Nucleotide-binding Domain And Leucine-rich Repeat Research Articles

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 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 non-flowering plants retain immune-related functions through trans-lineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to non-flowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable to motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the non-flowering 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 non-flowering 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.

Genetics of chilling response at early growth stage in rice: a recessive gene for tolerance and importance of acclimation.

Low-temperature adaptation in rice is mediated by the ability of a genotype to tolerate chilling temperatures. A genetic locus on chromosome 11 was analysed for chilling tolerance at the plumule stage in rice. The tolerant allele of A58, a japonica landrace in Japan, was inherited as a recessive gene (ctp-1A58), whereas the susceptible alleles from wild rice (Ctp-1W107) and modern variety (Ctp-1HY) were the dominant genes. Another recessive tolerant allele (ctp-1Silewah) was found in a tropical japonica variety (Silewah). Fine-mapping revealed that a candidate gene for the ctp-1 locus encoded a protein similar to the nucleotide-binding domain and leucine-rich repeat (NLR) protein, in which frameshift mutation by a 73 bp-deletion might confer chilling tolerance in ctp-1A58. Analysis of near-isogenic lines demonstrated that ctp-1A58 imparted tolerance effects only at severe chilling temperatures of 0.5 °C and 2 °C, both at plumule and seedling stages. Chilling acclimation treatments at a wide range of temperatures (8 °C-16 °C) for 72 h concealed the susceptible phenotype of Ctp-1W107 and Ctp-1HY. Furthermore, short-term acclimation treatment of 12 h at 8 °C was enough to be fully acclimated. These results suggest that the NLR gene induces a susceptible response upon exposure to severe chilling stress, however, another interacting gene(s) for acclimation response could suppress the maladaptive phenotype caused by the Ctp-1 allele. This study provides new insights for the adaptation and breeding of rice in a low-temperature environment.

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.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis

Quantitative disease resistance (QDR) remains the most prevalent form of plant resistance in crop fields and wild habitats. Genome-wide association studies (GWAS) have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR. To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum, we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R. solanacearum type III effector (T3E) mutants, identified as key pathogenicity determinants after a first screen on an A. thaliana core collection of 25 accessions. Although most quantitative trait loci (QTLs) were highly specific to the identity of the T3E mutant (ripAC, ripAG, ripAQ, and ripU), we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat (NLR) genes that exhibited structural variation. We functionally validated one of these NLRs as a susceptibility factor in response to R. solanacearum, named it Bacterial Wilt Susceptibility 1 (BWS1), and cloned two alleles that conferred contrasting levels of QDR. Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R. solanacearum effectors. In addition, we showed a direct interaction between BWS1 and RipAC T3E, and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b), the latter interaction being suppressed by RipAC. Together, our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC, mediating negative regulation of the SGT1-dependent immune response.

Genome-Wide Association Study Identifies a Rice Panicle Blast Resistance Gene Pb3 Encoding NLR Protein.

Rice blast is a worldwide fungal disease that seriously affects the yield and quality of rice. Identification of resistance genes against rice blast disease is one of the effective ways to control this disease. However, panicle blast resistance genes, which are useful in the fields, have rarely been studied due to the difficulty in phenotypic identification and the environmental influences. Here, panicle blast resistance-3 (Pb3) was identified by a genome-wide association study (GWAS) based on the panicle blast resistance phenotypes of 230 Rice Diversity Panel I (RDP-I) accessions with 700,000 single-nucleotide polymorphism (SNP) markers. A total of 16 panicle blast resistance loci (PBRLs) within three years including one repeated locus PBRL3 located in chromosome 11 were identified. In addition, 7 genes in PBRL3 were identified as candidate genes by haplotype analysis, which showed significant differences between resistant and susceptible varieties. Among them, one nucleotide-binding domain and Leucine-rich Repeat (NLR) gene Pb3 was highly conserved in multiple resistant rice cultivars, and its expression was significantly induced after rice blast inoculation. Evolutionary analysis showed that Pb3 was a typical disease resistance gene containing coiled-coil, NB-ARC, and LRR domains. T-DNA insertion mutants and CRISPR lines of Pb3 showed significantly reduced panicle blast resistance. These results indicate that Pb3 is a panicle blast resistance gene and GWAS is a rapid method for identifying panicle blast resistance in rice.

Genome-Wide Association Study Identifies a Rice Panicle Blast Resistance Gene, Pb2, Encoding NLR Protein.

