Piotrowski JS, Okada H, Lu F, Li SC, Hinchman L, Ranjan A, Smith DL, Higbee AJ

14 Pages·2015·2.29 MB·English

Plant-derived antifungal agent poacic acid
β
targets -1,3-glucan
JeffS.Piotrowskia,1,2,HirokiOkadab,1,FachuangLua,SheenaC.Lic,LiHinchmana,AshishRanjand,DamonL.Smithd,
AlanJ.Higbeee,ArneUlbriche,JoshuaJ.Coone,RaameshDeshpandef,YuryV.Bukhmana,SeanMcIlwaina,
IreneM.Onga,ChadL.Myersf,CharlesBoonec,g,RobertLandicka,JohnRalpha,MehdiKabbaged,andYoshikazuOhyab,2
aGreatLakesBioenergyResearchCenter,UniversityofWisconsin–Madison,Madison,WI53703;DepartmentsofdPlantPathologyandeChemistry,University
ofWisconsin–Madison,Madison,WI53706;bDepartmentofIntegratedBiosciences,GraduateSchoolofFrontierSciences,UniversityofTokyo,Kashiwa,
Chiba,Japan277-8561;cRIKENCenterforSustainableResourceScience,Wako,Saitama,Japan351-0198;fDepartmentofComputerScience
andEngineering,UniversityofMinnesota–TwinCities,Minneapolis,MN55455;andgTerrenceDonnellyCentreforCellularandBiomolecularResearch,
UniversityofToronto,Toronto,ON,CanadaM5S3E1
EditedbyDitervonWettstein,WashingtonStateUniversity,Pullman,WA,andapprovedFebruary11,2015(receivedforreviewJune13,2014)
Ariseinresistancetocurrentantifungalsnecessitatesstrategiesto but new compounds continue to be discovered (15). One under-
identifyalternativesourcesofeffectivefungicides.Wereportthe studied class of compounds found in grasses and their hydro-
discoveryofpoacicacid,apotentantifungalcompoundfoundin lysates is the dehydrodiferulates and compounds derived from
lignocellulosic hydrolysates of grasses. Chemical genomics using them (hereafter all simply termed diferulates) (16, 17). In
Saccharomyces cerevisiae showed that loss of cell wall synthesis grasses, diferulates are produced by radical dimerization of fer-
and maintenance genes conferred increased sensitivity to poacic ulatesthatacylatearabinoxylanpolysaccharidesandfunctionas
acid.Morphologicalanalysisrevealedthatcellstreatedwithpoacic powerfulcellwallcross-linkers(16);derivativesofdiferulatesare
acidbehavedsimilarlytocellstreatedwithothercellwall-target- releasedduringthehydrolysisofbiomass(16,18,19).Atpresent,
ing drugs and mutants with deletions in genes involved in pro- thestructuresofarangeofdiferulateshavebeendescribed(16,
cessesrelatedtocellwallbiogenesis.Poacicacidcausesrapidcell 18), but the biological activities of isolated diferulates (beyond
lysisandissynergisticwithcaspofunginandfluconazole.Thecel- their function in the plant cell wall) have not been explored.
lulartargetwasidentified;poacicacidlocalizedtothecellwalland Diferulatesmaybeexpectedtohaveeffectsonorganismsother
inhibitedβ-1,3-glucansynthesisinvivoandinvitro,apparentlyby
than plants. One study found a negative correlation between
directly binding β-1,3-glucan. Through its activity on the glucan
diferulateconcentrationandcolonizationbycorn-boringinsects
layer,poacicacidinhibitsgrowthofthefungiSclerotiniasclerotiorum
(20), but a direct effect of diferulates is unknown. Despite the
andAlternariasolaniaswellastheoomycetePhytophthorasojae.A
well-documented antifungal activity of ferulic acid and its
singleapplicationofpoacicacidtoleavesinfectedwiththebroad-
derivatives(13,21,22),nostudiesontheantifungalpropertiesof
range fungal pathogen S. sclerotiorum substantially reduced le-
diferulates havebeendescribed.
siondevelopment.Thediscoveryofpoacicacidasanaturalanti-
Wescreenedacollectionofdiferulatesfoundinlignocellulosic
fungal agent targeting β-1,3-glucan highlights the potential side
hydrolysatesforantifungalactivityusingtheyeastSaccharomyces
use of products generated in the processing of renewable bio-
cerevisiaeasadiscoverysystemforantifungalagents.Wefocused
mass toward biofuels as a source of valuable bioactive com-
on the diferulate 8–5-DC (16) derived during hydrolysis from
pounds and further clarifies the nature and mechanism of
a major diferulate in grasses; we name this compound here as
fermentationinhibitorsfoundinlignocellulosichydrolysates.
| | Significance
chemicalgenomics high-dimensionalmorphometrics
| |
lignocellulosichydrolysates fungalcellwall Saccharomycescerevisiae
Thesearchfornewantifungalcompoundsisstrugglingtokeep
Lignocellulosics are a potential sugar feedstock for biofuels pace with emerging fungicide resistance. Through chemo-
andbio-basedchemicals.Before plantmaterialscan becon- prospectingofanuntappedreservoirofinhibitorycompounds,
vertedtobiofuelsbyfermentation,theircellwallpolysaccharides lignocellulosic hydrolysates, we have identified a previously
mustbehydrolyzedtosugarmonomersformicrobialconversion undescribedantifungalagent,poacicacid.Usingbothchemical
(1).Thehydrolysisprocessesgenerates,inadditiontothesugars, genomicsandmorphologicalanalysistogetherforthefirsttime,
small acids, furans, and other compounds that affect microbial toourknowledge,weidentifiedthecellulartargetofpoacicacid
growthand inhibit fermentation (2–5). Theinhibitory effects of asβ-1,3-glucan.Throughitsactionontheglucanlayeroffungal
these compounds represent a challenge to efficient microbial cellwalls,poacicacidisanaturalantifungalagentagainsteco-
bioconversion. The primary focus of lignocellulosic-derived in- nomicallysignificantfungiandoomyceteplantpathogens.This
hibitor research has been to understand, evolve, and engineer work highlights the chemical diversity within lignocellulosic
tolerance in fermentative microbes (2). However, as natural hydrolysatesasasourceofpotentiallyvaluablechemicals.
antimicrobialagents,lignocellulosicfermentationinhibitorsoffer
anuntappedreservoir ofbioactivecompounds. Authorcontributions:J.S.P.,H.O.,A.R.,D.L.S.,R.L.,M.K.,andY.O.designedresearch;J.S.P.,
H.O.,F.L.,L.H.,A.R.,A.J.H.,A.U.,andM.K.performedresearch;J.S.P.,H.O.,F.L.,S.C.L.,
Oneincreasinglyimportantpotentialuseoftheseinhibitorsisas
D.L.S.,J.J.C.,C.L.M.,C.B.,J.R.,M.K.,andY.O.contributednewreagents/analytictools;J.S.P.,
antifungal agents. Worldwide, fungicide-resistant pathogens pose H.O.,A.J.H.,R.D.,Y.V.B.,S.M.,I.M.O.,andC.L.M.analyzeddata;andJ.S.P.,H.O.,C.B.,R.L.,
a threat to agricultural sustainability. Pathogen resistance to con- J.R.,andY.O.wrotethepaper.
ventional fungicides affects multiple crops (6, 7). Copper-based Theauthorsdeclarenoconflictofinterest.
fungicidesareeffectiveinorganicagriculturebutfacingrestrictions ThisarticleisaPNASDirectSubmission.
because of copper accumulation in soils (8, 9). Furthermore, cli-
FreelyavailableonlinethroughthePNASopenaccessoption.
matechangeisalteringtheglobaldistributionoffungalpathogens
1J.S.P.andH.O.contributedequallytothiswork.
