Contrasting demographic histories revealed in two invasive populations of the dry rot fungus Serpula lacrymans.

Globalization and international trade have impacted organisms around the world leading to a considerable number of species establishing in new geographic areas. Many organisms have taken advantage of human-made environments, including buildings. One such species is the dry rot fungus Serpula lacrymans, which is the most aggressive wood-decay fungus in indoor environments in temperate regions. Using population genomic analyses of 36 full genome sequenced isolates, we demonstrated that European and Japanese isolates are highly divergent and the populations split 3000-19,000 generations ago, probably predating human influence. Approximately 250 generations ago, the European population went through a tight bottleneck, probably corresponding to the fungus colonization of the built environment in Europe. The demographic history of these populations, probably lead to low adaptive potential. Only two loci under selection were identified using a Fst outlier approach, and selective sweep analyses identified three loci with extended haplotype homozygosity. The selective sweep analyses found signals in genes possibly related to decay of various substrates in Japan and in genes involved DNA replication and protein modification in Europe. Our results suggest that the dry rot fungus independently established in indoor environments in Europe and Japan and that invasive species can potentially establish large populations in new habitats based on a few colonizing individuals.

First production of Italian white truffle (Tuber magnatum Pico) ascocarps in an orchard outside its natural range distribution in France.

Truffles are ectomycorrhizal species forming edible ascocarps. The Italian white truffle (Tuber magnatum Pico) is the most famous and expensive species harvested to date; it comes exclusively from natural habitats in European countries. The annual production of T. magnatum is generally insufficient to respond to the high demands making its cultivation a research hotspot. The first attempt to cultivate T. magnatum started in the 1970s without success; only recently have mycorrhized plants been successfully produced. The aims of this study were (1) to assess the persistence of T. magnatum in the soil of plantations realized with mycorrhized plants and (2) to characterize the first T. magnatum orchard that produced ascocarps outside the known natural geographic range of this species. In 2018, five orchards were sampled in France, and T. magnatum was investigated in the soil. We confirmed that T. magnatum survived in the soil 3 to 8 years after planting. The key finding of this study was the harvest of T. magnatum ascocarps in 2019 and 2020 from one orchard. The production of ascocarps started 4.5 years after planting, and the ascocarps were harvested under different trees and during two consecutive seasons. A detailed analysis of the productive orchards (i.e., soil features, soil water availability, cultivation techniques) is presented. These results demonstrate the feasibility of T. magnatum cultivation worldwide by planting mycorrhized plants. The cultivation of T. magnatum could therefore become a real opportunity for farmers and could respond to the high demand of this high-priced food.

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).

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants.

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.

Two ectomycorrhizal truffles, Tuber melanosporum and T. aestivum, endophytically colonise roots of non-ectomycorrhizal plants in natural environments.

Serendipitous findings and studies on Tuber species suggest that some ectomycorrhizal fungi, beyond their complex interaction with ectomycorrhizal hosts, also colonise roots of nonectomycorrhizal plants in a loose way called endophytism. Here, we investigate endophytism of T. melanosporum and T. aestivum. We visualised endophytic T. melanosporum hyphae by fluorescent in situ hybridisation on nonectomycorrhizal plants. For the two Tuber species, microsatellite genotyping investigated the endophytic presence of the individuals whose mating produced nearby ascocarps. We quantified the expression of four T. aestivum genes in roots of endophyted, non-ectomycorrhizal plants. Tuber melanosporum hyphae colonised the apoplast of healthy roots, confirming endophytism. Endophytic Tuber melanosporum and T. aestivum contributed to nearby ascocarps, but only as maternal parents (forming the flesh). Paternal individuals (giving only genes found in meiotic spores of ascocarps) were not detected. Gene expression of T. aestivum in non-ectomycorrhizal plants confirmed a living status. Tuber species, and likely other ectomycorrhizal fungi found in nonectomycorrhizal plant roots in this study, can be root endophytes. This is relevant for the ecology (brûlé formation) and commercial production of truffles. Evolutionarily speaking, endophytism may be an ancestral trait in some ectomycorrhizal fungi that evolved from root endophytes.

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.

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.

Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists.

