Comparative Analysis of Fungal Genomes Reveals Different Plant Cell Wall Degrading Capacity in Fungi Zhongtao Zhao1, Huiquan Liu1*, Chenfang Wang1 and Jin-Rong Xu1,2

Zhao et al. BMC Genomics 2013, 14:274 http://www.biomedcentral.com/1471-2164/14/274

RESEARCH ARTICLE Open Access Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi Zhongtao Zhao1, Huiquan Liu1*, Chenfang Wang1 and Jin-Rong Xu1,2

Abstract EDITOR'S NOTE: Readers are alerted that there is currently a discussion regarding the use of some of the unpublished genomic data presented in this manuscript. Appropriate editorial action will be taken once this matter is resolved. Background: Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results: In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also revealed a complex history of lineage-specific expansions and attritions for the PL1 family. Conclusions: Our study provides insights into the variety and expansion of fungal CAZyme classes and revealed the relationship of CAZyme size and diversity with their nutritional strategy and host specificity. Keywords: Fungi, CAZymes, Glycoside Hydrolase, Polysaccharide Lyase, Carbohydrate Esterase, Pectinase, Cutinase, Lignocellulase

* Correspondence: [email protected] 1NWAFU-PU Joint Research Center and State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China Full list of author information is available at the end of the article

© 2013 Zhao et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhao et al. BMC Genomics 2013, 14:274 Page 2 of 15 http://www.biomedcentral.com/1471-2164/14/274

Background a complete and systematic comparative analysis of Carbohydrate-active enzymes (CAZymes) are respon- CAZymes across the fungal kingdom has not been sible for the breakdown, biosynthesis or modification of reported. In addition, it is still unclear whether the distri- glycoconjugates, oligo- and polysaccharides. Most im- bution of CAZymes in fungi is related to the plant cell portantly, the CAZymes produced by parasites play a wall components, although plant cell walls of dicots and central role in the synthesis and breakdown of plant cell monocot are known to be composed of different compo- wall as well as in host-pathogen interactions [1]. At present, nents particularly on pectins and hemicelluloses [5,19,20]. the CAZymes have been grouped into four functional clas- In this study, we identified and compared the full ses: glycoside hydrolases (GHs), glycosyltransferases (GTs), repertoires of CAZymes from representative fungi and polysaccharide lyases (PLs), and carbohydrate esterases performed a comprehensive comparison upon the (CEs) based on their structurally-related catalytic modules distribution and abundance of CAZyme families to ob- or functional domains [1]. Among them, the CAZymes of tain clues to their digestive potential, especially against classes CE, GH, and PL are often known as cell wall plant cell wall polysaccharides. Differences in the degrading enzymes (CWDEs) due to their important roles number and variety of CAZymes among saprophytic, in plant biomass decomposition by fungi and bacteria [2]. facultative parasitic, hemi-biotrophic, biotrophic, and In addition to the catalytic modules, around 7% of symbiotic fungi were analyzed. The relationship be- CAZymes also contain the carbohydrate-binding modules tween the number and variety of CAZymes and fungal (CBMs), which are the most common non-catalytic nutritional strategy and host specificity was also modules associated with enzymes active in cell-wall examined. hydrolysis [1]. Fungi can produce all kinds of CAZymes [1,3]. Among Results and discussion them, plant cell wall degrading enzymes received special The distribution of CAZyme families attentions because of their importance in fungal patho- The predicted proteomes of 103 fungi from Ascomycota, gens for penetration and successful infection of their Basidiomycota, Chytridiomycota,andZygomycota were hosts. Carbohydrates released from plant cell wall also systematically screened for different families of CAZymes can supply nutrition for fungal growth. As a matter of and CBMs based on family-specific HMMs [21]. These fact, some saprophytic fungi obtain nutrition for growth fungi represent five types of nutritional mode, saprophytic, and reproduction mainly by degrading plant cell wall facultative parasitic, hemi-biotrophic, biotrophic, and sym- materials with a variety of CWDEs. A number of studies biotic fungi, and include pathogens of plants, vertebrates, have revealed that activities of hydrolytic enzymes from nematodes, and insects. different fungi showed preferences for different types of In total, 187 CAZyme families were identified in fungal plant biomass and adaption to their lifestyles [4,5]. predicted proteomes. Over a half of the fungi analyzed When cultured on different substrates, various plant contain more than 300 CAZymes (Figure 1; Additional biomass degrading enzymes were shown to be produced file 1). Note that the ‘CAZymes’ referred here and below by different fungi, including the model filamentous indicates functional modules or domains not genes fungus Neurospora crassa [6-12]. The white-rot basidio- unless otherwise specified. Some CAZyme families, such mycete fungi such as Phanerochaete chrysosporium are as CE1, GH5, GH47, and GT2, were detected in all the found to be the main producers of ligninases for sub- fungal species examined (Figure 2), while some others, stantial lignin decay in wood [13,14]. For fungal patho- such as CE13, GH104, GH42, and GH77, occurred only gens, localized degradation of cell wall is necessary for in a few fungi (Enzymatic activities are listed in accessing plant cytoplasm and spreading across host Additional file 2). Interestingly, the distribution of some tissues. In several plant pathogenic fungi, CWDEs such CAZyme families appeared to be phylum-specific. For as pectinases and xylanases were demonstrated to be example, 28 families, including GH130, GH67, GH94, related to pathogenicity or virulence [15-17]. PL10, and PL11, were only found in the Ascomycetes. In To date, over a hundred of fungal genomes have contrast, 15 families, including GH44 and PL15, been sequenced and are publicly available, including appeared to be Basidiomycota-specific (Table 1). representative fungi from Ascomycota, Basidiomycota, Zygomycota, and Chytridiomycota. Most of fungi except Glycoside hydrolases (GHs) Saccharomycetes and Schizosaccharomycetes have a large GHs hydrolyze the glycosidic bond between two or more number of CWDE genes that are likely involved in plant carbohydrates, or between a carbohydrate and a non- infection or survival in the environments. Some genes carbohydrate moiety, such as a protein, or a lipid [1]. To coding polysaccharide degrading enzymes have expanded date, GHs are grouped into 127 families based on amino family members in certain fungi and gene redundancy has acid sequence in the CAZy database. Among the 127 been shown to guard critical functions [18]. However, families, 91 of them were detected in fungi examined, Zhao et al. 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Figure 1 Comparative analysis of fungal CAZymes. The numbers of CAZyme modules or domains were represented as horizontal bars. CBM, carbohydrate binding module; CE, carbohydrate esterase; GH, glycoside hydrolases; GT, glycosyltransferase; PL, polysaccharide lyase. Zhao et al. BMC Genomics 2013, 14:274 Page 4 of 15 http://www.biomedcentral.com/1471-2164/14/274

