Ferric reductase-like families of Eumycetes

f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 7 6 6 e7 7 7

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The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes Ibtissem GRISSAa,b,c, Fre´de´rique BIDARDb,c, Pierre GROGNETa,b,c, Sandrine GROSSETETEb,c, Philippe SILARa,b,c,* a

Univ Denis Diderot, UFR Sciences du Vivant, 75013 Paris, France Univ Paris-Sud, Institut de Ge´ne´tique et Microbiologie, UMR 8621, 91405 Orsay cedex, France c CNRS, Institut de Ge´ne´tique et Microbiologie, UMR 8621, 91405 Orsay cedex, France b

article info

abstract

Article history:

Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and

Received 19 April 2010

development of fungal structures. While the ROS-generating NADPH oxidases from

Received in revised form

filamentous fungi are under strong scrutiny, much less is known about the related integral

5 July 2010

Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29

Accepted 6 July 2010

fungal genomes covering the entire available range of fungal diversity. IMRs are present in

Available online 14 July 2010

all fungal genomes. They can be classified into at least 24 families, underscoring the high

Corresponding Editor: Michael Lorenz

diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Impor-

Keywords:

tantly, functional characterization of IMRs has been made on proteins belonging to only

Ferric reductase

two families, while nothing or very little is known about the proteins of the other 22

Fungal genomes

families.

NADPH oxidase

ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Reactive Oxygen Species

Introduction Reactive Oxygen Species (ROS) have been implicated in two major aspects of fungal biology. First, they participate in the degradation of the plant biomass, especially lignin lysis (ten Have & Teunissen 2001; Wesenberg et al. 2003; Martinez et al. 2005; Morel et al. 2009). Second, they are involved in signaling several developmental processes, including fruiting body maturation (Lara-Ortiz et al. 2003; Malagnac et al. 2004), spore germination (Malagnac et al. 2004; Cano-Dominguez et al. 2008; Lambou et al. 2008), mycelium growth (Leuthner et al. 2005; Semighini & Harris 2008), mutualistic symbiosis (Tanaka et al. 2006) and differentiation of appressorium and

appressorium-like structures (Egan et al. 2007; Segmuller et al. 2008; Brun et al. 2009). To date, the role of ROS in development has been mostly studied in ascomycetes, such as Aspergillus nidulans, Neurospora crassa, Epichloe festucae, Botrytis cinerea, Magnaporthe grisea and Podospora anserina. In these species, the major ROS producing enzymes evidenced as signaling development are NADPH oxidases (Nox, see Aguirre et al. 2005; Takemoto et al. 2007 for review), however a glyoxal oxidase (glo1) has also been shown to be necessary for normal hyphal morphology and filamentous growth in the basidiomycete Ustilago maydis (Leuthner et al. 2005). Plant biomass degradation is mostly studied in basidiomycetes, such as white and brown rots, which are able or not able to efficiently degrade

* Corresponding author. Institut de Ge´ne´tique et Microbiologie, UMR 8621 CNRS UPS, 91405 Orsay cedex, France. Tel.: þ33 1 69 15 46 58; fax: þ33 1 69 15 70 06. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.07.002

Nox and IMR of Eumycetes

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Table 1 e Fungal CDS used in this study. Species

Lifestyle

Nox and IMR

Chytridiomycota (Chytridiomycetes)

Batrachochytrium dendrobatidis

Frog pathogen

BDEG_06864 (Nox1) BDEG_07125 (Nox2) BDEG_01882 BDEG_02345 BDEG_07017

Chytridiomycota (Chytridiomycetes)

Spizellomyces punctatus

Soil saprobe

Blastocladiomycota (Blastocladiomycetes)

Allomyces macrogynus

Pond saprobe

AMAG_13933.1 (Nox1) AMAG_15568.1 (Nox2) AMAG_11331.1 AMAG_07049.1 AMAG_NA1a AMAG_NA2a

OM

Mucoromycotina

Mucor circinelloides

Soil saprobe

Mucci1_77308 Mucci1_95952 Mucci1_85232

JGI

Mucoromycotina

Phycomyces blakesleeanus

Soil saprobe

Phybl1_77767 Phybl1_76888

JGI

Mucoromycotina

Rhizopus oryzae

Soil saprobe

RO3G_05460 RO3G_04617 RO3G_10468 RO3G_13599

FGI

Ascomycota (Schizosaccharomycetes)

Schizosaccharomyces pombe

Yeast saprobe

Sp_Frp1 CAA17033 ¼ SPBC947.05c

NCBI/Sanger

Ascomycota (Saccharomycetes)

Ashbya gossypii

Yeast-like, cotton pathogen

NM_212056 NM_208455 NM_209529 NM_211017 NM_208618

AGD

Ascomycota (Saccharomycetes)

