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cGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus [1]

['Kenichi Nakayama', 'Program Of Biomedical Science', 'Graduate School Of Integrated Sciences For Life', 'Hiroshima University', 'Hiroshima', 'Hirokuni Hiraga', 'Aya Manabe', 'Program Of Basic Biology', 'Takahiro Chihara', 'Misako Okumura']

Date: 2024-12

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.

Author summary Nematodes are a highly diverse group of animals found in a wide variety of habitats and sensory systems. In particular, light-induced behavior has been found to differ among species. The photoreceptor protein and its downstream pathways in Caenorhabditis elegans have been identified, revealing unique and distinct characteristics compared to those in other animals. However, the mechanisms of photoreception in other nematodes remain largely unknown. This study focused on the analysis of the photoreception mechanisms in Pristionchus pacificus, a species for which many genetic and molecular tools are available. Similar to C. elegans, P. pacificus also exhibits light avoidance behavior towards short-wavelength light; however, known animal photoreceptor genes could not be identified in the P. pacificus genome using bioinformatic approaches. Using forward and reverse genetic approaches, we found that certain genes and neurons are required for light avoidance, some of which are conserved in C. elegans photoreception. These results suggest that the light-sensing mechanisms of C. elegans and P. pacificus are similar, yet there are differences between the two species. These findings highlight the various light-sensing mechanisms in nematodes.

Citation: Nakayama K, Hiraga H, Manabe A, Chihara T, Okumura M (2024) cGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus. PLoS Genet 20(11): e1011320. https://doi.org/10.1371/journal.pgen.1011320 Editor: Andrew D. Chisholm, University of California San Diego, UNITED STATES OF AMERICA Received: May 24, 2024; Accepted: September 30, 2024; Published: November 14, 2024 Copyright: © 2024 Nakayama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All materials generated in this study, including plasmids and worm strains, are available upon request to the corresponding author ([email protected]). The data underlying this article are available in the paper and its supporting information. Whole-genome sequencing data are available in DDBJ at https://www.ddbj.nig.ac.jp/index-e.html, and can be accessed with PRJDB 18138. 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. Competing interests: The authors have declared that no competing interests exist.

Introduction Light sensing is important for many animals that use visual information to avoid predators or unfavorable environments, and to find food sources or mating partners. Most animals, including Cnidaria, Ctenophora, and Bilateria, utilize opsins, which belong to the G protein-coupled receptor (GPCR) superfamily, as photoreceptors and downstream signaling pathways. For example, in vertebrate rods and cones, light is absorbed by the retinal chromophore, which binds to opsins. Isomerization of the retinal chromophore causes a conformational change in opsins, which activates downstream signaling pathways such as G proteins and phosphodiesterases (PDEs). This results in a decrease in cyclic GMP (cGMP) levels and the closure of cyclic nucleotide-gated (CNG) channels. Opsins and their downstream signaling pathways have been extensively studied in a wide range of animals, including aspects such as protein structure, signaling mechanisms, and evolution [1,2]. However, opsin-independent phototransduction mechanisms are limited in the animal kingdom, and the details of these mechanisms remain unclear. Some nematodes have been observed to exhibit photoresponses despite the absence of eyes [3–6]. In particular, the nematode Caenorhabditis elegans displays various responses to short-wavelength light, including avoidance behavior [7,8], stopping pharyngeal pumping [9], and spitting out food [10,11]. Furthermore, C. elegans can discriminate between colors [12,13]. Forward genetic screening using light-avoidance behavior has identified a novel photoreceptor protein, Cel-LITE-1 [14,15]. In silico prediction of the protein structure and in vivo ectopic expression analysis suggest that Cel-LITE-1 is a member of the 7-transmembrane-domain ion channels (7TMICs) and forms a tetramer [16,17]. Similar to GPCRs, 7TMICs have seven transmembrane domains, but their membrane topology is opposite to that of GPCRs, with the N- and C-termini located intracellularly and extracellularly, respectively [17–21]. Putative binding sites for the chromophore and the aromatic amino acids necessary for light absorption have been identified [16,19]. Although Cel-LITE-1 is not predicted to be a GPCR, downstream phototransduction of Cel-LITE-1 in C. elegans ASJ neurons, one of the photosensory cells, requires G proteins and the cGMP-dependent pathway (Fig 3C) [7,15]. It is predicted that Cel-LITE-1 transduces light stimuli mediated by G-protein α-subunits (Cel-GOA-1 and Cel-GPA-3) and guanylate cyclases (Cel-DAF-11 and Cel-ODR-1), resulting in the production of cGMP. Elevated cGMP levels lead to the opening of CNG channels (Cel-TAX-2 and Cel-TAX-4), causing an influx of calcium ions into the cell. In contrast to vertebrate rods and cones, PDEs (Cel-PDE-1, Cel-PDE-2, and Cel-PDE-5) are not necessary for light response in C. elegans [15], suggesting a unique opsin-independent mechanism of phototransduction. However, the mechanism of the light response in other nematode species that lack conventional opsins and LITE-1 remains unknown. The diplogastrid nematode Pristionchus pacificus has been established as a satellite model organism for comparison with C. elegans [22–24]. Several genetic tools have been developed for P. pacificus, such as an annotated genome [25,26], forward and reverse genetics [22,27], and synaptic connectome in pharynx and head neurons revealed using electron microscope [28–30], which provide a suitable model to understand its neural response and behavioral evolution [31–40]. Both C. elegans and P. pacificus have 12 pairs of amphid neurons, and putative amphid neuronal homologs have been identified between these two species [28]. However, it is likely that the functions of amphid neurons differ between the two species, which is supported by the fact that ciliary terminal structures and the expression of amphid neuron-specific genes vary between the two species [28]. It is currently unknown whether P. pacificus has the ability to sense and respond to light. Here, we found that P. pacificus avoids short-wavelength light, although we did not find a conventional opsin, cryptochrome/photolyase, or lite-1 in the P. pacificus genome. Forward genetic screening revealed that the cGMP-dependent pathway and a GPCR kinase (GRK) are necessary for light avoidance. In addition, a reverse genetic approach has shown that the neurotransmitters gamma-aminobutyric acid (GABA) and glutamate play a role in light avoidance. These genes were expressed in five amphid neurons, and the inhibition of neurotransmission in these amphid neurons reduced light avoidance.

