A Transcriptomic Atlas of the Ectomycorrhizal Fungus Laccaria bicolor.

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis.

Frequency of the two mating types in the soil under productive and non-productive trees in five French orchards of the Périgord black truffle (Tuber melanosporum Vittad.).

The Périgord black truffle (Tuber melanosporum Vittad.) is an ectomycorrhizal fungus forming edible fructifications. The production of T. melanosporum relies mainly on man-made plantations. T. melanosporum is a heterothallic species requiring the meeting of two partners of opposite mating types to fruit. It is common to have productive and non-productive trees in the same orchard. The aim of our study was to assess the distribution of T. melanosporum mating types in soil under productive and non-productive trees to test whether the presence or absence of one or two mating types could be an indicator of productivity. To achieve this aim, five orchards were selected in various French regions. Soils were harvested under productive and non-productive Quercus pubescens; soil characteristics and the distribution of the mating types in the soil were investigated. No significant differences between productive and non-productive soils according to soil parameters were detected. The total content of T. melanosporum DNA in the soil was significantly higher under productive trees compared with non-productive trees, and it was positively correlated only with soil available phosphorous. Under productive trees, it was more frequent to find both mating types than under non-productive trees. Soils with only one mating type were more frequent under non-productive trees than under productive ones. Moreover, no mating type was detected in the soil of 22% of the non-productive trees. These results suggest that the detection of T. melanosporum mating types in soil could be a tool to optimise the management of truffle orchards (e.g. by spore inoculation).

Soil temperature and hydric potential influences the monthly variations of soil Tuber aestivum DNA in a highly productive orchard.

Tuber aestivum, also known as the summer or Burgundy truffle, is an ectomycorrhizal Ascomycete associated with numerous trees and shrubs. Its life cycle occurs in the soil, and thus soil parameters such as temperature and water availability could influence it. T. aestivum cultivation has started in several countries, but ecological and agronomic requirements for the establishment and management of orchards are largely unknown. The aims of this work were: 1) to design a specific qPCR protocol using genomic data to trace and quantify T. aestivum DNA in the soil; and 2) to assess the monthly soil DNA dynamic according to soil parameters (i.e. soil hydric potential and temperature) in this orchard. The study was conducted in a highly productive T. aestivum orchard (hazels, oaks, pines, lime and hornbeam). The production started five years after the plantation and then increased exponentially to reach a maximum of 320 kg/ha in 2017. The soil hydric potential and temperature partially explained the monthly T. aestivum soil DNA variability. The data presented here offer new insights into T. aestivum ecology and cultivation.

Mediation of plant-mycorrhizal interaction by a lectin receptor-like kinase.

The molecular mechanisms underlying mycorrhizal symbioses, the most ubiquitous and impactful mutualistic plant-microbial interaction in nature, are largely unknown. Through genetic mapping, resequencing and molecular validation, we demonstrate that a G-type lectin receptor-like kinase (lecRLK) mediates the symbiotic interaction between Populus and the ectomycorrhizal fungus Laccaria bicolor. This finding uncovers an important molecular step in the establishment of symbiotic plant-fungal associations and provides a molecular target for engineering beneficial mycorrhizal relationships.

Identification of Populus Small RNAs Responsive to Mutualistic Interactions With Mycorrhizal Fungi, Laccaria bicolor and Rhizophagus irregularis.

Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant:microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms. In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions. We identified differential expression of sRNAs that were, to a large extent, (1) within the genomic regions lacking annotated genes in the Populus genome and (2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentially-expressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets. Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.

New insights into black truffle biology: discovery of the potential connecting structure between a Tuber aestivum ascocarp and its host root.