Figure 2 The distribution of each CAZyme family in 103 fungi. Blue bars showed the numbers of fungi that have the members of specific CAZyme family. Zhao et al. BMC Genomics 2013, 14:274 Page 5 of 15 http://www.biomedcentral.com/1471-2164/14/274

Table 1 Phylum specific CAZyme and CBM families (Figure 2). Ascomycetes and Basidiomycetes have no Family ABZCFamily ABZCobvious differences in the number of PLs. However, CBM15 0 0 1 0 GH44 0 6 0 0 families PL10, PL11, and PL17 are Ascomycota-specific CBM16 12 0 0 0 GH49 6 0 0 0 although they are present only in few Ascomycetes. CBM23 13 0 0 0 GH52 0 0 1 0 Some families, such as family PL15, are specific to Basidiomycota (Table 1). Among the 103 fungi exam- CBM26 1 0 0 0 GH67 31 0 0 0 ined, 21 lack any PL. The majority of them are sapro- CBM27 10 0 0 0 GH77 0 0 0 1 phytic or facultative parasitic, such as yeasts and fungi in CBM28 3 0 0 0 GH80 0 1 0 0 genus Trichophyton. The biotrophic barley powdery CBM37 12 0 0 0 GH82 4 0 0 0 mildew fungus Blumeria graminis is the only plant CBM39 0 1 0 0 GH94 11 0 0 0 pathogenic fungus that lacks any PL. CBM44 3 0 0 0 GT13 0 1 0 0 CBM47 0 1 0 0 GT18 0 4 0 0 Carbohydrate esterases (CEs) CBM53 0 0 0 1 GT43 0 3 0 0 CEs catalyze the de-O or de-N-acylation of esters or am- CBM57 1 0 0 0 GT51 1 0 0 0 ides and other substituted saccharides in which sugars CBM62 1 0 0 0 GT52 0 1 0 0 play the role of alcohol and amine [24]. Our results showed that fungi have 15 of the 16 CE families, with fam- CBM8 0 6 0 0 GT54 29 0 0 0 ilyCE11beingtheonlyonemissing.Thenecrotrophicpea CBM9 15 0 0 0 GT55 9 0 0 0 root pathogen Nectria haematococca has the most CEs CE11 2 0 0 0 GT73 0 1 0 0 (223). In general, Ascomycetes and Basidiomycetes have CE6 0 0 3 0 GT74 0 1 0 0 similar numbers of CEs, whereas Ascomycetes have more GH102 0 1 0 0 GT78 1 0 0 0 members of families CE3 (P < 0.01) and CE5 (P < 0.01) but GH103 1 0 0 0 GT82 1 0 0 0 fewer members of family CE16 (P < 0.01) than Basidiomy- GH117 3 0 0 0 GT93 0 1 0 0 cetes (Figure 3). Families CE1 and CE10 are present in all GH120 1 0 0 0 PL10 5 0 0 0 the fungi examined and family CE4 is absent only in the GH121 4 0 0 0 PL11 5 0 0 0 nematophagous facultative parasitic fungus Arthrobotrys GH124 0 1 0 0 PL15 0 15 0 0 oligospora. In contrast, families CE6 and CE13 were found GH130 8 0 0 0 PL17 2 0 0 0 only in 3 and 2 fungi, respectively (Figure 2). Members of Digits refer to the number of fungi which have enzymes in specific family. families CE1 and CE10 share the common activities of A, Ascomycota; B, Basidiomycota; Z, Zygomycota; C, Chytridiomycota. carboxylesterase and endo-1,4-β-xylanase. However, they have a great diversity in substrate specificity. For example, with the most prevalent families being GH5, GH13, vast majority of CE10 enzymes act on non-carbohydrate GH31, and GH61 (Figure 2). Our results showed that substrates [1]. GH families vary distinctly on distribution and abundance in fungi (Figure 2). For example, numerous Carbohydrate-binding modules (CBMs) members of families GH16 and GH18 are present in all CBMs are appended to carbohydrate active enzymes that fungi examined and 102 fungi, respectively. For families degrade insoluble polysaccharides [25]. Fifty two of 65 GH73, GH77, and GH104, only a single member each was CBM families were detected in fungi examined, with the identified in one predicted proteome (Figure 2). Interest- most prevalent families being CBM21 and CBM23 ingly, only the entomopathogenic fungus Cordyceps (Figure 2). Ascomycetes tend to have more CBM18 militaris and symbiotic fungus Laccaria amethystina have (P < 0.01) domains but fewer CBM12 (P < 0.01), CBM13 one member of family GH19, which is expanded in plants (P < 0.01) and CBM5 (P < 0.01) domains than Basidiomy- and bacteria [1,22]. Ascomycetes and Basidiomycetes differ cetes (Figure 3). Furthermore, many CBM families tend in the abundance of some families. For instance, Ascomy- to be Ascomycota-specific, such as CBM16, CBM23, cetes have more members of families GH2 (independent CBM27, and CBM37 (Table 1). Surprisingly, the faculta- samples t test, P < 0.01), GH72 (P < 0.01), and GH76 tive parasitic fungus A. oligospora has the most CBMs, (P < 0.01) but fewer members of families GH5 (P < 0.01) particularly CBM1 modules with the putative cellulose - and GH79 (P < 0.01) (Figure 3) than Basidiomycetes. binding function [1].