Candida albicans SC5314

Human commensal and pathogen

orf19.1264 orf19.1263 (¼CFL1) orf19.1415 (¼CFL95) orf19.1270 orf19.5634 orf19.1930 orf19.1932 orf19.701 orf19.6138 orf19.1844 orf19.3538 orf19.2312 orf19.7077 orf19.7112 orf19.867 orf19.4843

FGI

Ascomycota (Saccharomycetes)

Saccharomyces cerevisiae

Yeast saprobe

ScFRE1 ScFRE2 ScFRE3 ScFRE4 ScFRE5 ScFRE6 ScFRE7 ScFRE8 YGL160W

SGD

BDEG_03995 SPPG_07581 (Nox1) SPPG_07607 (Nox2) SPPG_00862 SPPG_0462 SPPG_05133 SPPG_06478 SPPG_07217 SPPG_08002

URL FGI

OM

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Table 1 e (continued) Species

Lifestyle

Nox and IMR

URL

Ascomycota (Saccharomycetes)

Yarrowia lipolytica

Yeast soil saprobe

YALI0D26081g YALI0B10846g YALI0B13134g YALI0B13112g YALI0E35112g YALI0E08096g YALI0B13090g YALI0F07040g YALI0B13046g YALI0F01386g YALI0F11825g YALI0F24299g YALI0E12551g

Genolevure

Ascomycota (Eurotiomycetes)

Emericalla nidulans (Aspergillus nidulans)

Soil saprobe

ANI05457 (NoxA) AN0773 AN3208 AN4906 AN6400 AN7518 AN8683 AN7662 AN10893 AN7981 AN9023

FGI

Ascomycota (Eurotiomycetes)

Neosartorya fumigata (Aspergillus fumigatus)

Soil saprobe, human opportunistic pathogen

Afu6g13350 (NoxA) Afu1g14340 Afu3g10820 Afu6g13750 Afu8g01310 Afu1g17270 Afu3g02980 Afu6g02170 Afu6g02820 Afu7g04970 Afu1g16040 Afu5g14940 Afu4g03940 Afu2g01270 Afu8g06210 AfuNA1a AfuNA2a

AspGD

Ascomycota (Eurotiomycetes)

Coccidioides immitis

Soil saprobe, human opportunistic pathogen

CIRG_09773 (Nox1) CIRG_00499 (Nox2) CIRG_08233 CIRG_07686 CIRG_07046 CIRG_09554 CIRG_02582 CIRG_NA1a CIRG_NA2a

FGI

Ascomycota (Eurotiomycetes)

Histoplasma capsulatum

Soil saprobe, human opportunistic pathogen

HCAG_05182 (Nox1) HCAG_01252 (Nox2) HCAG_03430 HCAG_01925 HCAG_02320 HCAG_05045 HCAG_03201 HCAG_01641 HCAG_11619 HCAG_03149 HCAG_07381

FGI

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Table 1 e (continued) Species

Lifestyle

Nox and IMR

URL

Ascomycota (Dothideomycetes)

Cochliobolus heterostrophus

Maize pathogen

CocheC5_1_95484 (Nox1) CocheC5_1_95158 (Nox2) CocheC5_1_117226 (Nox3) CocheC5_1_113323 CocheC5_1_115841 CocheC5_1_18297 CocheC5_1_36266 CocheC5_1_67300 CocheC5_1_67987 CocheC5_1_79855 CocheC5_1_86222 CocheC5_1_98199 CocheC5_1_59673 CocheC5_1_105418 CocheC5_1_96373 CocheC5_1_NA1a CocheC5_1_NA2a

JGI

Ascomycota (Leotiomycetes)

Sclerotinia sclerotiorum

Broad host range plant pathogen

SS1G_05661 (Nox1) SS1G_11172 (Nox2) SS1G_10691 SS1G_03147 SS1G_11881 SS1G_11821 SS1G_14206 SS1G_06988 SS1G_05414 SS1G_06624 SS1G_08043 SS1G_09140

FGI

Ascomycota (Sordariomycetes)

Fusarium verticillioides

Soil saprobe and corn pathogen

FVEG_00444 (Nox1) FVEG_11478 (Nox2) FVEG_00030 FVEG_00787 FVEG_01564 FVEG_01784 FVEG_02200 FVEG_02265 FVEG_07148 FVEG_14005 FVEG_13485 FVEG_05576 FVEG_04694 FVEG_02748 FVEG_08464 FVEG_10346 FVEG_10064 FVEG_12507 FVEG_01628 FVEG_12454 FVEG_NA1a

FGI

Ascomycota (Sordariomycetes)

