YFGBI 2781 No. of Pages 15, Model 5G 17 February 2015 Fungal Genetics and Biology xxx (2015) xxx–xxx 1 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi 2 Regular Articles 7 4 Evolution of novel wood decay mechanisms in Agaricales 8 5 revealed by the genome sequences of Fistulina hepatica and 6 Cylindrobasidium torrendii a,⇑ b c a d 9 Dimitrios Floudas , Benjamin W. Held , Robert Riley , Laszlo G. Nagy , Gage Koehler , d d c c c c 10 Anthony S. Ransdell , Hina Younus , Julianna Chow , Jennifer Chiniquy , Anna Lipzen , Andrew Tritt , c c c c e b 11 Hui Sun , Sajeet Haridas , Kurt LaButti , Robin A. Ohm , Ursula Kües , Robert A. Blanchette , c d a 12 Igor V. Grigoriev , Robert E. Minto , David S. Hibbett 13 a Department of Biology, Clark University, 950 Main St, Worcester 01610, MA, United States 14 b Department of Plant Pathology, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108-6030, United States 15 c US Department of Energy (DOE) Joint Genome Institute, United States 16 d Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, LD326, 402 N Blackford St, Indianapolis, IN 46202, United States 17 e Büsgen Department of Molecular Wood Biotechnology and Technical Mycology, University of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany 18 19 article info abstract 3521 22 Article history: Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed 36 23 Received 19 September 2014 white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present 37 24 Accepted 5 February 2015 the genome sequences of the white rot fungus Cylindrobasidium torrendii and the brown rot fungus Fis- 38 25 Available online xxxx tulina hepatica both members of Agaricales, combining comparative genomics and wood decay experi- 39 ments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica 40 26 Keywords: is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that 41 27 Agaricales C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same 42 28 Wood decay time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay 43 29 White rot 30 Brown rot and degrade all wood components but leave the middle lamella intact. Their gene content related to lign- 44 31 Reconciliation in degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes 45 32 Pseudogenes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have 46 33 Genome sequencing evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of 47 34 brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, 48 genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared 49 with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost 50 in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest 51 that transition toward a brown rot lifestyle could be an ongoing process in F. hepatica. Our results rein- 52 force the idea that wood decay mechanisms are more diverse than initially thought and that the dichoto- 53 mous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be 54 revisited. 55 Ó 2015 Published by Elsevier Inc. 56 57 58 59 1. Introduction 60 ⇑ Corresponding author. The plant cell wall (PCW) is a significant carbon pool in terres- 61 E-mail addresses: dfl[email protected] (D. Floudas), [email protected] (B.W. Held), trial ecosystems (Albersheim et al., 2011). saprotrophic Agari- 62 [email protected] (R. Riley), [email protected] (L.G. Nagy), [email protected] (G. Koehler), [email protected] (A.S. Ransdell), [email protected] comycotina exploit this pool as a carbon and energy source, 63 (H. Younus), [email protected] (J. Chow), [email protected] (J. Chiniquy), ALipzen@lbl. acting as wood or litter decomposers. Wood decomposers follow 64 gov (A. Lipzen), [email protected] (A. Tritt), [email protected] (H. Sun), [email protected] different strategies of decomposition termed white and brown 65 (S. Haridas), [email protected] (K. LaButti), [email protected] (R.A. Ohm), ukuees@ rot. White-rot fungi cause the degradation of all wood components 66 gwdg.de (U. Kües), [email protected] (R.A. Blanchette), [email protected] 67 (I.V. Grigoriev), [email protected] (R.E. Minto), [email protected] (D.S. Hibbett). including the recalcitrant lignin and crystalline cellulose mainly through enzymatic processes (Kersten and Cullen, 2007; Baldrian 68 http://dx.doi.org/10.1016/j.fgb.2015.02.002 1087-1845/Ó 2015 Published by Elsevier Inc. Please cite this article in press as: Floudas, D., et al. Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.02.002 YFGBI 2781 No. of Pages 15, Model 5G 17 February 2015 2 D. Floudas et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx 69 and Valaskova, 2008). In contrast, brown-rot fungi cause complete associated with white rot (Kaarik, 1965; Worrall et al., 1997). 135 70 degradation of polysaccharides, but only partial degradation of Brown rot is a rare nutritional strategy in Agaricales, associated 136 71 lignin (Blanchette, 1995; Worrall et al., 1997; Niemenmaa et al., with the small genera Fistulina, Ossicaulis, and Hypsizygus 137 72 2007; Yelle et al., 2008). (Redhead and Ginns, 1985). Ossicaulis and Hypsizygus are members 138 73 Enzymes implicated in lignin degradation by white-rot fungi of Lyophylleae and they seem to be closely related (Moncalvo et al., 139 74 include Class II peroxidases (POD), dye-decolorizing peroxidases 2002), but Fistulina is an isolated brown-rot genus closely related 140 75 (DyP) and laccases sensu stricto (Cullen and Kersten, 2004; to Schizophyllum, and the little-known Auriculariopsis and Porodis- 141 76 Martinez et al., 2005; Bourbonnais et al., 1995; Eggert et al., culus (Ginns, 1997; Binder et al., 2004). Until recently, sequenced 142 77 1996, 1997; Gronqvist et al., 2005; Liers et al., 2010). Enzymes genomes of Agaricales species related to PCW degradation includ- 143 78 involved in the degradation of crystalline cellulose by white-rot ed only the cacao pathogen Moniliophthora perniciosa (Mondego 144 79 fungi include mainly cellobiohydrolases (glycoside hydrolases et al., 2008), the litter decomposer Coprinopsis cinerea (Stajich 145 80 GH6 & GH7) and lytic polysaccharide monooxygenases (LPMO) et al., 2010) and the lignicolous S. commune (Ohm et al., 2010). This 146 81 (Harris et al., 2010). In addition to those enzymes, white-rot fungi picture has been changing with an increasing number of sequenced 147 82 employ diverse sets of other carbohydrate active enzymes (CAZY) Agaricales genomes (Morin et al., 2012; Wawrzyn et al., 2012; Bao 148 83 involved in the degradation of the PCW (Kirk and Cullen, 1998). et al., 2013; Aylward et al., 2013; Collins et al., 2013; Hess et al., 149 84 In brown-rot fungi, polysaccharide degradation takes place 2014). 150 85 through non-enzymatic processes, at least during the initial stages Here, we report the newly sequenced draft genomes of the 151 86 of degradation. Hydroxyl radicals generated through the Fenton ‘‘beefsteak fungus’’ Fistulina hepatica and Cylindrobasidium tor- 152 87 reaction have been suggested to be the major agent in non-enzy- rendii. Both species are members of the Agaricales, but the former 153 88 matic degradation of polysaccharides by brown-rot species (Kirk species causes brown rot on hardwood (Schwarze et al., 2000a), 154 89 and Highley, 1973; Illman, 1991). while the latter species is associated with white rot most frequent- 155 90 Recent genome investigations (Martinez et al., 2004, 2009; ly on hardwood (Ginns and Lefebvre, 1993). We compare the wood 156 91 Eastwood et al., 2011; Floudas et al., 2012) revealed that white- degradation strategies of each species with those of other wood- 157 92 rot species are enriched in genes related to the degradation of lign- degrading fungi and we explore the evolution of plant cell-wall 158 93 in (POD, DyP, laccases s.s.), crystalline cellulose (GH6, GH7, LPMO) degradation strategies in Agaricales based on gene tree/species 159 94 and other carbohydrates (GH43, GH74). Furthermore, white-rot tree reconciliation analyses. 160 95 species are rich in copies of the cellulose-binding module 1 96 (CBM1), which facilitates attachment of enzymes to crystalline cel- 2. Materials and methods 161 97 lulose (Boraston et al., 2004). In contrast, brown-rot fungi appear to 98 have few or no gene copies in these families and CBM1. It has been 2.1. Strain info and nucleic acid extraction 162 99 suggested that the role of hydroxyl radicals in carbohydrate degra- 100 dation renders extensive enzymatic lignin and carbohydrate degra- We sequenced the single spore isolates of F. hepatica (ATCC 163 101 dation redundant (Worrall et al., 1997). Thus, gene losses 64428, isolated from a sporophore growing on a Castanea dentata 164 102 accompanied the transitions from a white-rot to a brown-rot life- rootstock, North Carolina) and C. torrendii (HHB-15055, ss-10, iso- 165 103 style. Less is known regarding such processes in litter decom- lated from an Acer rubrum log, WI, USA, deposited at the Forest 166 104 posers, but it has been suggested that the latter group causes Products Laboratory culture collection).