Polysaccharide lyases (PLs) Plant cell wall degrading enzymes PLs mainly degrade glycosaminoglycans and pectin Plant cell walls are comprised mainly of pectins, cellu- [1,23]. They are classified into 21 families in CAZy loses, hemicelluloses, ligins, and other polysaccharides database. Our results showed that fungi encode 16 PL and proteins. We focus our detailed analysis on families, with the most populated family being PL1 pectinases, cellulases, and hemicellulases because they Zhao et al. BMC Genomics 2013, 14:274 Page 6 of 15 http://www.biomedcentral.com/1471-2164/14/274

99% A 95% 75% mean median 25% 5% 1%

CBM CE GH GT PL

B Number of CAZyme Number of CAZyme

CBM12 CBM13 CBM5 CE16 GH5 GH79 GT65 GT68

C Number of CAZyme

CBM18 CE3 CE5 GH2 GH72 GH76 GT71 Figure 3 Different numbers of CAZymes between Ascomycetes and Basidiomycetes. (A) The number of CAZymes in each class and CBMs were plotted for Ascomycetes and Basidiomycetes.(B) The number of CAZymes in the families which were more abundant in Basidiomycetes than in Ascomycetes (t test, P < 0.01). (C) The number of CAZymes in the families which were more abundant in Ascomycetes than in Basidiomycetes (t test, P < 0.01). See Figure 1 for abbreviations. Zhao et al. BMC Genomics 2013, 14:274 Page 7 of 15 http://www.biomedcentral.com/1471-2164/14/274

are the major plant cell wall degrading enzymes in fun- GH10, GH11, and GH39 [1,32]. At least 29 GH families gal pathogens. Although strictly speaking they are not cell are known to be involved in the degradation of plant wall degrading enzymes, cutinases are also included in this biomass [1,4,28,31] (Table 2). Among them, families section because they are often produced in early infection GH2, GH3, GH5, GH27, GH31, GH35, GH43, GH74, stages by phytopathogenic fungi to breach the plant and GH78 tend to be more populated or abundant since cuticle and function as important virulence factors in Table 2 Known substrates (most common forms) of some fungi [26]. CAZymes Pectin degrading enzymes (Pectinases) Family Substrates Family Substrates β Pectin can be broken down by pectin lyase, pectate lyase, GH1 CW ( -glycans) GH49 ESR (dextran) pectin esterase, and polygalacturonase (PGA) [20,27]. GH10 PCW (hemicellulose) GH5 CW (β-glycans) These enzymes mainly fall into nine CAZyme families, GH105 PCW (pectin) GH51 PCW (hemicellulose) including CE8, PL1, PL2, PL3, PL9, PL10, GH28, GH78, GH11 PCW (hemicellulose) GH53 PCW (hemicellulose) and GH88 [1,4,28]. Our results showed that fungi lack GH115 PCW (hemicellulose) GH54 PCW (hemicellulose) PL2 enzymes. Among 67 Ascomycetes examined, 31% GH12 PCW (cellulose) GH55 FCW (β-1,3-glucan) (21/67) lack enzymes belonging to these 9 CAZyme families. In contrast, the only one basidiomycete lacks any GH125 PG (N-glycans) GH6 PCW (cellulose) of them is Malassezia globosa, which is a facultative GH13 FCW + ESR (α-glucans) GH61* PCW (cellulose) parasitic fungus causing dandruff on human skin. In GH15 ESR (α-glucans) GH62 PCW (hemicellulose) lower fungi, only the saprophytic chytrid Allomyces GH16 FCW (β-glycans) GH63 PG (N-glycans) macrogynus has pectinases. Interestingly, many vascular GH17 FCW (β-1,3-glucan) GH64 CW (β-1,3-glucan) wilt and root pathogens, such as Verticillium albo- GH18 FCW (chitin) GH65 ESR (trehalose) atrum, Verticillium dahlia, N. haematococca, and Fusar- β ium oxysporum, tend to have more pectinases, which GH2 CW ( -glycans) GH67 PCW (hemicellulose) may be related to the blockage or collapse of vascular GH20 FCW (chitin) GH7 PCW (cellulose) bundles during disease development. GH23 BPG GH71 FCW (β-1,3-glucan) Polygalacturonases (family GH28) play a critical role GH24 BPG GH72 FCW (β-1,3-glucan) in pectin degradation in fungal pathogens. Several fungi, GH25 BPG GH74 PCW (cellulose) such as the necrotrophic white mold fungus Sclerotinia GH26 BPG GH75 FCW (chitin) sclerotiorum, gray mold fungus Botryotinia fuckeliana, and opportunistic human pathogen Rhizopus oryzae, GH27 PCW (hemicellulose) GH76 FCW (chitin) have an expanded family of PGAs (see Additional files 3, GH28 PCW (pectin) GH78 PCW (pectin) 4 and 5), suggesting that these fungi have high capacity of GH29 PCW (hemicellulose) GH8 CW pectin degradation. In contrast, all the Saccharomycetes GH3 CW (β-glycans) GH81 FCW (β-1,3-glucan) and Schizosaccharomycetes lack any PGA except the GH30 FCW GH85 FCW budding yeast Saccharomyces cerevisiae,whichhasasingle GH31 PG + ESR + PCW (hemicellulose) GH88 PCW (pectin) PGA gene. Pectinesterases (family CE8) catalyze the de- esterification of pectin to pectate and methanol. Most fungi GH32 ESR (sucrose/inulin) GH89 FCW contain only a small number (no more than 8) of pectin GH35 PCW (hemicellulose) GH9 CW esterases, which may play an auxiliary role in pectin GH36 PCW (hemicellulose) GH93 PCW (hemicellulose) breaking down. GH37 ESR (trehalose) GH94 PCW (cellulose) GH38 PG (N-/O-glycans) PL1 PCW (pectin) Lignocellulose degrading enzymes (lignocellulases) GH39 PCW (hemicellulose) PL11 PCW (pectin) Lignocellulose is a tight complex formed by cellulose, hemicellulose, and lignin, and is the most abundant GH4 ESR PL14 BEPS plant biomass on the planet. Lignocellulose degradation GH43 PCW (pectin + hemicellulose) PL3 PCW (pectin) is a complex process involving the cooperation of GH45 PCW (cellulose) PL4 PCW (pectin) heterogeneous groups of enzymes. For example, the GH46 FCW (chitin) PL7 BEPS thorough degradation of cellulose requires the collabor- GH47 PG (N-/O-glycans) PL9 PCW (pectin) ation of endoglucanase, cellobiohydrolase, and β-1, BEPS: Bacterial exopolysaccharides; BPG: Bacterial peptidoglycan; CW: Cell wall; 4-glucosidase [29-31]. The GH class contributes the ESR, energy storage and recovery; PCW: Plant cell wall; PG, protein most catalytic enzymes to the degradation of lignocellu- glycosylation; FCW: Fungal cell wall. *: Enzymes of GH61 family were not truly glycosidase, however, they are loses [3], such as cellulases in families GH1, GH3, GH5, important to degrade lignocellulose in conjunction with cellulases [34], and were GH45, and GH74 [1,4,31], xylanases in families GH3, kept as GH in the CAZy classification [1]. Zhao et al. BMC Genomics 2013, 14:274 Page 8 of 15 http://www.biomedcentral.com/1471-2164/14/274

