Cis-regulatory elements such as enhancers can be located even a million base pairs away from their cognate promoter and yet modulate gene transcription. Indeed, the 3D organisation of chromatin enables the establishment of long-range enhancer-promoter communication. The observation of long-range enhancer-promoter chromatin loops at active genes originally led to a model in which enhancers and promoters form physical contacts between each other to control transcription. Yet, recent microscopy data has challenged this prevailing activity-by-contact model of enhancer-promoter communication in transcriptional activation. Live single-cell imaging approaches do not systematically reveal a correlation between enhancer-proximity and transcriptional activation. We therefore discuss the need to move from a static to a dynamic view of enhancer-promoter relationships. We highlight recent studies that not only reveal considerable chromatin movement in specific cell types, but suggest links between chromatin compaction, chromatin movement and transcription. We describe the interplay between enhancer-promoter proximity within the context of biomolecular condensates and the need to understand how condensate microenvironments influence the chromatin binding kinetics of proteins that bind at cis-regulatory elements to activate transcription. Finally, given the complex multi-scale interplay between regulatory proteins, enhancer-promoter proximity and movement, we propose the need to integrate information from complementary single-cell next-generation sequencing and live-cell imaging approaches to derive unified 3D theoretical models of enhancer-promoter communication that are ultimately predictive of transcriptional output and cell fate. In time, improved models will shed light on how tissues grow and diseases emerge. Cis-regulatory sequences including enhancers (E) interact with transcription factors (TFs) and chromatin regulators within the three-dimensional (3D) chromatin landscape to precisely and dynamically modulate gene expression  and stripes corresponding to interactions between a loop anchor and contiguous genomic regions . DespiteTranscriptional regulation by enhancers is classically believed to be mediated by physical proximity between the promoter and its enhancer(s) . Indeed,Drosophila even-skipped (eve) enhancers relative to the promoter in Drosophila embryos during stripe patterning  was observed in the developing limb  (Sox2 Control Region and promoter (through targeting of fluorophore-tagged proteins to knock-in arrays of binding sites), and measuring Sox2 nascent transcription (through targeting of fluorophore-tagged proteins to knock-in MS2 sequences in nascent RNA) showed no correlation between E-P proximity and transcriptional activation in mouse pluripotent cells , did indeed correlate with nascent transcription monitored by MS2 tagging  . Yet, trnt cells . Instead tagging . Althouggagement    . This lengation) . In addingation) . These sFurthermore, our lab and others have uncovered several modes of chromatin movement: slow-diffusing tethered motion, fast-diffusing tethered motion and long-range untethered/directional motion  Figure . Slow moShh and eve genes during transcription (Sox2 locus during neuroectoderm differentiation supports the idea that chromatin compaction mediates changes in transcriptional programmes (These studies highlight the importance of identifying molecular mechanisms that control types of chromatin movement, including those that act beyond the transcriptional machinery. For example, chromatin movement may decrease within heterochromatin , whereascription . Decompacription . Furtherogrammes . Howeverogrammes . In addicis-regulatory elements observed during development (Recently, membraneless compartments called biomolecular condensates have gained traction as complementary models for gene regulation  Figure . They arelopment  (Figure elopment .Moreover, multivalent, low-affinity interactions can result in liquid-liquid phase separation in a concentration-dependent manner, with higher affinity or higher valency interactions sometimes leading to the formation of aggregates or thicker gel-like microenvironments . NeverthDrosophila mesectoderm enhancers, necessary for synchronised, sustained gene activity, or bookmarking of enhancers during epidermal differentiation (Drosophila mesoderm enhancers engage in stable chromosomal interactions days before detectable changes in gene expression (Shh regulation (There is growing evidence that establishment of lineage-specific chromatin landscapes may precede changes in gene transcription and cell lineage decisions . Examplentiation . In addipression . These egulation .cis-regulatory regions is transferred to promoters even at a distance, for example by diffusion of post-translationally modified TFs at enhancers (Nevertheless, the question remains of how pre-established chromatin changes can be temporally uncoupled from transcription, whilst preserving specificity and robustness of cell-fate decisions. Interestingly, recent papers propose that E-P interactions may be memorised into longer-lived promoter states that integrate past and current signals, suggesting that transcriptional output may vary between cells if they have been exposed to different signals . Neverthnhancers . Enhancenhancers . Althougin vivo studies have mostly been in zebrafish and Drosophila, the growing repertoire of tools will no doubt lead to similar studies within mammalian tissues.In summary, proximity-based methods and live-imaging approaches are beginning to provide both genome-wide and spatiotemporal information on E-P communication. However, there is no one approach that combines all of this information. As such, physical and computational models of chromatin that integrate datasets collected at different spatiotemporal scales will likely be needed to provide additional insights into the underlying chromatin organisation and regulation. Recent examples of such models reveal that E-P contact frequencies are sensitive to genomic distance, but that promoter activation is likely threshold-dependent . This tr


