Emilie Tisserant

Annegret Kohler

Pascale Dozolme

R Balestrini

K Benabdellah

A Colard

D Croll

C Da Silva

S K Gomez

R Koul

P Dozolme-Seddas

N Ferrol

V Fiorilli

D Formey

Ph Franken

N Helber

M Hijri

L Lanfranco

E Lindquist

Y Liu

M Malbreil

E Morin

J Poulain

H Shapiro

D Van Tuinen

A Waschke

C Azco ´n-Aguilar

G Be ´card

P Bonfante

M J Harrison

H Ku ¨ster

P Lammers

U Paszkowski

N Requena

S A Rensing

C Roux

I R Sanders

Y Shachar-Hill

G Tuskan

J P W Young

V Gianinazzi-Pearson

Francis Martin
email: fmartin@nancy.inra.fr

The transcriptome

Keywords: Glomeromycota, Glomus, meiosis and recombination genes, mycorrhiza, small secreted proteins, symbiosis, transcript profiling

Introduction

The arbuscular mycorrhizal (AM) symbiosis between fungi in the Glomeromycota [START_REF] Schu ¨ssler | A new fungal phylum, the Glomeromycota: phylogeny and evolution[END_REF] and plants involves over two-thirds of all known plant species, including important crop species, such as wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), soybean (Glycine max) and poplar (Populus spp.). This mutualistic symbiosis, involving one of the oldest fungal lineages, is arguably the most ecologically and agriculturally important symbiosis in terrestrial ecosystems [START_REF] Fitter | Nutritional exchanges in the arbuscular mycorrhizal symbiosis: implications for sustainable agriculture[END_REF]. The extraradical mycelium (ERM) of the symbiont acts as an extension of the root system and increases the uptake of key nutrients, particularly phosphorus (P) and zinc (Zn) and possibly also nitrogen (N) [START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[END_REF]. These fungi are therefore crucial to plant growth [START_REF] Smith | Mycorrhizal symbiosis[END_REF] and also define the diversity of plants in ecosystems [START_REF] Van Der Heijden | Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity[END_REF]. Furthermore, because the colonization of plants by AM fungi can also result in a 20% net increase in photosynthesis [START_REF] Smith | Mycorrhizal symbiosis[END_REF], these universal mycosymbionts make a very large, poorly understood contribution to the global carbon cycling budget of ecosystems.

The Glomeromycota are unique in that their spores and coenocytic hyphae contain multiple nuclei in a common cytoplasm. No sexual cycle is known, although anastomosis and nuclear movement between hyphae of the same species have been described [START_REF] Giovannetti | The occurrence of anastomosis formation and nuclear exchange in intact arbuscular mycorrhizal networks[END_REF], as well as genetic exchange between AM fungal individuals [START_REF] Croll | Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices[END_REF](Croll et al., , 2009)). Furthermore, the concept of an individual is unclear as nuclei within a single AM fungus appear to be genetically different in some species, which raises substantial questions about the natural selection, population genetics and gene expression of these highly unusual organisms [START_REF] Jany | Multinucleate spores contribute to evolutionary longevity of asexual Glomeromycota[END_REF]Sanders & Croll, 2010). AM fungi are thought to grow clonally, but controversy clouds their ploidy, their genome size, and whether or not they can reproduce sexually (for a review, see Sanders & Croll, 2010). Evidence for recombination has been provided, but whether mating and meiosis are involved is unknown (Croll & Sanders, 2009). There is therefore a need to identify genes whose products are required for proper completion of meiotic recombination.

Root colonization by AM fungi follows a series of distinct steps (for a review, see [START_REF] Bonfante | Mechanisms underlying beneficial plantfungus interactions in mycorrhizal symbiosis[END_REF] starting with a presymbiotic molecular dialogue that involves root-released strigolactones [START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF] and AM fungal signaling molecules, such as lipochitooligosaccharides [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF]. AM fungi are obligate mutualistic symbionts that can only grow for a limited time without colonizing a susceptible host root [START_REF] Be ´card | Partner communication in the arbuscular mycorrhizal interaction[END_REF]. This has been suggested to be a consequence of some nutritional deficiencies and loss of metabolic pathways, such as an absence of de novo fatty acid synthesis [START_REF] Bago | Carbon metabolism and transport in arbuscular mycorrhizas[END_REF], during the asymbiotic and presymbiotic phases related to putative genome erosion [START_REF] Ercolin | Successful joint ventures of plants: arbuscular mycorrhiza and beyond[END_REF]. After spore germination, the first contact at the root surface is marked by the differentiation of the fungal hyphae into an appressorium from which a penetration hypha invades the root epidermis [START_REF] Genre | Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection[END_REF]. After the fungus has traversed the outer cell layers it spreads in the inner cortex, that is, intraradical mycelium (IRM), and forms highly branched structures inside cortical cells, so-called arbuscules. Arbuscules are the main site of nutrient transfer from the fungus to the plant [START_REF] Javot | Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plantfungus interactions in mycorrhizal symbiosis[END_REF][START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[END_REF]. Arising from the colonized roots, the ERM proliferates in the growth medium, where it absorbs and assimilates the available nutrients before their transfer to the host plant. Major transcriptome shifts are thus expected during these different key developmental stages, but little is known of the repertoire of effector-like proteins, membrane transporters and assimilative enzymes involved in these different steps of symbiosis development and functioning.

The ability to establish a sophisticated zone of interaction, such as the haustorium in pathogenic oomycetes and fungi interacting with plants, requires sophisticated host defense suppression [START_REF] Dodds | Plant immunity: towards an integrated view of plant-pathogen interactions[END_REF], which is predominantly achieved via secreted proteins delivered into the host cell [START_REF] Kamoun | Groovy times: filamentous pathogen effectors revealed[END_REF]. The protein SP7 of Glomus intraradices is secreted and transferred to the plant cell nucleus in colonized roots of Medicago truncatula, where it binds to the host ethylene-responsive transcriptional factor regulating the expression of several defense-related genes [START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biothrophy[END_REF]. It remains to be determined whether G. intraradices expresses genes coding for additional secreted effector-like proteins during its interaction with the host plant.