Rice blast is one of the main diseases in rice and can occur in different rice growth stages. Due to the complicated procedure of panicle blast identification and instability of panicle blast infection influenced by the environment, most cloned rice resistance genes are associated with leaf blast. In this study, a rice panicle blast resistance gene, Pb2, was identified by genome-wide association mapping based on the panicle blast resistance phenotypes of 230 Rice Diversity Panel 1 (RDP1) accessions with 700,000 single-nucleotide polymorphism (SNP) markers. A genome-wide association study identified 18 panicle blast resistance loci (PBRL) within two years, including 9 reported loci and 2 repeated loci (PBRL2 and PBRL13, PBRL10 and PBRL18). Among them, the repeated locus (PBRL10 and PBRL18) was located in chromosome 11. By haplotype and expression analysis, one of the Nucleotide-binding domain and Leucine-rich Repeat (NLR) Pb2 genes was highly conserved in multiple resistant rice cultivars, and its expression was significantly upregulated after rice blast infection. Pb2 encodes a typical NBS-LRR protein with NB-ARC domain and LRR domain. Compared with wild type plants, the transgenic rice of Pb2 showed enhanced resistance to panicle and leaf blast with reduced lesion number. Subcellular localization of Pb2 showed that it is located on plasma membrane, and GUS tissue-staining observation found that Pb2 is highly expressed in grains, leaf tips and stem nodes. The Pb2 transgenic plants showed no difference in agronomic traits with wild type plants. It indicated that Pb2 could be useful for breeding of rice blast resistance.

Effect of Ultrasound-Guided Fascia Iliac Compartment Block on Serum NLRP3 and Inflammatory Factors in Patients with Femoral Intertrochanteric Fracture.

Objective To investigate the effects of ultrasound-guided fascia iliac compartment block (FICB) on patients' postoperative pain and inflammatory factors as well as nucleotide-binding domain and leucine-rich repeat (NLR) family, pyrin domain-containing 3 (NLRP3) in femoral intertrochanteric fracture. Methods This single-blind randomized controlled study included 231 patients with femoral intertrochanteric fracture treated in our hospital from January 2017 to December 2020. All patients were randomized into two groups, the FICB group (n = 116) and the general anesthesia group (control group, n = 115). The serum NLRP3 levels and inflammatory factors were evaluated. The heart rate (HR), mean arterial pressure (MAP), and SpO2 values were recorded. Pain condition was measured by the visual analogue scale (VAS) score. Harris score was performed for positive hip function. Results The values of HR and MAP were significantly lower after anesthesia induction in FICB groups compared with the control group. However, no significant difference was found for SpO2. Compared with the control group, the VAS scores within 72 h after surgery were all markedly lower in the FICB group than in the control group and showed no significant difference at 1 week after surgery. The levels of NLRP3 and interleukin 6 (IL-6) were significantly lower in FICB patients at 1 h, 6 h, 24 h, 48 h, and 72 h after surgery compared with the control group. Tumor necrosis factor-α (TNF-α) showed a significant lower level in the FICB group at 1 h and 6 h after surgery, and significant lower levels of C-reactive protein (CRP) were found at 1 h and 24 h after surgery compared with the control group. Positive correlation was found between NLRP3 and IL-6, as well as CRP and VAS scores after 1 h of the surgery. No significant difference was found for both Harris score and postoperative complications between the two groups. Conclusion Fascia iliac compartment block could reduce the postoperative pain, which might be associated with the decrease of the serum levels of NLRP3, CRP, IL-6, and TNF-α in femoral intertrochanteric fracture patients.

Cellular interorganelle crosstalk in health and disease states: A glimpse on nephrology-related conditions

Cellular interorganelle crosstalk in medical sciences is a new discussion of mechanisms and pathways of physiological functions and pathogenesis of diseases. The organelles (“mitochondria”, nucleus, lysosome, endoplasmic reticulum, Golgi apparatus) are members of such functional units that are needed to perform specific tasks. Mitochondria are essential metabolic organelles in cells, but they also contribute to iron and calcium homeostasis, as well as in the regulation of apoptosis, and they are increasingly recognized as key signaling platforms. In the kidney, crosstalk between mitochondria with endoplasmic reticulum is at mitochondria-associated endoplasmic reticulum membrane in regulating calcium homeostasis. Another crosstalk between these organelles is about autophagic mechanisms. Autophagy is triggered in the kidney in response to acute kidney injury and supports against kidney injury. High glucose-induced reactive oxygen species can be produced by both enzymatic and nonenzymatic pathways. The nucleotide-binding domain and leucine-rich repeat (NLR) family or nucleotide-binding and oligomerization domain (NOD)-like receptors family pyrin domain containing 3 (NLRP3) inflammasome plays a role in inducing infectious defenses via inflammatory cytokines. The NLRP3 inflammasome is activated by the mitochondria-associated endoplasmic reticulum membrane. It has a role in nephrocalcinosis-related chronic kidney disease. This review article is a summary of interorganelle crosstalk in health and disease states, especially in kidney and nephrology-related conditions.

