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Summary: Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown.By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER‐LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes.The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐dml and RNAi‐ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis).Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism. See also the Commentary on this article by Zimmermann & Gaillard, 238: 2259–2260. [ABSTRACT FROM AUTHOR]

Summary: Ectomycorrhizal (EcM) fungi play a crucial role in the mineral nitrogen (N) nutrition of their host trees. While it has been proposed that several EcM species also mobilize organic N, studies reporting the EcM ability to degrade N‐containing polymers, such as chitin, remain scarce.Here, we assessed the capacity of a representative collection of 16 EcM species to acquire 15N from 15N‐chitin. In addition, we combined genomics and transcriptomics to identify pathways involved in exogenous chitin degradation between these fungal strains.Boletus edulis, Imleria badia, Suillus luteus, and Hebeloma cylindrosporum efficiently mobilized N from exogenous chitin. EcM genomes primarily contained genes encoding for the direct hydrolysis of chitin. Further, we found a significant relationship between the capacity of EcM fungi to assimilate organic N from chitin and their genomic and transcriptomic potentials for chitin degradation.These findings demonstrate that certain EcM fungal species depolymerize chitin using hydrolytic mechanisms and that endochitinases, but not exochitinases, represent the enzymatic bottleneck of chitin degradation. Finally, this study shows that the degradation of exogenous chitin by EcM fungi might be a key functional trait of nutrient cycling in forests dominated by EcM fungi. [ABSTRACT FROM AUTHOR]

Summary: Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra‐species variation between four isolates of the ECM fungus Pisolithus microcarpus, in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis. We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate‐specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions. [ABSTRACT FROM AUTHOR]

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremulax alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography–tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone‐treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid‐stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development. The ectomycorrhizal fungus Laccaria bicolor affects sensitivity to jasmonate in roots of Populus tremulax alba, while promoting responses to salicylate, gibberellin, and ethylene. Adjusted accumulation and perception of these phytohormones is necessary for the development of ectomycorrhizae. [ABSTRACT FROM AUTHOR]

Summary: Microbial communities interplay with their environment through their functional traits that can be a response or an effect on the environment. Here, we explore how a functional trait—the decomposition of organic matter, can be addressed based on genetic markers and how the expression of these markers reflect ecological strategies of two fungal litter decomposer Gymnopus androsaceus and Chalara longipes. We sequenced the genomes of these two fungi, as well as their transcriptomes at different steps of Pinus sylvestris needles decomposition in microcosms. Our results highlighted that if the gene content of the two species could indicate similar potential decomposition abilities, the expression levels of specific gene families belonging to the glycoside hydrolase category reflected contrasting ecological strategies. Actually, C. longipes, the weaker decomposer in this experiment, turned out to have a high content of genes involved in cell wall polysaccharides decomposition but low expression levels, reflecting a versatile ecology compare to the more competitive G. androsaceus with high expression levels of keystone functional genes. Thus, we established that sequential expression of genes coding for different components of the decomposer machinery indicated adaptation to chemical changes in the substrate as decomposition progressed. [ABSTRACT FROM AUTHOR]

Summary: The black morel (Morchella importuna Kuo, O'Donnell and Volk) was once an uncultivable wild mushroom, until the development of exogenous nutrient bag (ENB), making its agricultural production quite feasible and stable. To date, how the nutritional acquisition of the morel mycelium is fulfilled to trigger its fruiting remains unknown. To investigate the mechanisms involved in ENB decomposition, the genome of a cultivable morel strain (M. importuna SCYDJ1‐A1) was sequenced and the genes coding for the decay apparatus were identified. Expression of the encoded carbohydrate‐active enzymes (CAZymes) was then analyzed by metatranscriptomics and metaproteomics in combination with biochemical assays. The results show that a diverse set of hydrolytic and redox CAZymes secreted by the morel mycelium is the main force driving the substrate decomposition. Plant polysaccharides such as starch and cellulose present in ENB substrate (wheat grains plus rice husks) were rapidly degraded, whereas triglycerides were accumulated initially and consumed later. ENB decomposition led to a rapid increase in the organic carbon content in the surface soil of the mushroom bed, which was thereafter consumed during morel fruiting. In contrast to the high carbon consumption, no significant acquisition of nitrogen was observed. Our findings contribute to an increasingly detailed portrait of molecular features triggering morel fruiting. [ABSTRACT FROM AUTHOR]

