Evolution and Functional Insights of Different Ancestral Orthologous Clades of Chitin Synthase

14 Pages·2016·2.92 MB·English

by  Huiquan Liu

ORIGINALRESEARCH
published:01February2016
doi:10.3389/fpls.2016.00037
Evolution and Functional Insights of
Different Ancestral Orthologous
Clades of Chitin Synthase Genes in
the Fungal Tree of Life
MuLi1†,CongJiang1†,QinhuWang1,ZhongtaoZhao2,QiaojunJin1,Jin-RongXu1,3and
HuiquanLiu1*
Editedby:
ThiagoMottaVenancio, 1StateKeyLaboratoryofCropStressBiologyforAridAreas,CollegeofPlantProtection,NorthwestA&FUniversity,Yangling,
UniversidadeEstadualdoNorte China,2SouthChinaBotanicalGarden,ChineseAcademyofSciences,Guangzhou,China,3DepartmentofBotanyand
Fluminense,Brazil PlantPathology,PurdueUniversity,WestLafayette,IN,USA
Reviewedby:
LakshminarayanM.Iyer, Chitin synthases (CHSs) are key enzymes in the biosynthesis of chitin, an important
NationalCenterforBiotechnology
structural component of fungal cell walls that can trigger innate immune responses in
Information,NationalLibraryof
Medicine,NationalInstitutesofHealth, hostplantsandanimals.MembersofCHSgenefamilyperformvariousfunctionsinfungal
USA
cellular processes. Previous studies focused primarily on classifying diverse CHSs into
RobsonFranciscoDeSouza,
UniversidadedeSãoPaulo,Brazil different classes, regardless of their functional diversification, or on characterizing their
A.MaxwellBurroughs, functions in individual fungal species. A complete and systematic comparative analysis
NationalCenterforBiotechnology
of CHS genes based on their orthologous relationships will be valuable for elucidating
Information,NationalLibraryof
Medicine,NationalInstitutesofHealth, the evolution and functions of different CHS genes in fungi. Here, we identified and
USA
compared members of the CHS gene family across the fungal tree of life, including
*Correspondence:
18 divergent fungal lineages. Phylogenetic analysis revealed that the fungal CHS gene
HuiquanLiu
[email protected] family is comprised of at least 10 ancestral orthologous clades, which have undergone
†Theseauthorshavecontributed multipleindependentduplicationsandlossesindifferentfungallineagesduringevolution.
equallytothiswork. Interestingly, one of these CHS clades (class III) was expanded in plant or animal
pathogenic fungi belonging to different fungal lineages. Two clades (classes VIb and
Specialtysection:
Thisarticlewassubmittedto VIc) identified for the first time in this study occurred mainly in plant pathogenic fungi
BioinformaticsandComputational from Sordariomycetes and Dothideomycetes. Moreover, members of classes III and
Biology,
VIb were specifically up-regulated during plant infection, suggesting important roles in
asectionofthejournal
FrontiersinPlantScience pathogenesis.Inaddition,CHS-associatednetworksconservedamongplantpathogenic
Received:11September2015 fungiareinvolvedinvariousbiologicalprocesses,includingsexualreproductionandplant
Accepted:11January2016
infection. We also identified specificity-determining sites, many of which are located at
Published:01February2016
oradjacenttoimportantstructuralandfunctionalsitesthatarepotentiallyresponsiblefor
Citation:
LiM,JiangC,WangQ,ZhaoZ,JinQ, functionaldivergenceofdifferentCHSclasses.Overall,ourresultsprovidenewinsights
XuJ-RandLiuH(2016)Evolutionand into the evolution and function of members of CHS gene family in the fungal kingdom.
FunctionalInsightsofDifferent
Specificity-determiningsitesidentifiedheremaybeattractivetargetsforfurtherstructural
AncestralOrthologousCladesof
ChitinSynthaseGenesintheFungal andexperimentalstudies.
TreeofLife.Front.PlantSci.7:37.
doi:10.3389/fpls.2016.00037 Keywords:chitinsynthase,fungi,evolution,functionaldiversification,plantinfection
FrontiersinPlantScience|www.frontiersin.org 1 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
INTRODUCTION daughtercellsaftercelldivisionasarepairenzyme(Cabibetal.,
1989). ScCHS2 participates in the processes of primary septum
Chitin, a polymer of N-acetylglucosamine, is the second most formationandcelldivision(Silvermanetal.,1988).Themajority
abundantbiomassinnatureaftercellulose(Merzendorfer,2011). of chitin is synthesized by ScCHS3, which is also responsible
Itisanimportantstructuralcomponentofthecellwallinfungi, for forming the ring of chitin at the budding site (Shaw et al.,
playing a crucial role in the maintenance of cell morphology 1991; Schmidt, 2004). Roles of CHSs are more complicated in
(Bowman and Free, 2006). As a classic pathogen-associated filamentousfungi.IntheplantpathogenicfungusMagnaporthe
molecular pattern (PAMP), fungal chitin can trigger innate oryzae, for example, CHSs also participate in processes related
immune responses in host plants and animals (Shibuya and to pathogenicity. The Mochs1 deletion mutant has a defect
Minami, 2001; Reese et al., 2007). A number of enzymes in conidiogenesis, which limits the infection of host plants
participate in the formation of chitin, but the chitin synthases (Kong et al., 2012). MoCHS7 is responsible for appressorium
(CHSs) are the crux of this process. CHSs are located in the penetration and invasive growth (Kong et al., 2012). Previous
plasmamembraneandtransferN-acetylglucosaminetogrowing studiesfocusedprimarilyonthefunctionsofCHSsinindividual
chitinchains(Merzendorfer,2011).Owingtothelackofchitinin fungalspecies.Acompleteandsystematiccomparativeanalysis
plantsandmammals,CHSsareconsideredattractivetargetsfor of CHS gene family across the fungal kingdom will be valuable
developingefficientantifungalagentsusedtocontrolpathogenic forelucidatingtheirevolutionaryandfunctionalrelationships.
fungi(Free,2013). In this study, we systematically identified members of the
CHSgeneshavebeenidentifiedinvariousfungi.Accordingto CHSgenefamilyacross109representativefungifrom18major
thephylogeneticpositionsandsimilarityindomainarchitecture, fungal lineages. The orthologous and paralogous relationships
these genes were divided into three divisions (Roncero, 2002; among various CHS genes were resolved. We found that one
Choquer et al., 2004; Mandel et al., 2006; Latgé, 2007). orthologous clade of CHS genes (class III) was expanded
CHSs in division I contain type I (CS1, PF01644) and type mainly in important animal or plant pathogenic fungi from
II (CS2, PF03142) chitin synthase domains, as well as a different fungal lineages. We also report the identification
chitin synthase N-terminal domain (CS1N, PF08407); CHSs in of two novel clades of CHSs (classes VIb and VIc) that
division II contain CS2 and other additional domains; CHSs are present mainly in pathogenic fungi from Sordariomycetes
in division III contain only the CS2 domain (Choquer et al., and Dothideomycetes. Further expression analyses showed
2004; Mandel et al., 2006). Division I was further divided that members of classes III and VIb were specifically up-
into three classes I, II, and III, and division II comprised regulated during plant infection, suggesting important roles in
three classes IV, V, and VII, whereas division III contained pathogenesis.Moreover,wefoundthatCHS-associatednetworks
the single class VI (Mandel et al., 2006). Previous studies are conserved in plant pathogenic fungi and involved in
focused primarily on classifying diverse CHSs into different various biological processes, including sexual reproduction and
classes (Choquer et al., 2004; Mandel et al., 2006; Ruiz-Herrera plant infection. In addition, we determined the specificity-
and Ortiz-Castellanos, 2010). The majority of these studies determining sites that are potentially responsible for functional
employed a limited number of fungal species from a narrow diversification of different CHS genes. Our results provide new
range of fungal lineages (mostly later-branching fungi), which insights into the evolution and function of the fungal CHS
could lead to difficulty in establishing the relationships among genes.
different classes and even ambiguously classifying some CHSs
into different or multiple classes. For example, class VI was
clustered with division I in the study by Odenbach et al.
RESULTS AND DISCUSSION
(2009) but with division II in the study by Niño-Vega et al.
(2004). In addition, some CHS genes from basidiomycetes of
The Fungal CHS Gene Family is Composed
class II in the study of Ruiz-Herrera and Ortiz-Castellanos
of at Least 10 Ancestral Orthologous
(2010) were classified into classes I and II in the study of
Munro and Gow (2001). More recently, Pacheco-Arjona and Clades
Ramirez-Prado classified CHSs from 54 fungal genomes into Based on the common conserved domains of CS1 and CS2,
theexistingclassificationscheme(Pacheco-ArjonaandRamirez- we identified 978 CHS genes in the predicted proteomes of
Prado, 2014). However, the established classes of CHSs may 109 representative fungi from 18 fungal lineages in phyla
contain distant ancestral paralogs with distinct functions in Cryptomycota, Microsporidia, Neocallimastigomycota,
fungi. Because theorthologs retainthesame function (Koonin, Chytridiomycota, Entomophthoramycotina, Zygomycota,
2005), evolutionary classification of CHS genes based on Glomeromycota, Ascomycota, and Basidiomycota (Figure1;
the orthologous relationship is more reliable for transferring Table S1). Among these, 373 genes contained CS1N, CS1, and
functionalannotation. CS2 domains, suggesting that these genes were members of
Different CHSs have been proved to play various roles, divisionI.FiveadditionalgeneslackedtheCS2domainandone
suggesting that members of this family have undergone of those also lacked the CS1N domain, which may be due to
functional diversification to a certain extent. For example, the genomeassemblygaps.Theremaining600genesharboredonly
buddingyeastSaccharomycescerevisiaehasthreeCHSgeneswith the CS2 domain, suggesting that these genes are members of
differentfunctions.ScCHS1replenisheschitininthecellwallof divisionIIorIII.