The mycorrhizal colonization leads to quantitative and qualitative changes in the host transcriptome. Plant genes that are specifically regulated during the establishment and ⁄ or functioning of the AM symbiosis have been identified by both targeted and high-throughput expression profiling in several sequenced model and crop plants [START_REF] Gu ¨imil | Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization[END_REF][START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Grunwald | Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots[END_REF]Guether et al., 2009a). In contrast, regulation of gene expression in the AM fungal symbionts caused by interactions with roots has so far been poorly explored, and studies have mainly been based on targeted approaches providing only narrow insights into fungal adaptation to the symbiotic mode [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Seddas | Symbiosisrelated plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF][START_REF] Tani | ATP-dependent but proton gradient-independent polyphosphate-synthesizing activity in extraradical hyphae of an arbuscular mycorrhizal fungus[END_REF][START_REF] Kuznetsova | Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices[END_REF]. Most studies have focused on P transport and metabolism [START_REF] Viereck | Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo 31 P NMR spectroscopy[END_REF][START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF][START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF][START_REF] Javot | Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles[END_REF][START_REF] Go ´mez-Ariza | Cell-specific gene expression of phosphate transporters in mycorrhizal tomato roots[END_REF][START_REF] Grunwald | Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots[END_REF] and primary carbon metabolism [START_REF] Bago | Carbon metabolism and transport in arbuscular mycorrhizas[END_REF][START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF][START_REF] Schaarschmidt | Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots[END_REF]. Further studies adopting a microarray or high-throughput sequencing approach are definitely warranted, because they could provide a wider understanding of how genetic changes in the plant affect overall patterns of fungal gene expression, as well as the impact of the fungal genotypes on symbiosis fitness and plant growth [START_REF] Angelard | Segregation in a mycorrhizal fungus alters rice growth and symbiosis-specific gene transcription[END_REF].

Biotrophs are widely accepted to intimately interact and coevolve with their hosts. The genetic changes that brought about the evolution of obligate biotrophy in the Glomeromycota are, however, unknown. Recent research on obligate biotrophic pathogens, such as the powdery mildew fungus Blumeria graminis [START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF], the downy mildew oomycete Hyaloperonospora arabidopsidis [START_REF] Baxter | Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome[END_REF], the oomycetous white rust Albugo laibachii [START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF], and the poplar and stem rusts Melampsora larici-populina and Puccinia graminis, respectively [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici[END_REF], reveals a close correlation between the biotrophic life style and gene losses in primary and secondary metabolism. In the oomycetes, all haustorium-forming species have lost the thiamine biosynthetic pathway, suggesting that haustorial oomycetes obtain thiamine from the host [START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF]. It appears that biotrophy in these obligate plant pathogens is also associated with a convergent loss of secondary metabolic enzymes and an extremely reduced set of carbohydrate-active enzymes devoted to plant cell wall depolymerization. Genes for nitrate and nitrite reductases, a nitrate transporter, and sulfite reductase are also often missing. This streamlining of metabolism presumably reflects adaptation to life within host cells, as it is not observed in nonobligate biotrophic pathogens. Whether similar events have also characterized AM fungal evolution remains to be determined.

As mentioned above, transcriptome studies have been pursued on AM roots in multiple plant species under a variety of experimental conditions, but they have mainly focused on the host plant transcriptome. No single study has yet brought together genome-wide transcriptomic data for the fungal component of this important symbiosis. The aim of this study was to establish a comprehensive, genome-wide inventory of gene expression in G. intraradices DAOM197198 by sequencing cDNA libraries from different fungal structures (germinated spores, extra-and intraradical mycelium, and arbuscules). Through transcriptomic analyses, we also wished to identify which of these genes were induced upon symbiosis and, further, to assign the differentially expressed transcripts to key symbiotic mechanisms, such as nutrient transport and assimilation, host colonization, and signaling pathways. Several factors have led to the choice of G. intraradices for the first large-scale transcriptome sequencing of an AM fungus. As a symbiont, G. intraradices is highly effective in mobilizing, taking up and transferring mineral nutrients, such as inorganic orthophosphate ions (Pi), N and sulfur (S), from soils to plants [START_REF] Govindarajulu | Nitrogen transfer in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Allen | Sulfur transfer through an arbuscular mycorrhiza[END_REF][START_REF] Tian | Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux[END_REF][START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[END_REF] and it readily colonizes many plants, including agriculturally important crop species (e.g. alfalfa (Medicago truncatula), poplar, rice and wheat) as well as model plants such as M. truncatula and Lotus japonicus. Glomus intraradices is one of the most studied AM fungi as it rapidly colonizes its host plants, and it is a model species for dissecting the molecular and cellular biology of the Glomeromycota [START_REF] Seddas | Symbiosisrelated plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]Sanders & Croll, 2010). It is readily amenable to in vitro culture on transformed host roots [START_REF] Chabot | The life cycle of Glomus intraradices in root organ culture[END_REF] and is the only species for which spores are available commercially in pure form in large quantities.

The comparative analyses of gene repertoires in G. intraradices, pathogenic obligate biotrophs and ectomycorrhizal fungi offer insights into genes that may be involved in obligate biotrophy and mycorrhizal symbioses. In the absence of a whole-genome sequence for any member of the phylum Glomeromycota (Martin et al., 2008a), the availability of large-scale expressed sequenced tag (EST) collections represents the core foundation for understanding genome functionality in the Glomeromycota. [START_REF] Be ´card | Early events of vesicular arbuscular mycorrhiza formation on Ri T-DNA transformed roots[END_REF]. Germinated spores were produced as described by [START_REF] Chabot | The life cycle of Glomus intraradices in root organ culture[END_REF]. Biological materials used for cDNA library construction and microarray transcript profiling are summarized in Table 1. Protocols for producing the biological materials, RNA purification and cDNAs are described in the online Supporting Information Methods S1. For array profiling, G. intraradices ERM was grown on liquid M medium without sucrose for 3 wk before harvesting. Glomus intraradices-and mock-inoculated roots of M. truncatula were prepared as described for the cDNA library AKNA (Methods S1). Germinated spores were sampled as described for the cDNA library AKND (Methods S1) [START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF]. Samples were snap-frozen in liquid N 2 . *The cDNA library AKNA has been constructed using mRNA from a mix of the three replicates also used for microarray transcript profiling.

Materials and Methods

Biological material

EST sequencing, filtering and assembly

Sanger sequencing was performed on ABI3730xl analyzers (Applied Biosystems, Nutley, NJ, USA) and pyrosequencing on the Genome Sequencer FLX System (454 Life Sciences ⁄ Roche, Nutley, NJ, USA). Reads (797 394) were filtered and trimmed for low quality, low complexity, and adaptor sequences using SeqClean (TIGR (The Institute for Genomic Research); http:// sourceforge.net/projects/seqclean/) (Supporting Information Fig. S1). Sequences £ 100 bp were discarded. The resulting high-quality sequences were screened to detect potential bacterial and fungal contaminant sequences, and plant sequences present in cDNAs from symbiotic tissues. Sequences having a GC% higher than that of G. intraradices (i.e. ‡ 45%) were removed (Fig. S2). Sequences with a high nucleotide sequence similarity (BLASTN e-value £ 1e-5, score ‡ 150, % identity ‡ 95) with identified contaminants, Aspergillus spp. and Chromobacterium violaceum, as well as the M. truncatula genome and transcripts, and other plant DNA (e.g. carrot) were also removed (Fig. S1).