that they are present in over a half of fungi examined It was reported that fungal α-arabinofuranosidases are and some of them are expanded in many fungi (Figure 2; mainly found in GH families 51 and 54 [38]. Our Additional files 3, 4 and 5). Our results showed that all results showed that over half of the fungi examined fungi examined have cellulose degrading enzymes such lack either GH51 or GH54 enzymes, and 43 fungi lack as members of family GH74. In contrast, only 38% of any member of these two families (Additional files 3, 4 the bacterial genomes were reported to code cellulase and 5). The saprophytic fungus Gymnopus luxurians genes [33]. has 5 GH51 enzymes, which is more than any other fungi. On the other hand, the facultative parasitic GH3 family: Enzymes of this family are classified based fungus Penicillium marneffei tends to have the most on substrate specificity into β-D-glucosidases, α-L- GH54 (4) enzymes. arabinofuranosidases, β-D-xylopyranosidases, and GH55, GH64, and GH81 families: Enzymes of families N-acetyl-β-D-glucosaminidases [35]. The most GH55, GH64, and GH81 display β-1,3-glucanase common form is β-D-glucosidase [4,28]. Our results activities [1,41]. Their known substrates are mainly the showed that GH3 enzymes were abundant in 97 of all fungal cell wall, which is enriched by β-1,3-glucan fungi examined (Figure 2). Two necrotrophic fungi, N. [11,41]. Callose is also a polysaccharide of β-1,3-glucan haematococca and F. oxysporum, have more GH3 in plant cell wall. It is involved in the plant defense enzymes (38 and 32, respectively) than any other fungi. responses during interaction with pathogenic fungi The tomato leaf mold fungus Cladosporium fulvum is [10]. Hence, we also investigated the variety of enzymes the only biotrophic fungus with a larger number (20) of belonging to these three families among plant GH3 enzymes. Among the Chytridiomycetes, only the pathogenic fungi. Our results showed that family GH55 amphibian pathogen Spizellomyces punctatus has the and GH81 enzymes showed no obvious variety among GH3 member. plant pathogenic fungi. Five out of six biotrophic fungi GH5 family: This is one of the largest GH families. It lack any GH64 member, whereas only two consists of a wide range of enzymes acting on different necrotrophic and one hemibiotrophic fungi lack any. substrates [36], with the most common forms being Interestingly, the hemibiotrophic fungus exo-/endo-glucanases and endomannanases [37,38]. Moniliophthora perniciosa lacks any member of these Among all the GHs, members belonging to the GH5 three families, distinctly deviating from other family are the most common ones and they are present hemibiotrophic fungi. in all fungi examined, suggesting that these enzymes play important roles in fungal degradation of Cutinases lignocellulose. In general, Basidiomycetes tend to have Cutin is composed of hydroxy and hydroxyepoxy fatty more GH5 enzymes than Ascomycetes. The acids. Cutinases (family CE5) catalyze the cleavage of saprophytic basidiomycete Jaapia argillacea has the ester bonds of cutin to release cutin monomers. Among largest number (34) of GH5 enzymes, closely followed the 103 fungi analyzed, 83 of them have cutinases by another saprophytic fungus Botryobasidium (Figure 2). In seven lower fungi belonging to Zygomycota botryosum (29). Interestingly, the biotrophic wheat rust and Chytridiomycota, only the zygomycete R. oryzae fungi Puccinia graminis and Puccinia triticina tend to has no cutinase. Interestingly, the necrotrophic fungus have more GH5 enzymes (28 and 27, respectively) than Fomitiporia mediterranea lacks any cutinase. The other fungi. Another biotrophic rust fungus hemibiotrophic rice blast fungus Magnaporthe oryzae Melampsora laricis-populina also has a large number has 19 cutinases, which is more than any other fungus. (21) of GH5 enzymes. Rhodotorula glutinis,a In M. oryzae, at least one cutinase gene is known to saprophytic basidiomycete, is the only fungus with a be important for plant infection [42,43]. Both two single GH5 member. necrotrophic fungi, Gaeumannomyces graminis and GH10 and GH11 families: Enzymes of families GH10 V. albo-atrum, have 15 cutinases. Interestingly, all and GH11 both display endoxylanase activities [1,39] biotrophic