FrontiersinPlantScience|www.frontiersin.org 2 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
Division I Division II Division III Total numbers
a b a b
Plant pathogen -----------------Trichophyton equinum 1 1 1 1 1 1 1
-------------Trichophyton verrucosum 1 1 1 1 1 1 1
Animal pathogen ---------------Trichophyton tonsurans 1 1 1 1 1 1 1
-------------------Trichophyton rubrum 1 1 1 1 1 1 1
-----------------Arthroderma gypseum 1 1 1 1 1 1 1
------------------------Arthroderma otae 1 1 1 1 1 1 1
--------------Arthroderma benhamiae 1 1 1 1 1 1 1
-------------------Coccidioides immitis 1 1 1 1 1 1 1
---------------Coccidioides posadasii 1 1 1 1 1 1 1
--------------------Uncinocarpus reesii 1 1 1 1 1 1 1
---------------Ajellomyces capsulatus 1 1 1 1 1 1 1
--------------Ajellomyces dermatitidis 1 1 1 1 1 1 1 Eurotiomycetes
-------Paracoccidioides brasiliensis 1 1 1 1 1 1 1
-------------------Aspergillus nidulans 2 1 2 1 1 1 1
----------------------Aspergillus terreus 1 1 2 1 1 1 1
------------------------Aspergillus niger 1 1 3 1 1 1 1
----------------------Aspergillus oryzae 1 1 4 1 1 1 1
-----------------------Aspergillus flavus 1 1 4 1 1 1 1 1
-----------------Aspergillus fumigatus 1 1 2 1 1 1 1
--------------Aspergillus fischerianus 1 1 3 1 1 1 1
--------------------Aspergillus clavatus 1 1 2 1 1 1 1
-------------Penicillium chrysogenum 1 1 3 1 1 1 1
------------------Penicillium marneffei 1 1 1 1 1 1 1
-----------------Talaromyces stipitatus 1 1 1 1 1 1 1
-------------------Metarhizium acridum 1 1 1 1 1 1 1
----------------Metarhizium anisopliae 1 1 1 1 1 1 1
---------------------Trichoderma reesei 1 1 1 1 1 1 1 1
---------------------Cordyceps militaris 1 1 1 1 1 1 1 1
----------------Fusarium verticillioides 1 1 3 1 1 1 3 2
------------------Fusarium oxysporum 1 1 2 1 1 1 1 3 1
---------------Fusarium graminearum 1 1 2 1 1 1 1 3 1
-----------------Nectria haematococca 1 1 3 1 1 2 1 1 1
-----------------Verticillium albo-atrum 1 1 1 1 1 1 1 2 Sordariomycetes
---------------------Verticillium dahliae 1 1 1 1 1 1 1 2
---------------Glomerella graminicola 1 1 1 1 1 1 1 1 1
--------------Neurospora tetrasperma 1 1 1 1 1 1 1
----------------------Neurospora crassa 1 1 1 1 1 1 1
---------------Chaetomium globosum 1 1 1 1 1 1 1
----------Chaetomium thermophilum 1 1 1 1 1 1 1
----------------------Magnaporthe poae 1 1 2 1 1 1
--------Gaeumannomyces graminis 1 1 1 1 1 1 1 1
--------------------Magnaporthe oryzae 1 1 1 1 1 1 1
------------------Botryotinia fuckeliana 1 1 2 1 1 1 1
---------------Sclerotinia sclerotiorum 1 1 1 2 1 1 1 Leotiomycetes
----------------------Blumeria graminis 1 1 1 1 1 1 1
---------Mycosphaerella graminicola 1 1 1 1 1 1 1 1
--------------Mycosphaerella fijiensis 1 1 1 1 1 1 1 1
------------------Cladosporium fulvum 1 1 1 1 1 1 1 1 1
-----------Baudoinia compniacensis 1 1 1 1 1 1 1
----------Pyrenophora tritici-repentis 1 1 1 1 1 1 1 1 Dothideomycetes
----------------------Pyrenophora teres 1 1 1 1 1 1 1 1
-------------------Cochliobolus sativus 1 1 1 1 1 1 1
-------------Phaeosphaeria nodorum 1 1 1 1 1 1 1
----------------Rhytidhysteron rufulum 1 1 1 1 1 1 1
----------------------Hysterium pulicare 1 1 1 1 1 1 1
--------Monacrosporium haptotylum 1 1 1 1 1 1 1 Orbiliomycetes
---------------Arthrobotrys oligospora 1 1 1 1 1 1 1
-----------------Tuber melanosporum 1 1 2 1 1 1 1 Pezizomycetes
-----------------------Candida albicans 2 1 1
------------------Candida parapsilosis 2 1 1
-----------------------Candida tropicalis 2 1 1
--------Lodderomyces elongisporus 2 1 2
--------------Debaryomyces hansenii 2 1 1 Saccharomycetes
----------------------Candida lusitaniae 2 1 1
----------------Candida guilliermondii 2 1 1
----------Saccharomyces cerevisiae 1 1 1
-----------------------Yarrowia lipolytica 1 1 1 1 1 2
-Schizosaccharomyces cryophilus 1 1
Schizosaccharomyces octosporus 1 1
--Schizosaccharomyces japonicus 1 1 Taphrinomycotina
------Schizosaccharomyces pombe 1 1
--------------------Taphrina deformans 1 1 1
--------------------Saitoella complicata 1 1 1 1 1 1 1 2
--------------------Coprinopsis cinerea 2 2 1 1 1 1 1
-------------Schizophyllum commune 2 1 1 1 1 1 1
--------------------------Laccaria bicolor 2 1 1 1 1 4 2
-------------Heterobasidion annosum 2 1 1 1 1 1 1
----------Ganoderma sp. 10597 SS1 2 1 1 1 1 1 2
-------------------Dichomitus squalens 2 1 1 1 1 1 1
---------------------------Postia placenta 3 1 1 2 1 Agaricomycotina
----------------Gloeophyllum trabeum 2 1 1 1 1 2 1
-------------------Coniophora puteana 2 1 1 1 1 1 2
----------------------Serpula lacrymans 2 1 1 1 1 3 2
-------------Fomitiporia mediterranea 3 1 1 1 1 1 1
------------Moniliophthora perniciosa 2 1 1 1 1
-----------Cryptococcus neoformans 3 1 1 1 1 1 1
---------------------Cryptococcus gattii 2 1 1 1 1 1 1
-------------------------Ustilago maydis 2 1 1 1 1 1 1 Ustilaginomycotina
---------------------Malassezia globosa 1 1 1 1 1 1
----------------------Puccinia striiformis 3 3 1 1 2 3
-----------------------Puccinia graminis 2 3 1 2 1 1 Pucciniomycotina
----------Melampsora larici-populina 2 2 2 1 1 1
--------------Rhizophagus irregularis 4 2 2 5 1 1 2 Glomeromycota
--------------------Coemansia reversa 1 1 1 3 4 2
---------------------Rhizopus chinensis 4 4 8 8 11 5 4 Zygomycota
----------------------Rhizopus delemar 4 2 6 5 6 3 3
--------------------Mucor circinelloides 4 2 4 4 6 3
--------------Conidiobolus coronatus 1 2 2 2 5 3 Entomophthoramycotina
------------------Gonapodya prolifera 4 1 2 2
---------------Allomyces macrogynus 11 6 1 23 Chytridiomycota
---Batrachochytrium dendrobatidis 3 1 4 2 6
-------------Spizellomyces punctatus 6 1 4 1 7
-----------------------Orpinomyces sp. 6 2 5 1 6 Neocallimastigomycota
-------------Enterocytozoon bieneusi 25 9
-------------Encephalitozoon cuniculi 1
-----------------------Anncaliia algerae 1 Microsporidia
---------------------------Vavraia culicis 1
-----------------------Edhazardia aedis 1
-----------------------Rozella allomycis 1 1 2 Cryptomycota
FIGURE1|DistributionofCHSgenesacrossdifferentfungallineages.ThecopynumberofeachorthologousCHSclassisindicated.Thespeciestreewas
constructedbasedonthephylogenetictreeofα-tubulins.ThehorizontalbarindicatesthetotalnumberofCHSsineachfungus.ForthedefinitionofI,II,III,IVa,IVb,V,
VII,VIa,VIb,VIc,VIII,andothers(OTS),pleaseseethetext.
FrontiersinPlantScience|www.frontiersin.org 3 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
We then performed maximum likelihood phylogenetic basidiomycetes and early-branching fungal lineages, including
analyses to determine the orthologous and paralogous Neocallimastigomycota, Chytridiomycota, Zygomycota, and
relationships of these identified CHS genes based on their Glomeromycota. This clade was identified for the first time in
common conserved domains. The phylogenetic tree of thisstudyanddefinedasclassVIII.
