Evolution and Functional Insights of Different Ancestral Orthologous Clades of Chitin Synthase Genes in the Fungal Tree of Life

ORIGINAL RESEARCH published: 01 February 2016 doi: 10.3389/fpls.2016.00037

Mu Li 1 †, Cong Jiang 1 †, Qinhu Wang 1, Zhongtao Zhao 2, Qiaojun Jin 1, Jin-Rong Xu 1, 3 and Huiquan Liu 1* Edited by: Thiago Motta Venancio, 1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Universidade Estadual do Norte China, 2 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China, 3 Department of Botany and Fluminense, Brazil Plant Pathology, Purdue University, West Lafayette, IN, USA Reviewed by: Lakshminarayan M. Iyer, Chitin synthases (CHSs) are key enzymes in the biosynthesis of chitin, an important National Center for Biotechnology Information, National Library of structural component of fungal cell walls that can trigger innate immune responses in Medicine, National Institutes of Health, host plants and animals. Members of CHS gene family perform various functions in fungal USA Robson Francisco De Souza, cellular processes. Previous studies focused primarily on classifying diverse CHSs into Universidade de São Paulo, Brazil different classes, regardless of their functional diversiﬁcation, or on characterizing their A. Maxwell Burroughs, functions in individual fungal species. A complete and systematic comparative analysis National Center for Biotechnology Information, National Library of of CHS genes based on their orthologous relationships will be valuable for elucidating Medicine, National Institutes of Health, 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: Huiquan Liu 18 divergent fungal lineages. Phylogenetic analysis revealed that the fungal CHS gene [email protected] family is comprised of at least 10 ancestral orthologous clades, which have undergone †These authors have contributed multiple independent duplications and losses in different fungal lineages during evolution. equally to this work. Interestingly, one of these CHS clades (class III) was expanded in plant or animal

Specialty section: pathogenic fungi belonging to different fungal lineages. Two clades (classes VIb and This article was submitted to VIc) identified for the first time in this study occurred mainly in plant pathogenic fungi Bioinformatics and Computational from Sordariomycetes and Dothideomycetes. Moreover, members of classes III and Biology, a section of the journal VIb were specifically up-regulated during plant infection, suggesting important roles in Frontiers in Plant Science pathogenesis. In addition, CHS-associated networks conserved among plant pathogenic Received: 11 September 2015 fungi are involved in various biological processes, including sexual reproduction and plant Accepted: 11 January 2016 Published: 01 February 2016 infection. We also identified specificity-determining sites, many of which are located at Citation: or adjacent to important structural and functional sites that are potentially responsible for Li M, Jiang C, Wang Q, Zhao Z, Jin Q, functional divergence of different CHS classes. Overall, our results provide new insights Xu J-R and Liu H (2016) Evolution and into the evolution and function of members of CHS gene family in the fungal kingdom. Functional Insights of Different Ancestral Orthologous Clades of Specificity-determining sites identified here may be attractive targets for further structural Chitin Synthase Genes in the Fungal and experimental studies. Tree of Life. Front. Plant Sci. 7:37. doi: 10.3389/fpls.2016.00037 Keywords: chitin synthase, fungi, evolution, functional diversification, plant infection

Frontiers in Plant Science | www.frontiersin.org 1 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family

INTRODUCTION daughter cells after cell division as a repair enzyme (Cabib et al., 1989). ScCHS2 participates in the processes of primary septum Chitin, a polymer of N-acetylglucosamine, is the second most formation and cell division (Silverman et al., 1988). The majority abundant biomass in nature after cellulose (Merzendorfer, 2011). of chitin is synthesized by ScCHS3, which is also responsible It is an important structural component of the cell wall in fungi, 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 filamentous fungi. In the plant pathogenic fungus Magnaporthe 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 plasma membrane and transfer N-acetylglucosamine to growing studies focused primarily on the functions of CHSs in individual chitin chains (Merzendorfer, 2011). Owing to the lack of chitin in fungal species. A complete and systematic comparative analysis plants and mammals, CHSs are considered attractive targets for of CHS gene family across the fungal kingdom will be valuable developing efficient antifungal agents used to control pathogenic for elucidating their evolutionary and functional relationships. fungi (Free, 2013). In this study, we systematically identified members of the CHS genes have been identified in various fungi. According to CHS gene family across 109 representative fungi from 18 major the phylogenetic positions and similarity in domain architecture, 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, we found that CHS-associated networks 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 (2010) were classified into classes I and II in the study of of at Least 10 Ancestral Orthologous 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, the existing classification scheme (Pacheco-Arjona and Ramirez- 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 the orthologs retain the same function (Koonin, Chytridiomycota, Entomophthoramycotina, Zygomycota, 2005), evolutionary classification of CHS genes based on Glomeromycota, Ascomycota, and Basidiomycota (Figure 1; the orthologous relationship is more reliable for transferring Table S1). Among these, 373 genes contained CS1N, CS1, and functional annotation. CS2 domains, suggesting that these genes were members of Different CHSs have been proved to play various roles, division I. Five additional genes lacked the CS2 domain and one 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 genome assembly gaps. The remaining 600 genes harbored only budding yeast Saccharomyces cerevisiae has three CHS genes with the CS2 domain, suggesting that these genes are members of different functions. ScCHS1 replenishes chitin in the cell wall of division II or III.

