Nucleotide-binding Leucine-rich Repeat Proteins Research Articles

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 from Nicotiana benthamiana accumulates in vivo as a homodimer that converts into a higher-order oligomer upon activation by its upstream virus disease resistance protein Rx. The cryo-EM structure of NbNRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation and contribute to immune receptor autoinhibition. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways, possibly to minimise undesired cross-activation and evade pathogen suppression of immunity. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

An Aegilops longissima NLR protein with integrated CC-BED module mediates resistance to wheat powdery mildew

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), reduces wheat yields and grain quality, thus posing a significant threat to global food security. Wild relatives of wheat serve as valuable resources for resistance to powdery mildew. Here, the powdery mildew resistance gene Pm6Sl is cloned from the wild wheat species Aegilops longissima. It encodes a nucleotide-binding leucine-rich repeat (NLR) protein featuring a CC-BED module formed by a zinc finger BED (Znf-BED) domain integrated into the coiled-coil (CC) domain. The function of Pm6Sl is validated via mutagenesis, gene silencing, and transgenic assays. In addition, we develop a resistant germplasm harbouring Pm6Sl in a very small segment with no linkage drag along with the diagnostic gene marker pm6sl-1 to facilitate Pm6Sl deployment in wheat breeding programs. The cloning of Pm6Sl, a resistance gene with BED-NLR architecture, will increase our understanding of the molecular mechanisms underlying BED-NLR-mediated resistance to various pathogens.

MiR472 deficiency enhances Arabidopsis thaliana defense without reducing seed production.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and Nucleotide-Binding Leucine Rich Repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two non-exclusive scenarios: miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction; or reduction of active miRNA levels in response to pathogen derived molecules (PAMPS and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen Plectospharaella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared to wild-type plants although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses.

Subfunctionalization of NRC3 altered the genetic structure of the Nicotiana NRC network.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play crucial roles in immunity against pathogens in both animals and plants. In solanaceous plants, activation of several sensor NLRs triggers their helper NLRs, known as NLR-required for cell death (NRC), to form resistosome complexes to initiate immune responses. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, molecular evolutionary events leading to the complex network architecture remained elusive. Here, we showed that solanaceous NRC3 variants underwent subfunctionalization after the divergence of Solanum and Nicotiana, altering the genetic architecture of the NRC network in Nicotiana. Natural solanaceous NRC3 variants form three allelic groups displaying distinct compatibilities with the sensor NLR Rpi-blb2. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility. These residues are positioned on multiple surfaces of the resting NRC3 homodimer, collectively contributing to their compatibility with Rpi-blb2. Upon activation, Rpi-blb2-compatible NRC3 variants form membrane-associated punctate and high molecular weight complexes, and confer resistance to the late blight pathogen Phytophthora infestans. Our findings revealed how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper compatibility and contributing to the increased complexity of the NRC network.

The Soybean Cyst Nematode Effector Cysteine Protease 1 (CPR1) Targets a Mitochondrial Soybean Branched-Chain Amino Acid Aminotransferase (GmBCAT1).

The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

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.

Selective Activation of G Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces ER Stress and Pyroptosis via AMPK Signaling Pathway in Experimental Subarachnoid Hemorrhage.

Neuroinflammation is a critical pathogenic event following hemorrhagic stroke. Endoplasmic reticulum (ER) stress-induced apoptosis and nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3(NLRP3)-associated pyroptosis can contribute to the escalation of neuroinflammatory responses, leading to increased brain damage. G protein-coupled estrogen receptor 1(GPER1), as the most extensively characterized brain-derived estrogen, was reported to trigger neuroprotective effects. However, the anti-apoptotic and anti-pyroptotic effect of GPER1 activation and the underlying mechanism has not been fully elucidated. We established the experimental SAH model by intravascular perforation. The GPER1 selective agonist G1 was intravenously administered 1h following SAH. For mechanistic exploration, the selective inhibitor of adenosine monophosphate-activated protein kinase (AMPK), dorsomorphin, was administered via intracerebroventricular injection 30min prior to SAH induction. Post-SAH assessments included SAH grade, the short-term and long-term neurological outcomes, brain edema, cerebral blood flow, transmission electron microscopy (TEM), western blot (WB), ELISA, TUNEL staining, Fluoro-Jade C staining (FJC), and immunofluorescence staining. The expression of GPER1 was observed to elevate at 6h and peaked at 24h subsequent to SAH, predominantly co-localized with neurons. Post-treatment with G1 markedly ameliorated both the short-term and long-term neurological deficits of SAH mouse, as well as inhibiting the expression of neuronal ER stress-associated apoptotic proteins (i.e., CHOP, GRP78, Caspase-12, Cleaved Caspase-3, Bax, Bcl2) and pyroptosis-associated proteins (i.e., NLRP3, ASC, Cleaved Caspase-1). Additionally, our research revealed that inhibition of AMPK with dorsomorphin attenuated the neuroprotective effects of G1. This was accompanied by modifications in the molecular pathways associated with ER stress-induced apoptosis and pyroptosis. These data herein elucidated that GPER1 exerted neuroprotective effects by mitigating neuroinflammation in an AMPK-dependent manner, which modulates neuronal ER stress-associated apoptosis and pyroptosis. Boosting the anti-apoptotic and anti-pyroptotic effect by activating GPER1 may be an efficient treatment strategy for SAH patients.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotianabenthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

A Coiled-Coil Nucleotide-Binding Domain Leucine-Rich Repeat Receptor Gene MeRPPL1 Plays a Role in the Replication of a Geminivirus in Cassava.

