Multiple fungal lineages have independently evolved carnivorous behaviors, preying on a diversity of nematodes as an adaptation for survival in low-nutrient environments. The edible oyster mushroom Pleurotus ostreatus is known to paralyze nematode prey, but the mechanism was unclear. We show that P. ostreatus triggers a massive calcium influx and rapid cell necrosis in the neuromuscular system of C. elegans via that nematode’s sensory cilia—a mode of action that is conserved across nematodes. Our study reveals a rapid killing mechanism that has not been described previously and is distinct from that employed by common anthelmintic drugs, representing a potential route for targeting parasitic nematodes. It also establishes a paradigm for studying cell death in C. elegans.

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.

Predators are known to evolve extraordinary adaptations to capture their prey. They employ diverse strategies, ranging from mechanical to chemical, to hunt effectively for their food. Athletic species such as leopards and cheetahs rely on their greater power and speed to catch their prey (1) whereas animals that are not athletic, such as cone snails, produce a large variety of peptide toxins that target ion channels and receptors in the neuromuscular systems of their prey (2).

Multiple fungal lineages of the Ascomycetes, Basidiomycetes, and Zygomycetes have independently evolved diverse strategies to prey on nematodes, the most abundant animals in soils (3), to supplement their nitrogen intakes. Nematode-trapping fungi such as Arthrobotrys oligospora (Ascomycota) and other closely related species are known to develop adhesive traps and constricting rings to mechanically catch their prey (4, 5). In contrast, the oyster mushroom Pleurotus ostreatus (Basidiomycota) produces chemicals to paralyze its nematode prey within a few minutes of contact (6, 7). Given the potent nematicidal activities of the Pleurotus species, these mushrooms may be used to control parasitic nematodes (8, 9). However, an effective management approach has not yet been established, partly because of the lack of knowledge of the basic biology of mushroom–nematode interactions in this microscopic predator–prey system. For instance, it had been unclear how P. ostreatus triggers paralysis in nematodes. Thus, we set out to investigate the cellular and molecular mechanisms of the Pleurotus-triggered paralysis in the model nematode Caenorhabditis elegans. We demonstrate that P. ostreatus paralyzes C. elegans via a previously unreported mechanism that is evolutionarily conserved across different nematode species. Through unbiased genetic screens, we found that the toxins produced by the Pleurotus mushrooms could only exert their nematicidal activity via the sensory cilia of C. elegans, triggering massive intracellular calcium influx and hypercontraction of the pharyngeal and body wall muscles, ultimately inducing cell necrosis in the neuromuscular system of the entire organism.