(10, 11). New sources of fungicides are a necessity to keep pace
2Towhomcorrespondencemaybeaddressed.Email:[email protected]@
withtheevolutionofresistantstrainsandemergingpathogens(12).
k.u-tokyo.ac.jp.
Theantifungalactivitiesofmanyoftheinhibitors(e.g.,ferulic
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
acid and furfural) in hydrolysates have been described (13, 14), 1073/pnas.1410400112/-/DCSupplemental.
E1490–E1497 | PNAS | PublishedonlineMarch9,2015 www.pnas.org/cgi/doi/10.1073/pnas.1410400112
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poacic acid, because it is found primarily in grasses (Poaceae). A P
By applying both chemical genomics and morphological ana-
lysis, we predicted and confirmed that poacic acid binds to cell
wall β-1,3-glucan. We showed its biological activity against not
only yeast but also, the economically important fungal and
oomycete pathogens Sclerotinia sclerotiorum, Alternaria solani,
and Phytophthora sojae.
Results
DiferulateswithAntifungalActivity.Wetestedacollectionofnine
diferulates known to occur in hydrolysates from corn stover for
their effects on S. cerevisiae growth (Fig. 1A and Table S1, de-
tailed nomenclature). Of these, only two had detectable bio-
activity at the tested concentration of 1 mg/mL. In particular,
poacic acid had the greatest antifungal activity, with an IC of
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111 μg/mL (324 μM) against our control yeast (Fig. 1B). This
inhibition is comparable with that of the widely used fungicides
picoxystrobin(IC50of308μM)andpolyoxinD(IC50of340μM) B
andsubstantiallylowerthanthatoftheprimaryfungicideusedin
organicagriculture,coppersulfate(IC of2.4mM) (23–25).
50
ChemicalGenomicsPredictThatPoacicAcidTargetstheFungalCell
Wall. To gain insight into the mode of action and the cellular
targetofpoacicacid,weconductedchemicalgenomicanalysis,a
method that uses genome-wide collections of viable gene-
deletion mutants to identify genes with deletions that confer
sensitivity or resistance to bioactive compounds (26, 27). The
resulting set of sensitive and resistant gene-deletion mutants
associatedwitharesponseyieldsfunctionalinsightintothemode
ofaction(26).Wechallengedapooledmixtureof∼4,000different
Fig.1. Bioactivityofdiferulates.Thebioactivityofninediferulatesagainst
yeast gene-deletion mutants with either poacic acid or a solvent S.cerevisiaeat1mg/mLwastested.Poacicacid(8–5-DC)had(A)thehighest
ceonnabtrloeld(uDsMtoSdOe)c.ipSheeqruthenecrienlgatiovfesftitrnaeinss-sopfeceiaficchDyeNasAtmbuatracnotdeins bioactivityand(B)anIC50of111μg/mL(mean±SE). LOGY
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thepresenceofthedrugrelativetothesolventcontrol(28). BI
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Deletion mutants for genes involved in cell wall and glyco- (Fig. 2C). Additionally, some other cell wall-related gene mu- R
C
sylation-related processes were present in the top significantly tants were resistant to poacic acid. A deletion mutant of NBP2 MI
sensitive and resistant strains (Fig. 2A, blue circles and Table (Nap1 binding protein) was resistant to poacic acid (Fig. 2A);
S2).Amongthetop10deletionmutantssensitivetopoacicacid, Nbp2p down-regulates cell wall biogenesis through an interac-
wedetectedenrichmentforgenesinvolvedinthegeneontology tionwithBck1p,whichisactivatedbyPkc1p(Fig.2B)(31).Thus,
(GO) category fungal-type cell wall organization (P < 0.01). it seems that defects in the PKC pathway confer sensitivity to
These mutants included deletion alleles of BCK1 (bypass of C poacic acid, whereas activation of the PKC pathway confers re-
kinase), which encodes an MAPKKK in the Pkc1p (protein ki- sistance.AdeletionmutantofDFG5(defectiveforfilamentous
nase C) cell wall integrity signaling pathway (PKC pathway); growth)alsowasresistanttopoacicacid;DFG5encodesaGPI-
CWH43,whichencodesamembraneproteininvolvedincellwall anchored protein involved in cell wall biogenesis that also has
biogenesisanditsnullmutationissyntheticallylethalwithPKC1 agenetic interaction withBck1p(32,33).
mutants(29);ROM2(Rho1multicopysuppressor),aGDP/GTP Because the chemical inhibitor of a gene product tends to
exchangefactorforRho1pandanothercomponentofthePKC mimictheloss-of-functionphenotypeofamutantthatinactivates
pathway;andACK1,whichseemstoencodeanupstreamactivator thegene,thechemical–genomicprofileforabioactivecompound
ofPkc1pandhasaphysicalinteractionwithRom2p.Overall,the can resemble the genetic interaction profile for the target (34).
PKC pathway was the most sensitive pathway to poacic acid PKC1 has a genetic interaction profile that is most significantly
(Pathwayzscore=−7.85)(Fig.2B).Thisprofileissimilartothe correlatedtothechemicalgenomicprofileofpoacicacid(Fig.2B)
chemicalgenomicprofilesofotheragentsthattargetthecellwall (P < 0.0001). PKC1 is an essential gene required for growth
andrelatedprocesses(e.g.,caspofungin)(26).Deletionmutantsof and response to cell wall stress, and it has been implicated as
BCK1arehypersensitivetoagentsthatcompromiseglycosylation a key mediator of cell wall-targeting drugs, such as the echi-
(tunicamycin) and cell wall β-1,3-glucan biosynthesis (caspo- nocandins (35). Together, these data narrowed our target
fungin)(26).Weconfirmedthesensitivityoftheindividualbck1Δ searchtothefungalcellwall.Poacicacidcoulddirectlydamage
mutant and found a sixfold reduction in the IC against poacic thecellwall,inhibitakeycellwallsynthesisenzyme,ordisrupt
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acidcomparedwiththecontrolstrain(WT)(Fig.2C). the PKC pathway.
