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.

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.

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.

DNA methylation of imprint control regions associated with Alzheimer's disease in non-Hispanic Blacks and non-Hispanic Whites.

Alzheimer's disease (AD) prevalence is twice as high in non-Hispanic Blacks (NHBs) as in non-Hispanic Whites (NHWs). The objective of this study was to determine whether aberrant methylation at imprint control regions (ICRs) is associated with AD. Differentially methylated regions (DMRs) were bioinformatically identified from whole-genome bisulfite sequenced DNA derived from brain tissue of 9 AD (5 NHBs and 4 NHWs) and 8 controls (4 NHBs and 4 NHWs). We identified DMRs located within 120 regions defined as candidate ICRs in the human imprintome ( https://genome.ucsc.edu/s/imprintome/hg38.AD.Brain_track ). Eighty-one ICRs were differentially methylated in NHB-AD, and 27 ICRs were differentially methylated in NHW-AD, with two regions common to both populations that are proximal to the inflammasome gene, NLRP1, and a known imprinted gene, MEST/MESTIT1. These findings indicate that early developmental alterations in DNA methylation of regions regulating genomic imprinting may contribute to AD risk and that this epigenetic risk differs between NHBs and NHWs.

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.

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.

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.

Paired plant immune CHS3-CSA1 receptor alleles form distinct hetero-oligomeric complexes.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.

Two adjacent NLR genes conferring quantitative resistance to clubroot disease in Arabidopsis are regulated by a stably inherited epiallelic variation.

Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassicaceae. Using a combination of quantitative trait locus (QTL) fine mapping, CRISPR-Cas9 validation, and extensive analyses of DNA sequence and methylation patterns, we revealed that the two adjacent neighboring NLR (nucleotide-binding and leucine-rich repeat) genes AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicae in Arabidopsis and that they are epigenetically regulated. The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited. Variations in DNA methylation at the Pb-At5.2 QTL are widespread across Arabidopsis accessions and correlate negatively with variations in expression of the two genes. Our study demonstrates that natural, stable, and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

Kinase fusion proteins: intracellular R-proteins in plant immunity.

Resistance (R) genes in the Triticeae tribe include not only genes encoding the canonical intracellular nucleotide-binding leucine-rich-repeat proteins (NLRs) but also genes encoding kinase fusion proteins (KFPs). Exploring these unconventional KFPs may expand the scope of effector-triggered immunity (ETI) and will have significant implications for crop improvement.

Identification of the NOD-like receptor family of golden pompano and expression in response to bacterial and parasitic exposure reveal its key role in innate immunity.

This study presents a genome-wide identification of NOD-like receptors (NLRs) in the golden pompano, key to its innate immunity. We identified 30 ToNLRs, analyzing their chromosomal positions, characteristics, evolutionary relationships, evidence of positive selection, and synteny with the yellowtail kingfish. Our findings categorize these NLRs into three main subgroups: NLRA, NLRC, and the distinct ToNLRX1. Post-exposure to Streptococcus agalactiae, most ToNLRs increased expression in the spleen, whereas NLRC3like13, NLRC3like16, and NLRC3like19 so in the kidneys. Upon Cryptocaryon irritans exposure, we categorized our groups based on the site of infection into the control group (BFS), the trophont-attached skin (TAS), and the nearby region skin (NRS). ToAPAF1 and ToNOD1 expressions rose in the NRS, in contrast to decreased expressions of ToNLRC5, ToNWD1 and ToCIITA. Other ToNLRs showed variable expressions in the TAS. Overall, this research lays the groundwork for further exploration of innate immunity in the golden pompano.

NLRP1- A CINDERELLA STORY: a perspective of recent advances in NLRP1 and the questions they raise.

NLRP1, while the first inflammasome described, has only recently begun to gain significant attention in disease pathology, inflammation research, and potentially, as a therapeutic target. Recently identified human variants provide key insights into NLRP1 biology while its unique expression in barrier cells such as keratinocytes and airway epithelial cells has aligned with new, human specific agonists. This differentiates NLRP1 from other inflammasomes such as NLRP3 and identifies it as a key therapeutic target in inflammatory diseases. Indeed, recent discoveries highlight that NLRP1 may be the predominant inflammasome in human barrier cells, its primary role akin to NLRP3, to respond to cellular stress. This review focuses on recent studies identifying new human-specific NLRP1 mechanisms of activation of, gain-of-function human variants and disease, its role in responding to cellular stress, and discuss potential advances and the therapeutic potential for NLRP1.

miR-122-3p Alleviates LPS-Induced Pyroptosis of Macrophages via Targeting NLRP1.

OBJECTIVE: Sepsis, a life-threatening organ dysfunction, is among the leading causes of mortality in intensive care units. Sepsis occurrence is associated with macrophage pyroptosis, and microRNAs (miRNAs) have emerged as key factors in this process. However, the specific role of miR-122-3p in pyroptosis during sepsis progression and its underlying mechanisms remain to be fully elucidated. METHODS: We established an in vitro sepsis model using lipopolysaccharide (LPS)-activated macrophages, followed by transfection of a miR-122-3p mimic into RAW264.7 macrophages. We subsequently determined the effects of miR-122-3p on cell viability and pyroptosis using cell viability, western blot, and qPCR assays. The binding affinity between miR-122-3p and NLR pyrin domain containing 1 (NLRP1) mRNA was then confirmed using a dual-luciferase reporter assay. Finally, the secretion of pro-inflammatory cytokines (interleukin (IL)-2, IL-6, and tumor necrosis factor-α (TNF-α) was determined using ELISA. RESULTS: The results revealed that LPS treatment lead to a significant increase in the production of pro-inflammatory cytokines including IL-2, IL-6, and TNF-α in RAW264.7 cells. We observed that overexpression of miR-122-3p effectively restored cell viability and attenuated the expression of key inflammatory markers promoted by LPS, such as caspase-1, pro-caspase-1, IL-18, IL-1ß, NLRP3, apoptosis-associated speck-like protein containing CARD, and cleaved- gasdermin-D. Our data indicate that miR-122-3p is capable of directly bounding to NLRP1 and inhibiting its expression. CONCLUSIONS: These results confirmed that miR-122-3p plays a crucial role in the inhibition of sepsis by suppressing macrophage pyroptosis in an NLRP1-dependent manner. Therefore, miR-122-3p presents as a promising therapeutic target for sepsis.

Methods to Activate the NLRP1 Inflammasome.

In addition to being the first NLR protein proposed to form inflammasome, NLRP1s have attracted much attention in their activation mechanism by post-translational auto-proteolysis to generate C-terminal CARD containing fragment to form inflammasome. Among NLRP1, mouse NLRP1B but not human NLRP1 is well studied for its activation by lethal toxin. As dissecting the cellular components involved in NLRP1-associated diseases is highly dependent on NLRP1 inflammasome activation, experiments that can lead to NLRP1 activation is of pivotal importance to elucidate the biological role and the activation mechanism of NLRP1 especially in human. In this chapter we describe methods commonly used for mouse NLRP1B inflammasome activation as well as activation of human NLRP1 inflammasome visualized by ASC speck formation in our laboratory.

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.

Regulation of plant immunity via small RNA-mediated control of NLR expression.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.