fungi have cutinases, in contrast to that two but GH10 enzymes have higher substrate specificity symbiotic fungi, Paxillus involutus and Paxillus than those of family GH11 [38]. Our results showed rubicundulus,lackany. that 59 and 43 fungi have GH10 and GH11 enzymes, respectively, 44 of 103 fungi examined lack members Comparing abundance of CAZymes among fungi belonging to these two families, including all The fungi examined in this study vary significantly in entomopathogenic fungi. the number of CAZymes. For example, nineteen of them GH51 and GH54 families: Enzymes of families GH51 have more than 500 CAZymes, twenty-two fungi have and GH54 mainly decompose hemicelluloses such as fewer than 200 CAZymes. In general, Dothideomycetes arabinoxylan, arabinogalactan, and L-arabinan [1,4,40]. and Sordariomycetes contain more CAZymes and Zhao et al. 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Saccharomycetes and Schizosaccharomycetes have fewer GH7, GH10, GH12, GH36, GH53, GH54, GH62, PL1, (Figure 1). For instance, there are 730 and 125 CAZymes and PL3 (Additional file 4). in F. oxysporum and Schizosaccharomyces cryophilus, respectively. Obligate parasitic fungi Obligate parasitic fungi depend on the presence of plant or animal hosts to complete their life cycle. In compari- Saprophytic fungi son with hemibiotrophic fungi, biotrophic fungi have the Based on the number of predicted CAZymes, sapro- least CAZymes and necrotrophic fungi have the most phytic fungi can be divided into two groups. The first CAZymes (Figure 1), although the numbers and variety group consists of 24 fungi that have more than 200 of CAZymes in each group are diverse. CAZymes from classes GH, CE and PL. However, they Biotrophic fungi derive nutrients from living tissues. lost several CAZyme families, including families CE11, Four of six biotrophic fungi analyzed are in phylum GH73, GH80, and GH82. Fungi of the second group, in- Basidiomycota, two are in phylum Ascomycota.In cluding 4 Schizosaccharomycetes, 2 Saccharomycetes, contrast to necrotrophic and hemibiotrophic fungi, one eurotiomycete Uncinocarpus reesii, and one basidio- biotrophic fungi lack GH6 enzymes, which are known to mycete R. glutinis, have fewer than 200 CAZymes, which display endoglucanase and cellobiohydrolase activities is fewer than the other saprophytes (Figure 1). Only [1] for plant cell wall degradation [4,28]. In general, R. glutinis of this group has PLs (Additional file 3). In biotrophic fungi tend to have fewer plant cell wall de- contrast to the first group, the latter lost many CAZyme grading enzymes than necrotrophic and hemibiotrophic families of GHs and CEs, including families CE7, CE8, fungi, such as enzymes of GH61, GH78, PL1 and PL3 GH1, GH6, GH10, GH11, GH30, and GH79. (Figure 4). Furthermore, they also have fewer enzymes belonging to family GH76 and CBM1, CBM18, and Facultative parasitic fungi CBM50 (Figure 4). Interestingly, CBM18 domains are Facultative parasitic fungi normally live as saprobes but present in various enzymes from families GH18, GH19, they are opportunistic pathogens of plants or animals. GH23, GH24, GH25, and GH73 [1]. Although it lacks Similar to saprophytic fungi, facultative fungal pathogens experimental supports, the absence or reduction of these can be divided into two groups based on the number families may be correlated to their biotrophic lifestyles. and type of CAZymes (Figure 1). The ten fungi in the Unlike other members of this group, the barley powdery first group have more CAZymes than the second group mildew fungus B. graminis lacks any PL enzyme. C. fulvum and mainly are saprobes. Twenty members of the second differs from most other members of Mycosphaerellaceae by group have fewer than 230 CAZymes. Most of them lack being a biotroph, while the others are hemibiotrophs or PL enzymes and are facultative vertebrate pathogenic necrotrophs [44]. Interestingly, our results showed C. fungi, such as Candida species. In contrast to the first fulvum has significantly more CWDEs of families GH3, group, they lost most families of CAZymes related to the GH31, GH43, and PL3 than any other biotrophic fungi plant biomass degradation, such as families CE5, GH6, (Additional file 5). Similar to biotrophic pathogens,

