Abstract: 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: 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 approximately 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.
Abstract: The transcriptional response in the parasitic fungus Monacrosporium haptotylum and its nematode host Caenorhabditis elegans were analysed during infection using cDNA microarrays. The array contained 2684 fungal and 372 worm gene reporters. Dramatic shifts occurred in the transcriptome of M. haptotylum during the different stages of the infection. An initial transcriptional response was recorded after 1 h of infection when the traps adhered to the cuticle, but before immobilization of the captured nematodes. Among the differentially expressed genes were two serine protease genes (spr1 and spr2), and several homologues to genes known to be regulated in other pathogenic fungi. After 4 h, when approximately 40% of the nematodes were paralysed, we identified an upregulated cluster of 372 fungal genes which were not regulated during the other phases of the infection. This cohort contained a large proportion (79%) of genes that appear to be specific for M. haptotylum and closely related species. These genes were of two different classes: those translating into presumably functional peptides and those with no apparent protein coding potential (non-coding RNAs). Among the infection-induced C. elegans genes were those encoding antimicrobial peptides, protease inhibitors and lectins.
Abstract: Hydrophobins are small, secreted proteins that play important roles in the development of pathogenic and symbiotic fungi. Evolutionary mechanisms generating sequence and expression divergence among members in hydrophobin gene families are largely unknown. Seven hydrophobin (hyd) genes and one hyd pseudogene were isolated from strains of the ectomycorrhizal fungus Paxillus involutus. Sequences were analysed using phylogenetic methods. Expression profiles were inferred from microarray experiments. The hyd genes included both young (recently diverged) and old duplicates. Some young hyd genes exhibited an initial phase of enhanced sequence evolution owing to relaxed or positive selection. There was no significant association between sequence divergence and variation in expression levels. However, three hyd genes displayed a shift in the expression levels or an altered tissue specificity following duplication. The Paxillus hyd genes evolve according to the so-called birth-and-death model in which some duplicates are maintained for a long time, whereas others are inactivated through mutations. The role of subfunctionalization and/or neofunctionalization for preserving the hyd duplicates in the genome is discussed.
Abstract: We have examined the variations in gene content and sequence divergence that could be associated with symbiotic adaptations in the ectomycorrhizal fungus Paxillus involutus and the closely related species Paxillus filamentosus. Strains with various abilities to form mycorrhizae were analysed by comparative genomic hybridizations using a cDNA microarray containing 1076 putative unique genes of P. involutus. To screen for genes diverging at an enhanced and presumably non-neutral rate, we implemented a simple rate test using information from both the variations in hybridizations signal and data on sequence divergence of the arrayed genes relative to the genome of Coprinus cinereus. C. cinereus is a free-living saprophyte and is the closest evolutionary relative to P. involutus that has been fully sequenced. Approximately 17% of the genes investigated were detected as rapidly diverging within Paxillus. Furthermore, 6% of the genes varied in copy numbers between the analysed strains. Genome rearrangements associated with this variation including duplications and deletions may also play a role in adaptive evolution. The cohort of divergent and duplicated genes showed an over-representation of either orphans, genes whose products are located at membranes, or genes encoding for components of stress/defence reactions. Some of the identified genomic changes may be associated with the variation in host specificity of ectomycorrhizal fungi. The proposed procedure could be generally applicable to screen for rapidly evolving genes in closely related strains or species where at least one has been sequenced or characterized by expressed sequence tag analysis.
Abstract: Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. The global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum were compared. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridized to a cDNA array containing probes of 2822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23.3 % (657 of 2822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Among them were several putative homologues for small GTPases, such as rho1, rac1 and ras1, and a rho GDP dissociation inhibitor (rdi1). Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. In the last category, a glycogen phosphorylase (gph1) gene homologue, one of the most upregulated genes in the knobs as compared to mycelium, was characterized. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant-pathogenic fungi. Among them, a gas1 (mas3) gene homologue (designated gks1), which is specifically expressed in appressoria of the rice blast fungus, was characterized.
Abstract: The genetic variation of Duddingtonia flagrans, which has become a promising biocontrol agent of animal parasitic nematodes, was investigated in a worldwide collection of 22 isolates. We analysed the sequence variation in four nuclear genes, tubA (beta-tubulin), CMD1 (calmodulin), EF1alpha (translation elongation factor 1alpha), and PII (extracellular serine protease). 1428 aligned base pairs (bp) were analysed from the four genes, including 709 bp of introns. In addition, the variations in three anonymous genomic regions comprising 1155 bp were examined. Three single nucleotide polymorphisms (SNPs) were detected in the seven loci, none of them in the protein encoding genes. The genetic variation was significantly higher in the nematode-trapping fungus Arthrobotrys oligospora, the closest evolutionary relative to D. flagrans. Analysis of 12 isolates of A. oligospora revealed 30 SNPs in tubA, CMD1, EF1alpha and PII. The genetic variation in the isolates of D. flagrans was further examined using AFLP analysis. Five primer combinations were used to detect 159 bands, of which 94 (59.1%) were polymorphic. A neighbour-joining tree based on the AFLP data showed no clear association between genotype and geographical origin. Furthermore, the AFLP data suggest that D. flagrans is mainly clonal and no recombination could be detected, not even within the same country. The low genetic variation in D. flagrans suggests that this fungus has recently diverged from a single progenitor. Based on estimations of mutation rates, it was calculated that this most recent common ancestor lived about 16,000-23,000 years ago.
Abstract: Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.
Abstract: BACKGROUND: Research involving expressed sequence tags (ESTs) is intricately coupled to the existence of large, well-annotated sequence repositories. Comparatively complete and satisfactory annotated public sequence libraries are, however, available only for a limited range of organisms, rendering the absence of sequences and gene structure information a tangible problem for those working with taxa lacking an EST or genome sequencing project. Paralogous genes belonging to the same gene family but distinguished by derived characteristics are particularly prone to misidentification and erroneous annotation; high but incomplete levels of sequence similarity are typically difficult to interpret and have formed the basis of many unsubstantiated assumptions of orthology. In these cases, a phylogenetic study of the query sequence together with the most similar sequences in the database may be of great value to the identification process. In order to facilitate this laborious procedure, a project to employ automated phylogenetic analysis in the identification of ESTs was initiated. RESULTS: galaxieEST is an open source Perl-CGI script package designed to complement traditional similarity-based identification of EST sequences through employment of automated phylogenetic analysis. It uses a series of BLAST runs as a sieve to retrieve nucleotide and protein sequences for inclusion in neighbour joining and parsimony analyses; the output includes the BLAST output, the results of the phylogenetic analyses, and the corresponding multiple alignments. galaxieEST is available as an on-line web service for identification of fungal ESTs and for download / local installation for use with any organism group at http://galaxie.cgb.ki.se/galaxieEST.html. CONCLUSIONS: By addressing sequence relatedness in addition to similarity, galaxieEST provides an integrative view on EST origin and identity, which may prove particularly useful in cases where similarity searches return one or more pertinent, but not full, matches and additional information on the query EST is needed.