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.

A KNOX Ⅱ transcription factor suppresses the NLR immune receptor BRG8-mediated immunity in rice

Nucleotide-binding site and leucine-rich repeat (NLR) proteins are activated by detecting pathogen effectors, which in turn trigger host defenses and cell death. Although many NLRs have been identified, the mechanisms responsible for NLR-triggered defense responses are still poorly understood. In this study, through a genome-wide association study approach, we identified a novel NLR gene, Blast Resistance Gene 8 (BRG8), which confers resistance to rice blast and bacterial blight diseases. BRG8 overexpression and complementation lines exhibit enhanced resistance to both pathogens. Subcellular localization assays showed that BRG8 is localized in both the cytoplasm and the nucleus. Additional evidence revealed that nuclear-localized BRG8 can enhance rice immunity without a hypersensitive response (HR)-like phenotype. We also demonstrated that the coiled-coil domain of BRG8 not only physically interacts with itself but also interacts with the KNOX Ⅱ protein HOMEOBOX ORYZA SATIVA59 (HOS59). Knockout mutants of HOS59 in the BRG8 background show enhanced resistance to Magnaporthe oryzae strain CH171 and Xoo strain CR4, similar to that of the BRG8 background. By contrast, overexpression of HOS59 in the BRG8 background will compromise the HR-like phenotype and resistance response. Further analysis revealed that HOS59 promotes the degradation of BRG8 via the 26S proteasome pathway. Collectively, our study highlights HOS59 as an NLR immune regulator that fine-tunes BRG8-mediated immune responses against pathogens, providing new insights into NLR associations and functions in plant immunity.

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.

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.

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.

Predicting the potential mechanism of radix chimonanthi pracecocis in treating osteoarthritis by network pharmacology analysis combined with experimental validation

Ethnopharmacological relevanceRadix Chimonanthi Pracecocis (RCP), also known as Tiekuaizi, widely used by the Miao community in Guizhou, exhibits diverse biological activities and holds promise for the treatment of osteoarthritis (OA). However, there is a lack of contemporary pharmacological research in this area. Aims of the studyThis study aims to explore the potential of targets and mechanisms of RCP in the treatment of OA. Materials and methodsThe chemical components of RCP were identified using UPLC-MS/MS, and active components were determined based on the Lipinski rule. RCP and OA-related targets were retrieved from public databases such as TCMSP and GeneCards. Network pharmacology approaches were employed to identify key genes. The limma package (version 3.40.2) in R 4.3.2 was used to screen for differentially expressed genes (DEGs) between OA and healthy individuals in GSE82107. DEGs were analyzed using an independent sample t-test and receiver operating characteristic analysis in GraphPad Prism 9.5.1. Additionally, molecular docking (SYBYL2.1.1) was used to analyze the binding interactions between the active components and target proteins. Finally, we established a papain-induced osteoarthritis (OA) rat model and treated it with RCP aqueous extract by gavage. We validated relevant indicators using real-time fluorescence quantitative polymerase chain reaction, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays. ResultsSeven active components and 53 targets were identified. The results of GO and KEGG enrichment analyses confirmed the significant role of RCP in the regulation of pyroptosis. Hypoxia-inducible factor-1α (HIF-1α) was identified as a key gene involved in the main biological functions. Molecular docking analysis revealed that Praecoxin, Isofraxidin, Esculin, and Naringenin can bind to the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) (T-Score >5). Additionally, Praecoxin can bind to HIF-1α (T-Score >5). In vivo experiments demonstrated that RCP significantly affects the NLRP3 inflammasome, which is regulated by the HIF-1α pathway. RCP inhibited pyroptosis and reduced synovial inflammation. ConclusionsThis study confirmed the efficacy of RCP aqueous extract in the treatment of OA and identified seven active components (esculin, dihydrokaempferol, naringenin, praecoxin, carnosol, hydroxyvalerenic acid, isofraxidin) that may play an anti-pyroptosis role in the treatment of OA by downregulating the expression of HIF-1α and NLRP3 inflammasome.

Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts.