A B

99% 95% 75% mean median 25% 5% 1% Number of CAZyme Number of CAZyme

CBM CE GH GT PL CBM1 CBM18 CBM50 GH3 GH61GH76 GH78 GT1 PL1 PL3 Figure 4 Comparison of CAZymes in different plant pathogenic fungi. (A) The number of CAZymes in each class and CBMs were plotted for biotrophic (red), hemibiotrophic (black), and necrotrophic (green) pathogens. (B) The number of CAZymes in the families that showed significant differences among biotrophic, hemibiotrophic, and necrotrophic pathogens. Differentiation was support by the t test at the significant level with P < 0.01. See Figure 1 for abbreviations. Zhao et al. BMC Genomics 2013, 14:274 Page 10 of 15 http://www.biomedcentral.com/1471-2164/14/274

symbiotic fungi contain small number of CAZymes and monocots, such as P. teres [46]. Cell wall components of di- also lack enzymes of family GH6. For example, Laccaria bi- cots and monocots are different, especially in the propor- color,amemberoftheTricholomataceae family that can tion of pectin and hemicellulose [5,20]. Activities of plant develop symbiotic associations with plant roots [45], con- biomass degrading enzymes in some fungi also are known tains a small number of CAZymes (Additional file 5). It to have preference of biomass type of monocot or dicot may be beneficial to symbiotic fungi to contain fewer plants [5]. To detect whether the CAZyme family diversity CAZymes for its symbiotic association with host plants. is correlated to the specificity of their hosts, we compared Necrotrophic plant pathogens derive nutrients from different pathogens that infect monocots or dicots. Because dead host cells. Most of the necrotrophic fungi sequenced biotrophic fungi lack most of plant cell wall degrading en- to date are from phyla Sordariomycetes, Dothideomycetes, zymes, they were excluded in this analysis. In general, dicot and Leotiomycetes. The wood rotting fungus Dichomitus pathogens have more pectinases belonging to families squalens is the only member of this group that lacks any GH28 (P < 0.01), GH88 (P < 0.01), and GH105 (P < 0.01) PL1 enzymes which expanded in other necrotrophic fungi. than fungi pathogenic to monocots (Figure 5), which agrees G. graminis, M. poae,andS. sclerotiorum have fewer PLs with the fact that cell walls of dicots are composed of than other fungi in this group (Figure 1; Additional file 5). higher levels of pectin than monocots [19]. Although the Hemibiotrophic fungi have the initial biotrophic phase but significance of the comparison between monocot and dicot switched to necrotrophic growth at late infection stages. pathogens with family PL1 and PL3 were not supported by In general, these fungi have more CAZymes than the t test, some dicot pathogens, such as N. haematococca, biotrophic fungi but similar to necrotrophic fungi in the V. albo-atrum,andV. dahlia,havemorePL1andPL3en- number and diversity of CAZymes. M. perniciosa, the zymes than monocot pathogens. causal agent of the witches’ broom disease of cocoa, con- Although dicot and monocot plants have different tains only two cutinases (CE5), which is fewer than any amounts of hemicelluloses in their cell wall, their patho- other hemibiotrophic fungi. The diversity of CAZymes in gens have no significant differences in the diversity or fungi with different lifestyles suggests that the adaptation number of enzymes related to the hemicellulose degrad- of fungal pathogens to different plant biomass and degrading. It should be noted that dicots and monocots have ing capabilities. different levels of xylans and mannans in the primary cell wall but approximately the same level in the second- The diversity of CAZymes between monocot and dicot ary cell wall [19]. The number of enzymes involved in pathogens cellulose degradation also has no significant differences Some fungi can infect both dicots and monocots such as between dicot and monocot pathogens, which agree with P. graminis, Melampsora larici-populin,andF. oxysporum. the fact that dicots and monocots are composed of simi- However, many fungi can only infect either dicots or lar levels of cutin and cellulose.

B A 99% 95% 75% mean Dicot pathogen median Monocot pathogen 25% 5% 1% Number of CAZyme Number of CAZyme

CBM CE GH GT PL GH105 GH28 GH88 PL1 PL3 Figure 5 Comparison of CAZymes among fungi pathogenic to monocots and dicots. (A) The number of members in each CAZyme class and CBMs in the predicted proteomes were plotted for fungi that infect dicots (red dots) or monocots (black dots). (B) The number of CAZymes which showed obvious differentiation among fungi that infect dicots or monocots. Dicot pathogens have more pectinases from families GH28 (P < 0.01), GH88 (P < 0.01) and GH105 (P < 0.01) than fungi pathogenic to monocots. Although the significance of the comparison was not supported by the t test (P > 0.05), some dicot pathogens tend to have much more PL1 and PL3 enzymes than monocot pathogens. See Figure 1 for abbreviations. Zhao et al. BMC Genomics 2013, 14:274 Page 11 of 15 http://www.biomedcentral.com/1471-2164/14/274

Saprophytic fungi have fewer CAZymes than plant of plant celluloses [4,28]. Furthermore, it contains pathogenic fungi members of families GH105, GH31, GH43, GH5, and In general, saprophytic fungi are considered to produce GH8, with which substrates are mainly plant lignocellu- a variety of CAZymes. Our results showed that loses [4,28]. The amphibian pathogen Batrachochytrium saprophytic fungi have fewer CAZymes belonging to dendrobatidis grows on amphibian skin [48] also has classes CE (P < 0.05), GH (P < 0.05), and PL (P < 0.05), lignocellulases and is not known for association with particularly families CE5 (P < 0.01), GT1 (P < 0.01), plants. PL1 (P < 0.01), and PL3 (P < 0.01), than plant patho- Unlike bacteria, these fungi produce enzymes belong genic fungi (Figure 6). to families GH5 and GH3 but not families GH6 and Interestingly, some fungi known for high capability of GH12. Whereas GH5 enzymes mainly include endo/ plant biomass degradation were found to contain fewer exo-glucanase, endo-1,6-glucanase, mannanase, and plant cell wall degrading enzymes in our analysis. For xylanase, GH3 enzymes include β-glucosidase, glucan example, the opportunistic human pathogen R. oryzae β-1,3-glucosidase, cellodextrinase, and exo-1,3-1,4-glucanase. has fewer lignocelluosic biomass degrading enzymes al- In contrast, GH6 enzymes include endoglucanase though it is known for its high degrading capacity [11]. and cellobiohydrolyase and GH12 enzymes include The high level of gene expression and enzyme activity endoglucanase, xyloglucan hydrolase, and β-1,3-1, may contribute to these fungi’s ability to degrade plant 4-glucanase. The predicted GH3 enzyme of M. globosa biomass. has the entire functional domain and conserved aspartic acid residue of the GFVISDW motif [35], The presence of plant cell wall degrading enzymes in suggesting that it is likely active. However, functional fungi associated with animals domains of some GH5 enzymes in M. globosa,suchas Some bacteria do not have a saprophytic lifestyle or cel- 164657103, 164657414, and 164655644, are truncated lulose degrading activity but contain cellulases belong to and unlikely to be active. Some cellulases are involved family GH6, GH12, and GH5 [33]. One example is the in cellulose biosynthesis in bacteria and plants [49,50] animal and human tuberculosis pathogen Mycobacter- but the fungal cell wall lacks cellulose. Thus, cellulase ium tuberculosis, which has two cellulase genes belong- genes in animal pathogenic fungi may be remnants of ing to family GH6 and GH12 but has no known ancestry genes, indicating that they may be evolved relationship with plants [33]. Our results showed that from plant pathogenic fungi. some fungal parasites of vertebrates in group 2 of facultative parasitic fungi have lignocellulases although they The expression profiles of CAZyme genes in Fusarium can live only as saprobes or parasites to animals. For ex- graminearum during plant infection ample, M. globosa is a lipid-dependent microorganism To investigate whether genes coding CAZymes play responsible for the onset of dandruff and other skin con- important roles during plant infection, we analyzed ditions in humans [47]. It has one member of family the microarray data of the hemibiotrophic fungus F. GH74, which is known to be involved in the degradation graminearum from spike infection of barley (FG1) and

