Xenomic networks variability and adaptation traits in wood decaying fungi.

1.4 MB·English

by  Morel, Melanie

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Xenomic networks variability and adaptation traits in
wood decaying fungi
MélanieMorel,1,2*EdgarMeux,1,2YannMathieu,1,2 Introduction
AnneThuillier,1,2KamelChibani,1,2LucHarvengt,3
Understanding the evolution of organisms and their
Jean-PierreJacquot1,2andEricGelhaye1,2
adaptation to environmental constraints remains a major
1UniversitédeLorraine,IAM,UMR1136,IFR110
challenge for biologists. The recent development of
EFABA,Vandoeuvre-lès-Nancy,F-54506,France.
sequencingtechnologieshasledtoanewscience‘Com-
2INRA,IAM,UMR1136,Vandoeuvre-lès-Nancy,
parative genomics’. Gene copy number variation (CNV)
F-54506,France.
has been observed from yeast (Carreto etal., 2008) to
3Laboratoiredebiotechnologie,PôleBiotechnologieet
human (Redon etal., 2006). These CNVs result from
SylvicultureAvancée,FCBA,CampusForet-Boisde
duplication/deletion events. Duplication events can also
Pierroton,33610Cestas,France.
be followed by functional diversification, this process
being one of the most important mechanisms leading to
Summary new functions (Ohta, 1991; Force etal., 1999). The cor-
relationbetweenthesegeneticvariationsandphenotypic
Fungal degradation of wood is mainly restricted to
consequencesremainsoftenunclear.Themostambitious
basidiomycetes, these organisms having developed
studiesassociatinggeneticandphenotypicvariationsare
complex oxidative and hydrolytic enzymatic sys-
currently conducted in mammals in connection with the
tems. Besides these systems, wood-decaying fungi
important implications in human health (Zhang etal.,
possessintracellularnetworksallowingthemtodeal
2009; Yalcin etal., 2011). In contrast few studies have
with the myriad of potential toxic compounds result-
been devoted to fungal CNVs (Tirosh and Barkai, 2007;
ing at least in part from wood degradation but also
Liti etal., 2009; Ellison etal., 2011;Taylor, 2011). During
more generally from recalcitrant organic matter deg-
the last years, several programmes have been launched
radation. The members of the detoxification path-
to describe the functional genomic diversity of fungi, the
ways constitute the xenome. Generally, they belong
relativelysmallsizeoftheirgenomebeingcompatiblewith
to multigenic families such as the cytochrome P450
the development of environmental genomic approaches
monooxygenases and the glutathione transferases.
(Sharptonetal.,2009;Martin,2010;Neafseyetal.,2010;
Taking advantage of the recent release of numerous
Ohm etal., 2010; Duplessis etal., 2011; Murat etal.,
genomes of basidiomycetes, we show here that
2011).
these multigenic families are extended and function-
Fungi are major actors in global geochemical cycles,
allyrelatedinwood-decayingfungi.Furthermore,we
particularly in the carbon and nitrogen cycles, because
postulate that these rapidly evolving multigenic
of their involvement in organic matter recycling. Fungi
families could reflect the adaptation of these fungi
play also major roles in different ecosystems through
to the diversity of their substrate and provide keys
their symbiotic or pathogenic interactions with other
to understand their ecology. This is of particular
organisms.
importance for white biotechnology, this xenome
During the last few years, many sequencing projects
being a putative target for improving degradation
have been devoted to wood-decaying fungi (Martinez
properties of these fungi in biomass valorization
etal., 2004; 2009; Eastwood etal., 2011), due to their
purposes.
potential applications in white biotechnology in general,
and in bioenergy production from biomass in particular.
Comparative genomic has confirmed the extraordinary
Received17September,2012;revised5November,2012;accepted diversity of enzymatic mechanisms developed by these
8 November, 2012. *For correspondence. E-mail mmorel@
fungi to degrade recalcitrant organic matter and in par-
scbiol.uhp-nancy.fr;Tel.[phone];Fax[phone].
MicrobialBiotechnology(2013)6(3),248–263 ticular lignocellulose. Recent research has mainly
doi:10.1111/1751-7915.12015 focused on the oxidative systems secreted by white-rot
Funding Information This work was supported by the Agence
and brown-rot fungi, and also on the myriad of fungal
Nationale de la Recherche (ANR) research grant (ANR-09-BLAN-
0012). enzymes involved in the polysaccharide-degrading
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd.Thisisanopenaccessarticleunder
thetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedthe
originalworkisproperlycited.