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Division I Division II Division III Total numbers

a b a b Plant pathogen ------Trichophyton equinum 111 1 111 ------Trichophyton verrucosum 111 1 111 Animal pathogen ------Trichophyton tonsurans 111 1 111 ------Trichophyton rubrum 111 1 111 ------Arthroderma gypseum 111 1 111 ------Arthroderma otae 111 1 111 ------Arthroderma benhamiae 111 1 111 ------Coccidioides immitis 111 1 111 ------Coccidioides posadasii 111 1 111 ------Uncinocarpus reesii 111 1 111 ------Ajellomyces capsulatus 111 1 111 ------Ajellomyces dermatitidis 111 1 111 ------Paracoccidioides brasiliensis 111 1 111 Eurotiomycetes ------Aspergillus nidulans 212 1 111 ------Aspergillus terreus 112 1 111 ------Aspergillus niger 113 1 111 ------Aspergillus oryzae 114 1 111 ------Aspergillus flavus 1 1 4 1 1 1 1 1 ------Aspergillus fumigatus 112 1 111 ------Aspergillus fischerianus 113 1 111 ------Aspergillus clavatus 112 1 111 ------Penicillium chrysogenum 113 1 111 ------Penicillium marneffei 111 1 111 ------Talaromyces stipitatus 111 1 111 ------Metarhizium acridum 111 1 111 ------Metarhizium anisopliae 111 1 111 ------Trichoderma reesei 111 1 1111 ------Cordyceps militaris 111 1 1111 ------Fusarium verticillioides 113 1 11 32 ------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 111 1 1112 ------Verticillium dahliae 111 1 1112 Sordariomycetes ------Glomerella graminicola 1 1 1 1 1 1 1 1 1 ------Neurospora tetrasperma 111 1 111 ------Neurospora crassa 111 1 111 ------Chaetomium globosum 111 1 111 ------Chaetomium thermophilum 111 1 111 ------Magnaporthe poae 112 1 1 1 ------Gaeumannomyces graminis 1 1 1 1 1 1 1 1 ------Magnaporthe oryzae 111 1 111 ------Botryotinia fuckeliana 112 1 111 ------Sclerotinia sclerotiorum 111 2 111 Leotiomycetes ------Blumeria graminis 111 1 111 ------Mycosphaerella graminicola 111 1 1111 ------Mycosphaerella fijiensis 1 1 1 1 1 1 1 1 ------Cladosporium fulvum 1 1 1 1 1 1 1 1 1 ------Baudoinia compniacensis 111 1 111 ------Pyrenophora tritici-repentis 111 1 1111 Dothideomycetes ------Pyrenophora teres 111 1 1111 ------Cochliobolus sativus 111 1 111 ------Phaeosphaeria nodorum 1 1 1 1 1 1 1 ------Rhytidhysteron rufulum 111 1 111 ------Hysterium pulicare 1 1 1 1 111 ------Monacrosporium haptotylum 111 1 111 ------Arthrobotrys oligospora 111 1 111 Orbiliomycetes ------Tuber melanosporum 112 1 111 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 ------Schizosaccharomyces pombe 1 1 Taphrinomycotina ------Taphrina deformans 1 1 1 ------Saitoella complicata 111 11112 ------Coprinopsis cinerea 2 2 1 1 1 1 1 ------Schizophyllum commune 2 111111 ------Laccaria bicolor 2 111142 ------Heterobasidion annosum 2 111111 ------Ganoderma sp. 10597 SS1 2 111112 ------Dichomitus squalens 2 1 1 1 1 1 1 ------Postia placenta 3 1 1 2 1 ------Gloeophyllum trabeum 2 111121 Agaricomycotina ------Coniophora puteana 2 111112 ------Serpula lacrymans 2 111132 ------Fomitiporia mediterranea 3 111111 ------Moniliophthora perniciosa 2 1 1 1 1 ------Cryptococcus neoformans 3 111111 ------Cryptococcus gattii 2 111111 ------Ustilago maydis 2 111111 ------Malassezia globosa 1 1 1111 Ustilaginomycotina ------Puccinia striiformis 3 3 1123 ------Puccinia graminis 2 3 1 2 1 1 Pucciniomycotina ------Melampsora larici-populina 2 2 2111 ------Rhizophagus irregularis 4 2 2 5 1 1 2 Glomeromycota ------Coemansia reversa 1 1 1 3 4 2 ------Rhizopus chinensis 44 88115 4 ------Rhizopus delemar 4 2 6 5 6 3 3 Zygomycota ------Mucor circinelloides 42 4463 ------Conidiobolus coronatus 1 2 2 2 5 3 Entomophthoramycotina ------Gonapodya prolifera 412 2 ------Allomyces macrogynus 11 6 1 23 Chytridiomycota ---Batrachochytrium dendrobatidis 341 2 6 ------Spizellomyces punctatus 6 1 4 1 7 ------Orpinomyces sp. 625 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