INR gene corresponded with ability to respond to In11 across ~53 million years (my) of evolution. In11 recognition is unique to the clade of Phaseoloid legumes, and only a single clade of INR homologs from Phaseoloids was functional in a heterologous model. The syntenic loci of several non-Phaseoloid outgroup species nonetheless contain non-functional INR-like homologs, suggesting that an ancestral gene insertion event and diversification preceded the evolution of a specific INR receptor function ~28 my ago. Chimeric and ancestrally reconstructed receptors indicated that 16 amino acid differences in the C1 leucine-rich repeat domain and C2 intervening motif mediate gain of In11 recognition. Thus, high PRR diversity was likely followed by a small number of mutations to expand innate immune recognition to a novel peptide elicitor. Analysis of INR evolution provides a model for functional diversification of other germline-encoded PRRs.As a first step in innate immunity, pattern recognition receptors (PRRs) recognize the distinct pathogen and herbivore-associated molecular patterns and mediate activation of immune responses, but specific steps in the evolution of new PRR sensing functions are not well understood. We employed comparative genomic and functional analyses to define evolutionary events leading to the sensing of the herbivore-associated peptide inceptin (In11) by the PRR inceptin receptor (INR) in legume plant species. Existing and de novo genome assemblies revealed that the presence of a functional  The health status of a plant depends on the immune system it inherits from its parents. Plants have many receptor proteins that can recognize distinct molecules from insects and microbes, and trigger an immune response. Inheriting the right set of receptors allows plants to detect certain threats and to cope with diseases and pests.Soybeans, chickpeas and other closely-related crop plants belong to a family of plants known as the legumes. Previous studies have found that, unlike other plants, some legumes are able to respond to oral secretions from caterpillars. These plants have a receptor known as INR that binds to a molecule called inceptin in the secretions. However, it remained unclear how or when INR evolved.To address this gap, Snoeck et al. tested immune responses to inceptin in the leaves of 22 species of legume. The experiments revealed that only members of a subgroup of legumes called the Phaseoloids were able to recognize the molecule.Analyzing the genomes of several legume species revealed that the gene encoding INR first emerged around 28 million years ago. Among the descendants of the legumes that first evolved this receptor, only the crop plant soybean and a few other species were unable to respond to inceptin. The genomic data indicated that these species had in fact lost the gene encoding INR over evolutionary time.Snoeck et al. then combined data from genes encoding modern-day receptors to reconstruct the sequence of building blocks that make up the 28-million-year-old version of INR. This ancestral receptor was able to respond to inceptin in the caterpillar secretion, whereas an older version of the protein, which had a slightly different set of building blocks, could not. This suggests that INR evolved the ability to respond to inceptin as a result of small mutations in the gene encoding a more ancient receptor.The work of Snoeck et al. reveals how the Phaseoloids evolved to respond to caterpillars, and how this ability has been lost in soybeans and other members of the subgroup. In the future, these findings may aid plant breeding or genetic engineering approaches for enhancing soybeans and other crops resistance to caterpillar pests. Arabidopsis thaliana genome contains about 223 LRR-RKs and 57 LRR-RLPs , and are restricted to the Solanum genus  eptitope pg9(At) derived from Botrytis cinerea -like proteins (NLPs) found in bacterial/fungal/oomycete species (Phytophthora sojae) and E02  (Leptosphaeria maculans in rapeseed (Xanthomonas (Cuscuta Receptor1 (CuRe1) is specific to Solanum lycopersicum and senses the peptide Crip21, which originates from parasitic plants of the genus Cuscuta  . LepR3  evolution of the INR locus. We show that In11 response is restricted to species in the ~28 my-old clade of the Phaseoloid legumes, which includes the agriculturally important subtribes Phaseolinae, Glycininae, and Cajaninae. Dynamic evolution of the syntenic INR receptor locus predates the divergence of Phaseoloids, but a single clade of INR homologs is active in recognition of In11. Finally, we used chimeric and ancestrally reconstructed LRR-RLPs to pinpoint key domains and amino acids (AA) involved in In11 recognition. Patterns of INR variation provide a framework for the evolution of novel functions within PRR gene families.In this study, we use the legume-specific LRR-RLP INR as a model to perform dense species phenotyping, comparative genomics, and functional validation to associate gain and loss of HAMP response with evolution of the contiguous Vigna unguiculata) and common bean (Phaseolus vulgaris), encode the corresponding INR receptor , soybean (Glycine max), yam bean (Pachyrhizus erosus), and calopo , although these species were able to respond to the unrelated bacterial PAMP flg22 , jack bean , and H. podocarpum , Medicago truncatula, and clover (Trifolium pratense) do not contain an LRR-RLP encoding gene at the locus, whereas all species within the Millettioids except for P. erosus contained at least one LRR-RLP, consistent with a gene insertion event of an LRR-RLP at the INR locus in the ancestor of extant Millettioids  also contained orthologs from plant species able to respond to In11  within this clade were cloned and transiently expressed in the non-legume model species N. benthamiana. Receptor function was measured using peptide-induced production of the plant defense markers ethylene and reactive oxygen species (ROS). In11-induced ethylene and ROS production were able to be conferred by each gene . Except for P. vulgaris G19833, all studied Phaseolinae have an LRR-RLP in each of the two clades discussed above . Finally, we tested the most closely related LRR-RLP to INR outside the contiguous INR locus for both V. unguiculata and P. vulgaris (Vigun07g039700 and Phvul.007g246600). No In11-induced responses could be observed upon heterologous expression of Vigun07g219700, Glyma.10G228000, Vigun07g039700, and Phvul.007g246600 . Thus, the ability to confer In11-induced ROS and ethylene production is strictly limited to members of the INR clade.To assess if more distantly related, INR-like homologs could also confer In11 recognition, we measured ethylene and ROS production upon expression of LRR-RLPs outside the e itself . Within ed above . The remsponsive  and sole7g246600 . ProteinINR locus throughout its evolution within the Millettioids using NOTUNG  and paralogous Vigun07g219700 , the Phaseolinae (N6) and the Phaseoloids (N4) conferred In11-induced ethylene and ROS response. In contrast, ancestral receptors of the INR-like 1 clade of the Phaseolinae (N16) and the Phaseoloids (N14) were not responsive to In11 (INR and its INR-like 1 sister clade (N3) is not responsive to In11  do not contain an LRR-RLP encoding gene at the locus, whereas all species investigated herein the Millettioids except for P. erosus contain at least one LRR-RLP , compared to other extensive studied LRR-RLPs which are generally studied within a single genus or species . Each of species . A phylothamiana . This inbination . In summbination  may drivINR receptor loci  may reflect the propensity of tandemly duplicated loci to lose functions through gene conversion and/or reciprocal gene loss  also provides insight into LRR-RLP function. Chimeric receptors formed by combining INR and an INR-like paralog revealed LRR-RLP subdomains required for In11 response. Across all chimeric receptors, those encoding the LRR (C1) and intervening motif (C2) domains of VuINR responded to In11 treatment with both an ethylene and ROS burst, suggestive of crucial elements for elicitor interaction in the C1-C2 