Unearthing the truffle genome

Special Feature Articles: Editorial Unearthing the truffle genome Francis Martin

Research reviews

Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum Paola Ceccaroli, Michele Buffalini, Roberta Saltarelli, Elena Barbieri, Emanuela Polidori, Simone Ottonello, Annegret Kohler, Emilie Tisserant, Francis Martin and Vilberto Stocchi

Truffle volatiles: from chemical ecology to aroma biosynthesis Richard Splivallo, Simone Ottonello, Antonietta Mello and Petr Karlovsky

Full papers Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum

Methods Deep RNA sequencing improved the structural annotation of the Tuber melanosporum transcriptome Emilie Tisserant, Corinne Da Silva, Annegret Kohler, Emmanuelle Morin, Patrick Wincker and Francis Martin

Andrea Rubini, Beatrice Belfiori, Claudio Riccioni, Emilie Tisserant, Sergio Arcioni, Francis Martin and Francesco Paolocci

Tuber melanosporum: mating type distribution in a natural plantation and dynamics of different strains of mating types on the roots of nursery-inoculated host plants Andrea Rubini, Beatrice Belfiori, Claudio Riccioni, Sergio Arcioni, Francis Martin and Francesco Paolocci

Genome-wide search and functional identification of transcription factors in the mycorrhizal fungus Tuber melanosporum Barbara Montanini, Elisabetta Levati, Angelo Bolchi, Annegret Kohler, Emmanuelle Morin, Emilie Tisserant, Francis Martin and Simone Ottonello

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Introduction The ‘black diamond’, the ‘mysterious product of the earth’, the ‘ultimate fungus’, ‘la grande mystique’ are some of the common names describing the delectable Périgord black truffle (Tuber melanosporum Vitt.). Truffle cultivation is notoriously difficult, in part because of its cryptic life cycle as an underground symbiont, in which the fungus trades nutrients with oaktree roots. Decreasing supply and rising market prices have provided a strong incentive for research on truffle cultivation.

This includes a better understanding of the fungus life cycle and the ecology of truffle grounds.

This Special Feature includes the description of the T. Melanosporum genome and a number of companion papers that will benefit scientists investigating plant–microbe interactions and the molecular ecology of the mycorrhizal symbiosis. Clearly, the analysis of the first genome sequence from an ectomycorrhizal fungus Laccaria bicolor (see New Phytologist Feature ‘Unwrapping the Laccaria genome’, Vol. 180, No. 2, 2008) and the present truffle genome have brought us to a point where we are faced with opportunities to develop understanding of the evolution of the mycorrhizal symbiosis in ways never anticipated.

The sequencing of the Périgord black truffle moves us a further step away from the past toward a future where we will be following multidimensional changes in gene networks and relating them to the ecology of the truffle ground. Genomics of T. melanosporum is also offering clues that could help a truffle industry that is fraught with unpredictable yields and a counterfeit market. The genome paper and its raft of companion papers are providing us with novel insight into the biology of this ‘ultimate’ fungus, yet this still leaves sufficient mystery in the area so that you can enjoy your truffled risotto. Bon appétit!

Research review Truffle volatiles: from chemical ecology to aroma biosynthesis

Author for correspondence: Richard Splivallo Tel: +49 551 39 3711 Email: ricsi17@hotmail.com

Richard Splivallo, Simone Ottonello, Antonietta Mello and Petr Karlovsky

Summary New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03523.x

Keywords: aroma biosynthesis, chemical ecology, ectomycorrhiza, plant–microbe interactions, truffles, Tuber borchii, Tuber melanosporum, volatile organic compounds.

Truffles (Tuber spp.) are symbiotic fungi that develop underground in association with plant roots. Food connoisseurs describe their scent as sensual, seductive and unique.

