The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens. The ability to induce defenses in response to pathogen attack is a critical feature of immunity in any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and have evolved to perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. The mechanism by which defense signaling is triggered by these NLRs is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, their complexes are highly heterometric. Also, in contrast to metazoan NLRs, their N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions is proposed to provide resilience to perturbation by pathogens.

Leucine-rich-repeats (LRRs) belong to an archaic procaryal protein architecture that is widely involved in protein–protein interactions. In eukaryotes, LRR domains developed into key recognition modules in many innate immune receptor classes. Due to the high sequence variability imposed by recognition specificity, precise repeat delineation is often difficult especially in plant NOD-like Receptors (NLRs) notorious for showing far larger irregularities. To address this problem, we introduce here LRRpredictor, a method based on an ensemble of estimators designed to better identify LRR motifs in general but particularly adapted for handling more irregular LRR environments, thus allowing to compensate for the scarcity of structural data on NLR proteins. The extrapolation capacity tested on a set of annotated LRR domains from six immune receptor classes shows the ability of LRRpredictor to recover all previously defined specific motif consensuses and to extend the LRR motif coverage over annotated LRR domains. This analysis confirms the increased variability of LRR motifs in plant and vertebrate NLRs when compared to extracellular receptors, consistent with previous studies. Hence, LRRpredictor is able to provide novel insights into the diversification of LRR domains and a robust support for structure-informed analyses of LRRs in immune receptor functioning.

Background: V(D) J recombination is essential for adaptive immunity in jawed vertebrates and is initiated by the RAG1-RAG2 endonuclease. The RAG1 and RAG2 genes are thought to have evolved from a RAGL (RAG-like) transposon containing convergently-oriented RAG1-like (RAG1L) and RAG2-like (RAG2L) genes. Elements resembling this presumptive evolutionary precursor have thus far only been detected convincingly in deuterostomes, leading to the model that the RAGL transposon first appeared in an early deuterostome. Results: We have identified numerous RAGL transposons in the genomes of protostomes, including oysters and mussels (phylum Mollusca) and a ribbon worm (phylum Nemertea), and in the genomes of several cnidarians. Phylogenetic analyses are consistent with vertical evolution of RAGL transposons within the Bilateria clade and with its presence in the bilaterian ancestor. Many of the RAGL transposons identified in protostomes are intact elements containing convergently oriented RAG1L and RAG2L genes flanked by terminal inverted repeats (TIRs) and target site duplications with striking similarities with the corresponding elements in deuterostomes. In addition, protostome genomes contain numerous intact RAG1L-RAG2L adjacent gene pairs that lack detectable flanking TIRs. Domains and critical active site and structural amino acids needed for endonuclease and transposase activity are present and conserved in many of the predicted RAG1L and RAG2L proteins encoded in protostome genomes. Conclusions: Active RAGL transposons were present in multiple protostome lineages and many were likely transmitted vertically during protostome evolution. It appears that RAGL transposons were broadly active during bilaterian evolution, undergoing multiple duplication and loss/fossilization events, with the RAGL genes that persist in present day protostomes perhaps constituting both active RAGL transposons and domesticated RAGL genes. Our findings raise the possibility that the RAGL transposon arose earlier in evolution than previously thought, either in an early bilaterian or prior to the divergence of bilaterians and non-bilaterians, and alter our understanding of the evolutionary history of this important group of transposons.

Summary: NLR (nucleotide‐binding [NB] leucine‐rich repeat [LRR] receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly modulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor‐like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana that allowed us to demonstrate that ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modelling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions modulate ZAR1 activity. We identified determinants required for the formation of the ZAR^CC oligomer and its activity. Lastly, we characterized intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

A series of “molecular domestication” events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events is not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, RAG2L-A proteins contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g., the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.