Finally, to avoid the loss of G. intraradices outlier sequences, removed sequences were aligned to the G. intraradices draft genome using BLASTN (e-value £ 1e-5, score ‡ 150) at the INRA GlomusDB website (http://mycor.nancy.inra.fr/IMGC/Glomus Genome/index3.html) and retained when sequence similarity was ‡ 95%. Filtered reads were then assembled using the MIRA assembler [START_REF] Chevreux | Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs[END_REF]. To choose the best assembly, several assemblies were constructed using different parameters and several indexes were measured, including the number of nonredundant virtual transcripts (NRVTs), the percentage of assembled reads and the percentage of NRVTs with hits against gene models from the taxonomically related, although distant, Mucoromycotina Rhizopus oryzae (http://genome.jgi-psf.org/ Rhior3/Rhior3.home.html) (Supporting Information Table S1).

The assembly generated by MIRASEARCHESTSNPS was selected.

The number of detected NRVTs increased with the number of reads and the rarefaction curve did not reach a plateau (Fig. S3a). However, many of these sequences may be the result of DNA pyrosequencing errors creating false transcripts. This is an acute problem for the AT-rich sequences of G. intraradices. In contrast, the number of protein sequences documented in the Swiss-Prot DNA database (http://www.uniprot.org/) and R. oryzae gene repertoire showing a similarity to G. intraradices NRVTs plateaued at approx. 5500 homologs (Fig. S3b), suggesting that most of the transcriptome of G. intraradices was covered. This was confirmed by a TBLASTN search (cut-off e-value of 1e-5) using the protein sequences of the core eukaryotic genes (CEGs) [START_REF] Parra | Assessing the gene space in draft genomes[END_REF]; 245 (98.7%) of the 248 CEG proteins were found in the current G. intraradices transcriptome. The missing protein sequences were a metal-binding protein, a spindle assembly checkpoint protein and a monooxygenase involved in coenzyme Q (ubiquinone) biosynthesis. Eighty-six per cent of NRVTs matched the current G. intraradices 52.5 Mb-genome assembly (Martin et al., 2008a), confirming that most filtered, high-quality ESTs were from G. intraradices. Single-nucleotide polymorphisms (SNPs) in NRVTs were identified with MIRASEARCHESTSNPS using the following parameters: minimum reads, 2 (Sanger) or 4 (454); minimum quality value, 30 (Sanger) or 25 (454); minimum neighbor quality value, 20.

For the digital northern gene expression, the number of reads for each NRVT in each library was counted and the relative frequency (reads of a given NRVT divided by the total number of reads) was obtained. Significant differences in gene expression between libraries for each NRVT were calculated [START_REF] Audic | The significance of digital gene expression profiles[END_REF].

EST sequences are available at the National Center for Biotechnology Information (NCBI) (accession numbers GW091323-GW125581 and GW086621-GW090678). A MySQL ⁄ PHP database compiling NRVT sequences and annotations is available at the INRA GlomusDB website. This server can also be used to query the ESTs and the draft genome assembly using BLAST programs.

Functional annotation

NRVTs were compared against Swiss-Prot using BLASTX with a significance threshold (e-value < 1e-5). Gene annotations were assigned to each NRVT based on the best BLAST hits. Sequences were searched against the G. intraradices draft genome contigs (version 'test14') at the INRA GlomusDB using BLASTN (e-value £ 1e-5). NRVTs were also compared with proteins of Basidiomycota (Cryptococcus neoformans, Laccaria bicolor, M. larici-populina, Phanerochaete chrysosporium and Ustilago maydis), Ascomycota (Aspergillus nidulans, B. graminis, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa and Tuber melanosporum) and Mucoromycotina (R. oryzae and Mucor circinelloides) using BLASTX (e-value £ 1e-5). To identify conserved protein domains in NRVTs, predicted protein sequences were compared with the Eukaryotic Orthologous Groups (KOG) database [START_REF] Tatusov | The COG database: an updated version includes eukaryotes[END_REF]. The counts of each KOG domain by species were transformed into a z-scores matrix to center the data, and visualization was performed using MeV (MultiExperiment Viewer) [START_REF] Saeed | TM4 microarray software suite[END_REF]. The gene ontology (GO) terms [START_REF] Ashburner | Gene ontology: tool for the unification of biology. The Gene Ontology Consortium[END_REF] were assigned to each sequence using BLAST2GO [START_REF] Conesa | Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research[END_REF]. Enrichment analysis of GO annotations was carried out with BINGO (http://www.psb. ugent.be/cbd/papers/BiNGO/Home.html) using the hypergeometric test and Benjamini and Hochberg's false discovery rate (FDR) multiple testing correction (P £ 0.05) [START_REF] Maere | BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks[END_REF].

To predict G. intraradices metabolic pathways, sequences were queried for Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) assignments [START_REF] Kanehisa | KEGG: Kyoto Encyclopedia of Genes and Genomes[END_REF] using the KEGG Automatic Annotation Server (KAAS) [START_REF] Moriya | KAAS: an automatic genome annotation and pathway reconstruction server[END_REF] and results were then plotted into the KEGG Global metabolic atlas (map01100) using iPATH [START_REF] Letunic | iPath: interactive exploration of biochemical pathways and networks[END_REF]. A highresolution version of this metabolic pathway map can be downloaded at: http://mycor.nancy.inra.fr/IMGC/GlomusGenome/ download/Gi_metabolic_map.pdf.

To identify sequences encoding secreted proteins, NRVTs were translated into protein using FrameFinder (http://www. ebi.ac.uk/~guy/estate/). In silico predictions of secreted proteins were then carried out using SIGNALP 3.0 [START_REF] Bendtsen | Improved prediction of signal peptides: SignalP 3.0[END_REF], TARGETP 1.1 [START_REF] Emanuelsson | Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[END_REF] and TMHMM 2.0 (prediction of transmembrane helices in proteins) [START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[END_REF] as previously reported in [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici[END_REF]. Small secreted proteins (SSPs) were selected based on an arbitrary cutoff of 300 amino acids [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici[END_REF].

Oligoarray design

For the design of custom oligoarrays, a MIRA assembly (v. 1.0) comprising 22 254 NRVTs was constructed using cDNA libraries CACE, CCHU, EXTA and EXTB. At the time, sequences from the AKNA, AKNB, AKNC and AKND libraries were not available. To ensure that all sequences were sufficiently long to allow the design of high-quality probes, we selected the subset of sequences ‡ 200 bp in length (n = 19 465). To remove redundancy among sequences that would produce cross-hybridation, the assembled NRVTs were clustered based on nucleotide sequence similarity using BLASTClust (http://www.ncbi. nlm.nih.gov/Web/Newsltr/Spring04/blastlab.html). Within each cluster, sequences were ranked by length, and the longer member of each cluster was selected (n = 15 932). We also selected singleton sequences with matches in NCBI databases and with length ‡ 200 bp (n = 2046). We then added 2327 NRVTs generated by the Paracel Transcript Assembler (Striking Development; http://www.paracel.com/) (S. Rensing, unpublished results) which are not found in the MIRA NRVT set. We also included 12 G. intraradices reference sequences downloaded from the NCBI, 23 G. intraradices mitochondrial sequences (C. Roux, unpublished results) and 70 G. intraradices sequences generated from the manual annotation of the G. intraradices genome and not found in the NRVTs. Finally, 4471 remaining singleton Sanger sequences without matches in public databases were added. For 2477 sequences of this set, no probe could be designed, probably because of their relative high A + T content, which resulted in a higher-than-usual prevalence of homopolymer runs and other forms of low-complexity sequences. This procedure produced a total set of 22 404 sequences containing 14 828 MIRA NRVTs, 1976 PARACEL NRVTs, 5496 singletons and 104 additional sequences. This set did not include NRVTs from symbiotic tissues not available when the custom array was constructed. However, c. 80% of the NRVTs used to construct the oligoarray were found by BLASTN in the 25 906 MIRASEARCHESTSNPS NRVTs.

