Leucine-rich Repeat-containing Proteins Research Articles

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase.

Chemical-inducible gene expression systems are commonly used to regulate gene expression for functional genomics in various plant species. However, a convenient system that can tightly regulate transgene expression in Nicotiana benthamiana is still lacking. In this study, we developed a tightly regulated copper-inducible system that can control transgene expression and conduct cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system exhibited the least concerns regarding leakiness in N. benthamiana. Although the copper-inducible system can control the expression of some tested reporters, it is not sufficiently tight to regulate certain tested hypersensitive cell death responses. Using the MoClo-based synthetic biology approach, we incorporated the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested leucine-rich repeat-containing proteins and their matching avirulence factors, and it can be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings offer new approaches for both fundamental and translational studies in plant functional genomics.

NRC immune receptor networks show diversified hierarchical genetic architecture across plant lineages.

Plants' complex immune systems include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, which help recognize invading pathogens. In solanaceous plants, the NRC (NLR required for cell death) family includes helper NLRs that form a complex genetic network with multiple sensor NLRs to provide resistance against pathogens. However, the evolution and function of NRC networks outside solanaceous plants are currently unclear. Here, we conducted phylogenomic and macroevolutionary analyses comparing NLRs identified from different asterid lineages and found that NRC networks expanded significantly in most lamiids but not in Ericales and campanulids. Using transient expression assays in Nicotiana benthamiana, we showed that NRC networks are simple in Ericales and campanulids, but have high complexity in lamiids. Phylogenetic analyses grouped the NRC helper NLRs into three NRC0 subclades that are conserved, and several family-specific NRC subclades of lamiids that show signatures of diversifying selection. Functional analyses revealed that members of the NRC0 subclades are partially interchangeable, whereas family-specific NRC members in lamiids lack interchangeability. Our findings highlight the distinctive evolutionary patterns of the NRC networks in asterids and provide potential insights into transferring disease resistance across plant lineages.

NLRP3 inflammasome activity and periodontal disease pathogenesis-A bidirectional relationship.

Periodontitis is an inflammatory oral disease that occurs as a result of the damaging effects of the immune response against the subgingival microflora. Among the mechanisms involved, the nucleotide-binding oligomerization domain, leucine-rich repeat-containing proteins family member NLRP3 (NLR family pyrin domain-containing 3), proposed as the key regulator of macrophage-induced inflammation, is strongly associated with periodontal disease due to the bacterial activators. This paper aimed to present key general concepts of NLRP3 inflammasome activation and regulation in periodontal disease. A narrative review was conducted in order to depict the current knowledge on the relationship between NLRP3 inflammasome activity and periodontal disease. Invitro and insitu studies were retrieved and commented based on their relevance in the field. The NLRP3 inflammasome activity stimulated by periodontal microbiota drive periodontal disease pathogenesis and progression. This occurs through the release of proinflammatory cytokines IL-1β, IL-18, and DAMPs (damage-associated molecular pattern molecules) following inflammasome activation. Moreover, the tissue expression of NLRP3 is dysregulated by oral microbiota, further exacerbating periodontal inflammation. The review provides new insights into the relationship between the NLRP3 inflammasome activity and periodontal disease pathogenesis, highlighting the roles and regulatory mechanism of inflammatory molecules involved in the disease process.

Changes in planta K nutrient content altered the interaction pattern between Nicotiana benthamiana and Alternaria longipes.