Trichoderma reesei

Soil saprobe

Trire2_79498 (Nox1) Trire2_81646 (Nox2) Trire2_106936 Trire2_110666 Trire2_111750 Trire2_111893 Trire2_22845 Trire2_23353 Trire2_54144 Trire2_6107 Trire2_61116 Trire2_81096

JGI

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Table 1 e (continued) Species

Lifestyle

Nox and IMR

URL

Ascomycota (Sordariomycetes)

Magnaporthe grisea

Rice pathogen

MGG_00750 (Nox1) MGG_06559(Nox2) MGG_08299 (Nox3) MGG_01018.6 MGG_02828.6 MGG_03667.6 MGG_07833.6 MGG_07853.6 MGG_08278.6 MGG_10760.6 MGG_12210.6 MGG_13426.6 MGG_14302.6 MGG_14647.6 MGG_14894.6

FGI

Ascomycota (Sordariomycetes)

Neurospora crassa

Soil saprobe

NCU02110 (Nox1) NCU10775 (Nox2) NCU00023 NCU00829 NCU00876 NCU02009 NCU02020 NCU02278 NCU03868 NCU08194

FGI

Ascomycota (Sordariomycetes)

Podospora anserina

Dung saprobe

Pa_1_2410 (Nox1) Pa_5_9580(Nox2) Pa_1_5020 (Nox3) Pa_5_11970 Pa_7_5660 Pa_5_10230 Pa_1_19630 Pa_1_16410 Pa_1_19550 Pa_4_2910 Pa_7_4470 Pa_1_15510 Pa_5_8960

Genoscope/IGM

Basidiomycota (Ustilaginomycetes)

Ustilago maydis

Maize pathogen

UM00382 UM00838 UM00839 UM01439 UM02395 UM05979 UM02730

FGI

Basidiomycota (Pucciniomycetes)

Puccinia graminis

Wheat pathogen

PGTG_01933 (Nox1) PGTG_04320 (Nox2) PGTG_12719 PGTG_16919 PGTG_01643 PGTG_08246 PGTG_03543 PGTG_00319

FGI

Basidiomycota (Microbotryomycetes)

Sporobolomyces roseus

Yeast, phylloplan saprobe

Sporo1_9097 (Nox1) Sporo1_31671 Sporo1_31662 Sporo1_33553 Sporo1_32130 Sporo1_27372 Sporo1_33967

JGI

Nox and IMR of Eumycetes

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Table 1 e (continued) Species

Lifestyle

Nox and IMR

URL

Basidiomycota (Tremellomycetes)

Cryptococcus neoformans

Yeast, soil and dropping saprobe, human and bird opportunistic pathogen

CNAG_00876 CNAG_06821 CNAG_06524 CNAG_06976 CNAG_07604 CNAG_03498 CNAG_07770 CNAG_07334

FGI

Basidiomycota (Tremellomycetes)

Tremella mesenterica

Mycoparasite

Treme1_68048 (Nox1) Treme1_32037 (Nox2) Treme1_12876 Treme1_74287 Treme1_58976 Treme1_38393 Treme1_63255 Treme1_32173

JGI

Basidiomycota (Agaricomycetes)

Coprinopsis cinerea

Dung and soil saprobe

CC1G_10525 (Nox1) CC1G_09830 (Nox2) CC1G_00426 CC1G_03087 CC1G_03568 CC1G_08381 CC1G_14424 CC1G_06499 CC1G_15165

FGI

Basidiomycota (Agaricomycetes)

Laccaria bicolor

Mycorhiza

Lacbi1_243586 (Nox1) Lacbi1_173015 (Nox2) Lacbi1_310387 Lacbi1_184285 Lacbi1_306020 Lacbi1_304910 Lacbi1_315189

JGI

URL: AGD: http://agd.vital-it.ch/index.html; AspGD: http://www.aspergillusgenome.org/; FGI: http://www.broadinstitute.org/science/projects/ fungal-genome-initiative/fungal-genome-initiative; Genolevure: http://www.genolevures.org/; Genoscope/IGM: http://podospora.igmors. u-psud.fr; JGI: http://genome.jgi-psf.org/; NCBI: http://www.ncbi.nlm.nih.gov/sites/entrez; OM: http://www.broadinstitute.org/annotation/ genome/multicellularity_project/MultiHome.html; Sanger: http://old.genedb.org/genedb/pombe/; SGD: http://www.yeastgenome.org/ a CDS not annotated in the genome project.