The G. intraradices expression array (4 • 72K) manufactured by Roche NimbleGen Systems Limited (Madison, WI, USA) (http://www.nimblegen.com/products/exp/index.html) contained three independent, nonidentical, 60-mer probes per sequence. Included in the oligoarray were G. intraradices sequences, and 5785 random 60-mer control probes.

Transcript profiling

Whereas biological samples used for cDNA sequencing were produced by several different collaborators, biological materials used for microarray transcript profiling were generated in triplicate in a small number of laboratories: spores at the Laboratoire de Recherche en Sciences Ve ´ge ´tales (Universite ´de Toulouse, France), M. truncatula ERM at the Departamento de Microbiologı ´a del Suelo y Sistemas Simbio ´ticos (Estacio ´n Experimental del Zaidı ´n, CSIC, Granada, Spain) and M. truncatula IRM at the Boyce Thompson Institute for Plant Research (Tower Road, Ithaca, NY, USA). cDNA synthesis was carried out at INRA-Nancy.

RNA was extracted from mycorrhizal roots with Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA) with additional phenol : chloroform (1 : 1, v ⁄ v) purification steps, from ERM and spores using the RNeasy Plant Mini Kit (Qiagen) with RLT lysis buffer. RNA from mycorrhizal roots was treated with Turbo DNase I (Ambion Inc., Austin, TX, USA) and purified with the RNeasy MinElute CleanUp kit (Qiagen). DNase treatment of RNA from ERM was performed using RNAse-free DNase (Qiagen). RNA quality was checked before cDNA synthesis using the Bio-Rad Experion analyzer. RNA preparations (three biological replicates) were amplified using the SMART PCR cDNA Synthesis Kit (Takara Bio Europe ⁄ Clontech, Saint-Germain-en-Laye, France) according to the manufacturer's instructions. Single dye labeling of cDNA samples, hybridization procedures and data acquisition were performed at the NimbleGen facilities (NimbleGen Systems, Reykjavik, Iceland) following their standard protocol. As ERM and IRM materials were not produced in strictly similar conditions, transcript profiles of ERM and IRM should be compared with caution.

Average expression levels were calculated for each gene from the independent probes on the array and were used for further analysis. Raw array data were normalized by the robust multiarray average (RMA) routine using the ARRAYSTAR software (Dnastar Inc., Madison, WI, USA). A transcript was deemed expressed when its signal intensity was three-fold higher than the mean signal-to-noise threshold (cut-off value) of the random oligonucleotide probes present on the array (50-100 arbitrary units). The maximum signal intensity values for the most abundant transcripts were c. 65 000 arbitrary units. A control hybridization was performed with cDNA from uninfected M. truncatula roots to evaluate any plant RNA cross-hybridization with G. intraradices sequences and to detect any plant-derived probes on the array. The later probe signals were discarded. A Student's t-test with FDR correction (Benjamini-Hochberg) was applied to the data using ARRAYSTAR. Transcripts with a significant P-value (< 0.05) and ‡ five-fold change in transcript level were considered to be differentially expressed. The complete expression data set is available as series (accession number GSE29866) at the Gene Expression Omnibus at NCBI (http:// www.ncbi.nlm.nih.gov/geo/).

Validation of oligoarray data

To obtain the biological material for the PCR validation of oligoarray data, 6000 arbuscule-containing cells from M. truncatula roots (2000 cells for each of three biological replicates) were microdissected using a laser microdissection system (LMD) according to [START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF]. ERM from monoxenic M. truncatula cultures and c. 6000 germinated spores were sampled, snap-frozen in liquid N 2 and stored at )80°C until used. Total RNA was extracted using the RNeasy Microarray Tissue Mini Kit (Qiagen, Courtaboeuf, France) following the manufacturer's instructions. All reverse transcriptase (RT)-PCR assays were carried out using the One Step RT-PCR kit (Qiagen). DNA contamination in RNA samples was evaluated using the G. intraradices elongation factor GintEF1a gene specific primers for G. intraradices. To determine the amount of extracted RNA, a semiquantitative RT-PCR using GintEF1a specific primers was performed. Reactions were carried out in a final volume of 25 ll. Amplification reactions were run for 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and an aliquot of the PCR reaction was taken after 35, 37 and 40 cycles.

Results and Discussion

An obligate biotroph with a large gene repertoire

The cDNA libraries of G. intraradices listed in Table 1 were sequenced to assess the size and diversity of the fungus gene repertoire and explore the responses of the AM fungus at key developmental stages, germinated spores, ERM and IRM. After sequencing, the de novo hybrid clustering of the 437 411 filtered reads produced 25 906 NRVTs (Table S1); 87% of the reads were in the assembly corresponding to 20.3 Mbp of nonredundant sequences. Only 10 823 NRVTs (41.8%) matched database sequences (cut-off e-value of 1e-5) in BLAST searches against genes from fungi other than Glomeromycota (Fig. S4). Homologous NRVTs corresponded to 8773 unique known genes in the DNA databases. Among the predicted peptide sequences, 9035 and 9265 showed significant sequence similarity to proteins from R. oryzae and M. circinelloides, respectively, distantly related fungi in the order Mucorales (Fig. S4). NRVTs that did not match any of the known genes still gave highly relevant hits against the G. intraradices draft genome, suggesting that these represent novel orphan genes. As many G. intraradices transcripts are represented by two NRVTs (i.e. aligning to the 5¢ or 3¢ end of the transcript) with an NRVT:gene ratio of 1.4, the number of nonredundant expressed transcripts in G. intraradices was c. 18 500 (25 906 ⁄ 1.4).

Based on BLASTClust analysis, the number of NRVTs found in multigene families was low, with 120 (90% sequence identity; 80% sequence coverage) to 500 (90% sequence identity; 50% sequence coverage) NRVTs, most of the gene families having only two members.