subdomain. Notably, a chimeric receptor with mixed C1 and C2, 219600-C1, is the sole construct that responded to the control treatment, suggestive of a critical role for C1-C2 compatibility. In addition, a chimeric receptor with mixed C2 and C3 (219600-C2) had delayed In11-induced ROS burst relative to all other In11-responsive constructs tested here  cytoplasmic tail to the extended version of VuINR-like 1 (Vigun07g219700), 219600 F, did not affect In11-response , whereas the common ancestor of both INR and INR-like (N3) did not confer In11 response  domain of the plant intracellular NOD-like receptor Pik-1 . SpecifiPlant species and accessions used in this study are listed in V. unguiculata cATPC sequence  was equally spread over the wounds with a pipette tip. After 1 hr, leaflets were excised and placed in sealed tubes for 2 hr before headspace sampling (1 mL). Ethylene was measured as previously described with a gas chromatographer  with flame ionization detection and quantified using a standard curve  is a host derived proteolytic fragment of the ATP synthase γ-subunit (cATPC), based on the sequence . The flgsequence . Both pe2O using individual cells of a white 96-well white bottom plate (BRANDplates F pureGrade S white [REF 781665]). After overnight incubation, ROS production was measured upon addition of a 100 μL assay solution which contained 10 μg/mL luminol-horseradish peroxidase, 17 μg/mL luminol and the treatment . Luminescence was quantified with a TECAN SPARK plate reader every minute for an hour using an integration time of 500 ms. Four technical replicates were quantified for each treatment, and significant differences were determined by performing a two-group Mann-Whitney U Test between both treatments. R and the R-packages dplyr (v1.0.7), ggpubr (v.0.4.0), and ggplot2 (v3.3.3) were used to analyze and plot the data. The resulting figure was edited in Corel-DRAW Home & Student x7.Leaf punches were taken with a 4 mm biopsy punch and floated in 150 μL of H2-chilled mortar and pestle. In contrast to P. erosus, nuclei isolation was first performed on frozen tissue for C. ensiformis, C. tetragonoloba, and H. podocarpum using the Bionano Plant Tissue Homogenization Buffer (Part number 20283). Nuclear DNA was extracted for all above mentioned species with a modified CTAB(Cetyltrimethyl ammonium bromide) protocol as described previously (Leaf tissue of legume plants was harvested and ground using an Neviously . ResultaA modified protocol using the Oxford Nanopore Technologies (ONT) Rapid barcoding kit (SQK-RBK004) was used for sequencing. Briefly, 27 μL of ~20 ng/μL HMW DNA was combined with 3 μL of Rapid barcoding fragmentation mix and incubated for 1 min at 30°C followed by 1 min at 80°C. Ampure beads were added at a 0.7× final concentration and bead clean-ups performed as described in the Rapid barcoding kit (SQK-RBK004). All remaining steps were performed as described in the Rapid barcoding kit protocol. Each sample was sequenced on a single MinION or PromethION flowcell (R9.4). High-accuracy base calling was performed in real time with MinKnow (v20.10.6).C. tetragonoloba and H. podocarpum from the same HMW DNA used for ONT long read sequencing. Paired end 2×150 bp sequence was generated on the Illumina NovaSeq6000 platform. In addition, we generated Illumina short reads for C. ensiformis, P. erosus, and Macroptylium lathyroides (Genewiz Inc). For these samples, genomic DNA was extracted using the Nucleospin Plant II kit (Macherey-Nagel). The Oxford Nanopore sequencing data and Illumina hiseq reads used in this study can be found in SRA(Short Read Archive) under Bioproject: PRJNA820752. The final genome assemblies are available on NCBI .Illumina short read sequence was generated for P. erosus genome was assembled using SPAdes (v3.15.4) using the meta option with both the Illumina and Oxford Nanopore readsets. Additional genome assemblies were produced with FlyE (v2.8.1), consensus was generated with three rounds of Racon (v1.3.1), and finally polished with Pilon (v1.22) three times with Illumina reads (2×150 bp). Final assembly quality was determined with assembly-stats and BUSCO (v5.3.0).The INR locus analysis can be found in INR locus, INR, and INR-like sequences. Similarly, if not yet annotated in publicly available genomes, syntenic INR and INR-like homologs were identified. First, BLASTN  was used to identify the INR syntenic locus by mining the genomes for homologs of the strongly conserved neighbor (anchor) genes of common bean INR (Phvul.007g077500); Phvul.007g077400 and Phvul.007g077600. Similarly, the genomes were mined for LRR-RLPs via BLASTN approach with a default e-value of 10, with exception of the Glycine pangenome where a BLASTP approach was used. The strongest LRR-RLP blast hits with INR were consistently identified in between the conserved neighbor genes. Finally, potential INR and INR-like ORFs(open reading frame) were determined by visual inspection in IGV (v2.10.3) and compared with the sequences of closely related annotated LRR-RLPs in other legume species. Additionally, INR homologs were identified in M. lathyroides by using an alternative method. First, M. lathyroides Illumina HiSeq paired end reads were mapped against both Phvul.007G077500 (INR) and PvUI111.07G078600 (INR-like) by using bwa (v0.7.17-r1188) , enzymatic clean-up using ExoSAP-IT (Thermo Fisher scientific), and Sanger sequencing of the entire amplified PCR product. Primers used in this process are listed in The analyzed genome assemblies, versions, and their sources for the 20 legume species included in the contiguous 7-r1188) . Second,7-r1188) . Third, INR loci and their corresponding annotation (P. erosus using the Q5 Hot Start High-Fidelity kit (NEB). Primers used in the reaction are listed in Locus comparison was performed using R (v4.0.3) and the R-package genoPlotR (v0.8.11) using the extracted contiguous notation . The resH. podocarpum was not incorporated in the phylogenetic analysis since the length of the sequence was about 79% of cowpea INR due to the absence of a part of the LRR domain in comparison to all other annotated receptors at the contiguous INR locus. A phylogenetic analysis was performed on the CIPRES web portal using RAXML-HPC2 on XSEDE (v8.2.12) with the automatic protein model assignment algorithm using ML criterion and 250 bootstrap replicates  INR and INR-like homologs were extracted, (2) C. tetragonoloba as it is the closest related legume species without a receptor at the contiguous INR locus, and (3) M. truncatula as the LRR-RLP used as an outgroup in the analysis was extracted from its genome. Third, the NOTUNG analysis was performed using the default options, including a duplication cost of 1.5 and a loss cost of 1 . RNA was extracted using the Nucleospin RNA Plant kit (Macherey-Nagel). cDNA was created using SuperScript IV Reverse Transcriptase (Invitrogen). All constructs were created using a hierarchical modular cloning approach facilitated by the MoClo toolkit and the MoClo Plant Parts kit (Phvul.007g246600 and Vigun07g039700) were amplified from cDNA. All primers used for amplification are