Intracellular Immune Receptors Research Articles

Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant.

Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici. The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50QCMJC, which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50QCMJC. Following an initial structural hypothesis driven by molecular docking, we identified the Sr50K711D single mutant, which induces an intermediate immune response against AvrSr50QCMJC without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50QCMJC with greater immune response intensities than Sr50K711D against the escape mutant. All effective mutations against AvrSr50QCMJC required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50K711D and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection.

Evolutionary and immune-activating character analyses of NLR genes in algae suggest the ancient origin of plant intracellular immune receptors.

Nucleotide-binding leucine-rich repeat (NLR) proteins are crucial intracellular immune receptors in plants, responsible for detecting invading pathogens and initiating defense responses. While previous studies on the evolution and function of NLR genes were mainly limited to land plants, the evolutionary trajectory and immune-activating character of NLR genes in algae remain less explored. In this study, genome-wide NLR gene analysis was conducted on 44 chlorophyte species across seven classes and seven charophyte species across five classes. A few but variable number of NLR genes, ranging from one to 20, were identified in five chlorophytes and three charophytes, whereas no NLR gene was identified from the remaining algal genomes. Compared with land plants, algal genomes possess fewer or usually no NLR genes, implying that the expansion of NLR genes in land plants can be attributed to their adaptation to the more complex terrestrial pathogen environments. Through phylogenetic analysis, domain composition analysis, and conserved motifs profiling of the NBS domain, we detected shared and lineage-specific features between NLR genes in algae and land plants, supporting the common origin and continuous evolution of green plant NLR genes. Immune-activation assays revealed that both TNL and RNL proteins from green algae can elicit hypersensitive responses in Nicotiana benthamiana, indicating the molecular basis for immune activation has emerged in the early evolutionary stage of different types of NLR proteins. In summary, the results from this study suggest that NLR proteins may have taken a role as intracellular immune receptors in the common ancestor of green plants.

A comprehensive review of soybean RNL and TIR domain proteins.

Both prokaryotic and eukaryotic organisms use the nucleotide-binding domain/leucine-rich repeat (NBD/LRR)-triggered immunity (NLR-triggered immunity) signaling pathway to defend against pathogens. Plant NLRs are intracellular immune receptors that can bind to effector proteins secreted by pathogens. Dicotyledonous plants express a type of NLR known as TIR domain-containing NLRs (TNLs). TIR domains are enzymes that catalyze the production of small molecules that are essential for immune signaling and lead to plant cell death. The activation of downstream TNL signaling components, such as enhanced disease susceptibility 1 (EDS1), phytoalexin deficient 4 (PAD4), and senescence-associated gene 101 (SAG101), is facilitated by these small molecules. Helper NLRs (hNLRs) and the EDS1-PAD4/SAG101 complex associate after activation, causing the hNLRs to oligomerize, translocate to the plasma membrane (PM), and produce cation-selective channels. According to a recent theory, cations enter cells through pores created by oligomeric hNLRs and trigger cell death. Occasionally, TNLs can self-associate to create higher-order oligomers. Here, we categorized soybean TNLs based on the protein domains that they possess. We believe that TNLs may help soybean plants effectively fight pathogens by acting as a source of genetic resistance. In summary, the purpose of this review is to elucidate the range of TNLs that are expressed in soybean.

The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of nonflowering plants retain immune-related functions through translineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to nonflowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable with motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the nonflowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent nonflowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

