Author response: Evolutionary gain and loss of a plant pattern-recognition receptor for HAMP recognition
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 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. Editor's evaluation 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. https://doi.org/10.7554/eLife.81050.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest 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. Introduction Plant and animal innate immunity is mediated by germline-encoded pattern recognition receptors (PRRs; Ronald and Beutler, 2010). PRRs serve as specific sensors of various pathogen-, herbivore-, and damage-associated molecular patterns (PAMPs, HAMPs, and DAMPs; Snoeck et al., 2022; Albert et al., 2020; Gust et al., 2017). In plants, PRRs in the large receptor kinase (RK) family comprise an extracellular domain, a single-pass transmembrane motif, and an intracellular kinase domain (DeFalco and Zipfel, 2021). Receptor-like proteins (RLPs) share structural similarities with RKs but lack a kinase domain (Wang et al., 2008; Fritz-Laylin et al., 2005). Extracellular domains of RK/RLPs include LysM, lectin, and malectin domains, but the most common ectodomain is a series of leucine-rich repeats (LRRs) which mediate PAMP binding and co-receptor association (Albert et al., 2020; Shiu and Bleecker, 2003; Shiu and Bleecker, 2001; Fischer et al., 2016; Hohmann et al., 2017; Restrepo-Montoya et al., 2020). In plants, LRR-RK/RLPs form a large gene family; for example, the Arabidopsis thaliana genome contains about 223 LRR-RKs and 57 LRR-RLPs (Wang et al., 2008; Fritz-Laylin et al., 2005; Shiu and Bleecker, 2001; Lehti-Shiu et al., 2009), and the number of annotated LRR-RK/RLPs per genome varies across plant species (Restrepo-Montoya et al., 2020; Ngou et al., 2022). Moreover, comparative genomic analyses of LRR-RK/RLPs involved in biotic interactions have revealed strong diversifying selection, lineage-specific expanded gene clusters, and immune receptor repertoire variation both within and between species (Fritz-Laylin et al., 2005; Jamieson et al., 2018; Tang et al., 2010; Pruitt et al., 2021; Steinbrenner, 2020), making the gene family a model for the evolution of innate immune systems. Evidence is accumulating for highly specialized roles of plant LRR-RLPs as immune sensors (Jamieson et al., 2018; Steinbrenner, 2020). 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 (Albert et al., 2020; Jamieson et al., 2018). For example, Cf-9, Cf-4, and Cf-2 interact with respective molecular patterns of Cladosporium fulvum, Avr9, Avr4, and Avr2 (via host protein Rcr3), and are restricted to the Solanum genus (Jones et al., 1994; Thomas et al., 1997; Kruijt et al., 2007; Dixon et al., 1996; Luderer et al., 2002; Krüger et al., 1979; Rooney et al., 2005). Arabidopsis RLP42 recognizes fungal endopolygalacturonases (PGs) eptitope pg9(At) derived from Botrytis cinerea (Zhang et al., 2014; Zhang et al., 2021). 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 (Jamieson et al., 2018; Albert et al., 2015). RXEG1 and RE02 were identified in wild tobacco and are respectively triggered by the fungal elicitors XEG1 (Phytophthora sojae) and E02 (Valsa mali) (Ma et al., 2015; Nie et al., 2021). LepR3 (AvrLm1) and RLM2 (AvrLm2) convey both race specific resistance to the fungal pathogen Leptosphaeria maculans in rapeseed (Larkan et al., 2015; Larkan et al., 2022). ReMAX is restricted to the Brassicaceae and triggered by the PAMP eMax originating from Xanthomonas (Jehle et al., 2013). Cuscuta Receptor1 (CuRe1) is specific to Solanum lycopersicum and senses the peptide Crip21, which originates from parasitic plants of the genus Cuscuta (Hegenauer et al., 2020; Hegenauer et al., 2016). 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 (Steinbrenner et al., 2020; Schmelz et al., 2006; Schmelz et al., 2012; Schmelz et al., 2007). Notably, all the above examples of LRR-RLPs are family-specific, restricted to the Solanaceae (Cf-2, Cf-4, Cf-9, RXEG1, RE02, and CuRe1), Brassicaceae (RLP23, RLP42, LepR3, RLM2, and ReMAX), or Leguminosae (tribe Phaseoleae; INR; Steinbrenner, 2020; Zhang et al., 2021). However, despite clear signatures of lineage-specific functions, specific evolutionary steps leading to novel LRR-RLP functions across multiple species have not been described. Mechanistic understanding of LRR-RLP sensing functions is also currently limited. Structural data exists for only one PAMP or HAMP sensing LRR-RLP, and genetic experiments have been limited to receptors from model species (Zhang et al., 2021; Albert et al., 2019; Kourelis et al., 2020; Hohmann and Hothorn, 2019; Sun et al., 2022). Ancestral sequence reconstruction (ASR) is a powerful approach for studying the evolution of protein function and specificity and has been applied to a limited number of receptor functions (Delaux et al., 2019; Białas et al., 2021; Scossa and Fernie, 2021; Carroll et al., 2011; Chantreau et al., 2019). However, ASR has not yet been applied to rapidly evolving LRR-RLPs functions, often encoded by complex multi-copy loci (Steinbrenner, 2020). Importantly, LRR-RLP recognition functions can be assessed through heterologous expression in a model plant, wild tobacco (Nicotiana benthamiana; Zhang et al., 2021; Steinbrenner et al., 2020; Albert et al., 2019; Goodin et al., 2008). 