Among the top significantly resistant strains, we detected
significantenrichmentfordeletionsofgenesinvolvedintheGO MorphologicalAnalysisRevealedThatPoacicAcidAffectstheFungal
categoryglycosphingolipidbiosyntheticprocess(P<0.01)driven Cell Wall. We investigated the morphological changes induced
by csg2Δ (calcium sensitive growth) and sur1Δ (suppressor of by poacic acid using high-dimensional microscopy (36, 37). Re-
Rvs161andrvs167mutations)(Fig.2A,yellowcirclesandTable cently, two morphological features (a wide neck and morpho-
S2). Deletion of glycosphingolipid genes has been shown to ac- logical heterogeneity) were reported as common phenotypes in
tivate the PKC pathway and cell wall biogenesis (Fig. 2B) (30). cells treated with agents known to affect the cell wall (38). The
Involvement of SUR1 in poacic acid sensitivity was confirmed morphologies of cells exposed to poacic acid had both features
when we isolated a spontaneous chain-termination mutant in (Fig.3AandB);theydisplayeddose-dependentincreaseinbud
SUR1abletoformcoloniesonagarwith500μgpoacicacid/mL neck size (Fig. 3C) and heterogeneous morphologies (Fig. 3D).
Piotrowskietal. PNAS | PublishedonlineMarch9,2015 | E1491
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Fig.2. Chemicalgenomicsofpoacicacid.(A)Treatmentoftheyeastdeletioncollectionwithpoacicacidrevealedthatmutantsinvolvedincellwallbio-
synthesisandglycosylationweresensitiveandresistanttothecompound(blue).Mutantsingenesinvolvedinglycosphingolipidbiogenesiswereamongthe
mostresistant(yellow).(B)ThePKCpathway,whichgovernscellwallintegritysignaling,wasthemostsensitivepathway(Pathwayzscore=−7.85),withmany
membersandinteractinggenesshowingsensitivitytopoacicacid(chemicalgeneticinteractionscoreinparentheses).Comparisonwiththeyeastgeneticin-
teractionnetworkindicatedthatthegeneticinteractionprofileoftheessentialgenePKC1wasmostsignificantlycorrelatedtothechemicalgenomicprofileof
poacicacid(P<0.001).(C)MutantsofthegeneencodingthecellwallsignalingkinaseBCK1weresixfoldmoresensitivetopoacicacid,whereasapoacicacid-
resistantmutant(PAr)withanSNPinSUR1hadincreasedresistancecomparedwiththecontrolstrain(meanandSEbarswereremovedforclarity).
Because mutants displaying a high correlation with a drug phe- bypoacicacidistothatinducedbyothercellwall-affectingdrugs,
notypecanhelpidentifytargetedprocesses,wenextcomparedthe we compared their morphological profiles. Two echinocandins
morphology of poacic acid-treated cells with the individual mor- (micafungin and echinocandin B), both of which bind Fks1p, had
phologiesof4,718yeastdeletionmutants(37,39,40).Forty-three morphologicalprofilesthatwerehighlycorrelatedwitheachother,
deletion mutants had morphological profiles statistically similar whereas poacic acid-treated cells had lower morphological corre-
(P<0.01)tothoseofpoacicacid(Fig.3EandTableS3).Within lationswiththeseandothercellwall-affectingcompounds(Fig.4C).
thetopcorrelations,wefoundsignificantenrichmentofgenesin Thus, although there is some morphological similarity with other
theGOcategorytransferaseactivity,transferringhexosylgroups cellwallagents,themorphologicalresponsetopoacicacidsuggests
(P < 0.001). This GO category contained genes responsible for thatitmayhaveamodeofactionthatisdifferentfromthatofother
key processes in the cell wall biogenesis, such as OCH1, which cellwall-targetingagents.
encodesamannosyltransferasethatinitiatespolymannoseouter
chain elongation, and FKS1 (FK506 sensitivity), which encodes PoacicAcid CausesRapid Cell Leakage.Cell wall-targeting agents,
a catalytic subunit of β-1,3-glucan synthase. Taken together, such as echinocandins, can lead to compromised cell integrity
thesedatafurtherindicatethatpoacicacidaffectstheyeastcell and ultimately, cell lysis from turgor pressure (45). We inves-
wall,consistentwiththe chemicalgenomicanalysis. tigatedwhetherpoacicacidcausedcelllysisinasimilarway.We
testedtheextentofcellpermeabilityafter4hoftreatmentwith
Poacic Acid Is Synergistic with Drugs That Target the Cell Wall and poacic acid, caspofungin, methyl methanesulfonate (MMS), or
MembraneIntegrity.Giventhatpoacicacidmaydirectlytargetthe DMSO using a propidium iodide dye that is taken up only by
cellwallortheintegritysignalingpathway,wetestedwhetherthe cells with compromised cell integrity. MMS, an agent that does
mode of action of poacic acid differed from that of the echino- not cause rapid cell wall damage, was included as a negative
candincaspofungin.Echinocandinsdamagetheyeastcellwallby control.Wefoundthatbothcaspofunginandpoacicacidcaused
noncompetitive bindingof theβ-1,3-glucan synthasecomplex at rapidcellleakage,whereasMMSandDMSOdidnot(Fig.4D).
the Fks1p subunit (41, 42). Synergistic interactions occur with When growth was arrested by depriving cells of a carbon
drugs targeting the same or a functionally related pathway but source,theeffectofcaspofunginwasdiminished,supportingthe
through different targets (43). We found significant synergistic known mode of action of echinocandins, which inhibit glucan
effects(Fig.4A)betweenpoacicacidandcaspofungin(P<0.05). synthesis.Theeffectsofpoacicacidwerereducedinnonactively
Thisinteractionsuggeststhatpoacicacidtargetsthecellwallbut growingcells,butleakagewasstillsignificantlygreater(P<0.05)
doessothroughamechanismdistinctfromthatofcaspofungin. thaninallothertreatments(Fig.4D).Themechanismbywhich
Becauseechinocandinsarealsosynergisticwithantifungalazoles poacic acid causes leakage is lessened without active growth,
that target ergosterol biosynthesis and compromise membrane showingthatthecompoundcanstillcauseleakageinnonactively
integrity (44), we tested and determined that poacic acid also growing cells, unlike with echinocandins. This result could in-
displayedsignificantsynergywithfluconazole(Fig.4B)(P<0.01). dicate a general disruption of cell wall integrity rather than an
enzymatic targetandthus,adifferentmode ofaction.
PoacicAcid-InducedMorphologiesAreUniqueComparedwithThose
from Other Cell Wall-Targeting Agents. Compounds that induce PoacicAcidLocalizestotheCellSurfaceandTargetsβ-1,3-Glucan.We
similar morphological responses can be indicative of similar next sought to determine to which cell wall component poacic
modesofaction.Todeterminehowsimilarthemorphologyinduced acidbindsbylocalizingthecompoundintreatedcells.Asaferulate
E1492 | www.pnas.org/cgi/doi/10.1073/pnas.1410400112 Piotrowskietal.