Methods Strains The strains used in this study are listed in S4 Table. C. elegans and P. pacificus were maintained at 20°C on Nematode Growth Medium (NGM) agar plates with Escherichia coli OP50 as previously described [23,70]. Light avoidance assay The light avoidance assay was conducted as previously described [7] with some modifications. One-day adult hermaphrodite worms were placed individually on NGM plates covered with a thin bacterial lawn of freshly seeded OP50 and left in the dark for at least 10 min before the assay. For head illumination, in Fig 1A, a fluorescence stereomicroscope (Leica, 165 FC) was connected to a mercury lamp (Leica, EL6000) and the head of the nematode that moved forward was illuminated using a fluorescence filter and an objective lens (Leica, 10450028). Light intensity was adjusted by manipulating the amount of light emitted from the mercury lamp. The following fluorescence filters and wavelengths were used: UV (350 nm), Leica ET UV LP, 1045609; blue (470 nm), Leica ET GFP, 10447408; green (545 nm), Leica ET DSR, 10447412. For other experiments, the LED light source from a fluorescence stereomicroscope (ZEISS, Discovery V20) was illuminated through a fluorescence filter (ZEISS, filter set 38 HE, 470±20 nm, 0.24 mW/mm2). For whole-body illumination, light from an LED source (Optocode, LED-EXSA) was delivered to the entire body of each nematode. To use light of different wavelengths, we changed the LED head accordingly (red, EX-660; green, EX-530; blue, EX-450; UV, EX-365). When nematodes ceased forward movement and began backward movement within 5 seconds after light irradiation, it was considered as “light-avoidance behavior.” In a previous C. elegans study [7], light-avoidance behavior was defined as backward movement within 3 s of light irradiation. However, in this study, we defined it as within 5 s because the locomotion speed of P. pacificus is slower than that of C. elegans. In Fig 3C, because only C. elegans was assayed, backward movement within 3 s was defined as light-avoidance behavior. For each individual, the light avoidance assay was performed five times at 10 min intervals after each assay. A red filter (Kenko, 158371) was used to minimize the impact of white light from the lower part of the microscope. All assays were performed in a blinded manner. The light intensity was measured using an optical power meter (HIOKI, 3664) with an optical sensor (HIOKI, 9742) divided by the illuminated area. Except for Fig 1, the intensity of blue light (470 nm) was 0.24 mW/mm2 (P. pacificus) or 1.83 mW/mm2 (C. elegans) for the light avoidance assay. Protein sequence collection We used previously published datasets and BLASTP sequence searches to collect a reference dataset for photoreceptor protein families. Specifically, for the opsin and cryptochrome/photolyase families, we utilized the data collected by Gühmann et al, 2022 [44] and Deppisch et al, 2022 [45]. For the LITE-1 family, we performed BLASTP (https://parasite.wormbase.org/Multi/Tools/Blast) searches on all nematode species registered in WormBase ParaSite (Version: WBPS18, https://parasite.wormbase.org) [46] using the C. elegans LITE-1 (WBGene00001803) protein sequence as query. From this result, we obtained 88 sequences using BioMart (https://parasite.wormbase.org/biomart/martview/). Furthermore, we added the sequences of Cel-EGL-47 (WBGene00001211), Cel-GUR-3 (WBGene00001804), and D. melanogaster Gr28b (FBgn0045495, from FlyBase; https://flybase.org) [71] which are registered as paralogs or orthologs of Cel-LITE-1 in WormBase (Version: WS291, https://wormbase.org) [72,73] to the LITE-1 family (S1 Data). These three protein sequence files were used as inputs for subsequent analyses. Exploration of putative photoreceptor proteins Protein domain searches were performed on the photoreceptor protein reference dataset and all proteins of P. pacificus based on the Pfam database (version 36.0) [74] using InterProScan (version 98.0, option: -dp -appl Pfam) [75]. Protein domains that were functionally important as photoreceptor proteins in the reference dataset were isolated and searched in all protein domain dataset of P. pacificus. Orthology clustering with OrthoFinder (Version: 2.5.5, option: -S blast -M msa) [76] was performed on the three photoreceptor protein reference datasets and the P. pacificus proteome (El paco