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. 2024 Nov 14;20(11):e1011320.
doi: 10.1371/journal.pgen.1011320. eCollection 2024 Nov.

cGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus

Affiliations

cGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus

Kenichi Nakayama et al. PLoS Genet. .

Abstract

Light sensing is a critical function in most organisms and is mediated by photoreceptor proteins and phototransduction. Although most nematodes lack eyes, some species exhibit phototaxis. In the nematode Caenorhabditis elegans, the unique photoreceptor protein Cel-LITE-1, its downstream G proteins, and cyclic GMP (cGMP)-dependent pathways are required for phototransduction. However, the mechanism of light-sensing in other nematodes remains unknown. To address this question, we used the nematode Pristionchus pacificus, which was established as a satellite model organism for comparison with C. elegans. Similar to C. elegans, illumination with short-wavelength light induces avoidance behavior in P. pacificus. Opsin, cryptochrome/photolyase, and lite-1 were not detected in the P. pacificus genome using orthology and domain prediction-based analyses. To identify the genes related to phototransduction in P. pacificus, we conducted forward genetic screening for light-avoidance behavior and isolated five light-unresponsive mutants. Whole-genome sequencing and genetic mapping revealed that the cGMP-dependent pathway and Ppa-grk-2, which encodes a G protein-coupled receptor kinase (GRK) are required for light avoidance. Although the cGMP-dependent pathway is conserved in C. elegans phototransduction, GRK is not necessary for light avoidance in C. elegans. This suggests similarities and differences in light-sensing mechanisms between the two species. Using a reverse genetic approach, we showed that gamma-aminobutyric acid (GABA) and glutamate were involved in light avoidance. Through reporter analysis and suppression of synapse transmission, we identified candidate photosensory neurons. These findings advance our understanding of the diversity of phototransduction in nematodes even in the absence of eyes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. P. pacificus exhibited avoidance behavior in response to light illumination.
(A, B) Results of light avoidance assays using P. pacificus (straight line) and C. elegans (dashed lines). UV, blue, green, or red (only B) light illuminated the head (A) or whole body (B) of the worms. C. elegans and P. pacificus responded to short wavelength light.
Fig 2
Fig 2. Conserved photoreceptor proteins were not identified in P. pacificus using a combination of orthology and domain prediction analyses.
(A) Search pipeline for known photoreceptors within the P. pacificus multiome. Using InterProScan/Pfam, protein domains were identified in P. pacificus proteomes and three photoreceptor families in other animals (Green). CRY/PL; Cryptochrome/Photolyase. Clustering of each photoreceptor family and all P. pacificus proteins into orthogroups was performed using OrthoFinder (Blue). PSI-/DELTA-BLAST and tBLASTn were performed to search for known photoreceptor proteins in the P. pacificus multiome (Blue). Finally, data outputs from OrthoFinder and BLAST were utilized to identify P. pacificus proteins that possess Pfam domains associated with photoreceptors. (B) Sequence alignment of candidate photoreceptor proteins identified by OrthoFinder to the opsin family. While the retinal-binding lysine K296 (Bos taurus, red box) is highly conserved among opsins, the candidate GPCRs in P. pacificus do not have the conserved lysine. The NPxxY motif, which is highly conserved among GPCRs in their seventh transmembrane domain, is colored with green, blue and orange. (C) Blue light avoidance assay for Ppa-sro-1 mutants. These mutants showed normal percentage of light avoidance. One-way ANOVA, Dunnett’s multiple comparison tests, compared with wild type. n.s. = not significant. (D) Sequence alignment of Cel-LITE-1/GUR-4/5 and Ppa-GUR-4/5 in the LITE-1 family. Critical amino acid residues (W77, R222, S226, C300, and W328) for photoreception in Cel-LITE-1 are not conserved in Ppa-GUR-4/5. Circles indicate candidate genes detected in the pipeline.
Fig 3
Fig 3. Light avoidance requires the cGMP-dependent pathway in P. pacificus.