FIGURE 1 | Distribution of CHS genes across different fungal lineages. The copy number of each orthologous CHS class is indicated. The species tree was constructed based on the phylogenetic tree of α-tubulins. The horizontal bar indicates the total number of CHSs in each fungus. For the deﬁnition of I, II, III, IVa, IVb, V, VII, VIa, VIb, VIc, VIII, and others (OTS), please see the text.

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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 this study and defined as class VIII. division I displayed four distinct ancestral orthologous clades Phylogenetic analysis showed that members of divisions II and (Figures 2,4A). Three of these orthologous clades correspond III are distantly related to each other (Figures 3,4B). 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; Choquer et al., 2004; Pacheco-Arjona and Ramirez-Prado, Pacheco-Arjona and Ramirez-Prado, 2014), but our study 2014) was actually comprised of two orthologous clades: 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

S.oct|SOCG00496T0 S.cry|SPOG02009T0 Saccharomycotina Y.lip|YALI0D25938p C

S.pom|gi19113750 h.D .C 913532941ig|ole.L . 4 S.jap|gi213410345 S.cer|330443698

ul 7032 Sa.co|Saico54082

|iu |

2i 2

D.squ|gi395323237 7

G.tra|jgi100384 9 6

552i

4 641 L.bic|gi170112698 0 S.com|gi302696351C.cin|gi299744074

91 54

|ort.

8 P.gra|PGTG10666T0C.neo|gi58264402 3 ubme7239 C.gat|gi321254707 6 8365 T

5 0 Eurotiomycetes 17 P.str|CQM14412T0 C.par|gi354548408

B.fuc|gi154315738 T

0 .

09695

F.ver|FVEG10778T0 g.B

e F.oxy|FOXG12345T0 12 a

F.gra|FGSG03418T0N.hae|gi302886924 l

| |

g uT F.oxy|FOXG19023T0 0 06 F.ver|FVEG02839T0

emb

C.mil|gi346324114 P

V.dah|gi346974805V.alb|gi302421586 8

2 G.gra|gi310792794C.glo|CHGT00596 2

M 1 C.the|gi340923659 N.tet|gi350293588 |ao

N.cra|NCU03611T0

B.fuc|gi154296226

N.hae|gi302886234 M.ory|MGG01802T0

Ga.gr|GGTG06861T0 .M M.poa|MAPG05453T0 F.ver|FVEG00084T0

FIGURE 2 | Maximum likelihood phylogeny of division I CHSs. The phylogenetic tree was constructed using PhyML3.1 (Guindon et al., 2010). The SH-like support of approximate likelihood ratios (aLRT-SH) are plotted as circles on the branches (only SH-like support >0.8 are indicated). Colors of branches indicate corresponding classes. The scale bar corresponds to 0.1 amino acid substitution per site. For abbreviations of fungi, please see Table S1.

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Color ranges: Protein domains: Chytridiomycota Myosin motor-like domain Zygomycota Cyt- b5 Ustilaginomycotina Agaricomycotina CS2 Pucciniomycotina DEK_C Taphrinomycotina Saccharomycotina

M.poa|MAPG00522T0

Ga.gr|GGTG04108T0 Orbiliomycetes N 0T9 V.dah|gi346970242

M.ory|MGG06064T0 C G.gra|gi310792055 N V.alb|gi302422504 .

5 .C

6 t

54 rc S.scl|gi156063542 g t. B.fuc|gi154302175 h 4 80 a

e 4

e 91 o

P.ter|gi330907029 t P.tri|gi189198009 3

2 Dothideomycetes N| | | B.gra|bgh00811 5 9

C| 1

0 2

C.sat|jgi221014 H M.gra|gi398390950 3 UC

2 5 43ig

6 G

|i g |ar 40T2 9 rca T.ton|gi326469906 |l T.equ|gi326479129

8 8 9 i A.gyp|gi315056703 n

Leotiomycetes 3

g. A.ota|gi296827214 . m.C

5 0T86227 C.pos|gi303320149 56 C.imm|gi119183988 N.hae|gi302882365 F.oxy|FOXG04179T0

F M M

4 5

3 m 2 M.ory|MGG13014T0 B.gra|bgh00325 .

m. f B.fuc|gi154289813 9 5 g.