lignin, respectively. In these species several ROS producing enzymes have been implicated in plant biomass degradation, including laccases, GMC oxidoreductases, galactose oxidases, copper radical oxidases (e.g. glyoxal oxidase), quinone reductases, pyranose oxidases and cellobiose dehydrogenases (ten Have & Teunissen 2001; Wesenberg et al. 2003; Martinez et al. 2005; Morel et al. 2009). From one fungus to the other the enzymology of plant biomass degradation appears different. For example, in the white rot Phanerochaete chrysosporium plant biomass degradation relies on a large set of ROS-generating oxidative enzymes that degrade lignin directly or through the activation of small weight mediators, coupled to glycoside hydrolases that break down cellulose (Martinez et al. 2004; Kersten & Cullen 2007). However, P. chrysosporium has no laccase. On the contrary, Pycnoporus cinnabarinus does rely essentially on these enzymes for lignocellulose degradation (Eggert et al. 1996). In the brown rot Postia placenta, the paucity in glycoside hydrolases genes typically found in cellulolytic microbes and the up-regulation of various oxidoreductases suggest that cellulose degradation probably relies on Fenton chemistry in which Fe2þ and oxygen peroxide react to form

hydroxyl radicals, a powerful reactant able to hydrolyze biopolymers (Martinez et al. 2009). Unlike basidiomycetes, ascomycetes, except Xylariales (Pointing et al. 2003), are not thought to be able to degrade lignin. However, the genome sequence of P. anserina, a Sordariales, shows that this species possesses many ROS-generating enzymes endowing this fungus with the ability to degrade lignin (Espagne et al. 2008). Moreover, P. anserina is able to efficiently complete its cycle with sawdust as sole nutrient source, suggesting that it is able to efficiently retrieve nutrients from wood (P.S., pers. obser.). To date, the complete array of fungal enzymes able to produce ROS for signaling development or degrading plant biomass is unknown. Similarly, the respective role of ROS and ROS-generating enzymes in development and/or biomass degradation is not understood. For example, the glo1 enzyme from U. maydis has a glyoxal oxidase activity and generates H2O2, yet it is involved in signaling (Leuthner et al. 2005). Similarly, we reported (Brun et al. 2009) that deletion of the Nox genes of P. anserina results in developmental defect associated with modification of cellulose degradation and with an apparent

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Fig 1 e Nox and IMR tree based on complete sequence alignment. CDS names have been colored according to the phylogeny using the code depicted at the bottom. Fungal Nox and IMR families have been colored arbitrarily to facilitate analysis.

augmentation of ROS production, a phenomenon also observed in M. grisea (Egan et al. 2007) and A. nidulans (Semighini & Harris 2008). The enzymes responsible for increased ROS production are unknown. Preliminary mining of the P. anserina genome suggested that integral membrane reductases (IMRs,

also known as Ferric Reductases or Metal reductases, (Kosman 2003)) could be some of the redox enzymes up-regulated in the P. anserina Nox mutants, as the genome of this species contains ten coding sequences (CDS) with similarity to IMRs (Brun et al. 2009). Like Nox, IMRs oxidize cytoplasmic

Nox and IMR of Eumycetes

NADPH and transfer the electron across the plasma membrane to reduce small molecules, dioxygen in the case of Nox (yielding thus the superoxide anion) and mostly metal (Fe3þ and Cu2þ) in the case of IMRs. However, it has been shown that the ScFRE1 Saccharomyces cerevisiae IMR may reduce other small molecules such as paraquat, albeit inefficiently (Hassett & Kosman 1995). IMRs are similar in sequence to Nox, suggesting a common origin (Dancis et al. 1992; Roman et al. 1993; Shatwell et al. 1996). If the Nox proteins of filamentous fungi have recently been under strong scrutiny, little is known about IMRs in these organisms, which have been essentially studied in the yeasts S. cerevisiae (Dancis et al. 1992; Georgatsou & Alexandraki 1994; Shatwell et al. 1996; Georgatsou & Alexandraki 1999; Yun et al. 2001; Singh et al. 2007), Candida albicans (Hammacott et al. 2000; Knight et al. 2002) and Schizosaccharomyces pombe (Roman et al. 1993). Additional studies on these enzymes have been performed on algae (Allen et al. 2007), plants (Wu et al. 2005; Feng et al. 2006; Mukherjee et al. 2006) and parasites (see Sutak et al. 2008 for a review). Because of their involvement in the generation of Fe2þ and possibly other small reduced molecules, IMRs could participate in plant biomass degradation through Fenton reactions or radical attacks. Additional roles in signaling are possible, as demonstrated for the glyoxal oxidase glo1 of U. maydis. Here, we present the results of genome analysis for the presence of Nox and IMRs in 29 fungal genomes, covering the entire known diversity of the Eumycetes. We report that the genomes of these organisms usually contain several IMR isoforms, belonging to a large array of very divergent families. The evolution of fungal Nox/IMR appears very complex. Importantly, proteins demonstrated as having a metal reductase activity in S. cerevisiae and S. pombe cluster within a restricted set of families, questioning the actual enzymatic activities of the other IMRs.