Nutrient transfer is a key feature of the arbuscular mycorrhizal (AM) symbiosis. Valuable mineral nutrients are transferred from the AM fungus to the plant, increasing its fitness and productivity, and, in exchange, the AM fungus receives carbohydrates as an energy source from the plant. Here, we analyzed the transcriptome of the Populus trichocarpa- Rhizophagus irregularis symbiosis using RNA-sequencing of non-mycorrhizal or mycorrhizal fine roots, with a focus on the effect of nitrogen (N) starvation. In R. irregularis, we identified 1,015 differentially expressed genes, whereby N starvation led to a general induction of gene expression. Genes of the functional classes of cell growth, membrane biogenesis and cell structural components were highly abundant. Interestingly, N starvation also led to a general induction of fungal transporters, indicating increased nutrient demand upon N starvation. In non-mycorrhizal P. trichocarpa roots, 1,341 genes were differentially expressed under N starvation. Among the 953 down-regulated genes in N starvation, most were involved in metabolic processes including amino acids, carbohydrate and inorganic ion transport, while the 342 up-regulated genes included many defense-related genes. Mycorrhization led to the upregulation of 549 genes mainly involved in secondary metabolite biosynthesis and transport; only 24 genes were down-regulated. Mycorrhization specifically induced expression of three ammonium transporters and one phosphate transporter, independently of the N conditions, corroborating the hypothesis that these transporters are important for symbiotic nutrient exchange. In conclusion, our data establish a framework of gene expression in the two symbiotic partners under high-N and low-N conditions. [ABSTRACT FROM AUTHOR]

To clarify the early molecular interaction between ectomycorrhizal partners, we performed a RNA-Seq study of transcriptome reprogramming of the basidiomycete Hebeloma cylindrosporum before symbiotic structure differentiation with Pinus pinaster. Mycorrhiza transcriptome was studied for comparison. By reference to asymbiotic mycelium, 47 and 46 genes were specifically upregulated over fivefold ( p ≤ 0.05) upon rhizosphere colonization and root adhesion respectively. Other 45 were upregulated throughout the symbiotic interaction, from rhizosphere colonization to differentiated mycorrhizas, whereas 274 were specifically upregulated in mycorrhizas. Although exoproteome represents 5.6% of H. cylindrosporum proteome, 38.5% of the genes upregulated upon pre-infectious root colonization encoded extracellular proteins. The proportion decreased to 23.5% in mycorrhizas. At all studied time points, mycorrhiza-induced small secreted proteins (MiSSPs), representing potential effectors, were over-represented among upregulated genes. This was also the case for carbohydrate-active enzymes (CAZymes). Several CAZymes were upregulated at all studied stages of the interaction. Consistent with a role in fungal morphogenesis and symbiotic interface differentiation, CAZymes over-expressed before and upon root attachment targeted fungal and both fungal and plant polysaccharides respectively. Different hydrophobins were upregulated upon early root adhesion, in mycorrhizas or throughout interaction. The functional classification of genes upregulated only in mycorrhizas pointed to intense metabolic activity and nutritional exchanges. [ABSTRACT FROM AUTHOR]

Background: Transporter proteins are predicted to have an important role in the mycorrhizal symbiosis, due to the fact that this type of an interaction between plants and fungi requires a continuous nutrient and signalling exchange. ABC transporters are one of the large groups of transporter proteins found both in plants and in fungi. The crucial role of plant ABC transporters in the formation of the mycorrhizal symbiosis has been demonstrated recently. Some of the fungal ABC transporter-encoding genes are also induced during the mycorrhiza formation. However, no experimental evidences of the direct involvement of fungal ABC transporters in this process are available so far. To facilitate the identification of fungal ABC proteins with a potential role in the establishment of the mycorrhizal symbiosis, we have performed an inventory of the ABC protein-encoding genes in the genomes of 25 species of mycorrhiza-forming fungi. Results: We have identified, manually annotated and curated more than 1300 gene models of putative ABC protein-encoding genes. Out of those, more than 1000 models are predicted to encode functional proteins, whereas about 300 models represent gene fragments or putative pseudogenes. We have also performed the phylogenetic analysis of the identified sequences. The sets of ABC proteins in the mycorrhiza-forming species were compared to the related saprotrophic or plant-pathogenic fungal species. Our results demonstrate the high diversity of ABC genes in the genomes of mycorrhiza-forming fungi. Via comparison of transcriptomics data from different species, we have identified candidate groups of ABC transporters that might have a role in the process of the mycorrhiza formation. Conclusions: Results of our inventory will facilitate the identification of fungal transporters with a role in the mycorrhiza formation. We also provide the first data on ABC protein-coding genes for the phylum Glomeromycota and for orders Pezizales, Atheliales, Cantharellales and Sebacinales, contributing to the better knowledge of the diversity of this protein family within the fungal kingdom. [ABSTRACT FROM AUTHOR]

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of E laphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the E laphomyces lineage is most closely related to T alaromyces and Trichocomaceae s.s. The genome of E . granulatus is reduced in carbohydrate-active enzymes, despite a large expansion in genome size, both of which are consistent with what is seen in T uber melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E . granulatus genome, especially Gypsy-like long terminal repeats, and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue, this study has provided insights into E laphomyces phylogenetics, genomics, metagenomics and the evolution of the ectomycorrhizal association. [ABSTRACT FROM AUTHOR]