6 7

R.ruf|jgi8676 if.M

04 4 j |

T | g

P.ter|gi330922468 77 g

u i

5 1 i 4 3511i

3 b

2ig|ra 7 9 8 P.nod|gi169620245 8

9 m

6ig 61i 8

3365

A.ben|gi302495767T.ver|gi302664948 2

T.ton|gi326474546 2 | 5

46 4e

g 0

g T.equ|gi326478152 g|r d | T.rub|gi327299264 3

| 2 9 i

A.gyp|gi315052390 yr A.ota|gi296815292 0

l 9

A.cap|gi154275092 A.der|gi261199334 et

f 60

A.fis|gi119481549 o C.pos|gi303322685 .A A.fum|gi71001990 .A .A U.ree|gi258565649 A

C.imm|gi119189947 A.cla|gi121716016 98 A.nig|gi145231807

P.chr|gi255933041 08

FIGURE 3 | Maximum likelihood phylogeny of divisions II and III CHSs. The phylogenetic tree was constructed using PhyML3.1. The SH-like support of approximate likelihood ratios (aLRT-SH) are plotted as circles on the branches (only SH-like support >0.8 are indicated). Colors of branches indicate corresponding classes. The scale bar corresponds to 0.5 amino acid substitution per site. For abbreviations of fungi, please see Table S1. were temporarily labeled as “others” (OTS) (Figure 1). 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 (Figure 2). 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 members in the same class are more closely related to each other, also contain the Cytochrome b5-like (Cyt-b5, PF00173) domain but distantly related to members in other classes (Figure S1). Each (Figure 3). This domain is absent from some members of classes class formed a distinct group. These results are consistent with IVa and IVb in division II. The Cyt-b5 domain can bind heme those of our phylogenetic analysis. CHSs of division III are more and steroids in electron transfer (Schenkman and Jansson, 2003), similar to those of division II than division I. but the ligand for this domain in fungal CHSs is unknown. An

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FIGURE 4 | Maximum likelihood phylogenies of CHS genes from early-branching fungi. (A) Division I. (B) Divisions II and III. Phylogenetic tree was constructed using PhyML3.1. The SH-like support of approximate likelihood ratios (aLRT-SH) are plotted as circles on the branches (only SH-like support >0.8 are indicated). Colors of branches indicate corresponding classes. For abbreviations of fungi, please see Table S1.

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N-terminal myosin motor-like domain (MMD, PF00063) and a together (Figures 2–4), suggesting that they were derived from DEK C-terminal (DEK_C, PF08766) domain also exist in classes species-specific expansions. In fact, the genome of Rhizopus V and VII CHSs. The MMD assiststhe localizationof CHSs tothe delemar underwent a whole-genome duplication and recent gene hyphae via interaction with the actin cytoskeleton, and facilitates duplications (Ma et al., 2009). polarized exocytosis (Tsuizaki et al., 2009; Schuster et al., 2012). Notably, duplications in members of class III were most The DEK_C domain is related to the C-terminal of animal DEK common in plant or animal pathogens within Aspergillus spp., protein, which is responsible for self-multimerization and DNA Fusarium spp., and Pucciniomycotina. Since the class III CHS binding (Kappes et al., 2004). Nevertheless, the role of the DEK_C has been experimentally demonstrated to play important roles domain in fungi remains to be determined. 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. Fungal Lineages Furthermore, the classes VIb and VIc CHSs occurred mainly in plant pathogens from Sordariomycetes and Dothideomycetes. Yeasts generally contain few CHSs (Figure 1). For example, 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 yeast S. cerevisiae harbors three CHSs. Filamentous fungi usually 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; number of copies among the later-branching fungi, 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, the shifts at specificity-determining sites (Gu, 2001). Therefore, and glomeromycetes, generally possess more CHSs than the we examined CS domains among the orthologous classes for later-branching fungi (Figure 1), likely because the cell walls of functional specificity-determining sites. early-branching fungi contain a higher percentage of chitin than In division I, we identified 15 specificity-determining sites those of later-branching fungi (Ma et al., 2009). Unexpectedly, (Figure 5). 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 largest number of CHS genes discovered to date. 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 consistent with a previous report (Pacheco-Arjona and Ramirez- functional divergence. Residues at these sites are conserved in Prado, 2014). different clades but their properties are dissimilar, which could lead to functional specifications within a gene family (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 branching fungi and later-branching fungi (Figure 1), 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 Saitoella complicata. The class IVa was lost in individual fungal 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, contained most of the orthologous classes. 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 CHSs identified by ConSurf (Figure 5; Figures S2–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 branching fungi, and those from one species generally clustered (Figure 5). This shift probably affects the chemical conformation

Frontiers in Plant Science | www.frontiersin.org 7 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family

