Rice (Oryza sativa L.) is a major staple food crop worldwide. Drought stress induced by extreme weather severely limits its growth and yield. Proteins containing domains of unknown function (DUF) play important roles in plant stress responses, but their regulatory mechanisms remain largely unclear. In this study, we demonstrate that the plasma membrane-localized protein OsDUF1223 functions as a negative regulator of drought tolerance in rice seedlings. Under drought stress, OsDUF1223-overexpressing lines exhibited lower survival rates, higher water loss rates, and reduced antioxidant enzyme activities and osmolyte accumulation. Transcriptomic and physiological analyses revealed that OsDUF1223 modulates drought responses through ABA signaling and nitrogen response pathways. Collectively, this study elucidates that OsDUF1223 regulates drought tolerance at the seedling stage in rice through functional association with the ABA and nitrogen signaling pathways, thereby providing a candidate gene resource for the genetic improvement of drought tolerance in rice.
Post-transcriptional control by RNA binding proteins (RBPs) and microRNAs play central roles in mRNA stability and translation, yet how RBPs and microRNAs coordinate in developmental time to regulate cell fate remains poorly understood. Here, we demonstrate that post-transcriptional regulation of the Profilin 2 (Pfn2) transcript is essential for differentiation of embryonic stem cells (ESCs) into the primary germ layer lineages. The Pfn2 3’untranslated region has both an Iron Regulatory Protein binding site (IRE) and a nearby binding site for ESC enriched microRNAs. Deletion of this microRNA site leads to increased PFN2 and reduced FGF signaling during pluripotency transition prior to germ layer formation. In contrast, deletion of the IRE leads to decreased PFN2, a Wnt signaling defect, reduced nuclear beta-catenin, and a subsequent block in mesendodermal lineages during early germ layer formation. We further find that loss of the IRE site results in a cell autonomous defect in Wnt signaling and mesendodermal differentiation. The IRE site acts to stabilize beta-catenin, as disruption of the site leads to reduced nuclear beta-catenin levels. Together, these findings reveal the Pfn2 microRNA-IRE regulatory axis as a critical post-transcriptional regulatory node governing the switch from pluripotency to somatic differentiation.
A major determinant of the exceptional intrinsic resistance of M. abscessus is the lipid-rich cell envelope, yet the regulatory systems that remodel envelope-associated pathways remain poorly defined. Here, we determine the σ D regulon in M. abscessus and establish its role in cell envelope homeostasis and intrinsic resistance to hydrophobic antibiotics. RNA-Seq analysis of a MabΔ sigD mutant identified 447 differentially expressed genes, while ChIP-Seq mapped 72 σ D binding sites and defined a conserved promoter motif (GTAACA/G-N 16 -CGAT). Using a combination of σ D binding, motif orientation and expression data, we identified a core set of directly regulated genes, distinct from what was previously observed in M. tuberculosis , many of which encode proteins involved in envelope-associated functions. These include loci involved in trehalose polyphleate (TPP) biosynthesis, the antigen 85 complex and peptidoglycan remodeling enzymes. Deletion of sigD resulted in a significant reduction in TPPs in the cell envelope and an increase in ethidium bromide accumulation. Consistent with these changes, loss of σ D selectively sensitized M. abscessus to hydrophobic antibiotics, including rifampicin and tigecycline. Deletion of mmpL10 , which is required for transport of TPP precursors, recapitulated the drug sensitivity of MabΔ sigD , implicating envelope composition as a key effector of the phenotype. Expression of the σ D regulon further increased during starvation and in response to SDS, isoniazid, and ethambutol, mediated by degradation of RsdA, consistent with a role in stress-responsive envelope adaptation. Together, these findings demonstrate σ D is active during logarithmic growth in rich media where it regulates the expression of envelope-associated genes that influence envelope permeability and basal level susceptibility to hydrophobic antibiotics; its activity further increases in response to cell envelope stress, presumably promoting envelope remodeling to counteract damage.
Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.