(A) Blue light avoidance assay for light-unresponsive mutants that were isolated by forward genetic screening. These mutants had decreased light-induced avoidance response. (B) List of predicted causal genes. (C) Scheme of phototransduction in the C. elegans ASJ neurons. (D) Blue light avoidance assay for mutants of cGMP-dependent pathway genes. Mutants of guanylate cyclases, CNG channels, and PDEs had defect in light avoidance in P. pacificus. G-protein α-subunit mutants displayed normal light avoidance. One-way ANOVA, Dunnett’s multiple comparison tests, compared with wild type. n.s = not significant, **P < 0.01, ****P < 0.0001.
Fig 4
Fig 4. GPCR kinase, Ppa-GRK-2 is required for light avoidance.
(A) Schematic of GPCR desensitization by GRKs. After a ligand binds to a GPCR, G proteins are released from the activated GPCR to transmit signaling. GRKs phosphorylate the activated GPCR, promoting the binding of arrestin to the receptor. GPCR is endocytosed and desensitized. (B) Blue light avoidance assay for GRK and arrestin mutants in C. elegans. These mutants displayed normal light avoidance. The Cel-lite-1 mutants were used as a negative control. (C) Blue light avoidance assay for GRK and arrestin mutants in P. pacificus. Ppa-grk-2 mutants had decreased light avoidance. One-way ANOVA, Dunnett’s multiple comparison tests, compared with wild type. n.s = not significant, ***P < 0.001, ****P < 0.0001.
Fig 5
Fig 5. GABA and glutamate are required for light avoidance.
Blue light avoidance assay for neurotransmitter-related mutants. Ppa-unc-25 and Ppa-eat-4 mutants exhibit decreased light avoidance. One-way ANOVA Dunnett’s multiple comparison tests, compared with wild type. n.s = not significant, *P < 0.05, ****P < 0.0001.
Fig 6
Fig 6. Amphid neurons expressing guanylate cyclase, CNG channels, and GRK-2 are important for light avoidance.
(A–C) Representative fluorescence images of Ppa-tax-2p::RFP (A), Ppa-tax-4p::RFP (B), and Ppa-grk-2p::GFP (C) in the head region. Anterior is left and dorsal is up. (A–C) are maximum projection images. (C′) and (C′′) are single focal plane images. (D) Summary of the expression pattern of phototransduction genes. Check marks indicate that the reporter fluorescent proteins were expressed in the corresponding cells in more than 80% individuals. All four phototransduction genes we examined were expressed in AM1, 3, 4, 5, and 8 neurons (orange rows). The expression of Ppa-daf-11 was examined in a previous study [62]. Ppa-tax-2p::RFP; n = 17 (excbh23), n = 20 (excbh24), Ppa-tax-4p::RFP; n = 18, Ppa-grk-2p::GFP; n = 19. (E) Blue light avoidance assay for worms expressing tetanus toxin in AM1, 3, 4, 5, and 8 neurons using the Ppa-daf-11 promotor. The transgenic animals had decreased light avoidance. Student’s t-test. *P < 0.05.
Fig 7
Fig 7. Proposed regulatory models of light avoidance behavior in C. elegans and P. pacificus.
(A) In C. elegans, Cel-LITE-1, a photoreceptor protein in photosensory neurons (ASJ and ASK) receives light and transmits the signal to the downstream cGMP-dependent pathway. Signals from the photosensory neurons are transmitted to the interneurons and motor neurons. (B) In P. pacificus, unknown photoreceptor proteins in the photosensory neurons (AM1, 3, 4, 5, or 8) detect light and transmit the signal to the downstream cGMP-dependent pathway. Signals from photosensory neurons are transmitted to interneurons and motor neurons via glutamate or GABA, leading to the muscle contraction and avoidance behavior. GRKs may phosphorylate photoreceptor proteins.

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Grants and funding

This work was supported by JSPS KAKENHI (grant number 20K15903), AMED (grant number JP19gm6310003), JST FOREST Program (grant number JPMJFR214M), Tomizawa Jun-ichi & Keiko Fund of Molecular Biology Society of Japan for Young Scientist, Research Encouragement Award for Young Scientists (Hiroshima University), RIKEN-Hiroshima Univ Science & Technology Hub Collaborative Research Program, The Mitsubishi Foundation, Research grants in the Natural Sciences, Narishige Zoological Science Award, and Yamada Science Foundation to M.O., JSPS KAKENHI (grant numbers 21H02479 and 21K18236) to T.C., JSPS Research Fellows (grant number 21J21628) to K.N. and JST, the establishment of university fellowships towards the creation of science technology innovation, Grant Number JPMJFS2129 to H.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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