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 INR receptor locus. By leveraging both existing high-quality assemblies and long read, de novo assemblies of key legume species, we were able to analyze the ~53 million year (my) 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. Results In11 perception is restricted to certain legume species within the Phaseoloid clade Response to the peptide HAMP In11 was previously thought to be highly restricted since only species in a subtribe of legumes, the Phaseolinae, which includes the species cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), encode the corresponding INR receptor (Steinbrenner et al., 2020). To understand the emergence of INR function within legumes with a higher precision, we measured ethylene accumulation triggered by In11 as a defense marker across a set of 22 legume species of the NPAAA papilionoids (Figure 1a, Supplementary file 5a, b). Response to In11 was observed in plant species within the broader monophyletic clade of the Phaseoloids. Hence, phylogenetic evidence suggests a single origin of In11 response at the base of the Phaseoloid legumes ~28 my ago (mya; Figure 1b, ★). Several of the tested plant species/accessions within this clade were unable to respond to In11, namely Hylodesmum podocarpum, winged bean (Psophocarpus tetragonolobus), soybean (Glycine max), yam bean (Pachyrhizus erosus), and calopo (Calopogonium mucunoides), although these species were able to respond to the unrelated bacterial PAMP flg22 (Figure 1—figure supplement 1). These observations suggest the occurrence of multiple independent losses of In11 response throughout Phaseoloid evolution after the initial emergence of this function. Figure 1 with 1 supplement see all Download asset Open asset Induced ethylene response to In11 is limited to Phaseoloid legumes. (a) Individual trifoliate leaflets were scratch wounded and treated with 1 μM In11 or H2O. The ratio of ethylene production for leaflets within the same leaf is shown (x-axis). The vertical orange line shows a ratio equal to one, i.e., no In11-induced ethylene burst. Biologically replicated plants are shown as separate dots. Significant differences between the control and the treatment of interest are indicated (paired Wilcoxon signed-rank test; ns non-significant, * p≤0.05, ** p≤0.01, and *** p≤0.001). Plant species names are colored blue (significant response to In11) or red (insignificant response). Species/accessions and resulting response data can respectively be found in Supplementary file 5a and Supplementary file 5b. (b) A summary chronogram representing time-based phylogenetic relationships within the tested lineages, with colors representing In11 response phenotypes as in (a). The star symbol indicates the node containing all In11-responsive species at the base of Phaseoloid legumes. Divergence time is shown in million years ago (mya) and represents a composite average (Egan et al., 2016; Li et al., 2013; Lavin et al., 2005; Stefanović et al., 2009). The contiguous INR locus shows LRR-RLP copy number variation in the Millettioids To explore gain and loss of In11 response in relation to its defined PRR, we analyzed the evolution of its cognate receptor INR and its genomic locus in existing reference genomes and high quality de novo genome assemblies of Phaseoloid and non-Phaseoloid species. We focused de novo sequencing efforts on key nodes separating In11-responsive and unresponsive species in the phylogeny for comparative genomic analysis (Figure 1b). The contiguous INR locus, which is flanked by conserved anchor genes, was extracted from 16 existing legume genomes and four de novo assemblies: P. erosus, guar bean (Cyamopsis tetragonoloba), jack bean (Canavalia ensiformis), and H. podocarpum (Figure 2, Supplementary file 6a; Steinbrenner et al., 2020). For de novo assemblies obtained in this study, a combination of Oxford Nanopore and Illumina sequence data facilitated the assembly of a contiguous INR locus in all sequenced species (Supplementary file 6b). Figure 2 with 2 supplements see all Download asset Open asset Leucine-rich repeat (LRR)-receptor-like protein (RLP) copy number variation at the INR locus in Millettioid and non-Millettioid legume genomes. Anchor, LRR-RLP, and other genes are colored as per legend. (a) Locus comparison of the contiguous INR locus of 20 NPAAA papilionoid species. Blast hits between loci are indicated with lines (e-value <1e-04) with score according to grayscale gradient and darker grays indicating higher similarity. Genes are labeled LRR-RLP if a complete coding sequence is present (≥875 AA). Species names followed by superscript (#) were newly sequenced and assembled for this project. (b) Locus comparison of the contiguous INR locus of P. vulgaris and P. erosus. P. vulgaris has a functionally validated inceptin receptor (INR) homolog (LRR-RLP) (Steinbrenner et al., 2020), while P. erosus lacks a full-length LRR-RLP. The disruption of the P. erosus LRR-RLP was validated by PCR followed by Sanger sequencing as indicated with light green bars. 