division I displayed four distinct ancestral orthologous clades PhylogeneticanalysisshowedthatmembersofdivisionsIIand
(Figures2,4A). Three of these orthologous clades correspond III are distantly related to each other (Figures3,4B). Division
to classes I, II and III defined previously (Munro and Gow, II is comprised of four distinct orthologous clades. Two clades
2001; Choquer et al., 2004). Therefore, we define them as correspond to classes V and VII defined previously (Mandel
classes I, II and III, respectively. Notably, previous studies et al., 2006) and are designated as classes V and VII in this
showed ambiguous phylogenetic positions of classes I and study. Notably, class IV defined previously (Munro and Gow,
II CHS genes from basidiomycetes (Munro and Gow, 2001; 2001;Choqueretal.,2004;Pacheco-ArjonaandRamirez-Prado,
Pacheco-Arjona and Ramirez-Prado, 2014), but our study 2014)wasactuallycomprisedoftwoorthologousclades:classes
clearly suggested that these CHS genes should be within class IVa and IVb. Orthologous relationships of some CHS genes
I. A fourth orthologous clade contained CHS genes from from early-branching fungi were not determined. These genes
Color ranges: Protein domains:
Chytridiomycota CS1N
Zygomycota CS1
Ustilaginomycotina CS2
Agaricomycotina
Pucciniomycotina
Taphrinomycotina
SODELSPeauoeorobrcrztdohitcilziatiiohdoioormaemimmoroyoymyymcmccceyeyeeycttcteteceeeesostssteteisnsa AAAAAAAAAAA.......S...mmm.mmmmSmmmm.Sp.aaaaaaaaaUSpaaC.BuScccccccpccCBucSc..C.||n|.||||.cu|mB|AAAg||AAdnSAAp.DL.A.AAA|od.pnnSa.e|MuMMcM.M.aMMMMd.SFmMMeubp|eAtsninPSy|enA.nAAnPiuAAoAAqg.AGAA||c|AmAA|SPPonM|BgS|g|n|AuGGGPiGgG|GGSB.GGGg3.G.Gr|g.CiGPefi|DPHn|t..Bi3lyl7GASi1P00gD22r11apo10ia.d015iA.01G1|aP00E.s1D.gpi2529c1gPe20r|0P80a|04fE83.57A7|pg2y4lg0|Gut0|Ti3r69923j2E7A4n5Pa16g2Gg93|.2g0eiT0||6i8A2t3fg207495g635.j3|5ni606Ti|9i1651rGi3g4P0f09.43g30gi0|19s3i584095108|7050u019337c.02igi908|j841355i40010l8g6322TT0T3TcTAi6mT5Tg63T613T5TT4aA47674T191129i4hi2.338990000T00|940840|724.0f0410301084r1403i41T9g8127g11|9n766808021i3sA6356i3Ai5910g648056|T60185.878.ig9473T4850g9829|n222o902901Ai485i009gr8976P.55117991i9df131.y3l576|5|1703889c569ag0A5g9911857|i4hi.132rfA4497g653i0|.i8M6fA705R7s11g72.|iuM6423.547.c3gRc2dlim038621AR.8i5ePa..|rc18648|.4d4|l.5nRig10|c9i1cg3rieA08Ri9.|93hh.dl5730641c||ro5O41Ri|0r381A|hR52g0.j9i2gy39O7gf3ii16|.l8A|603GdR9M.3ji2g753ag1t8i72Te|A855.00G.e.i58Md159lg01c9r5T|i86n|5eR62i16i52.92rT002glc39g4.1P|iO|953|3.09R5is97119r4tg883796i|iO7m3T10|0034.G4T1a751g0R03e9U9ri44P00|83T7GqT.5294.5..g0c99m4ig0ut411T2|h5A7R042ro.23a64gi0a23i|1nT.b84y2j|2Mc|2g352Pe02|Tgh31g.Tii2.25nPG21li|04via3|6047.20TjgeTT2sgr.2i46r144r||tCG0i6g388r06u67gA|4i0.3i05.C030bgC8|2736o31329aQtAg.5108.i3n89t4a|M082|g132eg81Cy265gC2o5i60i0.3p3667.F|T|215png278C3.g9o220.eimi1i1180s608o3|236e4m897|1g6g4di02U1T5m5.i1|3|1r15g00701g8e88ii463A39Ge28.15|9830d631.3g31tHi7eA92r02.r52a0.|151ca156|Dg95jain7g801.P8p2n4i5.s|1716|qb62gji3r91gu401a491i||74g58P9Pg.85ii4t763C.r3022ipG90|.P697p.l51g.at5i8u0s39e|S16pt7rs3||86.g|f33ljg6a9g8iiS031c1i35.P893|c.939Cg22.o1n005i95sm3o89a93C3dt34|||3g26.j08gcii33g33ii1n37003L6|212g5R3.9.7bi6r216i38cu060fU5|1|597gj.6Hi80mg1.i6407pa13M660uy1Ml6.|0|.5jfggi99j9gil|i87o2j3C7|13gM.giMf40.3i2.u19gl0l5a0r|64j3r3aP4g||8ig3.6g8i1si736t6390r219|4BFC8.6P.85c8v0Q4.2soe6911Mrt|0r2mP||FF19jC.7.g4g3Vo6Qirx46E8a3yM0||8GP5F151FGT.410OCg04T77rX.1gGa7|Ga281F1tTT|02gG006N3Ci.S364h.2Gn65a10eTTeF|23o.F00g54|io.gm14x3iy5870|e82T0dF2807|OgFF68.iX.463vm49eG9re2013|d9422F|0g1FVS.2i23Eg.3cr99GoTa43|m0002F2|1g8G2i3S3CN9.90G4.hcT211ai60n0e9||1g6g1ii2363L95M0T..91b2a07i9nci4||1g4g4ii018D37762M.04.s421aq7c1ur12||g06gii13939982T55.2r36e2G9e3|.8s2gi2p338|7j49gC0Ci..15p1m1ui6l5t||62ggi19i334449V6S2.35d.l2aa94hc2|1|3ggi10i334443669V3.76al4G9b8|5.0tgir35a36|0jg2i41G.20g1H0r53a.|a88gn6i4n3|1jg0i7192C2.27gl89o0|44CPH.pGlTa0|s0C.f5t19h0e6|7gi34P0.p92la3|6sSN5f.6ta9e5.tc|ogi|3S05a0ic2o93554N80.8c8r2a|NT.CdUe0f|3T6D112MT5.0p7oS0a|.jaM.pA|PgiG20153445130T304Ga5.gSr1|.pGoGmT|Gg0i16986111T30750SM..ocrry|y|MSGPGO0G1800220T009T0SB..ofucct||gSi1O5C42G9062024696T0N.Yh.aliep|g|iY3A02L8I806D23245938pF.ver|FVSE.cGe00r0|3834T00443698M.poa|MACPG.1lu05s2|1gTi0260945371S.scl|gi1560C596.g90ui|gi146419865B.fuc|gi154315738D.han|gi294656380B.gar|bghP00822C.par|gi354548408T.mel|Tubme1214L.elo|gi149235319T.mel|TubmCe.tr2o|7gi42555723074M.hap|jgi1C.3al3b|4gi68478998A.oli|gi3C4.g5u5i|6gi7174664520848Y.lip|YAD.LhIa0n|Dgi02391476594p652T.def|TC.lDu2s|5gi72360947162Sa.coL.|Selao|icgio114796224558G16.sp|Cj.gpi1ar1|4gi93558454D73.s3q5uC.t|rgoi|3g9i52535370P343.1p060lCa.0|aslfb1|7gi684S7.6la41cA.0|golii|3g3i634356C54.67p72u5M4.t8|hg8ai3p|9j2gi5H496.a21T7n2.2nm7|jegl|i1Tu4Lb6.7mb0eicM97.|2go3ri1y9|70MC0G9.c0GiG8n0a69|.g65gir52|19MGT90G7.pT4Me7.Gr5p1|g8o4i40a2|S337M.8cTA5oN0.P9mc4Gr|50ag|67Gi388N00.Ct02rNUa.T6t00|9ej4gt6|2i7C1g5i931.37c5T5Ci09.n09g2|lg49oi|F416C6.m39HC9.8etG5dhT6|e0Cg|17ig3.3i0n4939Ge.343og02r|91gCa|15i.5g46giV895.a3205at1l|620gbU7|7i34.g9im25V33.125a02d6y25aM6|49gh|2.3ig7gC02i.1l1o3308m|4iPg2l96|3i.19sg1i6M7t0.r347|9a4C66Pc605rQ.|344gMg1M9ir.8a430a8|M820Pn6i26|.G5Tl69ga.iT91rr34G|TePg200e1.i|293s7Ng72.t2i8r1h83|1PC0a8453.Qe50gTF|6.75r0Mg4iag81Mr312|4Pa0531|.FlG21.0aF997oTrPG1|6xGg3S.yTs|iF403.0GtF02r2v1P|358OeC0r.28|3XgBQ7.52FrGTMag07TV0r0|4T.15PaEt07B|o50.G0Gbf7n1T0guT88|.g3chGeT7|S0i.00q3Ts25As0c2u008c3.l16|8|gT.Bg0.4301gy0iiTc3795p1o.25T5|v5gM760me.6A|i74j3rg058|r.g1g5ibC3a5.5|i1efT2302g5nu5i0.l32r1||33M2jug90.5A96fgb9ii8i3j8.54||61oH0jg45.19Ct2gi11ai3p042.52|2uRp4lg958.|0C74ojr8i62328gsu7.4iP5f9i9|.m|2U1gj868n32gi28m1.i3oCr016.A5e0d|4|85sg83e.45gPdai43i|1.tAg6t|e321j1i3.er126Pgc9r|i.P|0g59at11rg76i.8i7pi2P3b|10.A59|362rgg47bi9a3.18riAo97161A0.|18ag6r898|59d29.y7iA9f9g421eA2.|il9rga1229c|52.i|97Un7530aAgg9.i262i6r81p5ig.2|2660Ct5eA76|.e6g639ig8ie16.1r|983C2if1A5|83m.i1gg5s4i547.1p9Ai0|f3Am24917.g7u|5o852771.oi09sgmAAn6t1i|87.55138ia11g50.|g3dT|5i4Pgc6.1966y8|tg36lii.g9444a7TpT5c0o0.|21i888|076h3.2ngegT99P5|s6i94.73riq167rtg91|38.751iPiAgmu18|u3.212|5g43i.00T3P2bb151gta512i.|i14r25075e.1r76vCigP37t|4i|541.1n7eg34eg|20.P2A397r4ms77rii.|g6412724|i33.RTa0gng4na.87915ii638r4tHiA.7os|9|234233d001r.t0j|8iMudgAg.10153082|ci3pg.5lf|i690Ci3A3204fg22g|.u8ia.j3f0456699MuA|g15f.ii6l2i12jf18|7.852mgB2|A6iuji4s5.j36g72n4531.||gg5M5Ail.1f229|i98161c2lggi.3Mr28Ajdii2172g6.76a7o|a9o6C.A7a17|19912i.6rg0|9mnT7P1.7aingg.10760iyt03FiRm2.8.|65ciig|9e91715rF7|R32c8jg..|67grerg8014FRi7vi5h9|c3|.g.l3N5iMgei|o78891ei1g8.c3rh.788gVM0|g3ii129|d7x6iVrh.R2.3534gig|1h4|.G6rR34ci44jy9.e9iF2dM2R979|.ai.1icga7jlG|4T332a597d.r7.|Ma238VFi9iS|g5c9.|geCS6145.2FRrl8.1eCS0ci0a1602hbp|OdNES3h.2|r3N8503.Sl3G03.j.hS9YOg0793.||aoB|So.i.a9epBDtTGC.2g79X25gg.2MC|cRA92CgCiLCa9h7.9i32oj.t|S.ra8|pfo33...c96426i4.girrgsejl.24Gyl|3.0..fcem93O.3.7ci.g.g.6|.ho|yT|GGme0lrcu7i1po42g10cpiaMt|t5Gh76gu9e3|47t0a63i|1r5M|r|ao23Dlg8c95e5l||l7uSag0l06Caia0oos8|r|i6S1TY21|4Gb02Ang|8G||g75i8|lN4i0Gr42Ti|9p|SP|3g|g|963|40H043|1OAgb||gi9g7PTi9gT9g9467100gg|1C4ii75307aO4G53i31jg1iGiLui8GC4G8i21Tg8912ii50176193i60U423654hGcIb023868i104T5690G4090T950882095345Po04mTT601950600967B1135045914140253510010922748043346801e78521653961240T253238791221258996954492653002055186181257003458182T9561540751TT74142423311T083T35750001468T444078608p280000001001
FIGURE2|MaximumlikelihoodphylogenyofdivisionICHSs.ThephylogenetictreewasconstructedusingPhyML3.1(Guindonetal.,2010).TheSH-like
supportofapproximatelikelihoodratios(aLRT-SH)areplottedascirclesonthebranches(onlySH-likesupport>0.8areindicated).Colorsofbranchesindicate
correspondingclasses.Thescalebarcorrespondsto0.1aminoacidsubstitutionpersite.Forabbreviationsoffungi,pleaseseeTableS1.