Although the vast majority of genes encoding enzymes of primary metabolism are retained in obligate biotrophic pathogens (downy and powdery mildews, white rust, and poplar and stem rusts), notable exceptions include anaerobic fermentation, biosynthesis of glycerol from glycolytic intermediates, biosynthesis of thiamine, and nitrate and sulfate assimilation [START_REF] Baxter | Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome[END_REF][START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF][START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici[END_REF][START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF]. Out of the 25 906 G. intraradices NRVTs, 5296 (20.4%) had significant matches in the KEGG pathway database and were assigned to 175 KEGG pathways. Most KEGG enzymes were mapped to NRVTs (see the metabolic pathway map online at http://mycor.nancy.inra.fr/IMGC/GlomusGenome/download3. php?select=anno), indicating the occurrence of these active metabolic processes in G. intraradices mycelium. In contrast to oomycete and fungal biotrophic pathogens, transcripts coding for nitrate and nitrite reductases, nitrate transporter, and sulfite reductase were highly expressed in G. intraradices. However, G. intraradices has lost the thiamine biosynthetic pathway (Table S2), as have most haustorium-forming species [START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF]. We infer that G. intraradices obtains thiamine from the host. There is no invertase transcript in the G. intraradices transcriptome (Table S2), implying that this fungus depends on the plant to both provide and hydrolyse sucrose, with the glucose moiety then preferentially transferred to the mycobiont. This is consistent with earlier observations [START_REF] Schaarschmidt | Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots[END_REF]. Additional genes missing from biotrophic pathogens [START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF], such as those coding for the allantoin permease DAL4p, the uracil permease FUR4p, and several enzymes involved in aromatic amino acid metabolism and detoxification, were also missing from G. intraradices (Table S2). Although some metabolic pathways are missing in G. intraradices, we hypothesize that evolution to biotrophy in G. intraradices was not initiated by massive loss of metabolic complexity as observed in obligate biotrophic pathogens [START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF]. The mycobiont retains the ability to take up and assimilate nutrients from its soil environment.

Protein domain distributions

About half (5056) of the 10 823 peptide sequences from G. intraradices with sequence similarity to documented proteins in databases were shared with all other fungal species and can thus be considered ubiquitous among fungi. The comparison of protein sets of G. intraradices with those of R. oryzae and M. circinelloides showed that 1076 proteins were unique to these basal lineages of aseptate, coenocytic fungi. Interestingly, a small set of G. intraradices protein sequences were only shared with one or other of the sequenced mycorrhizal symbionts, the ascomycete T. melanosporum (49 NRVTs) or the basidiomycete L. bicolor (107 NRVTs). They mainly code for hypothetical proteins with no known function, although a few predicted proteins belong to signaling pathways. A total of 9321 NRVTs contained at least a part of a conserved protein domain in the KOG database. Compared with other fungal gene repertoires, G. intraradices showed an overrepresentation of proteins involved in signaling pathways and ubiquitin-related metabolism (Fig. 1). The expansion of the tyrosine kinase-encoding gene family involved in signaling pathways is also a feature of the ectomycorrhizal L. bicolor genome (Fig. 1; Martin et al., 2008b).

Sequence polymorphism of transcripts

There were a total of 43 872 SNPs in 3963 NRVTs (15.3% of the total number of NRVTs; 2.1 SNP ⁄ kb); 1102 NRVTs contained < 5 SNPs (Fig. 2a). When this SNP analysis was conducted on NRVT regions having a similarity with known protein coding sequences, 846 polymorphic NRVTs (8%; 1.3 SNP ⁄ kb) were identified among 10 823 homologs. Most polymorphic NRVTs contained < 5 SNPs (Fig. 2b). The NRVTs with known function having the highest SNPs were a 60S ribosomal protein L17 (15 SNPs) and a Ras-related Rab (11 SNPs).

The presence of multiple SNPs in hundreds of NRVTs confirmed the within-isolate DNA sequence polymorphism that has repeatedly been reported for a number of genes in G. intraradices [START_REF] Croll | Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices[END_REF]Sanders & Croll, 2010). The transcriptome data show that this polymorphism is widespread in the genome, and that > 1 variant of each polymorphic gene is transcriptionally active. This implies that the sequence variation may be of functional importance.

Symbiosis induces alterations in the G. intraradices transcriptome

Little is known about the impact of symbiosis on the mycobiont transcriptome and molecular factors driving developmental pathways [START_REF] Gu ¨imil | Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization[END_REF][START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Seddas | Symbiosisrelated plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF][START_REF] Kuznetsova | Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices[END_REF]Sanders & Croll, 2010). Our transcript profiling thus provides the first large-scale discovery of fungal symbiosis-related genes. Of the 18 751 coding sequences detected by oligoarrays, 395 (2.1%) and 569 (3.0%) were upand down-regulated ( ‡ five-fold; P-value £ 0.05), respectively, in IRM in comparison to germinated spores, whereas 202 (1.1%) and 74 (0.4%) were up-and down-regulated, respectively, in ERM in comparison to germinated spores. Highly up-regulated genes in IRM and ERM are shown in Tables 2, S3 and S4. In this study, mRNA concentrations were used as a proxy of protein concentrations. Gene expression is, however, a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins, and transcript levels are not always related to protein levels and enzyme activities [START_REF] Schwanha ¨usser | Global quantification of mammalian gene expression control[END_REF]. It remains to be determined whether the observed changes in transcripts lead to alteration of the proteome, enzyme activities and ⁄ or metabolic fluxes. In addition, regulatory enzymes exhibiting allosteric properties are likely to be effective agents for fine tuning of metabolic fluxes in symbiotic tissues.

The majority of IRM-and ERM-induced genes were lineagespecific genes, as 80% of the most highly up-regulated transcripts coded for orphan proteins (Tables 2,S3). Differentially expressed genes coding for proteins of known function were categorized into functional classes (Tables 3,S4). Genes overrepresented in IRM vs germinated spores showed a GO enrichment (FDR-corrected P £ 0.05) for several categories of biological processes associated with ion transport, lipid ⁄ steroid metabolism and DNA replication (data not shown). Out of 25 transcripts that appeared to be highly up-regulated in IRM, 17 were validated by RT-PCR (data not shown). While 13 out of 17 transcripts were detected in at least one other stage of the life cycle besides arbuscules, four were arbuscule-specific transcripts (Table S5). Three were orphan genes, while the fourth showed similarity to a multidrug resistance protein (ATP Binding Cassette (ABC) transporter). Table 2 Most highly up-regulated Glomus intraradices transcripts (nonredundant virtual transcripts (NRVTs)) in the intraradical mycelium (IRM) compared with germinated spores identified using expression oligoarrays and ranked by decreasing fold changes

As the oligoarray was constructed before the 454 ESTs from IRM and LMD arbuscule-containing cells were obtained, we also carried out a digital northern analysis based on the differential frequency of 454 reads [START_REF] Audic | The significance of digital gene expression profiles[END_REF]. This transcript profiling confirmed the above oligoarray expression patterns (data not shown). However, we detected a significant set of IRM transcripts coding for SSPs not detected in ERM and spores. These SSPs showed a striking up-regulation in IRM and arbusculecontaining cells (Table 4). The most highly up-regulated IRM orphan transcript 'step3_c3163' coded for an SSP of 280 amino acids of unknown function with similarity to proteins of several biotrophic basidiomycetes (e.g. L. bicolor and M. larici-populina). This transcript was only detected in mycorrhizal roots and arbuscules.