Abstract 2461: Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy

Abstract Boosting anti-tumor immune responses can be achieved by activating the innate immune system. Despite the ongoing development of numerous STING and TLR agonists for this purpose, none of them have progressed to late-stage clinical evaluation. Alpha protein kinase-1 (ALPK1) is an intracellular innate immune receptor newly identified for its ability to detect ADP-heptose, a metabolite released during the biosynthesis and degradation of lipopolysaccharides (LPS). Since heptose is not naturally present in mammalian cells, the recognition of heptose metabolites by ALPK1 serves as a reliable mechanism to identify bacterial infections. When ADP-heptose binds to ALPK1, it initiates the phosphorylation of TIFA, thereby triggering a cascade of immune responses. Systemic administration with ADP-heptose induces robust ALPK1-dependent cytokine responses in mice. While high doses of ADP-heptose demonstrate anti-tumor effects, its instability and low in vivo potency hinder its clinical development. Through a SBDD medicinal chemistry effort, a novel, stable, and potent ADP-heptose analog, designated PTT-936, has been discovered and is progressing into clinical evaluation in cancer patients. PTT-936 exhibits remarkable nanomolar potencies for ALPK1 activation in various cellular assays. Notably, in human PBMCs, PTT-936 induces both concentration-dependent release of cytokines and activation of NK and T cells. Consistent with in vitro studies, oral administration of PTT-936 triggered significant cytokine responses in mice at microgram dose levels. However, overt toxicities were observed only at milligram dose levels, indicating a wide therapeutic window for this compound. In syngeneic tumor models, oral administration of PTT-936 induced dose-dependent tumor growth inhibition, which correlated with elevations in several cytokine biomarkers. Mechanistic studies show that PTT-936, working exclusively through the ALPK1 pathway, elicited strong innate and adaptive immune responses, as evidenced by robust cytokine induction and immune cell activation and recruitment. In syngeneic mouse models, the anti-tumor effects of PTT-936 were significantly hindered by either CD8+ T cell depletion or neutralization of select cytokines. Besides monotherapy studies, compelling evidence demonstrates strong synergistic anti-tumor effects when combining PTT-936 with immune checkpoint inhibitors. Intriguingly, PTT-936 exhibits favorable tolerability and a wider therapeutic index compared to certain clinical-stage STING and TLR agonists. Furthermore, PTT-936 does not enhance cytokine elevations induced by checkpoint inhibitors, which have been linked to adverse events (including CRS) observed in clinical combination studies. The dosing frequency of PTT-936 in the upcoming first-in-human clinical trial is carefully designed to prioritize both tolerability and patient compliance. Citation Format: Yupeng Wang, Nana Du, Xiaona Dong, Xuebing Sun, Yuan Hong, Xiaolong Yang, Zhijiang Ji, Qianyu Wang, Yun Lv, Zhichen Shi, Yajuan Gao, Xiaofu Hou, Yaxin Zhang, Juan Wang, Rui Mao, Yinxin Liu, Liang Li, Jingjin Ding, Hexiang Wang, Nan Hu, Counde O’Yang, Zhihong Li, Keith Bley, Feng Shao. Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2461.

Extracellular perception of multiple novel core effectors from the broad host-range pear anthracnose pathogen Colletotrichum fructicola in the nonhost Nicotiana benthamiana.

Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in a wide range of horticultural crops, particularly fruits. Exploitation of nonhost resistance (NHR) represents a robust strategy for plant disease management. Perception of core effectors from phytopathogens frequently leads to hypersensitive cell death and resistance in nonhost plants; however, such core effectors in C. fructicola and their signaling components in non-hosts remain elusive. Here, we found a virulent C. fructicola strain isolated from pear exhibits non-adaptation in the model plant Nicotiana benthamiana. Perception of secreted molecules from C. fructicola appears to be a dominant factor in NHR, and four novel core effectors-CfCE4, CfCE25, CfCE61, and CfCE66-detected by N. benthamiana were, accordingly, identified. These core effectors exhibit cell death-inducing activity in N. benthamiana and accumulate in the apoplast. With a series of CRISPR/Cas9-edited mutants or gene-silenced plants, we found the coreceptor BAK1 and helper NLRs including ADR1, NRG1, and NRCs mediate perceptions of these core effectors in N. benthamiana. Concurrently, multiple N. benthamiana genes encoding cell surface immune receptors and intracellular immune receptors were greatly induced by C. fructicola. This work represents the first characterization of the repertoire of C. fructicola core effectors responsible for NHR. Significantly, the novel core effectors and their signaling components unveiled in this study offered insights into a continuum of layered immunity during NHR and will be helpful for anthracnose disease management in diverse horticultural crops.