CS1N CS1 Partial CS2 ① ② ③ ④ ⑤

Class ൕ (101 seqs)

Class ൖ (62 seqs)

Class ൗ DivisionI (96 seqs)

Class ൜ (19 seqs)

f s s f f f f f f f ss f f f f f f f s s ss f f f f s f s f s sssss f sss f s f f f f sssss f f sss f f f f f ss f ss f ss ss s s f f f f f f

Class ൘a (97 seqs)

Class ൘b (36 seqs)

Class ൙ Division II Division (93 seqs)

Class ൚, (90 seqs)

f f ff ss s s f f ff s f f ss f f s f f s f ss f s f s sssss s f f ss ss f f f s f s f f f s f ss f s s f s sss s s f fs f

Class 9, a (49 seqs)

Class 9, b (21 seqs) Division III Division Class 9, c (7 seqs)

f ff ss f f ff f ss f s s f f f f s sf f s f f f f f f s s s f s ff f f f s ss f f f f f

FIGURE 5 | Conservation and diversification of CS domains in the fungal CHS gene family. Sequence logos indicate conserved sequence patterns of CHS domain in each class. Black horizontal bars indicate regions of domains that are defined by the Pfam database. Red horizontal lines indicate conserved functional motifs according to the CDD database of NCBI and the previous study (Zakrzewski et al., 2014): , ligand binding; , metal ion binding site; , donor saccharide binding; , acceptor saccharide binding; , product binding. Green shaded regions represent sites that are conserved across all classes. Red and blue triangles indicate type I and type II specificity-determining sites identified by SPEER-SERVER (Chakraborty et al., 2012), respectively. The letters f and s represent functional sites and structural sites identified by ConSurf (Ashkenazy et al., 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 two functionally important motifs EDRXL and QRRRW of CHSs expanded in important pathogenic fungi, we examined the that are responsible for acceptor saccharide binding and product expression of these CHSs during infection. In the wheat scab binding in yeast (Nagahashi et al., 1995; Choquer et al., 2004; fungus Fusarium graminearum, one of two paralogs of class III Zakrzewski et al., 2014). These two motifs are well conserved in CHS genes (FGSG_10116) was specifically up-regulated during all classes of CHSs, confirming their critical roles. Interestingly, spike infection of barley and wheat (Figure 6). This class III gene four functional specificity-determining sites are adjacent to these had the highest expression change during host infection relative two motifs. The site 409 upstream three residues of the EDRXL 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) was responsible for virulence (Kong et al., 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 functional divergence of classes III and the other three classes of the three copies (PGTG_02527) of class III CHSs was also highly division I. induced during infection of wheat and barley (Figure S5A). These

Frontiers in Plant Science | www.frontiersin.org 8 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family

A 1.5 B 2.5

1.0 2.0 0.5 1.5 0.0 -0.5 1.0 -1.0 0.5

-1.5 0.0 Expression change Expression Expression change change Expression -2.0 -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

C 4.0 D 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 Expression change change Expression change Expression 0.0 0.0 -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

FIGURE 6 | Expression profiles of CHS genes during different stages in Fusarium graminearum. (A) Conidia germination. (B) Sexual development in vitro. (C) Infection of wheat. (D) Infection of barley spikes. In each condition, the mean values of RMA normalized expression data of three independent biological duplicates were analyzed. All time-series were transformed to (0, v_1-v_0, v_1-v_0,..., v_n-v_0) so that the time series begins at 0. results indicate that the orthologous class III in pathogenic fungi enrichment analysis showed that, beyond chitin biosynthesis, may perform important functions during host infection. 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 acid were significantly enriched in these conserved orthologous of infection (Figure 6). Its ortholog (GI: 310801203) in the genes (Figure 7C; Table S3). These results indicated that the maize anthracnose fungus Glomerella graminicola was also CHS-associated network is likely related to fungal pathogenicity. 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, suggesting that this class VIb CHS is important for pathogenesis. 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 Previous studies have demonstrated that the classes V and/or VII colonization success (Rabe et al., 2013). CHSs are required for pathogenicity in many plant pathogens, We identified the putative protein complexes or parts of such as F. graminearum, F. verticillioides, Fusarium oxysporum, pathways hidden in the CHS network of F. graminearum M. oryzae, G. graminicola, and Ustilago maydis (Madrid et al., constructed from the evidence of genomic context, 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; interaction clusters were detected and found to be comprised of Larson et al., 2011; Kong et al., 2012). 20, 34, 17, 7, 5, and 3 genes, respectively (Figure 8A; Table S4). Based on the expression data downloaded from PLEXdb (Dash The CHS-Associated Network is et al., 2012), most of the genes in these clusters were up-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 Although some genes exhibited the opposite expression pattern, 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 (Figure 8B). These results suggested that genes in these clusters pathogenic fungi. A total of 57, 39, and 36 genes were determined might play important roles in the necrotrophic phase of F. to be functionally associated with the CHSs in F. graminearum, graminearum. Further functional enrichment analysis indicated M. oryzae, and U. maydis, respectively (Figure 7A; Table S2). that genes in cluster 2 are involved in various processes, such as In the three CHS-associated networks, about half of the pathogenesis, the MAPK cascade, small GTPase-mediated signal genes were conserved (Figures 7A,B). Gene ontology (GO) transduction, transmembrane receptor protein serine/threonine