V3, http://pristionchus.org) [77] to obtain orthogroup data (S1 Table). Orthogroups from OrthoFinder and domain prediction data from InterProScan were integrated to search for photoreceptor protein candidates from all proteins of P. pacificus. Given that BLASTP, as used in OrthoFinder, may not detect remotely homologous genes, we performed PSI- and DELTA-BLAST (BLAST+, Version: 2.15.0; option: -comp_based_stats 1) for searching photoreceptor protein families in the proteome of P. pacificus (S2 Table). PSI- and DELTA-BLAST searches were iterated three times with the threshold set at 1e-3. To determine whether any traces of known photoreceptor proteins remained in the genome, we performed tBLASTn (BLAST+, Version: 2.15.0) (option: -evalue 1e-3) on the genome and transcriptome of P. pacificus, using each photoreceptor protein reference dataset as a query. Sequence alignment The protein sequences used for sequence alignment were candidate protein sequences from a homology search using OrthoFinder/BLAST and protein domain prediction using InterProScan. Sequences were aligned with MAFFT (version 7.525) [78] using the "—auto" option, specifically employing the L-INS-i method. The aligned sequences were visualized in R (Version 4.3.2) using the ggmsa package (Version: 1.3.4) [79]. Genetic screen for light-unresponsive mutants P. pacificus PS312 was mutagenized with ethyl methanesulfonate (EMS), as described previously [80]. Two methods were used to screen the light-unresponsive mutants. In the first method, the F1 worms were individually transferred onto E. coli plates. When the F2 animals reached the adult stage, a light-avoidance assay was conducted once per individual, with 10 individuals per plate. F2 strains exhibiting light avoidance at a frequency of 40% or less were selected, and F2 worms were individually transferred to E. coli plates. After a few days, the F3 worms were again tested for light avoidance. The mutants with impaired locomotion were excluded. In the second method, plates containing P0 were left for several days and F2 or F3 individuals were transferred individually to E. coli plates. Subsequently, primary and secondary screening were conducted in the same manner as in the first method. We screened more than 20,000 mutagenized F2 strains. Whole genome sequence Five 6 cm NGM plates containing many adult worms were prepared. After collecting and washing the nematodes with M9 buffer, genomic DNA was purified using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, G1N10). Whole genome sequencing (WGS) was performed by BGI JAPAN. The WGS data were mapped based on the method described by Rödelsperger et al, 2020 [81] to identify mutation sites. Briefly, Illumina read data were aligned to the El Paco genome assembly [26,77] using the BWA Mem program [82]. The initial variant call was generated using the mpileup command in BCFtools [83]. Genetic mapping Recombinant lines used for genetic mapping were obtained by crossing light-unresponsive mutants (derived from PS312) with a male wild-type strain RSA076. Light-unresponsive F2 individuals were isolated using a light avoidance assay. After laying eggs, F2 individuals were lysed using a worm lysis buffer. To confirm the light insensitivity of the F2 individuals, a light avoidance assay was repeated on the F3 individuals. Primers were designed around marker sequences from Pristionchus.org (http://pristionchus.org), which contained insertions or deletions in PS312 and RSA076. PCR with Dream Taq Green PCR Master Mix (Thermo Fisher Scientific, K1081) was used to determine the genotype. The primers used for chromosome mapping are listed (S5 Table). Some primers were adapted from a previous study [84]. CRISPR/Cas9 mutagenesis To generate CRISPR knock-out mutants, we followed previously described co-injection marker methods [85–87]. The CRISPR target sequences were designed using CHOP-CHOP v3 (http://chopchop.cbu.uib.no/) [88]. All tracrRNAs, crRNAs, and Cas9 proteins were synthesized by Integrated DNA Technologies (IDT). We mixed 0.5 μl of the Cas9 protein (10 μg/μl), 0.95 μl of crRNA (100 μM), and 0.9 μl of tracrRNA (100 μM), and incubated the mixture at 37°C for 15 minutes. Using the co-CRISPR system, we combined each RNP complex containing the gRNA of Ppa-prl-1 and a target gene. For the fluorescence marker method, we added Ppa-egl-20p::turboRFP or Ppa-eft-3p::turboRFP (50 