Using the newly available genome for Eucalyptus grandis, we sought to determine the genome-wide traits that enable this host to form mutualistic interactions with ectomycorrhizal ( ECM) Pisolithus sp. and to determine how future predicted concentrations of atmospheric carbon dioxide ( CO2) will affect this relationship., We analyzed the physiological and transcriptomic responses of E. grandis during colonization by different Pisolithus sp. isolates under conditions of ambient (400 ppm) and elevated (650 ppm) CO2 to tease out the gene expression profiles associated with colonization status., We demonstrate that E. grandis varies in its susceptibility to colonization by different Pisolithus isolates in a manner that is not predictable by geographic origin or the internal transcribed spacer ( ITS)-based phylogeny of the fungal partner. Elevated concentrations of CO2 alter the receptivity of E. grandis to Pisolithus, a change that is correlated to a dramatic shift in the transcriptomic profile of the root., These data provide a starting point for understanding how future environmental change may alter the signaling between plants and their ECM partners and is a step towards determining the mechanism behind previously observed shifts in Eucalypt-associated fungal communities exposed to elevated concentrations of atmospheric CO2. [ABSTRACT FROM AUTHOR]

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes. [ABSTRACT FROM AUTHOR]

The transcriptome is defined as the sum of all transcripts in a given cell, tissue or organ and can be assessed by different approaches. The most recent ones, microarrays and RNA sequencing, are compared in this chapter with a focus on plant-microbe interactions. The advantages and the specific problems to monitor both partners at the same time by sequencing are discussed. We show that microdissection in combination with transcriptomics is a powerful tool to study interactions. Further, we present an overview of bioinformatic approaches and available algorithms to process RNA-Seq data. Finally, we summarize the transcriptome analyses from mycorrhizal fungi in the genome era, in particular Laccaria bicolor, Tuber melanosporum, Rhizophagus irregularis and Paxillus involutus. [ABSTRACT FROM AUTHOR]

Phlebiopsis gigantea has been routinely used as the biological control agent for the conifer pathogen Heterobasidion annosum sensu lato, but the actual mechanism for the biocontrol process is not known. To investigate the effect of secreted molecules from culture filtrate produced by P. gigantea on the gene expression profile of H. annosum s.s., microarray analysis was used. Analysis of the differentially expressed genes led to the identification of genes with diverse functions. A major proportion of the up- and downregulated genes were either uncharacterized or genes whose functions were not known. A number of genes coding for proteins involved in metabolism, transport, and signal transduction were differentially downregulated; comparatively lower number of such genes were upregulated. Some genes involved in transport (polyamine transporters, 2573-fold, P = 0.002) and metabolism (endoglucanase, 622.5-fold, P = 0.002, cytochrome P450, 133.2-fold, P = 0.05) showed high transcript fold changes and were statistically significantly upregulated. Genes encoding defense-related proteins such as hydrophobins were either downregulated or expressed at relatively low levels. Further analysis of the effect of the culture filtrate on glucose metabolism showed downregulation of some key enzymes at the early stage of the glycolytic pathway while some genes were upregulated at the later stage of the pathway. A subset of the genes were selected and used to validate the micro-array result by quantitative real time polymerase chain reaction (qPCR) method. Generally, the high transcript levels of genes encoding several biochemically important genes (protein kinases, major facilitator superfamily polyamine transporters, endoglucanase, cytochrome P450, endoglucanase) suggests their potential functional relevance in signal perception, stress tolerance, cell defenses, and detoxification of toxic molecules during competitive interaction. These results have provided further insights into possible molecular and genetic factors underlying the response of H. annosum to metabolites from P. gigantea during interspecific interaction. [ABSTRACT FROM AUTHOR]

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 ∼125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼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. [ABSTRACT FROM AUTHOR]

The early phase of the interaction between tree roots and ectomycorrhizal fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We aimed to identify gene networks that regulate LR development during the early signal exchanges between poplar (Populus tremula × Populus alba) and the ectomycorrhizal fungus Laccaria bicolor with a focus on auxin transport and signaling pathways. Our data demonstrated that increased LR development in poplar and Arabidopsis (Arabidopsis thaliana) interacting with L. bicolor is not dependent on the ability of the plant to form ectomycorrhizae. LR stimulation paralleled an increase in auxin accumulation at root apices. Blocking plant polar auxin transport with 1-naphthylphthalamic acid inhibited LR development and auxin accumulation. An oligoarray-based transcript profile of poplar roots exposed to molecules released by L. bicolor revealed the differential expression of 2,945 genes, including several components of polar auxin transport (PtaPIN and PtaAUX genes), auxin conjugation (PtaGH3 genes), and auxin signaling (PtaIAA genes). Transcripts of PtaPIN9, the homolog of Arabidopsis AtPIN2, and several PtaIAAs accumulated specifically during the early interaction phase. Expression of these rapidly induced genes was repressed by 1-naphthylphthalamic acid. Accordingly, LR stimulation upon contact with L. bicolor in Arabidopsis transgenic plants defectiv in homologs of these genes was decreased or absent. Furthermore, in Arabidopsis pin2, the root apical auxin increase during contact with the fungus was modified. We propose a model in which fungus-induced auxin accumulation at the root apex stimulates LR formation through a mechanism involving PtaPIN9-dependent auxin redistribution together with PtaIAA-based auxin signaling. [ABSTRACT FROM AUTHOR]