These mysterious fungi, however, do not produce their aroma for the mere pleasure of humans. Truffle volatiles act as odorant cues for mammals and insects which are thus able to locate the precious fungi underground and spread their spores. They also freely diffuse in the soil and mediate interactions with microorganisms and plant roots, potentially regulating a complex molecular dialogue among soil fauna and flora. The aim of this review is to synthesize 30 yr of research on truffle volatiles, spanning fields of study from chemical ecology to aroma biosynthesis. Specific aspects of truffle volatile ecology and biology will be discussed, including which species have been studied so far and for what purpose, what ecological role has been demonstrated or speculated to exist for specific truffle volatiles, which volatiles are common or unique to certain species and what their biosynthetic route might be. Future challenges in truffle aroma research will also be addressed, focusing on how high-throughput postgenomic technologies may advance our understanding of truffle aroma biosynthesis and chemical ecology.

Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum Andrea Rubini, Beatrice Belfiori, Claudio Riccioni, Emilie Tisserant, Sergio Arcioni, Francis Martin and Francesco Paolocci

Summary Author for correspondence: Francesco Paolocci Tel: +39 075 5014861 Email: francesco.paolocci@igv.cnr.it

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The genome of Tuber melanosporum has recently been sequenced. Here, we used this information to identify genes involved in the reproductive processes of this edible fungus. The sequenced strain (Mel28) possesses only one of the two master genes required for mating, that is, the gene that codes for the high mobility group (HMG) transcription factor (MAT1-2-1), whereas it lacks the gene that codes for the protein containing the α-box- domain (MAT1-1-1), suggesting that this fungus is heterothallic.

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A PCR-based approach was initially employed to screen truffles for the presence of the MAT1-2-1 gene and amplify the conserved regions flanking the mating type (MAT) locus. The MAT1-1-1 gene was finally identified using primers designed from the conserved regions of strains that lack the MAT1-2-1 gene.

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Mating type-specific primer pairs were developed to screen asci and gleba from truffles of different origins and to genotype single ascospores within the asci. These analyses provided definitive evidence that T. melanosporum is a heterothallic species with a MAT locus that is organized similarly to those of ancient fungal lineages.

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A greater understanding of the reproductive mechanisms that exist in Tuber spp. allows for optimization of truffle plantation management strategies.

New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03492.x

KEYWORDS: ascomycetes, ectomycorrhizal fungi, heterothallism, mating type, reproductive system, Tuber melanosporum.

Tuber melanosporum: mating type distribution in a natural plantation and dynamics of different strains of mating types on the roots of nurseryinoculated host plants Andrea Rubini, Beatrice Belfiori, Claudio Riccioni, Sergio Arcioni, Francis Martin and Francesco Paolocci

Summary Author for correspondence: Francesco Paolocci Tel: +39 075 5014861 Email: francesco.paolocci@igv.cnr.it

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In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions.

New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03493.x

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In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation.

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In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months.

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The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.

KEYWORDS: ascospores, ectomycorrhiza, genet distribution, gleba, mating type, simple sequence repeats (SSRs), truffles, Tuber.

Genome-wide search and functional identification of transcription factors in the mycorrhizal fungus Tuber melanosporum Barbara Montanini, Elisabetta Levati, Angelo Bolchi, Annegret Kohler, Emmanuelle Morin, Emilie Tisserant, Francis Martin and Simone Ottonello

Summary Author for correspondence: Simone Ottonello Tel: +39 0521 905646 Email: s.ottonello@unipr.it

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Developmental transitions associated with the life cycle of plant-symbiotic fungi, such as the ascomycete Tuber melanosporum, are likely to require an extensive reprogramming of gene expression brought about by transcription factors (TFs). To date, little is known about the transcriptome alterations that accompany developmental shifts associated with symbiosis or fruiting body formation.

New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03525.x

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Taking advantage of the black truffle genome sequence, we used a bioinformatic approach, coupled with functional analysis in yeast and transcriptome profiling, to identify and catalogue T. melanosporum TFs, the so-called ‘regulome’.

KEYWORDS: mycorrhiza, regulome, transcription factors, transcriptional activator trap assay, truffles, tuber, yeast.