Plant organ development involves coordinated cell fate transitions across multiple tissues, yet the cellular programs underlying organ-specific differentiation in woody plants remain poorly understood, particularly the mechanisms limiting efficient root development during vegetative propagation of oak species. Here, we generated a comprehensive single-cell transcriptomic landscape of leaf, stem, and root tissues of Quercus robur to resolve developmental trajectories at cellular resolution. A total of 41,471 high-quality cells were classified into 30 distinct clusters, enabling the identification of major cell types and organ-specific transcriptional features across three vegetative organs. Pseudotime analyses exhibited the developmental programs related to guard cell differentiation in leaves, vascular formation in stems, and root tissue development. Additionally, combining scRNA-seq, bulk transcriptome profiling, and phytohormone investigations, we identified auxin signaling as an important regulator during adventitious root development process. Notably, QrIAA14-1, an IAA14 homolog, was preferential enrichment in root hair, near-root hair cells and root cap along root developmental trajectories, which was further supported by RT-qPCR and in situ hybridization assays. Furthermore, the overexpression of QrIAA14-1 significantly inhibited oak root elongation, resulting in around 64.46% reduction in adventitious root length compared with control plants, providing mechanistic insight into the limitations of root development in oak. Together, this study provides the first high-resolution single-cell atlas of cellular organization and developmental dynamics across oak vegetative organs and identifies the candidate regulator genes associated with root development, offering new insights into the regulatory mechanisms of woody plant root regeneration and clonal propagation.
Polyurethane (PU) is a synthetic polymer characterized by highly stable urethane linkages that hinder biological turnover. Although fungi have been implicated in PU degradation, the molecular mechanisms that couple polymer surface sensing to enzymatic depolymerization remain largely undefined. Here we show that the marine-derived fungus Alternaria alternata FB1 employs a surface-sensing signaling pathway that drives appressorium-mediated degradation of both polyester and polyether PUs. Contact with hydrophobic polymer surfaces rapidly induces melanized appressoria that mechanically penetrate the polymer matrix and promote its oxidative and hydrolytic depolymerization. Integrative transcriptomic analysis and targeted gene disruption identify the mucin-like surface sensor Msb2 as an upstream component of the polyurethane surface-sensing machinery. Loss of Msb2 disrupts MAPK and Ca2+ signaling, impairs appressorium differentiation, and reduces expression of degradative enzymes. Biochemical profiling further reveals multiple urethane-hydrolyzing enzymes that expand the known catalytic repertoire for PU bond cleavage. Together, these findings establish a mechanistic framework linking surface recognition, appressorium development, and polymer degradation, providing insight into how fungi transform recalcitrant polyurethane materials.
Biological clocks allow organisms to anticipate cyclical environmental changes, yet in high-latitude or deep-sea habitats, the diel cues that entrain these rhythms are often seasonally diminished or absent. Fishes of the order Perciformes have rapidly diversified across these arrhythmic ecosystems, raising the question of whether changes to circadian rhythms and biological clock genetic architecture are a component of their evolutionary success. Here, we used a comparative genomic approach to investigate patterns of core biological clock gene loss across 96 perciform and five outgroup species. We found widespread and lineage-specific loss in core clock genes, particularly in the convergently evolving polar and deep-sea suborders Notothenioidei and Cottoidei. This trend of clock gene loss was significantly amplified with higher-latitude species. To determine if these genomic signatures reflect a functional loss of rhythmicity, we performed metabolic phenotyping on three notothenioid species. We found a consistent lack of circadian metabolic oscillations during the late austral fall across all notothenioids, including the sub-Antarctic sister lineage to the cryonotothenioid adaptive radiation, Eleginops maclovinus. Experimental data across Perciformes, combined with suborder-wide patterns of gene loss, suggest that a release from circadian constraints occurred early in their diversification, potentially facilitating the repeated expansion of these fishes into polar and deep-sea habitats.