• The ectomycorrhizal fungus Laccaria bicolor has the largest genome of all fungi yet sequenced. The large genome size is partly a result of an expansion of gene family sizes. Among the largest gene families are protein kinases and RAS small guanosine triphosphatases (GTPases), which are key components of signal transduction pathways. • Comparative genomics and phylogenetic analyses were used to examine the evolution of the two largest families of protein kinases and RAS small GTPases in L. bicolor. Expression levels in various tissues and growth conditions were inferred from microarray data. • The two families possessed a large number of young duplicates (paralogs) that had arisen in the Laccaria lineage following the separation from the saprophyte Coprinopsis cinerea. The protein kinase paralogs were dispersed in many small clades and the majority were pseudogenes. By contrast, the RAS paralogs were found in three large groups of RAS1-, RAS2- and RHO1-like GTPases with few pseudogenes. • Duplicates of protein kinases and RAS small GTPase have either retained, gained or lost motifs found in the coding regions of their ancestors. Frequent outcomes during evolution were the formation of pseudogenes (nonfunctionalization) or proteins with novel structures and expression patterns (neofunctionalization). [ABSTRACT FROM AUTHOR]

• Sequencing of the Laccaria bicolor and Phanerochaete chrysosporium genomes, together with the availability of many fungal genomes, allow careful comparison to be made of these two basidiomycetes, which possess a different way of life (either symbiotic or saprophytic), with other fungi. Central to the antioxidant systems are superoxide dismutases, catalases and thiol-dependent peroxidases (Tpx). The two reducing systems (thioredoxin (Trx) and glutathione/glutaredoxin (Grx)) are of particular importance against oxidative insults, both for detoxification, through the regeneration of thiol-peroxidases, and for developmental, physiological and signalling processes. Among those thiol-dependent antioxidant systems, special emphasis is given to the redoxin and methionine sulfoxide reductase (Msr) multigenic families. • The genes coding for these enzymes were identified in the L. bicolor and P. chrysosporium genomes, were correctly annotated, and the gene content, organization and distribution were compared with other fungi. Expression of the Laccaria genes was also compiled from microarray data. • A complete classification, based essentially on gene structure, on phylogenetic and sequence analysis, and on existing experimental data, was proposed. • Comparison of the gene content of fungi from all phyla did not show huge differences for multigenic families in the reactive oxygen species (ROS) detoxification network, although some protein subgroups were absent in some fungi. [ABSTRACT FROM AUTHOR]

To understand key processes governing defense mechanisms in poplar (Populus spp.) upon infection with the rust fungus Me/am psora larici-populina, we used combined histological and molecular techniques to describe the infection of Papulus trichocarpa X Populus deltoides 'Beaupré' leaves by compatible and incompatible fungal strains. Striking differences in host-tissue infection were observed after 48-h postinoculation (hpi) between compatible and incompatible interactions. No reactive oxygen species production could be detected at infection sites, while a strong accumulation of monolignols occurred in the incompatible interaction after 48 hpi, indicating a late plant response once the fungus already penetrated host cells to form haustorial infection structures. P. trichocarpa whole-genome expression oligoarrays and sequencing of cDNAs were used to determine changes in gene expression in both interactions at 48 hpi. Temporal expression profiling of infection-regulated transcripts was further compared by cDNA arrays and reverse transcription-quantitative polymerase chain reaction. Among 1,730 significantly differentially expressed transcripts in the incompatible interaction, 150 showed an increase in concentration ≥3-fold, whereas 62 were decreased by ≥3-fold. Regulated transcripts corresponded to known genes targeted by R genes in plant pathosystems, such as inositol-3-P synthase, glutathione S-transferases, and pathogenesis-related proteins. However, the transcript showing the highest rust-induced up-regulation encodes a putative secreted protein with no known function. In contrast, only a few transcripts showed an altered expression in the compatible interaction, suggesting a delay in defense response between incompatible and compatible interactions in poplar. This comprehensive analysis of early molecular responses of poplar to M. larici-populina infection identified key genes that likely contain the fungus proliferation in planta. [ABSTRACT FROM AUTHOR]

The ectomycorrhizal symbiosis involving trees and soil fungi is a process of major ecological importance in forest ecosystems. The establishment of an effective symbiosis encompasses a series of complex and overlapping developmental processes in the colonizing mycelium and roots of host trees. Regulated gene expression is an important mechanism for controlling ectomycorrhizal symbiosis development and functioning. Gene profiling studies led to the identification of genes that are required for fungal attachment, plant defense, and symbiosis-related metabolism. They showed that changes in morphology associated with mycorrhizal development were accompanied by changes in transcript patterns, but no ectomycorrhiza-specific genes were detected. Comparison of the genomes of pathogenic and saprobic fungi with the recently released ectomycorrhizal Laccaria genome is providing crucial insights into the genetic makeup of plant–fungus interactions. [Copyright &y& Elsevier]

• In an effort to understand processes that are related to heavy metal sequestration, we characterized six metallothionein genes ( PtdMTs) in the hybrid cottonwood, Populus trichocarpa ×  deltoides. • cDNA microarrays and reverse transcriptase-polymerase chain reaction were used to examine PtdMT expression in poplar tissues. They displayed differential gene expression patterns, which may be associated with the diverse roles and functions PtdMTs have in coping with particular developmental (e.g. root development and leaf senescence) and environmental cues. • The heterologous expression in a cadmium (Cd)-hypersensitive yeast mutant showed the ability of PtdMT cDNAs to confer Cd tolerance. The concentration of PtdMT mRNAs were increased by zinc, but not by copper and Cd. • Further studies will help to clarify the role of metallothionein genes in metal homeostasis and poplar development, and help to isolate poplar genotypes particularly tolerant to stress for use in experiments of phytoremediation. New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01168.x © New Phytologist (2004) [ABSTRACT FROM AUTHOR]

Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. Here, we describe the identification, characterization, and localization of PtdMTP1, a member of the CDF family from the hybrid poplar Populus trichocarpa x Populus deltoides. PtdMTP1 is expressed constitutively and ubiquitously, although at low levels. Heterologous expression in yeast showed that PtdMTP1 was able to complement the hypersensitivity of mutant strains to Zn but not to other metals, including Cd, Co, Mn, and Ni. PtdMTP1 fused to green fluorescent protein localized to the vacuolar membrane both in yeast and in plant cells, consistent with a function of PtdMTP1 in zinc sequestration. Overexpression of PtdMTP1 in Arabidopsis confers Zn tolerance. We show that PtdMTP1, when expressed in yeast and Arabidopsis, forms homooligomers, a novel feature of CDF members. Oligomer formation is disrupted by reducing agents, indicating possible disulfide bridge formation. PtdMTP1 also contains a conserved Leu zipper motif. Although not necessary for oligomer formation, Leu residues within this motif are required for PtdMTP1 functional activity. [ABSTRACT FROM AUTHOR]

To date, most poplar expressed sequence tags (ESTs) are from above-ground tissues such as wood, leaf and buds. Here, we present a large-scale production of ESTs from roots of the hybrid cottonwood, Populus trichocarpa×deltoides. cDNA libraries were generated from the root system of 2-month-old rooted cuttings, and roots of 2.5-month-old cuttings water-stressed for 19 days. Partial sequences obtained from 7013 clones were assembled into 1347 clusters and 3527 singletons. This set of ESTs represents 4874 unique transcripts expressed in roots. Putative functions could be assigned to 3021 (62%) of the transcripts. A significant portion of the ESTs encode proteins of common metabolic pathways; energy and metabolism represented 5% and 8% of total transcripts, respectively. Of specific interest to root functions are the 6% of ESTs involved in signalling pathways and hormone metabolism, and 4% encoding transporters and channels. The current poplar root ESTs and the aspen root ESTs present in public databases represent 6700 unique transcripts. The Unigene set was selected from the ESTs and used to generate nylon microarrays. Changes in aquaporins and transporter transcripts were then studied during adventitious root development. [Copyright &y& Elsevier]

Summary: The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships.Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed.MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2‐regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis.Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis. [ABSTRACT FROM AUTHOR]

Summary: Ectomycorrhizal (ECM) fungi associated with plants constitute one of the most successful symbiotic interactions in forest ecosystems. ECM support trophic exchanges with host plants and are important factors for the survival and stress resilience of trees. However, ECM clades often harbour morpho‐species and cryptic lineages, with weak morphological differentiation. How this relates to intraspecific genome variability and ecological functioning is poorly known. Here, we analysed 16 European isolates of the ascomycete Cenococcum geophilum, an extremely ubiquitous forest symbiotic fungus with no known sexual or asexual spore‐forming structures but with a massively enlarged genome. We carried out whole‐genome sequencing to identify single‐nucleotide polymorphisms. We found no geographic structure at the European scale but divergent lineages within sampling sites. Evidence for recombination was restricted to specific cryptic lineages. Lineage differentiation was supported by extensive copy‐number variation. Finally, we confirmed heterothallism with a single MAT1 idiomorph per genome. Synteny analyses of the MAT1 locus revealed substantial rearrangements and a pseudogene of the opposite MAT1 idiomorph. Our study provides the first evidence for substantial genome‐wide structural variation, lineage‐specific recombination and low continent‐wide genetic differentiation in C. geophilum. Our study provides a foundation for targeted analyses of intra‐specific functional variation in this major symbiosis. [ABSTRACT FROM AUTHOR]

SUMMARY: Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree‐microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non‐ectomycorrhizal P. trichocarpa cuttings to short‐term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short‐term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi‐omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa‐L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non‐mycorrhizal roots to abiotic challenges. Significance Statement: Current research on tree physiology primarily focuses on tree responses to abiotic stress, with little emphasis on root‐associated mutualistic fungi. Our results provide insights into how and to what extent the presence of ectomycorrhizas modifies host responses to abiotic changes: symbiosis has local and systemic impacts on host signaling pathways. A better understanding of the molecular mechanisms underpinning root‐microbe mutualistic interactions under abiotic stress will allow for robust modeling of tree adaptation to environmental challenges. [ABSTRACT FROM AUTHOR]

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita, a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls [ABSTRACT FROM AUTHOR]