Frontiers in Plant Science | www.frontiersin.org 9 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family

FGSG_01273 FGSG _08653

FGSG _12297

FGSG _12039

FGSG _10619

UM00474

pp UM04290

pp MGG_04172

pp MGG_01780 pp

ppp

MGG_01868

ppp MGG_05023

pp UM00462 pp FGSG_02729

FGSG _10116 UM03052 pp

pp pp ppp

FGSG _01949 pp pp FGSG _05847

pp pp ppp pp FGSG _05663 pp ppp pp

pp pp MGG_09159 pp pp pp pp pp pp ppp pp pp pp pp pp pp

pp FGSG_02022 pp

pp pp pp ppp pp pp pp pp pp pp pp pp pp pp pp pp pp

pp pp pp pp pp

pp pp pp MGG_01150 pp pp FGSG _07503 pp pp pp pp pp pp pp pp pp pp pp ppp pp

pp ppp FGSG _03418 ppp pp FGSG _00452 pp UM00126 pp pp pp pp pp pp UM05637 pp pp pp pp pp pp pp MGG_06064 ppp ppp

pp pp pp pp pp

pp pp pp pp pp pp

pp pp FGSG _04146

pp MGG_13014 pp pp ppp MGG_08774 UM01143

MGG_09962 pp pp ppp pp pp UM00638 pp pp pp pp pp pp MGG_12117 pp pp pp FGSG_10145 FGSG _10992 pp UM04583 pp pp pp ppp pp pp pp pp

pp pp

pp pp pp pp pp pp pp pp pp pp

pp UM01788 pp pp

pp pp pp pp MGG_04145 pp pp pp FGSG _06550 pp pp pp pp pp FGSG _06452 pp pp FGSG _10327 pp FGSG _12515 pp

MGG_09551

pp pp pp pp pp

pp pp UM02019 MGG_05828 FGSG_07946 pp pp pp pp pp pp

pp pp pp MGG_01802

UM02689

pp ppp pp pp pp pp pp pp pp pp pp pp pp pp pp pp MGG_11497 pp pp MGG_14966 pp UM05792

ppp pp

pp pp pp pp pp FGSG_06998 pp pp pp

pp ppp

FGSG _03170 pp UM02406 pp pp pp pp

ppp pp pp pp pp FGSG _06549 pp pp pp pp pp ppp pp pp pp pp pp pp pp FGSG _03544 pp

ppp pp FGSG _01272 pp FGSG_06957 pp pp UM05014 pp ppp pp ppp pp pp pp

ppp

pp pp pp pp pp pp MGG_08356 pp pp

pp pp pp pp

pp pp pp pp pp pp

MGG_01922

pp pp pp pp

MGG_12939

pp pp

pp UM03465 FGSG _02483 pp pp

MGG_00307 pp pp pp

pp pp pp

FGSG_10861 pp pp pp pp MGG_03985 MGG_13013 FGSG_07343 pp pp pp pp pp MGG_03461 MGG_06389

pp FGSG_09492

MGG_04704

UM05480 UM02569 pp pp UM03204 UM04588

pp pp pp pp FGSG _08692

pp pp

pp FGSG _01964

pp pp pp FGSG_10941 pp ppp pp

pp pp pp pp pp pp pp pp pp pp pp

pp pp

pp pp pp pp MGG_06044 pp pp FGSG_08768 pp

UM02840

pp UM06155 pp pp pp pp pp pp

FGSG _01948 pp pp pp pp

pp pp pp pp pp pp

pp pp pp pp pp pp pp

pp MGG_09887 UM04250

UM06398 pp pp pp pp FGSG_02014 ppp

pp UM01748 pp

pp pp pp MGG_06759 pp

pp FGSG _10034 FGSG_08719 pp pp pp

pp pp ppp pp pp pp pp pp pp FGSG _08475 pp UM04556 pp

FGSG_07443 pp

pp pp

pp pp pp pp pp pp ppp

MGG_01282 ppp pp pp

pp pp pp

pp pp pp UM00923

FGSG _11793 pp pp pp

pp pp pp pp pp pp pp

FGSG_10048 pp pp

pppp

pp FGSG_01956 UM03213 pp UM03286 pp MGG_06320 UM01640

pp pp UM02073 pp pp pp

MGG_08078 pp MGG_07928

pp pp pp

pp MGG_01656 pp

FGSG _00746

pp pp

pp pp FGSG _10895 MGG_03876 pp UM05228 pp pp pp

FGSG_10061 pp

FGSG_07311 pp pp

pp pp UM03004 FGSG _06999

MGG_01318 FGSG _01118

pp UM01639 pp

UM02554

FGSG_07054 FGSG_06767 pp

MGG_12821

MGG_00865

UM04417

FGSG _11977

FGSG_09582

FGSG _08486

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

GO:0008361 regulation of cell size Fg Mo Number of genes GO:0045807 positive regulation of endocytosis

