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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
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
1
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
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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
7
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
SpeakerPresentations
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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
SpeakerPresentations
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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.
SpeakerPresentations
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
SpeakerPresentations
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
SpeakerPresentations
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.
SpeakerPresentations
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.
SpeakerPresentations
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.
SpeakerPresentations
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
SpeakerPresentations
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.