Key processes required for the different stages of fungal carnivory by a nematode-trapping fungus.

Nutritional deprivation triggers a switch from a saprotrophic to predatory lifestyle in soil-dwelling nematode-trapping fungi (NTF). In particular, the NTF Arthrobotrys oligospora secretes food and sex cues to lure nematodes to its mycelium and is triggered to develop specialized trapping devices. Captured nematodes are then invaded and digested by the fungus, thus serving as a food source. In this study, we examined the transcriptomic response of A. oligospora across the stages of sensing, trap development, and digestion upon exposure to the model nematode Caenorhabditis elegans. A. oligospora enacts a dynamic transcriptomic response, especially of protein secretion-related genes, in the presence of prey. Two-thirds of the predicted secretome of A. oligospora was up-regulated in the presence of C. elegans at all time points examined, and among these secreted proteins, 38.5% are predicted to be effector proteins. Furthermore, functional studies disrupting the t-SNARE protein Sso2 resulted in impaired ability to capture nematodes. Additionally, genes of the DUF3129 family, which are expanded in the genomes of several NTF, were highly up-regulated upon nematode exposure. We observed the accumulation of highly expressed DUF3129 proteins in trap cells, leading us to name members of this gene family as Trap Enriched Proteins (TEPs). Gene deletion of the most highly expressed TEP gene, TEP1, impairs the function of traps and prevents the fungus from capturing prey efficiently. In late stages of predation, we observed up-regulation of a variety of proteases, including metalloproteases. Following penetration of nematodes, these metalloproteases facilitate hyphal growth required for colonization of prey. These findings provide insights into the biology of the predatory lifestyle switch in a carnivorous fungus and provide frameworks for other fungal-nematode predator-prey systems.

Nematode-Trapping Fungi Produce Diverse Metabolites during Predator-Prey Interaction.

Nematode-trapping fungi are natural antagonists of nematodes. These predatory fungi are capable of switching their lifestyle from a saprophytic to predatory stage in the presence of nematodes by developing specialized trapping devices to capture and consume nematodes. The biochemical mechanisms of such predator-prey interaction have become increasingly studied given the potential application of nematode-trapping fungi as biocontrol agents, but the involved fungal metabolites remain underexplored. Here, we report a comprehensive liquid-chromatography mass spectrometry (LC-MS) metabolomics study on one hundred wild isolates of nematode-trapping fungi in three different species, Arthrobotrys oligospora, Arthrobotrys thaumasia, and Arthrobotrys musiformis. Molecular networking analysis revealed that the fungi were capable of producing thousands of metabolites, and such chemical diversity of metabolites was notably increased as the fungi switched lifestyle to the predatory stage. Structural annotations by tandem mass spectrometry revealed that those fungal metabolites belonged to various structural families, such as peptide, siderophore, fatty alcohol, and fatty acid amide, and their production exhibited species specificity. Several small peptides (<1.5 kDa) produced by A. musiformis in the predatory stage were found, with their partial amino acid sequences resolved by the tandem mass spectra. Four fungal metabolites (desferriferrichrome, linoleyl alcohol, nonadecanamide, and citicoline) that were significantly enriched in the predatory stage were identified and validated by chemical standards, and their bioactivities against nematode prey were assessed. The availability of the metabolomics datasets will facilitate comparative studies on the metabolites of nematode-trapping fungi in the future.

Sensory cilia as the Achilles heel of nematodes when attacked by carnivorous mushrooms.

Fungal predatory behavior on nematodes has evolved independently in all major fungal lineages. The basidiomycete oyster mushroom Pleurotus ostreatus is a carnivorous fungus that preys on nematodes to supplement its nitrogen intake under nutrient-limiting conditions. Its hyphae can paralyze nematodes within a few minutes of contact, but the mechanism had remained unclear. We demonstrate that the predator-prey relationship is highly conserved between multiple Pleurotus species and a diversity of nematodes. To further investigate the cellular and molecular mechanisms underlying rapid nematode paralysis, we conducted genetic screens in Caenorhabditis elegans and isolated mutants that became resistant to P. ostreatus We found that paralysis-resistant mutants all harbored loss-of-function mutations in genes required for ciliogenesis, demonstrating that the fungus induced paralysis via the cilia of nematode sensory neurons. Furthermore, we observed that P. ostreatus caused excess calcium influx and hypercontraction of the head and pharyngeal muscle cells, ultimately resulting in rapid necrosis of the entire nervous system and muscle cells throughout the entire organism. This cilia-dependent predatory mechanism is evolutionarily conserved in Pristionchus pacificus, a nematode species estimated to have diverged from C. elegans 280 to 430 million y ago. Thus, P. ostreatus exploits a nematode-killing mechanism that is distinct from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, representing a potential route for targeting parasitic nematodes in plants, animals, and humans.

Natural diversity in the predatory behavior facilitates the establishment of a robust model strain for nematode-trapping fungi.

Nematode-trapping fungi (NTF) are a group of specialized microbial predators that consume nematodes when food sources are limited. Predation is initiated when conserved nematode ascaroside pheromones are sensed, followed by the development of complex trapping devices. To gain insights into the coevolution of this interkingdom predator-prey relationship, we investigated natural populations of nematodes and NTF that we found to be ubiquitous in soils. Arthrobotrys species were sympatric with various nematode species and behaved as generalist predators. The ability to sense prey among wild isolates of Arthrobotrys oligospora varied greatly, as determined by the number of traps after exposure to Caenorhabditis elegans While some strains were highly sensitive to C. elegans and the nematode pheromone ascarosides, others responded only weakly. Furthermore, strains that were highly sensitive to the nematode prey also developed traps faster. The polymorphic nature of trap formation correlated with competency in prey killing, as well as with the phylogeny of A. oligospora natural strains, calculated after assembly and annotation of the genomes of 20 isolates. A chromosome-level genome assembly and annotation were established for one of the most sensitive wild isolates, and deletion of the only G-protein ß-subunit-encoding gene of A. oligospora nearly abolished trap formation. In summary, our study establishes a highly responsive A. oligospora wild isolate as a model strain for the study of fungus-nematode interactions and demonstrates that trap formation is a fitness character in generalist predators of the nematode-trapping fungus family.

Nematode-free agricultural system of a fungus-growing termite.

Fungus-growing termites forage dead plant materials from the field to cultivate symbiotic Termitomyces fungi in the nest. Termite foraging behavior and the entry of symbiotic arthropod inquilines may transfer nematodes into a nest and adversely affect fungus production. To test whether nematodes were transferred to fungus gardens by termites and inquilines, we examined the occurrence of nematodes in fungus gardens, five termite castes, and nine species of inquilines of a fungus-growing termite, Odontotermes formosanus. Our results revealed that nematodes were commonly carried by foraging termites and beetle inquilines. Numerous nematodes were found under the beetle elytra. No nematodes were found on termite larvae, eggs, and wingless inquilines. In addition, nematodes rarely occurred in the fungus garden. By observing the response of nematodes to three species of Termitomyces spp. and the fungus gardens, we confirmed that the fungus and fungus gardens are not actually toxic to nematodes. We suggest that nematodes were suppressed through grooming behavior and gut antimicrobial activity in termites, rather than through the antimicrobial activity of the fungus.