listed in Aequorea victoria; pICSL50008), and the OCS1 terminator (pICH41432) (Leaf tissue of legume plants was harvested and ground using an Narts kit . LRR-RLPCH41432) .Agrobacterium tumefaciens strain GV3101 (pMP90 and pSOUP). Overnight cultures were resuspended in 150 μM acetosyringone in 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.6, and 10 mM MgCl2. After 3 hr of incubation at room temperature, N. benthamiana leaves of 5-week-old plants were infiltrated at an optical density of 0.45 at 600 nm (OD600).Constructs were electroporated into N. benthamiana leaves to validate the expression of the constructs in N. benthamiana by western blot. Ground, frozen tissue was homogenized in a 3× lamellae buffer  and then cleared by centrifugation . Subsequently, proteins in the supernatant were separated by performing an SDS-PAGE on an 8% acrylamide gel. Finally, a western blot was performed to visualize the GFP-tagged heterologously expressed proteins and actin as a loading control with respectively an α-GFP polyclonal  primary antibody at a 1:2000 dilution and an anti-Actin  primary antibody at a 1:5000 dilution. α-rabbit  was used as a secondary antibody for both at 1:10,000 dilution.Leaf punches were taken 48 hr after infiltration of the Agrobacterium infiltration for receptor expression (24 hr), leaf punches were taken with a 4 mm biopsy punch and floated in 150 μL of H2O using individual cells of a white 96-well white bottom plate (BRANDplates F pureGrade S white). Subsequently, the same procedure was followed as outlined for ROS production in legumes. Four biological replicates were quantified (n=4 plants), with each biological replicate representing six technical replicates. R and the R-packages dplyr (v1.0.7), ggpubr (v.0.4.0), and ggplot2 (v3.3.3) were used to analyze and plot the data. The resulting figure was edited in Corel-DRAW Home & Student x7.Following N. benthamiana, a fully expanded leaf of 5-week-old plants was infiltrated with H2O or 1 μM In11 with a blunt syringe. Subsequently, four leaf discs within the infiltrated area were immediately excised with a no. 5 cork borer and sealed in tubes , resulting in an L0 vector. Primers used in these reactions are listed in N. benthamiana was validated with western blot.Ancestral reconstructed LRR-RLP constructs were generated using the MoClo toolkit . MoClo oN. benthamiana with western blot.N4 ancestral receptors with a single AA were created with two different approaches due to relatively low cloning efficiencies. N4 Y91F, N4 H239D, N4 A359S, and N4 H404N were created with the NEB Q5 Site-Directed Mutagenesis kit with as input the L1 vector of the N4 ancestral receptor. Primers used in these reactions are listed in A protein structure prediction of Vigun07g219600 was created using the ColabFold platform v1.3.0; . The alp This manuscript, of interest to those studying the evolution of immunity, investigates the evolutionary history of a recently described herbivore-associated molecular pattern (HAMP) receptor, INR, which perceives the caterpillar-derived peptide HAMP, In11. The authors compare INR homologs to identify evolutionarily conserved residues and use chimeric fusion proteins to investigate specificity. The findings presented are valuable and supported by convincing experiments and analysis. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor (me!) and Detlef Weigel as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Daniil M Prigozhin (Reviewer #1).Thank you for submitting your article "Evolutionary gain and loss of a plant pattern-recognition receptor for HAMP recognition" for consideration by We have discussed your manuscript. Overall, we are all very positive about your work. The paper is nicely constructed and was fun to read. However, both reviewers would like to see a bit more analysis/interpretation of the predicted structure and I had one technical concern. Please see below for detail. I have drafted the following to help you prepare a revised submission. Please feel free to reach out should you require any clarification.Essential revisions:1) Please include additional analysis/interpretation based on structural predictions . In addition, this may allow additional interpretation of the data including the auto activity of 219600-C1.2) Both reviewers made several suggestions for ways to improve clarity and correct minor typos.3) I had one small technical question – I notice that the ROS responses are cyclic for some receptors and a single burst for others. Any thoughts on this? Also, in a few cases, you see a response with ethylene but not ROS or the other way around. My guess is that these are false positives. Do you agree? For example Glyma_10G228000. Perhaps a sentence or two in the discussion is warranted.Reviewer #1 (Recommendations for the authors):1. Independent losses of INR are important to the overall understanding of the story. Following up on the likely genomic events that lead to several independent losses is advisable.2. Evolution of protein receptor recognition is best interpreted in the context of protein structure. It is up to the authors whether this makes main figures or not, but including a predicted protein model with N3 to N4 and N3 to N14 changes highlighted should at least make a supplemental figure. Alphafold/Rosettafold results are well accepted as a best guess estimates absent an experimental structure.3. For the ROS time courses, unifying the Y-axis across figures would improve sample-to-sample comparison. Same for the X-axis of the ethylene subfigures of Figure 3 to Figure 5.4. The use of "autoactivity" (in line 374) with regards to the mutant 219600-c1 is surprising, and the nature of the differences between 219600-F, C3, C2, and C1 is somewhat underexplored. Would the 219600-c1 receptor signal in the absence of scratch wounding etc? Is there a phenotype in the transient assay after protein expression but before any other manipulations are performed? I would agree with a "responded to control treatment only" characterization absent more details.Reviewer #2 (Recommendations for the authors):Line 44 – include Benthamiana RLPs – several have been identified here e.g. RXEG1 and RE02.Whilst LRR-RLP structures have not currently been solved, based upon what we know from other LRR domains predictions of structure should be quite accurate. It would be nice to highlight the residues that you identify as being important for the recognition of a structural prediction. This could help the reader.Figure 1 – it could be interesting to show the correlation between In11-induced ethylene and flg22-induced ethylene . Is there a strong correlation?158 – 'contained a orthologs'.Figure 4 b – why does mock give a response in 219600? Would you expect a burst if it is constitutive autoactivity as suggested (374)? Does this RLP induce cell death? Is the burst in the mock reproducible? Also, the burst seems weaker following treatment in the rep shown. If it was autoactive I think it would not matter whether it is mock or treatment.For expression western blots it would have been nice to include a -ve control of an untransformed leaf – however, this is not strictly required as some of the receptors have slightly different sizes.Whilst perception seems to have been lost in G. max/G. soja it would be interesting to know how widely INR is present in the pangenome of a responsive species. In Arabidopsis, it seems that many RLPs have significant presence/absence polymorphism. It would be interesting to know if this is the case with INR, however, I understand that the availability of genomic/germplasm resources may be limiting. E.g. what proportion of cowpea accessions are In11 responsive?Whilst many of the receptors are not responsive to In11, they could also be functional receptors for an unknown ligand. I think this could be made more apparent in the text. One option could be polymorphisms within In11, is it known if these exist? E.g. 327-328.Does INR1-like form a constitutive interaction with SOBIR1? I would expect it can be given the activity of the C2 chimera, however, it could be nice to demonstrate this via Co-IP.It would be nice to test each of the 5 substitutions between N3 and N4 for a loss of In11 perception in Benth, however, I do not think this is necessary.316 – 'unusually long evolutionary history' – please provide more context what are you comparing with? It would be nice to add context to what is currently known.Personally, I think the discussion could be made more concise.326 – I would not agree that it represents an 'alternative' mechanism – I don't think there is currently a paradigm for how these receptors evolve. I think this work represents one of the few attempts to characterise the origin of the receptor in detail.349 – gives the impression that EFR/XPS1/CORE is RLPs – rephrase. I would also argue that for some of the receptors there has been some extensive phenotyping than others – e.g. for RLP42.356 – I would argue that this paper suggests that RLPs have dynamic evolutionary patterns – similar to NLRs, unlike RKs . They highlight two examples that are conserved, but I would not say that these examples are representative.I would prefer that P-values are also shown on the graphs .I am not qualified to comment on the quality of the sequencing – but I believe it represents a valuable resource of the community and is sufficient to support the claims in the manuscript.364 – I would consider TMM a ligand binding LRR-RLP (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458759/) although I appreciate it is significantlyI would recommend citing this paper for the lack of RLP structure https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503760/. Essential revisions:1) Please include additional analysis/interpretation based on structural predictions . In addition, this may allow additional interpretation of the data including the auto activity of 219600-C1.We have generated an AlphaFold-based structural prediction of INR. In addition, we have included clarified interpretations of the 219600-C1 construct, which showed ROS response to water-only treatment. Please see specific responses to reviewers for additional details.2) Both reviewers made several suggestions for ways to improve clarity and correct minor typos.We appreciate the suggestions and corrections of both reviewers and incorporated them in the manuscript.3) I had one small technical question – I notice that the ROS responses are cyclic for some receptors and a single burst for others. Any thoughts on this? Also, in a few cases, you see a response with ethylene but not ROS or the other way around. My guess is that these are false positives. Do you agree? For example Glyma_10G228000. Perhaps a sentence or two in the discussion is warranted.N. benthamiana signaling machinery to activate RBOHD. We added the following sentences to address the observation in the results:We are also interested in cyclic ROS responses but unable at this stage to account for differences. We tested if cyclic ROS was a function of the C-terminal GFP tag. Although more subtle, cyclic ROS responses were still observed when two receptor homologs did not have a C-terminal GFP tag. Both constructs also gave equivalent ethylene burst to the original GFP tagged constructs, so it is not clear that cyclic ROS translates into different downstream response. We have added this data on the effect of C-terminal tag in a new Figure 3—figure supplement 2. Our overall model is that each ortholog or tagged variant interfaces slightly differently with “Intriguingly, cyclic ROS responses were observed for three out of five In-11 responding receptors . Corresponding constructs without a C-terminal GFP tag reduced cyclic characteristics of the ROS response but resulted in equivalent ethylene bursts .”Regarding ROS versus ethylene correspondence -- ROS responses were previously shown with scaled y-axes, which inflated the apparent size of certain responses. This is now adjusted to only use one of 3 scales throughout all figures. In11-induced ROS and ethylene responses were complementary for all constructs. Nevertheless, the results for Glyma_10G228000 are indeed remarkable as our control treatment resulted in a low and late ROS burst relative to any of the other ROS bursts observed in this study for other constructs. Similarly, the control treatment resulted in a higher ethylene production for this construct.Reviewer #1 (Recommendations for the authors):1. Independent losses of INR are important to the overall understanding of the story. Following up on the likely genomic events that lead to several independent losses is advisable.We agree that the INR loss events, potentially mediated via TE activity, are intriguing and of interest for further investigation. We therefore used the CENSOR tool to annotate repetitive elements in INR loci using the Repbase database with Arabidopsis as a sequence source.Pachyrhizus erosus), multiple fragments of an LRR-RLP receptor can be found by BLAST analysis as indicated by Figure 2b. Transposon annotation with CENSOR revealed a hit with a partial transposable element of the DNA/Mariner class (117 bp) in between two LRR-RLP fragments, possibly consistent with an ancient full transposition interrupting an LRR-RLP and/or INR function . We added the following sentence to the results:In the INR locus of jicama .”Beyond the example in jicama, we found various TE hits in all INR loci of the Millettioids. This finding aligns well with structural variation and variable number of LRR-RLPs found at the corresponding INR loci . We adapted the result section as follows:“The organization of the INR locus is highly diverse among legumes, with zero to seven LRR-RLP encoding-genes per species in addition to extensive repetitive sequence and transposable element content .”And the discussion as follows:P. vulgaris and M. pruriens lost INR-like, and P. erosus seems to have lost both INR and INR-like as it does not contain any complete coding sequence of an LRR-RLP at the INR locus. Repetitive elements and transposable elements within the INR locus may have disrupted ancestral INR and INR-like genes in these lineages, and can be found between LRR-RLP genes and gene fragments . Loss of INR (and INR-like) may reflect the propensity of tandemly duplicated loci to lose functions through gene conversion, and/or reciprocal gene loss (62).”