Eva Friman

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.

Infection-related surface proteins on conidia of the nematophagous fungus Drechmeria coniospora

The adhesion of conidia of Drechmeria coniospora to the nematode Panagrellus redivivus was reduced after treatment of the conidia with Pronase E, or the detergents sodium dodecyl sulphate (SDS) and dodecyltrimethylammonium bromide (DTAB) suggesting involvement of proteinaceous compounds in the adhesion process. In the TEM the thick extracellular pad covering the adhesive bud of the conidia was completely removed after treatment with Pronase E. After treatment with SDS or DTAB the proteinaceous compounds appeared to be dissolved leaving mainly carbohydrates in the pad as observed on OsO4 without and OsO4 with Ruthenium red-stained material, respectively. The detergent extracts after SDS and DTAB treatments contained nine and seven peptides, respectively, with molecular masses in the range from 6 to 80 kDa on SDS-PAGE gels, and five biotinylated peptides were found in each extract, after blotting to nitrocellulose membranes, indicating that these were surface proteins. None of the detergent extracts was able to reduce adhesion of the conidia after treatment of the nematodes. The detergent extracts contained protease- and phosphatase activity. The protease inhibitor, chymostatin, inhibited infection of nematodes and growth of the conidia, suggesting the involvement of chymotrypsin-like proteases in the infection process. On gelatin-containing substrate gel electrophoresis two proteases were clearly chymostatin sensitive.

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.

Interspecific and host-related gene expression patterns in nematode-trapping fungi

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.

Influence of phosphate on development of the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora captures and digests nematodes by means of adhesive networks. These traps are formed in the presence of nematodes but can also be induced by low nutrient media containing amino acids. Influence of phosphate on growth and development of A. oligospora was studied in a liquid culture system known to allow heavy trap formation. Substrate-induced but not nematode-induced trap, formation was inhibited by phosphate at concentrations above 30 mu M. High numbers of chlamydospores were formed in aging cultures irrespective of phosphate concentration.