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Fig.3. Morphologicalcharacteristicsofpoacicacid-treatedcells.Poacicacidtreatmentcaused(AandB)abnormalcellmorphologyand(CandD)mor- O
BI
phologicalcharacteristicssimilartothosecausedbyothercellwall-targetingagents.Poacicacid-treatedcellshad(C)adose-dependentincreasedbudneck O
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sizeand(D)heterogeneityofcellmorphology.(E)Thephenotypeofpoacicacid-treatedcellswashighlycorrelatedwiththephenotypesofmutantsingenes C
involvedintransferaseactivitytransferringhexosylgroups(P<0.001).TheredlineindicatesanadjustedPvalueof<0.01. MI
derivative, poacic acid is fluorescent, enabling us to visualize buds,itdoesnotchangemannoproteinstainingwithfluorescent
its accumulation at the cell surface (Fig. 5A). Based on this dye-conjugatedConA(Fig.S1),whichsuggeststhatpoacicacid
result together with poacic acid’s chemical genomic profile, acts primarilyonthe formation of theglucan fibrils rather than
morphological profile, phenotypic similarity to fks1Δ, and ability byinhibiting mannoprotein assemblyinthecell wall.
tocausecellleakage,wehypothesizedthatpoacicacidtargetsthe
β-1,3-glucan layer and thus, rapidly compromises cell integrity, PoacicAcidIsanInhibitorofFungalandOomycetePlantPathogens.
leadingtocelllysiswhenturgorpressureburststheweakenedcell As a plant-derived natural product, poacic acid may have a po-
wall. The absence of chitin-related genes in both the chemical tential use in organic agriculture, which is presently lacking in
genomic and morphological profiles and the low correlation be- fungicide diversity beyond copper sulfate mixtures. We initially
tween the morphological profile of poacic acid and the chitin testedtheeffectsofpoacicacidonS.sclerotiorum,anascomycete
targetingcompoundnikkomycinZledustobelievethatthechitin fungalpathogenwithanextremelybroadhostplantrange(>400
layer is not the cell wall target of poacic acid. Furthermore, species)andworldwidedistribution.Insoybeans,S.sclerotiorum
theuniformstainingpatternwithpoacicacidisdifferentfromthe
causes Sclerotinia stem rot or white mold of soybean. The in-
calcofluorwhitestainingofchitin,whichpreferentiallybindsthe
corporationofpoacicacidintoculturemediacausedasignificant
budneck andbudscar. (P < 0.01) dose-dependent decrease in fungal growth both on
Wesuggestthatthemodeofpoacicacidactionisdistinctfrom
agar plates and in liquid cultures, which was evidenced by
that of the echinocandins, acting through direct binding to the
decreasesincolonyradialgrowthandfungalmass(Fig.6A).We
glucan fibrils rather thaninhibition ofglucan synthase. This hy-
further investigated whether poacic acid could inhibit lesion de-
pothesis is supported by observations that poacic acid does not
velopmentinplantaondetachedsoybeanleaves.Solvent(DMSO)
localizespecificallytothesiteofbudgrowth,likeFks1p(46),but
control or poacic acid (500 μg/mL) solutions were applied to de-
rather,bindsacrosstheentirecellsurface(Fig.5A).Poacicacid
caninhibitβ-1,3-glucansynthesisinvivoasshownbysignificantly tachedleavesbeforeinoculationwithagarplugscontainingactively
decreased glucan staining in buds (Fig. 5B and Fig. S1) and growingmyceliaofS.sclerotiorum.Lesiondevelopmentwasmoni-
significantlyless14C-glucoseincorporationintotheβ-1,3-glucan tored daily up to 120 h postinoculation. Poacic acid treatment
layer after poacic acid treatment (Fig. 5C). We also observed markedlyreducedlesiondevelopmentoverthistimecoursecom-
an in vitro inhibition of β-1,3-glucan synthesis after poacic acid paredwiththecontrol(Fig.6BandC).Wealsofoundpoacicacid
treatment (Fig. 5D). By incubating purified yeast glucan with to be similarly effective against the ascomycete A. solani, which
poacic acid and observing fluorescence, we found that poacic causesearlyblightintomatoandpotatocrops(Fig.S2).Thesedata
aciddirectlybindsβ-1,3-glucan(Fig.5E).Furthermore,although showthatpoacicacidinhibitsfungalgrowthinvitroandinplanta,
poacic acid can reduce aniline blue staining of β-1,3-glucan in withpromisingagriculturalapplications.
Piotrowskietal. PNAS | PublishedonlineMarch9,2015 | E1493
A B and therefore, represents a previously undescribed compound tar-
getingβ-1,3-glucan.Althoughwefoundno effects of poacic acid
on mannoprotein assembly, direct binding of glucan fibrils
outsidetheplasmamembranemayalsoresultininhibitionofcell
wall assemblages, such as the glucan-transglycolase Gas1p and
thechitintransglycosylasesCrh1pandUtr2p,thatconnectchitin
chainstoglucans,whichrequireglucanasasubstrateorcosubstrate.
In nature, diferulates strengthen monocot cell walls by cross-
linking polysaccharides (arabinoxylans) to each other and poly-
saccharides to lignin—in both cases by radical coupling mecha-
nisms (16, 17). It remains unknown if poacic acid has a similar
interaction with the fungal cell wall polysaccharides. However,
this work raises the question about whether diferulates may
C D haveadualroleasantifungalsecondarymetabolitesthatconfer
protectiontocertainplants.Otherdiferulatesmayhavethesame
potential to bind glucan, but the size/physical properties of
poacicacidmaycontribute toitsmoreoptimalbioactivity.
Against yeast, the bioactivity of poacic acid is similar to the
widelyusedfungicidepicoxystrobin(IC of308μM),lowerthan
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thiabendazole (IC of 607 μM), and considerably more toxic
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thancoppersulfate(IC of2.4mM)(25).Poacicacidmayalso
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have potential to be used combined with agricultural azoles
throughitsdocumentedsynergismtoslowthedevelopmentof
azole resistance. Although there are conventional fungicides
effective at lower doses(e.g., captan at IC of 19μMand pro-
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chloraz at IC of 132 μM) (25), most conventional agents are
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Fig. 4. Synergisms and the mode of action of poacic acid. (A) Poacic specific to either the Eumycota or Oomycota, whereas poacic
acid(125μg/mL)issignificantlysynergisticwithcaspofungin(12.5ng/mL). acid affects both. Options for organic agriculture are limited to
(B)Poacicacid(125μg/mL)isalsosynergisticwithfluconazole(3.8μg/mL).