GO:0036166 phenotypic switching 16 12 4 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

00 4 6 8

FIGURE 7 | CHS-associated networks in Fusarium graminearum, Magnaporthe oryzae, and Ustilago maydis. (A) CHS-associated networks in three plant pathogens. Nodes and edges represent genes and functional links, respectively. Node colors indicate the range of ortholog groups that appear in three networks. (B) Venn diagram for the distribution of ortholog groups in the three networks. Numbers correspond to ortholog groups, while the numbers in brackets represent the total number of genes (Ordering: Fg-Mo-Um). (C) Selected GO terms enriched in ortholog genes that are conserved in the three networks. The circle colors indicate the number of genes with a relevant GO term in the test set. The circle size indicates the ratio of the percentage of genes with relevant GO terms in the test set to that in the reference set (enrichment factor). Fg, F. graminearum; Mo, M. oryzae; Um, U. maydis. Please see Tables S2, S3 for more detailed information. kinase signaling pathway, and ascospore formation (Figure 8C; 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; Xu et al., 1998; Beyer and Verreet, 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 survival of fungi due to their crucial roles in the biosynthesis of genes in classes III and VIb have expanded or have been retained 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 is involved in the adaptation to pheromone during conjugation that CHS-associated networks are conserved among important with cellular fusion, which is essential for normal vegetative pathogenic fungi, and participate in various important processes, growth in Neurospora crassa (Kothe and Free, 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 the CHS gene family. These specificity-determining sites may be 109 representative fungi from Ascomycota, Basidiomycota, attractive targets for further structural and experimental studies. 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 insights into the evolution and function of the fungal CHS genes.

Frontiers in Plant Science | www.frontiersin.org 10 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family

ABC 0h 24h 48h 72h 96h 144h192h 2 1 Score (1) protein serine/threonine kinase activity; 0 (1) V (2) transmembrane transporter activity; 1 logFC Z logFC (3) acetyl-CoA carboxylase activity; 2 (4) aspartate carbamoyltransferase activity.

(20 genes, 14.11 scores)

(1) pathogenesis; (2) activation of MAPK activity; (3) small GTPase mediated signal transduction; (4) transmembrane receptor protein serine/ (2) II threonine kinase signaling pathway; I (5) conidium & ascospore formation; (6) chitin biosynthetic process.

(34 genes, 9.27 scores)

IV a (1) response to salicylic acid; (2) small GTPase mediated signal transduction; (3) (3) cell morphogenesis; (4) ascospore formation; (5) phenotypic switching. (17 genes, 6.38 scores)

(1) response to pheromone involved (4) in conjugation with cellular fusion; (2) chitin biosynthetic process. (7 genes, 5.00 scores)

(1) small GTPase mediated signal transduction; (5) (2) organelle organization.

(5 genes, 3.00 scores)

(6) NA 0h 24h 48h 72h 96h 144h192h (3 genes, 3.00 scores)

FIGURE 8 | Expression profiles of interaction clusters in CHS-associated networks in F. graminearum. (A) Interaction clusters identified in the network of CHSs in F. graminearum. The square nodes represent CHSs of F. graminearum. Numbers in the brackets correspond to gene numbers and scores calculated by MCODE (Bader and Hogue, 2003), respectively. Please see Table S4 for more detailed information. (B) Dynamic expression profiling of interaction clusters during infection of wheat. Each row in the heatmap indicates a single gene and each column represents a time point after wheat was inoculated with F. graminearum. The scale bar represents the Z-scores of log2-fold change. (C) Selected GO terms enriched in five interaction clusters. Please see Table S5 for more detailed information.