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 (Figure 2a, Figure 2—figure supplement 1, Supplementary file 6c). For all species, BLASTN search did not identify any regions with higher score than sequences at the contiguous INR locus of the respective species, strongly suggesting that all potential INR homologs are encoded at this syntenic locus. Legume species within the Hologalegina, namely Lotus angustifolius, chickpea (Cicer arietinum), 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 (Figure 1a). To investigate this emergence, we analyzed the de novo assembly of C. tetragonoloba, a close outgroup of Millettioid legumes (Figure 1b; Cardoso et al., 2015; Zhao et al., 2021). As with all species in the outgroup Hologalegina, neither LRR-RLP was present between the anchor genes in C. tetragonoloba nor did we find an LRR-RLP with greater than 68% similarity in the whole genome, strengthening support for a single ancestral LRR-RLP gene insertion event ~32 mya (Figure 2a). In contrast to other closely related legume species, BLASTN analysis of the contiguous INR locus of P. erosus revealed only partial coding sequence fragments with homology to LRR-RLPs, and consequently the absence of an INR homolog (Figure 2b). The de novo assembly of the locus was validated by performing PCR spanning the complete disruption and Sanger sequencing of the resulting amplicon. A Mariner-like transposase element exists between the LRR-RLP gene fragments (Figure 2—figure supplement 1,Supplementary file 6c). The absence of an INR homolog in P. erosus corresponds with species phenotype, namely the lack of induced ethylene response after In11 treatment (Figure 1a). In11-induced functions are conferred by a single clade of LRR-RLPs (INR clade) To associate INR locus variation with the variable In11 responses (Figure 1), we next investigated the function and relationship of individual LRR-RLP homologs at the contiguous INR locus. We performed a maximum-likelihood phylogenetic analysis on the protein sequences of the LRR-RLPs within the locus across 16 Millettioid species (Figure 2). This analysis was supplemented with the closest related LRR-RLP genes outside the contiguous INR locus from V. unguiculata and P. vulgaris: Phvul.007g246600 and Vigun07g039700 (Supplementary file 2). A single well-supported clade which includes the previously characterized functional INR from V. unguiculata (Vigun07g219600) also contained orthologs from plant species able to respond to In11 (Figure 3a; Steinbrenner et al., 2020). Hence, we hypothesized that this clade contains functional INR homologs which can confer In11-induced functions. Figure 3 with 2 supplements see all Download asset Open asset Phylogenetic analysis of leucine-rich repeat (LRR)-receptor-like proteins (RLPs) at the contiguous INR locus in 16 diverse species and subsequent heterologous expression reveals a clade of functional receptors. (a) A phylogenetic analysis of LRR-RLPs from 16 Millettioid species is shown. Maximum likelihood analysis bootstrap values are indicated, and only values higher than 65 are shown. The scale bar represents 0.5 AA substitutions per site. Filled dots indicate species of origin according to legend, where different colors indicate different subtribes. A T. pratense LRR-RLP was used as an outgroup to root the phylogenetic gene tree and is underlined. One asterisk (*) highlights the LRR-RLPs which are not part of a contiguous INR locus. Two asterisks (**) highlight the LRR-RLPs used to create the chimeric receptors. The functionally validated inceptin receptors (INRs) of P. vulgaris, V. unguiculata, M. lathyroides, C. cajan, and M. pruriens are highlighted in blue as they confer induced reactive species and ethylene functions in response to In11 heterologous expression in as shown respectively in and C. These validated within the labeled receptors which were not to In11 are highlighted in (b) are after treatment with or the peptide In11 indicate for four independent with representing The shows the of ethylene after with 1 μM In11 or independent was tested by performing a Wilcoxon signed-rank non-significant, * To the INR genes INR, and within this clade were and in the model species function was measured production of the plant defense ethylene and reactive species In11-induced ethylene and production were able to be conferred by gene (Figure consistent with novel In11 recognition by the INR clade (Figure blue responses were observed for of In11 receptors and a