Potassium (K) fertilisation has frequently been shown to enhance plant resistance against pathogens, though the mechanisms remain elusive. This study investigates the interaction dynamics between Nicotiana benthamiana and the pathogen Alternaria longipes under different planta K levels. On the host side, adding K activated the expressions of three NLR (nucleotide-binding domain and leucine-rich repeat-containing proteins) resistance genes, including NbRPM1, NbR1B23 and NbNBS12. Silencing these NLRs attenuated resistance in high-K (HK, 40.8 g/kg) plant, whereas their overexpression strengthened resistance in low-K (LK, 23.9 g/kg) plant. Typically, these NLRs mainly strengthened plant resistance via promoting the expression of pathogenesis-related genes (PRs), ROS burst and synthesis of antifungal metabolites in HK plant. On the pathogen side, the expression of effectors HKCSP1, HKCSP2 and LKCSP were shown to be related to planta K content. A. longipes mainly expressed effectors HKCSP1 and HKCSP2 in HK plant to interfere host resistance. HKCSP1 physically interacted with NbRPM1 to promote the degradation of NbRPM1, then attenuated related resistance in HK N. benthamiana. Meanwhile, HKCSP2 directly interacted with NbPR5 to suppress resistance in HK plant. In LK plant, A. longipes mainly deployed LKCSP that interacted with NbR1B23 to interfere reduce resistance in N. benthamiana. Overall, our research insights that both pathogen and host mobilise distinct strategies to outcompete each other during interactions in different K nutrient environments.

Targeting NLRP3 inflammasome for neurodegenerative disorders.

Neuroinflammation is a key pathological feature in neurological diseases, including Alzheimer's disease (AD). The nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) belong to the pattern recognition receptors (PRRs) family that sense stress signals, which play an important role in inflammation. As a member of NLRs, the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is predominantly expressed in microglia, the principal innate immune cells in the central nervous system (CNS). Microglia release proinflammatory cytokines to cause pyroptosis through activating NLRP3 inflammasome. The active NLRP3 inflammasome is involved in a variety of neurodegenerative diseases (NDs). Recent studies also indicate the key role of neuronal NLRP3 in the pathogenesis of neurological disorders. In this article, we reviewed the mechanisms of NLRP3 expression and activation and discussed the role of active NLRP3 inflammasome in the pathogenesis of NDs, particularly focusing on AD. The studies suggest that targeting NLRP3 inflammasome could be a novel approach for the disease modification.

Comprehensive analysis of LRR-RLKs and key gene identification in Pinus massoniana resistant to pine wood nematode.

Pinus massoniana is a pioneer tree widely planted for afforestation on barren hills in southern China where the total planted area is 8.04 million ha. The invasive pine wood nematode (Bursaphelenchus xylophilus) poses a serious threat to the survival of P. massoniana. Plant resistance genes encoded by leucine-rich repeat-containing transmembrane-receptor proteins play important roles in plant defense. Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest subfamily of the RLK protein family, play an important role in sensing stress signals in plants. However, the LRR-RLKs of P. massoniana have not been characterized previously, and their role in resistance to B. xylophilus is unknown. In this study, 185 members of the LRR-RLK subfamily were identified in P. massoniana and were categorized into 14 subgroups. Transcriptomic and quantitative real-time RT-PCR analyses showed that PmRLKs32 was highly expressed in the stem tissue after inoculation with B. xylophilus. The gene exhibited high homology with AtFLS2 of Arabidopsis thaliana. PmRLKs32 was localized to the plasma membrane and was significantly upregulated in nematode-resistant and nematode-susceptible individuals. The transient expression of PmRLKs32 resulted in a burst of reactive oxygen species production in P. massoniana and Nicotiana benthamiana seedlings. These results lay a foundation for further exploration of the regulatory mechanism of LRR-RLKs in response to biotic stress in P. massoniana.

COMPARISON OF MACHINE LEARNING TECHNIQUES FOR PREDICTING NLR PROTEINS

The nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins plays significant role in the intestinal tissue repair and innate immunity. It recently added to the members of innate immunity effectors molecules. It also plays an essential role in intestinal microbiota and recently emerged as a crucial hit for developing ulcerative colitis (UC) and colitis-associated cancer (CAC). A machine learning-based approach for predicting NLR proteins has been developed. In this study, we present a comparison of three supervised machine learning algorithms. Using ProtR and POSSUM Packages, the features are extracted for the dataset used in this work. The models are trained with the input compositional features generated using dipeptide composition, amino acid composition, etc., as well as Position Specific Scoring Matrix (PSSM) based compositions. The dataset consists of 390 proteins for the negative and positive datasets. The five-fold cross-validation (CV) is used to optimize Sequential Minimal Optimization (SMO) library of Support Vector Machine (LIBSVM) and Random Forest (RF) parameters, and the best model was selected. The proposed work performs rationally well with an accuracy of 90.91% and 93.94% for RF as the best classifier for the Amino Acid Composition (AAC) and PSE_PSSM-based model. We believe that this method is a reliable, rapid and useful prediction method for NLR Protein.