Results

Pleurotus Mushrooms Can Paralyze a Diversity of Nematode Species and Cause Massive Calcium Influx in C. elegans Pharyngeal and Body Wall Muscles. To assess how conserved is the predator–prey interaction between Pleurotus mushrooms and nematodes, we tested 15 species of Pleurotus for their ability to paralyze C. elegans. We cultured Pleurotus species on low-nutrient medium (LNM) and directly exposed C. elegans to the fungal hyphae. We observed that C. elegans were quickly paralyzed within a few minutes of contacting the fungal hyphae (Fig. 1A and SI Appendix, Fig. S1A). Next, we investigated if the oyster mushroom P. ostreatus could paralyze a diversity of nematode species and found that nematodes of the genera Caenorhabditis, Diploscapter, Oscheius, Rhabditis, Pristionchus, Panagrellus, Acrobeloides, Cephalobus, Mesorhabditis, and Pelodera were all paralyzed and ultimately consumed (Fig. 1 B and C and SI Appendix, Fig. S1B). These results demonstrate that the predator–prey relationship between Pleurotus fungi and nematodes is highly conserved. Fig. 1. Pleurotus mushrooms trigger paralysis and muscle hypercontraction in nematodes, including C. elegans. (A) Phylogenetic tree of the ITS region of 15 Pleurotus species. “+” indicates paralysis and “−” indicates no effect on C. elegans wild-type strain N2. Coprinopsis cinerea was selected as an outgroup. (B) Phylogenetic tree of the small-subunit rDNA regions of 17 nematode species, all of which were paralyzed by P. ostreatus. (C) Adult N2 nematode interacting with P. ostreatus and A. oligospora hyphae. (Scale bars, 50 µm.) (D) Pharyngeal pumping rate of adult N2 on P. ostreatus (mean ± SEM; n = 15). (E) Quantification of the rate of paralysis for different developmental stages of N2. Each dot represents 15 to 20 animals (mean ± SEM, n = 8). (F) GCaMP6 signal of the pharyngeal corpus of adult N2 in response to P. ostreatus and A. oligospora hyphae (mean ± SEM; n shown above the x axis). We then used the model nematode C. elegans to dissect the molecular mechanisms underlying P. ostreatus-induced paralysis. When C. elegans came into contact with fungal hyphae, we observed marked hypercontraction of the head muscles and cessation of pharyngeal pumping. Nematodes became immobilized almost immediately after the nose of the nematode touched the spherical droplet-like structure on the fungal hyphae (Fig. 1 C and D and Movie S1). This acute paralysis response differs considerably from the outcome when C. elegans encounters the nematode-trapping fungus A. oligospora, whereby the nematodes are attracted to the fungal hyphae and continue to move for several hours after triggering fungal trap morphogenesis via their ascaroside pheromones (10, 11). We found that all developmental stages of C. elegans are sensitive to P. ostreatus and they become paralyzed upon contacting the P. ostreatus hyphae (Fig. 1E). To further characterize the observed hypercontraction of the head muscle cells and cessation of pharyngeal pumping, we expressed in C. elegans the calcium indicator GCaMP6 (12) under the myo-2 and myo-3 promoters. We found that the calcium levels were massively increased (ΔF/F 0 > 10) in the corpus region of the pharynx and in the head muscles (ΔF/F 0 > 2.5) upon nematodes coming into contact with the fungal hyphae (Fig. 1F, SI Appendix, Fig. S1C, and Movies S2 and S3).