FrontiersinPlantScience|www.frontiersin.org 4 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
Color ranges: Protein domains:
Chytridiomycota Myosin motor-like domain
ZUysgtiolamgiyncoomtaycotina Cyt-b5
Agaricomycotina CS2
Pucciniomycotina DEK_C
Taphrinomycotina
Saccharomycotina
ODELSPeuoeorborrztohditilziitiaodoioormemmimooyymymycccceyeyeetctcetteeeeesststseess PMTTTSABP.TT.ACnAP.CMC..g..M.e.Br.t.sbCof.tNU..v.orur.FC.ugqe.AotFiCA.Cpceae.mdNamern.bc..a.cyuPrNttl..iF.oce|rn.g|.cr.||a|o|hMsoH|d.|gpcngige|mgfgRgg|.rgAsh|gl..|rrgAuag|bemx|egaavt|gAiiileaG|.iigii|gi.A3a|1igeo.. ig31|3pylG3igtr|e1rf.gpi|3iMMfr1||V|gV3.ig|3i|faiNe396it||.iA.gFj|i32uc82u3ji2jjr3u5|isgC|Ag2g3oF5aig30|gi.34.g|||08g.9.l1i90jGf3C.96l4d7|agmgF14iapo2r6ri2Ag.6|.Oif|02iHg220Ai2i1461lag1jyt1jP694146a12li3i52o|rV5Ug42Sgai.e03b623i26|5g48.0|42yrX9|GP8081n029h.375g950a8g8n|gi76ir|E3|3690GT0c17i1|298i78g901G.2|g81192|7G2441ii09M|i59T5ig89g1gh7.25m424MG111d7i30561T6528i709855492255s222|28G6i30130ir316289|3G692.61g57421731t49|144a4920Y617A90g49363i150m44540g9768i459T940|2A6014175r779.i988G798976599382869Pi7gl62|98516034286723i.67G8e56924g612861i018434S441opG060l712337T32i953M95|9548l64S|079982273ia252964T4488080Y75|.5T0S0.0306.41l204T9g1762uC2h1409c.A1Ta21707il25102.0b85a8o02323ca748LS60593p|2|m17p2873I8.4c461S3ug4T9||09c9046j5it7eT0ga|60LFgo33HT35ii.43g01im.92L33ci96b099.7ia|3o86C6314b0c8.ngGi9166|F30i07ccn2g8537i.3.|5|i1vgj5n8652g0D01Ve|igrG9.62p92ia7rg1.si0|6054t6|0GF6grq2N659.48F1V4iaCu92s344|.3|.1F6jhPEg9p1.10g5.6F|fg3i.arj7.Giu0o9p5a1F35elg41lx7mPi8|17|SM.9|7a.GFj6y4v8g|1ge1C.0j.5p6|G.t5e.1lpi92Fid6Cf3rg318|nr.i7iau25S0Oj6||a02P8gP|egj1F|3n9i.7j9j8G2g9X3oag.0|Vs833|it0gnSTt8i38i|0G3M1r9EPg91o|9.0Pi5g143l723i01Ud.G3513CP.9t1a639250|ePr380g97|MG3Q214mr03.21.8i6|0g1t61ai6g20Mg6r0M43lT2y36i3i10||8034o706gC92.2|T2g69g3R3iFi064.8gA.810r70i0T7f.2d1a8uR88g.R6319F1.0f4e.|85lr9lTlVg642c|.a0|ad49jo810ig47hT|1|3eiRRx8gF5lF9|i.6308|j91yi69.5GOc253v2|g8F9R5iF3h03e854Si3.7|1OOg98r5FjG18G0|73r44F3.g1XM8a0io2.18V81G1G|31xcF90F8i10Ey94r.106G3|R64Gv|G93.F4130e54RISca3.61O93r04Th.Gc|33iTg0FX30|Mhj45.1ri06VTG||gVc5jT2Gi3iE0cr2g9.205|iTGVaRGR0T.91.010l.Tb5c1d07d346R|G4h0a.Te6Tg7iR5l|.3|chM3j0i02.0.3drTh|3gR89eic7gi0eCi|2l0j9erTi|9O23|.Tg9|0m83i4gR40G09460i64GiO3.Tl36g7|90134Vg05r4740R0ia.G.389d0755|1c4Nga476T15hM6ihi.880.35F|h43j|c9T14g.ai82ggrR00Ri3ie|.5.3r0F816|ca0dT4g2.03h|o3e06iiRF4l3|1.9x|8j9G2Fc0y289gR7i|.h2S082iFvR6|1O.83jeGOT00d39gr0i20eX|7M3lGF.1|7S62G11cV0i5a1R97r0E7|5.6OB1c21G103o8.440Td0|71GS4Me05.T01b9anA5c0T4i6|i.9rcB6o|0RT7o.8Dl00icT1|82gEhM60i8TR.i|PT.31j40acM.407g5nmihi58i9M|.T1|.ha5j0g0i1aar6g9Ti|c38pg0r81.2|C8|si8.j212gg9ti6Ai177mi|i395gl2.61|Tc26i.5622glr29ia344e6A39|262geN94|..427if316guhi574F23am.355A172eo4|1|.795gxf0g5ii00yis5|72P3F19|.10Fg0.47g7c9i2rO61h798a17X|r759|F9Fg1.GA744vi0G.2859ne457rS05i|A1d594GF6|.9Ng1n3V.33i2i76hgET707a|03AGg3e5|70Vi.913.tg57eiT7d12r30a77|8Ag0h20|1i2.931VfgTi8.l11a03a8l5|0Ag41b42|.i692o90Gg9.3i3r17yg3188rA|005ga1.|22c0i93g84Cai0.152tp3372h1|P6g1e501|.i59b17gi0Cr259.a3784g|l49g22Ao0i858|2.9d159C5eN85.H9rt8|69geUG6t58|iT2.9gr3N0i6.e734c1e8r592C|9ag00|0.ii2312MmN.965oCm2r683Uy65||Cg80G7i.6M41ap0.31Go5g5r9s9|G0|19g1GT8Ai330G3.00oT91B3t8.Ga4f380|Tu2g3c0A0|i27g.1i4g94S15y6.95psT84|c02lg2|T7i8g3.i2e9 11q8154u150|36Bg5.0i6g3T3r72.1at0|6o33bn4P8.g|7t9grh9ii|301Tg20i2.r6139u842Pb.965t|1g9er9i9|3A80g2i.06b730e321n09|94gC2.6i3s2T80a4.9t2v|6Pj.e58ginr0|o1g0d2i2|31g80i1S0212a665.95c68o20|S082a0R4.icTr5o.umf|8je7gil7|8T56uH4M7.b6p.mgulrAe|.aj7b|ggi9ei23n3|09g5i83130B92.T0c4.9ov95em5r0|7|gj6ig73i407226T5.6tM24oV09.nf|4ijg8|ijg3i2966T4.C07e.5q4fu5u5|l|4gjig63i12963487T.38ru18Hb5|.2pgiu3l2|j7g2i997A9.R62g.y6rpu4|fgi|j3g1i1500I582C73A.6.s9oa0tta||jggii229261P80.1t1e5A4r.2|cg9ai23p|3agi01950472072P59.0tr9iA|.2gdi1er8|9gi1296811090993B3.4Cg.rpao|sb|ggih3000383B1221.f6u8cC5|.igim15m|4gi310129117859S9.4s7cl|gUi.1r5ee6|0gi62355845625M64.o9ry|MA.Gfis|Ggi016190468415T409Ga.gr|GGA.fTuGm|0g4i7110080T10990M.poa|MAA.PclGa|0gi015221721T60016G.gra|Ag.in3ig1|0gi714952203518507V.alb|Pg.ic3hr0|2gi422552953034041V.dah|gi346A.9ni7d|0gi26472540294C.the|gi3409A.o5r9y|gi916659774655VC.glo|CHGTAf.0la|gi4238849637635N.cra|NCU05268T0A.ter|gi1153842720N.tetg|i350292872T.sti|gi242819391N.hae|gi302882365P.mar|gi212530180.IF.oxy|FOXG04179T0M.fi|jgji563325IF.gra|CF.GSfGu0l1|9j4g9T1i088642M.acr|gMi3.2g2r6a93|4g6i3798407989M.anBi|gi.3c22o71m245|2jgi70429TC..mmile|lgi|T3u46b3m25e0489506T.ree|Mgi.3h4a0p5|1jg39i984400S.pAu.no|lSi|PgPi3G4057576861T0051A.Yma.lci|pA|YMAALGI000E68262T1098pA.Ym.alicp||AYMAALIG00E2118074T107pSRa..dcelo||SRaO3icGo0181723714T