“In contrast, Glycine max and tight correlation with TE variation. Nevertheless, we agree that this could be an interesting and achievable project in the near future upon release of additional legume genomes.While intriguing, we believe we cannot draw firm conclusions about the role of these elements with respect to the loss of INR in specific species. For example, high-quality genomes of close Glycininae outgroups would be needed to identify specific losses of et.al. 2022, https://doi.org/10.1186/s13100-021-00259-7). Fine-scale tracking of TE behavior within the Glycininae would allow genome-wide analysis of both NLR and RLP evolution.We are also hesitant to draw stronger conclusions due to the TE source used for our CENSOR analysis. Genome specific pipelines for TE annotation would improve the analysis as the use of a TE database from a distantly related species (Arabidopsis) decreases annotation quality . Manual curation of automated predictions is also advisable as discussed by  which includes a structural prediction of INR (Vigun07g219600). In this structure we highlight 5 AA of interest based on analysis of N4 vs N3, the respective ancestors of the INR clade and the combined INR+INR-like clade. We also added additional functional data which shows that H404 and R406 are essential for function in the N4 ancestrally reconstructed construct. For more details, please see the adapted manuscript .3. For the ROS time courses, unifying the Y-axis across figures would improve sample-to-sample comparison. Same for the X-axis of the ethylene subfigures of Figure 3 to Figure 5.We thank the reviewer for this suggestion. We agree that a consistent use of x- and y-axes limits improves the figure readability. We now use three y-axes for ROS with small changes in upper limit for readability  rather than previous mixed axes with scales as fine as 0-400 RLU. We changed the main Figures 3-5 and the newly added figures  to the manuscript accordingly for both ROS and ethylene data.4. The use of "autoactivity" (in line 374) with regards to the mutant 219600-c1 is surprising, and the nature of the differences between 219600-F, C3, C2, and C1 is somewhat underexplored. Would the 219600-c1 receptor signal in the absence of scratch wounding etc? Is there a phenotype in the transient assay after protein expression but before any other manipulations are performed? I would agree with a "responded to control treatment only" characterization absent more details.We agree with the reviewer and changed the text accordingly (new text underlined):“(Line 244) Intriguingly, the chimeric receptor 219600−C1, containing C1 of VuINR and C2 of VuINR-like, responded to the control treatment.”(Line 445) “Notably, a chimeric receptor with mixed C1 and C2, 219600-C1, is the sole construct that responded to the control treatment, suggestive of a critical role for C1-C2 compatibility.”2O, 1 µM In11 and 1 µM flg22. Similarly, no phenotypes were observed for 7 days after construct infiltration . We added the following sentences to the results:Luminescence for the ROS assays is measured in leaf disk punches. Hence, we are unable to measure ROS without creating leaf damage which potentially has an influence on the observed ROS response to control treatment. We attempted to further explore the heightened responsiveness of 219600-C1 using two additional approaches. First, we expressed chimeric receptor 219600-C1 and parent receptors without any subsequent manipulations to check for a potential phenotype by eye, such as cell death or chlorosis. No phenotype was observed 7 days after infiltration. We show this lack of response in a new panel, Figure 4—figure supplement 2a. Secondly, we hypothesized that heightened ROS responses to control treatment might manifest as cell death responses to PAMPs or HAMPs. We therefore repeated the above-described set-up except that 24h post infiltrations, we infiltrated the following treatments: HN. benthamiana with or without subsequent infiltration of flg22, In11 or H2O one day post the construct infiltration .”“Intriguingly, the chimeric receptor 219600−C1, containing C1 of VuINR and C2 of VuINR-like, responded to the control treatment. Nonetheless, no visible phenotype was observed seven days post infiltration of 219600-C1 in We agree that multiple factors could result in what we observed and thank reviewer 1 for suggesting other hypotheses. We changed the text accordingly:“Notably, a chimeric receptor with mixed C1 and C2, 219600-C1, is the sole construct that responded to the control treatment, suggestive of a critical role for C1-C2 compatibility.”The interface between C1 and C2 is also informed by structural modeling. The AlphaFold prediction of INR  suggests an interaction between C2 and the N-terminal part of the C1 LRR. Additionally, one of the five conserved AA (Y91) identified by our ASR analysis is predicted to play a part in this interaction. Y91F did not completely abolish ROS-burst although it was relatively low. We now note this C1-C2 predicted interaction in the Results section (lines 314-317).Finally, we discussed in our response to the editor above that the cyclic ROS response seems to be somewhat dependent on the C-terminal tag and specific homologue used . We therefore do not want to overinterpret the single peak of 219600-C2. However, we have added the following sentence acknowledging cyclic responses and the role of C-terminal tags.“Intriguingly, cyclic ROS responses were observed for three out of five In-11 responding receptors . Corresponding constructs without a C-terminal GFP tag resulted in equivalent ethylene bursts, but ROS bursts only had relatively subtle cyclic characteristics .”Reviewer #2 (Recommendations for the authors):Line 44 – include Benthamiana RLPs – several have been identified here e.g. RXEG1 and RE02.Nicotiana benthamiana and Brassica napus. The introduction changed as follows (new text underlined):We included a few extra examples of LRR-RLPs with identified molecular patterns for both Cladosporium fulvum, Avr9, Avr4 and Avr2 (via host protein Rcr3), and are restricted to the Solanum genus (19–25). Arabidopsis RLP42 recognizes fungal endopolygalacturonases (PG) eptitope pg9(At) derived from Botrytis cinerea . Similarly, RLP23 is an Arabidopsis-specific LRR-RLP, which recognizes nlp20 peptide from the NECROSIS AND ETHYLENE-INDUCING PEPTIDE1 (NEP1)-LIKE PROTEINS (NLPs) found in bacterial/fungal/oomycete species . RXEG1 and RE02 were identified in wild tobacco and are respectively triggered by the fungal elicitors XEG1 (Phytophthora sojae) and E02  . LepR3 (AvrLm1) and RLM2 (AvrLm2) convey both race specific resistance to the fungal pathogen Leptosphaeria maculans in rapeseed . ReMAX is restricted to the Brassicaceae and triggered by the MAMP eMax originating from Xanthomonas (33). Cuscuta Receptor1 (CuRe1) is specific to Solanum lycopersicum and senses the peptide Crip21 which originates from parasitic plants of the genus Cuscuta . Finally, the inceptin receptor (INR) appears to be specific to the legume tribe Phaseoleae and recognizes inceptin (In11), a HAMP found in the oral secretions of multiple caterpillars (36–39). Notably, all the above examples of LRR-RLPs are family-specific, restricted to the Solanaceae , Brassicaceae  or Leguminosae (tribe Phaseoleae) (INR) . However, despite clear signatures of lineage-specific functions, specific evolutionary steps leading to novel LRR-RLP functions across multiple species have not been described.”