Dag Ahrén

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

Mycorrhizal symbioses—the union of roots and soil fungi—are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains ∼20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

Expansion of the BioCyc collection of pathway/genome databases to 160 genomes

The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB in the BioCyc collection describes the genome and predicted metabolic network of a single organism, inferred from the MetaCyc database, which is a reference source on metabolic pathways from multiple organisms. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms. This paper discusses the computational methodology by which the BioCyc collection has been expanded, and presents an aggregate analysis of the collection that includes the range of number of pathways present in these organisms, and the most frequently observed pathways. We seek scientists to adopt and curate individual PGDBs within the BioCyc collection. Only by harnessing the expertise of many scientists we can hope to produce biological databases, which accurately reflect the depth and breadth of knowledge that the biomedical research community is producing.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus)

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 108 synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue

In order to obtain information on genes specifically expressed in the ectomycorrhizal symbiosis, 3,555 expressed sequence tags (ESTs) were analyzed from a cDNA library constructed from ectomycorrhiza formed between the basidiomycete Paxillus involutus and birch (Betula pendula). cDNA libraries from saprophytically growing fungus (3,964 ESTs) and from axenic plants (2,532 ESTs) were analyzed in parallel. By clustering all the EST obtained, a nonredundant set of 2,284 unique transcripts of either fungal or plant origin were identified. The expression pattern of these genes was analyzed using cDNA microarrays. The analyses showed that the plant and fungus responded to the symbiosis by altering the expression levels of a number of enzymes involved in carbon metabolism. Several plant transcripts with sequence similarities to genes encoding enzymes in the tricarboxylic cycle and electron transport chain were down regulated as compared with the levels in free-living roots. In the fungal partner, a number of genes encoding enzymes in the lipid and secondary metabolism were down regulated in mycorrhiza as compared with the saprophytically growing mycelium. A substantial number of the ESTs analyzed displayed significant sequence similarities to proteins involved in biotic stress responses, but only a few of them showed differential expression in the mycorrhizal tissue, including plant and fungal metallothioneins and a plant defensin homologue. Several of the genes that were differentially expressed in the mycorrhizal root tissue displayed sequence similarity to genes that are known to regulate growth and development of plant roots and fungal hyphae, including transcription factors and Rho-like GTPases.

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors

Summary Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose‐degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition ‘toolbox’ has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Measuring genome conservation across taxa: divided strains and united kingdoms

Species evolutionary relationships have traditionally been defined by sequence similarities of phylogenetic marker molecules, recently followed by whole-genome phylogenies based on gene order, average ortholog similarity or gene content. Here, we introduce genome conservation—a novel metric of evolutionary distances between species that simultaneously takes into account, both gene content and sequence similarity at the whole-genome level. Genome conservation represents a robust distance measure, as demonstrated by accurate phylogenetic reconstructions. The genome conservation matrix for all presently sequenced organisms exhibits a remarkable ability to define evolutionary relationships across all taxonomic ranges. An assessment of taxonomic ranks with genome conservation shows that certain ranks are inadequately described and raises the possibility for a more precise and quantitative taxonomy in the future. All phylogenetic reconstructions are available at the genome phylogeny server: <>.

Genomic Mechanisms Accounting for the Adaptation to Parasitism in Nematode-Trapping Fungi

Orbiliomycetes is one of the earliest diverging branches of the filamentous ascomycetes. The class contains nematode-trapping fungi that form unique infection structures, called traps, to capture and kill free-living nematodes. The traps have evolved differently along several lineages and include adhesive traps (knobs, nets or branches) and constricting rings. We show, by genome sequencing of the knob-forming species Monacrosporium haptotylum and comparison with the net-forming species Arthrobotrys oligospora, that two genomic mechanisms are likely to have been important for the adaptation to parasitism in these fungi. Firstly, the expansion of protein domain families and the large number of species-specific genes indicated that gene duplication followed by functional diversification had a major role in the evolution of the nematode-trapping fungi. Gene expression indicated that many of these genes are important for pathogenicity. Secondly, gene expression of orthologs between the two fungi during infection indicated that differential regulation was an important mechanism for the evolution of parasitism in nematode-trapping fungi. Many of the highly expressed and highly upregulated M. haptotylum transcripts during the early stages of nematode infection were species-specific and encoded small secreted proteins (SSPs) that were affected by repeat-induced point mutations (RIP). An active RIP mechanism was revealed by lack of repeats, dinucleotide bias in repeats and genes, low proportion of recent gene duplicates, and reduction of recent gene family expansions. The high expression and rapid divergence of SSPs indicate a striking similarity in the infection mechanisms of nematode-trapping fungi and plant and insect pathogens from the crown groups of the filamentous ascomycetes (Pezizomycotina). The patterns of gene family expansions in the nematode-trapping fungi were more similar to plant pathogens than to insect and animal pathogens. The observation of RIP activity in the Orbiliomycetes suggested that this mechanism was present early in the evolution of the filamentous ascomycetes.