Recent studies of plant-microbe interactions involving pathogenic and ectomycorrhizal fungi have shown that many lineage-specific orphan genes code for effector proteins playing key roles in host colonization and in planta accommodation by controlling the plant immune system (De [START_REF] Wit | Fungal effector proteins: past, present and future[END_REF]Plett et al., 2011). The set of differentially expressed G. intraradices orphan genes included the effector protein SP7, which interacts with the pathogenesis-related transcription factor Ethylene Response Factor (ERF19) in the host nucleus [START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biothrophy[END_REF] (Table S4). Given the key results obtained for SP7, the role played by other mycorrhiza-up-regulated SSPs of G. intraradices should be elucidated, as well as the identity of plant-based signals that may control their expression within the root space.

Stealth colonization to evade host defenses

Several obligate biotrophic pathogens and ectomycorrhizal symbionts have a decreased repertoire of carbohydrate-acting enzymes involved in the degradation of plant cell wall (PCW) polysaccharides (Martin et al., 2008b[START_REF] Martin | Pe ´rigord Black Truffle genome uncovers evolutionary origins and mechanisms of symbiosis[END_REF][START_REF] Baxter | Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome[END_REF][START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF][START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici[END_REF]. We identified 139 NRVTs encoding carbohydrate-active enzymes in the G. intraradices transcriptome (Table S6). They included enzymes that are involved in acquiring and converting carbohydrates and in maintaining fungal cell wall plasticity. Genes encoding glycosyl hydrolases (GHs) involved in degrading PCW lignocellulosic polymers were not found in the IRM predicted proteome. No genes encoding GH from families GH6 and GH7 (which include cellobiohydrolases and are involved in the attack of crystalline cellulose), polysaccharide lyases or proteins with cellulose-binding motif 1 (CBM1) were identified (Table S6). Enzymes that attack amorphous cellulose, hemicellulose, and pectin (e.g. b-glucosidases and cellobiose dehydrogenases of family GH3, GH10 xylanases, and GH28 pectinases) were absent. Genes encoding carbohydrate esterases and secreted feruloyl esterase which could hydrolyze cross-links in PCW were also not expressed. However, genes encoding GH5 endoglucanases (which act on mannan and ⁄ or cellulose) and a GH9 cellobiohydrolase were identified (Table S6). As observed in ectomycorrhizal symbionts (Martin et al., 2008b[START_REF] Martin | Pe ´rigord Black Truffle genome uncovers evolutionary origins and mechanisms of symbiosis[END_REF][START_REF] Nagendran | Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei[END_REF], the minimal set of PCW-degrading enzymes in the transcriptome of G. intraradices might be an evolutionary adaptation to avoid the release of polysaccharide fragments and their detection by the host immune system during the obligate biotrophic phase of the fungus. By contrast, a large set of genes encoding enzymes involved in chitin metabolism were expressed, including chitin synthases, chitin deacetylases, and chitinases (Table S6). They probably play a role in remodeling the fungal cell wall during growth and symbiosis. The abundance of transcripts coding for dolichyl-related mannosylation enzymes suggests an intense biosynthesis of cell wall mannans and mannoproteins.

Nutrient assimilation

We identified numerous transcripts coding for different predicted proteins associated with the uptake and assimilation of major soil nutrients, such as nitrate and Pi, and conversion of metabolites shuttled between partners (amino acids and carbohydrates) (Table S7). Although a few genes involved in nutrient transport and assimilation have previously been identified in G. intraradices and Glomus mosseae [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF][START_REF] Govindarajulu | Nitrogen transfer in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Javot | Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles[END_REF][START_REF] Tian | Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux[END_REF], there is no comprehensive molecular description of N and P assimilation pathways, despite the crucial importance of metabolite exchange in this symbiosis [START_REF] Kiers | Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis[END_REF][START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[END_REF]. The striking induction of several genes coding for metal transporters suggests that metals, such as Zn, are important for plant colonization, and the expression pattern of genes coding for assimilative enzymes fully supports rapid uptake, translocation and transfer of metabolites, as discussed in the following sections.

Nitrogen metabolism In the G. intraradices-colonized M. truncatula roots, transcripts for transporters and enzymes involved in N uptake and assimilation showed high constitutive expression, supporting the contention that a high N turnover is taking place in the symbiosis [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF]Guether et al., 2009b). The up-regulation of transcripts for nitrate permease and nitrate reductase (Table S8), together with the higher expression of the gene coding for the ammonium transporter gene GintAMT2 (Table S8; Pe ´rez-Tienda et al., 2011), might favor the accumulation of NH 4 + for incorporation into amino acids and further translocation of N to the host cells. The IRM transcript profile is consistent with assimilation of NH 4 + via the glutamine synthetase ⁄ glutamate synthase (GS ⁄ GOGAT) cycle. Transcripts encoding enzymes of arginine biosynthesis and degradation were highly expressed in spores, ERM and IRM (Table S8), indicating an intense cycling of N through the arginine pathway [START_REF] Tian | Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux[END_REF]. The higher expression of genes coding for arginase, urease and urease accessory protein (UreG) in IRM might lead to a greater release of NH 4 + able to feed the GS ⁄ GO-GAT cycle and ⁄ or the passive efflux of ammonia to the interfacial apoplast and the uptake of NH 4 + by the host ammonium transporter(s) [START_REF] Govindarajulu | Nitrogen transfer in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Tian | Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux[END_REF]. High expression of amino acid transporter genes in ERM and IRM (Table S7) suggests that the mycobiont can taken up amino acids present in both soil and the interfacial apoplast.

Phosphate metabolism Glomus intraradices expressed a wide spectrum of secreted phosphatase transcripts, including those coding for the p-nitrophenylphosphatase Pho13p, acid phosphatase Pho3p, repressible alkaline phosphatase Pho8p, and magnesium-dependent phosphatase, able to act on various phosphate esters (Table S9). No phytase transcript was detected. Glomus intraradices has a combination of low-affinity and highaffinity transporter genes, including the high-affinity Pi transporter gene GintPT (equivalent to Pho84p from Saccharomyces cerevisiae), and genes encoding the low-affinity Pi transporter Pho91p and the Na + ⁄ Pi symporter Pho89p (Table S9). The transcripts coding for the interactor protein Pho88p which promotes maturation and trafficking of Pho84p in yeast have also been identified. Pi transporter genes are expressed in spores, ERM and IRM. The expression pattern of GintPT is in agreement with the results obtained on GmosPT in G. mosseae [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF] and the observed expression of GmosPT in arbuscules [START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF][START_REF] Go ´mez-Ariza | Cell-specific gene expression of phosphate transporters in mycorrhizal tomato roots[END_REF]. Although high net transfer of P from soil to the plant takes place via the mycobiont [START_REF] Kiers | Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis[END_REF][START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales[END_REF], our findings suggest that the IRM may re-absorb some of the Pi released in the symbiotic apoplastic space. As shown by [START_REF] Kiers | Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis[END_REF], the fungus might exert some control on the delivery of nutrients to its host plant, and reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis.