MATERIALS AND METHODS as the cutoff. Only the sequences marked as CHSs (best match) were submitted to the Pfam database and the SMART database Collection of Fungal Genomes (Letunic et al., 2012) to confirm the chitin synthase domains. The predicted proteomes of 109 public fungal genomes (Table S1) were collected from GenBank of National Center for Sequence Alignment and Phylogenetic Biotechnology Information (NCBI), Joint Genome Institute Analysis (JGI) site of DOE (http://genome.jgi.doe.gov/programs/fungi/ Multiple sequence alignments were generated with the M-Coffee index.jsf), Fungal Genome Initiative (FGI) site of Broad Institute program (Di Tommaso et al., 2011) using the combination of T- (http://www.broadinstitute.org/science/projects/projects/ Coffee, MAFFT, MUSCLE, and ProbCons methods and further fungal-genome-initiative), UniProt database (http://www. edited manually. CHSs in division I were aligned based on the uniprot.org/proteomes/), and Blugen (http://www.blugen.org/). continuous CS1N, CS1, and CS2 domains, whereas CHSs in divisions II and III were aligned based only on the CS2 domain. Identification of Putative CHSs in Fungi Phylogenies were subsequently constructed by the Maximum The Hmmscan program in HMMER 3.0 package (Eddy, 2011) likelihood (ML) method using PhyML3.1 (Guindon et al., 2010) was used to search each of the fungal proteomes with the with eight categories of γ-distributed substitution rates and SPRs domain-specific HMM profiles of chitin synthases downloaded algorithms, based on the best-fit model LG + G estimated by from the Pfam database (Finn et al., 2014) as queries, including ProtTest2.4 (Abascal et al., 2005). SH-like approximate likelihood Chitin_synth_1 (PF01644) and Chitin_synth_2 (PF03142) ratios (aLRT-SH) supports were used to evaluate the reliability of domains. The primary result was filtered with the score of 20 internal branches. The trees were further edited using the ITOL

Frontiers in Plant Science | www.frontiersin.org 11 February 2016 | Volume 7 | Article 37 Li et al. Fungal Chitin Synthase Gene Family tool (Letunic and Bork, 2007). The identity scores of alignment (version 10) database (Szklarczyk et al., 2015) with a confidence were extracted with BioEdit software and then the heat map was score of 0.4. Then CHS-associated networks were visualized constructed with the gplot package in R. in Cytoscape (version 3.2.0) software (Shannon et al., 2003). Interaction clusters were detected using MCODE (version Prediction of Specificity-Determining Sites 1.4.1) plugin (Bader and Hogue, 2003) with the default in the Fungal CHS Gene Family parameters. Functional enrichment analysis was performed using Complete sequences of CHSs in each division were aligned Ontologizer 2.1 software (Bauer et al., 2008) with the whole F. using COBALT (Papadopoulos and Agarwala, 2007). Specificity- graminearum annotation as the reference set. The term-for-term determining sites were identified using SPEER-SERVER (http:// approach and Benjamini and Hochberg FDR correction (adjusted www.hpppi.iicb.res.in/ss/index.html) with highest scores. The p < 0.05) options were used. Heatmaps were constructed with options of 20% gap allowed per column and a weight of 1.0 for the R package gplot. Hierarchical clustering was carried out with relative entropy and physico-chemical (PC) properties were used Pearson correlation distance measure and the pairwise average- (Chakraborty et al., 2012). A sequence logo of each class of CHSs linkage method. was created with WebLogo 3 (Crooks et al., 2004). Structural sites and functional sites were identified using the ConSurf Server AUTHOR CONTRIBUTIONS (Ashkenazy et al., 2010). The default Bayesian calculation method and JTT evolutionary substitution model were used. JRX conceived the study; HL designed and coordinated the Analysis of Gene Expression Profiles of study; ML and CJ performed bioinformatic analyses; QW and ZZ contributed to the data collection; ML, CJ, QW, QJ, JX, and HL CHSs in Plant Pathogens interpreted the results; ML and HL wrote the paper. All authors The mean values of Robust Multi-array Average (RMA) read, corrected and approved the final manuscript. normalized expression data of F. graminearum were obtained from the Plant Expression Database (PLEXdb) (http://www. plexdb.org/index.php) with four individual experiments, ACKNOWLEDGMENTS including conidia germination (Experiment FG7), sexual We thank Dr. Larry Dunkle at Purdue University for development (Experiment FG5) and spike infection of barley proofreading. This work was supported by the National (Experiment FG1) and wheat (Experiment FG15). For each Major Project of Breeding for New Transgenic Organisms experiment, all time-series were transformed to (0, v_1-v_0,..., (2012ZX08009003), grant 2013CB127702 from the National v_n-v_0) so that the time series begins at 0. The normalized gene Basic Research Program of China (973 program) and the expression data of G. graminicola (O’Connell et al., 2012) and Special Fund for Agro-scientific Research in the Public Interest P. graminis f. sp. tritici (Duplessis et al., 2011) were downloaded (201303016). from NCBI GEO (accession no. GSE34632 and GSE25020). The data of three biological replicates were averaged and further transformed to log2 ratios. SUPPLEMENTARY MATERIAL Network Analysis The Supplementary Material for this article can be found We used CHSs as baits to extract all the functional protein- online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. associated networks using different evidence in the STRING 00037

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