Fishes host a diverse microbiome in their gills, but a broad characterization of this microbiome at the metagenomic level is lacking. Here, we apply genome-resolved metagenomics to the gills of the hamlets (Hypoplectrus spp), a group of reef fishes from the Greater Caribbean. The analysis of 353 gill samples from 15 hamlet species collected at eight locations over 13 years revealed a stark contrast between the gill microbiota and reef water microbial communities, indicating a distinct and specific gill microbiome. A total of 70 gill-associated metagenome-assembled genomes (MAGs) were recovered. These MAGs belong to 17 lineages, most of which are novel. They relate to known fish gill pathogens, fish gut microbes, free-living and biofilm-associated taxa, indicating that the gill microbiome was assembled from a collection of distinct eco-evolutionary trajectories. The MAGs harbor diverse metabolic modules, involved notably in nitrogen cycling, antibiotic production and biofilm formation, revealing a highly dynamic microbial ecosystem. One lineage in the Burkholderiaceae family was outstandingly prevalent across fish host species, sampling locations and years. Its genome encoded complete metabolic modules for carbon fixation and sulfur oxidation, indicating chemosynthetic potential. To the best of our knowledge, this is the first line of evidence that fishes may host sulfur-oxidizing chemosynthetic bacteria in their gills. The functional significance of this chemosynthetic potential for the fish host or other members of the gill microbiome remains to be established. The high prevalence of this lineage allowed to build a pangenome. It revealed large-scale geographic structure (western Caribbean, eastern Caribbean and Gulf of Mexico), which parallels the phylogenomic pattern observed in the hamlets. Overall, our findings point to complex fish host-microbe and microbe-microbe eco-evolutionary interactions in the gills that may influence fish physiology, homeostasis and immune response.
The SKN-1/Nrf transcription factor family is integral to metabolic homeostasis. There are three SKN-1 isoforms in Caenorhabditis elegans , each with different functions. One of these, SKN-1B, is expressed specifically in neurons and controls hermaphrodite behaviour in response to food. In a laboratory environment C. elegans hermaphrodites must allocate energy efficiently between exploratory food-seeking behaviour, reproduction, and survival, whereas males have to balance both food-seeking with mate-seeking exploratory behaviours. Here, we evaluate the role of SKN-1B in male behaviour and show that SKN-1B acts in a sexually dimorphic manner to control exploratory behaviour, mitochondrial morphology, and mitochondrial function. Moreover, we show that SKN-1B is expressed in a sexually dimorphic pattern, and that SKN-1B influences gene expression on a much broader scale in males compared to hermaphrodites. This analysis identified a neuronal signalling pathway, specifically in males between SKN-1B, the TGF-β ligand DAF-7 and the chemosensory regulator ODR-10, and that this pathway contributes to increased exploratory behaviour in skn-1b mutant males. Taken together our findings identify SKN-1B as a sex-specific neuronal switch to control food searching and mating behaviours. Sexual dimorphism is essential for optimizing survival and reproductive success and our findings establish SKN-1B as a regulator of sex-specific behavioural and metabolic traits. This work provides new insights into neuronal regulation of metabolism and behaviour, with implications for sex-specific physiological adaptations in other species.
Ongoing climate change is driving unprecedented environmental fluctuations, with extreme events predicted to increase in frequency, intensity and duration. Among climatic parameters, temperature is expected to fluctuate the most by the end of the century and its elevation has already demonstrated to pose a major challenge to plant health. In this context, understanding how temperature modulates plant-pathogen interactions and their underlying genetic architecture is critical. Bacterial wilt, caused by strains of the Ralstonia solanacearum species complex, is a devastating disease affecting many plant species. Genetic resistance remains the most effective control strategy. In tomato, resistance is quantitative and mostly relies on quantitative trait loci (QTLs) bwr-6 and bwr-12 . However, as in other crops, high temperature and humidity can compromise this resistance in commercial tomato cultivars. We investigated temperature-dependent quantitative disease resistance (QDR) using a panel of 189 wild tomato accessions, predominantly Solanum pimpinellifolium , representing genetic diversity from contrasting ecological conditions. Disease progression was monitored from three to ten days post-inoculation at 28 °C and 32 °C, using a time-course phenotyping approach. Genome-wide association (GWA) analyses were performed, based on daily symptom scores and two reference genomes, to account for structural variations and improve QTL detection. This strategy identified 44 candidate genes and revealed a temporally dynamic genetic architecture of the plant response. Strikingly, no candidate genes were shared between temperatures, supporting distinct genetic determinants under different temperature conditions. Many candidate genes were expressed in roots and belong to gene families involved in plant immunity, with two candidates co-localizing with bwr-6 and bwr-12 , whose causal genes remain unknown. Altogether, our findings demonstrate that resistance to bacterial wilt in wild tomato is flexible, environment-dependent, and temporally dynamic, highlighting the importance of integrating environmental context and genomic diversity to better understand plant-pathogen interactions.