Identification and Fine-Mapping of a New Bacterial Blight Resistance Gene, Xa47(t), in G252, an Introgression Line of Yuanjiang Common Wild Rice (Oryza rufipogon).

Bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae is a common, widespread, and highly devastating disease that affects rice yield. Breeding resistant cultivars is considered the most effective measure for controlling this disease. The introgression line G252 derived from Yuanjiang common wild rice (Oryza rufipogon) was highly resistant to all tested strains, including C5, C9, PXO99, PB, T7147Y8, Hzhj19, YM1, YM187, YJdp-2, and YJws-2. To identify the BB resistance gene(s) of G252, we developed an F2 population from the cross between G252 and 02428. A linkage analysis was performed for the phenotype and genotype of the population. A segregation ratio of 3:1 was observed between the resistant and susceptible individuals in the F2 progeny, indicating a dominant resistance gene, Xa47(t), in G252. The resistance gene was mapped within an approximately 26.24-kb physical region on chromosome 11 between two InDel markers, R13I14 and 13rbq-71. Moreover, one InDel marker, Hxjy-1, co-segregated with Xa47(t). Three genes were predicted within the target region, including a promising candidate gene encoding a nucleotide-binding domain and leucine-rich repeat (NLR) protein (LOC_Os11g46200) by combining the structure and expression analysis. Physical mapping data suggested that Xa47(t) is a new broad-spectrum BB resistance gene without identified allelic genes.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

The Tomato Interspecific NB-LRR Gene Arsenal and Its Impact on Breeding Strategies.

Tomato (Solanum lycopersicum L.) is a model system for studying the molecular basis of resistance in plants. The investigation of evolutionary dynamics of tomato resistance (R)-loci provides unique opportunities for identifying factors that promote or constrain genome evolution. Nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors belong to one of the most plastic and diversified families. The vast amount of genomic data available for Solanaceae and wild tomato relatives provides unprecedented insights into the patterns and mechanisms of evolution of NB-LRR genes. Comparative analysis remarked a reshuffling of R-islands on chromosomes and a high degree of adaptive diversification in key R-loci induced by species-specific pathogen pressure. Unveiling NB-LRR natural variation in tomato and in other Solanaceae species offers the opportunity to effectively exploit genetic diversity in genomic-driven breeding programs with the aim of identifying and introducing new resistances in tomato cultivars. Within this motivating context, we reviewed the repertoire of NB-LRR genes available for tomato improvement with a special focus on signatures of adaptive processes. This issue is still relevant and not thoroughly investigated. We believe that the discovery of mechanisms involved in the generation of a gene with new resistance functions will bring great benefits to future breeding strategies.

Weighted Gene Co-Expression Network Coupled with a Critical-Time-Point Analysis during Pathogenesis for Predicting the Molecular Mechanism Underlying Blast Resistance in Rice