Some soil fungi in the Leotiomycetes form ericoid mycorrhizal (ERM) symbioses with Ericaceae. In the harsh habitats in which they occur, ERM plant survival relies on nutrient mobilization from soil organic matter (SOM) by their fungal partners. The characterization of the fungal genetic machinery underpinning both the symbiotic lifestyle and SOM degradation is needed to understand ERM symbiosis functioning and evolution, and its impact on soil carbon (C) turnover. We sequenced the genomes of the ERM fungi Meliniomyces bicolor, M. variabilis, Oidiodendron maius and Rhizoscyphus ericae, and compared their gene repertoires with those of fungi with different lifestyles (ecto- and orchid mycorrhiza, endophytes, saprotrophs, pathogens). We also identified fungal transcripts induced in symbiosis. The ERM fungal gene contents for polysaccharide-degrading enzymes, lipases, proteases and enzymes involved in secondary metabolism are closer to those of saprotrophs and pathogens than to those of ectomycorrhizal symbionts. The fungal genes most highly upregulated in symbiosis are those coding for fungal and plant cell wall-degrading enzymes (CWDEs), lipases, proteases, transporters and mycorrhiza-induced small secreted proteins (MiSSPs). The ERM fungal gene repertoire reveals a capacity for a dual saprotrophic and biotrophic lifestyle. This may reflect an incomplete transition from saprotrophy to the mycorrhizal habit, or a versatile life strategy similar to fungal endophytes.

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.

Diversity and Structure of Fungal Communities in Neotropical Rainforest Soils: The Effect of Host Recurrence.

The patterns of the distribution of fungal species and their potential interactions with trees remain understudied in Neotropical rainforests, which harbor more than 16,000 tree species, mostly dominated by endomycorrhizal trees. Our hypothesis was that tree species shape the non-mycorrhizal fungal assemblages in soil and litter and that the diversity of fungal communities in these two compartments is partly dependent on the coverage of trees in the Neotropical rainforest. In French Guiana, a long-term plantation and a natural forest were selected to test this hypothesis. Fungal ITS1 regions were sequenced from soil and litter samples from within the vicinity of tree species. A broad range of fungal taxa was found, with 42 orders and 14 classes. Significant spatial heterogeneity in the fungal communities was found without strong variation in the species richness and evenness among the tree plots. However, tree species shaped the fungal assemblages in the soil and litter, explaining up to 18 % of the variation among the communities in the natural forest. These results demonstrate that vegetation cover has an important effect on the structure of fungal assemblages inhabiting the soil and litter in Amazonian forests, illustrating the relative impact of deterministic processes on fungal community structures in these highly diverse ecosystems.

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis.

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Reconstructing the evolutionary history of gypsy retrotransposons in the Périgord black truffle (Tuber melanosporum Vittad.).

Truffles are ascomycete fungi belonging to genus Tuber, and they form ectomycorrhizal associations with trees and shrubs. Transposable elements constitute more than 50 % of the black Périgord truffle (Tuber melanosporum) genome, which are mainly class 1 gypsy retrotransposons, but their impact on its genome is unknown. The aims of this study are to investigate the diversity of gypsy retrotransposons in this species and their evolutionary history by analysing the reference genome and six resequenced genomes of different geographic accessions. Using the reverse transcriptase sequences, six different gypsy retrotransposon clades were identified. Tmt1 and Tmt6 are the most abundant transposable elements, representing 14 and 13 % of the T. melanosporum genome, respectively. Tmt6 showed a major burst of proliferation between 1 and 4 million years ago, but evidence of more recent transposition was observed. Except for Tmt2, the other clades tend to aggregate, and their mode of transposition excluded the master copy model. This suggests that each new copy has the same probability of transposing as other copies. This study provides a better view of the diversity and dynamic nature of gypsy retrotransposons in T. melanosporum. Even if the major gypsy retrotransposon bursts are old, some elements seem to have transposed recently, suggesting that they may continue to model the truffle genomes.

Fine-scale genetic structure of natural Tuber aestivum sites in southern Germany.