Tetraploid potato is the most important vegetable food crop in the world. However, genetic gains from modern breeding efforts have been limited due to tetrasomic inheritance, the accumulation of deleterious mutations during domestication, and subsequent clonal propagation during modern breeding. In this study, we analyzed previously identified evolutionarily constrained genomic regions in the potato genome relative to a breeding panel of tetraploid potato clones derived from elite cultivars (the MASPOT panel). We demonstrate a significant association between derived allele frequency (DAF) and both positive (constrained) and negative (excess of substitutions relative to neutrality, i.e., hypervariable) nucleotide conservation scores (GERP scores) in the breeding panel, indicating that negative GERP scores can annotate sites affected by positive selection or other non-neutral processes affecting population allele frequency. The deep phylogeny approach can be leveraged to alleviate inbreeding in potatoes through the qualification of causative alleles in tetraploids, offering a valuable resource for potato genetic improvement by molecular breeding. Estimates of genetic load within the tetraploid clones reveal that heterozygous loads vastly exceed homozygous loads for deleterious alleles, consistent with masking of recessive mutations. Deleterious load was significantly correlated with several trait phenotypes but did not improve prediction accuracy of any traits in the tetraploid panel. This could be due to the increased complexity of tetrasomic inheritance, including more effective load complementation, and/or a higher proportion of heterozygosity in tetraploid compared to diploid potato. Intriguingly, the hypervariable load correlated significantly with several phenotypes and improved genomic prediction accuracy of yield by 5% relative to a negative control (permuted loads). This indicates that the inferred hypervariable sites from Solanaceae evolutionary information may contain genetic information relevant for breeding, and that, in contrast to current practice, it is relevant to include and further explore sites with negative GERP scores in general.
Previous research suggests that social isolation induces changes in gene expression that encode a starvation-like brain state and reduce sleep. However, the extent to which social isolation alters behaviors via sex-specific brain changes is unclear. Here, we use Drosophila melanogaster to explore sex differences in isolation-induced behavioral and transcriptomic changes. Male and female adult flies were isolated for seven days, and multiple behavioral sex-based differences were identified through tests of activity, motivation, aggression, and sugar preference. RNA-seq analysis also identified several candidate genes that were associated with sex differences in isolation-induced behavioral changes. Takeout (to) expression and sucrose selection were upregulated exclusively in females following social isolation. Following to knockdown in to-expressing cells, sucrose preference decreased in socially isolated females but increased in males. Overall, our results suggest that manipulating to expression influences sucrose choice in opposite directions between females and males following social isolation. It is possible that isolation-induced to overexpression in non-neuronal cells in the female head could contribute to sex differences in this behavior.
Acting as the central endocrine hub, the pituitary gland is closely related to the mechanism of adaptation to high-altitude hypoxia. Here, by integratively combining long-read (Oxford Nanopore) and short-read (Illumina) single-cell sequencing approaches, we profiled pituitaries from Diqing Tibetan pigs, inhabiting high-altitude environments (3,200 m) and Diannan small-ear pigs from low-altitude regions (500 m), thereby generating a full-length single-cell atlas, which in turn enabled the identification of molecular mechanisms potentially underlying adaptation to high-altitude hypoxia stress. Systematically delineating pituitary structure and transcriptional dynamics, we profiled 27,339 single cells encompassing 28,932 expressed genes. Leveraging unsupervised clustering coupled with marker-based annotation, we identified ten major cell types. Expression profiling of 20 canonical marker genes revealed pronounced cell-type-specific expression patterns, ten of which were independently validated by immunofluorescence, thus substantiating the accuracy of cell-type annotation. Gene-ontology enrichment analysis further suggested that upregulated genes were predominantly involved in oxidative metabolism and energy production, whereas downregulated genes were significantly associated with protein biosynthesis and translation processes, indicating a functional reprogramming of metabolic pathways. Moreover, comparative analyses between breeds and cell-cell communication analyses highlighted pathway shifts, including broad upregulation of the collagen family and four ligand-receptor pairs that may mediate pituitary intercellular coordination. In parallel, transcription-factor activity analysis nominated several regulators, including EBF3, DBX2, and TCF21, which may collectively contribute to altitude-associated adaptation. Finally, integrating long-read sequencing data revealed widespread transcript isoform diversity, with ~28% novel annotations, indicating considerable isoform complexity in pigs. Our findings delineate cellular heterogeneity, inferred intercellular communication networks, and transcript-isoform diversity in the porcine pituitary, thereby providing a cell-resolved resource for understanding pituitary features associated with high-altitude environments.