New insight into Ca2+ -permeable channel in plant immunity.

Calcium ions (Ca2+ ) are crucial intracellular second messengers in eukaryotic cells. Upon pathogen perception, plants generate a transient and rapid increase in cytoplasmic Ca2+ levels, which is subsequently decoded by Ca2+ sensors and effectors to activate downstream immune responses. The elevation of cytosolic Ca2+ is commonly attributed to Ca2+ influx mediated by plasma membrane-localized Ca2+ -permeable channels. However, the contribution of Ca2+ release triggered by intracellular Ca2+ -permeable channels in shaping Ca2+ signaling associated with plant immunity remains poorly understood. This review discusses recent advances in understanding the mechanism underlying the shaping of Ca2+ signatures upon the activation of immune receptors, with particular emphasis on the identification of intracellular immune receptors as non-canonical Ca2+ -permeable channels. We also discuss the involvement of Ca2+ release from the endoplasmic reticulum in generating Ca2+ signaling during plant immunity.

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato

BackgroundGeminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV).ResultsWe showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes.ConclusionWe have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

ROS and RNS production, subcellular localization, and signaling triggered by immunogenic danger signals.

Plants continuously monitor the environment to detect changing conditions and to properly respond, avoiding deleterious effects on their fitness and survival. An enormous number of cell surface and intracellular immune receptors are deployed to perceive danger signals associated with microbial infections. Ligand binding by cognate receptors represents the first essential event in triggering plant immunity and determining the outcome of the tissue invasion attempt. Reactive oxygen and nitrogen species (ROS/RNS) are secondary messengers rapidly produced in different subcellular localizations upon the perception of immunogenic signals. Danger signal transduction inside the plant cells involves cytoskeletal rearrangements as well as several organelles and interactions between them to activate key immune signaling modules. Such immune processes depend on ROS and RNS accumulation, highlighting their role as key regulators in the execution of the immune cellular program. In fact, ROS and RNS are synergic and interdependent intracellular signals required for decoding danger signals and for the modulation of defense-related responses. Here we summarize current knowledge on ROS/RNS production, compartmentalization, and signaling in plant cells that have perceived immunogenic danger signals.

The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis.

In the presence of pathogenic bacteria, plants close their stomata to prevent pathogen entry. Intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogenic effectors and activate effector-triggered immune responses. However, the regulatory and molecular mechanisms of stomatal immunity involving NLR immune receptors are unknown. Here, we show that the Nicotiana benthamiana RPW8-NLR central immune receptor ACTIVATED DISEASE RESISTANCE 1 (NbADR1), together with the key immune proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (NbEDS1) and PHYTOALEXIN DEFICIENT 4 (NbPAD4), plays an essential role in bacterial pathogen- and flg22-induced stomatal immunity by regulating the expression of salicylic acid (SA) and abscisic acid (ABA) biosynthesis or response-related genes. NbADR1 recruits NbEDS1 and NbPAD4 in stomata to form a stomatal immune response complex. The transcription factor NbWRKY40e, in association with NbEDS1 and NbPAD4, modulates the expression of SA and ABA biosynthesis or response-related genes to influence stomatal immunity. NbADR1, NbEDS1, and NbPAD4 are required for the pathogen infection-enhanced binding of NbWRKY40e to the ISOCHORISMATE SYNTHASE 1 promoter. Moreover, the ADR1-EDS1-PAD4 module regulates stomatal immunity in Arabidopsis (Arabidopsis thaliana). Collectively, our findings show the pivotal role of the core intracellular immune receptor module ADR1-EDS1-PAD4 in stomatal immunity, which enables plants to limit pathogen entry.