“Evidence is accumulating for highly specialized roles of plant LRR-RLPs as immune sensors . Various LRR-RLPs from Arabidopsis, tomato, wild tobacco, rapeseed and cowpea have been shown to detect molecular patterns from fungi, bacteria, parasitic weeds and herbivores . For example, Cf-9, Cf-4 and Cf-2 interact with respective molecular patterns of Whilst LRR-RLP structures have not currently been solved, based upon what we know from other LRR domains predictions of structure should be quite accurate. It would be nice to highlight the residues that you identify as being important for the recognition of a structural prediction. This could help the reader.We thank the reviewer for the suggestion which was also given by reviewer 1. We agree and our response to reviewer 1 is reproduced here:We now include a new main figure  which includes a structural prediction of INR (Vigun07g219600). In this structure we highlight 5 AA of interest from analysis of N4 vs N3, the respective ancestors of the INR clade and the combined INR+INR-like clade. We also added additional functional data which shows that H404 and R406 are essential for function in the N4 ancestrally reconstructed construct. For more details, please see the adapted manuscript .Figure 1 – it could be interesting to show the correlation between In11-induced ethylene and flg22-induced ethylene . Is there a strong correlation?V. radiata, P. vulgaris, M. pruriens and L. purpureus) do not respond to flg22 with an ethylene burst . Hence, ethylene responses to both elicitors are uncorrelated for those four species. Nevertheless, all four of them do respond to flg22 with a ROS burst.We thank the reviewer for this interesting idea. Of 12 total In11-responding species, 4 species . However, a strong correlation between the ethylene ratio of response against both elicitors is absent for these species as can be seen in In contrast, 8/12 species respond to both flg22 and In11 with an ethylene burst ? Does this RLP induce cell death? Is the burst in the mock reproducible? Also, the burst seems weaker following treatment in the rep shown. If it was autoactive I think it would not matter whether it is mock or treatment.We do agree with reviewer 2 and agree that autoactivity is not the correct way to describe the behavior of this specific construct (219600-C1). We changed the text accordingly as suggested by reviewer 1 in both the result and the Discussion section:“Intriguingly, the chimeric receptor 219600−C1, containing C1 of VuINR and C2 of VuINR-like, responded to the control treatment.”The ROS burst following the mock treatment was consistent over multiple biological replicates measured on different days (i.e. different plant batches) and was not observed for any other constructs tested on the same leaves.Reviewer 1 raised similar questions regarding 219600-C1 and we reproduce our response here:2O, 1 µM In11 and 1 µM flg22. Similarly, no phenotypes were observed for 7 days after construct infiltration . We added the following sentences to the results:Luminescence for the ROS assays is measured in leaf disk punches. Hence, we are unable to measure ROS without creating leaf damage which potentially has an influence on the observed ROS response to control treatment. We attempted to further explore the heightened responsiveness of 219600-C1 using two additional approaches. First, we expressed chimeric receptor 219600-C1 and parent receptors without any subsequent manipulations to check for a potential phenotype by eye, such as cell death or chlorosis . No phenotype was observed 7 days after infiltration. Secondly, we hypothesized that heightened ROS responses to control treatment might manifest as cell death responses to PAMPs or HAMPs. We therefore repeated the above-described set-up except that 24h post infiltrations, we infiltrated the following treatments: HN. benthamiana with or without subsequent infiltration of flg22, In11 or H2O one day post the construct infiltration .”“Intriguingly, the chimeric receptor 219600−C1, containing C1 of VuINR and C2 of VuINR-like, responded to the control treatment. Nonetheless, no visible phenotype was observed seven days post infiltration of 219600-C1 in For expression western blots it would have been nice to include a -ve control of an untransformed leaf – however, this is not strictly required as some of the receptors have slightly different sizes.Our revised manuscript includes an additional western blot for mutated ancestral sequence reconstructed (ASR) receptors. This time, we included an infiltration media only control .Whilst perception seems to have been lost in G. max/G. soja it would be interesting to know how widely INR is present in the pangenome of a responsive species. In Arabidopsis, it seems that many RLPs have significant presence/absence polymorphism. It would be interesting to know if this is the case with INR, however, I understand that the availability of genomic/germplasm resources may be limiting. E.g. what proportion of cowpea accessions are In11 responsive?Vigna unguiculata accessions were In11 responsive (https://doi.org/10.1073/pnas.2018415117 – Table S2). Additional Phaseolus vulgaris and Vigna unguiculata reference genomes are now available on Phytozome, and all encode INR. We added the following sentence to the manuscript:We agree that the degree of conservation of INR is a very interesting aspect of its evolution. In a previous study, all 364 tested V. unguiculata are able to respond to In11 and our analysis now extends this conservation across legume species (36).”“Each of 364 tested varieties of Glycine max and G. soja. None have INR. We note this observation in the results and the discussion:The recently published soybean pangenome (https://doi.org/10.1016/j.cell.2020.05.023) provided 26 de novo assemblies for Glycine and not just in reference assemblies, we performed BLASTP searches using Vigun07g219700 AA sequence against 26 G. soja and G. max de novo assemblies from a recent pangenome analysis (56).”“To confirm that INR was lost in G. soya and G. max, do not encode INR (56).”