Plant NLRs: Evolving with pathogen effectors and engineerable to improve resistance.

Pathogens are important threats to many plants throughout their lifetimes. Plants have developed different strategies to overcome them. In the plant immunity system, nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) are the most common components. And recent studies have greatly expanded our understanding of how NLRs function in plants. In this review, we summarize the studies on the mechanism of NLRs in the processes of effector recognition, resistosome formation, and defense activation. Typical NLRs are divided into three groups according to the different domains at their N termini and function in interrelated ways in immunity. Atypical NLRs contain additional integrated domains (IDs), some of which directly interact with pathogen effectors. Plant NLRs evolve with pathogen effectors and exhibit specific recognition. Meanwhile, some NLRs have been successfully engineered to confer resistance to new pathogens based on accumulated studies. In summary, some pioneering processes have been obtained in NLR researches, though more questions arise as a result of the huge number of NLRs. However, with a broadened understanding of the mechanism, NLRs will be important components for engineering in plant resistance improvement.

The role of NLRP3 inflammasome in colorectal cancer: potential therapeutic target.

All phases of carcinogenesis are affected by inflammation. Activation of the inflammasome is a crucial signaling mechanism that leads to acute and chronic inflammation. When specific nucleotide-binding domains, leucine-rich repeat-containing proteins (NLRs) are activated, inflammasomes are formed. The NLRP3 is one of the NLR family members with the most functional characterization. NLRP3 can modulate the immune systems, apoptosis, growth, and/or the gut microbiome to impact cancer development. Colorectal cancer (CRC) is one of the most common cancers, and it begins as a tissue overgrowth on the internal part of the rectum or colon. In vivo and in vitro studies showed that the NLRP3 inflammasome has a role in CRC development due to its broad activity in shaping immune responses. Here, onwards, we focus on the NLRP3 inflammasome role in CRC development, as well as the therapeutic prospective of modifying NLRP3 inflammasome in the context of anti-cancer therapy.

Genetic deletion of aryl hydrocarbon receptor activates cardiac NLRP3-inflammasome and suppresses apoptosis in mice with acute myocardial infarction

Abstract Background and purpose There is great interest in understanding the role of immune cells in the inflammatory mechanisms associated with acute myocardial infarction (AMI). Resident cardiac immune cells are activated by damage-associated molecular patterns interacting with specific pattern recognition receptors (PRRs), triggering inflammatory responses. Activated immune cells and fibroblasts then promote prohypertrophic and profibrotic signalling, which induces cardiac hypertrophy, fibrosis and remodelling after AMI. Nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) are PRRs that have emerged as key therapeutic targets in cardiovascular disease. NLRP3 assembles the inflammasome, which serves as molecular signalling platform to activate caspase-1 and regulate maturation of the potent proinflammatory cytokine IL-1β. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic Helix-Loop-Helix-PER-ARNT-SIM superfamily that mediates responses against environmental pollutants. Beyond its role in metabolising exogenous toxins as part of an adaptive chemical response, there is growing evidence that AhR is activated by endogenous ligands under physiological conditions. AhR has been proposed as a negative regulator of NLRP3-inflammasome activation. Here we studied the effects of defective AhR signalling on apoptosis, NLRP3-inflammasome activation and survival in a mouse model of AMI. Methods and results AMI was induced in 8–10-weeks-old AhR+/+ and AhR−/− mice by ligation of the left anterior descending coronary artery. Immunofluorescence analysis revealed that the number of TUNEL-positive nuclei and the expression of the apoptotic effector cleaved caspase-3 was higher in hearts from infarcted AhR+/+ mice than from sham-operated AhR+/+ mice 24 h after surgery. A similar analysis in AhR−/− mice revealed the absence of apoptotic markers after AMI. Gene analysis showed that cardiac levels of Nlrp3, apoptosis-associated speck-like protein (Asc) and Il1b were all significantly higher in AhR−/− mice than in AhR+/+ mice 24 h after AMI. Finally, survival analysis showed that 25% of AhR+/+ mice died 72 h after AMI surgery whereas the mortality in AhR−/− mice was 100%. Conclusion Our results show that loss of AhR inhibits cardiac apoptosis and increases NLRP3-mediated inflammation 24 h after AMI, highlighting a possible role for this nuclear receptor in cardiac remodelling post-AMI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund

The Small GTPase OsRac1 Forms Two Distinct Immune Receptor Complexes Containing the PRR OsCERK1 and the NLR Pit.

Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we revealed that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane (PM). Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the PM and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.

Expression analysis of genes related to cold tolerance in Dendroctonus valens.

Pine beetles are well known in North America for their widespread devastation of pine forests. However, Dendroctonus valens LeConte is an important invasive forest pest in China also. Adults and larvae of this bark beetle mainly winter at the trunks and roots of Pinus tabuliformis and Pinus sylvestris; larvae, in particular, result in pine weakness or even death. Since the species was introduced from the United States to Shanxi in 1998, its distribution has spread northward. In 2017, it invaded a large area at the junction of Liaoning, Inner Mongolia and Hebei provinces, showing strong cold tolerance. To identify genes relevant to cold tolerance and the process of overwintering, we sequenced the transcriptomes of wintering and non-wintering adult and larval D. valens using the Illumina HiSeq platform. Differential expression analysis methods for other non-model organisms were used to compare transcript abundances in adults and larvae at two time periods, followed by the identification of functions and metabolic pathways related to genes associated with cold tolerance. We detected 4,387 and 6,091 differentially expressed genes (DEGs) between sampling dates in larvae and adults, respectively, and 1,140 common DEGs, including genes encoding protein phosphatase, very long-chain fatty acids protein, cytochrome P450, and putative leucine-rich repeat-containing proteins. In a Gene Ontology (GO) enrichment analysis, 1,140 genes were assigned to 44 terms, with significant enrichment for cellulase activity, hydrolase activity, and carbohydrate metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment analyses showed that the lysosomal and purine metabolism pathways involved the most DEGs, the highly enriched terms included autophagy—animal, pentose and glucuronate interconversions and lysosomal processes. We identified 140 candidate genes associated with cold tolerance, including genes with established roles in this trait (e.g., genes encoding trehalose transporter, fructose-1,6-bisphosphatase, and trehalase). Our comparative transcriptome analysis of adult and larval D. valens in different conditions provides basic data for the discovery of key genes and molecular mechanisms underlying cold tolerance.

Receptors in the Induction of the Plant Innate Immunity.

Plants adjust amplitude and duration of immune responses via different strategies to maintain growth, development, and resistance to pathogens. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) play vital roles. Pattern recognition receptors, comprising a large number of receptor-like protein kinases and receptor-like proteins, recognize related ligands and trigger immunity. PTI is the first layer of the innate immune system, and it recognizes PAMPs at the plasma membrane to prevent infection. However, pathogens exploit effector proteins to bypass or directly inhibit the PTI immune pathway. Consistently, plants have evolved intracellular nucleotide-binding domain and leucine-rich repeat-containing proteins to detect pathogenic effectors and trigger a hypersensitive response to activate ETI. PTI and ETI work together to protect plants from infection by viruses and other pathogens. Diverse receptors and the corresponding ligands, especially several pairs of well-studied receptors and ligands in PTI immunity, are reviewed to illustrate the dynamic process of PTI response here.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Insight Into Inflammasome Signaling: Implications for Toxoplasma gondii Infection

