalexpressionof

biosyntheticgeneclusters(BGCs)andlackofefficienttoolsfortranslatingBGCstometabolites.Guided

byestablishedbiosyntheticlogics,weusedacombinationoffunctionalgenomics,syntheticbiology,and

chemicalecologytoolstouncoverthesecrypticmetabolitesandtheirfunctions.Inparticular,theBGCs

wereprioritizedbasedongeneexpressiondataofpathogensduringinfectionofplanthostsand

heterologoushosts(A.nidulansorS.cerevisiae)wereusedforreconstructionofselectedBGCs.Some

examplesfromourgroup’srecentstudiesarepresentedhere,includingthediscoveryofmellein,

elsinochrome,andcytochalasin(inprogress)pathwaysinthewheatpathogenParastagonosporanodorum,andtheirpossiblerolesinplant-microbeinteractions.

ChemicaldiversitygenerationusingRiPPpathways

Schmidt,EricW([email protected])1;Gu,Wenjia

1

1DepartmentofMedicinalChemistry,UniversityofUtah,SaltLakeCity,UT,USA.

RiPPs(ribosomallysynthesizedandpost-translationallymodifiedpeptides)areaubiquitousfamilyof

naturalproductsderivedfromribosomallysynthesizedpeptides.Assuch,RiPPbiosyntheticpathways

havebeenusedinthesynthesisoflargelibrariesofderivatives.Here,Iwilldescribehowcyanobactin

RiPPpathwayscanbeexploitedtogeneratedesignedderivativesthroughrationalengineering.These

derivativescanbesynthesizedinvitroorincellsforsyntheticbiologyapplications.

PosterPresentations

40

ImpactofHLBonthemetallomeandmetabolomeofCitrus1H

McNeil,ChristopherJ([email protected])1;Chin,Elizabeth

1;Lombardi,Rachel

1;Mishchuk,Darya

1;

Slupsky,Carolyn1,2

1DepartmentofFoodScienceandTechnology,UniversityofCalifornia,Davis,CA,USA.

2Departmentof

NutritionalBiology,UniversityofCalifornia,Davis,CA,USA.

NMR-basedmetabolomicshasrecentlybeenusedtostudythecitrushostresponsetoinfectionby

CandidatusLiberibacterasiaticus(CLas),thebacteriumassociatedwiththedestructivecitrusdisease

Huanglongbing(HLB;syn.citrusgreeningdisease).Metabolomicsisapromisingmethodfortheearly

detectionofCLasinplants.Metalions,includingzinc,magnesium,potassium,andiron,arerichincitrus

planttissuesandsomeofthesecanbindtocertainmetabolites,broadeningtheirsignalsandmaking

quantificationdifficultvia1HNMR.Herewelongitudinallysampledandanalyzedleavesfromnavel

orangetreesgraft-inoculatedwithCLaswithinductivelycoupledplasma-massspectroscopy(ICP-MS).

WeobservedchangesinMg2+,Ca

2+,Cu

2+,Fe

2+,K

+,andZn

2+concentrationwithinfectionovertime,with

thegreatestdifferencebetweencontrolandCLas+plantsoccurringwithsevereHLBsymptoms.

Additionally,tocharacterizetheimpactofvaryingconcentrationsofmetalionsonthemetaboliteNMR

spectrum,thesesameleaveswereanalyzedbyICP-MSand1HNMR.HighconcentrationsofMg2+,Ca

2+,

andFe2+broadenedsomemetabolites,makingidentificationandquantificationmoredifficult.We

thereforeinvestigatediftheadditionofethylenediaminetetraaceticacid(EDTA),acommonchelating

agent,couldimprovethequalityoftheNMRspectrumandhelpquantifythemetalconcentrationin

citrusleafsamples.OurobservationsshowthattheadditionofEDTAremovestheimpacttheseions

haveonidentifyingandquantifyingmetaboliteNMRresonances,andallowstheconcentrationsofMg2+,

Ca2+,andFe

2+tobemeasuredwithouttheneedforusingICP-MS.

Investigatingagenome-to-phenotypepipelineformodelgrasses

Ahkami,AmirH([email protected])1;Lindenmaier,Rodica

1;Myers,GabrielL

1;Chrisler,William

B1;Fang,Yilin

1;Yabusaki,SteveB

1;Hixson,Kim

1;Lipton,Mary

1;Cruz,Jeffrey

2;Savage,Linda

2;Tessmer,

OliverL2;Kramer,David

2;Jansson,Christer

1EnvironmentalMolecularScienceLaboratory,Richland,WA,USA.

