The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like Arthrobotrys oligospora or Duddingtonia flagrans produce adhesive trapping networks to catch and immobilize nematodes. After penetration of the cuticle, hyphae grow and develop inside the worm and secrete large amounts of hydrolytic enzymes for digestion. In many microbial pathogenic interactions small-secreted proteins (SSPs) are used to manipulate the host. The genome of D. flagrans encodes more than 100 of such putative SSPs one of which is the cysteine-rich protein CyrA. We have chosen this gene for further analysis because it is only found in NTF and appeared to be upregulated during the interaction. We show that the cyrA gene was transcriptionally induced in trap cells, and the protein accumulated at the inner rim of the hyphal ring before Caenorhabditis elegans capture. After worm penetration, the protein appeared at the fungal infection bulb, where it is likely to be secreted with the help of the exocyst complex. A cyrA-deletion strain was less virulent, and the time from worm capture to paralysis was extended. Heterologous expression of CyrA in C. elegans reduced its lifespan. CyrA accumulated in C. elegans in coelomocytes where the protein possibly is inactivated. This is the first example that SSPs may be important in predatory microbial interactions.

Orsay Virus Infection in Caenorhabditis elegans.

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: Synchronize C. elegans development by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

The cysteine-rich virulence factor NipA of Arthrobotrys flagrans interferes with cuticle integrity of Caenorhabditis elegans.

Animals protect themself from microbial attacks by robust skins or a cuticle as in Caenorhabditis elegans. Nematode-trapping fungi, like Arthrobotrys flagrans, overcome the cuticle barrier and colonize the nematode body. While lytic enzymes are important for infection, small-secreted proteins (SSPs) without enzymatic activity, emerge as crucial virulence factors. Here, we characterized NipA (nematode induced protein) which A. flagrans secretes at the penetration site. In the absence of NipA, A. flagrans required more time to penetrate C. elegans. Heterologous expression of the fungal protein in the epidermis of C. elegans led to blister formation. NipA contains 13 cysteines, 12 of which are likely to form disulfide bridges, and the remaining cysteine was crucial for blister formation. We hypothesize that NipA interferes with cuticle integrity to facilitate fungal entry. Genome-wide expression analyses of C. elegans expressing NipA revealed mis-regulation of genes associated with extracellular matrix (ECM) maintenance and innate immunity.

Caenorhabditis elegans neuropeptide NLP-27 enhances neurodegeneration and paralysis in an opioid-like manner during fungal infection.

The nervous system of metazoans is involved in host-pathogen interactions to control immune activation. In Caenorhabditis elegans, this includes sleep induction, mediated by neuropeptide-like proteins (NLPs), which increases the chance of survival after wounding. Here we analyzed the role of NLP-27 in the infection of C. elegans with the nematode-trapping fungus Arthrobotrys flagrans. Early responses of C. elegans were the upregulation of nlp-27, the induction of paralysis (sleep), and neurodegeneration of the mechanosensing PVD (Posterior Ventral Process D) neurons. Deletion of nlp-27 reduced neurodegeneration during fungal attack. Induction of nlp-27 was independent of the MAP kinase PMK-1, and expression of nlp-27 in the hypodermis was sufficient to induce paralysis, although NLP-27 was also upregulated in head neurons. NLP-27 contains the pentapeptide YGGYG sequence known to bind the human µ- and κ-type opioid receptors suggesting NLP-27 or peptides thereof act on opioid receptors. The opioid receptor antagonist naloxone shortened the paralysis time like overexpression of NLP-27.

Fatal attraction of Caenorhabditis elegans to predatory fungi through 6-methyl-salicylic acid.

Salicylic acid is a phenolic phytohormone which controls plant growth and development. A methyl ester (MSA) derivative thereof is volatile and involved in plant-insect or plant-plant communication. Here we show that the nematode-trapping fungus Duddingtonia flagrans uses a methyl-salicylic acid isomer, 6-MSA as morphogen for spatiotemporal control of trap formation and as chemoattractant to lure Caenorhabditis elegans into fungal colonies. 6-MSA is the product of a polyketide synthase and an intermediate in the biosynthesis of arthrosporols. The polyketide synthase (ArtA), produces 6-MSA in hyphal tips, and is uncoupled from other enzymes required for the conversion of 6-MSA to arthrosporols, which are produced in older hyphae. 6-MSA and arthrosporols both block trap formation. The presence of nematodes inhibits 6-MSA and arthrosporol biosyntheses and thereby enables trap formation. 6-MSA and arthrosporols are thus morphogens with some functions similar to quorum-sensing molecules. We show that 6-MSA is important in interkingdom communication between fungi and nematodes.