Abscisic acid (ABA) acts as a key signalling molecule that mediates plant responses to environmental cues as well as plant growth and development. Stress-induced and developmental changes in ABA content trigger a myriad of post-transcriptional and transcriptional events. Yet, ABA-dependent transcriptional responses are context dependent, and their temporal dynamics in roots under non-stress conditions remain poorly resolved. In this study, we characterised the hallmarks of ABA signalling and responses in the poplar root transcriptome (Populus nigra L.). We disturbed ABA homeostasis by exogenous ABA treatments, and we combined time-resolved transcriptomics with unsupervised gene network analysis to identify ABA-activated and ABA-inactivated gene co-expression modules. Considering the temporal dynamics of transcriptional events, we predicted the primary targets of the ABA signal, characterised early responding ABA-dependent processes, identified hub genes and revealed their putative functional links. We demonstrated that the properties of a master ABA-activated module induced by exogenous treatments were preserved in the transcriptome response to osmotic stress, revealing a core gene set of ABA-dependent stress responses. Our work sheds light on ABA repression of gene expression, the reprogramming of metabolism and the leaf-senescence pathway. Based on current functional knowledge and phylogenetic information, including poplar-specific features, we proposed a working model of ABA action on root transcriptome in poplar that integrates master genes, key responsive processes, and their putative regulatory architecture.
The most common genetic cause of both familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G 4 C 2 repeat in the first intron of the gene C9orf72. The C9orf72 repeat expansion is bidirectionally transcribed into sense and anti-sense RNA foci, and also produces dipeptide repeats (DPRs) via a non-canonical translation mechanism known as repeat-associated (RAN) translation. Each of these components of the G 4 C 2 repeat expansion cause neurodegenerative effects in animal models when expressed in neurons, but impacts from glial expression are more poorly understood. Here, we use glial cell type-specific expression of individual DPRs, of RNA repeat-only, or of the G 4 C 2 repeat, that produces both DPRs and RNA repeats, to systematically investigate glial toxicity of each component. We find that as with neurons, the GR and G 4 C 2 transgenes produce the highest degree of toxicity when expressed in glia. Each of these transgenes are capable to produce the GR DPR, which also is the most toxic factor in neurons. We demonstrate that both the GR and G 4 C 2 transgenes cause activation of mdg4, an endogenous retrovirus (ERV). Such ERV expression is a hallmark of TDP-43 dysfunction that is commonly observed in C9orf72 patients. We find that glial expression of either the GR or the G 4 C 2 transgene is toxic to glial cells, but such expression does not cause loss nearby neurons. However, blocking apoptotic signaling within glia that express either GR or G 4 C 2 via expression of the p35 caspase inhibitor further exacerbates effects on lifespan and ablating such glia via expression of the proapoptotic reaper gene partially ameliorates these effects. Together, these results indicate that expression of toxic C9orf72 components in glia produces deleterious effects on lifespan, though potentially through different mechanisms than seen in TDP-43 models of ALS/FTD.
