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The nematode-trapping fungus Arthrobotrys oligospora detects prey pheromones via G protein-coupled receptors

Nature Microbiology (2024)Cite this article

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Abstract

The ability to sense prey-derived cues is essential for predatory lifestyles. Under low-nutrient conditions, Arthrobotrys oligospora and other nematode-trapping fungi develop dedicated structures for nematode capture when exposed to nematode-derived cues, including a conserved family of pheromones, the ascarosides. A. oligospora senses ascarosides via conserved MAPK and cAMP–PKA pathways; however, the upstream receptors remain unknown. Here, using genomic, transcriptomic and functional analyses, we identified two families of G protein-coupled receptors (GPCRs) involved in sensing distinct nematode-derived cues. GPCRs homologous to yeast glucose receptors are required for ascaroside sensing, whereas Pth11-like GPCRs contribute to ascaroside-independent nematode sensing. Both GPCR classes activate conserved cAMP–PKA signalling to trigger trap development. This work demonstrates that predatory fungi use multiple GPCRs to sense several distinct nematode-derived cues for prey recognition and to enable a switch to a predatory lifestyle. Identification of these receptors reveals the molecular mechanisms of cross-kingdom communication via conserved pheromones also sensed by plants and animals.

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Fig. 1: Genomic and transcriptomic analyses of GPCRs in A. oligospora during nematode predation.
Fig. 2: The endocytosis of Gpr2 and Gpr3 is triggered by ascr#3 and ascr#7.
Fig. 3: Overexpression of Gpr2 or Gpr3 enhanced trap development in response to ascarosides.
Fig. 4: Gpr2 and Gpr3 physically interact with Gpa2 in vivo.
Fig. 5: Gin1, Gin3 and Gin4 sense nematode-derived cues and regulate trap development.

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Data availability

The A. oligospora TWF154 genome database used in this study is from National Center for Biotechnology Information GenBank under the accession number SOZJ00000000. RNA sequencing data used in this study are from the National Center for Biotechnology Information Gene Expression Omnibus database under the accession number GSE233568. References to these accession numbers can also be found throughout this paper. Source data are provided with this paper.

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Acknowledgements

We thank S.-C. Cheng for providing the anti-HA antibody and J.-Y. Leu for assistance with yeast transformation. We thank C.-Y. Lin and T.-H. Chang at the Metabolomics Core Facility, Agricultural Biotechnology Research Center at Academia Sinica for assisting with UPLC–tandem mass spectrometry parameter optimization and data analysis. We thank S.-P. Lee at the Imaging Core at the Institute of Molecular Biology, Academia Sinica for technical assistance with imaging. We are grateful to L.-M. Hsu and A.-M. Yang for their technical assistance in the laboratory. Funding for this work was provided by the Academia Sinica Investigator Award (to Y.-P.H., AS-IA-111-L02); the Ministry of Science and Technology MOST grant (to Y.-P.H., 110-2311-B-001-047-MY3); the Academia Sinica Postdoctoral Scholar Program (to R.J.T., AS-PD 11301-L06); National Science and Technology Council NSTC grant (to Y.-C.C., 108-2113-M-038-002-MY3); and the National Institutes of Health (to F.C.S., R35GM131877). We also thank support from the EMBO YIP and GIN programs.

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Authors and Affiliations

  1. Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan

    Chih-Yen Kuo, Sheng-Chian Juan & Yen-Ping Hsueh

  2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

    Chih-Yen Kuo, Rebecca J. Tay, Hung-Che Lin, Sheng-Chian Juan, Guillermo Vidal-Diez de Ulzurrun & Yen-Ping Hsueh

  3. Department of Biochemistry and Molecular Cell Biology, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Yu-Chu Chang

  4. Boyce Thompson Institute, Cornell University, Ithaca, NY, USA

    Jason Hoki & Frank C. Schroeder

  5. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Jason Hoki & Frank C. Schroeder

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Contributions

C.-Y.K. and Y.-P.H. conceived and designed the research. C.-Y.K., H.-C.L. and S.-C.J. performed the experiments. C.-Y.K., H.-C.L., S.-C.J., G.V.-D.U., Y.-C.C. and Y.-P.H. analysed the data. Y.-C.C., J.H. and F.C.S contributed reagents, materials and analysis tools. C.-Y.K., R.J.T., F.C.S. and Y.-P.H. wrote the paper.

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Correspondence to Yen-Ping Hsueh.

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Extended data

Extended Data Fig. 1 Phylogenetic tree of GPCRs from carbon receptor family.

A maximum-likelihood phylogenetic tree of GPCR protein sequences belonging to carbon receptor family from A. oligospora and orthologs from model fungi. Ani: Aspergillus nidulans. Ao: A. oligospora. Cal: Candida albicans. Cne: Cryptococcus neoformans. Fgr: F. graminearum. Mor: M. oryzae. Ncr: Neurospora crassa. Sce: S. cerevisiae. Uma: Ustilago maydis.

Extended Data Fig. 2 Expression of GINs and GPRs is up-regulated after exposure to C. elegans.

a, Transcripts per kilobase million (TPM) values of the differentially expressed Pth11-like GPCRs, with arrows indicating the five highly upregulated Pth11-like GPCRs. (The values represent the average of three independent biological replicates.). b, TPM values of the differentially expressed GPR2 and GPR3. (The values represent the average of three independent biological replicates.). c, d, Protein sequence alignment of Gins (c) and Gprs (d). Three levels of shading and three different symbols are used to indicate degrees of sequence similarity: black background with asterisk (*) indicates identical amino acids, intermediate grey background with colon (:) indicates conserved amino acids, and light grey with single dots (.) indicates semi-conserved amino acids.