A B

99% 95% 75% mean median 25% 5% 1% Number of CAZyme Number of CAZyme

CBM CEGH GT PL CBM18 CE5 GT1 PL1 PL3 Figure 6 Comparison of CAZymes between saprophytic fungi and plant pathogenic fungi. (A) The number of members in each CAZyme class and CBM in the predicted proteomes were plotted for plant pathogenic fungi (red dots) and saprophytic fungi (black dots). (B) The number of CAZymes which showed obvious differentiation supported by t test among plant pathogenic fungi and saprophytic fungi. Zhao et al. BMC Genomics 2013, 14:274 Page 12 of 15 http://www.biomedcentral.com/1471-2164/14/274

wheat head (FG15), as well as conidia germination (FG7) during conidium germination, indicating that they play downloaded from PLEXdb database (www.plexdb.org). less important roles in this process. Interestingly, genes All of the CAZyme genes (CEs, PLs, and GHs) identified in cluster 5 were up-regulated during germination but in F. graminearum were expressed in these experiments. showed no obvious changes during wheat or barley in- The expression profiles of these genes could be catego- fection, suggesting that they play more important roles rized into nine models by k-means clustering algorithm in conidium germination than in plant infection. implemented in program Mayday [51]. The expression profiles of spike infection of barley and wheat head were Evolution of fungal polysaccharide lyase family 1 (PL1) similar to each other but different from those of conid- Family PL1 mainly displays activities of pectate lyases ium germination. Most CAZyme genes were up- and pectin lyases, and is one of the largest families of PL regulated during plant infection (Additional file 6) but class in fungi. Members in PL1 are important for plant the majority of cluster 9 genes were down-regulated. infection and may be related to fungal virulence [17]. These genes generally encode CEs and fungal cell wall We found that saprophytic and pathogenic fungi differ decomposing enzymes, such as CEs FGSG_03012, significantly in the number of PL1 enzymes (Figure 6). FGSG_00784, and FGSG_11578 and GHs FGSG_03827, To investigate their evolution in fungi, we reconstructed FGSG_04648, and FGSG_09648 (Additional file 7). In the phylogenetic tree for the PL1 enzymes (Figure 7 and contrast to plant infection, conidium germination Additional file 8). Many clades containing entries from showed different gene expression models in cluster 1, 2, different fungal taxa in the phylogenetic tree, suggesting 5, and 7. Genes in cluster 1 and 7 were down-regulated that the last common fungal ancestor possessed

Chytridiomycota Basidiomycota Dothideomycetes Pezizomycetes Eurotiomycetes Orbiliomycetes Leotiomycetes Sordariomycetes

0.2

Figure 7 The maximum likelihood tree of family PL1. The phylogenetic tree was constructed with PhyML3.0 based on a multiple sequence alignment generated with PSI-Coffee [54]. The midpoint rooted base tree was drawn using Interactive Tree Of Life Version 2.1.1 (http://itol.embl. de/). The p-values of approximate likelihood ratios (SH-aLRT) plotted as circle marks on the branches (only p-values >0.5 are indicated) and circle size is proportional to the p-values. Scale bars correspond to 0.2 amino acid substitutions per site. A detailed version of this tree is found in Additional file 8. Zhao et al. BMC Genomics 2013, 14:274 Page 13 of 15 http://www.biomedcentral.com/1471-2164/14/274

numerous paralogous PL1 genes. The clades contain Institute (http://www.broadinstitute.org/science/projects/ only one or some of fungal taxa and none of the taxa re- projects), 30 were obtained from GenBank of NCBI, 23 tains representatives of all ancestral paralogs, indicating were downloaded from DOE Joint Genome Institute that different subsets of ancestral paralogs may have (JGI) site [52], and 1 was downloaded from BluGen been lost in certain fungal taxa during evolution. For ex- (http://www.blugen.org/) (Additional file 1). ample, Basidiomycetes may have lost most of the ances- We used the Hmmscan program in HMMER 3.0 pack- tral PL1 genes whereas Sordariomycetes have retained age [53] to search each of fungal predicted proteomes most of them (Figure 7). Furthermore, lineage or with the family-specific HMM profiles of CAZymes species-specific gene duplication (gain) events also have downloaded from dbCAN database [21] as queries. The occurred within many fungal taxa, particularly in plant primary results were processed by the hmmscan-parser pathogens. For example, the Fusarium species, which script supplied by the dbCAN. are necrotrophic pathogens, contain many closely related paralogous PL1 genes, suggesting of the recent gene du- Cluster analysis of gene expression profiles of Fusarium plication and divergence events. In all, the phyletic dis- graminearum CAZymes tribution and phylogenetic relationship of PL1 genes The expression data were downloaded from Plant Expres- within different fungal taxa revealed a complex history sion Database (PLEXdb) (http://www.plexdb.org/index.php). of lineage-specific gene expansions and attritions which For each experiment, three biological replicates were ana- may be related to their nutritional strategies. lyzed. The expression data of RMA treatment means for all probesets were used. The software Mayday 2.13 [51] was Conclusions used to construct the k-means clustering with Pearson cor- In conclusion, we systemically identified glycoside hydro- relation distance measure. The CAZymes in GT class were lases, polysaccharide lyases, carbohydrate esterases, and not included in this analysis. glycosyltransferases, as well as carbohydrate-binding modules from predicted proteomes of 103 representative fungi Phylogenetic analysis belonging to Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis revealed that fungi Multiple alignments of protein sequences were constructed exhibit tremendous diversity in the number and variety of using PSI-Coffee [54] and the regions of large gap and CAZymes. Among them, some families of GH and CE are ambiguous alignments were removed manually. Maximum likelihood (ML) phylogeny were estimated with PhyML3.0 themostprevalentCAZymesthatarepresentinallthe γ fungi analyzed. Importantly, cellulases of some GH families assuming 8 categories of -distributed substitution rate and are present in fungi that are not known to degrade cellu- SPRs algorithms, based on amino acid sequence alignment loses. Our results also showed that plant pathogenic fungi, andthebest-fitmodelLG+FselectedbyProtTest2.4[55]. in general, contain more CAZymes than saprophytic, The reliability of internal branches was evaluated based on symbiotic, and animal pathogens. Among plant pathogens, SH-like approximate likelihood ratios (aLRT) supports. biotrophic fungi have fewer CAZymes in comparison with The resulting alignment and phylogenetic tree have been necrotrophic and hemibiotrophic fungi. In addition, deposited in treeBASE under URL (http://purl.org/phylo/ fungi infecting dicots contain more pectinases than fungi treebase/phylows/study/TB2:S13822?x-access-code=5adfa7 infecting monocots. Interestingly, several non-yeast sapro- c8af1e503811a8adfcec7f769f&format=html). phytic fungi, including R. oryzae, contain fewer CAZymes although they have high capacity of plant biomass degrad- Statistical analysis ation. Furthermore, analysis of the gene expression profiles We used the program OriginPro 8.5 (http://originlab. of the wheat scab fungus F. graminearum revealed that com/index.aspx?go=PRODUCTS/OriginPro) to generate most of the CAZyme genes were up-regulated during plant statistical box charts. Independent samples t tests were infection. Phylogenetic analysis of the PL1 family revealed a performed by the program SPSS Statistic 17.0 (www.01. complex history of lineage-specific gene expansions and ibm.com/software/analytics/spss/). attritions. Results from this study provide insights into the variety and expansion of fungal CAZyme families and Additional files revealed the relationships of CAZyme size and diversity of fungi with their nutritional strategy and host specificity. Additional file 1: Distribution of CAZymes in 103 fungi. Additional file 2: Known activities of each enzyme/domain family. Methods Additional file 3: Distribution of CAZymes and CBMs in saprophytic Data collection and CAZyme annotation fungi. The predicted proteomes of 49 fungi were downloaded Additional file 4: Distribution of CAZymes and CBMs in facultative pathogenic fungi. from Fungal Genome Initiative (FGI) site of Broad Zhao et al. BMC Genomics 2013, 14:274 Page 14 of 15 http://www.biomedcentral.com/1471-2164/14/274