Materials and methods Fungal genomes and deduced protein sets from complete sequencing projects were searched for the presence of putative Nox and IMR genes by TBLASTN and BLASTP, respectively (Altschul et al. 1990). Because in many instances automatic annotation may not give accurate intron splicing boundaries, each putative IMR gene was manually re-annotated (Table 1). A few genes were incomplete due to gaps in the genomic sequences. Additional genes not called during the annotation were discovered (Table 1). The deduced protein sequences for complete bona fide genes were then aligned with the MAFFT algorithm (Katoh et al. 2005) using the L-INS-i parameters recommended for sequences with one conserved domain and long gaps, (Supplemental Fig 1). The alignment was then manually edited to remove poorly conserved regions (Supplemental Fig 2). Trees for the complete and edited proteins were constructed and bootstrapped (100 replicates) with PhyML with the following parameters (data type: 1, sequence format: i, number of data sets: 1, number of bootstrapped data sets: 100, substitution model: WAG, prop. invariable sites: 0.0, number categories: 4, gamma parameter: 1.0, starting tree optimized topology: BIONJ, optimized branch lengths: y and rate parameters: y) (Guindon & Gascuel 2003). Trees were visualized with the iToL server (Letunic & Bork 2007).

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Results and discussion Preliminary analysis of available fungal genome sequences by BLAST (Altschul et al. 1990) and FUNGIpath (Grossetete et al. 2010) suggested that fungi are endowed with many isoforms of Nox and IMR genes (often more than 10 per genomes). We thus focused our analysis on 29 species that would cover the whole range of fungal evolution (Table 1). These live either as yeast or mycelium and have diverse lifestyles (Table 1). As seen in Table 1, all investigated fungi are endowed with at least one IMR gene, while some lack Nox genes (Lalucque & Silar 2003). S. pombe has the simplest complement of Nox/IMR genes with only two IMR genes, including the previously characterized Frp1 IMR gene (Roman et al. 1993). At the other end of the spectrum, Fusarium verticillioides has 19 IMR and two Nox genes. There seems to be no obvious correlation between the number of Nox/IMR and the lifestyle (Table 1). Of interest is the presence in the choanoflagellate Monosiga brevicollis of one Nox (Monbr1_33523) and one IMR (Monbr1_37160). IMR are not found in animals such as Trichoplax adherens, Nematostella vectensis (a sea anemone), Drosophila melanogaster, Caenorhabiditis elegans and Human. They have thus been specifically lost in the metazoan lineage. In total, 276 fungal proteins were used for phylogenetic analysis. The 276 coding sequences (CDS) along with representative Nox and IMRs from other phyla (animals, plants and various protists) were aligned and manually edited to remove poorly aligned regions. Phylogenetic trees on complete and edited protein alignments were then constructed by maximum likelihood. Both complete and edited version yielded similar trees that permit to delimit Nox and IMR families with high statistical confidence (Fig 1 and Supplemental Fig 3). However the branching order of the different families changed between the two analyses, emphasizing the large diversity of these enzymes and precluding the elucidation of the evolution of the various Nox and IMRs. As seen in Fig 1, Nox and IMR enzymes are separated with nearly 100 % confidence, confirming that IMRs and Nox are different proteins (DeLeo et al. 1999; Kawahara et al. 2007). Presently, three Nox isoforms have been described in fungi: Nox1/NoxA, Nox2/NoxB and Nox3/NoxC (Lara-Ortiz et al. 2003). These three families are recovered in both our analyses with 100 % confidence (Fig 1). Interestingly, Spizellomyces punctatus is endowed with two CDS (SPPG_08002 and SPPG_05133) and Allomyces macrogynus one CDS (AMAG_11331.1) that cluster with the NOX enzymes and that do not clearly belong to the three previously defined families. Domain analysis of the CDS with Interproscan (Hunter et al. 2009) reveals that the three CDS possess the NAD, FAD and haem binding domains, always found in Nox. SPPG_08002 and AMAG_11331.1 contain calcium binding sites (EF-hand) in the N-terminus of the protein, as observed for the Nox3 isoforms of ascomycetes, the mammalian Nox5 isoforms and the plant enzymes. These proteins may thus be distant relatives of the other fungal Nox3 enzymes, in line with the high divergence between the Ascomycota, the Blastocladiomycota and the Chytridiomycota. On the contrary, SPPG_05133 lacks EF-hand domain and has a plasma membrane targeting signal followed by a long N-terminus extension. This N-terminus is highly

