The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Our results support the hypothesis that cytoplasmic proteins abundant in fungal fruiting bodies are involved in fungal resistance against predation. The toxicity of these proteins toward stylet-feeding nematodes, which are also capable of feeding on plants, and the abundance of these proteins in edible mushrooms, may open possible avenues for biological crop protection against parasitic nematodes, e.g., by expression of these proteins in crops.

Fungi are an attractive source of nutrients for predators. As part of their defense, some fungi are able to induce the production of anti-predator protein toxins in response to predation. A previous study on the interaction of the model mushroom Coprinopsis cinerea by the fungivorous nematode Aphelenchus avenae on agar plates has shown that the this fungal defense response is most pronounced in the part of the mycelium that is in direct contact with the nematode. Hence, we hypothesized that, for a comprehensive characterization of this defense response, an experimental setup that maximizes the zone of direct interaction between the fungal mycelium and the nematode, was needed. In this study, we conducted a transcriptome analysis of C. cinerea vegetative mycelium upon challenge with A. avenae using a tailor-made microfluidic device. The device was designed such that the interaction between the fungus and the nematode was confined to a specific area and that the mycelium could be retrieved from this area for analysis. We took samples from the confrontation area after different time periods and extracted and sequenced the poly(A)+ RNA thereof. The identification of 1229 differentially expressed genes (DEGs) shows that this setup profoundly improved sensitivity over co-cultivation on agar plates where only 37 DEGs had been identified. The product of one of the most highly upregulated genes shows structural homology to bacterial pore-forming toxins, and revealed strong toxicity to various bacterivorous nematodes. In addition, bacteria associated with the fungivorous nematode A. avenae were profiled with 16S rRNA deep sequencing. Similar to the bacterivorous and plant-feeding nematodes, Proteobacteria and Bacteroidetes were the most dominant phyla in A. avenae. The use of a novel experimental setup for the investigation of the defense response of a fungal mycelium to predation by fungivorous nematodes resulted in the identification of a comprehensive set of DEGs and the discovery of a novel type of fungal defense protein against nematodes. The bacteria found to be associated with the fungivorous nematode are a possible explanation for the induction of some antibacterial defense proteins upon nematode challenge.

Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.