Fungalxenomicnetworks 249
enzymes (Cazymes). Besides these extracellular probably important (Cornwell etal., 2009). For instance,
systems, it appears that gene families involved in the the decomposition of softwood is slower than hardwood
detoxification pathways are particularly extended in in a given environment, suggesting that the chemical
wood-decaying fungi. These genes constitute the composition, which is highly variable between these
xenome, being defined as the biosystem responsible for wood types, could be an important factor governing
the detection, transport and metabolism of xenobiotics wood decay (Weedon etal., 2009; Freschet etal.,
(Edwards etal., 2005). Fungi as other organisms have 2012). Wood is a complex structure, its composition
to deal with potential toxic compounds resulting from depending both on the species considered and on envi-
organic matter degradation, secondary metabolism of ronmentalfactors,whichhaveinfluencedthetreegrowth
antagonist organisms, and human activities. It is parti- (Chave etal., 2009). Wood susceptibility to fungal
cularly true for wood-interacting fungi, which are able degradation is indeed governed at least in part, by its
to degrade recalcitrant lignin and to cope with the high content in lignin and other phenolic derivatives
myriad of complex compounds synthesized by plant compounds.
species. The efficient fungal degradation of lignin is mainly
This article presents the fungal xenomic network with restricted to specialized basidiomycetes. These latter
a comparative genomic analysis of two xenomic multi- are able to modify/decompose lignin through oxidative
genic families found in wood decaying fungi. We have processes (see below), which could lead to aromatic
focused on the cytochrome P450 (CytP450) family, radicalscatalysingsubsequentdegradationandalsofor-
which is involved in the first step of compound modifi- mation of potential toxic molecules. Besides the well-
cation mainly through oxidation reactions (phase I) and known effect linked to lignin, secondary metabolites
the glutathione transferase (GST) family, which belongs involved in plant defence could also play a major role in
to the phase II conjugating enzymes and for which func- the durability of the various wood species (Chave etal.,
tional data are becoming available (Meux etal., 2011; 2009; Freschet etal., 2012). The tree primary defence
Thuillier etal., 2011; Mathieu etal., 2012). In the litera- involves usually bark that provides a physical and
ture, studying GST is usually justified because of the chemical barrier against abiotic and biotic stresses.
considerable interest of these enzymes in medicine, Besides these constitutive defences, induced systems
agriculture and analytical biotechnology. For example, in are activated after injury or infection (Franceschi etal.,
medicine, GSTs could be molecular targets for the 2005). For instance in conifers, induced defence con-
designofnewanticancerdrugs.Inagriculture,GSTsare cerns cell wall re-enforcements and production of
exploited in the development of transgenic plants with secondary metabolites as terpenes, phenols, stilbenes,
increased resistance to biotic and abiotic stresses, or flavonoids and lignans (Ralph etal., 2006; Danielsson
they could be used as biosensors for monitoring envi- etal., 2011). These changes in chemical composi-
ronmental pollutants, such as herbicides and insecti- tion and their heterogeneity have potentially important
cides (Chronopoulou and Labrou, 2009). In this review, ‘afterlife’ effects on the fungal efficiency in wood
we focus the analysis on fungal GSTs, which have been degradation.
far less studied than their plant and mammals counter-
parts, postulating that these enzymes and more globally
the xenome could be new targets of investigations in
Fungalxenome:thephaseInetwork
white biotechnology.
Facing this large diversity of nutriments and potential
toxic component sources, fungi, which mainly control
Woodcompositionanddegradation
wood decomposition in forest ecosystems, have also
Wood is mainly constituted of cellulose, a variety of concomitantly evolved modified detoxification pathways,
hemicelluloses, lignin and to a lesser extent of second- defining the xenome (Edwards etal., 2005). As in other
ary chemicals (wood extractives). Wood decomposition eukaryotic organisms, the detoxification pathways
is a complex process, which, like litter degradation, dealing with recalcitrant/toxic compounds can be divided
involves biotic and abiotic factors (Cornwell etal., 2009; into three different steps in fungi, the first one corre-
Weedon etal., 2009; Freschet etal., 2010). Numerous sponding to an activation of the molecule often through
studies suggest that plant traits have predictable long- an oxidation stage, the second one to a conjugation
termeffectsonlitterdecomposition(Hobbie,1996;Corn- stage, and the third one either to transport or to storage
well etal., 2008) and wood degradation (Cornwell etal., (Fig.1). In this article, we will focus mainly on the oxi-
2009; Weedon etal., 2009; Cornelissen etal., 2012). dation and conjugation steps found in wood-decaying
Among the factors, which lead to ‘afterlife effects’ on its fungi through the determination of their xenomic gene
degradation, wood chemical and anatomical traits are content in relation to their environment.