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similar to proteins from various animals (e.g. 44 % identical with BRAFLDRAFT_120072 from Branchiostoma floridae) and protozoa (e.g. 41 % identical with XP_002673156 from Naegleria gruberi). No CDS containing this domain is found in the other Eumycetes. Provided that SPPG_05133 is not a sequencing/annotation artifact, it may thus define a novel family of Nox (Nox4) resulting from the fusion of a typical Nox coding sequence with another domain of unknown activity. Supplementary Table 1 describes the complement of Nox and NoxR genes found in fungal genomes. NoxR is a regulator of Nox genes (Takemoto et al. 2006) and is surprisingly found in fungi lacking Nox genes, i.e. the Mucoromycotina and Yarrowia lipolytica (Takemoto et al. 2007). A possible explanation is that these NoxR genes activate some IMRs. Fungal IMRs are more complex than Nox as they can be classified into at least 24 families (IeXXIV; Fig 1). All these families are well separated from IMRs identified in other phylogenetic groups, i.e. plants and protozoa. Some contain a single member (families XV and XXIV), while others contain many (family V has 24 representatives). Likely, as more fungi are analyzed, the singleton will be rejoined by other members. For example, analysis of additional fungal genomes shows that F. verticillioides CDS FEVG_NA1, the single representative of family XXIV analyzed here, has orthologues in Fusarium oxysporum and Trichoderma virens; a pseudogene is also present in Trichoderma atroviride. What these IMR families reflect is not clear. Indeed, some are found in only one clade of fungi: families I and XVII are found only in Saccharomycotina, families VI, XI and XVI only in Pezizomycotina, family IV only in Ustilaginomycotina, family XIX only in Taphrinomycotina, etc. These families could thus result from the diversification in the various groups of fungi of an ancestral gene. However some families are found in both Ascomycota and Basidiomycota, such as families II, III and V. Yet, none is present in the whole diversity of fungi. From the trees of Figs 1 and 2, there is no clear evidence of horizontal gene transfer. Each fungal species has its own set of proteins belonging to particular families, even for closely related species. P. anserina, M. grisea and N. crassa are three Sordariomycetes. P. anserina has ten IMRs (two from families III, VI and VII and one from families V, VIII, IX and XI), N. crassa has eight IMRs (two from families VI and VII and one from families V, VIII, IX and XI) and M. grisea has 12 IMRs (three from family III, two from family VII, VIII and XVI, one from families V, VI and XI). Overall, the data suggest that IMR distribution is the result from a complex set of gene gains by duplication and gene losses by deletion and

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mutations (we have detected several pseudogenes during our manual gene annotation). A similar situation is not unprecedented and is found for other protein families (Espagne et al. 2008). Fungal IMRs have so far been biochemically or genetically characterized in two Saccharomycotina (S. cerevisiae, C. albicans) and one Taphrinomycotina (S. pombe) (Kosman 2003). Interestingly, the ScFre1 to ScFRE6 S. cerevisiae isoforms induced by metal deficiency and whose activities are well characterized all belong to family I (Dancis et al. 1992; Georgatsou & Alexandraki 1994; Shatwell et al. 1996; Georgatsou & Alexandraki 1999; Yun et al. 2001; Singh et al. 2007). The CFL1 (Hammacott et al. 2000) and CFL95 (Knight et al. 2002) proteins from C. albicans also belong to family I. The two S. pombe proteins define family XIX. Although a reductase activity can be predicted for the proteins of the other 22 families based on the protein domains, the actual electron acceptors for these proteins are not known. It is therefore possible that molecules other than dioxygen and metals are the true electron acceptors for some of these proteins, as it has been shown that ScFRE1 can reduce organic compounds, such as paraquat (Hassett & Kosman 1995). Additionally, these proteins could be involved in developmental processes, since the S. cerevisiae mutants lacking ScFRE8 have defective bud morphology (Watanabe et al. 2009). ScFRE8, along with other IMRs from Saccharomycotina, define IMR family XVII. To gain some insight onto the potential roles of the IMRs of the other families, we analyzed the expression profiles of the P. anserina and N. crassa proteins, as microarray data are available for these two fungi. N. crassa global transcriptome changes are available during colony development/conidiation (Kasuga & Glass 2008), and during degradation of plant cell wall (Tian et al. 2009). Data are available for all IMRs and Nox1, but lack for Nox2. As seen in Fig 2A, while Nox1 expression is not affected by the carbon source, six out of eight N. crassa IMRs are induced during growth on avicel (microcrystalline cellulose) and ground Miscanthus stem, as compared to growth on sucrose, a feature compatible with a role for IMRs in plant biomass degradation. Similarly, a developmental role in N. crassa is possible for IMRs as most are regulated during N. crassa colony development (Fig 2B). Five are up-regulated after 3 h of growth and one is down-regulated during conidiation (21 and 27 h of growth). P. anserina global transcriptome changes (Bidard et al. 2010) are available for colony development (F.B. and P.S., unpubl.), fruiting body