BackgroundRice blast, caused by the ascomycete fungus M. oryzae, is one of the most important diseases of rice. Although many blast resistance (R) genes have been identified and deployed in rice varieties, the molecular mechanisms responsible for the R gene-mediated defense responses are yet not fully understood.ResultsIn this study, we used comparative transcriptomic analysis to explore the molecular mechanism involved in Piz-t-mediated resistance in a transgenic line containing Piz-t (NPB-Piz-t) compared to Nipponbare (NPB). Clustering and principal component analysis (PCA) revealed that the time-point at 24-h post inoculation (hpi) was the most important factor distinguishing the four time-points, which consisted of four genes of mitogen-activated protein kinases (MAPKs) signaling pathway, one gene related to WRKY DNA-binding domain containing protein, five pathogenesis-related protein (OsPR1s) genes, and three genes of R proteins involving in the most significant protein-protein interaction (PPI) pathway. Using weighted gene co-expression network analysis (WGCNA) to investigate RNA-seq data across 0, 24, 48, and 72 hpi, nine modules with similar patterns expression pattern (SEP) and three modules with differential expression pattern (DEP) between NPB-Piz-t and NPB across 0, 24, 48, and 72 hpi with KJ201 (referred to as Piz-t-KJ201 and NPB-KJ201) were identified. Among these the most representative SEP green-yellow module is associated with photosynthesis, and DEP pink module comprised of two specific expressed nucleotide-binding domain and leucine-rich repeat (NLR) genes of LOC_Os06g17900 and LOC_Os06g17920 of Pi2/9 homologous, three NLR genes of LOC_Os11g11810, LOC_Os11g11770, and LOC_Os11g11920 which are putatively associated with important agronomic traits, and a B3 DNA binding domain containing protein related genes (LOC_Os10g39190). Knockout of LOC_Os10g39190 via CRISPR-Cas9 resulted in plant death at the seedling stage.ConclusionsThe research suggested that Piz-t and multiple NLR network might play important roles in the regulation of the resistance response in the Piz-t-KJ201 interaction system. The identified genes provide an NLR repository to study the rice-M. oryzae interaction system and facilitate the breeding of blast-resistant cultivars in the future.

Variation Patterns of NLR Clusters in Arabidopsis thaliana Genomes

The nucleotide-binding domain and leucine-rich repeat (NLR) gene family is highly expanded in the plant lineage with extensive sequence and structure polymorphisms. To survey the landscape of NLR expansion, we mined the published long-read data generated by the resistance gene enrichment sequencing of 64 diverse Arabidopsis thaliana accessions. We found that the hot spots of massive multi-gene NLR cluster expansion did not typically span the whole cluster; instead, they were restricted to a handful of, or only one, dominant radiation(s). All sequences in such a radiation were distinct from other genes in the cluster but not from each other in the clade, making it difficult to assign trustworthy reference-based orthologies when multiple reference genes were present in the radiation. Consequently, NLR genes can be broadly divided into two types: radiating or high-fidelity, where high-fidelity genes are well conserved and well separated from other clades. A similar distinction could be made for NLR clusters, depending on whether cluster size was determined primarily by extensive radiation or the presence of numerous high-fidelity genes. We also identified groups of well-conserved NLR clades that were missing from the Columbia-0 reference genome. This suggests that the classification of NLRs using gene IDs from a single reference accession can rarely capture all major paralogs in a cluster accurately and representatively and that a reference-agnostic perspective is required to properly characterize these additional variations. Finally, we present a quantitative visualization method for differentiating these situations in a given clade of interest.

Structures of plant resistosome reveal how NLR immune receptors are activated.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins make up the largest immune receptor family in plants. Although many studies have put effort into revealing the working mechanism of NLRs, the activation details of plant NLRs still remain obscure. Recently, two remarkable works resolved the structures of a plant NLR protein, the Arabidopsis thaliana HOPZ-ACTIVATED RESISTANCE1 (ZAR1), both in resting and activation states. The activated ZAR1 with its partner proteins form a wheel-like pentamer called resistosome that is thought to be able to trigger cell death by perturbing plasma membrane integrity. These findings greatly further our understanding of plant immune system.

HSC70 regulates cold-induced caspase-1 hyperactivation by an autoinflammation-causing mutant of cytoplasmic immune receptor NLRC4

NLRC4 [nucleotide-binding domain and leucine-rich repeat (NLR) family, caspase recruitment domain (CARD) containing 4] is an innate immune receptor, which, upon detection of certain pathogens or internal distress signals, initiates caspase-1-mediated interleukin-1β maturation and an inflammatory response. A gain-of-function mutation, H443P in NLRC4, causes familial cold autoinflammatory syndrome (FCAS) characterized by cold-induced hyperactivation of caspase-1, enhanced interleukin-1β maturation, and inflammation. Although the H443P mutant shows constitutive activity, the mechanism involved in hyperactivation of caspase-1 by NLRC4-H443P upon exposure of cells to lower temperature is not known. Here, we show that heat shock cognate protein 70 (HSC70) complexes with NLRC4 and negatively regulates caspase-1 activation by NLRC4-H443P in human cells. Compared with NLRC4, the structurally altered NLRC4-H443P shows enhanced interaction with HSC70. Nucleotide binding- and leucine-rich repeat domains of NLRC4, but not its CARD, can engage in complex formation with HSC70. Knockdown of HSC70 enhances apoptosis-associated speck-like protein containing a CARD (ASC)-speck formation and caspase-1 activation by NLRC4-H443P. Exposure to subnormal temperature results in reduced interaction of NLRC4-H443P with HSC70, and an increase in its ability to form ASC specks and activate caspase-1. Unlike the NLRC4-H443P mutant, another constitutively active mutant (NLRC4-V341A) associated with autoinflammatory diseases, but not FCAS, showed neither enhanced interaction with HSC70 nor an increase in inflammasome formation upon exposure to subnormal temperature. Our results identify HSC70 as a negative regulator of caspase-1 activation by the temperature-sensitive NLRC4-H443P mutant. We also show that low-temperature-induced hyperactivation of caspase-1 by NLRC4-H443P is due to loss of inhibition by HSC70.

