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

Abstract

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.

Fig 1. A. oligospora responds to the presence of prey with a dynamic transcriptomic response.

(A) Overview of time-course RNA-seq experiment. hpe, hours post-exposure. (B) Principal component analysis of A. oligospora transcript expression upon exposure to C. elegans of 3 biological replicates for the indicated time points, as analyzed by RNA-seq. The data underlying this Figure can be found in S1 Data. (C) Venn diagrams depicting the number of A. oligospora transcripts that are up-regulated (left) or down-regulated (right) upon exposure to C. elegans. (D) The total number of transcripts with altered expression for each of the time points (“size of each list”) is shown. (E) Cluster analysis of transcript expression profiles revealed 5 common expression profiles. The 2,693 differentially expressed genes were subjected to soft clustering to identify 5 common expression profiles. The top enriched GO term for each cluster is listed, respectively. The data underlying this Figure can be found in S1 Data.

Fig 2. Ribosome biosynthesis is up-regulated in early time point.

(A) Expression profile of structural constituents of ribosome upon exposure to C. elegans. The data underlying this Figure can be found in S1 Data. (B) Trap induction and quantification of WT A. oligospora grown on LNM and LNM with 4 μg/ml rapamycin (scale bar, 500 μm). The data underlying this Figure can be found in S1 Data. (C) Survival rate of C. elegans on WT A. oligospora grown on LNM and LNM with 4 μg/ml rapamycin. The data underlying this Figure can be found in S1 Data. (D) Trap induction and quantification of WT A. oligospora on LNM and LNM with 200 mM HU (scale bar, 500 μm). The data underlying this Figure can be found in S1 Data. (E) Close-up images and quantification of the nuclei of the H2B::mCherry strain in the traps of A. oligospora grown on LNM and LNM with 4 μg/ml rapamycin or 200 mM HU (scale bar, 20 μm). The arrowhead indicates the trap cell; the arrow indicates vegetative hyphae. The data underlying this Figure can be found in S1 Data. hpe, hours post-exposure; HU, hydroxyurea; LNM, low-nutrient medium; WT, wild-type.

Fig 3. A. oligospora‘s response to C. elegans is dependent on protein secretion.

(A-D) Percentage and expression pattern analysis of the predicted secretome (A, B) and effectorome (C, D) that showed differential expression upon exposure to C. elegans. The data underlying this Figure can be found in S1 Data. (E) High-resolution image of traps stained by SR2200. Images were taken 24 h after induction (scale bar, 50 μm). The arrowhead indicates the trap cell; the arrow indicates vegetative hyphae. (F, G) Quantification of trap numbers (F) induced by exposure to 30 C. elegans nematodes and nematode escaping rate. (G) Among trapping of 50 C. elegans (mean ± SEM). The data underlying this Figure can be found in S1 Data.

Fig 4. The TEP family is an important component of the A. oligospora response to C. elegans.

(A) Expression profiles of TEP transcripts upon exposure to C. elegans. The data underlying this Figure can be found in S1 Data. (B) Histogram depicting the number of genes containing the DUF3129 domain encoded in the 2,512 fungal genomes (S5 Table). The data underlying this Figure can be found in S1 Data. (C) Orthogroup analysis cloud map. Each dot represents a DUF3129 protein from the indicated species, with lines connecting proteins belonging to the same orthogroup. The data underlying this Figure can be found in S1 Data. (D) High-resolution confocal imaging of 4 highly expressed TEP transcripts in WT traps. The WT strain was induced by nematodes for 24 h. Hairpins were conjugated with Alexa Fluor 647, 594, 546, and 514 (red) (scale bar, 20 μm). The arrowhead indicates the trap cell; the arrow indicates vegetative hyphae. (E) An image of trap induction of the TEP1-GFP strain (scale bar, 200 μm). The arrowhead indicates the trap cell; the arrow indicates vegetative hyphae. (F) Confocal images of the localization of TEP1-GFP (scale bar, 100 μm). (G) Images of traps of the WT, tep1, tep4, tep5, and tep1 TEP1 following a 6-h exposure to 30 C. elegans nematodes; the images were captured 24 h later (scale bar, 200 μm). The arrowhead indicates the trap cell. (H) Quantification of the nematode escaping rate of 50 C. elegans (mean ± SEM). The data underlying this Figure can be found in S1 Data. BF, bright field; hpe, hours post-exposure; NTF, nematode-trapping fungi; TEP, Trap Enriched Protein; WT, wild-type.

Fig 5. Late time point induced protease and its potential role in digesting nematodes.

(A) Expression profiles of differentially expressed protease transcripts upon exposure to C. elegans. The data underlying this Figure can be found in S1 Data. (B) TPM of the differentially expressed proteases. Arrows indicate the 4 highly up-regulated proteases. The data underlying this Figure can be found in S1 Data. (C) Image and quantification of hyphal colonization after penetration for 6–18 h and treatment with 1× PIC (scale bar, 100 μm). The data underlying this Figure can be found in S1 Data. (D) Image and quantification of hyphal colonization in C. elegans after being captured by traps for 6 h in WT, mcp1, and amp1 (scale bar, 100 μm). The data underlying this Figure can be found in S1 Data. hpe, hours post-exposure; PIC, protease inhibitor cocktail; TPM, Transcript Per Million; WT, wild type.