Transcriptome analysis of Stagonospora nodorum: gene models, effectors, metabolism and pantothenate dispensability

The wheat pathogen Stagonospora nodorum, causal organism of the wheat disease Stagonospora nodorum blotch, has emerged as a model for the Dothideomycetes, a large fungal taxon that includes many important plant pathogens. The initial annotation of the genome assembly included 16,586 nuclear gene models. These gene models were used to design a microarray that has been interrogated with labelled transcripts from six cDNA samples: four from infected wheat plants at time points spanning early infection to sporulation, and two time points taken from growth in artificial media. Positive signals of expression were obtained for 12,281 genes. This represents strong corroborative evidence of the validity of these gene models. Significantly differential expression between the various time points was observed. When infected samples were compared with axenic cultures, 2882 genes were expressed at a higher level in planta and 3630 were expressed more highly in vitro. Similar numbers were differentially expressed between different developmental stages. The earliest time points in planta were particularly enriched in differentially expressed genes. A disproportionate number of the early expressed gene products were predicted to be secreted, but otherwise had no obvious sequence homology to functionally characterized genes. These genes are candidate necrotrophic effectors. We have focused attention on genes for carbohydrate metabolism and the specific biosynthetic pathways active during growth in planta. The analysis points to a very dynamic adjustment of metabolism during infection. Functional analysis of a gene in the coenzyme A biosynthetic pathway showed that the enzyme was dispensable for growth, indicating that a precursor is supplied by the plant.

Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production.

Moths generally rely on pheromone communication for mate finding. The pheromone components of most moths are produced by a common pathway of fatty‐acid biosynthesis coupled with species‐specific modifications of the final products. Some genes involved in moth pheromone production have previously been described, whereas others remain to be characterized and thus the molecular mechanisms accounting for the production of species‐specific blends are far from understood. The turnip moth, Agrotis segetum, has a multicomponent pheromone, consisting of at least four components derived from palmitic and stearic acid. Different populations produce and respond to different pheromone blends, which makes this species an excellent model for research on genes and molecular mechanisms involved in moth pheromone production. For this purpose, we performed an expressed sequence tag (EST) analysis of two cDNA libraries, one representing the female pheromone gland and the other representing the remainder of the insect body. Among 2285 ESTs analysed altogether, we identified a unigene set of 707 putative gene representatives. The comparative distribution of those in the two libraries showed the transcriptomes of the tissues to be clearly different. One third of the gene representatives were exclusively found in the pheromone gland. From sequence homology to public database information we assigned putative functional roles for a majority of the unigenes and then compared functional profiles of the two tissues. In the set of ESTs more abundant in the pheromone gland library, we found homologues of an acyl‐CoA Δ11‐desaturase, a G‐protein subunit, a chemosensory protein as well as a juvenile hormone binding protein.

Proteome of the Nematode-Trapping Cells of the Fungus Monacrosporium haptotylum

ABSTRACT Many nematophagous fungi use morphological structures called traps to capture nematodes by adhesion or mechanically. To better understand the cellular functions of adhesive traps, the trap cell proteome of the fungus Monacrosporium haptotylum was characterized. The trap of M. haptotylum consists of a unicellular structure called a knob that develops at the apex of a hypha. Proteins extracted from knobs and mycelia were analyzed using SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC–MS-MS). The peptide sequences were matched against predicted gene models from the recently sequenced M. haptotylum genome. In total, 336 proteins were identified, with 54 expressed at significantly higher levels in the knobs than in the mycelia. The upregulated knob proteins included peptidases, small secreted proteins with unknown functions, and putative cell surface adhesins containing carbohydrate-binding domains, including the WSC domain. Phylogenetic analysis showed that all upregulated WSC domain proteins belonged to a large, expanded cluster of paralogs in M. haptotylum. Several peptidases and homologs of experimentally verified proteins in other pathogenic fungi were also upregulated in the knob proteome. Complementary profiling of gene expression at the transcriptome level showed poor correlation between the upregulation of knob proteins and their corresponding transcripts. We propose that the traps of M. haptotylum contain many of the proteins needed in the early stages of infection and that the trap cells can tightly control the translation and degradation of these proteins to minimize the cost of protein synthesis.