of the response but in ethylene (Figure supplement 2). To if more INR-like homologs could also confer In11 we measured ethylene and production expression of LRR-RLPs outside the INR the clade to the INR clade contains LRR-RLPs from of species which also have a INR homolog in the INR clade (Figure this we and the LRR-RLP of V. unguiculata for P. vulgaris all have an LRR-RLP in of the above (Figure 2). The species, H. podocarpum, C. and were (Figure and encode LRR-RLP receptors which outside the INR clade and its this we and the soybean LRR-RLP Finally, we tested the most closely related LRR-RLP to INR outside the contiguous INR locus for both V. unguiculata and P. vulgaris and In11-induced responses could be observed heterologous expression of and Phvul.007g246600 (Figure of these LRR-RLPs were or more strongly in to the LRR-RLP INR; Figure supplement 1), except for the which with its closest related gene in P. vulgaris AA Thus, the ability to confer In11-induced and ethylene production is limited to members of the INR We identified and potential gene and losses at the contiguous INR locus throughout its evolution within the Millettioids (Figure 2—figure supplement et al., analysis between gene and species revealed a complex evolutionary history gene and 20 gene losses in the a single event to the INR clade containing all INR homologs and its clade which contains the closest related INR-like P. vulgaris and pruriens contain an INR clade homolog but not members of the INR-like 1 consistent with independent gene In the analysis the ancestral loss of an INR homolog within the INR clade for and H. podocarpum and clade specific events resulting in LRR-RLP of To that INR was lost in and not in reference we performed AA sequence and de novo assemblies from a analysis et al., 2020). the reference no hits to for any of the de novo assemblies were identified that had an AA similarity higher than suggesting that INR was lost the of and i.e., to soybean The C1 and C2 of the ectodomain mediate In11-induced functions To understand receptor to the functional INR we assembled chimeric receptors and AA Supplementary file genes contain a LRR-RLP extracellular domain with by a intervening motif (Figure Steinbrenner et al., 2020). Chimeric receptors were at a in and response to In11 was assessed by ethylene and (Figure Figure supplement 1). Chimeric receptors with the C1 domain were not to In11. In all chimeric receptors containing the of to In11 treatment with both an ethylene and and Figure the chimeric receptor containing C1 of and C2 of to the control no was observed of in with or subsequent of In11, or 1 the (Figure supplement the chimeric receptor, to In11 but has a to all other In11-responsive In the C1 and C2 are both for In11 response in chimeric receptors. Figure with 2 supplements see all Download asset Open asset Chimeric receptors indicate that the C1 and C2 mediate inceptin receptor (INR) recognition function. (a) of and the chimeric receptors used for analysis. Leucine-rich repeat (LRR)-receptor-like protein (RLP) as per Fritz-Laylin et al., 2005; one or of that may structural multiple with an intervening motif domain, domain, transmembrane and sequences of the chimeric receptors can be found in Supplementary file (b) reactive species production the heterologous expression of receptors in are after treatment with or the peptide In11 indicate for four independent with representing production the heterologous expression of receptors in production was after with or the peptide In11 independent was tested by performing a Wilcoxon signed-rank non-significant, * ASR of the INR domain To understand the molecular for functional divergence between INR and unresponsive INR-like homologs, we multiple ancestral sequences of the monophyletic In11-responsive INR clade and its INR-like 1 clade (Figure Figure supplement 1, Supplementary file To perform we first that nodes of interest for reconstruction were well supported by both and likelihood gene (Figure supplement 1). we reconstructed the ancestral sequences for the domain (Supplementary file et al., and the resulting domains with domains and from to complete the ancestral receptor in Figure expression in was across all ASR (Figure supplement 2). Figure with 2 supplements see all Download asset Open asset analysis of ancestrally reconstructed leucine-rich repeat domains of inceptin receptor (INR) and INR-like receptors. (a) of the phylogenetic analyses of the of proteins (RLPs) from the contiguous INR locus used for the ancestral sequence reconstruction (ASR) phylogeny can be found in Figure supplement reconstructed nodes are with a star and The scale bar represents AA substitutions per site. sequences of the domain can be found in Supplementary file (b) of and the ASR receptors used for analysis. LRR-RLP as per Fritz-Laylin et al., 2005; one or of that may
Source: eLife
Language: English
Subfield: Insect Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4312261890