60LR..bcihic|j|ggii215730108631L3R..bdiecl||gRi1O634G6151162953T2C0R..cdienl||gRi2O399G7014988S6R8.2.Tcc20hoi|mjgi|g1i435022069HR.3.ca3hni8|jn1g|ijg4i737418G0S..ptruan||jgSi6P9P8G20G9A8..6s5mp6a|Tcj|g0iA1M15A5GDA1.1.72sm1qa1uc6||gTA0i3M9AS5AG.3.0l2am79ca5c|8|g134iA35T3M06AC3AG.6.1p66mu65at16c||g64iAT309MS2AA.5.lGa81mc76a|19gc3|2i305A3TCM60AA.3.p7Gum20at|07cg|51i3A4898CMA2T.A.50nmG9e0a3o7c8||5g6A4i159CMA8T..A20gmG5a1a8t|7c8g|35iA635F92MA..T1Am02mGe4a1d9c7|5|3gA6P7iA32.M6.s9TAmt30raG|2Cc1|17Q6AP3S2M..M8s5p61At8ruT1G|n0C1|036QSP21BPMT.6.gP90d0rTeG2a0n04||P30MB2G3S.8DT.Tl9paE0GuTrT0|n00|g42iSU343PM2..98m8Pc73i8raGT|50y008M3|5g8R0.1.i887gc0841lh0oiT0|Tj|01g0Rgi7iR1..29c6c23h4h67ii6|1|jj6Rggi0Mi..12d3c2i2e2r98|l0|450RR03RO..1d3d2eGe5ll||MTR1R0R3..Oc0cOi33hr3i|G3|1RjGT1R0g0..i0c044c1h187h6ii504||9Rjj13gT7gTM.iTi0.6c7080ch8i7ri2||0Mj20g4R..9ic2c5i6rh4i|0R|06j3RI0T.g.ic04c2h9h3iiR1||j4jRgT..g4cii0d3h6e8ilM9V||9j2Rg..R3cid2iOre1A|3l|03S.G.22mR0p9Oa7u8A37cn2B|..4|mGTAd6S10MaeTAP20cnB3AP.|.|m4AGBdG7aeM0AD0Tcn5S9E.|0Ab|m4aA7T.BG240acMAD6051oc.MT|AE7T7.m|0ATS7Gc50iaA0Ma56ri0|Rc8.5.c9mA0|5TcAo73G30aAh98Mi24|c0R5.3j8.mA|78TgA6ci71GA0ahTM1i10Rc|.000.j9mA|99AdgTiAG7a6eM01lR6.c60|.m45A|7dRA3ATa7Ge6lMO0.3c|Mm03.35|AR5ABgTalGG5MOU.0co1.d1|30|2A3AegB1mi4GTGnM3M.a171.d0|l2y16B63A|Be2a442TDgPGrn.iT.|6610d|E7s105g48BSeti12T6r6TnD3|P.071.0T0p|2EC22B8g1B0u8r262TP7DnQ.8.a7Td90||E6gMSS90e93rP3T15CP.n3a.6p7|G0|6PgB9Bu36TP9a4CG.1ntD1.d|GG1|S70nEeS0gTTi26e.Hn9TPp.031Go|9B0|au2BP303G3g.1nn88iDG.d9t3S2n|T75re|TSE0F0j630.8.a0n6pPT6g0|02ijA|6um70PBT61gC.5in3e3.09mGD6A955|d3p99S|E0TPa8.0u7.3gmtP0Si0c6T8|87p9l|3P9Sa.0gA7.ip3a90Nl1cG|M3u7j3a|4D.0A9Tn.g2hcA0iF|32sM|01a18SGg.G45qi7eg.9FA4P91u73s|r5|S.g1G3a76P3.op8Fg|Ti9i74|6cj31xG3F.C30303ogv0y7.MiG00997e|c23L8iFm75T..1S|r59Ma3n6O|3b02|g42GLFin8i6.29.gTTX2cVi73ia09|b59|1i0B.Gg200Ec1Tg.3rCci1e9i2r|201Gd03S41|.9e6.7gg3Vme02i6078|2pi1An2g1.341|.i50VdTul177i26|4143BAa0gn3m.10.24|0G7a94hiTDa103763A8S6lm0|..Mbc04E914T9gg8|5Pa|9A26T6m40ir.gG.A2c3Pp0a160|36aiA04m3aoM|M7.112GcAg36|0a.1a7A000AmNig.5.9M32Ac|5o79|MrS9aG7.m14N9A.r65|Mtc1AG8ye01A|M2p3a.CG4A|2ct71.M0cGuMPA|1R|84C.rcg94GT.ntiAa5GTM4|9GAhRr31i00c.S3|3|.1GgGe0S92N8AMhGR51I1.cBiTl190|.4s|1PoGC0g0R0jAh006.S67ic1d2|.Pf|12Ti7g9UCM9Gju38li6eB.1|d40l91Gs98.gg294c|10THR8iP.e8V63c6c70li0Tg|.0069Ri9R1|9Gg6P6l.T2r0T2rd0|953.Tt|5R8R1Oig4aC0rT.08T91ec21200.ti06lMT4i|RO|730e25|01.h344bcg5400iRs.7GrHT4R0|5.6T3ghi0j6.a|crRi1TP0i27g61G0|0huO.3gd6trj.R80pTi89i||07.2Pf9e131gjcMn3.9T.2|u7gl00i58jmT2|0Rh31cGgo2T2.l.5i72i79|R1090rhR70|.Ticd0j4aej.018i00u53gicR83A2e9|.O3|2g.rlj29936bR9TigTh||tci205.q11.1gToMi581530A0|04dbi.hi37|.T7ug1U13junAviT8dG5545ee.14.0|6C|gAi961.jbeg|l8ge30g8.C68inA06g|09mg7o9l.l.MmTryP12iR673|i0|dg3oi.33P|.t6C3gpR042nga7cP86a.|0C.0eaeO20.Fbl2Uig|55aie2y.T0O3|gGt3.agA5r36pi.2g|r|6H3s.A99|p9o0.0i0Dr71r1mgaiAg04gm3.o5ri.tG34G|aA2|08|12f..iiS2TtAr6e0|g28gigG.L0siaf.rA371|7|ge200LssA9m.0uc.504iC5eiaCP6.5A.i|9.Cnns025P13iSTd5601c0|1.qgo.l6b6P0mS62||.6M5gYafGbCp.|QiSn.1j100g2M1otC|81015.DBiul..rd81Cg.Mci7ngC5AC9CLLe9gC1.3aaip|ccsil|y.|0|2148i1m24.1.m4T.cg|aMi61|jji1ig9l..i25i.5..0.rf0c.|n|hf.t.ug1ggg.|.i.1g8.64.cg038h21e7e9g|2ugglicio|7piGgc8a9ta1ih5p69eug|j|31a|griti1i0i70r|o440u95|6a0g97glol381rii1gl6T||l|6i6|lg1la86oajo2o12|ris232ig3rgi9Yb7abg0865mg983Ti14j|i74n3i91|15i7|70|9i|55pg3|r|2|g|6321S216i11i7|1|g|7i968g5iA6g2g78gg07837|35|143592ggg1|570|i4i54g891g737084i948a1ij21j3i60i2ii48339355L225ii1ig6g26281128261i49i16340116513i483420316632134I215c4i7i45066446785879T80293987256165985553265o01296625099T93123592C49840424740255444069519534317792207006436539764119928665257256311692444021509797877124665044868816488668359027349357640530885779770105345649975438261010044505558089p15
FIGURE3|MaximumlikelihoodphylogenyofdivisionsIIandIIICHSs.ThephylogenetictreewasconstructedusingPhyML3.1.TheSH-likesupportof
approximatelikelihoodratios(aLRT-SH)areplottedascirclesonthebranches(onlySH-likesupport>0.8areindicated).Colorsofbranchesindicatecorresponding
classes.Thescalebarcorrespondsto0.5aminoacidsubstitutionpersite.Forabbreviationsoffungi,pleaseseeTableS1.
weretemporarilylabeledas“others”(OTS)(Figure1).Division Domain Architectures of CHSs are
III is comprised of three distinct orthologous clades. One of Consistent in Divisions I and III but Diverse
them (class VIa) corresponds to class VI defined previously in Division II
(Munro and Gow, 2001). The other two orthologous clades
Besides the three CS1N, CS1, and CS2 domains, no additional
(classes VIb and VIc) are reported for the first time in this domains were identified in CHSs of division I (Figure2).
study.