Inflammasomes are multimeric protein complexes regulating the innate immune response to invading pathogens or stress stimuli. Recent studies have reported that nucleotide-binding leucine-rich repeat-containing (NLRs) proteins and DNA sensor absent in melanoma 2 (AIM2) serve as inflammasome sentinels, whose stimulation leads to the proteolytic activation of caspase-1, proinflammatory cytokine secretion, and pyroptotic cell death. Toxoplasma gondii, an obligate intracellular parasite of phylum Apicomplexans, is reportedly involved in NLRP1, NLRP3 and AIM2 inflammasomes activation; however, mechanistic evidence regarding the activation of these complexes is preliminary. This review describes the current understanding of inflammasome signaling in rodent and human models of T. gondii infection.

A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity.

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and β-subunit (BKβ1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

PALs: Emerging Key Players in Broad-Spectrum Disease Resistance.

Phenylalanine ammonia lyases (PALs) are involved in resistance to plant pathogens, but the mechanism by which they contribute to plant immunity is unclear. Two recent studies provide direct evidence for PALs' contributions to rice broad-spectrum resistance (BSR) mediated by nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins and RNA-binding proteins (Zhai et al., Mol. Cell, 2019; Zhou et al., Proc. Natl. Acad. Sci. U. S. A., 2018).

Genome-wide association study of rice genes and loci conferring resistance to Magnaporthe oryzae isolates from Taiwan

BackgroundRice blast, caused by Magnaporthe oryzae, is an important rice disease occurring in all rice-growing areas. To manage blast disease effectively and in an environmentally friendly way, it is important to continually discover diverse resistant resources for breeding. In this study, genome-wide association study (GWAS) was used to map genes/loci resistant to rice blast in the open-access rice diversity panel 1 (RDP1), previously genotyped with a 44K single-nucleotide polymorphism array. Two geographically and genetically different M. oryzae isolates from Taiwan, D41-2 and 12YL-DL3-2, were used to challenge RDP1. Infected leaves were visually rated for lesion type (LT) and evaluated for proportion of diseased leaf area (%DLA) by image analysis software.ResultsA total of 32 quantitative trait loci (QTLs) were identified, including 6 from LT, 30 from DLA, and 4 from both LT and DLA. In all, 22 regions co-localized with previously reported resistance (R) genes and/or QTLs, including two cloned R genes, Pita and Ptr; 19 mapped R loci, and 20 QTLs. We identified 100 candidate genes encoding leucine-rich repeat-containing proteins, transcription factors, ubiquitination-related proteins, and peroxidases, among others, in the QTL intervals. Putative resistance and susceptibility haplotypes of the 32 QTL regions for each tested rice accessions were also determined.ConclusionsBy using Taiwanese M. oryzae isolates and image-based phenotyping for detailed GWAS, this study offers insights into the genetics underlying the natural variation of blast resistance in RDP1. The results can help facilitate the selection of desirable donors for gene/QTL validation and blast resistance breeding.

Multiple strategies for pathogen perception by plant immune receptors.

Contents Summary 17 I. Introduction 17 II. Pathogen perception by NLRs: from direct recognition to integrated decoys 18 III. Multiple activation and signaling pathways for NLRs 18 IV. How to engineer NLR-mediated disease resistance? 21 V. Conclusion 23 Acknowledgements 23 References 23 SUMMARY: Plants have evolved a complex immune system to protect themselves against phytopathogens. A major class of plant immune receptors called nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) is ubiquitous in plants and is widely used for crop disease protection, making these proteins critical contributors to global food security. Until recently, NLRs were thought to be conserved in their modular architecture and functional features. Investigation of their biochemical, functional and structural properties has revealed fascinating mechanisms that enable these proteins to perceive a wide range of pathogens. Here, I review recent insights demonstrating that NLRs are more mechanistically and structurally diverse than previously thought. I also discuss how these findings provide exciting future prospects to improve plant disease resistance.

NLR network mediates immunity to diverse plant pathogens

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that "sensor" NLR proteins are paired with "helper" NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.