2MSU-DOEPlantResearch

Laboratory,MichiganStateUniversity,EastLansing,MI,USA. AlthoughtheeffectsofelevatedCO2onplantgrowth,physiology,andmetabolismhasbeeninvestigated

thoroughly,theunderlyingintegratedorganismal,cellular,andmolecularmechanismsofthesechanges

arelessunderstood.Also,twomajorplantphotosynthesistypes,C3andC4plants,areofteneach

affectedindifferentwaysbyallglobalchangeparameters.Inthisstudy,accessionsofBrachypodiumdistachyonBd21(C3modelgrass)andSetariaviridisA10.1(C4modelgrass)weregrownundercurrent

andelevatedCO2levelsingrowthchambers.Detailedgrowth-stage-basedphenotypicanalysisrevealed

differentaboveandbelow-groundmorphologicalandphysiologicalresponsesofC3andC4grassestothe

enhancedCO2levelscondition.Basedonourpreliminaryresultsandscreeningvaluesoftotalbiomass,

wateruseefficiency(WUE),roottoshootratio,rootsystemarchitecture(RSA)parameters,andnet

assimilationrates,wepostulatedathree-phasephysiologicalmechanism(RootPlus,BiomassPlus,and

YieldPlusphases)forgrassgrowthundertheelevatedCO2condition.Tocharacterizeadditional

physiologicalchanges,weusedanovelnon-invasive,image-baseddynamicenvironmental

photosynthesisimager(DEPI)chambercapableofrevealingnewtransientorenvironment-specific

phenotypes.Thegeneratedimagesandrevealedphotosynthesis-specificparametersincluding

PosterPresentations

41

photosystemII(PSII)quantumefficiency(ϕII),light-drivenlinearelectronflow(LEF),dissipativenon-photochemicalquenching(NPQ)ofabsorbedlightenergy,anditscomponents(qEandqIresponses)will

bepresented.Moreover,thesecomprehensivesetsofmorphologicalandprocess-basedobservations

arecurrentlyinusetodevelop,test,andcalibratebiophysicalwholeplantmodelsandinparticularto

simulateleaf-levelphotosynthesisatvariousdevelopmentalstagesofC3andC4usingthemodelBioCro.

Also,thewholeplantphenotypicobservationswillbecomplementedwiththestomataldensity,

stomatalarea,epidermalcellarea,andomicsdatatofurtherlinktheobservedphenotypictraitsatthe

organismalleveltotissueandmolecularlevels.

Establishingagenome-to-phenotypepipelineformodelgrasses

Ahkami,AmirH([email protected])1;Shilling,John

1;Guenther,Alex

2;Gu,Dasa

1;Lindenmaier,

Rodica1;Myers,GabrielL

1;Chrisler,WilliamB

1;Fang,Yilin

1;Yabusaki,SteveB

1;Hixson,Kim

1;Lipton,

Mary1;Cruz,Jeffrey

3;Savage,Linda

3;Tessmer,OliverL

3;Kramer,David

3;andJansson,Christer

1

1EnvironmentalMolecularScienceLaboratory,PacificNorthwestNationalLaboratory,Richland,WA,

USA.2AirUCI,UniversityofCalifornia,Irvine,CA,USA.

3MSU-DOEPlantResearchLaboratory,Michigan

StateUniversity,EastLansing,MI,USA.

TheintegratedPlant-Atmosphere-SoilSystems(iPASS)InitiativeisaPacificNorthwestNational

Laboratory(PNNL)Laboratory-DirectedResearchandDevelopment(LDRD)projectaimedatdeciphering

fundamentalprinciplesthatgoverntheplantecosystem,fromplantgenotypethroughmultiplescalesto

ecosystemtraitsandresponses.Akeytoobtainingmechanisticunderstandingofplantresponsesto

environmentalperturbationsistolinkphenotypictraitsatorganismalandecosystemscalesto

molecular-scalephenotypesunderavarietyofconditions.Plantsandassociatedmicrobiotaemita

diversearrayofvolatileorganiccompounds(VOCs)intotheatmosphere.VOCsareavitalelementofa

plant’sphenotypeandareacentralcharacterintheplantecosystemduetotheirroleasecological

signalsandtheirinfluenceonatmosphericchemistry.VOCsareverydiverseandconsistofvarious

organicclassessuchasisoprenes,terpenes,fattyacidderivatives,alcohols,alkanes,alkenes,esters,and

acids.VOCsactivelyparticipateinplantgrowthandprotectionagainstbioticandabioticstresses,and,