Following Pi uptake, PolyP accumulates in G. intraradices hyphae and is stored in the vacuolar compartments, where it buffers cytoplasmic Pi concentrations. PolyP is translocated along hyphae toward the interfacial apoplast [START_REF] Viereck | Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo 31 P NMR spectroscopy[END_REF][START_REF] Javot | Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles[END_REF][START_REF] Hijikata | Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus[END_REF]. The rapid synthesis of PolyP is thus crucial for the maintenance of effective hyphal Pi uptake [START_REF] Tani | ATP-dependent but proton gradient-independent polyphosphate-synthesizing activity in extraradical hyphae of an arbuscular mycorrhizal fungus[END_REF]. Transcripts coding for the PolyP polymerase ⁄ Vacuolar Transporter Chaperone Complex Vtc4p protein involved in the synthesis and transfer of PolyP to the vacuole [START_REF] Hothorn | Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase[END_REF], and for its accessory proteins, such as Vtc1p, were expressed in G. intraradices (Table S9). Transcripts for vacuolar endopolyphosphatase Ppn1p and the exopolyphosphatase Ppx1p were also identified. The endopolyphosphatase transcript was strikingly up-regulated in IRM. Several components of the complex phosphate regulatory system (PHO regulon) found in S. cerevisiae and N. crassa were expressed in G. intraradices, including the ankyrin-repeat-containing NUC2related protein (Pho81p), the cyclin-dependent protein kinase regulator Pho80p and the mitogen-activated serine ⁄ threonine Cdc2 (Cell Cycle Control 2) cyclin-dependent protein kinase Pho85p (Table S9), suggesting putative regulatory similarities.

Lipid metabolism

The profuse development of fungal membranes associated with arbuscule development in host cells dramatically increases the need for plasma membrane fatty acids and sterols. The oligoarray analysis identified transcripts in IRM coding for the subunits a and b of the fatty acid synthase complex, together with the acetyl-CoA carboxylase transcript (Table S10), confirming that the mycobiont does possess fatty acid synthetic capacities (Table S10). Similarly, mycorrhizainduced genes predicted to be involved in fatty acid metabolism are up-regulated in M. truncatula [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF], suggesting a complementary regulation of fungal and plant lipid metabolism. In this host plant, the gene MtMSBP1, encoding a membrane steroid (progesterone)-binding protein, is also induced early by a diffusible AM fungal signal produced by G. intraradices branched hyphae [START_REF] Kuhn | Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula[END_REF]. The high activity of enzymes involved in sterol and steroid metabolism in IRM, together with the AM fungal induction of MtMSBP1, might be related to the need to alter sterol metabolism (e.g. lanosterol) to allow plasma membrane invagination and intracellular accommodation of the fungal symbiont in the cortical cells.

An expanded inventory of conserved meiotic genes provides evidence for cryptic sex

The lack of an observed sexual stage in any member of the Glomeromycota led to the suggestion, which has been debated, that AM fungi were ancient asexuals. Results from Croll & Sanders (2009) strongly suggest that recombination occurred among some G. intraradices genotypes in the field, although the majority of populations examined so far have been clonal [START_REF] Rosendahl | Communities, populations and individuals of arbuscular mycorrhizal fungi[END_REF]. As Glomeromycota could represent one of the earliest diverging fungal lineages, their meiotic processes could represent an ancestral state. We surveyed the transcriptome of G. intraradices for a set of sex and meiotic genes conserved among eukaryotes [START_REF] Malik | An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis[END_REF] (Table S11) and identified several 'meiosis-specific' genes (HOP2 (Homologous-pairing protein 2) and MND1 (Meiotic nuclear division protein 1)) which are only known to function in meiosis in other eukaryotes [START_REF] Malik | An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis[END_REF]. These genes are hypothesized to be present in organisms with sexual ancestry (Table S11). The homolog of a transcript coding for the key meiotic recombinase SPO11 (REC12) was not found, but has been identified in the genomic sequences of Glomus diaphanum (MUCL 43196), G. intraradices (DAOM 197198), Glomus clarum (DAOM 234281) and Glomus cerebriforme (DAOM 227022) [START_REF] Halary | Conserved meiotic machinery in Glomus spp., a putatively ancient asexual fungal lineage[END_REF]. Transcripts coding for high-mobility group (HMG) domain-containing transcriptional factors with a significant similarity (55%) to the sexP and sexM genes from Phycomyces blakesleeanus [START_REF] Idnurm | Identification of the sex genes in an early diverged fungus[END_REF] were also retrieved. These genes are master switches controlling mating type in fungi. Mechanisms controlling the G. intraradices sexual cycle need to be further examined within AM fungal populations as they are likely to allow the mixing of nuclei and subsequent recombination among different individuals of this fungus interacting in soil.

In summary, induced expression of genes coding for membrane transporters and SSPs during the symbiotic interaction and the lack of expression of hydrolytic enzymes acting on PCW polysaccharides are hallmarks of G. intraradices. These results extend conserved patterns of gene expression profiles observed in obligate biotrophic pathogens [START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF][START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF] and ectomycorrhizal symbionts (Plett & Martin, 2011) to the Glomeromycota lineage. By contrast, obligate biotrophy in G. intraradices is not associated with a striking reduction of metabolic complexity (e.g. lack of N and S assimilation pathways), as observed in many obligate biotrophic pathogens, so that the ability to interact with the soil environment with respect to nutrient uptake is maintained in the symbiotic fungus. Finally, we can hypothesize that biotrophy in AM fungi has evolved through a series of steps requiring effectors, such as the secreted SP7 [START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biothrophy[END_REF], coupled with a reduced inventory of PCW-hydrolyzing enzymes to suppress or attenuate host defense reactions, and weak selection forces to maintain certain biosynthetic pathways if products (e.g. thiamine and sucrose) can be directly obtained from the host. The present comprehensive repertoire of G. intraradices genes, the first for Glomeromycota, provides a basis for future research in environmental genomics and for accessing symbiosis-related functional features in other members of this unique phylum.

Table S8 Expression of nonredundant virtual transcripts (NRVTs) coding for transporters and enzymes involved in nitrogen metabolism in the Glomus intraradices transcriptome Table S9 Expression of nonredundant virtual transcripts (NRVTs) coding for transporters and enzymes involved in phosphate metabolism in the Glomus intraradices transcriptome Table S10 Expression of nonredundant virtual transcripts (NRVTs) coding for lipid metabolism enzymes in the Glomus intraradices transcriptome Table S11 Expression levels and regulation ratio of transcripts related to meiosis in the Glomus intraradices transcriptome Methods S1 Construction of the cDNA libraries.