Helper NLRs Nrc2 and Nrc3 act codependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato.

Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), detect pathogen effector proteins and activate NLR-triggered immunity (NTI). Recently, 'sensor' NLRs have been reported to function with 'helper' NLRs to activate immunity. We investigated the role of two helper NLRs, Nrc2 and Nrc3, on immunity in tomato to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) mediated by the sensor NLR Prf and the Pto kinase. An nrc2/nrc3 mutant no longer activated Prf/Pto-mediated NTI to Pst containing the effectors AvrPto and AvrPtoB. An nrc3 mutant showed intermediate susceptibility between wild-type plants and a Prf mutant, while an nrc2 mutant developed only mild disease. These observations indicate that Nrc2 and Nrc3 act additively in Prf-/Pto-mediated immunity. We examined at what point Nrc2 and Nrc3 act in the Prf/Pto-mediated immune response. In the nrc2/3 mutant, programmed cell death (PCD) normally induced by constitutively active variants of AvrPtoB, Pto, or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active Nrc3 was also abolished in a Nicotiana benthamiana line with reduced expression of Prf. MAPK activation triggered by expression of AvrPto in the wild-type tomato plants was completely abolished in the nrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for PCD triggered by Ptr1, another sensor NLR-mediating Pst resistance, although these helper NLRs do appear to be involved in resistance to certain Pst race 1 strains.

Cell-type-specific responses to fungal infection in plants revealed by single-cell transcriptomics

Pathogen infection is a dynamic process. Here, we employ single-cell transcriptomics to investigate plant response heterogeneity. By generating an Arabidopsis thaliana leaf atlas encompassing 95,040 cells during infection by a fungal pathogen, Colletotrichum higginsianum, we unveil cell-type-specific gene expression, notably an enrichment of intracellular immune receptors in vasculature cells. Trajectory inference identifies cells that had different interactions with the invading fungus. This analysis divulges transcriptional reprogramming of abscisic acid signaling specifically occurring in guard cells, which is consistent with a stomatal closure dependent on direct contact with the fungus. Furthermore, we investigate the transcriptional plasticity of genes involved in glucosinolate biosynthesis in cells at the fungal infection sites, emphasizing the contribution of the epidermis-expressed MYB122 to disease resistance. This work underscores spatially dynamic, cell-type-specific plant responses to a fungal pathogen and provides a valuable resource that supports in-depth investigations of plant-pathogen interactions.

Interplay between cell-surface receptor and intracellular NLR-mediated immune responses.

The functional link between cell-surface receptors and intracellular NLR immune receptors is a critical aspect of plant immunity. To establish disease, successful pathogens have evolved mechanisms to suppress cell-surface immune signalling. In response, plants have adapted by evolving NLRs that recognize pathogen effectors involved in this suppression, thereby counteracting their immune-suppressing function. This ongoing co-evolutionary struggle has seemingly resulted in intertwined signalling pathways in some plant species, where NLRs form a separate signalling branch downstream of activated cell-surface receptor complexes essential for full immunity. Understanding these interconnected receptor networks could lead to novel strategies for developing durable disease resistance.

NLR receptors in plant immunity: making sense of the alphabet soup.

Plants coordinately use cell-surface and intracellular immune receptors to perceive pathogens and mount an immune response. Intracellular events of pathogen recognition are largely mediated by immune receptors of the nucleotide binding and leucine rich-repeat (NLR) classes. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction, usually accompanied by a form of programmed cell death termed the hypersensitive response. Some plant NLRs act as multifunctional singleton receptors which combine pathogen detection and immune signaling. However, NLRs can also function in higher order pairs and networks of functionally specialized interconnected receptors. In this article, we cover the basic aspects of plant NLR biology with an emphasis on NLR networks. We highlight some of the recent advances in NLR structure, function, and activation and discuss emerging topics such as modulator NLRs, pathogen suppression of NLRs, and NLR bioengineering. Multi-disciplinary approaches are required to disentangle how these NLR immune receptor pairs and networks function and evolve. Answering these questions holds the potential to deepen our understanding of the plant immune system and unlock a new era of disease resistance breeding.