therefore,theintensityandcompositionofVOCemissionsarestronglydependentonenvironmental

conditions.ManystudieshaveshownproductionofVOCsisstronglyregulatedbygenetics,makingVOC

emissionshighlyspeciesspecific.AlsoseveralstudieshaveshownvariabilityamongVOCemissionswith

genotypesofcultivatedandwildplants.Inthisstudy,accessionsofBrachypodiumdistachyonBd21(C3

modelgrass)andSetariaviridisA10.1(C4modelgrass)weregrownundercurrentandelevatedCO2

levelsingrowthchambers.Detailedgrowth-stage-basedphenotypicanalysisrevealeddifferentabove-

andbelow-groundmorphologicalandphysiologicalresponsesinC3andC4grassestoenhancedCO2

levelscondition.Basedonourpreliminaryresultsandbyscreeningvaluesoftotalbiomass,wateruse

efficiency(WUE),root:ratioallometry,rootsystemarchitecture(RSA)parameters,andnetcarbon

assimilationrates,wepostulatedathree-phasephysiologicalmechanism(RootPlus,BiomassPlus,and

YieldPlusphases)forgrassgrowthundertheelevatedCO2condition.Tocharacterizeadditional

physiologicalchanges,weusedanovelnon-invasive,image-baseddynamicenvironmental

photosynthesisimager(DEPI)chambercapableofrevealingnewtransientorenvironment-specific

phenotypes.Moreover,thesecomprehensivesetsofmorphologicalandprocess-basedobservationsare

currentlyinusetodevelop,test,andcalibratebiophysicalwhole-plantmodelsandinparticularto

simulateleaf-levelphotosynthesisatvariousdevelopmentalstagesofC3andC4grassesusingtheBioCro

model.Also,thewhole-plantphenotypicobservationswillbecomplementedwithdataforstomatal

density,stomatalarea,epidermalcellarea,andomicstofurtherlinktheobservedphenotypictraitsat

PosterPresentations

42

theorganismalleveltotissueandmolecularlevels.ThepreliminarydataofidentifiedVOCmetabolites

emittedbyBrachypodiumplantsmeasuredbydynamicvegetationenclosurewillbealsopresented.

CharacterizationofthemicroviridinbiosyntheticgeneclusterfromChryseobacterium

Blair,Patricia1([email protected])

1OakRidgeNationalLaboratory,OakRidge,TN,USA.

Therapidityandeaseofgenomesequencinghasenabledbioinformatics-guidednaturalproduct

discoveryandcharacterization.ThePlant-MicrobeInterfacesprojectatOakRidgeNationalLaboratory

aimstoidentifynaturalproductsthatinfluenceinteractionsinthePopulusManymicrobialgenomes

encodethemachinerytoproducediversebioactivemoleculesthatcanbeusedinhealthcare,

agricultureandfood.Itsnaturalmodularitymakesthismachineryaparticularlyattractivetargetfor

syntheticbiology.There-engineeringofthebiosyntheticcapacityofmicrobesrequiresthedevelopment

ofawiderangeofexperimentalandcomputationaltools.Theserangefromorthogonaltranscriptional

controlcircuitsandbacterialmicrocompartments,tocomputationaltoolsforthedetectionandanalysis

ofsecondarymetabolitebiosynthesisgeneclustersthatenrichourlibraryofpartsandbuildingblocks

forpathwayengineering,andhigh-resolutionmassspectrometryanalysisforthedebuggingofthe

engineeredsystems.

InthistalkIwillexplorethepossibilitiescreatedbytheapplicationofthedesign/build/test/learncycle

ofsyntheticbiologytotheengineeringofmicrobialmetabolismfortheproductionofhigh-value

chemicals,asimplementedinthehigh-throughputplatformoftheBBSRC/EPSRC-fundedManchester

SyntheticBiologyResearchCentre,SYNBIOCHEM.Iwillalsodiscussthegrowingtoolboxoftechniques

andapproaches,illustratedwithconcreteapplicationcasestudies.microbiome,andtodeterminehow

microbiomestructureaffectsthehealthoftheplanthost.IncollaborationwiththeJointGenome

Institute,254bacterialisolatesfromtherhizosphereofPopulustrichocarpaandPopulusdeltoideshavebeensequenced;thishasenabledabioinformatics-drivencharacterizationofthebiosyntheticpotential

ofthePopulusmicrobiome.Thebiosyntheticgeneclusterresponsibleformicroviridinbiosynthesiswas

foundwithinnearly75%ofthesequencedChryseobacteriumstrains,butisabsentinallother

sequencedorganisms.Here,wecharacterizethesecondarymetaboliteclusterswithinthe18

ChryseobacteriumisolatesfromthePopulusrhizosphereanddrawcomparisonstoknownmicroviridins.