copper-basedfungicides,whicharefacingincreasingrestrictions
(C)Morphologicalsimilaritybetweenpoacicacidandothercellwall-affecting
because of copper accumulation in soil ecosystems (8, 9). Fur-
agentswasmeasuredbasedonthecorrelationcoefficientvalue(R)oftheir
morphological profiles. (D) Poacic acid causes cell leakage within 4 h of thermore, as a natural plant-based phenolic acid, poacic acid
treatment, similar to the cell wall-targeting compound caspofungin. The would likely be rapidly broken down in the soil and would not
leakage is most apparent in actively growingcells [yeast extract peptone accumulate (50). Field trials, application strategies (e.g., seed
dextrose(YPD)]comparedwithcellsarrestedwithoutacarbonsource[yeast treatmentvs.foliarspray),andmorediversepathogentestswill
extractpeptone(YP)].DMSOandMMSwereincludedascontrolagentsthat be necessary to determine its performance as an agricultural
donot directly affect cellwallintegrity. Inarrested cells, poacic acidhad
significantlygreatercellleakagethanothertreatments.One-wayANOVA
andTukey’stestwereusedtocalculatethedifferencesbetweentreatments
(mean±SE).PA,poacicacid. A B C
Fungi generally have 30–80% glucan in their cell walls (47);
similarly,oomyceteshaveacellwallcontainingβ-1,3-glucanand
β-1,6-glucan,butunlikefungi,oomycetewallscontainacellulose
layer rather than chitin. Oomycetes are broadly distributed,
economicallysignificantpathogens,andanaturalfungicidethat
couldaffectbothtruefungiandoomycetes bydisruption ofthe
glucan layer could be of high value. We found that poacic acid
significantlyreducescolonygrowthoftheoomyceteP.sojae(Fig. D E
6D)(P<0.01),awidespreadpathogenthatcausesrootandstem
rot of soybeans. Given its effectiveness against both fungi and
oomycetes, poacic acid may have potential as a plant-derived
fungicidewithbroadaction.
Discussion
Through chemoprospecting of lignocellulosic hydrolysates, we
have identified a promising antifungal agent. Combining chem-
ical genomic and morphological analyses, we determined that
poacic acid targets β-1,3-glucan within fungal cell walls. In-
hibition of glucan synthesis in vivo and in vitro and cell-wide Fig.5. Poacicacidtargetsβ-1,3-glucan.(A)Poacicacidisfluorescentand
localization and direct binding of purified glucan indicate that accumulates on the cell wall. Poacic acid inhibits β-1,3-glucan in vivo as
the compound can bind to β-1,3-glucans in the growing glucan shownby(B)thedecreaseinsignalfromanilinebluestaining(arrowheads)
fibrilsaswellasthematurewall.ThecellwalldyeCongoredmay and(C)theincorporationof14C-labeledglucoseintotheβ-1,3-glucanlayer
ofthecellwall(P<0.05).Concentrationsofpoacicacid,echinocandinB,and
also bind growing glucan fibrils (48), but it also binds to chitin,
hydroxyureawere250μg/mL,4μg/mL,and30mM,respectively.(D)Poacic
and biochemical evidence indicates that the primary target of
Congo red is chitin (49). Poacic acid targets the β-1,3-glucan (aEc)idPoinahciibciatsciβd-1d,i3r-egcltulycabninsdysntphuarsifeieadctyiveiatystignluvictarno.wStituhdeannt’IsC5t0teosft3w1aμsgu/mseLd.
layeroffungalcellwallsinamannerdistinctfromthatofother to determine significant differences (mean ± SD). DIC, differential inter-
cell wall-affecting agents (e.g., caspofungin and nikkomycin Z) ferencecontrast.
E1494 | www.pnas.org/cgi/doi/10.1073/pnas.1410400112 Piotrowskietal.
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DetailedmethodsareprovidedinSIMethods.
Chemical Genomic Analysis. Chemical genomic analysis of poacic acid was
performed asdescribed previously (26, 53). Thetested yeast deletion col-
lection had ∼4,000 strains using the genetic background described by
Andrusiak (54). The optimal inhibitory concentration of poacic acid for
chemical genomic profiling [70–80% growth vs. solvent control in yeast
extract-peptone-galactosemediumafter24hofgrowth]wasdetermined
using an eight-point dose curve. A concentration of 88 μg/mL inhibited
C D growthwithinthisrange;200-μLculturesofthepooleddeletioncollection
ofS.cerevisiaeweregrownwith88μg/mLpoacicacid(n=3)oraDMSO
controlintriplicatefor48hat30°C.GenomicDNAwasextractedusingthe
EpicentreMasterPureYeastDNAPurificationKit.Mutant-specificmolecular
barcodeswereamplifiedwithspeciallydesignedmultiplexprimers(28).The
barcodesweresequencedusinganIlluminaMiSeq.Replicatesofeachcon-
dition,poacicacid(n=3)orDMSO(n=2),weresequenced.Thebarcode
countsforeachyeastdeletionmutantinthepresenceofpoacicacidwere
compared to the DMSO control conditions to determine sensitivity or re-
sistanceofindividualstrains(thechemicalgeneticinteractionscore)(26).To
determineaPvalueforeachtopsensitiveandresistantmutant,weusedthe
EdgeRpackage(55,56).ABonferroni-correctedhypergeometricdistribution
testwasusedtosearchforsignificantenrichmentofGOtermsamongthe
Fig. 6. Poacic acid inhibits the growth of fungal and oomycete plant
top 10 sensitive and resistant deletion mutants (57). To understand the
pathogens. (A) Colony growth on plates and mycelia weight of S. scle-
pathwaysthatweremostaffectedbypoacicacid,wedevelopedaprotein
rotiorum(strain1980)inliquidcultureweresignificantlyinhibitedbypoacic
complex/pathwayscorebasedonthesummationofthezscores foreach
acidinadose-dependentmanner.(BandC)Asingleaerosoltreatmentof
poacicacid(500μg/mL)beforeinoculationinhibitedwhitemoldlesionde- complex/pathway (Pathway z score). Correlation of the chemical genomic
profileofpoacicacidwiththeyeastgeneticinteractionnetworkwasper-
velopment on soybean leaves in planta. (D) Representative photographs
weretaken96hpostinoculation.Poacicacidsignificantlyinhibitedcolony formedaspreviouslydescribed(34).
growthofP.sojae(fieldisolate7dofgrowth).DashedcirclesinInsetin-
dicatethemyceliumfrontafter2dofgrowth.One-wayANOVAandTukey’s Multivariate Morphological Analysis. Cells of budding yeast S. cerevisiae
testwereusedtocalculatethedifferencebetweendrugtreatmentsamong (BY4741his3Δ::KanMX;hereafter,his3Δ)wereculturedin2mL1%Bacto
treatments(mean±SE). yeastextract(BDBiosciences),2%Bactopeptone(BDBiosciences),and2%
glucose(YPD)with0,25,50,75,100,or125μg/mLpoacicacidoraDMSO Y
G
controlat25°Cfor16huntiltheearlylogphase.Themaximumconcen- O
L
fungicideandevaluateitspersistenceintheenvironment,butit trationofthedrug(125μg/mL)wasdeterminedbasedonthegrowthin- BIO
may also provide an important and abundant lead compound hibitionrates(10%).Cellfixation,staining,andobservationwereperformed RO
thatcouldbemodified forincreasedefficacy. (n=5)asdescribedpreviously(37).Imagesofcellshape,actin,andnuclear MIC
Although it was identified from lignocellulosic hydrolysates DNAwereanalyzedusingtheimageprocessingsoftwareCalMorph(version
1.2),whichextractedatotalof501morphologicalquantitativevaluesfrom
for bioethanol fermentation, poacic acid alone is not likely to
atleast200individualcellsineachexperiment(37).Imageswereprocessed
beaprimaryinhibitoraffectingfermentation givenitsrelatively
usingPhotoshopCS2(AdobeSystems)forillustrativepurposes.