ng/μl) to the RNP complex and diluted with nuclease-free water up to 20 μl. The injection mixtures were microinjected into the gonads of young adult worms. The injected worms (P0) were placed individually on NGM plates. Approximately 24–48 hours later, P0 worms were removed from the plate. After 3–4 days, the F1 worms were screened for the presence of a roller phenotype or fluorescent worms. For mutation screening, a heteroduplex mobility assay was performed using microchip electrophoresis on MultiNA (Shimazu, MCE-202) or the DNA gel separation improvement agent Loupe 4 K/20 (GelBio). Sanger sequencing by Eurofins Genomics was used to determine the genotype. The identified mutants were subsequently backcrossed with the original wild-type strain (PS312) for at least three generations to eliminate off-target effects. The target sequences of the gRNA and primers are listed in S5 Table. Generating transgenic lines The promoter regions were amplified using KOD One PCR Master Mix (TOYOBO, KMM-101). The lengths of the promoter sequences were as follows: Ppa-daf-11: 794 bp; Ppa-tax-2: 2401 bp; Ppa-tax-4: 3001 bp; Ppa-grk-2: 3001 bp. The promoter for Ppa-daf-11 was constructed as described in a previous study [62]. For Ppa-tax-2, we used a region predicted to be a promoter in a previous study [89]. For Ppa-tax-4 and Ppa-grk-2, we obtained a sequence of 3001 bp sequence upstream from the start codon. These promotors were cloned into vector containing codon optimized GFP, TurboRFP or tetanus toxin and Ppa-rpl-23 3’UTR [86]. The plasmids and genomic DNA of PS312 were digested using HindIII (pMO56, pMO59, and pMO81) or PstI (pMO74). These transgenes (3–5 ng/μl), Ppa-egl-20p::RFP or Ppa-egl-20p::GFP (50 ng/μl) as co-injection markers, and genomic DNA (60 ng/μl) were injected into the gonad of young adult worms. The transgenic animals were screened under a fluorescence microscope (Leica, M165 FC or ZEISS, Discovery V20). Dye-filing To identify the cell type of the amphid neurons (Figs 6A–6D and S1A–S1D) and assess dye-filing defects (S2A–S2F Fig) in amphid neurons, we followed previously described staining methods using the lipophilic dye DiI Stain (Thermo Fisher Scientific, D3911) or Fast DiO Solid (Thermo Fisher Scientific, D3898) [28,39,40]. Well-fed J2 or J3 larvae (for cell identification) or adult (for dye-filing assay) were collected in M9 buffer and centrifuged at 1500 × g for 2 min. After discarding the supernatant, worms were incubated with 150 μl of M9 containing a 1:150 dilution of FastDiO or a 1:74 dilution of DiI for 1.5–3 h at 20°C. The nematodes were washed three times with 1 ml of M9 buffer and crawl freely on E. coli seeded NGM plates for more than 1 h. Worms were immobilized on 2% agarose pads containing 5 mM levamisole or 0.3% sodium azide and covered with a cover slip. Z-stack images were obtained using a confocal microscope (Zeiss, LSM900). The cell type of the amphid neurons was identified by analyzing the positional relationship between the stained cells and cells expressing the fluorescent protein. Reporter-positive cells were defined if the reporter fluorescence was observed in those cells in more than 80% of the individuals. To evaluate the dye-filing defect, maximum projections were generated using Fiji software [90]. A clear DiI signal in the cell bodies of amphid neurons was counted as a positive staining. Statistical analysis The Prism software package GraphPad Software 9 was used for statistical analyses. Information about the statistical tests, p-values, and n numbers is provided in the respective figures and figure legends. All error bars show SEM.

Acknowledgments We thank Ms. Masako Shigemori and Ms. Satoko Okazaki (Hiroshima University) for their technical support. We thank Dr. Kozue Hamao (Hiroshima University), Dr. Toshiaki Kozuka (Kanazawa University), and Dr. Takuma Sugi (Hiroshima University) for their advice regarding this study. Nematode strains and plasmids were obtained from the Caenorhabditis Genetics Center and Dr. Ralf J. Sommer (Max Planck Institute for Biology Tübingen, Germany). This study was conducted at the Natural Science Center for Basic Research and Development at Hiroshima University. We also thank all the members of the Chihara laboratory for their kind support and Editage (www.editage.jp) for English language editing.

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