The release of mature interleukin (IL-) 1β from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals. Temporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data. Exposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1β was unaffected after contact with CoCr particles. Although CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

Mapping and characterization of rust resistance genes Lr53 and Yr35 introgressed from Aegilops species

Key messageThe rust resistance genes Lr53 and Yr35 were introgressed into bread wheat from Aegilops longissima or Aegilops sharonensis or their S-genome containing species and mapped to the telomeric region of chromosome arm 6BS.Wheat leaf and stripe rusts are damaging fungal diseases of wheat worldwide. Breeding for resistance is a sustainable approach to control these two foliar diseases. In this study, we used SNP analysis, sequence comparisons, and cytogenetic assays to determine that the chromosomal segment carrying Lr53 and Yr35 was originated from Ae.longissima or Ae. sharonensis or their derived species. In seedling tests, Lr53 conferred strong resistance against all five Chinese Pt races tested, and Yr35 showed effectiveness against Pst race CYR34 but susceptibility to race CYR32. Using a large population (3892 recombinant gametes) derived from plants homozygous for the ph1b mutation obtained from the cross 98M71 × CSph1b, both Lr53 and Yr35 were successfully mapped to a 6.03-Mb telomeric region of chromosome arm 6BS in the Chinese Spring reference genome v1.1. Co-segregation between Lr53 and Yr35 was observed within this large mapping population. Within the candidate region, several nucleotide-binding leucine-rich repeat genes and protein kinases were identified as candidate genes. Marker pku6B3127 was completely linked to both genes and accurately predicted the absence or presence of alien segment harboring Lr53 and Yr35 in 87 tetraploid and 149 hexaploid wheat genotypes tested. We developed a line with a smaller alien segment (< 6.03 Mb) to reduce any potential linkage drag and demonstrated that it conferred resistance levels similar to those of the original donor parent 98M71. The newly developed introgression line and closely linked PCR markers will accelerate the deployment of Lr53 and Yr35 in wheat breeding programs.

TSPO deficiency exacerbates acute lung injury via NLRP3 inflammasome-mediated pyroptosis.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in many critically ill patients. Although inflammasome activation plays an important role in the induction of acute lung injury (ALI) and ARDS, the regulatory mechanism of this process is still unclear. When cells are stimulated by inflammation, the integrity and physiological function of mitochondria play a crucial part in pyroptosis. However, the underlying mechanisms and function of mitochondrial proteins in the process of pyroptosis are largely not yet known. Here, we identified the 18-kDa translocator protein (TSPO), a mitochondrial outer membrane protein, as an important mediator regulating nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages during ALI. TSPO gene knockout (KO) and lipopolysaccharide (LPS)-induced ALI/ARDS mouse models were employed to investigate the biological role of TSPO in the pathogenesis of ARDS. Murine macrophages were used to further characterize the effect of TSPO on the NLRP3 inflammasome pathway. Activation of NLRP3 inflammasome was preformed through LPS+adenosine triphosphate (ATP) co-stimulation, followed by detection of mitochondrial membrane potential, reactive oxygen species (ROS) production, and cell death to evaluate the potential biological function of TSPO. Comparisons between two groups were performed with a two-sided unpaired t -test. TSPO- KO mice exhibited more severe pulmonary inflammation in response to LPS-induced ALI. TSPO deficiency resulted in enhanced activation of the NLRP3 inflammasome pathway, promoting more proinflammatory cytokine production of macrophages in LPS-injured lung tissue, including interleukin (IL)-1β, IL-18, and macrophage inflammatory protein (MIP)-2. Mitochondria in TSPO -KO macrophages tended to depolarize in response to cellular stress. The increased production of mitochondrial damage-associated molecular pattern led to enhanced mitochondrial membrane depolarization and pyroptosis in TSPO -KO cells. TSPO may be the key regulator of cellular pyroptosis, and it plays a vital protective role in ARDS occurrence and development.

A pair of atypical NLR-encoding genes confers Asian soybean rust resistance in soybean

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

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.

Downregulation of otulin induces inflammasome activation in neutrophilic asthma

Neutrophilic asthma (NA) is a severe asthma phenotype associated with steroid resistance and IL-1β overproduction; however, the exact mechanism remains unclear. Moreover, the dysfunction of TNF-α signaling pathway, a regulator of IL-1β production, was associated with the deficiency of ovarian tumor protease deubiquitinase with linear linkage specificity (otulin) in autoimmune patients. We hypothesized that otulin downregulation in macrophages (Mφ) could trigger Mφ activation via the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway. We assessed the expressions of otulin in blood monocyte subsets from NA patients and in alveolar Mφ from NA mice. Additionally, we evaluated the functional consequences of otulin deficiency in bone marrow-derived Mφ. The effects of inhibiting receptor-interacting protein kinase (RIPK)-1 and RIPK-3 on neutrophils and group 3 innate lymphoid cells (ILC3s) were assessed invitro and invivo. When comparing nonclassical monocytes, a significant downregulation of otulin in the intracellular components was observed in NA patients compared to healthy controls (P= .005). Moreover, isolated alveolar Mφ from the NA mice exhibited lower otulin expression compared to those from control mice. After otulin knockdown in bone marrow-derived Mφ, we observed spontaneous IL-1β production depending on NLRP3 inflammasome. Moreover, the infiltrated neutrophils and ILC3s were significantly decreased by combined treatment of RIPK-1 and RIPK-3 inhibitors through blocking IL-1β release in NA. IL-1β overproduction caused by a deficiency of otulin, an upstream triggering factor, could be a promising diagnostic and therapeutic target for NA.