low concentration (0.1 μM) (Table S4). However, it could be
To assess the morphological similarity between the cells treated with
synergisticwithotherinhibitoryagents(e.g.,furfuralandphenolics)
poacicacidandnonessentialdeletionmutantsorcellstreatedwithothercell
or other diferulates (e.g., 8–5-C) (2, 16). The combined effects of wall-affecting drugs, their morphological profiles were compared as de-
diferulates with other inhibitors could significantly affect lignocel- scribed previously (36). To identify functional gene clusters, the most sig-
lulosic biofuel synthesis by fungi, but such combinatorial effects nificant similar mutants (43 genes, P < 0.01 after Bonferroni correction,
haveyettobetested. ttest)wereselectedasaqueryforGOtermanalysis(GOtermfinderinthe
The complementary profiling methodologies that we applied Saccharomycesgenomedatabase).
to the analysis of poacic acid’s effects, including chemical ge- Toextractindependentandcharacteristicfeaturesofmorphologyinduced
nomic profiling and morphological profiling, are powerful and bypoacicacid,atwo-stepprincipalcomponentanalysiswasperformedas
can provide high-resolution predictions of targeted processes; described previously (39). Tocompare phenotypic noise in the yeast pop-
thisworkhighlightsthepowerofthecombinedapproach.Given ulation(poacicacidvs.DMSO),thenoisescorewascalculatedasdescribed
previously(58).
theincreasingthroughputofbothtechniquesthankstoadvances
in next generation sequencing and automated microscopy, the
MeasurementofinVivoβ-1,3-GlucanSynthesis.Inhibitionofinvivoβ-1,3-glucan
useofbothgeneticandmorphologicalapproachesinlarge-scale
synthesis was measured as described previously with slight modification
screeningofdruglibrariesmayallowunbiasedwhole-celltarget (n=3)(59).Yeastcells(his3Δ)weregrowninYPDtoearlylogphaseat25°C.
identification with less reliance on target-centric high-through- Theculturedcellsweredilutedto1×107cellsper1mLwith1mLof
putscreening methods. YPDmediumcontainingone-tenththeglucosecontaining23.125kBq
Thisstudywasdesignedtoidentifynovelbioactivecompounds [14C]glucose (ARC0122; American Radiolabeled Chemicals) and test com-
from lignocellulosic hydrolysates. Given the goal of cellulosic pounds[250μg/mLforpoacicacid,4μg/mLforechinocandinB,30mMfor
ethanol production [60 billion L/y by 2022, requiring 0.6–1.2 hydroxyurea(negativecontrol),or0.4%(vol/vol)DMSOasasolventcontrol].
trillion L hydrolysate/y assuming 5–10% (vol/vol) ethanol before Thecellswereradiolabeledbyculturingat25°Cfor2h.Thelabeledcells
distillation] (51, 52), even low-abundance compounds within wereharvestedandincubatedwith1NNaOHat80°Cfor30min.Theinsoluble
hydrolysates could be available in significant quantities. We have pellets were resuspended in 10 mM Tris·HCl, pH 7.5, containing 5 mg/mL
zymolyase100T(Seikagaku)andincubatedat37°Cfor18h.Afterdigestion,
detected monomeric ferulate in hydrolysates at markedly higher
thezymolyase-resistantmaterialwasremovedbycentrifugation(15,000×g
levels (up to 1.7 mM in alkaline H O -treated corn stover). If
2 2 for15min),andthezymolyase-degradationproduct(mostlyβ-1,3-glucan)
synthesizedfromtherecoveredferulatecomponentposthydrolysis,
waspurifiedbyultrafiltrationwithacentrifugalfiltermembrane(Amicon
poacicacidmayconfergreatervaluetolignocellulosicconversion.
Ultra0.5mL;molecularweightcutoffis10,000;Millipore).Theflow-through
Our results highlight the chemical diversity within lignocellulosic fractionwasmixedwithscintillationmixture(UltimaGold;PerkinElmer),and
hydrolysatesasasourceofpotentiallyvaluablechemicals. radioactivitywasmeasuredbyascintillationcounter(LSC-6100;Aloka).The
Piotrowskietal. PNAS | PublishedonlineMarch9,2015 | E1495
differencesintheincorporationratesinsampleswerenormalizedbyΔOD600 was measured. Inhibition of S. sclerotiorum in planta was tested by in-
measuredbeforeandafterthelabelingperiod. oculating detached soybean leaves of the commercial variety Williams 82
withanagarplugofactivelygrowingS.sclerotiorummycelia.Leaveswere
Measurement of β-1,3-Glucan Synthase Activity in the Membrane Fraction. treatedonetimebeforeinoculationwitheitheranaerosolsprayofwater
After the membrane fraction was prepared from S. cerevisiae BY4741 as withDMSO(control)ora500μg/mLsolutionofpoacicacid.Leaveswerein-
describedpreviously(n=3)(60),β-1,3-glucansynthaseactivitywasmeasured cubatedinamoistenvironment,andlesiondevelopmentwasmonitoredupto
asdescribedpreviously(n=3)(61)withslightmodification.Briefly,20μL 120hpostinoculation.FieldstrainsofP.sojaeandA.solaniweregrownon
membranefraction(∼70μgtotalprotein)wasaddedtoareactionmixture cornmealandpotatodextroseagarplates,respectively,atroomtemperature
(finalvolumeof100μL)containing50mMTris·HCl,pH7.5,10mMpotassium for 7 and5 d,respectively,beforemeasurement. The growthinhibition of
fluoride, 1 mM EDTA, 0.2 mM UDP-Glc (with 89 Bq UDP-[Glucose-14C]; poacicacidwasassessedat0,500,1,000,and1,500μg/mLinreplicateplates
NEC403;PerkinElmer),anddifferentconcentrationsofpoacicacid(1.25,2.5, (n=3).Agarplugsfromactivelygrowingcultureswereplacedatthecenterof
5,10,20,40,80,160,or320μg/mL).Thereactionmixturewasincubatedat theplatesandallowedtogrowatroomtemperature.Colonydiameterwas
25°Cfor30minandstoppedbytheadditionofethanol.Totrapreaction monitoredforeachtreatmentinatime-courseexperiment.One-wayANOVA
product(β-1,3-glucanpolymer),thereactionmixturewasfilteredthrough andTukey’stestwereusedtocalculatethedifferencesbetweendrugtreat-
themembranefilter(mixedcelluloseesters;0.2μminporesize;ADVANTEC), mentsamongtreatments.
washedonetimewith2mLdistilledwater,anddriedatroomtemperature.