Summary: Extensive evidence showed that the efficiency of fungal wood degradation is closely dependent on their ability to cope with the myriad of putative toxic compounds called extractives released during this process. By analysing global gene expression of Phanerochaete chrysosporium after short oak extractive treatment (1, 3 and 6 h), we show that the early molecular response of the fungus concerns first mitochondrial stress rescue followed by the oxidation and finally conjugation of the compounds. During these early responses, the lignolytic degradative system is not induced, rather some small secreted proteins could play an important role in cell protection or signaling. By focusing on the functional characterization of an hitherto uncharacterized glutathione transferase, we show that this enzyme interacts with wood molecules suggesting that it could be involved in the detoxification of some of them, or act as a scavenger to prevent their cytosolic toxicity and favour their transport. [ABSTRACT FROM AUTHOR]

This article documents the public availability of transcriptomic resources for (i) the Hazelnut tree (Corylus avellana L.) and (ii) the oriental rat flea and primary plague vector, Xenopsylla cheopis. [ABSTRACT FROM AUTHOR]

Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer., We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources., Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface., This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments. [ABSTRACT FROM AUTHOR]

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin‐encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin‐encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web. [ABSTRACT FROM AUTHOR]

Linking community composition to ecosystem function is a challenge in complex microbial communities. We tested the hypothesis that key biological features of fungi - evolutionary history, functional guild, and abundance of functional genes - can predict the biogeochemical activity of fungal species during decay. We measured the activity of 10 different enzymes produced by 48 model fungal species on leaf litter in laboratory microcosms. Taxa included closely related species with different ecologies (i.e. species in different "functional guilds") and species with publicly available genomes. Decomposition capabilities differed less among phylogenetic lineages of fungi than among different functional guilds. Activity of carbohydrases and acid phosphatase was significantly higher in litter colonized by saprotrophs compared to ectomycorrhizal species. By contrast, oxidoreductase activities per unit fungal biomass were statistically similar across functional guilds, with white rot fungi having highest polyphenol oxidase activity and ectomycorrhizal fungi having highest peroxidase activity. On the ecosystem level, polyphenol oxidase activity in soil correlated with the abundance of white rot fungi, while soil peroxidase activity correlated with the abundance of ectomycorrhizal fungi in soil. Copy numbers of genes coding for different enzymes explained the activity of some carbohydrases and polyphenol oxidase produced by fungi in culture, but were not significantly better predictors of activity than specific functional guild. Collectively, our data suggest that quantifying the specific functional guilds of fungi in soil, potentially through environmental sequencing approaches, allows us to predict activity of enzymes that drive soil biogeochemical cycles. [ABSTRACT FROM AUTHOR]

Recalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. To address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root), and used transgenic approaches to verify the role of several transcription factors in the control of adventitious rooting. Increased peroxidase activity was positively correlated with better rooting. We found differentially expressed genes encoding reactive oxygen species scavenging proteins to be enriched in OP42 compared with T89. A greater number of differentially expressed transcription factors in cambium cells of OP42 compared with T89 was revealed by a more intense transcriptional reprograming in the former. PtMYC2 , a potential negative regulator, was less expressed in OP42 compared with T89. Using transgenic approaches, we demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. Our results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process. [ABSTRACT FROM AUTHOR]

Ectomycorrhizal fungi, living in soil forests, are required microorganisms to sustain tree growth and productivity. The establishment of mutualistic interaction with roots to form ectomycorrhiza (ECM) is not well known at the molecular level. In particular, how fungal and plant cell walls are rearranged to establish a fully functional ectomycorrhiza is poorly understood. Nevertheless, it is likely that Carbohydrate Active enZymes (CAZyme) produced by the fungus participate in this process. Genome-wide transcriptome profiling during ECM development was used to examine how the CAZome of Laccaria bicolor is regulated during symbiosis establishment. CAZymes active on fungal cell wall were upregulated during ECM development in particular after 4 weeks of contact when the hyphae are surrounding the root cells and start to colonize the apoplast. We demonstrated that one expansin-like protein, whose expression is specific to symbiotic tissues, localizes within fungal cell wall. Whereas L. bicolor genome contained a constricted repertoire of CAZymes active on cellulose and hemicellulose, these CAZymes were expressed during the first steps of root cells colonization. L. bicolor retained the ability to use homogalacturonan, a pectin-derived substrate, as carbon source. CAZymes likely involved in pectin hydrolysis were mainly expressed at the stage of a fully mature ECM. All together, our data suggest an active remodelling of fungal cell wall with a possible involvement of expansin during ECM development. By contrast, a soft remodelling of the plant cell wall likely occurs through the loosening of the cellulose microfibrils by AA9 or GH12 CAZymes and middle lamella smooth remodelling through pectin (homogalacturonan) hydrolysis likely by GH28, GH12 CAZymes. [ABSTRACT FROM AUTHOR]

Summary: In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell‐wall‐degrading enzymes.Here, we characterize the biochemical properties of the symbiosis‐induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance of LbGH28A was confirmed by generating RNA interference (RNAi)‐silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots.Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front.These findings suggest that the symbiosis‐induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization. [ABSTRACT FROM AUTHOR]

