The ‘black diamond’, the ‘mysterious product of the earth’, the ‘ultimate fungus’ and ‘la grande mystique’ are some of the common names describing the delectable Périgord black truffle (Tuber melanosporum Vitt.). The culture, harvesting and marketing of this highly prized ectomycorrhizal fungus is a world that retains some of the secrets and intrigue of the past (Hall et al., 2007). Truffle cultivation is notoriously difficult, in part because of its cryptic life cycle as an underground symbiont, in which the fungus trades nutrients with oak-tree roots. By the end of the 1960s, there had been some success in devising new methods for producing truffle-infected seedlings under controlled conditions in glasshouses by inoculating plants with truffle cultures and spores. After successful plantation in orchards, reliable information on truffle yields and production is very difficult to obtain as a result of under-reporting of harvests, under-the-table marketing practices and a lack of administration records. It appears, however, that the production of truffles, as with other mushrooms, is erratic from year to year (depending on the weather conditions) and tends to decline as a result of global climate change. 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. Scientists investigating both plant–microbe interactions and the molecular ecology of the mycorrhizal symbiosis will benefit from the description of the T. melanosporum genome (Martin et al., 2010) and equally so from the companion papers compiled in this issue of New Phytologist (Ceccaroli et al., 2011; Montanini et al., 2011; Rubini et al., 2011a,b; Splivallo et al., 2011; Tisserant et al., 2011) and in Fungal Genetics and Biology (Bolchi et al., 2011; Murat et al., 2011; Zampieri et al., 2011). The authors of these papers have used the freshly minted truffle genome to begin to probe some of the most intriguing questions in both the biology and ecology of this iconic fungus.
What has the initial analysis of the truffle genome sequence revealed? The Tuber genome, of 125 megabases, is one of the largest fungal genomes sequenced so far, but it contains fewer than 7500 protein-coding genes – a very compact gene space (Martin et al., 2010). It would appear, based on the different gene repertoires and symbiosis transcriptomes of T. melanosporum and the ectomycorrhizal basidiomycete Laccaria bicolor (Martin et al., 2008), that the evolution of the symbiotic lifestyle is quite divergent (Plett & Martin, 2011). The two symbionts use very different molecular ‘toolboxes’ to interact with their respective hosts. The Tuber genome comprises > 55% retrotransposons and DNA transposons (Martin et al., 2010). Born in bursts, retrotransposons mainly dispersed in the genome of Tuber during the last 5 million yr. Whether these retrotransposons are generating new genes through rearranging gene fragments in T. melanosporum remains to be investigated. Simple sequence repeats (SSR) are also highly abundant in the genome and have been used to generate multilocus SSR fingerprints of several geographic accessions (Murat et al., 2011). This survey showed that T. melanosporum is a species with a high genetic diversity, which is in agreement with its recently uncovered heterothallic mating system (Rubini et al., 2011b, pp. 710–722).
While looking at the gene repertoire of T. melanosporum and classifying the function of its genes can tell us what it is capable of, it does not tell us whether that species ever takes advantage of this capability. Transcriptomics gives us a snapshot of which genes are expressed, and by comparing the transcriptomic profile of T. melanosporum as it experiences different environments, we can start to understand how the truffle will respond to changes within its host plant and ecosystem environment. To complement the genomic sequencing effort, Tisserant et al. (pp. 883–891) used Illumina-based RNA ultrasequencing to generate a picture of the repertoire of genes expressed during symbiosis and fruiting body formation. Having this picture proved to be very valuable to the community in the annotation process. Genome analysis and transcriptomics is only one power tool in the toolbox. To fully understand the biology of the truffle symbiosis, we need to perform multi -’omic observations using genomics, proteomics and metabolomics to determine changes in the genomic potential, protein inventory or metabolite profile. Montanini et al. (pp. 736–750) used functional analysis in yeast to identify and catalogue T. melanosporum transcriptional factors (TFs). They identified several strikingly upregulated TFs in the mycelium colonizing the root, suggesting that they are controlling both the development and functioning of the symbiosis. Comprehensive annotation and transcript profiling of genes involved in carbohydrate metabolism (Ceccaroli et al., pp. 751–764) and metal homeostasis (Bolchi et al., 2011) are providing novel insights on the functioning of T. melanosporum during truffle formation and symbiosis. One point to always consider is that without information about the environment from which these -’omic observations are made, we can reach no meaningful conclusions regarding the relationship of these observations to the ecosystem. Contextual information, such as temperature, rainfall and pH, may help us to build up a picture of the conditions which have acted to create the transcriptome in the first place. Thus, the identification of cold-responsive genes (Zampieri et al., 2011) is providing new markers to study the truffle biology in situ.
Understanding how T. melanosporum functions and interacts in an ecosystem is vital. That issue is addressed directly in a breakthrough study of mating-type gene distribution and dynamics in a truffle ground by Rubini et al. (2011a, pp. 723–735). The observed biased distribution of mating types in a truffle ground is a factor that may severely limit truffle fructification and production, and a thorough understanding of this mechanism will probably benefit truffle ground management. Moreover, the availability of collections of several geographic accessions should allow for next-generation DNA and RNA sequencing to characterize the functional impact of genome variability. This will allow unique questions about genome organization and plasticity and gene expression, including sex-related genes.
Truffle gourmands describe the scent of the Périgord truffle as sensual, seductive and unique. As pinpointed by Splivallo et al. (pp. 688–699), these aromatic volatile organic compounds (VOCs) are not synthesized 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. VOCs also play a role in the ecology of the symbiont in controlling the interactions with the plants and rhizospheric microorganisms. Simultaneous analysis of VOCs and transcript profiles in environmental samples is currently underway and will become increasingly meaningful for understanding the synthesis of the complex cocktail of truffle aromatic volatiles.
Clearly, the analysis of the first genome sequence from an ectomycorrhizal fungus, L. bicolor (Martin et al., 2008), and the present truffle genome have brought us to a point where we are faced with opportunities to develop an 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 towards 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!