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

Abstract

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 β-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.

Keywords: G-protein signaling; natural population; nematode-trapping fungi; predator–prey interaction; trap morphogenesis.

Fig. 1.

High diversity of nematodes and nematode-trapping fungus species are sympatric in soil samples. (A) Geographic distribution of the sampling sites in Taiwan. (B) Nematodes and NTF are sympatric in more than 60% of our sampling sites. (C) Phylogeny of the different species of NTF isolated in this study based on their ITS sequences. The numbers represent the prevalence of the difference species of NTF in soil samples. Trap types and images of conidia are shown (Right). AC, adhesive columns; AN, adhesive networks; AK, adhesive knobs; CR, constricting rings. (D) Prevalence and diversity of nematode species in Taiwanese soil samples.

Fig. 2.

A. oligospora, A. musiformis, and A. thaumasia are generalist predators. (A) NTF preying on different sympatric nematode species. (Scale bar, 200 µm.) (B) NTF preying on allopatric nematode species.

Fig. 3.

Prey-sensing ability varies considerably among wild isolates of A. oligospora. (A) Quantification of trap number induced by C. elegans WT strain N2 among wild isolates of A. oligospora (mean ± SEM, n = 6; different letters represent significant differences from Tukey test). (B) Quantification of trap number induced by ascarosides among wild isolates of A. oligospora (mean ± SEM, n = 6; different letters represent significant differences from Tukey test). (C) Quantification of trap number induced by C. elegans in wild isolates of A. musiformis and A. thaumasia (mean ± SEM, n = 6). (D) Time of trap emergence after exposition to C. elegans among different A. oligospora wild isolates. (Mean ± SEM; n = 4; different letters represent significant differences from Tukey test.) (E) Percentage of live C. elegans after exposition to different strains of A. oligospora over 12 h. (Mean ± SEM.) (F) Competition fitness assay of A. oligospora isolates TWF154 and TWF106, computed as the percentage of conidia produced by TWF154 against the total number of conidia in the presence and absence of C. elegans (N2). (Mean ± SEM; n = 7; asterisks represent significance levels of unpaired t test.) (G) Phylogeny of 19 A. oligospora wild isolates sequenced and assembled for this study. The phylogenetic tree was constructed using 500 random single orthologs and the heatmap summarizes the trapping response of the isolates toward N2 and ascarosides.

Fig. 4.

G-protein signaling is required for prey sensing in A. oligospora. (A) Genome architecture of A. oligospora strain TWF154. Tracks (outer to inner) represent the distribution of genomic features: 1) positions (in Mb) of the 10 TWF154 contigs, with numbers indicating the order of scaffold size; 2) gene density (along a 100-kb sliding window); 3) distribution of transposable elements (along a 10-kb sliding window); 4) telomere repeat frequency (along a 1-kb sliding window), showing that 8 contigs have 2 telomeric ends; and 5), A. oligospora species-specific gene content (along a 100-kb sliding window). (B) Predicted function of genes in the TWF154 genome cataloged using the cluster of orthologous groups database. (C) Circular map of the mitochondrial genome of A. oligospora TWF154. Tracks (outer to inner) show: 1) annotation of mitochondrial DNA-encoded genes: subunits of NADH dehydrogenase/complex I (yellow), cytochrome c oxidase/complex IV (red), and ATP synthase/complex V (blue); apocytochrome b COB (green), ribosomal protein S3 RPS3 (pink), and ribosomal RNA genes RNS and RNL (orange); 2) approximate location of LAGLI-DADG (light gray) and GIY-YIG endonucleases (dark gray); 3) annotation of transfer RNA, where “t” stands for “tRNA” followed by the respective amino acid 1-letter code and number of copies.

Fig. 5.

G-protein β-subunit Gpb1 plays an important role in trap induction in A. oligospora. (A) Images of the traps induced by C. elegans laboratory strain N2 or ascarosides (ascr#7 and ascr#18) in the WT (TWF154), gpb1 mutants, and gpb1 GPB1 rescued strain. (Scale bar, 200 µm.) (B) Quantification of the trap numbers induced by C. elegans WT strain N2 or ascarosides (ascr#7 and ascr#18) in the WT (TWF154), gpb1 mutants, or gpb1 GPB1 reconstituted strain (n shown along the x axis). (Mean + SEM; n shown along the x axis; asterisks represent significance levels of unpaired t test compared to the WT.) (C) Fungal colonies developed by the WT (TWF154), gpb1 mutants, and gpb1 GPB1 rescued strain after 5 d of growth on PDA plates (5-cm diameter).