NLR immune receptors: structure and function in plant disease resistance.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are a diverse family of intracellular immune receptors that play crucial roles in recognizing and responding to pathogen invasion in plants. This review discusses the overall model of NLR activation and provides an in-depth analysis of the different NLR domains, including N-terminal executioner domains, the nucleotide-binding oligomerization domain (NOD) module, and the leucine-rich repeat (LRR) domain. Understanding the structure-function relationship of these domains is essential for developing effective strategies to improve plant disease resistance and agricultural productivity.

Effects of AIM2 and IFI16 on Infectious Diseases and Inflammation.

Both absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16) are intracellular innate immune receptors that recognize double-stranded DNA released during pathogenic infection, leading to the assembly of the inflammasome. The assembly of the inflammasome results in the secretion of bioactive interleukin (IL)-1β and IL-18 and induces cell death through an inflammatory process called pyroptosis. Although the AIM2 inflammasome is generally harmful in the context of some aseptic inflammatory illnesses, it plays a protective role in infectious diseases. During inflammatory processes, there is competition between IFI16 and AIM2. In this review, we explore the impacts of IFI16 and AIM2 in infectious disease and aseptic inflammation, respectively, and how they compete.

Is localized acquired resistance the mechanism for effector-triggered disease resistance in plants?

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) are intracellular immune receptors that are activated by their direct or indirect interactions with virulence effectors. NLR activation triggers a strong immune response and consequent disease resistance. However, the NLR-driven immune response can be targeted by virulence effectors. It is thus unclear how immune activation can occur concomitantly with virulence effector suppression of immunity. Recent observations suggest that the activation of effector-triggered immunity does not sustain defence gene expression in tissues in contact with the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato. Instead, strong defence was observed on the border of the infection area. This response is reminiscent of localized acquired resistance (LAR). LAR is a strong defence response occurring in a ~2 mm area around cells in contact with the pathogen and probably serves to prevent the spread of pathogens. Here we propose that effector-triggered immunity is essentially a quarantining mechanism to prevent systemic pathogen spread and disease, and that the induction of LAR is a key component of this mechanism.

Plasma membrane association and resistosome formation of plant helper immune receptors

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

Taking the lead: NLR immune receptor N-terminal domains execute plant immune responses.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are important intracellular immune receptors that activate robust plant immune responses upon detecting pathogens. Canonical NLRs consist of a conserved tripartite architecture that includes a central regulatory nucleotide-binding domain, C-terminal leucine-rich repeats, and variable N-terminal domains that directly participate in immune execution. In flowering plants, the vast majority of NLR N-terminal domains belong to the coiled-coil, Resistance to Powdery Mildew 8, or Toll/interleukin-1 receptor subfamilies, with recent structural and biochemical studies providing detailed mechanistic insights into their functions. In this insight review, we focus on the immune-related biochemistries of known plant NLR N-terminal domains and discuss the evolutionary diversity of atypical NLR domains in nonflowering plants. We further contrast these observations against the known diversity of NLR-related receptors from microbes to metazoans across the tree of life.

Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities.

Engineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine-rich repeat (NLR) receptor superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defense responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signaling networks that coevolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. The sensor and helper NLRs of the rice (Oryza sativa) NLR pair Pik have coevolved, and mismatching between noncoevolved alleles triggers constitutive activation and cell death. This limits the extent to which protein modifications can be used to engineer pathogen recognition and enhance disease resistance mediated by these NLRs. Here, we dissected incompatibility determinants in the Pik pair in Nicotiana benthamiana and found that heavy metal-associated (HMA) domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely coevolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities, overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic domains with novel and enhanced recognition specificities. Taken together, our results reveal a strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.