The cAMP-PKA pathway regulates prey sensing and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Abstract

Sensing environmental factors and responding swiftly to them is essential for all living organisms. For instance, predators must act rapidly once prey is sensed. Nematode-trapping fungi (NTF) are predators that use "traps" differentiated from vegetative hyphae to capture, kill, and consume nematodes. These traps undergo drastic and rapid morphological changes upon nematode induction. Multiple signaling hubs have been shown to regulate this remarkable process. Here, we demonstrate that the conserved cAMP-PKA signaling pathway exerts a crucial role in trap morphogenesis of the nematode-trapping fungi Arthrobotrys oligospora. A gene deletion mutant of the PKA catalytic subunit TPK2 proved insensitive toward nematode presence. Moreover, we show that the G protein alpha subunit GPA2 acts upstream of adenylate cyclase, with GPA2 deletion resulting in substantially reduced trap formation, whereas exogenous provision of cAMP rescued the prey-sensing and trap morphogenesis defects of a gpa2 mutant. Thus, we show that cAMP production triggered by G protein signaling and downstream PKA activity are vital for prey-sensing and trap development in A. oligospora, demonstrating that this highly conserved signaling pathway is critical for nematode-trapping fungi and nematode predator-prey interactions.

Keywords: Arthrobotrys oligospora; PKA; cAMP signaling pathway; nematode-trapping fungi; predator–prey interaction.

Fig. 1.

Disruption of the catalytic subunit Tpk2 of PKA results in defective trap morphogenesis in A. oligospora. a) Domain description of PKA catalytic subunits TPK1 and TPK2 in A. oligospora. Positions of the protein kinase domain (PROSITE entry #PS50011) and AGC-kinase C-terminal domain (AGC; PROSITE entry #PS51285) in the Tpk1 and Tpk2 protein sequences were determined using ScanProsite. b) A neighbor-joining phylogenetic tree of PKA catalytic subunit protein sequences from A. oligospora and orthologs from model fungi. Ani: A. nidulans. Ao: A. oligospora. Mor: M. oryzae. Ncr: N. crassa. Sce: S. cerevisiae. Uma: U. maydis. c) Normalized transcripts per million (TPM) of TPK1 and TPK2 in a time-course transcriptomic analysis in response to C. elegans nematodes. d) Colony morphologies of wild-type (ku70), tpk2 mutant, and the tpk2 TPK2-complemented strain after 4 days at 25°C on PDA plates (5 cm diameter). e) Conidiation by mutant and complemented strains was recorded after culturing for 4 days on PDA plates (scale bar, 200 μm). f) Trap induction of mutant and complemented strains. Trap formation was induced by adding 30 C. elegans nematodes to fungal cultures on LNM plates (2.5 cm), and images were taken 24 h after induction (scale bar, 200 μm). g) Close-up images of A. oligospora traps after 24 h of continuous nematode exposure. Vegetative hyphae and traps of A. oligospora were stained with SR2200, which specifically binds to fungal cell walls (scale bar, 20 μm). h) Nematode survival assay of mutant and complemented strains. Survival rate of nematodes for each timepoint was calculated by dividing the number of living nematodes by the total number of nematodes at timepoint zero.

Fig. 2.

G protein alpha subunit Gpa2 is vital for formation of a complete adhesive network. a) A neighbor-joining phylogenetic tree of Gα subunit protein sequences from A. oligospora and orthologs from model fungi. Ani: A. nidulans. Ao: A. oligospora. Bci: Botrytis cinerea. Cne: C. neoformans. Ncr: N. crassa. Pgr: Pyricularia grisea. Sce: S. cerevisiae. Uma: U. maydis. b) Colony morphologies of wild-type (ku70), gpa2 mutant, and gpa2 GPA2 complemented strains after 4 days on PDA plates. c) Conidiation by mutant and complemented strains after 4 days on PDA plates (scale bar, 200 μm). d) Trap induction of gpa2 mutant and complemented strains. Images were taken 24 h after induction (scale bar, 200 μm). e) Close-up images of traps of gpa2 mutant and complemented strains after 24 h of continuous nematode exposure. Vegetative hyphae and traps were stained with SR2200 (scale bar, 20 μm). f) Nematode survival assay of gpa2 mutant and complemented strains. Survival rate of nematodes for each timepoint was calculated by dividing the number of living nematodes by the total number of nematodes at timepoint zero.

Fig. 3.

Exogenous cAMP restores trap formation in the gpa2 mutant line. a) Trap induction by gpa2, tpk2, and gpb1 mutants on LNM and LNM supplemented with 5 mM cAMP or 5 mM IBMX (a nonspecific inhibitor of cAMP phosophodiesterases; 2.5 cm plates). Caenorhabditis elegans (n = 30) nematodes were added to each plate for 6 h and then washed away. Images were taken 24 h after nematode induction (scale bar, 200 μm). b) Trap quantification of the mutant strains on LNM and LNM supplemented with 5 mM cAMP or 5 mM IBMX (mean ± SEM). c) Close-up images of traps after 24 h of continuous nematode exposure. Vegetative hyphae and traps were stained with SR2200 (scale bar, 40 μm).

Fig. 4.

Hypothetical model of cAMP-PKA signaling during prey sensing and trap development in A. oligospora. Nematode-derived signals activate unidentified GPCRs of A. oligospora, leading to dissociation of the heterotrimeric G-protein complex and release of the Gα Gpa2. Gpa2 then activates the downstream adenylate cyclase Cyr1, leading to an increase in cAMP levels. cAMP binds to regulatory Bcy1 subunits of PKA to activate its Tpk2 catalytic subunits that phosphorylate the downstream substrates required for trap morphogenesis.