Fig 2 e IMR gene expression profiles in N. crassa and P. anserina. (A) Expression profile of N. crassa IMR genes at 16 h of growth on minimal medium containing sucrose as carbon source (MM 16 h), compared to growth on avicel and Miscanthus at various times (16 h, 40 h, 5 d and 10 d). Data are taken from Tian et al. (2009). (B) Expression profile of N. crassa IMR genes during colony development corresponding to 1 h, 3 h, 9 h, 15 h, 21 h and 27 h of growth. At the two last time points, N. crassa differentiates conidias. Data are taken from Kasuga & Glass (2008). Because NCU02020 has a much higher expression level than the other IMR genes, it is depicted by itself in the insert. (C) Expression profile of P. anserina IMR genes during colony development at 24 h, 48 h and 72 h of growth, during fruiting body maturation 0 h, 12 h, 24 h and 96 h after maturation and in the PaNox1 null mutant grown for 24 h. After 72 h of mycelium growth P. anserina gametes are formed and await fertilization. After 96 h of maturation, fruiting bodies are ripe and start ejecting ascospores. Because Pa_7_5660 has a much higher expression level, it is depicted in the insert. N. crassa transcriptomic data are the mean of SNR ((signal-background)/background standard deviation of the biological replicates). P. anserina transcriptomic data are mean intensity of four biological replicates.

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(perithecium) maturation (F.B. and V. Berteaux-Lecellier, unpubl.) and the Nox1 mutant (Brun et al. 2009). Nox genes are not differentially regulated in P. anserina (Brun et al. 2009), however differential expression of some IMR genes during develoment is evidenced in P. anserina. As seen in Fig 2C, four IMRs are up-regulated during P. anserina colony development. Similarly, five IMRs are down-regulated during fruiting body development, two of them (Pa_1_16410 and Pa_5_11970) having their expression increased again during the late stages of perithecium maturation. Finally, three IMRs have their expression increased in the Nox1 mutants, which have been shown to degrade cellulose more efficiently than wild type and to produce more ROS (Malagnac et al. 2004; Brun et al. 2009). The IMRs differentially regulated in N. crassa and P. anserina may or may not belong to the same family, a feature easily accounted for by the very different culture conditions used for the two fungi.

Conclusion Fungal genomes contain several IMR genes. These can be classified into at least 24 families, underscoring the high diversity of these enzymes in Eumycetes. Many are differentially regulated by the carbon source present in the growth medium and/ or during colony and fruiting body development. Those that have been genetically or biochemically investigated belong to only two of the 24 families. We therefore advocate performing targeted gene deletion and biochemical characterization of representative IMRs belonging to the other 22 families not yet investigated to gain insight into their cellular roles.

Acknowledgments This work was supported by grant ANR-05-Blan-0385. Ibtissem Grissa is the recipient of an ATER fellowship from Universite´ of Paris Diderot Paris 7.

Supplemental data Supplementary data associated with this article can be found in online version at doi:10.1016/j.funbio.2010.07.002.

references

Aguirre J, et al., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends in Microbiology 13: 111e118. Allen MD, et al., 2007. FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in irondeficient Chlamydomonas reinhardtii. Eukaryotic Cell 6: 1841e1852. Altschul SF, et al., 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403e410. Bidard F, et al., 2010. A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina. BMC Research Notes 3: 171.

I. Grissa et al.

Brun S, et al., 2009. Functions and regulation of the Nox family in the filamentous fungus Podospora anserina: a new role in cellulose degradation. Molecular Microbiology 74: 480e496. Cano-Dominguez N, et al., 2008. NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa. Eukaryotic Cell 7: 1352e1361. Dancis A, et al., 1992. Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron. Proceedings of the National Academic of Sciences of the United States of America 89: 3869e3873. DeLeo FR, et al., 1999. Despite structural similarities between gp91phox and FRE1, flavocytochrome b558 does not mediate iron uptake by myeloid cells. Journal of Laboratory and Clinical Medicine 134: 275e282. Egan MJ, et al., 2007. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. Proceedings of the National Academic of Sciences of the United States of America 104: 11772e11777. Eggert C, et al., 1996. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Applied and Environmental Microbiology 62: 1151e1158. Espagne E, et al., 2008. The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biology 9: R77. Feng H, et al., 2006. Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)chelate reductase gene AtFRO6. Plant Physiology 140: 1345e1354. Georgatsou E, Alexandraki D, 1994. Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae. Molecular and Cellular Biology 14: 3065e3073. Georgatsou E, Alexandraki D, 1999. Regulated expression of the Saccharomyces cerevisiae Fre1p/Fre2p Fe/Cu reductase related genes. Yeast 15: 573e584. Grossetete S, et al., 2010. FUNGIpath: a tool to assess fungal metabolic pathways predicted by orthology. BMC Genomics 11: 81. Guindon S, Gascuel O, 2003. Simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696e704. Hammacott JE, et al., 2000. Candida albicans CFL1 encodes a functional ferric reductase activity that can rescue a Saccharomyces cerevisiae fre1 mutant. Microbiology 146: 869e876. Hassett R, Kosman DJ, 1995. Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. Journal of Biological Chemistry 270: 128e134. ten Have R, Teunissen PJ, 2001. Oxidative mechanisms involved in lignin degradation by white-rot fungi. Chemical Reviews 101: 3397e3413. Hunter S, et al., 2009. InterPro: the integrative protein signature database. Nucleic Acids Research 37: D211eD215. Kasuga T, Glass NL, 2008. Dissecting colony development of Neurospora crassa using mRNA profiling and comparative genomics approaches. Eukaryotic Cell 7: 1549e1564. Katoh K, et al., 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511e518. Kawahara T, et al., 2007. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evolutionary Biology 7: 109. Kersten P, Cullen D, 2007. Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Fungal Genetics and Biology 44: 77e87. Knight SA, et al., 2002. Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator. Microbiology 148: 29e40. Kosman DJ, 2003. Molecular mechanisms of iron uptake in fungi. Molecular Microbiology 47: 1185e1197.