Additional file 5: Distribution of CAZymes and CBMs in plant novel alpha-glucan acting enzymes with unexpected expression profiles. – pathogenic fungi. Mol Genet Genomics 2008, 279(6):545 561. 8. Paper JM, Scott-Craig JS, Adhikari ND, Cuomo CA, Walton JD: Comparative Additional file 6: Cluster profiles of Fusarium graminearum CAZyme proteomics of extracellular proteins in vitro and in planta from the genes during infection of wheat or barley and conidium pathogenic fungus Fusarium graminearum. Proteomics 2007, 7(17):3171–3183. germination. 9. Mastronunzio JE, Tisa LS, Normand P, Benson DR: Comparative secretome Additional file 7: Expression changes of Fusarium graminearum analysis suggests low plant cell wall degrading capacity in Frankia CAZyme genes during wheat infection, barley infection, and symbionts. BMC Genomics 2008, 9(1):47. conidium germination. 10. Brown NA, Antoniw J, Hammond-Kosack KE: The predicted secretome of Additional file 8: The maximum likelihood tree of family PL1. the plant pathogenic fungus Fusarium graminearum: a refined comparative analysis. PLoS One 2012, 7(4):e33731. 11. Battaglia E, Benoit I, van den Brink J, Wiebenga A, Coutinho PM, Henrissat B, Abbreviations de Vries RP: Carbohydrate-active enzymes from the zygomycete fungus CA: Cutinase; CAZyme: Carbohydrate activity enzyme; CBM: Carbohydrate Rhizopus oryzae: a highly specialized approach to carbohydrate binding module; CE: Carbohydrate esterase; GH: Glycoside hydrolases; degradation depicted at genome level. BMC Genomics 2011, 12:38. GT: Glycosyltransferase; CWDE: Cell wall degrading enzyme; 12. Tian C, Beeson WT, Iavarone AT, Sun J, Marletta MA, Cate JHD, Glass NL: PGA: Polygalacturonase; PL: Polysaccharide lyase. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proc Natl Acad Sci 2009, Competing interests 106(52):22157–22162. The authors declare that they have no competing interests. 13. Dashtban M, Schraft H, Syed TA, Qin W: Fungal biodegradation and enzymatic modification of lignin. Int J Biochem Mol Biol 2010, 1(1):36–50. Authors’ contributions 14. Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B, Martinez ZZ performed bioinformatic analyses, participated in the interpretation of the AT, Otillar R, Spatafora JW, Yadav JS, et al: The Paleozoic origin of results and drafted the manuscript. HL designed and coordinated the study, enzymatic lignin decomposition reconstructed from 31 fungal genomes. participated in the bioinformatic analyses, in the interpretation of the results Science 2012, 336(6089):1715–1719. and in the writing of the manuscript. CW participated in the coordination of 15. Douaiher MN, Nowak E, Durand R, Halama P, Reignault P: Correlative the study. JRX conceived the study, participated in the interpretation of the analysis of Mycosphaerella graminicola pathogenicity and cell wall‐ results and in the writing of the manuscript. All authors read, corrected and degrading enzymes produced in vitro: the importance of xylanase and approved the final manuscript. polygalacturonase. Plant pathology 2007, 56(1):79–86. 16. Ferrari S, Galletti R, Pontiggia D, Manfredini C, Lionetti V, Bellincampi D, Acknowledgements Cervone F, De Lorenzo G: Transgenic Expression of a Fungal endo- We thank Jun Guo from Northwest A&F University for the supporting of Polygalacturonase Increases Plant Resistance to Pathogens and Reduces – fungal physiology knowledge and Lin An from Georgetown University for Auxin Sensitivity. Plant Physiol 2007, 146(2):669 681. the data collection. We are grateful to the anonymous reviewers for their 17. Kikot GE, Hours RA, Alconada TM: Contribution of cell wall degrading comments on the submitted manuscript. 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