Although the Burgundy truffle (Tuber aestivum) is an ectomycorrhizal fungus of important economic value, its subterranean life cycle and population biology are still poorly understood. Here, we determine mating type and simple sequence repeat (SSR) maternal genotypes of mapped fruiting bodies to assess their genetic structure within two naturally colonized forest sites in southern Germany. Forty-one genotypes were identified from 112 fruiting bodies. According to their mating types, the maternal genotypes were aggregated only in one population. Genotypic diversity of individuals that mostly were small and occurred in 1 out of 2 years of sampling was high. Although these results suggested a ruderal colonization strategy, some genets spread several hundred meters. This result indicates that, besides sexual spore dispersal, vegetative growth or spreading by mycelial propagules contributes to dissemination. In one site, fewer individuals with a tendency to expand genets belonging to only one genetic group were observed. In the second site, numerous small individuals were found and were grouped into two clearly differentiated genetic groups that were spatially intermingled. Forest characteristics and disturbances are possible reasons for the observed genetic patterns. Our findings contribute to a better understanding of the biology of one of the most widespread and commercially important truffle species. This knowledge is critical for establishing and maintaining sustainable long-term truffle cultivations.

SSR-based identification of genetic groups within European populations of Tuber aestivum Vittad.

Tuber species are ectomycorrhizal ascomycetes establishing relationships with different host trees and forming hypogeous fruiting bodies known as truffles. Among Tuber species, Tuber aestivum Vittad. has a wide distributional range being found naturally all over Europe. Here, we performed large-scale population genetic analyses in T. aestivum to (i) investigate its genetic diversity at the European scale, (ii) characterize its genetic structure and test for the presence of ecotypes and (iii) shed light into its demographic history. To reach these goals, 230 ascocarps from different populations were genotyped using 15 polymorphic simple sequence repeat markers. We identified 181 multilocus genotypes and four genetic groups which did not show a clear geographical separation; although, one of them was present exclusively in Southeast France, Italy and Spain. Fixation index values between pairs of genetic groups were generally high and ranged from 0.29 to 0.45. A significant deficit of heterozygosity indicated a population expansion instead of a recent population bottleneck, suggesting that T. aestivum is not endangered in Europe, not even in Mediterranean regions. Our study based on a large-scale population genetic analysis suggests that genetically distinct populations and likely ecotypes within T. aestivum are present. In turn, this study paves the way to future investigations aimed at addressing the biological and/or ecological factors that have concurred in shaping the population genetic structure of this species. Present results should also have implications for the truffle market since defining genetic markers are now possible at least for some specific T. aestivum genetic groups.

Fine-scale spatial genetic structure analysis of the black truffle Tuber aestivum and its link to aroma variability.

Truffles are symbiotic fungi in high demand by food connoisseurs. Improving yield and product quality requires a better understanding of truffle genetics and aroma biosynthesis. One aim here was to investigate the diversity and fine-scale spatial genetic structure of the Burgundy truffle Tuber aestivum. The second aim was to assess how genetic structuring along with fruiting body maturation and geographical origin influenced single constituents of truffle aroma. A total of 39 Burgundy truffles collected in two orchards were characterized in terms of aroma profile (SPME-GC/MS) and genotype (microsatellites). A moderate genetic differentiation was observed between the populations of the two orchards. An important seasonal and spatial genetic structuring was detected. Within one orchard, individuals belonging to the same genet were generally collected during a single season and in the close vicinity from each other. Maximum genet size nevertheless ranged from 46 to 92 m. Geographical origin or maturity only had minor effects on aroma profiles but genetic structuring, specifically clonal identity, had a pronounced influence on the concentrations of C8 - and C4 -VOCs. Our results highlight a high seasonal genetic turnover and indicate that the aroma of Burgundy truffle is influenced by the identity of single clones/genets.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and ß-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Forty years of inoculating seedlings with truffle fungi: past and future perspectives.

The first commercialization of seedlings inoculated with truffle fungi occurred in 1973. Over the last 40 years, considerable progress has been made relative to quality control for inoculated seedlings. A recently published paper by Andrés-Alpuente and colleagues (Mycorrhiza 24:29-37, 2014) reviewed and tested the different methods of mycorrhization assessment currently used in Europe. The aim of this paper is to augment their findings by adding information to the discussion about the most important steps in seedling quality control. Additionally, the history of seedlings inoculated with truffles, procedures currently used in France for seedling control quality, and a reflection on future research aimed at increasing truffle production will be presented.

The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.

BACKGROUND: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. RESULTS: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. CONCLUSIONS: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant-fungus relationship.

Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.