The role of NLRP3 in traumatic brain injury and its regulation by pioglitazone.

Perilesional edema is a predominant mechanism underlying secondary brain injury after traumatic brain injury (TBI). Perilesional edema is characterized by inflammation, production of proinflammatory cytokines, and migration of peripheral immune cells into the brain. The nucleotide-binding domain and leucine-rich repeat (NLR) family pyrin domain-containing 3 protein (NLRP3) is a key component of secondary injury. Pioglitazone regulates NLRP3 and other inflammatory cytokines. In the present study, the role of NLRP3 and the pharmacological effects of pioglitazone were investigated in animal TBI models. Brain contusion was induced in a weight drop model involving 3 groups of mice: C57 BL/6 (sham group), NLRP3 knockout (K/O group), and pioglitazone-treated mice (treatment group). The percentage of brain water content of the 3 groups of mice was compared over a period of time. Western blot, immunohistochemistry, and immunofluorescence analyses were conducted to investigate NLRP3-related inflammasomes and the effects of pioglitazone in the TBI models. Brain edema was the highest on day 3 after TBI in the sham group. Brain edema in both the K/O and the treatment groups was lower than in the sham group. In Western blot, the expression of inflammasomes was higher after TBI in the sham group, but the expression of interleukin-1β, caspase-1, and NLRP3 was decreased significantly following treatment with pioglitazone. The expression of GFAP (glial fibrillary acidic protein) and Iba1 was decreased in both the K/O and treatment groups. In addition, confocal microscopy revealed a decrease in microglial cell and astrocyte activation following pioglitazone therapy. The inflammasome NLRP3 plays a pivotal role in regulating cerebral edema and secondary inflammation. Interestingly, pioglitazone reduced cerebral edema and immune response after TBI by downregulating the effects of NLRP3. These results suggest that the clinical application of pioglitazone may be a neuroprotective strategy in TBI.

Characterization of genes required for both Rpg1 and rpg4-mediated wheat stem rust resistance in barley

BackgroundPuccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an “integrated sensory domain” resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9).ResultsThe rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F6:7 recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein.ConclusionGenetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.

Multiple pairs of allelic MLA immune receptor-powdery mildew AVRA effectors argue for a direct recognition mechanism.

Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors.

Subfamily-Specific Specialization of RGH1/MLA Immune Receptors in Wild Barley.

The barley disease resistance (R) gene locus mildew locus A (Mla) provides isolate-specific resistance against the powdery mildew fungus Blumeria graminis hordei and has been introgressed into modern cultivars from diverse germplasms, including the wild relative Hordeum spontaneum. Known Mla disease resistance specificities to B. graminis hordei appear to encode allelic variants of the R gene homolog 1 (RGH1) family of nucleotide-binding domain and leucine-rich repeat (NLR) proteins. Here, we sequenced and assembled the transcriptomes of 50 H. spontaneum accessions representing nine populations distributed throughout the Fertile Crescent. The assembled Mla transcripts exhibited rich sequence diversity, linked neither to geographic origin nor population structure, and could be grouped into two similar-sized subfamilies based on two major N-terminal coiled-coil (CC) signaling domains that are both capable of eliciting cell death. The presence of positively selected sites located mainly in the C-terminal leucine-rich repeats of both MLA subfamilies, together with the fact that both CC signaling domains mediate cell death, implies that the two subfamilies are actively maintained in the population. Unexpectedly, known MLA receptor variants that confer B. graminis hordei resistance belong exclusively to one subfamily. Thus, signaling domain divergence, potentially as adaptation to distinct pathogen populations, is an evolutionary signature of functional diversification of an immune receptor. Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .