A viral E3 ubiquitin ligase produced by herpes simplex virus 1 inhibits the NLRP1 inflammasome.

Guard proteins initiate defense mechanisms upon sensing pathogen-encoded virulence factors. Successful viral pathogens likely inhibit guard protein activity, but these interactions have been largely undefined. Here, we demonstrate that the human pathogen herpes simplex virus 1 (HSV-1) stimulates and inhibits an antiviral pathway initiated by NLRP1, a guard protein that induces inflammasome formation and pyroptotic cell death when activated. Notably, HSV-1 infection of human keratinocytes promotes posttranslational modifications to NLRP1, consistent with MAPK-dependent NLRP1 activation, but does not result in downstream inflammasome formation. We identify infected cell protein 0 (ICP0) as the critical HSV-1 protein that is necessary and sufficient for inhibition of the NLRP1 pathway. Mechanistically, ICP0's cytoplasmic localization and function as an E3 ubiquitin ligase prevents proteasomal degradation of the auto-inhibitory NT-NLRP1 fragment, thereby preventing inflammasome formation. Further, we demonstrate that inhibiting this inflammasome is important for promoting HSV-1 replication. Thus, we have established a mechanism by which HSV-1 overcomes a guard-mediated antiviral defense strategy in humans.

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.

Focus on negatively regulated NLRs in inflammation and cancer.

Nucleotide-binding and oligomerization structural domain (NOD)-like receptors (NLRs) play an important role in innate immunity as cytoplasmic pattern recognition receptors (PRRs). Over the past decade, considerable progress has been made in understanding the mechanisms by which NLR family members regulate immune system function, particularly the formation of inflammasome and downstream inflammatory signals. However, recent studies have shown that some members of the NLRs, including Nlrp12, NLRX1, and NLRC3, are important in the negative regulation of inflammatory signaling and are involved in the development of various diseases, including inflammatory diseases and cancer. Based on this, in this review, we first summarize the interactions between canonical and non-canonical nuclear factor-κB (NF-κB) signaling pathways that are mainly involved in NLRs, then highlight the mechanisms by which the above NLRs negatively regulate inflammatory signaling responses as well as their roles in tumor progression, and finally summarize the synthetic and natural derivatives with therapeutic effects on these NLRs, which are considered as potential therapeutic agents for overcoming inflammatory diseases.

The soybean plasma membrane GmDR1 protein conferring broad-spectrum disease and pest resistance regulates several receptor kinases and NLR proteins.

Overexpression of Glycine max disease resistant 1 (GmDR1) exhibits broad-spectrum resistance against Fusarium virguliforme, Heterodera glycines (soybean cyst nematode), Tetranychus urticae (Koch) (spider mites), and Aphis glycines Matsumura (soybean aphids) in soybean. To understand the mechanisms of broad-spectrum immunity mediated by GmDR1, the transcriptomes of a strong and a weak GmDR1-overexpressor following treatment with chitin, a pathogen- and pest-associated molecular pattern (PAMP) common to these organisms, were investigated. The strong and weak GmDR1-overexpressors exhibited altered expression of 6098 and 992 genes, respectively, as compared to the nontransgenic control following chitin treatment. However, only 192 chitin- and 115 buffer-responsive genes exhibited over two-fold changes in expression levels in both strong and weak GmDR1-overexpressors as compared to the control. MapMan analysis of the 192 chitin-responsive genes revealed 64 biotic stress-related genes, of which 53 were induced and 11 repressed as compared to the control. The 53 chitin-induced genes include nine genes that encode receptor kinases, 13 encode nucleotide-binding leucine-rich repeat (NLR) receptor proteins, seven encode WRKY transcription factors, four ethylene response factors, and three MYB-like transcription factors. Investigation of a subset of these genes revealed three receptor protein kinases, seven NLR proteins, and one WRKY transcription factor genes that are induced following F. virguliforme and H. glycines infection. The integral plasma membrane GmDR1 protein most likely recognizes PAMPs including chitin and activates transcription of genes encoding receptor kinases, NLR proteins and defense-related genes. GmDR1 could be a pattern recognition receptor that regulates the expression of several NLRs for expression of PAMP-triggered immunity and/or priming the effector triggered immunity.

ATR2C ala2 from Arabidopsis-infecting downy mildew requires 4 TIR-NLR immune receptors for full recognition.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

The nucleotide-binding domain of NRC-dependent disease resistance proteins is sufficient to activate downstream helper NLR oligomerization and immune signaling.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins with pathogen sensor activities have evolved to initiate immune signaling by activating helper NLRs. However, the mechanisms underpinning helper NLR activation by sensor NLRs remain poorly understood. Although coiled coil (CC) type sensor NLRs such as the Potato virus X disease resistance protein Rx have been shown to activate the oligomerization of their downstream helpers NRC2, NRC3 and NRC4, the domains involved in sensor-helper signaling are not known. Here, we used Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana to show that the nucleotide-binding (NB) domain within the NB-ARC of Rx is necessary and sufficient for oligomerization and immune signaling of downstream helper NLRs. In addition, the NB domains of the disease resistance proteins Gpa2 (cyst nematode resistance), Rpi-amr1, Rpi-amr3 (oomycete resistance) and Sw-5b (virus resistance) are also sufficient to activate their respective downstream NRC helpers. Using transient expression in the lettuce (Lactuca sativa), we show that Rx (both as full length or as NB domain truncation) and its helper NRC2 form a minimal functional unit that can be transferred from solanaceous plants (lamiids) to Campanulid species. Our results challenge the prevailing paradigm that NLR proteins exclusively signal via their N-terminal domains and reveal a signaling activity for the NB domain of NRC-dependent sensor NLRs. We propose a model in which helper NLRs can perceive the status of the NB domain of their upstream sensors.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

Co-expression network analysis and identification of core genes in the interaction between wheat and Puccinia striiformis f. sp. tritici.

The epidemic of stripe rust, caused by the pathogen Puccinia striiformis f. sp. tritici (Pst), would reduce wheat (Triticum aestivum) yields seriously. Traditional experimental methods are difficult to discover the interaction between wheat and Pst. Multi-omics data analysis provides a new idea for efficiently mining the interactions between host and pathogen. We used 140 wheat-Pst RNA-Seq data to screen for differentially expressed genes (DEGs) between low susceptibility and high susceptibility samples, and carried out Gene Ontology (GO) enrichment analysis. Based on this, we constructed a gene co-expression network, identified the core genes and interacted gene pairs from the conservative modules. Finally, we checked the distribution of Nucleotide-binding and leucine-rich repeat (NLR) genes in the co-expression network and drew the wheat NLR gene co-expression network. In order to provide accessible information for related researchers, we built a web-based visualization platform to display the data. Based on the analysis, we found that resistance-related genes such as TaPR1, TaWRKY18 and HSP70 were highly expressed in the network. They were likely to be involved in the biological processes of Pst infecting wheat. This study can assist scholars in conducting studies on the pathogenesis and help to advance the investigation of wheat-Pst interaction patterns.

Unveiling the Role of RNA Recognition Motif Proteins in Orchestrating Nucleotide-Binding Site and Leucine-Rich Repeat Protein Gene Pairs and Chloroplast Immunity Pathways: Insights into Plant Defense Mechanisms.

In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function.

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.

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.

NLRP inflammasomes in health and disease.

NLRP inflammasomes are a group of cytosolic multiprotein oligomer pattern recognition receptors (PRRs) involved in the recognition of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) produced by infected cells. They regulate innate immunity by triggering a protective inflammatory response. However, despite their protective role, aberrant NLPR inflammasome activation and gain-of-function mutations in NLRP sensor proteins are involved in occurrence and enhancement of non-communicating autoimmune, auto-inflammatory, and neurodegenerative diseases. In the last few years, significant advances have been achieved in the understanding of the NLRP inflammasome physiological functions and their molecular mechanisms of activation, as well as therapeutics that target NLRP inflammasome activity in inflammatory diseases. Here, we provide the latest research progress on NLRP inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRP7, NLRP2, NLRP9, NLRP10, and NLRP12 regarding their structural and assembling features, signaling transduction and molecular activation mechanisms. Importantly, we highlight the mechanisms associated with NLRP inflammasome dysregulation involved in numerous human auto-inflammatory, autoimmune, and neurodegenerative diseases. Overall, we summarize the latest discoveries in NLRP biology, their forming inflammasomes, and their role in health and diseases, and provide therapeutic strategies and perspectives for future studies about NLRP inflammasomes.

High allelic diversity in Arabidopsis NLRs is associated with distinct genomic features.

Plants rely on Nucleotide-binding, Leucine-rich repeat Receptors (NLRs) for pathogen recognition. Highly variable NLRs (hvNLRs) show remarkable intraspecies diversity, while their low-variability paralogs (non-hvNLRs) are conserved between ecotypes. At a population level, hvNLRs provide new pathogen-recognition specificities, but the association between allelic diversity and genomic and epigenomic features has not been established. Our investigation of NLRs in Arabidopsis Col-0 has revealed that hvNLRs show higher expression, less gene body cytosine methylation, and closer proximity to transposable elements than non-hvNLRs. hvNLRs show elevated synonymous and nonsynonymous nucleotide diversity and are in chromatin states associated with an increased probability of mutation. Diversifying selection maintains variability at a subset of codons of hvNLRs, while purifying selection maintains conservation at non-hvNLRs. How these features are established and maintained, and whether they contribute to the observed diversity of hvNLRs is key to understanding the evolution of plant innate immune receptors.

Complete genome assembly provides a high-quality skeleton for pan-NLRome construction in melon.

Melon (Cucumis melo L.), being under intensive domestication and selective breeding, displays an abundant phenotypic diversity. Wild germplasm with tolerance to stress represents an untapped genetic resource for discovery of disease-resistance genes. To comprehensively characterize resistance genes in melon, we generate a telomere-to-telomere (T2T) and gap-free genome of wild melon accession PI511890 (C. melo var. chito) with a total length of 375.0 Mb and a contig N50 of 31.24 Mb. The complete genome allows us to dissect genome architecture and identify resistance gene analogs. We construct a pan-NLRome using seven melon genomes, which include 208 variable and 18 core nucleotide-binding leucine-rich repeat receptors (NLRs). Multiple disease-related transcriptome analyses indicate that most up-regulated NLRs induced by pathogens are shell or cloud NLRs. The T2T gap-free assembly and the pan-NLRome not only serve as essential resources for genomic studies and molecular breeding of melon but also provide insights into the genome architecture and NLR diversity.

Increased NLRP1 mRNA and Protein Expression Suggests Inflammasome Activation in the Dorsolateral Prefrontal and Medial Orbitofrontal Cortex in Schizophrenia.

Schizophrenia is a complex mental condition, with key symptoms marked for diagnosis including delusions, hallucinations, disorganized thinking, reduced emotional expression, and social dysfunction. In the context of major developmental hypotheses of schizophrenia, notably those concerning maternal immune activation and neuroinflammation, we studied NLRP1 expression and content in the postmortem brain tissue of 10 schizophrenia and 10 control subjects. In the medial orbitofrontal cortex (Brodmann's area 11/12) and dorsolateral prefrontal cortex (area 46) from both hemispheres of six schizophrenia subjects, the NLRP1 mRNA expression was significantly higher than in six control brains (p < 0.05). As the expression difference was highest for the medial orbitofrontal cortex in the right hemisphere, we assessed NLRP1-immunoreactive pyramidal neurons in layers III, V, and VI in the medial orbitofrontal cortex in the right hemisphere of seven schizophrenia and five control brains. Compared to controls, we quantified a significantly higher number of NLRP1-positive pyramidal neurons in the schizophrenia brains (p < 0.01), suggesting NLRP1 inflammasome activation in schizophrenia subjects. Layer III pyramidal neuron dysfunction aligns with working memory deficits, while impairments of pyramidal neurons in layers V and VI likely disrupt predictive processing. We propose NLRP1 inflammasome as a potential biomarker and therapeutic target in schizophrenia.

An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome.

Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1ß, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1ß, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Conserved transcription factors NRZ1 and NRM1 regulate NLR receptor-mediated immunity.

Plant innate immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors plays an important role in defense against various pathogens. Although key biochemical events involving NLR activation and signaling have been recently uncovered, we know very little about the transcriptional regulation of NLRs and their downstream signaling components. Here, we show that the Toll-Interleukin 1 receptor homology domain containing NLR (TNL) gene N (Necrosis), which confers resistance to Tobacco mosaic virus, is transcriptionally induced upon immune activation. We identified two conserved transcription factors, N required C3H zinc finger 1 (NRZ1) and N required MYB-like transcription factor 1 (NRM1), that activate N in an immune responsive manner. Genetic analyses indicated that NRZ1 and NRM1 positively regulate coiled-coil domain-containing NLR- and TNL-mediated immunity and function independently of the signaling component Enhanced Disease Susceptibility 1. Furthermore, NRZ1 functions upstream of NRM1 in cell death signaling, and their gene overexpression induces ectopic cell death and expression of NLR signaling components. Our findings uncovered a conserved transcriptional regulatory network that is central to NLR-mediated cell death and immune signaling in plants.