Forward Genetic Screens Revealed That Mutants Defective in Ciliogenesis Are Resistant to P. ostreatus-Induced Paralysis. To gain molecular insights into the acute paralysis response of C. elegans, we conducted random ethyl methanesulfonate mutagenesis screens to identify C. elegans mutants resistant to P. ostreatus-induced paralysis. We conducted several rounds of mutagenesis to attain genome coverage of ∼200,000 and isolated a dozen mutants that exhibited locomotory ability on P. ostreatus hyphae. Quantitative measurements of the locomotion demonstrated that these mutants moved with a speed on P. ostreatus hyphae comparable to movements on the nonparalyzing nematode-trapping fungus A. oligospora (Fig. 2A and Movie S4). To identify the mutations responsible for this phenotype, we employed single-nucleotide polymorphism (SNP) mapping (13) and whole-genome sequencing (WGS) by CloudMap (14) and identified 9 independent alleles representing loss-of-function mutations in the C. elegans dyf-7 (15), daf-6 (16), osm-6 (17), osm-1 (18), and che-13 (19) genes (Fig. 2B and SI Appendix, Fig. S2). All of these genes have been extensively studied in C. elegans. dyf-7, osm-6, osm-1, and che-13 are required for the development of sensory cilia, whereas daf-6 is expressed in glial socket and sheath cells and is required for amphid channel morphogenesis (20, 21). A phenotypic hallmark of C. elegans mutants defective in ciliogenesis is the lack of dye uptake in the amphid sensory neurons when stained with the lipophilic dye DiI (22). Accordingly, when we applied DiI to our mutants, they failed to incorporate this fluorescent dye (Fig. 2C). By individually expressing the genomic fragments containing the respective wild-type genes in our mutants, we recovered their susceptibility to P. ostreatus-induced paralysis, as well as their ability to take up DiI dye in ciliated sensory neurons, demonstrating that resistance to fungal-induced paralysis was indeed caused by the loss-of-function mutations in these genes (Fig. 2 C and D). We further tested other C. elegans mutants that are known to exhibit defects in the development of ciliated sensory neurons (20)—namely the che-11, daf-10, osm-5, che-2, dyf-2, and daf-19 mutants—and found that all of these mutants exhibited various degrees of resistance to the paralysis induced by contact with P. ostreatus hyphae (Fig. 2E), supporting our conclusion that intact cilia of nematode sensory neurons are required for the fungus to induce paralysis in C. elegans. To establish if the signaling function of these sensory neurons is required to trigger the paralysis response, we examined the sensitivity to P. ostreatus of mutants deficient in signaling and function of the ciliated sensory neurons. We found that mutant lines exhibiting impaired neuronal signaling (23)—such as due to mutations of the cyclic nucleotide gated channels tax-2 and tax-4, G proteins odr-3 and gpa-3, and transient receptor potential (TRP) channels osm-9 and trp-1 or the kinesin-like protein klp-6—were all paralyzed by P. ostreatus to the same extent as the wild-type nematodes (Fig. 2E). These results demonstrate that the structure of nematode sensory cilia must be intact for the acute paralysis response but the signaling function of the sensory cilia is dispensable for responding to P. ostreatus. Furthermore, we also observed that the calcium level in the pharyngeal muscles of mutants resistant to P. ostreatus-induced paralysis (i.e., dyf-7 and osm-6) did not massively increase upon hyphal contact (Fig. 2F). Fig. 2. Genetic screens reveal that mutants defective in ciliogenesis are resistant to P. ostreatus-induced paralysis. (A) Movement of wild-type (N2) and P. ostreatus-resistant C. elegans mutants on P. ostreatus and A. oligospora (mean ± SD; n > 8). (B) Genetic mapping and whole-genome sequencing reveal the causative mutations in P. ostreatus-resistant mutants (marked by asterisks). Boxes and lines represent exons and introns, respectively. (C) Images of DiI-stained P. ostreatus-resistant mutants and the respective complemented strains. (+ indicates mutants harboring the reintroduced wild-type genomic locus; Materials and Methods). (Scale bars, 20 µm.) (D and E) Quantification of rates of paralysis for wild-type, mutant, and complemented strains of C. elegans on P. ostreatus hyphae. Each dot represents 15 to 20 animals (mean ± SEM; n shown below the x axis). (F) GCaMP6 signal of the pharyngeal corpus of P. ostreatus-resistant mutants in response to P. ostreatus and A. oligospora hyphae (mean ± SEM; n shown above the x axis). *P < 0.05, ***P < 0.001, ****P < 0.0001; ns, not significant.

Restoring Ciliogenesis of a Single Class of Nematode Neurons Whose Cilia Endings Are Externally Exposed Is Sufficient to Trigger Paralysis. C. elegans possess ∼60 ciliated sensory neurons. To investigate if a single or multiple neurons are involved in triggering paralysis, we performed cell-specific rescue experiments in the osm-6(yph3) mutant background by using different promoters to drive expression of wild-type osm-6 complementary (c)DNA in various neurons (Fig. 3A and SI Appendix, Fig. S3). We found that if osm-6 expression was recapitulated in 1 or multiple head sensory neurons that have externally exposed ciliated dendritic endings (such as the IL2, ADF, ASH, and ASI neurons), the nematodes regained susceptibility to paralysis by P. ostreatus (Fig. 3A). In contrast, expressing osm-6 in the phasmid neurons of the tail, or in the olfactory AWB and AWC neurons that are enclosed in sheath cells, failed to recover the paralysis response to P. ostreatus hyphae (Fig. 3A). We then used laser ablation to kill the IL2 and ASH neurons to determine if they are required for paralysis and found that the neuron-ablated nematodes were still sensitive to P. ostreatus (Fig. 3B). These results demonstrate that multiple ciliated sensory neurons are involved in the paralysis triggered by P. ostreatus, and that certain neurons possessing externally exposed ciliated dendritic endings (such as IL2) are sufficient for the paralysis response. To monitor the activity of the IL2 neurons, we expressed GCaMP6 under the klp-6 promoter. We observed that after only 2 min of C. elegans touching P. ostreatus hyphae, there was a 2-fold increase in the GCaMP6 signals of nematode IL2 neurons (Fig. 3C and Movie S5), suggesting that the IL2 neurons had been activated by the fungal hyphae. However, activation of the IL2 neurons was not due to the touch response, because the GCaMP6 signal of IL2 neurons did not increase in response to touching A. oligospora hyphae. Furthermore, the activation of IL2 was dependent on osm-6, and could be complemented in a respective mutant line by expressing osm-6 cDNA under an IL2-specific promoter (Fig. 3D). Fig. 3. Multiple ciliated sensory neurons mediate responses to P. ostreatus hyphae. (A) Quantification of the rates of paralysis in wild-type C. elegans (N2), the osm-6 mutant, and cell-specific rescue lines expressing osm-6 cDNA under various promoters (mean ± SEM; n shown along the x axis). (B) Quantification of P. ostreatus-induced paralysis in mock and laser-ablated nematodes (n shown along the x axis). (C) GCaMP6 signal of IL2 neurons in adult N2 in response to P. ostreatus or A. oligospora hyphae (mean ± SEM; n shown above the x axis). (D) GCaMP6 signal of IL2 neurons in the osm-6 mutant (Left) and a cell-specific rescue line (Right) in response to P. ostreatus hyphae (mean ± SEM; n shown above the x axis). *P < 0.05, ****P < 0.0001.