Likewise, no other domain was found in CHSs of division
Comparison of sequence identity of CHSs showed that
III besides the CS2 domain, but most CHSs of division II
membersinthesameclassaremorecloselyrelatedtoeachother, alsocontaintheCytochromeb5-like(Cyt-b ,PF00173)domain
5
butdistantlyrelatedtomembersinotherclasses(FigureS1).Each (Figure3).Thisdomainisabsentfromsomemembersofclasses
class formed a distinct group. These results are consistent with IVa and IVb in division II. The Cyt-b domain can bind heme
5
thoseofourphylogeneticanalysis.CHSsofdivisionIIIaremore andsteroidsinelectrontransfer(SchenkmanandJansson,2003),
similartothoseofdivisionIIthandivisionI. buttheligandforthisdomaininfungalCHSsisunknown.An
FrontiersinPlantScience|www.frontiersin.org 5 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
A G.pro|jgi209559 B S.pun|SPPG 00288T0
G.pro|jgi98390 F.gra|FGSG 03170T0
G.pro|jgi57357 F.gra|FGSG 06550T0
G.pro|jgi113727 FF..ggrraa||FFGGSSGG 1102621997TT00
A.mac|AMAG 05239T0 B.den|BDET 08098 VI
A.mac|AMAG 00190T0 O.sp|jgi1182300
A.mac|AMAG 10750T0 A.ory|gi 317150795
A.mac|AMAG 02955T0 M.ory|MGG 06064T0
A.mac|AMAG 10238T0 F.gra|FGSG 01949T0
A.mac|AMAG 08465T0 AN..mcraac|N (C6 Ug0e5n2e6s)8T0
A.mac|AMAG 14613T0 C.cor|jgi80336
A.mac|AMAG 17040T0 A.mac (2 genes)
A.mac|AMAG 12350T0 C.rev|jgi79855
A.mac|AMAG 05690T0 R.del (3 genes)
A.mac|AMAG 12344T0 G.pro|jgi157224
C.rev|jgi79909 O.sp|jgi1187618
S.pun|SPPG 05446T0 CR..rirer|vjg|jgi6i970875039
S.pun|SPPG 07609T0 VIII C.cor|jgi43134
O.sp|jgi1189321 R.all|jgi3847
O.sp|jgi1191081 O.sp|jgi1174053
B.den|BDET 08281 R.irr|jgi344952
S.pun|SPPG 04845T0 R.all|jgi3453
B.den|BDET 08415 EV..bcuiel| V(2C5U gGe n0e0s6)84T0
S.pun|SPPG 05605T0 E.cun|ECU01 1390.1
O.sp|jgi1181464 A.alg|H312 02655T0
O.sp|jgi1174333 E.aed|EDEG 01376T0
O.sp|jgi1190690 A.mac (6 genes)
S.pun|SPPG 09498 G.pro|jgi45170
B.den|BDET 05228 O.sp (2 genes)
R.irr|jgi225626
S.pun|SPPG 03909T0 R.irr|jgi95067
C.cor|jgi43661 R.del (2 genes)
G.int|jgi121158 C.cor|jgi7537 IVa
G.int|jgi318355 C.cor|jgi21721
U.may|gi 71020413 C.rev|jgi46483
F.med|gi 393218266 P.gra|PGTG 21586T0
S.com|gi 302694783 SU..cmoamy||ggii 37012061947495467
R.del|RO3G 00942T0 F.med|gi 393213109
R.del|RO3G 08861T0 R.del (4 genes)
L.elo|gi 149235438 S.cer|6319497
AS..oceryr||g6i3 31197511527775 II FA..gorray||FgiG 3S1G71 0319257920T0
F.gra|FGSG 02483T0 NM..corray||NMCGUG0 90392946T20T0
M.ory|MGG 04145T0 R.all|jgi926
N.cra|NCU05239T0 S.pun|SPPG 07620T0
R.del|RO3G 08099T0 B.den|BDET 01759
R.del|RO3G 11900T0 R.del (3 genes)
R.del|RO3G 00522T0 A.mac|AMAG 06169T0
R.del|RO3G 16230T0 G.pro|jgi70638
S.pun|SPPG 05426T0
GG..iinntt||jjggii332389522401 CB..dreevn| jg(2i3 g0e1n2e6s) IVb
G.int|jgi67365 C.cor|jgi58605
G.int|jgi35412 R.irr (5 genes)
F.med|gi 393217420 C.cor|jgi78977
S.com|gi 302681663 R.del (2 genes)
P.gra|PGTG 03325T0 PP..ggrraa||PPGGTTGG 0098973390TT00
U.may|gi 71003910 I U.may|gi 71022795
U.may|gi 71003886 S.com|gi 302680496
P.gra|PGTG 10666T0 F.med|gi 393217767
S.com|gi 302696351 O.sp (2 genes)
F.med|gi 393212940 O.sp (3 genes)
F.med|gi 393212941 GS..ppuron||jSgiP1P19G8 0832289T0
S.cer|330443698 S.pun|SPPG 03084T0
L.elo|gi 149235319 B.den|BDET 02397
L.elo|gi 149245816 A.mac (3 genes)
A.ory|gi 169783242 G.pro|jgi289059
F.gra|FGSG 10327T0 S.pun|SPPG 07669T0
N.cra|NCU04251T0 A.mac (2 genes)
O.sp|jgi1183098
M.ory|MGG 09551T0 C.cor|jgi42612
P.gra|PGTG 02527T0 R.all|jgi3114
P.gra|PGTG 20839T0 A.mac (10 genes)
P.gra|PGTG 17815T0 O.sp|jgi1183097
U.may|gi 71023109 S.pun|SPPG 03441T0
F.med|gi 393215655 S.pun|SPPG 00937T0
S.com|gi 302696797 SB..pduenn| S(2P gPeGn e0s6)659T0
A.ory|gi 317140613 III B.den|BDET 03308
A.ory|gi 169762508 B.den|BDET 03306
A.ory|gi 317146829 S.pun|SPPG 06138T0
A.ory|gi 169777897 B.den (2 genes)
M.ory|MGG 01802T0 C.rev (3 genes)
N.cra|NCU03611T0 RR..idrre|lj g(i63 4g8e5n4e6s)
F.gra|FGSG 03418T0 S.com|gi 302683558
0.2 F.gra|FGSG 10116T0 F.med|gi 393216324 V
Color ranges: U.may|gi 71018241
A.ory|gi 169774657
N.cra|NCU04352T0
Zygomycota M.ory|MGG 13013T0
Entomophthoramycotina F.gra|FGSG 01964T0
C.rev (4 genes)
Chytridiomycota C.cor (5 genes)
R.irr|jgi346613
Cryptomycota R.del (2 genes)
Neocallimastigomycota U.may|gi 71017937
Glomeromycota SP..cgoram|P|gGi T3G02 0649438338T10 VII
Ascomycota FA..moreyd|g|gi i1 36993727146625558
Basidiomycota N.cra|NCU04350T0
Microsporidia 0.5 MF..gorray||FMGGSGG 1 132001349TT00
FIGURE4|MaximumlikelihoodphylogeniesofCHSgenesfromearly-branchingfungi.(A)DivisionI.(B)DivisionsIIandIII.Phylogenetictreewas
constructedusingPhyML3.1.TheSH-likesupportofapproximatelikelihoodratios(aLRT-SH)areplottedascirclesonthebranches(onlySH-likesupport>0.8are
indicated).Colorsofbranchesindicatecorrespondingclasses.Forabbreviationsoffungi,pleaseseeTableS1.
FrontiersinPlantScience|www.frontiersin.org 6 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
N-terminalmyosinmotor-likedomain(MMD,PF00063)anda together (Figures2–4), suggesting that they were derived from
DEKC-terminal(DEK_C,PF08766)domainalsoexistinclasses species-specific expansions. In fact, the genome of Rhizopus
VandVIICHSs.TheMMDassiststhelocalizationofCHSstothe delemarunderwentawhole-genomeduplicationandrecentgene
hyphaeviainteractionwiththeactincytoskeleton,andfacilitates duplications(Maetal.,2009).
polarizedexocytosis(Tsuizakietal.,2009;Schusteretal.,2012). Notably, duplications in members of class III were most
TheDEK_CdomainisrelatedtotheC-terminalofanimalDEK common in plant or animal pathogens within Aspergillus spp.,
protein,whichisresponsibleforself-multimerizationandDNA Fusarium spp., and Pucciniomycotina. Since the class III CHS
binding(Kappesetal.,2004).Nevertheless,theroleoftheDEK_C has been experimentally demonstrated to play important roles
domaininfungiremainstobedetermined. during host infection in the rice blast fungus M. oryzae (Kong
et al., 2012), the expansion of this class in pathogenic fungi
Variable Numbers of CHSs in Different may be related to infection and pathogenicity of these fungi.
Furthermore,theclassesVIbandVIcCHSsoccurredmainlyin
Fungal Lineages
plantpathogensfromSordariomycetesandDothideomycetes.
YeastsgenerallycontainfewCHSs(Figure1).Forexample,the
fission yeasts possess only two CHSs, the fewest copies among
all fungal lineages, which may be related to the absence of Specificity-Determining Sites Responsible
chitin in their vegetative cell walls (Free, 2013). The budding for Functional Divergence in the CHS Gene
yeastS.cerevisiaeharborsthreeCHSs.Filamentousfungiusually
Family
contain a larger number of CHSs. For example, the cereal
Previous studies have revealed that CHSs of several classes
pathogen Fusarium verticillioides has 13 CHSs, the highest
perform different functions in fungi (Silverman et al., 1988;
numberofcopiesamongthelater-branchingfungi,ascomycetes
Cabib et al., 1989; Shaw et al., 1991; Kong et al., 2012). It
and basidiomycetes. On the other hand, the early-branching
is known that functional divergence commonly results from
fungi, chytridiomycetes, entomophthoromycetes, zygomycetes,
theshiftsatspecificity-determiningsites(Gu,2001).Therefore,
and glomeromycetes, generally possess more CHSs than the
we examined CS domains among the orthologous classes for
later-branchingfungi(Figure1),likelybecausethecellwallsof
functionalspecificity-determiningsites.
early-branchingfungicontainahigherpercentageofchitinthan
In division I, we identified 15 specificity-determining sites
those of later-branching fungi (Ma et al., 2009). Unexpectedly,
(Figure5). Four of them are likely related to type I functional
the zygomycete Rhizopus chinensis has 44 CHS genes, the
divergence. Residues at these sites are conserved in one
largestnumberofCHSgenesdiscoveredtodate.Microsporidia
orthologous clade but highly variable in the other clades,
generally contain one CHSs, but Enterocytozoon bieneusi has
indicating that functional constraints have changed between
many more CHS genes than other species in this lineage,
them (Gu, 2001). The remaining 11 sites are related to type II
consistentwithapreviousreport(Pacheco-ArjonaandRamirez-
functional divergence. Residues at these sites are conserved in
Prado,2014).
different clades but their properties are dissimilar, which could
leadtofunctionalspecificationswithinagenefamily(Gu,2001).
Frequent Duplications and Losses of CHS
Ten specificity-determining sites are potentially responsible for
Genes in Different Fungal Lineages the functional divergence between class III and the other three
The classes IVa, IVb, VIa, VIb, and VIII occur in both early- classes. In addition, the residue at site 180 is Phe (F) in classes
branchingfungiandlater-branchingfungi(Figure1),suggesting III and VIII, while the corresponding residue in classes I and
that they were generated in fungal ancestors. However, during II is Tyr (Y). Functional divergence of class II from the other
subsequent evolution, the class VIII was lost in all ascomycetes three classes may be due to a shift at site 460 from Ser (S)
and most zygomycetes. In addition, the class III was lost in to Ala (A). In division II, we identified 12 type II specificity-
all early-branching fungal lineages, in agreement with previous determining sites. Residues at all of these sites in classes IVa
reports (Ruiz-Herrera and Ortiz-Castellanos, 2010; Pacheco- and IVb are distinct from those in classes V and VII. At site
Arjona and Ramirez-Prado, 2014). The class IVb was lost 123, functional divergence of class IVa from class IVb may be
in all ascomycetes but not in the archiascomycetous yeast due to a shift from His (H) to Phe (F). At site 344, functional
Saitoellacomplicata.TheclassIVawaslostinindividualfungal divergence of class V from class VII may result from a Ser
lineages, such as fission yeasts. The classes VIa and VIb were (S) to His (H) shift. In division III, we identified four type
lost in basidiomycetes and most of early-branching fungi. II specificity-determining sites. Residues at these sites differ
Unexpectedly, in contrast to other yeasts that retained only among the three classes. For example, the residues at site 209
the classes I, II, and IVa, Yarrowia lipolytica and S. complicata are Cys (C), Thr (T), and Ser (S) in classes VIa, VIb, and VIc,
containedmostoftheorthologousclasses. respectively.
In addition to gene losses, duplications of the CHS Most of the specificity-determining sites are adjacent to or
genes occurred frequently in different fungal lineages during located at the functionally or structurally important sites of
evolution. For example, members of class I were duplicated CHSsidentifiedbyConSurf(Figure5;FiguresS2–S4;Ashkenazy
in Basidiomycota, Zygomycota, and Candida species. CHS et al., 2010). For example, in division I, type II specificity-
genes in each class were duplicated multiple times in early- determining site 80 is directly involved in ligand binding
branchingfungi,andthosefromonespeciesgenerallyclustered (Figure5).Thisshiftprobablyaffectsthechemicalconformation
FrontiersinPlantScience|www.frontiersin.org 7 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
CS1N CS1 Partial CS2
① ② ③ ④ ⑤
Class (cid:3413)
(101 seqs)
n I Class (cid:3414)
o (62 seqs)
si
vi
DiClass (cid:3415)
(96 seqs)
Class (cid:3420)
(19 seqs)
f s sffff fff ss f ff f fffs s ssff ffsfs f s sssssfsss fsfff fsssssffsss ffff fssfssfss ss s s ffffff
Class (cid:3416)a
(97 seqs)
n IIClass (cid:3416)b
o (36 seqs)
si
vi
Di Class (cid:3417)
(93 seqs)
Class (cid:3418)(cid:44)
(90 seqs)
f fff ss s s ffffs f f ssff sff s f ss fs f s sssss sf fss ssfff s f sf ffsfssfs sf s ss s s s ff sf
Class (cid:57)(cid:44)a
n III (49 seqs)
sio Class (cid:57)(cid:44)b
vi (21 seqs)
Di
Class (cid:57)(cid:44)c
(7 seqs)
f f f ss fffff ssf s s ff ff s sffs f f ffff s s s f sf ffffs ss fff ff
FIGURE5|ConservationanddiversificationofCSdomainsinthefungalCHSgenefamily.SequencelogosindicateconservedsequencepatternsofCHS
domainineachclass.BlackhorizontalbarsindicateregionsofdomainsthataredefinedbythePfamdatabase.Redhorizontallinesindicateconservedfunctional
motifsaccordingtotheCDDdatabaseofNCBIandthepreviousstudy(Zakrzewskietal.,2014):À,ligandbinding;Á,metalionbindingsite;Â,donorsaccharide
binding;Ã,acceptorsaccharidebinding;Ä,productbinding.Greenshadedregionsrepresentsitesthatareconservedacrossallclasses.Redandbluetriangles
indicatetypeIandtypeIIspecificity-determiningsitesidentifiedbySPEER-SERVER(Chakrabortyetal.,2012),respectively.Thelettersfandsrepresentfunctional
sitesandstructuralsitesidentifiedbyConSurf(Ashkenazyetal.,2010),respectively.
of CHSs. Additionally, in division II, the structural site 346, Members of Classes III and VIb CHS Genes
which may have undergone type II functional divergence from are Specifically Up-Regulated during
division I, is adjacent to the acceptor saccharide binding Infection
sites. This shift may affect the interaction between CHSs and
acceptors. Previous studies have experimentally demonstrated Because classes III, VIb, and VIc were mainly retained or
twofunctionallyimportantmotifsEDRXLandQRRRWofCHSs expanded in important pathogenic fungi, we examined the
thatareresponsibleforacceptorsaccharidebindingandproduct expression of these CHSs during infection. In the wheat scab
binding in yeast (Nagahashi et al., 1995; Choquer et al., 2004; fungus Fusarium graminearum,oneoftwoparalogsofclassIII
Zakrzewskietal.,2014).Thesetwomotifsarewellconservedin CHS genes (FGSG_10116) was specifically up-regulated during
allclassesofCHSs,confirmingtheircriticalroles.Interestingly, spikeinfectionofbarleyandwheat(Figure6).ThisclassIIIgene
fourfunctionalspecificity-determiningsitesareadjacenttothese hadthehighestexpressionchangeduringhostinfectionrelative
twomotifs.Thesite409upstreamthreeresiduesoftheEDRXL to other CHS genes, in agreement with the results of a recent
motif underwent alteration in class III CHSs from Tyr (Y) study (Cheng et al., 2015). Deletion of this gene was lethal to
to Phe (F). Likewise, the site 462 downstream six residues F. graminearum (Cheng et al., 2015). Its ortholog in M. oryzae
of the QRRRW motif underwent alteration in class III CHSs (MGG_01802)wasresponsibleforvirulence(Kongetal.,2012).
from Phe (F) to Ala (A). These sites may be responsible for In the stem rust fungus Puccinia graminis f. sp. tritici, one of
functionaldivergenceofclassesIIIandtheotherthreeclassesof thethreecopies(PGTG_02527)ofclassIIICHSswasalsohighly
divisionI. inducedduringinfectionofwheatandbarley(FigureS5A).These
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Lietal. FungalChitinSynthaseGeneFamily
A1.5 B2.5
1.0 2.0
Expression change ----002110......050505 Expression change0011....0505
-0.5
-2.5
-7.0 -1.0
-7.5 -1.5
0 h 2 h 8 h 24 h 0 h 24 h 48 h 72 h 96 h 144 h
C4.0 D3.5
3.5 3.0
ession change 11223.....05050 ession change1122....0505
Expr00..05 Expr00..05
-0.5 -0.5
-1.0 -1.0
-1.5 -1.5
0 h 24 h 48 h 72 h 96 h 144 h 192 h 0 h 24 h 48 h 72 h 96 h 144 h
a a b b b
FIGURE6|ExpressionprofilesofCHSgenesduringdifferentstagesinFusariumgraminearum.(A)Conidiagermination.(B)Sexualdevelopmentinvitro.
(C)Infectionofwheat.(D)Infectionofbarleyspikes.Ineachcondition,themeanvaluesofRMAnormalizedexpressiondataofthreeindependentbiologicalduplicates
wereanalyzed.Alltime-seriesweretransformedto(0,v_1-v_0,v_1-v_0,...,v_n-v_0)sothatthetimeseriesbeginsat0.
resultsindicatethattheorthologousclassIIIinpathogenicfungi enrichment analysis showed that, beyond chitin biosynthesis,
mayperformimportantfunctionsduringhostinfection. genes involved in ascospore formation, phenotypic switching,
One CHS gene of class VIb (FGSG_06550) from positive regulation of endocytosis, and response to salicylic
F. graminearum was highly induced during the later phases acidweresignificantlyenrichedintheseconservedorthologous
of infection (Figure6). Its ortholog (GI: 310801203) in the genes (Figure7C; Table S3). These results indicated that the
maize anthracnose fungus Glomerella graminicola was also CHS-associatednetworkislikelyrelatedtofungalpathogenicity.
up-regulated during infection and had the highest expression In F. graminearum, ascospores are the primary inoculum for
level in the necrotrophic phase among all CHSs (Figure S5B), infecting wheat and barley (Trail et al., 2002). In addition,
suggestingthatthisclassVIbCHSisimportantforpathogenesis. salicylic acid (SA) is considered to be a key plant defense
In addition, genes in classes V (FGSG_01964) and VII hormone. A previous study demonstrated that U. maydis
(FGSG_12039) were highly induced during host infection. could sense and restrict the SA-levels of host plants to assure
PreviousstudieshavedemonstratedthattheclassesVand/orVII colonizationsuccess(Rabeetal.,2013).