In plants, the response to photoperiod is marked by global reprogramming of gene expression that drives extensive developmental changes. Small RNAs (sRNAs) play important roles in this process, but a comprehensive characterization of micro RNAs (miRNAs) and the more recently recognized transfer RNA fragments (tRFs) within this context is lacking. Herein, we characterize the patterns of miRNAs and tRFs in Arabidopsis by performing time-course sRNA-sequencing across three photoperiods. By comparing with our previous messenger RNA (mRNA) sequencing time-courses, we identified positively- or inversely-correlated miRNA-mRNA pairs between the two co-sequenced datasets that suggest photoperiodic sRNA regulations. Furthermore, we revealed 20 patterns of photoperiodic tRFs. These patterns are linked to the transfer RNA (tRNA) isotypes and positions they derive from, suggesting that tRFs are subjected to photoperiodic regulation. Finally, we present a major update to our web-app "Photo-Graph," (http://gendron-lab.shinyapps.io/PhotoGraph) featuring new visualizations of this mRNA-sRNA co-sequencing dataset. In summary, our findings indicate that plants regulate sRNAs within a diel cycle in a photoperiodic manner and form highly-correlated pairs with mRNAs.
Genome-wide association studies (GWAS) have identified thousands of variants associated with complex traits, but many are non-causal. Statistical fine-mapping methods aim to pinpoint the most likely causal variants among the many associated ones. While most fine-mapping methods were originally limited to single ancestry analysis, multi-ancestry fine-mapping methods are now available, leveraging differences in linkage disequilibrium (LD) and minor allele frequencies (MAFs) across ancestries to improve fine-mapping resolution. However, the biological relevance of the putative causal variants identified through fine-mapping often remains unclear. Colocalization methods improve interpretability by integrating GWAS data with other functional genomics datasets to assess whether two traits share the same causal variants. Despite the growing availability of multi-ancestry data, there are currently no established methods for multi-ancestry colocalization. In this study, we propose multi-ancestry colocalization approaches through the integration of multi-ancestry fine-mapping methods, SuSiEx and MsCAVIAR, with single ancestry colocalization methods, coloc and eCAVIAR. We introduce coloc_SuSiEx, eCAVIAR_SuSiEx, eMsCAVIAR and coloc_MsCAVIAR. The performance of the proposed approaches is evaluated and compared through simulation studies. In loci with a single causal variant, credible set sizes across the four approaches were comparable, as was the prioritization of the true causal variant. MsCAVIAR-based approaches were more computationally expensive compared to SuSiEx-based approaches, which is an important consideration for the analysis of regions with multiple causal variants. Compared to the coloc-based approaches, the eCAVIAR-based approaches tended to report lower loci level colocalization posterior probabilities. For the analysis of loci with multiple causal variants, coloc_SuSiEx is the preferred approach. We apply the proposed approaches to perform a colocalization analysis of multi-ancestry T2D GWAS data from the DIAMANTE Consortium and European pQTL data from the INTERVAL study. This work addresses the increasing need for multi-ancestry approaches to colocalization analysis as more multi-ancestry data become available.
Insulin / insulin-like growth factor signaling (IIS) in C. elegans is mediated by the DAF-2 receptor and insulin-like peptides (ILP) that can act as agonists or antagonists. DAF-2 signaling is also affected by DAF-2B, a truncated, non-signaling, secreted isoform of DAF-2 that acts as a decoy receptor by sequestering ILPs. In this study, we performed a forward genetic screen for modifiers of DAF-2B protein expression and identified a mutation in unc-31 that increased DAF-2B. UNC-31 is involved in dense core vesicle docking and is required for neuropeptide secretion, including ILPs. As a result, unc-31 mutants constitutively enter the dauer larval stage and are long-lived due to reduced IIS. We find that increased nervous system DAF-2B accumulation is associated with reduced agonist ILP availability, in both unc-31 mutants and wild type worms. Using auxin-induced degradation (AID) and fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET), we find that a significant fraction of nervous system DAF-2B is in the form of a heterodimeric complex with a full-length DAF-2 receptor isoform, representing a new class of DAF-2 hybrid receptor. In unc-31 mutants, DAF-2B also undergoes endocytosis in neurons in an AP2-dependent manner and genetic manipulation of daf-2b in the unc-31 mutant suggests that DAF-2B homodimers may function to reinforce a reduced insulin signaling state by clearing agonist ILPs from the extracellular space. These findings indicate that DAF-2B not only forms homodimers, but also hybrid receptors with full-length DAF-2, to regulate the activity of the large and diverse family of ILPs in C. elegans.