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The T. melanosporum regulome contains 102 homologs of previously characterized TFs, 57 homologs of hypothetical TFs, and 42 putative TFs apparently unique to Tuber. The yeast screen allowed the functional discovery of four TFs and the validation of about one-fifth of the in silico predicted TFs. Truffle proteins apparently unrelated to transcription were also identified as potential transcriptional regulators, together with a number of plant TFs.

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Twenty-nine TFs, some of which associated with particular developmental stages, were found to be up-regulated in ECMs or fruiting bodies. About one-quarter of these up-regulated TFs are expressed at surprisingly high levels, thus pointing to a striking functional specialization of the different stages of the Tuber life cycle.

Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum Paola Ceccaroli, Michele Buffalini, Roberta Saltarelli, Elena Barbieri, Emanuela Polidori, Simone Ottonello, Annegret Kohler, Emilie Tisserant, Francis Martin and Vilberto Stocchi

Summary Author for correspondence: Paola Ceccaroli Tel: +39 0722 305262 Email: paola.ceccaroli@uniurb.it

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Primary carbohydrate metabolism plays a special role related to carbon/nitrogen exchange, as well as metabolic support of fruiting body development, in ectomycorrhizal macrofungi. In this study, we used information retrieved from the recently sequenced Tuber melanosporum genome, together with transcriptome analysis data and targeted validation experiments, to construct the first genomewide catalogue of the proteins supporting carbohydrate metabolism in a plantsymbiotic ascomycete.

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More than 100 genes coding for enzymes of the glycolysis, pentose phosphate, tricarboxylic acid, glyoxylate and methylcitrate pathways, glycogen, trehalose and mannitol metabolism and cell wall precursor were annotated. Transcriptional regulation of these pathways in different stages of the T. melanosporum lifecycle was investigated using whole-genome oligoarray expression data together with real -time reverse transcription-polymerase chain reaction analysis of selected genes.

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The most significant results were the identification of methylcitrate cycle genes and of an acid invertase, the first enzyme of this kind to be described in a plantsymbiotic filamentous fungus.

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A subset of transcripts coding for trehalose, glyoxylate and methylcitrate enzymes was up-regulated in fruiting bodies, whereas genes involved in mannitol and glycogen metabolism were preferentially expressed in mycelia and ectomycorrhizas, respectively. These data indicate a high degree of lifecycle stage specialization for particular branches of carbohydrate metabolism in T. melanosporum.

New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03520.x

KEYWORDS: carbohydrate metabolism, ectomycorrhizal fungi, genome-wide repertoire, invertase, mannitol, trehalose, Tuber melanosporum.

Methods Deep RNA sequencing improved the structural annotation of the Tuber melanosporum transcriptome Emilie Tisserant, Corinne Da Silva, Annegret Kohler, Emmanuelle Morin, Patrick Wincker and Francis Martin

Summary Author for correspondence: Francis Martin Tel: +33 383 39 40 80 Email: fmartin@nancy.inra.fr

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The functional complexity of the Tuber melanosporum transcriptome has not yet been fully elucidated. Here, we applied high-throughput Illumina RNAsequencing (RNA-Seq) to the transcriptome of T. melanosporum at different major developmental stages, that is free-living mycelium, fruiting body and ectomycorrhiza.

New Phytologist (2010) doi: 10.1111/j.1469-8137.2010.03597.x

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Sequencing of cDNA libraries generated a total of c. 24 million sequence reads representing > 882 Mb of sequence data. To construct a coverage signal profile across the genome, all reads were then aligned to the reference genome assembly of T. melanosporum Mel28.

KEYWORDS: ectomycorrhiza, fruiting body, high-throughput Illumina RNAsequencing (RNA-Seq), transcriptome, Tuber.

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We were able to identify a substantial number of novel transcripts, antisense transcripts, new exons, untranslated regions (UTRs), alternative upstream initiation codons and upstream open reading frames.

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This RNA-Seq analysis allowed us to improve the genome annotation. It also provided us with a genome-wide view of the transcriptional and post-transcriptional mechanisms generating an increased number of transcript isoforms during major developmental transitions in T. melanosporum.

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