The Ganoderma species in Polyporales are ecologically and economically relevant wood decayers used in traditional medicine, but their genomic traits are still poorly documented. In the present study, we carried out a phylogenomic and comparative genomic analyses to better understand the genetic blueprint of this fungal lineage. We investigated seven Ganoderma genomes, including three new genomes, G. australe, G. leucocontextum, and G. lingzhi. The size of the newly sequenced genomes ranged from 60.34 to 84.27 Mb and they encoded 15,007 to 20,460 genes. A total of 58 species, including 40 white-rot fungi, 11 brown-rot fungi, four ectomycorrhizal fungi, one endophyte fungus, and two pathogens in Basidiomycota, were used for phylogenomic analyses based on 143 single-copy genes. It confirmed that Ganoderma species belong to the core polyporoid clade. Comparing to the other selected species, the genomes of the Ganoderma species encoded a larger set of genes involved in terpene metabolism and coding for secreted proteins (CAZymes, lipases, proteases and SSPs). Of note, G. australe has the largest genome size with no obvious genome wide duplication, but showed transposable elements (TEs) expansion and the largest set of terpene gene clusters, suggesting a high ability to produce terpenoids for medicinal treatment. G. australe also encoded the largest set of proteins containing domains for cytochrome P450s, heterokaryon incompatibility and major facilitator families. Besides, the size of G. australe secretome is the largest, including CAZymes (AA9, GH18, A01A), proteases G01, and lipases GGGX, which may enhance the catabolism of cell wall carbohydrates, proteins, and fats during hosts colonization. The current genomic resource will be used to develop further biotechnology and medicinal applications, together with ecological studies of the Ganoderma species. [ABSTRACT FROM AUTHOR]

Summary: The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae.We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens.The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall‐degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE 'nests', or dense aggregations of TEs. Some PCWDEs have been retained or even expanded, mostly in a species‐specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters).Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch. [ABSTRACT FROM AUTHOR]

Summary: We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown‐rot relatives.Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae.Ectomycorrhizal Boletales have a reduced set of plant cell‐wall‐degrading enzymes (PCWDEs) compared with their brown‐rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown‐rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector‐like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay.This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes. [ABSTRACT FROM AUTHOR]

The success of many wood decaying fungi lies in their ability to overcome unfavourable environmental conditions within and outside of litter and wood debris. Although so much has been learned about the ecology, taxonomy and physiology of several wood decaying basidiomycete fungi, the molecular basis for their survival in a diverse range of substrates and ecological habitats has been very little studied. Using the wood decay fungus ( Heterobasidion annosum s.s.) as a model, we investigated its transcriptomic response when exposed to several environmental stressors (high and low temperature, osmotic stress, oxidative stress and nutrient starvation) and during growth on specific pine wood compartments (bark, sapwood and heartwood). Among other genes and pathways, we documented the specific induction of the major facilitator superfamily 1 and cytochrome P450 families at low temperature, and protein kinases together with transcription factors during starvation. On the other hand, during saprotrophic growth, we observed the induction of many glycosyl hydrolases, three multi-copper oxidases ( MCO), five manganese peroxidases ( MnP) and one oxidoreductase which are specific for wood degradation. This is the first study providing insights on the potential mechanisms for adaptation to abiotic stresses and pine heartwood degradation in H . annosum s.s. [ABSTRACT FROM AUTHOR]

Background: Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results: In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions: The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples. [ABSTRACT FROM AUTHOR]

In this review, we highlight the main insights that have been gathered from recent developments using large-scale genomics of fungal saprotrophs and symbiotrophs (including ectomycorrhizal and orchid and ericoid mycorrhizal fungi) inhabiting forest ecosystems. After assessing the goals and motivations underlying our approach, we explore our current understanding of the limits and future potential of using genomics to understand the ecological roles of these forest fungi. Comparative genomics unraveled the molecular machineries involved in lignocellulose decomposition in wood decayers, soil and litter saprotrophs, and mycorrhizal symbionts. They also showed that transitions from saprotrophy to mutualism entailed widespread losses of lignocellulose-degrading enzymes; diversification of novel, lineage-specific symbiosis-induced genes; and convergent evolution of genetic innovations that facilitate the accommodationof mutualistic symbionts within their plant hosts. We also identify the major questions that remain unanswered and propose new avenues of genome-based research to understand the role of soil fungi in sustainable forest ecosystems. [ABSTRACT FROM AUTHOR]

A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components. [ABSTRACT FROM AUTHOR]

Abstract: Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants. [Copyright &y& Elsevier]

Abstract: Filamentous polarized growth involves a series of events including polarization of the cytoskeleton to selected growth sites, and the transport of secretory vesicles containing the components required for growth. The availability of fungal genome sequences has recently led to the identification of a large number of proteins involved in these processes. We have explored the Tuber melanosporum genome sequence by searching for homologs of genes known to play crucial roles in the morphogenesis and cell polarity of yeasts and filamentous fungi. One hundred and forty-nine genes have been identified and functionally grouped according to the deduced amino acid sequences (44 genes involved in cell polarity/morphogenesis, 39 belonging to the actin cytoskeleton and 66 involved in membrane dynamics, septation and exocytosis). A detailed gene annotation has shown that most components of the cell polarity machinery, morphogenesis and cytoskeleton found in yeasts and filamentous fungi are conserved, although the degree of similarity varies from strong to weak. Microscopic analysis of quick-frozen truffle hyphae detected the characteristic subcellular components of the hyphal tip in septate filamentous fungi, while transcript profiles revealed a moderately variable pattern during the biological cycle. [Copyright &y& Elsevier]