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  Glomus intraradicesSchenck & Smith DAOM 197198 (recently reassigned to G. irregulare and then Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schu ¨ßler comb. nov.; see Stockinger et al., 2009) was produced in monoxenic cultures maintained on Agrobacterium rhizogenestransformed carrot (Daucus carota; clone DC2) roots (Be ´card &

Fig. 1

 1 Fig. 1 Most abundant Eukaryotic Orthologous Groups (KOG) domains in Glomus intraradices compared with representative saprotrophic (black circle), pathogenic (black square) and mutualistic fungi (open circle). The heatmap is based on the relative z-score of KOG conserved domains. The top 55 KOG domains found in G. intraradices were selected. Colors indicate abundance: from dark green (highly abundant) to white (weakly abundant). An, Aspergillus nidulans; Bc, Botrytis cinerea; Bg, Blumeria graminis; Cn, Cryptococcus neoformans; Lb, Laccaria bicolor; Ml, Melampsora larici-populina; Mg, Magnaporthe grisea; Mc, Mucor circinelloides; Nc, Neurospora crassa; Pc, Phanerochaete chrysosporium; Ro, Rhizopus oryzae; Tm, Tuber melanosporum; and Um, Ustilago maydis.

Fig. 2

 2 Fig. 2 Single nucleotide polymorphisms (SNPs) in nonredundant virtual transcript (NRVT) sequences from Glomus intraradices. (a) SNPs were identified by the MIRASEARCHESTSNPS program by aligning the complete nucleotide sequences from NRVTs, including 5¢ and 3¢ untranslated regions (UTRs). (b) SNPs were calculated in the coding regions of NRVTs having a homolog documented in the Swiss-Prot database.

Table 1

 1 Glomus intraradices tissues used in the extraction of transcripts for cDNA library construction and oligoarray profiling

		Sanger	454	Array
	Samples	sequencing	sequencing	profiling
	Germinated spores	CCHU		Three
				replicates
	Germinated spores		EXTA
	mix = spores
	+ spores in GR24 +
	spores in API
	Germinated spores		AKNC
	in GR24
	Germinated spores		AKNB
	in API
	Daucus carota ERM	CACE	EXTB
	Medicago truncatula			Three
	ERM			replicates
	M. truncatula IRM		AKNA*	Three
				replicates
	LMD-microdissected		AKND
	arbuscule-containing cells
	GR24, strigolactone GR24; API, apigenin; ERM, extraradical mycelium;
	IRM, intraradical mycelium; AKNA, AKNB, AKNC, AKND, CACE, CCHU,
	EXTA and EXTB stand for cDNA library IDs. AKNA, M. truncatula IRM;
	AKNB, germinated spores incubated in apigenin for 24 h; AKNC, germinated
	spores incubated in the strigolactone GR24 for 24 h; AKND, laser-microdis-
	sected M. truncatula arbusculed cells; CACE, M. truncatula ERM; CCHU,
	germinated spores; EXTA, germinated spore mix; EXTB, M. truncatula ERM;
	LMD, laser microdissection system.

Table 4

 4 The transcripts coding for mycorrhizal-induced small secreted proteins (MiSSPs) showing up-regulation in Glomus intraradices

	intraradical mycelium (IRM) compared with germinated spores based on
	454 read frequency
		IRM	Arbuscules	Spores	Putative	Size
	NRVT ID	(reads)	(reads)	(reads)	function	(aa) # Cys
	step3_c3163	62	4	0	Hypothetical	280 12
					protein
	remain_c2375 24	16	0	No hit	156 3
	step3_c3587	15	2	0	No hit	149 0
	remain_c4548 12	4	0	MD-2-related	173 4
					lipid
					recognition
					domain
	step3_c2834	11	19	0	No hit	61 9
	remain_c2379 11	7	0	No hit	120 0
	step3_c3282	10	1	0	No hit	86 2
	remain_c8131	9	0	0	Hypothetical	150 3
					protein
	remain_c2779	7	20	0	No hit	67 0
	remain_c8897	7	1	0	No hit	71 7
	remain_c12045 5	3	0	No hit	122 4
	These sequences have no probe on oligoarrays.
	aa, amino acids; # Cys, number of cysteine residues; NVRT, nonredundant
	virtual transcript.

Ó 2011 The Authors New Phytologist Ó 2011 New Phytologist Trust New Phytologist (2012) 193: 755-769 www.newphytologist.com

New Phytologist(2012) 193: 755-769www.newphytologist.com

Acknowledgements E.T. is supported by a scholarship from the Re ´gion Lorraine and the European Commission ENERGYPOPLAR project (FP7-211917), and Y.L. by a scholarship from the Education Ministry of China. This work was supported by the Network of Excellence EVOLTREE (FP6-016322) and the US Department of Energy (DOE) -Oak Ridge National Laboratory Scientific Focus Area for Genomics Foundational Sciences (grant to F.M.). Ph.F. was supported by the German Research Foundation and the Ministries of Food, Agriculture, and Consumer Protection of the FRG and the Brandenburg and Thu ¨ringen States. P.B. received funding from the Regional Project Converging Technologies-BI-OBIT, and N.F. and C.A.A. from the project AGL2009-08868 from the Spanish Ministry of Science and Education. Support for development of mycorrhizal M. truncatula root materials was provided by the US National Science Foundation (grant IOS-0842720 to M.J.H.). A portion of the writing of this manuscript was sponsored by the DOE Genomic Science Program (DE-AC05-00OR22725). EST sequencing was funded by INRA, Genoscope, New Mexico University, and Michigan State University. The genomic and Sanger EST sequencing was conducted by JGI (DE-AC02-05CH11231). We would like to thank Nicolas Corradi for helpful discussions on the meiotic genes, and three anonymous referees for their insightful comments and suggestions.

Supporting Information

Additional supporting information may be found in the online version of this article. Table S1 The number of assembled nonredundant virtual transcripts (NRVTs) was affected by the parameters used for assembling the Glomus intraradices expressed sequence tags (ESTs) using the MIRA assembler Table S2 Genes missing in the Glomus intraradices transcriptome, and gene repertoires of Blumeria graminis [START_REF] Spanu | Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism[END_REF] and other obligate biotrophic pathogens [START_REF] Kemen | Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana[END_REF] Table S3 Most highly up-regulated Glomus intraradices transcripts in the extraradical mycelium (ERM) vs germinated spores identified using expression oligoarrays and ranked by decreasing fold changes

Table S4 All transcripts with functional annotations that were up-regulated by at least 10-fold in Glomus intraradices extraradical mycelium (ERM) compared with germinated spores based on oligoarray profiling (ratio ‡ 10; P-value < 0.05)