According to isotopic labeling experiments, most of the carbon used by truffle (Tuber sp.) fruiting bodies to develop underground is provided by host trees, suggesting that trees and truffles are physically connected. However, such physical link between trees and truffle fruiting bodies has never been observed. We discovered fruiting bodies of Tuber aestivum adhering to the walls of a belowground quarry and we took advantage of this unique situation to analyze the physical structure that supported these fruiting bodies in the open air. Observation of transversal sections of the attachment structure indicated that it was organized in ducts made of gleba-like tissue and connected to a network of hyphae traveling across soil particles. Only one mating type was detected by PCR in the gleba and in the attachment structure, suggesting that these two organs are from maternal origin, leaving open the question of the location of the opposite paternal mating type.

Influence of annual climatic variations, climate changes, and sociological factors on the production of the Périgord black truffle (Tuber melanosporum Vittad.) from 1903-1904 to 1988-1989 in the Vaucluse (France).

From 1903-1904 to 1988-1989, the two World Wars and sociological factors as rural desertification and changes in land uses mainly explained the decline of black truffle production in the Vaucluse department, which well reflects that of the whole of France. These can be correlated with the annual climatic variations as well as, from 1924-1925 to 1948-1949, the raw production rates of the managed truffle orchard of Pernes-les-Fontaines located in Vaucluse. The two methods used (correlation coefficients and Bayesian functional linear regression with Sparse Step functions) gave consistent results: the main factor explaining the annual variations of truffle production was the summer climatic water deficit of the year n. A general model including the rural exodus and the cumulated climatic water deficit of summer months both allowed to well explain the evolution of truffle production from 1903-1904 to 1988-1989 in the Vaucluse and its huge decrease. During that period, global warming had little effect. However, in the twenty-first century, all the scenarios predict increased summer water stress for the Mediterranean region, which could greatly affect black truffle production.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.

Fungi as a Source of Food.

In this article, we review some of the best-studied fungi used as food sources, in particular, the cheese fungi, the truffles, and the fungi used for drink fermentation such as beer, wine, and sake. We discuss their history of consumption by humans and the genomic mechanisms of adaptation during artificial selection.

Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies.

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small-scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small-scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two-hundred forty-one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small-scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.

Temporal changes of bacterial communities in the Tuber melanosporum ectomycorrhizosphere during ascocarp development.

Ectomycorrhizae create a multitrophic ecosystem formed by the association between tree roots, mycelium of the ectomycorrhizal fungus, and a complex microbiome. Despite their importance in the host tree's physiology and in the functioning of the ectomycorrhizal symbiosis, detailed studies on ectomycorrhiza-associated bacterial community composition and their temporal dynamics are rare. Our objective was to investigate the composition and dynamics of Tuber melanosporum ectomycorrhiza-associated bacterial communities from summer to winter seasons in a Corylus avellana tree plantation. We used 16S ribosomal RNA (rRNA)-based pyrosequencing to compare the bacterial community structure and the richness in T. melanosporum's ectomycorrhizae with those of the bulk soil. The T. melanosporum ectomycorrhizae harbored distinct bacterial communities from those of the bulk soil, with an enrichment in Alpha- and Gamma-proteobacteria. In contrast to the bacterial communities of truffle ascocarps that vastly varies in composition and richness during the maturation of the fruiting body and to those from the bulk soil, T. melanosporum ectomycorrhiza-associated bacterial community composition stayed rather stable from September to January. Our results fit with a recent finding from the same experimental site at the same period that a continuous supply of carbohydrates and nitrogen occurs from ectomycorrhizae to the fruiting bodies during the maturation of the ascocarps. We propose that this creates a stable niche in the ectomycorrhizosphere although the phenology of the tree changes.

Climatic variations explain annual fluctuations in French Périgord black truffle wholesale markets but do not explain the decrease in black truffle production over the last 48 years.