Nox and IMR of Eumycetes

Lalucque H, Silar P, 2003. NADPH oxidase: an enzyme for multicellularity? Trends in Microbiology 11: 9e12. Lambou K, et al., 2008. A crucial role for the Pls1 tetraspanin during ascospore germination of the saprophytic fungus Podospora anserina. Eukaryotic Cell 7: 1809e1818. Lara-Ortiz T, et al., 2003. Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Molecular Microbiology 50: 1241e1255. Letunic I, Bork P, 2007. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23: 127e128. Leuthner B, et al., 2005. A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis. Molecular Genetics and Genomics 272: 639e650. Malagnac F, et al., 2004. Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina. Fungal Genetics and Biology 41: 982e997. Martinez AT, et al., 2005. Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. International Microbiology 8: 195e204. Martinez D, et al., 2009. Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proceedings of the National Academic of Sciences of the United States of America 106: 1954e1959. Martinez D, et al., 2004. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotechnology 22: 695e700. Morel M, et al., 2009. Reactive oxygen species in Phanerochaete chrysosporium relationship between extracellular oxidative and intracellular antioxidant systems. Advances in Botanical Research 52: 153e186. Mukherjee I, et al., 2006. Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper. Planta 223: 1178e1190. Pointing SB, et al., 2003. Production of wood-decay enzymes, mass loss and lignin solubilization in wood by tropical Xylariaceae. Mycological Research 107: 231e235. Roman DG, et al., 1993. The fission yeast ferric reductase gene frp1þ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human

777

NADPH phagocyte oxidoreductase. Molecular and Cellular Biology 13: 4342e4350. Segmuller N, et al., 2008. NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea. Molecular PlanteMicrobe Interactions 21: 808e819. Semighini CP, Harris SD, 2008. Regulation of apical dominance in Aspergillus nidulans hyphae by reactive oxygen species. Genetics 179: 1919e1932. Shatwell KP, et al., 1996. The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase. Journal of Biological Chemistry 271: 14240e14244. Singh A, et al., 2007. The metalloreductase Fre6p in Fe-efflux from the yeast vacuole. Journal of Biological Chemistry 282: 28619e28626. Sutak R, et al., 2008. Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence. Trends in Microbiology 16: 261e268. Takemoto D, et al., 2006. A p67Phox-like regulator is recruited to control hyphal branching in a fungal-grass mutualistic symbiosis. The Plant Cell 18: 2807e2821. Takemoto D, et al., 2007. NADPH oxidases in fungi: diverse roles of reactive oxygen species in fungal cellular differentiation. Fungal Genetics and Biology 44: 1065e1076. Tanaka A, et al., 2006. Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. The Plant Cell 18: 1052e1066. Tian C, et al., 2009. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proceedings of the National Academic of Sciences of the United States of America 106: 22157e22162. Watanabe M, et al., 2009. Comprehensive and quantitative analysis of yeast deletion mutants defective in apical and isotropic bud growth. Current Genetics 55: 365e380. Wesenberg D, et al., 2003. White-rot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnology Advances 22: 161e187. Wu H, et al., 2005. Molecular and biochemical characterization of the Fe(III) chelate reductase gene family in Arabidopsis thaliana. Plant and Cell Physiology 46: 1505e1514. Yun CW, et al., 2001. The role of the FRE family of plasma membrane reductases in the uptake of siderophoreeiron in Saccharomyces cerevisiae. Journal of Biological Chemistry 276: 10218e10223.