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
250 M.Moreletal.
ppllaanntt mmaatteerriiaallss hhuummaannaaccttiivviittiieess
RReeccaallcciittrraanntt//TTooxxiicc
ccoommppoouunnddss
EExxttrraacceelllluullaarrooxxiiddaattiioonn
HHOO pphhaassee II:: iinnttrraacceelllluullaarraattttaacckk
22 22
EExx:: CCyyttoocchhrroommee PP445500
:: PPeerrooxxiiddaasseess,, llaaccccaasseess,,
FFee22++ OOxxiiddaattiioonn,, rreedduuccttiioonn,,
ooxxiiddaasseess hhyyddrroollyyssiiss,,ddeehhaallooggeennaattiioonn
FFee22++//HH OO :: FFeennttoonn rreeaaccttiioonn
22 22
AAccttiivvaatteeddrreeccaallcciittrraanntt
ccoommppoouunnddss
FFuurrtthheerr
phase II: conjugation
ccaattaabboolliissmm
Ex: GST
GGlluuttaatthhiioonnyyllaattiioonn,, aacceettyyllaattiioonn,,
MMeetthhyyllaattiioonn,, gglluuccoorroonniiddaattiioonn
IInntteerrmmeeddiiaattee
mmeettaabboolliitteess
vacuole
pphhaassee IIIIII:: eexxccrreettiioonn,,
ssttoorraaggee
FFuunnggaallcceellll
EExxccrreettoorryyddeerriivvaattiivveess
Fig.1. Thefungaldetoxificationsystemdefinedas‘xenome’.
Theextracellularnetwork via the Fenton reaction (Goodell etal., 1997; Eastwood
etal., 2011).These radicals are involved in lignin modifi-
Inthecaseofwooddecayingfungi,thefirstphasecould cation and in polysaccharide degradation. In contrast,
be mediated by the extracellular oxidative network white-rotfungiareabletomineralizelignin,theoxidative
involvedinlignin/woodmodification.Wood-decayingfungi degradation of the polymer being mainly mediated
could be mainly divided into two main functional groups, through the production of various oxidases and peroxi-
brownandwhiterot,dependingontheirabilitytodegrade/ dases(Hofrichteretal.,2010;Harmsetal.,2011).Glyco-
mineralize lignin. The brown-rot fungi are able to modify syl hydrolases are mainly involved in polysaccharide
lignin to gain access to cellulose and hemicellulose.The degradationthroughhydrolyticprocesses(Martinezetal.,
recent release of the genomes of Postia placenta and 2004; 2009). Whatever the functional group considered,
Serpula lacrymans has confirmed previous biochemical the oxidative stage exhibits low substrate specificity,
worksdemonstratingthatthisstepismainlymediatedby explaining the well-studied potential of these fungi for
non-enzymatic reactions in these fungi (Martinez etal., bioremediation purposes (Harms etal., 2011). In particu-
2009; Eastwood etal., 2011). Brown-rot fungi use small lar,white-rotbasidiomycetesareabletodegradealarge
phenolic compounds to reduce iron and produce hydro- panel of environmental pollutants (Asgher etal., 2008).
genperoxide,prerequisitestepstoformhydroxylradicals Further information concerning the extracellular enzy-
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
Fungalxenomicnetworks 251
matic networks involved in lignin breakdown can be In P.chrysosporium, it is assumed that these versatile
obtained in recent reviews dealing with this topic CytP450s have emerged from different ancestral genes
(Sanchez, 2009; Wong, 2009; Hofrichter etal., 2010; due to extensive gene duplication events and intragen-
Buggetal.,2011;Hernandez-Ortegaetal.,2012). omic recombinations suggesting an evolution driven by
the adaptation of the fungus to wood degradation (Dod-
dapaneniandYadav,2005;Hirosueetal.,2011).Froma
ThecytochromeP450monooxygenases
generalpointofviewmainlyresultingfromgenomicdata,
The oxidative degradation of wood components leads to itisnowbecomingobviousthatthenumberandrangeof
the formation of a variety of compounds with potential CytP450s within an individual organism reflect, to some
toxic effects depending on the initial substrate composi- extent, its lifestyle (Schuler, 2011).Transcriptional analy-
tion and also on the oxidative attack. To deal with these ses confirm the role of various CytP450s in wood
compounds, fungi possess a large number of CytP450. degradation/mineralization for both white-rot and brown-
These enzymes, found in all life forms, are able to rotfungi.Forinstance,inthebrown-rotP.placenta,where
catalysevariousreactionsashydroxylation,dealkylation, 230genesarerelatedtoCytP450,differentialinductionof
sulfoxidation etc. and could be involved in primary, sec- 15encodingCytP450geneswasobserveddependingof
ondary and detoxification metabolisms. These haem- thewoodspeciesincubatedwiththefungalculture(aspen
thiolate proteins have first been discovered in liver versus pine) (Vanden Wymelenberg etal., 2010). In a
microsomes (Omura and Sato, 1962) through their similar way, in the white-rot Phanerochaete carnosa, 21
involvement in complex insertions of molecular oxygen genes encoding CytP450s are found strongly regulated
into different substrates. General reviews on CytP450s according to wood species (coniferous or deciduous
have been published (Paine, 1995; Mizutani and Ohta, wood) (MacDonald etal., 2011). All these data are in
2010;NelsonandWerck-Reichhart,2011;Schuler,2011; agreementwiththefollowingstatementmadebyHirosue
Ichinose,2012). andco-workers:‘molecularevolutionofbasidiomycetous
In fungi as well as in other organisms, CytP450s have CytP450 has been, at least in part, vigorously driven by
been shown to be involved in primary metabolism as survivalstrategiestoprovideasuperiormetabolicsystem
CYP61andCYP51inergosterolbiosynthesis(Lepesheva to degrade exogenous chemicals, presumably involving
andWaterman,2007)andalsoinsecondarymetabolism plant-relatedcompoundsincludingligninand/oritsderiva-
asforinstanceinaflatoxinsynthesisinaspergilli(Yuetal., tives’(Hirosueetal.,2011).
2004). Fungal CytP450s are also involved in detoxifica-
tion pathways as shown in the basidiomycete Phanero-
Fungalxenome:thephaseIInetwork
chaetechrysosporiumwherevariousisoformshavebeen
shown to be involved in polycyclic aromatic hydrocarbon Tocopewiththecompoundsresultingfromtheactivityof
oxidation (Syed etal., 2010; Hirosue etal., 2011). In phaseIenzymes,fungipossesslargenetworksofphase
basidiomycetes, the CytP450ome ranges from four (the II conjugative enzymes. It is widely admitted that conju-
pathogen Cryptococcus neoformans) to over 250 gation enhances compound solubility and usually
sequences(thebrown-rotP.placenta),thenumberofiso- decreasestheirreactivity.Theresultingconjugatescould
forms apparently increasing with the ability to form myc- be excreted, further degraded or stored. These phase II
orrhizae and to degrade plant litter (Cresnar and Petric, enzymescatalysetheformationofsugar(glucoside,xylo-
2011). It is assumed that the high number of CytP450 sideorglucuronide),sulfate,glutathione,acetylormethyl
isoforms found in wood decaying fungi could reflect the conjugates.Thedifferentconjugates(and/orthedifferent
ability of these fungi to metabolize and mineralize aro- activities) have been detected during the degradation of
maticcompoundsresultingfromwoodextracellularoxida- variouscompoundsinvariousfungalspecies(Sutherland
tion. The functional diversity of CytP450s has been etal., 1991; Bezalel etal., 1997; Hundt etal., 2000;
investigated in particular in P.chrysosporium, where 154 Reddy and Gold, 2000; Myung etal., 2008; Campoy
CytP450-encoding genes have been identified in this etal.,2009;Ningetal.,2010).Furthermore,asproposed
white-rot basidiomycete (Martinez etal., 2004; Doddap- fortheirplantcounterparts,thedifferentconjugatescould
aneniandYadav,2005;Chiguetal.,2010;Hirosueetal., be rapidly processed preventing their detection (Dixon
2011).FourteenPcCytP450sisoformsareabletooxidize etal., 2010). Conjugation enzymes exhibit a broad spe-
anthracenetoanthroquinoneandnumerousisoformsare cificityacceptingvarioussubstrates,whicharestructurally
versatileenzymesacceptingabroadrangeofsubstrates different.Despitethislowspecificity,transferasesbelong
with varying three-dimensional structures (Hirosue etal., to multigenic families suggesting a specialization of the
2011). In contrast to CytP450 essential to primary various isoforms. For instance, more than 100 putative
metabolism, the isoforms involved in detoxification path- glycosyltransferase-encoding genes are found in the
waysareusuallylessspecific(CresnarandPetric,2011). brown-rot P.placenta (Eastwood etal., 2011). Neverthe-
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
252 M.Moreletal.
Fig.2. Phylogenetictreeofglutathione
GGGTTTTTT222 transferasesfromBasidiomycetes.Various
PPPHHHIII subclassescouldbedistinguished:Ure2p,
GSTFuA,Omega(GSTO),GlutathionylHyd-
roquinoneReductase(GHR),Phi,GTT1and
GTT2.Thesequenceswereretrievedfrom
genomesavailableontheJointGenome
Institute(http://www.jgi.doe.gov/).Sequence
GGGTTTTTT111 alignmentsweredonebyCLUSTALWandthe
treewasconstructedwiththeneighbour-
joiningmethodinMEGA5.0software
(Tamuraetal.,2011).Therobustnessofthe
brancheswasassessedbythebootstrap
methodwith500replications.Thescale
markerrepresents0.2substitutionsper
residue.
UUUrrreee222ppp
GGGSSSTTTFFFuuuAAA
GGGHHHRRR
GGGSSSTTTOOO
0.2
less, such a high number of glycosyltransferases is not domain, which is more or less conserved, is involved in
specific to wood-decaying basidiomycetes since similar glutathione binding (G-site). The C-terminal domain is
contentsarefoundinpathogenic/symbioticascomycetes morevariableandisinvolvedinthebindingofsubstrates
(Klosterman etal., 2011), with nevertheless differential (H-site).ThenomenclatureofcytosolicGSTsisbasedon
distributionwithinspecificclassesaccordingtothewayof amino acid sequence identity and at least eight classes
life (Sprockett etal., 2011). Similarly, GSTs form a large have been described to date in fungi and named GTT1,
ubiquitous multigenic family. The number of isoforms GTT2, URE2p, MAK16, EFb1, Etherase-like recently
found in fungi range from six in Saccharomyces cerevi- renamedGSTFuA(Mathieuetal.,2012),GSTOandGHR
siaetomorethan50,thehighestnumberbeingfoundin (McGoldricketal.,2005,Moreletal.,2009)(Fig.2).Tra-
the wood/litter decaying fungi (Morel etal., 2009). ditionally, two proteins belong to the same class if they
Besides these large families, the number of fungal sharemorethan40%identityandisoenzymesbelonging
arylamineN-acetyltransferases(NAT)rangedfromoneto to different classes share less than 20% (Hayes etal.,
five paralogues in fungi, NAT genes being lost in many 2005). Nevertheless, based on these primary sequence
species of the higher Agaricomycotina (Martins etal., criteria only, many ‘non-canonical’ GST groups have
2010). Concerning sulfotransferases, few studies have emerged especially in bacteria and fungi, increasing the
been devoted to the diversity of these enzymes in fungi, complexityoftheGSTsclassification.Afewproteinfamilies
butneverthelessthisdetoxificationpathwayseemstobe areusuallyclassifiedasGSTs(EF1Bg,MAK16),butthisis
restrictedtoascomycetes. ratherbasedonstructuralsimilarities,notontheexistence
of a glutathione-dependent activity. In addition, further
immunologic,genetic,structuralandfunctionalinvestiga-
Fungalglutathionetransferases
tions could reveal unexpected similarities between
Soluble GSTs are usually dimeric enzymes, each mono- enzymesfirstlistedindifferentgroups(Meuxetal.,2012).
mer being composed of two domains. The N-terminal ThisemphasizesthechallengeofGSTclassification.
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
Fungalxenomicnetworks 253
otot PP.. pplalacceennttaa
n rn r
ww
oo
BrBr CC.. ppuutteeaannaa
PP.. ccaarrnnoossaa
otot
e re r FF. .mmeeddiitteerrrraanneeaa
hithit
WW
PP.. cchhrryyssoossppoorriiuumm
ss
erer CC.. cciinneerreeaa
ss
oo
pp
mm
oo
ecec AA.. bbisisppoorruuss
DD
cc HH.. ccyylliinnddrroossppoorruumm
otioti
bibi
mm
yy
SS LL.. bbiiccoolloorr
nn MM.. llaarriicciiss--ppooppuulliinnaa
ee
gg
oo
hh
atat
PP CC.. nneeooffoorrmmaannss
0 5 10 15 20 25 30 35 40 45
Gene Copy number
GSTFuA Ure2p GTT1 GTT2 PHI GHR GSTO
Fig.3. OrganizationoftheGSTomeinselectedfungi.GSTisoformnumbersforeachsubclassarereported.Therepartitionhasbeendone
basedonthesequencesretrievedfromtheJGIcoupledtophylogeneticanalyses.
It is largely admitted than the main activity of these Fig.3. We have investigated the diversity of GSTs in
enzymes is the catalysis of a glutathione adduct onto saprophytic, symbiotic and pathogenic fungi (number of
electrophilic molecules, the specificity of these enzymes genesaregiveninsupplementarydata)andhighlighteda
remaining usually very weak. However, two classes of huge diversity in term of GST number and repartition
GSTs called omega (GSTO) and glutathionyl hydroqui- within the different classes. While the number of GST is
none reductase (GHR) possess in their catalytic site a higherinsaprophyticfungi,nocorrelationcouldbeestab-
cysteinylresidueconferringtotheseproteinstheabilityto lished between the trophic mode of the fungi and their
removeglutathionefromvariousstructurallydifferentmol- GSTcontent.
ecules(Garceraetal.,2006;Xunetal.,2010;Meuxetal.,
2011). These enzymes are highly versatile being able to
GTTsandphiclasses
switchactivitywhenchangingonlyoneorfewaminoacids
in the protein sequence (Mannervik etal., 2009). This The GTTclass has been defined for GlutaThione Trans-
propertyresultsinalargediversityofactivitiesevenwithin ferase, since they correspond to the first characterized
asingleclass,theenzymesbeingabletoexhibitglutath- GST in S.cerevisiae (Choi etal., 1998). In yeast, it is
ionylation, deglutathionylation or peroxidase activity with composedofGTT1andGTT2.Bothproteinsexhibitactiv-
variousstructurallydifferentsubstrates. ity against classical GST substrates as 1-chloro-2,4-
Taking advantage of the recent release of several dinitrobenzene (CDNB). ScGTT2 also catalyses the
basidiomycete genome sequences provided by the Joint formation of glutathione-Cd conjugates (Adamis etal.,
GenomeInstitute(http://www.jgi.doe.gov),wedescribein 2004),whileScGTT1catalysesthereductionofhydroper-
this review the occurrence of the main fungal cytosolic oxides, in particular cumene hydroperoxide probably in
GSTsclasseswithinfungiwithvariouswaysoflife.Some the endoplasmic reticulum (Choi etal., 1998; Herrero
selectedspeciesandtheirGSTdistributionareshownin etal., 2006). ScGTT1 and ScGTT2 have overlapping
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
254 M.Moreletal.
functions with glutaredoxins (ScGrx1 and ScGrx2) being Ure2p4 and Ure2p6 of P.chrysosporium have been bio-
involvedintheresponsetooxidativestress(Marianietal., chemically characterized (Thuillier etal., 2011). The cor-
2008). Phylogenetic analyses concerning ascomycetes, responding genes are recent duplicates, the sequences
zygomycetesandbasidiomyceteshaveshownthatGTT1 beingtandemlyorganizedinthegenomewith83%amino
and GTT2 homologues cluster separately (Fig.2) (Choi acid sequence identity. However the proteins do not
etal., 1998; McGoldrick etal., 2005; Morel etal., 2009). exhibit the same activity pattern, Ure2p6 being much
ThenumberofGTT1andGTT2isoformsrangesfrom0to more active than Ure2p4 in both GST and thiol disulfide
4forGTT1and0to5forGTT2(supplementalTableS1). oxidoreductaseactivities.
A separate cluster has been identified in the present Ure2pB is less expanded and more ubiquitous than
phylogenetic analysis (Fig.2). Since the amino acid Ure2pA (from one in most of the organisms to four
sequences show similarity to the plant-related phi class, sequences in Agaricus bisporus, Coniophora puteana,
wenamedthisnewgroupGSTFforphi.Inparticular,the PhlebiabrevisporaandBotryobasidiumbotryosom)andis
N-terminalpartofsomebasidiomycetesequencesexhibit also present in bacterial genomes. Two isoforms (YfcG
around 75% of similarity with Poplar and Arabidopsis and YchU) have been characterized in Escherichia coli
GSTFs. In fungi, the number of GSTF-encoding genes (Kanaietal.,2006).Bothhavethioldisulfideoxidoreduct-
differs considerably depending on the genome consid- aseactivityandcontrarytotheyeastisoform,weakGST
ered, ranging from 0 to 10. The higher number (10 and peroxidase activities (Wadington etal., 2009; Stour-
isoforms) is found in Botryobasidium botryosum, Hydno- manetal.,2011).
meruliuspinastri,ConiophoraputeanaandSerpulalacry- TheexpansionanddiversificationofUre2pinAgarico-
mans (supplemental Table S1). A proteomic study mycotina could be linked to environmental conditions.
revealed upregulation of P.chrysosporium phi isoform Transcriptomicanalysesrevealedspecificupregulationof
uponadditionofexogenousapplicationofvanillin,oneof someUre2p-encodinggenesinresponsetovariouswood
the key intermediates found during lignin degradation species or aromatic compounds.As examples, P.chrys-
(Shimizuetal.,2005).However,sincenoisoformofGTT osporiumUre2p9andUre2p6geneexpressionisinduced
and phi has been characterized to date in basidiomyc- in presence of aspen and pine respectively (Vanden
etes,theirphysiologicalroleremainsunclear. Wymelenberg etal., 2011). In the same species Ure2p4
andUre2p6showspecificexpressionafterpolycyclicaro-
matic compound treatments (Thuillier etal., 2011). Simi-
Ure2pclass
larly, 8 Ure2p genes are upregulated by fir in P.carnosa
GSTs belonging to this class have been found only in (MacDonaldetal.,2011).
fungiandbacteria(exceptinStramenopilesandDictyos-
teliida).InS.cerevisiae,Ure2pactsbothasaregulatorof
GSTFuAclass
nitrogencatabolicrepression,andasastress-responsive
protein(CoschiganoandMagasanik,1991;Blinderetal., This class is only found in microbes (bacteria and fungi)
1996;Cooper,2002).Theproteinhasbeenwellstudiedin (Morel etal., 2009). The proteins are related to Lig pro-
yeast for its particularity to form prions (Wickner, 1994). teinsofSphingobiumsp.SYK-6(Masaietal.,1989).The
The additional amino acids in the N-terminal part of the Lig coding genes are organized in operons, which
protein responsible for this property have been exclu- containsLigE,LigFandLigG.Thecorrespondingproteins
sivelyfoundinsomeyeastspecies,suggestingfunctional are involved in the cleavage of b-aryl ether linkages,
specializationofUre2pintheseorganisms. whichisthemostabundantbondfoundinlignin.SpLigE
By analysing ascomycete and basidiomycete and SpLigF are enantioselective GSTs involved in
sequences, it has been shown that this class could be cleavageofb-arylethercompounds,likeguaiacylglycerol
split into two subclasses called Ure2pA and Ure2pB. In b-O-4-methylumbelliferone (GOU) to produce b-
each subclass, the sequences cluster according to the hydroxypropiovanilloneand4-methylumbelliferoneinvitro
taxonomy suggesting a recent diversification of Ure2p (Otsukaetal.,2003).SpLigGisaglutathionelyasecata-
sequences (Thuillier etal., 2011). The analysis of the lysingglutathioneremovalfromtheglutathioneconjugate
fungal genomes reveals that only Schizophyllum producedbySpLigF(Masaietal.,2003).
commune does not possess any Ure2 sequence, sug- The number of isoforms is highly variable within and
gestinganimportantroleofthisclassamongbasidiomyc- amongfungalphylaanditsdistributionisnotcorrelatedto
etes (supplemental Table S1). Ure2pA is fungal specific fungal taxonomy but rather to the trophic state of fungi.
and the number of isoforms is highly variable among Thisclassisindeedhighlyrepresented(between4and19
species ranging from 0 in many species to 20 in P.car- isoforms) in wood interacting fungi (saprotrophic, litter-
nosa. It is also interesting to note that species with the decomposingandnecrotrophicfungi)whereasitisabsent
highernumberofUre2pAisoformsaresaprophyticfungi. intheanalysedbiotrophicpathogens(supplementalTable
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
Fungalxenomicnetworks 255
S1).Interestinglyectomycorrhizalfungialsoexhibitahigh ScGTO2 is active towards several hydroquinone deri-
numberofisoforms(from3to10)inaccordancewiththe vates (Lam etal., 2012) and its expression is strongly
observationthatsomeofthesefungimayliveasfaculta- induced after exposure with stress agents, such as
tive saprobes (Talbot etal., 2008). Unlike other fungal cadmium,hydroperoxideordiamide(Barretoetal.,2006),
GSTs depicted here, the sequences cluster according to suggesting an involvement in oxidative stress response.
theorganismandnospecificsubclasscouldbeidentified Based on sequence similarity and functional data
(Fig.2). obtained with quinone derivatives, the yeast GSTOs iso-
forms could in fact be part of the glutathionyl hydroqui-
nonereductases.
Cysteine-containingGSTs
Cysteine-containingGSTsarerelativelyancientmembers
AdirectlinkbetweenGSTomeandP450ome
of the cytosolic GST superfamily widespread in several
kingdoms and phyla [Omega (O) in mammals, fungi and Comparative analysis of genome sequence data is an
insect; S-glutathionyl hydroquinone reductase (GHR) in important tool to reveal adaptation of organisms to spe-
fungiandprokaryotes;Lambda(L)andDHARinplants]. cificenvironments.Networksinvolvedindrugmetabolism
Although they exhibit the canonical thioredoxin fold, all and by extension in recalcitrant compound degradation/
these subclasses have a cysteine residue in their active detoxificationareparticularlyconcernedbyCNVs.
site,whereotherGSTsexhibitaserineoratyrosine.This Considering fungi from various phyla, a significant
cysteine changes dramatically the reactivity profiles of expansion of the total number of GST has been high-
these enzymes compared with the classical GSTs. They lighted in Agaricomycotina (Fig.4A and supplemental
do not catalyse conventional conjugation reactions and TableS2).ThisCNVisindependentoftheglobalnumber
instead use GSH as a cofactor rather than co-substrate. of gene models. This result can be correlated with a
Cysteine-containing GSTs have been described to previousstudyperformedontheCytP450family(Cresnar
catalyse glutathione-dependent reductions and thiol and Petric, 2011). These authors showed that predomi-
transferase reactions. They have been identified in all nantly yeast-form fungi such as Saccharomycotina have
basidiomycete genomes studied here, whatever their a small P450ome, while mycorrhizal relationships and
trophicproperties. complexnutrientdegradationmainlyperformedbyAgari-
GSTOs are subdivided into two subclasses (II and III) comycotina seem to enhance the P450ome size. Based
andsequencesarefoundinallsymbioticandsaprophytic onthisobservationandthedataobtainedfromtheFungal
basidiomycete genomes with variability in their isoform Cytochrome P450 database (http://p450.riceblast.snu.
number, while they were not detected in biotrophic fungi ac.kr/species.php),adirectcorrelationbetweenP450ome
like Melampsora larici populina, Puccinia graminis or and GSTome contents can be observed suggesting a
Malassezia globosa suggesting an evolution of the GST close link between phase I and phase II detoxification
drivenbytheadaptationofthefungustoitshabitat(sup- processes (Fig.4B). However, this correlation with
plemental Table S1). GHR sequences cluster separately CytP450 does not exist when considering the GST
and have been recently described as a new subclass of classes individually. This suggests that compensatory
enzymescatalysingtheremovalofglutathionefromhyd- effects between GST gene copy numbers could exist to
roquinoneconjugates(Xunetal.,2010;BelchikandXun, relaythefirstdetoxificationstep.Bothproteinfamiliesare
2011;Meuxetal.,2011).OnetofourGHRisoformshave largelyexpandedinwood-interactingfungi.Forinstance,
beendetectedinthestudiedfungalgenomes,exceptfor the symbiotic Laccaria bicolor exhibits a weak ability to
M.globosaandP.graminis,forwhichnoGHRsequence degrade recalcitrant organic matter in correlation with
hasbeenfound(supplementalTableS1). relativelyrestrictedxenomiccontent.Incontrast,Paxillus
In contrast to higher eukaryotes, fungal cysteine- involutus, another symbiotic basidiomycete, possesses
containing GSTs have been poorly studied, and most of an extracellular oxidative system (Rineau etal., 2012)
the investigations have focused on yeast. The S.cerevi- and also an expanded xenome. The most expanded
siaegenomeencodesthreeproteinscalledGTO1,GTO2 xenomes are found in fungi interacting with wood as the
and GTO3 that display similarities with human GSTO necrotrophic Heterobasidion annosum and of course the
class(Garceraetal.,2006).Theseenzymesareactiveas whiteandbrown-rotfungi.
