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.

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.

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. N ematode-trapping fungi have the unique ability to capture and infect free-living nematodes (1). Given their potential use as biological control agents for plant-and animal-parasitic nematodes (2), there is much interest in studying their infection biology. To enter the parasitic stage, nematode-trapping fungi develop a unique morphological structure called traps. These traps develop from hyphal branches, either spontaneously or in response to signals from the environment, such as peptides or other compounds released by the host nematode (3). Molecular phylogeny studies have shown that the majority of nematode-trapping fungi belong to a monophyletic group in the order Orbiliales (Ascomycota). Within this clade, the trapping mechanisms have evolved along two major lineages, one leading to species with constricting rings and the other to species with adhesive traps, including three-dimensional networks, knobs, and branches (4-6).Despite large variation in their morphology, adhesive traps share a unique ultrastructure that clearly separates them from vegetative hyphae (3). One feature that is common to all traps is the presence of numerous cytosolic organelles called dense bodies. These organelles have catalase and D-amino acid oxidase activities, which indicates that they are peroxisome-like organelles (3). However, the function of these organelles is not yet fully understood. Another feature of th...

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.

BackgroundNematode-trapping fungi are soil-living fungi that capture and kill nematodes using special hyphal structures called traps. They display a large diversity of trapping mechanisms and differ in their host preferences. To provide insights into the genetic basis for this variation, we compared the transcriptome expressed by three species of nematode-trapping fungi (Arthrobotrys oligospora, Monacrosporium cionopagum and Arthrobotrys dactyloides, which use adhesive nets, adhesive branches or constricting rings, respectively, to trap nematodes) during infection of two different plant-pathogenic nematode hosts (the root knot nematode Meloidogyne hapla and the sugar beet cyst nematode Heterodera schachtii).ResultsThe divergence in gene expression between the fungi was significantly larger than that related to the nematode species being infected. Transcripts predicted to encode secreted proteins and proteins with unknown function (orphans) were overrepresented among the highly expressed transcripts in all fungi. Genes that were highly expressed in all fungi encoded endopeptidases, such as subtilisins and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors; and transcripts containing the Ricin-B lectin domain. Differentially expressed transcripts among the fungal species encoded various lectins, such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain; and proteins containing a DUF3129 domain. A small set of transcripts were differentially expressed in infections of different host nematodes, including peptidases, WSC domain proteins, tyrosinases, and small secreted proteins with unknown function.ConclusionsThis is the first study on the variation of infection-related gene expression patterns in nematode-trapping fungi infecting different host species. A better understanding of these patterns will facilitate the improvements of these fungi in biological control programs, by providing molecular markers for screening programs and candidates for genetic manipulations of virulence and host preferences.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-968) contains supplementary material, which is available to authorized users.