Afteradditionofscintillationmixture(Econofluor-2;PerkinElmer),radioac- ACKNOWLEDGMENTS. We thank T. K. Sato, E. Hendel, S. Morford, and
tivitywasmeasuredbyascintillationcounter(LSC-6100;Aloka).Inhibition N. Keller for critical discussions of the manuscript. J.S.P., F.L., L.H., A.J.H.,
A.U., J.J.C., S.M., I.M.O., and J.R. are funded by Department of Energy
curves and IC50 values were determined using R software (ver. 3.0.1) by (DOE)GreatLakesBioenergyResearchCenterDOEBiologicalandEnviron-
sigmoidalcurvefittingwiththeglmfunction.
mentalResearchOfficeofScienceGrantDE-FC02-07ER64494.J.S.P.,F.L.,and
M.K. are supported by Wisconsin Alumni Research Foundation Award
Growth Inhibition of Plant Pathogens. To test inhibition in liquid culture, MSN178899.H.O.isaresearchfellowoftheJapanSocietyforthePromotion
adosecurveof0,125,250,and500μg/mLpoacicacidin100mLpotato ofScience.S.C.L.issupportedbyaRIKENForeignPostdoctoralFellowship.
dextrosebroth(n=3)wasused.CulturesofS.sclerotiorumstrain1980were A.R.,D.L.S.,andM.K.aresupportedbyWisconsinSoybeanMarketingBoard
inoculatedwith100μLhomogenizedmyceliaandgrownat25°Cfor48h. GrantMSN172403.R.D.andC.L.M.aresupportedbyNationalInstitutesof
HealthGrants1R01HG005084-01A1,1R01GM104975-01,andR01HG005853
Themyceliaintheliquidmediaweredriedandweighed.Thegrowthin-
and National Science Foundation Grant DBI 0953881. C.L.M. and C.B. are
hibition of poacic acid on solid agar cultures (potato dextrose agar) was
supportedbytheCanadianInstituteforAdvancedResearchGeneticNetworks
assessedbygeneratingreplicateplates(n=3)containing0,125,250,and Program.M.K.issupportedbyUnitedSoybeanBoardGrantMSN143317.Y.O.
500μg/mLpoacicacid.Plateswereinoculatedwithanactivelygrowingplug issupportedbyMinistryofEducation,Culture,Sports,ScienceandTechnology,
ofS.sclerotiorumandgrownat25°C.Themycelialradialgrowthafter48h JapanGrantforScientificResearch24370002.
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Piotrowskietal. PNAS | PublishedonlineMarch9,2015 | E1497
Supporting Information
Piotrowski et al. 10.1073/pnas.1410400112
SIMethods 0.6.2) (3) using the default parameters, with the exception of
Compounds,InitialScreening,andGrowth.Thediferulatecompounds thefractionofmissingalignmentsthreshold,whichwassetat
tested were synthesized as described by Lu et al. (1) and resus- 0.08(–ninbwaaln).SNPandindeldetectionwereperformed
pended in DMSO. Caspofungin, nikkomycin Z, and MMS were with the Genome Analysis Toolkit (GATK version 1.4) (4)
purchased from Sigma-Aldrich. Echinocandin B was a gift from followingtheirbestpracticevariant-callingworkflow(https://www.
O.Kondo(ChugaiPharmaceuticals,Tokyo,Japan).Micafungin broadinstitute.org/gatk/).Duplicatereadsweremarkedfollowedby
was provided by Astellas Pharma. Diferulates were initially base quality recalibration using a single nucleotide polymorphism
screenedataconcentrationof1mg/mLtodeterminebioactivity. databasedesignedfor S.cerevisiae.Tominimizefalse-positivevar-
CellsofSaccharomycescerevisiae(MATαpdr1Δ::natMXpdr3Δ:: iant calls, stringent parameters were used: namely, the minimum
KI.URA3snq2Δ::KI.LEU2can1Δ::STE2pr-Sp_his5lyp1Δhis3Δ1 base quality required to consider a base for calling was 30, and
leu2Δ0ura3Δ0met15Δ0),referredtoasthecontrolstrain,were the minimum phred-scaled confidence threshold for genotype
grownin200-μLculturesat30°CinYPDwithadrugorDMSO calling was 50 (–mbq and –scc in the UnifiedGenotyper tool).
CustomPerlscriptswereusedtofurtherfiltercallsonthebasis
control.PlateswerereadonaTECANM1000overa48-hgrowth
of read depth, mapping quality, and strand bias. This analysis
period.ThespecificgrowthratewascalculatedusingGCATanal-
revealedanSNPinthegeneSUR1(glutamate>stopcodon)in
ysis software (https://gcat3-pub.glbrc.org/) (2). When presented,
the poacicacid-resistant mutant.
IC values for growth rate inhibition were calculated from trip-
50
licate eight-point dose curves and SigmaPlot 12.0. When pre-
sented,errorbarsaremeans±SEsofatleastthreereplicates. CellLeakageAssays.AFungaLight CellViability Assay(L34952;
Invitrogen)usingaGuavaFlowCytometer(Millipore)wasused
to determine if poacic acid caused membrane damage. The
DeterminingtheMostSensitivePathwayThroughChemicalGenomics.
populationofstainedcells(damagedintegrity)vs.nonstained
A complex/pathway score based on chemical genomic data to
cellscanbedeterminedbyflowcytometry.Caspofungin(50ng/mL)
identifyproteincomplexesorpathwayswasdevelopedbasedon
wasincludedasapositivecontrol.MMSandDMSOwereincluded
which members showed significant deviation in their chemical
asanoncellwall-targetingcontrolandasolventcontrol,respectively.
genetic interactions in the presence of a compound. For each
To test the effects of the compounds on both active and arrested
complex,thechemicalgeneticinteractionscoreofthegenesinthe
cells,log-phasecultureswerewashedwith1×PBSandresuspended
complex with the compound was summed.To determine signif-
toanOD ineitherYPDmediumorYP(nocarbonsource)inthe
icance,theexpectationsforsuchasumforrandomsetsofgenesof 0.5
presenceofthedrugs(n=3)for4hat30°C.Thecellswerethen
equal size were calculated. The random sets of equal size were
stainedandimmediatelyreadbyflowcytometry.One-wayANOVA
expectedtohavemeansequaltothebackgroundmeanandSDs andTukey’stestwereusedtocalculatethedifferencebetweendrug
equal to the background SD/sqrt(n). With this information, a z
treatmentsamongcellswitharrestedgrowth.
score (number of SDs from the expected mean) for each com-
plexor pathwaycan be computed: SynergyScreening.To test for synergy, a 6 × 6-dose matrix was
initially used to identify potentially synergistic dose combina-
Pathway z score=ðΣ=n−μÞ=ðσ×sqrtðnÞÞ;
tions, and these points were then confirmed in triplicate. Cul-
tures (200 μL) were grown with combinations of poacic acid
where Σ = sum of the chemical genetic interaction scores of (125μg/mL),caspofungin(12.5ng/mL),andfluconazole(3.8μg/mL),
genesinthecomplex,μ=meanofthechemicalgeneticinterac-
and the ODs of relevant single-agent and solvent controls were
tion scores of the compounds with all genes studies, σ = SD of
measured after 24h. Synergy was determined by comparing ac-
chemical genetic interaction scores of the compounds with all
tual OD in the presence of compound combinations with an
genesinthe study, andn =size of the complex.
expected value calculated using the multiplicative hypothesis.
Thismethodassumesthat,intheabsenceofaninteraction,each
Isolation, Sequencing, and Evaluation of Drug-Resistant Mutants.
compound would decrease the OD of the cell culture by the
Agar containing 500 μg/mL poacic acid was inoculated with ∼1
samefractioninthepresenceoftheothercompoundasitdoes
million cells of yeast (control strain). After 1 wk, two colonies when applied alone (that is, E = A × B/C, where E is the ex-
were found growing on the agar. Single-colony isolates were pected OD, A is OD when compound A is applied alone, B is
obtained and found to be resistant to poacic acid. For whole- OD when compound B is applied alone, and C is OD of the
genome sequencing, single-colony isolates of poacic acid-
control culture (DMSO). In the presence of synergy, the actual
resistant mutant, the caspofungin-resistant mutant, and the con- OD value is lower than the expected OD. A paired t test was
trol strain (WT) were grown in triplicate 200-μL cultures and
usedtoconfirmstatisticalsignificanceofthisdifferenceinthree
pooled for genomic DNA extraction (Epicentre MasterPure
replicates ofthe experiment.
Yeast Kit; MPY80200). The genomic DNA was prepared for
Illuminawhole-genomesequencingusingtheIlluminaTruSeqKkit Staining of Cells with Poacic Acid. Log-phase yeast cells (his3Δ)
(FC-121-3001) and sequenced by 150-bp paired-end reads on the were harvested by centrifugation, washed two times with PBS,
MiSeqplatform. sonicatedmildly,andthen,incubatedwith0.25%(wt/vol)poacic
To determine mutations in the drug-resistant mutants, read acid for 5 min. A small aliquot of the cells was mounted on
qualityanalysiswasperformedusingFastQC(www.bioinformatics. a glass slide and observed under an Axioimager M1 Fluores-
babraham.ac.uk/projects/fastqc/). Short reads were examined for cenceMicroscope (CarlZeiss)using theXF09FilterSet(Opto
quality and trimmed at the 3′ end when average base quality in a Science; excitation wavelength, 340–390 nm; emission wave-
3-nt window fell below Q30. Short reads were mapped to the length,517.5–552.5 nm).
standard S. cerevisiae reference genome, strain S288c (obtained
from the National Center for Biotechnology Information RefSeq MannoproteinandGlucanStaining.β-1,3-Glucan was stained with
repository),usingBurrows-WheelerAlignment(BWAversion anilineblue(016-21302;WakoChemicals)asdescribedpreviously
Piotrowskietal.www.pnas.org/cgi/content/short/1410400112 1of6
(5)withslightmodification.Briefly,log-phaseyeastcells(his3Δ) and observed under a fluorescent microscope using a regular
were cultured in YPD with poacic acid (125 μg/mL) at 25 °C. DAPI filter set (Carl Zeiss).
Then,cellswerecollectedat0,2,4,and6haftertreatmentand
stainedwithanilinebluewithoutfixationasdescribedpreviously Determination of Ferulate and Diferulates by Reverse-Phase HPLC–
(6).CellsmountedonaglassslidewereexposedtoUVfor30s High-Resolution/AccurateMSinHydrolysates.Ammoniafiberexpan-
to bleach out poacic acid fluorescence before acquiring images. sion treated corn stover hydrolysates samples were diluted 1:10,
Staining of chitin or mannoproteins with calcofluor white (F3543; and20-μLsampleswereanalyzedbyreverse-phase(C18)HPLC–
Sigma-Aldrich)orAlexa594-ConA(C11253;LifeTechnologies), high-resolution/accurate MS. Peak areas of peaks matching in
respectively, was performed as described previously (6). For retentiontimeandaccuratemass±10ppmofauthenticreference
cell-free glucan staining, yeast glucan (G0331; Tokyo Chem- standards were used to calculate concentrations by comparison
ical Industry) was suspended to 0.125% (wt/vol) poacic acid withanexternalstandardcurve.
1.LuF,WeiL,AzarpiraA,RalphJ(2012)Rapidsynthesesofdehydrodiferulatesviabiomimetic 4.DePristoMA,etal.(2011)Aframeworkforvariationdiscoveryandgenotypingusing
radicalcouplingreactionsofethylferulate.JAgricFoodChem60(34):8272–8277. next-generationDNAsequencingdata.NatGenet43(5):491–498.
2.SatoTK,etal.(2014)HarnessinggeneticdiversityinSaccharomycescerevisiaeforim- 5.WatanabeD,AbeM,OhyaY(2001)YeastLrg1pactsasaspecializedRhoGAPregu-
provedfermentationofxyloseinhydrolysatesofalkalinehydrogenperoxidepre- lating1,3-β-glucansynthesis.Yeast18(10):943–951.
treatedbiomass.ApplEnvironMicrobiol80(2):540–554. 6.OkadaH,OhyaY(2015)ColdSpringHarborProtocols(ColdSpringHarborLabPress,
3.LiH,DurbinR(2009)FastandaccurateshortreadalignmentwithBurrows-Wheeler Plainview,NY).
transform.Bioinformatics25(14):1754–1760.
Fig.S1. Poacicacidtreatmentreducesglucanstainingwithanilinebluebuthasnoeffectonmannoproteinstraining.Thecontrolstrainyeastcells(his3Δ)
weregrowninYPDat25°Cuntilearlylogphase,transferredtofreshYPDmediumcontainingpoacicacid(125μg/mL)orDMSO[0.125%(vol/vol)]asasolvent
control,andculturedfor6h.Thecellswerecollected,andthecellwallcomponentsmannoproteinswerestainedwithAlexa594-conjugatedConAfollowedby
β-1,3-glucanstainingwithanilineblue.Thecellswereobservedunderafluorescentmicroscope,andover150buddingcellswerecountedaccordingtothe
stainingsignalfromthreeindependentexperiments.AStudent’sttestwasusedtodeterminesignificantdifferences(mean±SE;n=3).
Piotrowskietal.www.pnas.org/cgi/content/short/1410400112 2of6