InitialMS-basedscreeningdemonstratedtheproductionofthepredictedcyclicdepsipeptidesinvitro.

Antagonismwasobservedinspot-on-lawnassays,whichmayinpartbetheresultofmicroviridin

production.

SurveyofthebiosyntheticpotentialofthePopulusmicrobiome

Pelletier,Dale1;([email protected])

1OakRidgeNationalLaboratory,OakRidge,TN,USA.

Thousandsofbacterialspecieshavebeenisolatedfromplantrootmicrobiomes,makingthestudyof

microbe-microbeandmicrobe-plantinteractionsachallenge.BacilliandStreptomycesareubiquitousinsoilandmakeupalargepercentageofthemicrobialcommunityintherhizosphere.Thesebacteriahave

beenstudiedforuseasbiocontrolagentsduetotheirgeneticpotentialtoproducecomplexnatural

productswithantibioticactivity;however,theroleofthesecompoundsinmicrobe-microbeandplant-

microbeinteractionsremainslargelyunknown.RhizosphereisolatesfromPopuluswereanalyzedforbiosyntheticgeneclustersandtheproductionofbioinformaticallypredictednaturalproducts,and

PosterPresentations

43

Streptomycesstrainswereselectedforfurtheranalysisbasedonanabundanceofpredictedclusters.Screeningofbacterialextractsshowedanumberofbioinformaticallypredictedcompoundswere

producedunderlaboratoryconditions,includingonelassopeptide,siamycinI.Usingchemicalimaging,

productionofthelassopeptideswasobservedinPlantae.Significantplantgrowtheffectswereobservedwhenplantsweretreatedwiththebacterialisolates,butconstructedbacterialcommunities

onplantrootswerenotdramaticallyalteredwiththeadditionofthesestrains,suggestinganuanced

andmultifacetedroleforthesenaturalproductsintherhizosphere.

EngineeringthebiocatalyticselectivityofiridoidproductioninSaccharomycescerevisiae

Billingsley,John([email protected])

UniversityofCalifornia,LosAngeles,CA,USA

Monoterpeneindolealkaloids(MIAs)representastructurallydiverse,medicinallyessentialclassof

plant-derivedsecondarymetabolitesthathaverecentlybeenproducedinSaccharomycescerevisiae.However,theirreversiblereductionofα,β-unsaturatedcarbonylpathwayintermediatesresultsinanon-

recoverablelossofcarbon,whichhasastrongnegativeimpactonmetabolicflux.Inthisstudy,we

soughttocharacterizeandengineerthedeterminantsofbiocatalyticselectivitythatcontrolfluxtowards

theiridoidscaffoldfromwhichMIAsarederived.Invitroreconstitutionofpreviouslyuncharacterized

shuntpathwaysenabledtheidentificationoftwodistinctroutestoareducedshuntproductincluding

endogenous“ene”-reductionandnon-productivereductionbyiridoidsynthase.Tothisend,deletionof

fivegenesinvolvedinα,β-unsaturatedcarbonylmetabolismresultedinafive-foldincreasein

biocatalyticselectivityofthedesirednepetalactoloverreducedshuntproduct.Weanticipatethatour

engineeringstrategieswillplayanimportantroleinthedevelopmentofS.cerevisiaeforsustainableproductionofiridoidsandMIAs.

Genomeminingoffungalnaturalproducts

YanYan([email protected])

UniversityofCalifornia,LosAngeles,CA,USA.

Naturalproductsaremajorsourcesfordrugdiscovery;however,becauseofincreasingdrugresistance

toexistingmoleculesandadwindlingpipelineofnewdrugleads,ourneedtouncovernovelnatural

productsisbecomingevermorecritical.Hereweproposedandappliedanewstrategy,target-guided

mining,todiscovernewnaturalproductswithdesiredbioactivities.Glyceraldehyde-3-phosphate

dehydrogenase,knownasatargetforanti-anaerobicbacteriaandantimalarialandanti-canceragents,

wasusedasaninputtocarryouttarget-guidedgenomemining,andwesuccessfullydiscoveredand

verifiedaknowninhibitorofthistargetasanoutput.Thisexampleshowsthefeasibilityoftarget-guided

genomeminingofbioactivenaturalproducts.

presentations and meeting abstracts€¦ · deciphering the role of fungal secondary metabolites...

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