“Two reference genomes as well as 26 de novo assemblies of the closely related Glycininae, Whilst many of the receptors are not responsive to In11, they could also be functional receptors for an unknown ligand. I think this could be made more apparent in the text. One option could be polymorphisms within In11, is it known if these exist? E.g. 327-328.We agree with reviewer 2 and assume that they are functional receptors as they seem to be similarly conserved in comparison the In11-responsive INR-clade. However, so far, we can only hypothesize about the ligand it interacts with. Several polymorphisms do exist for In11 as shown in V. unguiculata also responds quantitatively similar to the In11 variant from the monocot Zea mays (ICDVNGVCVDA), the exact same In11 variant which we identified in multiple Millettioids as shown in the attached alignment .Second, Schmelz 2007  investigated the effect of alanine substitutions on the elicitor potential of In11. Only mutations of positions 3, 8, and 10 of the mature In11 peptide  lost elicitation activity, and these are different from the polymorphic sites 4, 7, and 9 across legumes.First, Schmelz 2006  showed that We have added a sentence to add our thinking about the function of INR-like:“Certain Phaseoloids and earlier diverged non-Phaseoloids also contain INR-like homologues at the contiguous INR locus; thus, INR most likely arose from an existing LRR-RLP, ~28 mya ago. The role of INR-like homologues is not clear, but they may detect a related, In11-like ligand.”Does INR1-like form a constitutive interaction with SOBIR1? I would expect it can be given the activity of the C2 chimera, however, it could be nice to demonstrate this via Co-IP.Vigna unguiculata INR associates with both AtSOBIR1 and VuSOBIR1 (Vigun09g096400). However, no SOBIR1 association data is available for Vigna unguiculata INR-like1. We agree with reviewer 2 that this would be a valuable experiment and believe that Co-IP could potentially give us valuable insights not only for INR-like 1 but also for multiple chimeric and even ancestrally reconstructed constructs. However, we believe these experiments would be best performed in a full study of SOBIR1 association, BAK1/SERK association, and ligand binding – especially if a ligand for INR-like1 can be identified.We thank the reviewer for this idea and interest in INR1-like signaling mechanisms. As described in Steinbrenner 2020, It would be nice to test each of the 5 substitutions between N3 and N4 for a loss of In11 perception in Benth, however, I do not think this is necessary.We thank the reviewer for the interest in these sites, which we agree are interesting with AlphaFold structural context. We tested these mutations and now place data alongside the INR structural model. We added an extra main figure  which provides ROS and ethylene data of N4 constructs with the five single AA switches.316 – 'unusually long evolutionary history' – please provide more context what are you comparing with? It would be nice to add context to what is currently known.We have rephrased this sentence as follows. We specifically cite RLP42 and Cf-2 as examples where a broader comparative approach has been taken, but where the receptors seem to be present only in a single genus.“Analysis of INR provides an example of a PRR with conserved recognition function across relatively long evolutionary history (since the emergence of the Phaseoloids ~28 mya), compared to other extensive studied LRR-RLPs which are generally studied within a single genus or species .”Personally, I think the discussion could be made more concise.We have simplified where possible .326 – I would not agree that it represents an 'alternative' mechanism – I don't think there is currently a paradigm for how these receptors evolve. I think this work represents one of the few attempts to characterise the origin of the receptor in detail.We thank the reviewer for this comment, and agree that additional case studies for receptors are needed to reveal broader patterns in PRR evolution. We do not aim to draw a sharp contrast with Cf-2 but still would like to highlight its evolution within the Solanum genus. We changed the cited text fragment accordingly:“This evolutionary pattern is reminiscent of the evolution of LRR-RLP Cf-2 which evolved <6 mya within a genus Solanum, potentially by intergenic recombination (41). In summary, an ancestral gene insertion event is the likely source of extant gene and copy number variation at the INR locus, which preceded the evolution of a specific peptide recognition function.”349 – gives the impression that EFR/XPS1/CORE is RLPs – rephrase. I would also argue that for some of the receptors there has been some extensive phenotyping than others – e.g. for RLP42.We thank the reviewer for the helpful feedback on the paragraph regarding receptor evolution. We have re-written the paragraph to focus on examples where phenotyping has been performed and comparative genomic analysis could be helpful to define evolutionary patterns. We removed mention of RK for simplicity.“Our detailed analysis of the emergence of INR provides a roadmap for understanding evolution of recognition specificity for other lineage-specific PRRs. For analogous RLP-mediated responses within the Brassicaceae, extensive phenotyping was earlier performed for several PAMPs -- eMax, nlp20, SCFE1, IF1 and pg13 – across Arabidopsis accessions and related species . However, analysis of the respective PRR loci across Brassicaceae has not yet been conducted, and may reveal processes leading to their dynamic evolution.”356 – I would argue that this paper suggests that RLPs have dynamic evolutionary patterns – similar to NLRs, unlike RKs . They highlight two examples that are conserved, but I would not say that these examples are representative.The comments about complex vs conserved RLPs in the Arabidopsis pangenome are very interesting. We agree that Pruitt 2021 data (https://doi.org/10.1038/s41586-021-03829-0) did indeed show that many RLPs show rapid evolving patterns, just like NLRs. We have rewritten the paragraph to highlight the broader finding, while still pointing out these specific exceptions.A. thaliana varieties (17). Further cross-species analysis may reveal a similar trajectory to INR, via ancestral duplications preceding fixation in the Arabidopsis lineage. In summary, additional case studies for specific receptors are needed to reveal broader patterns in PRR evolution.”“A recent pangenomic analysis of Arabidopsis indicates that while many RLPs occur in complex or copy-number variable loci , three PAMP sensing LRR-RLPs  are conserved across I would prefer that P-values are also shown on the graphs .We opted to add the p-values and other statistical information in an extra supplementary for aesthetic reasons (supplementary table 7).I am not qualified to comment on the quality of the sequencing – but I believe it represents a valuable resource of the community and is sufficient to support the claims in the manuscript.We thank reviewer 2 for the appreciation of the assemblies provided in the manuscript.364 – I would consider TMM a ligand binding LRR-RLP (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458759/) although I appreciate it is significantlyWe agree with the reviewer and changed the sentence accordingly:“Despite multiple intense attempts, and in contrast to LRR-RKs , structural information for PAMP or HAMP sensing LRR-RLPs is not available, including INR .”I would recommend citing this paper for the lack of RLP structure https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503760/.We added the reference as suggested by reviewer 2 in both the introduction and the discussion.