thiol transferases, dehydroascorbate reductases and as As proposed for CytP450s, the diversification of GSTs
dimethylarsinic reductases trough a monothiol mecha- isalsorecentandcouldbedrivenbytheadaptationofthe
nism(Garceraetal.,2006).Otherexperimentssuggesta fungitotheirenvironment.Ananalysisoftherapiddiver-
roleofScGTO1insulfurmetabolismintheperoxisomes, gent genes associated in particular with duplication/
whichcouldberelatedtotheredoxregulationoftheStr3 deletion events has been conducted in various isolated
cystathionine b-lyase protein (Barreto etal., 2006). strains of the symbiotic fungus P.involutus (Le Quere
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
256 M.Moreletal.
A
0.0035
MSP P U A
0.003 ucoromycotinaaccharomycotinaezizomycotina uccinomlycotina stilaginomycotina garicomycotina
0.0025
s
el
d
o 0.002
m
e
n
e
g 0.0015
/
T
S
G
0.001
0.0005
0
B
300
RR22==00..7799
250
r
e
b
m 200
u
n

0 ss
5 150 eerr
dd
4 aa
P ggrr
ee
yt dd dd
C 100 oooo
WW
50
0
0 10 20 30 40 50
GST number
Fig.4. GSTomeexpansioninAgaricomycotina.A.RelativeGSTomesizeoffungalspeciesbelongingtovariousphyla.Theratiobetween
numberofGSTandnumberofgenemodelsisreported.B.RelationshipbetweenGSTandCytP450copynumbersintheJGIavailablefungal
genomes.WooddegradersexhibitthehighestnumbersofGSTandCytP450isoforms.Speciesnamesanddataaregiveninsupplemental
TableS2.
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263
Fungalxenomicnetworks 257
AA BB Fig.5. HeatmapsforselectedP.carnosa
transcriptscodingforCytP450(A)andGSTs
(B),duringgrowthonYMPG(Y)ormedium
containingwoodfromfir(F),pine(P),
spruce(S)ormaple(M),asdeterminedby
mRNA-Seq.Thecolourscalerepresentsthe
readsperkilobaseofgenemodelpermillion
mappedreads.Thefigurehasbeen
adaptedfromMacDonaldandcolleagues
(2011).
0 50000
etal., 2006). Among the identified CNVs, the authors induces strongly three CytP450 and a Ure2p-encoding
reported at least three CytP450 and one GST-encoding genes (Subramanian and Yadav, 2009). In the context
genes demonstrating that the variation in gene copy of wood degradation, induction of different CytP450s-
number occurring in this fungus at the isolate level con- encoding genes has been observed in P.chrysosporium
cerns these gene families (Hedh etal., 2009). In other and P.placenta in a wood species-dependent manner
organisms, CytP450s and GSTs are also concerned by (VandenWymelenbergetal.,2011)andP.carnosa(Mac-
CNVs as shown for instance for the human cytochrome Donald etal., 2011). In P.carnosa, heat maps revealed
P4502D6,thispolymorphismbeingassociatedwithdrug variable expression of CytP450 and GST genes during
metabolizing systems (He etal., 2011). In a similar way, growthonfir,pine,spruceormaple(Fig.5adaptedfrom
the human genome encodes 17 soluble GSTs, two of MacDonald etal., 2011). Some transcripts have been
them missing in some individuals (Mannervik, 2012).All found to be abundant in all wood degrading conditions,
these data agree with a rapid evolution of the xenomic while others seem to accumulate in specific ones.As an
network driven by the molecular environment of the example CytP450 (Gene ID 516) and GST (Gene ID
organisms. 1262)exhibitthesametranscriptprofile,i.e.aninduction
byallwoodsubstrates.Similarly,CytP450(GeneID3626)
andGST(GeneID2315)arebothinducedspecificallyby
CoexpressionofP450omeandGSTome
fir (Fig.5). Concomitant analysis of CytP450 and GST
The direct correlation between the P450ome and expression could be a starting point to establish a func-
GSTomewasalsoinvestigatedusingthegrowingnumber tionallinkbetweenisoforms.
oftranscriptionalstudies.Forexample,specificCytP450-
encodinggenesfoundinP.chrysosporiumareinducedin
FunctionallinksbetweenGSTomeandP450ome
presence of polycyclic aromatic hydrocarbons (Syed
etal.,2010)anditisalsothecasefortwoUre2pisoforms ThedirectcorrelationobservedbetweentheCytP450and
(Thuillier etal., 2011). In the same fungus, nonylphenol GST contents raises the question of the functions of the
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,248–263