Neuronal Activity Is Not Required for the Muscle Hypercontraction Triggered by P. ostreatus. To establish if neuronal activity is required for the phenotypes we observed when C. elegans encountered P. ostreatus hyphae, we examined head muscle hypercontraction and monitored the pharyngeal calcium levels in tph-1 (serotonin), cat-2 (dopamine), eat-4 (glutamate), unc-46 (GABA), unc-17 (acetylcholine), unc-13 (synaptic vesicle fusion), and unc-31 (dense-core vesicle fusion) mutants. We found that these mutants exhibited comparable phenotypes compared with the wild-type animals, suggesting that these neurotransmitters are not required for the calcium influx observed in the pharyngeal muscle cells (Fig. 4 A and B). Moreover, when we blocked synaptic transmission by expressing tetanus toxin (TeTx) in ciliated sensory neurons, we found that it did not significantly affect paralysis or the increase in pharyngeal calcium levels (Fig. 4 C and D). These results suggest that neuronal activities are dispensable for the observed calcium influx in the pharynx muscles upon contacting Pleurotus hyphae. Fig. 4. Neuronal activity is not required for the paralysis induced by P. ostreatus hyphae. (A) Quantification of the head muscle hypercontraction phenotype in wild-type C. elegans (N2) and various neurotransmitter mutant lines (mean ± SEM; n shown along the x axis). (B) Quantification of the GCaMP6 signal in the pharyngeal corpus of the neurotransmitter mutant lines in response to P. ostreatus hyphae. (C) Quantification of paralysis in wild-type N2, osm-6 mutants, and mutants expressing osm-6 cDNA and the tetanus toxin under the osm-6 promoter (mean ± SEM; n shown along the x axis). (D) GCaMP6 signal of the pharyngeal corpus in wild-type N2 expressing TeTx in ciliated sensory neurons in response to P. ostreatus hyphae (n = 6). ****P < 0.0001.

P. ostreatus Triggers Rapid Cell Necrosis in Multiple Tissues of C. elegans. When we monitored the GCaMP6 signals in the IL2 neurons in response to contact with P. ostreatus hyphae, we observed that the neuronal processes became fragmented within a few minutes of exposure to the fungal hyphae. Therefore, we systematically examined different types of neurons by exposing multiple C. elegans neuronal reporter lines to P. ostreatus hyphae for 10 min and then observed the resulting neuronal morphology. Strikingly, we observed massive neuron degeneration across the entire nervous system, including of the amphid and phasmid ciliated sensory neurons, cholinergic motor neurons, mechanosensory neurons, as well as glia cells (Fig. 5A and Movie S6). Moreover, all developmental stages of C. elegans showed pronounced fragmentation of the neuronal processes and swollen cell bodies, representing morphological features of neuronal necrosis (24, 25), in all developmental stages of C. elegans, and this massive neuronal necrosis was independent of neurotransmission (SI Appendix, Fig. S4 A and B). Furthermore, the muscle cells also exhibited prominent signs of necrosis after contacting the P. ostreatus hyphae (Fig. 5B). Approximately 90% of C. elegans presented signs of necrosis in the head sensory neurons with externally exposed ciliated dendrites 5 min after coming into contact with P. ostreatus hyphae, suggesting that the fungus exerts its paralytic activity very rapidly (Fig. 5C). We do not consider that the observed cell death is a form of apoptosis because ced-3 is not required (Fig. 5D). Next, we introduced the Posm-6::GFP reporter into the P. ostreatus-resistant dyf-7, che-13, and osm-6 mutants isolated from our genetic screens, and found that the neurons were largely intact even after 10 min of exposure to P. ostreatus hyphae (Fig. 5E). We again observed massive cell necrosis when we complemented osm-6 expression in a single class of ciliated sensory neurons (either IL2 or ADF), supporting our conclusion that the intact cilia structure of a single class of neurons is sufficient to trigger paralysis (Fig. 5E). Fig. 5. P. ostreatus triggers rapid cell necrosis via a mechanism that requires intact ciliated sensory neurons. (A) Images of cell necrosis in various neurons (indicated at the bottom left of each image) and glia cells. (Scale bars, 20 µm.) (B) Images of cell necrosis in the head or body wall muscle (Pmyo-3::mitoGFP). (Scale bars, 20 µm.) (C) Rates of cell fragmentation in various reporter lines that label specific neurons and head muscle (n > 20 for each time point). (D and E) Quantification of necrosis of ciliated sensory neurons (Posm-6::GFP) in wild-type N2 and ced-3 mutants (D), or Pleurotus-resistant mutants and cell-specific rescue lines expressing osm-6 cDNA under various promoters (E). (F) GCaMPer signal of the pharyngeal corpus of adult N2 in response to P. ostreatus hyphae (mean ± SEM; n shown above the x axis). (G) GCaMP6 signal of the pharyngeal corpus of adult N2 and unc-68(e540) mutants in response to P. ostreatus hyphae (mean ± SEM; n shown above the x axis). (H) Quantification of necrosis of ciliated sensory neurons in mutants in which calcium release is modulated from the ER. Multiple pathways can contribute to necrotic cell death in C. elegans. The hyperactive degenerin ion channels (26, 27) and other stress signals converge to markedly increase intracellular Ca2+ levels (28), signaling cell death. Since we had observed a greater than 10-fold increase in GCaMP6 signal in nematode pharyngeal muscles, we tested if this massive calcium influx is the major factor that led to cell death. To examine if the source of Ca2+ influx originated from the sarcoplasmic reticulum in the pharyngeal muscles, we imaged the sarcoplasmic reticulum Ca2+ store by expressing a low-affinity GCaMP3 variant (GCaMPer) that had been designed to attach to the lumen of the endoplasmic reticulum (ER) (29). We observed that calcium levels decreased in the sarcoplasmic reticulum upon hyphal contact (Fig. 5F). Furthermore, when we monitored the Pmyo2::GCaMP6 signals in the unc-86 (ryanodine receptor) mutant background, we observed that the massively increased GCaMP signal upon hyphal contact was reduced to less than 50% of the wild-type nematodes, demonstrating that calcium influx into the pharynx was considerably diminished (Fig. 5G). Next, we examined if the ciliated sensory neurons and pharyngeal muscles of the unc-68 mutant also underwent necrosis upon contact with P. ostreatus hyphae and found that the level of cell necrosis was comparable to that of the wild-type C. elegans (Fig. 5H and SI Appendix, Fig. S4C). In addition, cell necrosis was also not affected in the itr-1, crt-1, and cnx-1 mutants that are known to regulate and block the necrotic cell death induced by the dominant MEC-4(d) channel (Fig. 5H and SI Appendix, Fig. S4 C and D) (30). Together, these results suggest that although the ER is the source of Ca2+ influx, the downstream mechanism may be distinct from previously well-characterized hyperactivated channel-mediated neurotoxicity in C. elegans (30).