Production of the black truffle (Tuber melanosporum Vittad.) has experienced a decline in France over the last century. Different sociological factors as well as climate change have been suggested as possible explanations for this decline. The aims of this study were to assess the effects of annual climatic variations on black truffle sales by analysing reliable data. Over the past 25 years, almost 90% of French truffle sales occurred in the southeastern region of France and, despite a decrease in southwestern France, for the last 25 years, sales were stable for France as a whole. An analysis of the two main southeastern wholesale markets (Richerenches and Carpentras) revealed that the main factor explaining the huge annual variations was the cumulative hydric balance from May to August of the year n. For the first time, frost days were also identified as an important factor in Richerenches. Using the model established for the past 25 years and the climatic data for the Richerenches and Carpentras basins, the truffle sales would have been stable from 1965 to nowadays. This simulation suggested that the production decline observed since 48 years could be attributed more to the change of rural world than to the climatic changes. The stability of production or the slight increase observed during the last 25 years could reflect the input of truffle orchards recently planted.

Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles.

Although truffles are cultivated since decades, their life cycle and the conditions stimulating ascocarp formation still remain mysterious. A role for bacteria in the development of several truffle species has been suggested but few is known regarding the natural bacterial communities of Périgord Black truffle. Thus, the aim of this study was to decipher the structure and the functional potential of the bacterial communities associated to the Black truffle in the course of its life cycle and along truffle maturation. A polyphasic approach combining 454-pyrosequencing of 16S rRNA gene, TTGE, in situ hybridization and functional GeoChip 3.0 revealed that Black truffle ascocarps provide a habitat to complex bacterial communities that are clearly differentiated from those of the surrounding soil and the ectomycorrhizosphere. The composition of these communities is dynamic and evolves during the maturation of the ascocarps with an enrichment of specific taxa and a differentiation of the gleba and peridium-associated bacterial communities. Genes related to nitrogen and sulphur cycling were enriched in the ascocarps. Together, these data paint a new picture of the interactions existing between truffle and bacteria and of the potential role of these bacteria in truffle maturation.

Carbon transfer from the host to Tuber melanosporum mycorrhizas and ascocarps followed using a 13C pulse-labeling technique.

Truffles ascocarps need carbon to grow, but it is not known whether this carbon comes directly from the tree (heterotrophy) or from soil organic matter (saprotrophy). The objective of this work was to investigate the heterotrophic side of the ascocarp nutrition by assessing the allocation of carbon by the host to Tuber melanosporum mycorrhizas and ascocarps. In 2010, a single hazel tree selected for its high truffle (Tuber melanosporum) production and situated in the west part of the Vosges, France, was labeled with (13)CO2. The transfer of (13)C from the leaves to the fine roots and T. melanosporum mycorrhizas was very slow compared with the results found in the literature for herbaceous plants or other tree species. The fine roots primarily acted as a carbon conduit; they accumulated little (13)C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated (13)C prior to ascocarp development. Then, the mycorrhizas transferred (13)C to the ascocarps to provide constitutive carbon (1.7 mg of (13)C per day). The ascocarps accumulated host carbon until reaching complete maturity, 200 days after the first labeling and 150 days after the second labeling event. This role of the Tuber ascocarps as a carbon sink occurred several months after the end of carbon assimilation by the host and at low temperature. This finding suggests that carbon allocated to the ascocarps during winter was provided by reserve compounds stored in the wood and hydrolyzed during a period of frost. Almost all of the constitutive carbon allocated to the truffles (1% of the total carbon assimilated by the tree during the growing season) came from the host.

Fine-scale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes.

The genetic structure of ectomycorrhizal (ECM) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies. Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas. A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types. Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards.

Characterization of transposable elements in the ectomycorrhizal fungus Laccaria bicolor.

BACKGROUND: The publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TE-specific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome. METHODOLOGY/PRINCIPAL FINDINGS: TEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copy elements distributed within 171 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs exhibits signs of ancient transposition except some intact copies of terminal inverted repeats (TIRS), long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TE expansion in L. bicolor: the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 0.5 Mya ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea. CONCLUSIONS: This analysis 1) represents an initial characterization of TEs in the L. bicolor genome, 2) contributes to improve genome annotation and a greater understanding of the role TEs played in genome organization and evolution and 3) provides a valuable resource for future research on the genome evolution within the Laccaria genus.

Survey and analysis of simple sequence repeats in the Laccaria bicolor genome, with development of microsatellite markers.

It is becoming clear that simple sequence repeats (SSRs) play a significant role in fungal genome organization, and they are a large source of genetic markers for population genetics and meiotic maps. We identified SSRs in the Laccaria bicolor genome by in silico survey and analyzed their distribution in the different genomic regions. We also compared the abundance and distribution of SSRs in L. bicolor with those of the following fungal genomes: Phanerochaete chrysosporium, Coprinopsis cinerea, Ustilago maydis, Cryptococcus neoformans, Aspergillus nidulans, Magnaporthe grisea, Neurospora crassa and Saccharomyces cerevisiae. Using the MISA computer program, we detected 277,062 SSRs in the L. bicolor genome representing 8% of the assembled genomic sequence. Among the analyzed basidiomycetes, L. bicolor exhibited the highest SSR density although no correlation between relative abundance and the genome sizes was observed. In most genomes the short motifs (mono- to trinucleotides) were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. In the L. bicolor genome, most of the SSRs were located in intergenic regions (73.3%) and the highest SSR density was observed in transposable elements (TEs; 6,706 SSRs/Mb). However, 81% of the protein-coding genes contained SSRs in their exons, suggesting that SSR polymorphism may alter gene phenotypes. Within a L. bicolor offspring, sequence polymorphism of 78 SSRs was mainly detected in non-TE intergenic regions. Unlike previously developed microsatellite markers, these new ones are spread throughout the genome; these markers could have immediate applications in population genetics.

Gene organization of the mating type regions in the ectomycorrhizal fungus Laccaria bicolor reveals distinct evolution between the two mating type loci.

In natural conditions, basidiomycete ectomycorrhizal fungi such as Laccaria bicolor are typically in the dikaryotic state when forming symbioses with trees, meaning that two genetically different individuals have to fuse or 'mate'. Nevertheless, nothing is known about the molecular mechanisms of mating in these ecologically important fungi. Here, advantage was taken of the first sequenced genome of the ectomycorrhizal fungus, Laccaria bicolor, to determine the genes that govern the establishment of cell-type identity and orchestrate mating. The L. bicolor mating type loci were identified through genomic screening. The evolutionary history of the genomic regions that contained them was determined by genome-wide comparison of L. bicolor sequences with those of known tetrapolar and bipolar basidiomycete species, and by phylogenetic reconstruction of gene family history. It is shown that the genes of the two mating type loci, A and B, are conserved across the Agaricales, but they are contained in regions of the genome with different evolutionary histories. The A locus is in a region where the gene order is under strong selection across the Agaricales. By contrast, the B locus is in a region where the gene order is likely under a low selection pressure but where gene duplication, translocation and transposon insertion are frequent.

Phylogenetic and populational study of the Tuber indicum complex.

When examined using SEM, Chinese samples of Tuber indicum and T. sinense displayed the same ascospore ornamentation as that of T. pseudohimalayense, T. indicum, collected in India by Duthie in 1899, and samples renamed T. himalayense in 1988. The different authors who named the four taxa (T. indicum, T. himalayense, T. sinense, T. pseudohimalyense) described differences in the surface of the peridium which could be considered as usual variations within a single species. We consider T. indicum, T. himalayense, T. sinense and T. pseudohimalayense as one species, T. indicum. Within this T. indicum complex, according to ITS and beta-tubulin sequences, there are two groups in China, which could be considered as geographical ecotypes. This study is the first to identify a genetic and phylogeographical structure within the Chinese Tuber species.

Phylogenetic relationships between Tuber pseudoexcavatum, a Chinese truffle, and other Tuber species based on parsimony and distance analysis of four different gene sequences.

Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, beta-tubulin, protein kinase C and elongation factor 1alpha. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.