Source data

Extended Data Fig. 3 Expression of GPR3 and GINs is regulated by Ste12.

a, b, The expression level of GPR (a) and GIN (b) genes in the WT and ste12 mutant was evaluated using qPCR under with or without exposure to C. elegans. GPD1 was used as normalization control. (Data represent mean ± SEM; n shown along the x axis; two-tailed unpaired Student’s t-test; P values are unadjusted.).

Source data

Extended Data Fig. 4 The Gins and Gprs are not required for fungal growth.

a, Colonies of the WT, gin, and gpr mutants grown on PDA plates (5.5-cm diameter) for 3 days (Scale bar, 1 cm; the images are representative of three independent biological repeats.). b, Quantification of colony diameter for the A. oligospora WT and gpcr mutants. (Data represent mean ± SEM; n shown along the x axis; two-tailed unpaired Student’s t-test; P values are noted.).

Source data

Extended Data Fig. 5 Protein structural alignment of Gpr2, Gpr3, and SRBC-66.

a, b, TM-align was utilized to compare the protein structure of Gpr3 (depicted in green) with Gpr2 (depicted in blue) (a) and SRBC-66 (depicted in red) (b). (0.5 < TM-score < 1.00, in about the same fold). An overall map of the protein structural alignment between Gpr2 (blue), Gpr3 (green), and SRBC-66 (red) was based on the structural predictions from AlphaFold.

Extended Data Fig. 6 The prediction of ascaroside binding pocket in Gpr3.

Using SwissDock to predict the ascaroside binding pocket in Gpr3. An overall map of the docking interactions between Gpr3 (green) and and ascr#3 (a) and ascr#7 (b), based on the structural predictions from AlphaFold. The structural display of the amino acid region from 212 to 264 has been omitted due to its prediction as a random coil. Additionally, an enlarged schematic diagram highlights the potential ascaroside docking region in Gpr3.

Extended Data Fig. 7 Western blot analysis with S. cerevisiae transformants expressing each GPCR-3×HA and Gpa-Myc.

Total proteins were extracted from S. cerevisiae expressing each GPCR-3×HA or each Gpa-Myc construct. The expected 48-kD GPCR-3×HA and 42-kD Gpa-Myc bands were detected in each transformants. Detection with the anti-tubulin antibody was used as the loading control. The blots are representative of two independent biological repeats.

Source data

Extended Data Fig. 8 The Gins are not required for ascaroside sensing.

a, Quantification of the trap numbers induced by ascarosides for the A. oligospora WT and gin mutants. (Data represent mean ± SEM; n shown along the x axis; two-tailed unpaired Student’s t-test; P values are noted.). b, Representative brightfield images of the traps induced by C. elegans in the A. oligospora WT and gin mutants. (Scale bar, 200 μm. Black arrow indicates trap; the images are representative of three independent biological repeats.). c, Quantification of the trap numbers induced by C. elegans for the A. oligospora WT and gin mutants. (Data represent ± SEM; n shown along the x axis; two-tailed unpaired Student’s t-test; P values are noted.). d, Images of traps formed by the A. oligospora WT and gin mutants after 24 hours of continuous induction with 400 C. elegans. Vegetative hyphae and traps of A. oligospora were stained with SR2200, which specifically bound to fungal cell walls. (Scale bar, 20 μm; the images are representative of three independent biological repeats.). e, The localization of Gin1-GFP was displayed at 0, 2, and 10 hours after C. elegans induction. Merged image shows the GFP channel, FM4-64, and CMAC. (Scale bar, 10 μm; the images are representative of three independent biological repeats.) f, The expression level of GIN genes in the WT and gin3 mutant was evaluated using qPCR under with or without exposure to C. elegans. GPD1 was used as normalization control. (Data represent ± SEM; n shown along the x axis; two-tailed unpaired Student’s t-test; P values are noted.).

Source data

Extended Data Fig. 9 Distribution of GPCRs across the nine A. oligospora chromosomes.

GPCR-encoding genes from Pth11-like family (pink), carbon receptor (blue), and other classes (green) distribute across the nine A. oligospora chromosomes.

Extended Data Fig. 10 Hypothetical model of GPCRs-governed prey sensing and trap development in A. oligospora.

When Gpr2 and Gpr3 recognize ascr#3 and ascr#7, Gpa2 dissociates from the GPCRs and subsequently activates the downstream cAMP-PKA pathway. Additionally, Gin3 is activated by other unknown nematode-derived signals and also operates upstream of the cAMP-PKA pathway. PKA then phosphorylates the downstream substrates required for trap morphogenesis. Additionally, Gpr2, Gpr3, and Gin3 may be involved in the modulation of phosphorylation of Hog1. Gin3 may also partially activate the pheromone response MAPK pathway to induce the expression of GINs and GPR3, which may indicate their potential role as nematode-responsive genes and as receptors for unidentified nematode signals in A. oligospora.

Supplementary information

Supplementary Information

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41564_2024_1679_MOESM4_ESM.xlsx

Supplementary Table 1 Manual annotation of 83 putative GPCR genes. Supplementary Table 2 Wilcoxon test result of Pth11-like GPCR expression. Supplementary Table 3 The primers used in this study. Supplementary Table 4 The strains and plasmids used in this study.

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Kuo, CY., Tay, R.J., Lin, HC. et al. The nematode-trapping fungus Arthrobotrys oligospora detects prey pheromones via G protein-coupled receptors. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01679-w

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