CHSs are required for pathogenicity in many plant pathogens, We identified the putative protein complexes or parts of
suchasF. graminearum,F. verticillioides,Fusarium oxysporum, pathways hidden in the CHS network of F. graminearum
M. oryzae, G. graminicola, and Ustilago maydis (Madrid et al., constructedfromtheevidenceofgenomiccontext,experiments
2003; Garcerá-Teruel et al., 2004; Werner et al., 2007; Martín- and text-mining. Among the 171 genes in the network, six
Urdíroz et al., 2008; Kim et al., 2009; Treitschke et al., 2010; interactionclustersweredetectedandfoundtobecomprisedof
Larsonetal.,2011;Kongetal.,2012). 20,34,17,7,5,and3genes,respectively(Figure8A;TableS4).
Based ontheexpressiondata downloaded from PLEXdb(Dash
The CHS-Associated Network is
etal.,2012),mostofthegenesintheseclusterswereup-regulated
Conserved in Pathogenic Fungi during the later phases (72, 96, or 144h) of wheat infection.
To better understand the functional conservation of CHSs in Althoughsomegenesexhibitedtheoppositeexpressionpattern,
pathogenic fungi, we compared functional protein-associated the entire clusters seemed to be co-regulated. For example,
networks involved in chitin biosynthesis, constructed based on contrary to most genes in cluster 2 that were induced along
co-expression evidence and experimental evidence from the the timeline, five genes in the same cluster were repressed
STRING database (Szklarczyk et al., 2015) in three important (Figure8B).Theseresultssuggestedthatgenesintheseclusters
pathogenicfungi.Atotalof57,39,and36genesweredetermined might play important roles in the necrotrophic phase of F.
tobefunctionallyassociatedwiththeCHSsinF.graminearum, graminearum.Furtherfunctionalenrichmentanalysisindicated
M. oryzae, and U. maydis, respectively (Figure7A; Table S2). thatgenesincluster2areinvolvedinvariousprocesses,suchas
In the three CHS-associated networks, about half of the pathogenesis,theMAPKcascade,smallGTPase-mediatedsignal
genes were conserved (Figures7A,B). Gene ontology (GO) transduction,transmembranereceptorproteinserine/threonine
FrontiersinPlantScience|www.frontiersin.org 9 February2016|Volume7|Article37
Lietal. FungalChitinSynthaseGeneFamily
A
FGSG_08635 FGSG_01273
FGSG_08629 FGSG_11793FGSG_06550FGSpGp_03F481GSG_10161pppppppppppppppppppppFGSG_10691ppppFGSG_01948pppFpGSG_01p9p94ppppppppppFGSG_pp0pp3p5p4p4pppppppFGSG_1p2p551ppFGSG_108pp59ppppppppppppppppppppppppFpGSG_04164pppppppppppppFGSG_120p93ppppppppppppppppppppppppppppppppFGSGp_pppp0ppp6594pppppFpGSpppppppG_pppp06425ppppppppppFGSppGp_p07p53p0ppppppppppppppppppppppFGSG_1p2pp279ppppppppppppppppppFppppGpppSFpGG_S0G5_613609p29pppppppFGSGpp_pp1p0ppp0pp43pppppppppppppFpGSG_02438ppppFGSG_0ppp3pp107ppppFpGpSG_0042p5pppppFGSGp_p011pp81ppppppFGSG_05874ppppppppppppppppppppppppppppFFGGSSGG__10p01pp327p227ppppFGSG_00764pppppppppFpGSG_0845p7pppppppppFGSG_01946ppFGSppGp_pp1pp1p977pppppppppppppFGSG_06999ppppppppppppppppppppppppppppppppppppppppppFGSGF_G0pF1SpFG9GG5S_6pSG0pG8_7p0_p670897p43ppFp64G3SppG_06767ppppppFGSpGp_02022pppppppppppppppppppppppFGSGp_p09582ppppFGSGp_p10061ppppppppppppFGSG_08719pppFpGSG_0F69G5Sp7pGp_pp109pp41ppppppppppppppFGpSpG_10145FGSG_0705p4pppppppFGSG_10048FFGGSSGG__10028762p1p9ppppppppFGSG_02pp014ppppFGSG_06998FGSG_07311 FGSG_09492 FGSG_07443 MG_G06389MGG_09878 MGG_07928 MGG_03958MGG_01105ppppMGG_11479ppppppppppppMGG_0M1G3G1_08pp6pp044MGGp_0p1p2p82ppppppppMppGG_00307ppMGG_01708ppppppppppppppMppGG_12117ppppMpGGp_01656ppppMGG_00865ppppppppMGG_06759ppppppMGG_13031ppppMGG_03876pppppMpGG_08747ppppMGG_14966MGG_09195ppppppppppppppppMMGGGp_Gp0_1091M228G6G_812812ppMGGp_p04154ppppppppMGG_06p3p0p2ppppppppppppppppppppppppppMpppMGpGppG_G0p_41p7340014pppppppppMGG_018Mp0pG2G_04172pppppppppppppppppppppppppppppppppppMGG_1p2p93pp9ppppMGG_0604pp6ppppMGG_0955ppp1ppppppppppppppppMGG_08087ppppppppppppMpGG_03M46GG1_05032pppMpGG_09926ppppppppppppppMGG_p0p836p5pppMGG_05882 UM05401 UM04250UM05228ppppUM04358ppUMp0p4588UM00392ppppUM01963pppppUpM06155ppppppppppppppppppppppppppppUM04741ppppUM02p4p06UMpp0207p3pUMpp04655ppUM03204ppppppppppppppUM00462UM02554UpMp0p0p63p8ppppppppppppppppppppppUpM02p6pp8p9UM0p1p748UM03p62p8ppppppppppppppppppUM0p0p447UM00612ppppppppppppppUM029p5pp6pUM01788ppppppUppMpp03400ppppppppppppppppppppppppppppppppUMpp0pp5279ppppppUUMM0054428900ppppppUM01143pppppUpM01p06p4ppppppppppppppppUpppMp06398ppppUM02019pUpM03p20p5pppppppppppppppppppUpMp05763 UM03213UM02840 UM03465
FGSG_08468
Fg Mo Um
Orthologs found in: All networks Networks of Fg & Mo Networks of Fg & Um Only one network
B C
GO:0009751 response to salicylic acid 1 17
Fg Mo GO:0008361 regulation of cell size
Number of genes
GO:0045807 positive regulation of endocytosis
16 12 4 GO:0036166 phenotypic switching
GO:0071704 organic substance metabolism Enrichment factor
GO:0006807 nitrogen compound metabolism
18
(23,23,22) GO:0051704 m
6 0
GO:0006031 chitin biosynthetic process
GO:0034605 cellular response to heat
GO:0022607 cellular component assembly
8 GO:0005975 carbohydrate metabolism
GO:0030476 ascospore wall assembly
Um
GO:0003774 motor activity
GO:0004100 chitin synthase activity
MF
ransferase activity
GO:0016810 hydrolase activity, acting on
but not peptide) bonds
0 0 4 6 8
FIGURE7|CHS-associatednetworksinFusariumgraminearum,Magnaportheoryzae,andUstilagomaydis.(A)CHS-associatednetworksinthreeplant
pathogens.Nodesandedgesrepresentgenesandfunctionallinks,respectively.Nodecolorsindicatetherangeoforthologgroupsthatappearinthreenetworks.(B)
Venndiagramforthedistributionoforthologgroupsinthethreenetworks.Numberscorrespondtoorthologgroups,whilethenumbersinbracketsrepresentthetotal
numberofgenes(Ordering:Fg-Mo-Um).(C)SelectedGOtermsenrichedinorthologgenesthatareconservedinthethreenetworks.Thecirclecolorsindicatethe
numberofgeneswitharelevantGOterminthetestset.ThecirclesizeindicatestheratioofthepercentageofgeneswithrelevantGOtermsinthetestsettothatin
thereferenceset(enrichmentfactor).Fg,F.graminearum;Mo,M.oryzae;Um,U.maydis.PleaseseeTablesS2,S3formoredetailedinformation.
kinase signaling pathway, and ascospore formation (Figure8C; the number of CHS genes among different fungal lineages.
Table S5). Many of these processes are related to fungal Phylogenetic analysis revealed that the fungal CHS gene family
pathogenicity(Bölker,1998;Xuetal.,1998;BeyerandVerreet, is comprised of at least 10 ancestral orthologous clades, which
2005). In addition, cluster I includes acetyl-CoA carboxylase have undergone frequent duplications and losses in different
and aspartate carbamoyltransferase, which are essential for the fungal lineages during evolution. Our results showed that CHS
survivaloffungiduetotheircrucialrolesinthebiosynthesisof genesinclassesIIIandVIbhaveexpandedorhavebeenretained
fatty acids and pyrimidines (Simmer et al., 1990; Tong, 2005). in pathogenic fungi and that they likely perform important
Furthermore, calcineurin subunit B (FGSG_07404) in cluster 4 functions during host infection. Comparative analysis revealed
isinvolvedintheadaptationtopheromoneduringconjugation that CHS-associated networks are conserved among important
with cellular fusion, which is essential for normal vegetative pathogenicfungi,andparticipateinvariousimportantprocesses,
growthinNeurosporacrassa(KotheandFree,1998). including sexual reproduction and plant infection. In addition,
many specificity-determining sites are located at or adjacent to
CONCLUSIONS important sites such as ligand binding and acceptor saccharide
sites of CHSs and probably lead to functional diversification in
We systematically identified and compared CHS genes across theCHSgenefamily.Thesespecificity-determiningsitesmaybe
109 representative fungi from Ascomycota, Basidiomycota, attractivetargetsforfurtherstructuralandexperimentalstudies.
Cryptomycota, Microsporidia, Chytridiomycota, Zygomycota, Results from this study elucidated orthologous and paralogous
Neocallimastigomycota, Entomophthoramycotina, and relationships among various CHS genes and provided new
Glomeromycota. Comparative analysis revealed diversity in insightsintotheevolutionandfunctionofthefungalCHSgenes.
FrontiersinPlantScience|www.frontiersin.org 10 February2016|Volume7|Article37