Abstract: The Tuber melanosporum genome has been analysed with the aim of identifying and characterizing the genes involved in the environmental stress response. A whole genome array (7496 genes/probe) was used to verify the fungal transcriptional profiling upon a cold temperature period (7days at 4°C). A total of 423 genes resulted to be differentially expressed in a significant manner (>2.5-fold; p-value<0.05) in the mycelia exposed to cold, compared to the control ones: 187 of these genes were up-regulated, while 236 were down-regulated. Sixty-six and fifty-one percent, respectively, of the up- or down-regulated transcripts had no KOG classification and were clustered as unclassified proteins, which was the most abundant category in the both up- and down-regulated genes. A gene subset, containing a range of biological functions, was chosen to validate the microarray experiment through quantitative real time PCR (qRT-PCR). The analysis confirmed the array data for 16 out of 22 of the considered genes, confirming that a cold temperature period influences the truffle global gene expression. The expressed genes, which mostly resulted to be genes for heat shock proteins (HSPs) and genes involved in cell wall and lipid metabolism, could be involved in mechanisms, which are responsible for fungal adaptation. Since truffle ascomata develop during the winter period, we hypothesize that these differentially expressed genes may help the truffle to adapt to low temperatures and/or perceive environmental signals that regulate the fructification. [Copyright &y& Elsevier]

Abstract: An analysis of the Populus root transcriptome was carried out in order to improve our understanding of molecular mechanisms underlying the response of trees to copper (Cu) stress. In a first stage, cuttings of nine Populus species were grown hydroponically and screened for differential phenotypical response to Cu. In a second stage, plants of the most Cu tolerant clone (Populus deltoides) were exposed to 30 and 60μM CuSO4 for 12 and 24h. RNA isolated from root tissues was analyzed with 4.6k cDNA arrays in order to determine alterations in transcription. Copper exposures increased significantly Cu concentration in roots. A series of transcripts differentially accumulated in a general or a specific way under the different stress conditions. Most of the transcripts encode proteins related to defense against biotic stress, antioxidant mechanisms, metal homeostasis, and water flux control. Expression of several transcripts involved in signaling, such as MAP kinases, calcium and ethylene-related enzymes were also altered under Cu exposure. Our results suggest the Cu accumulation in roots and a general defensive response against oxidative stress. [Copyright &y& Elsevier]

• Here, the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor was explored with the aim of constructing a genome-wide inventory of genes involved in fatty acid metabolism. • Sixty-three genes of the major pathways were annotated and validated by the detection of the corresponding transcripts. Seventy-one per cent belonged to multigene families of up to five members. In the mycelium of L. bicolor, 19 different fatty acids were detected, including at low concentrations palmitvaccenic acid (16:1(11Z)), which is known to be a marker for arbuscular mycorrhizal fungi. • The pathways of fatty acid biosynthesis and degradation in L. bicolor were reconstructed using lipid composition, gene annotation and transcriptional analysis. Annotation results indicated that saturated fatty acids were degraded in mitochondria, whereas degradation of modified fatty acids was confined to peroxisomes. • Fatty acid synthase (FAS) was the second largest protein annotated in L. bicolor. Phylogenetic analysis indicated that L. bicolor, Ustilago maydis and Coprinopsis cinerea have a vertebrate-like type I FAS encoded as a single protein, whereas in other basidiomycetes, including the human pathogenic basidiomycete Cryptococcus neoformans, and in most ascomycetes FAS is composed of the two structurally distinct subunits α and β. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]

• Ectomycorrhizal interactions established between the root systems of terrestrial plants and hyphae from soil-borne fungi are the most ecologically widespread plant symbioses. The efficient uptake of a broad range of nitrogen (N) compounds by the fungal symbiont and their further transfer to the host plant is a major feature of this symbiosis. Nevertheless, we far from understand which N form is preferentially transferred and what are the key molecular determinants required for this transfer. • Exhaustive in silico analysis of N-compound transporter families were performed within the genome of the ectomycorrhizal model fungus Laccaria bicolor. A broad phylogenetic approach was undertaken for all families and gene regulation was investigated using whole-genome expression arrays. • A repertoire of proteins involved in the transport of N compounds in L. bicolor was established that revealed the presence of at least 128 gene models in the genome of L. bicolor. Phylogenetic comparisons with other basidiomycete genomes highlighted the remarkable expansion of some families. Whole-genome expression arrays indicate that 92% of these gene models showed detectable transcript levels. • This work represents a major advance in the establishment of a transportome blueprint at a symbiotic interface, which will guide future experiments. [ABSTRACT FROM AUTHOR]

Background: Tetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea. Results: The exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Δtsp3 and Δtpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Δpls1 mutants, which are completely non-pathogenic on barley and rice. Conclusion: A new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi. [ABSTRACT FROM AUTHOR]