Functional diversification of a wild potato immune receptor at its center of origin.

Plant cell surface pattern recognition receptors (PRRs) and intracellular immune receptors cooperate to provide immunity to microbial infection. Both receptor families have coevolved at an accelerated rate, but the evolution and diversification of PRRs is poorly understood. We have isolated potato surface receptor Pep-13 receptor unit (PERU) that senses Pep-13, a conserved immunogenic peptide pattern from plant pathogenic Phytophthora species. PERU, a leucine-rich repeat receptor kinase, is a bona fide PRR that binds Pep-13 and enhances immunity to Phytophthora infestans infection. Diversification in ligand binding specificities of PERU can be traced to sympatric wild tuber-bearing Solanum populations in the Central Andes. Our study reveals the evolution of cell surface immune receptor alleles in wild potato populations that recognize ligand variants not recognized by others.

Danggui-Shaoyao-San Promotes Amyloid-ß Clearance through Regulating Microglia Polarization via Trem2 in BV2 Cells.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder currently without satisfactory therapeutic treatments. Triggering receptors expressed on a myeloid cells-2 (Trem2) gene mutation has been reported as a powerful AD risk factor that induces Trem2 gene deletion aggravated microglia disfunction and Amyloid-ß (Aß) aggregation in the brain. The traditional Chinese medicine (TCM) formula Danggui-Shaoyao-San (DSS) has shown therapeutic effect on alleviating the symptoms of AD. However, the neuroprotective effect and underlying mechanism of DSS against AD is still far from fully understood. METHODS: Double-label immunofluorescence and Western blotting were employed to evaluate the different polarization states of mouse BV2 microglial (BV2) cells after lipopolysaccharide (LPS) or interleukin (IL)-4 treatment. Trem2 over-expression lentiviral vector and Trem2 siRNA were used respectively to evaluate the effect of Trem2 on microglia polarization via detecting the proteins expression of iNOS and arginase1 (Arg1) by Western blotting while the Aß-scavenging capacity of BV2 cells was assessed by flow cytometry. Cell counting kit-8 (CCK8) assay was performed to assess the effect of DSS on the viability of BV2 cells. Flow cytometry was used to investigate the effect of DSS on the Aß-scavenging capacity of BV2 cells treated with corresponding concentration of DSS-containing serum. Protein of Trem2 and the gene expression of the M1 or M2 phenotype in BV2 cells treated with DSS after Trem2 over-expression or silence were detected by Western blot and RT-qPCR, respectively. RESULTS: In vitro experiments. DSS exhibited anti-inflammatory and neuroprotective functions. It was found that Trem2 had an effect on inducing a shift of M1 microglia towards the M2 phenotype and enhanced the Aß-scavenging capacity of BV2 cells, further that DSS administration relieved inflammation by engulfing Aß through the activities of Trem2. Importantly, DSS treatment effectively increased the Aß-scavenging capacity of BV2 cells through accelerating the shift of M1 microglia towards an M2 phenotype via increasing Trem2 expression. CONCLUSIONS: Results demonstrated that DSS promoted the clearance of Aß through the regulation of microglia polarization via increased expression of Trem2 in BV2 cells.

Regulation of TREM2 expression by transcription factor YY1 and its protective effect against Alzheimer's disease.

TREM2 encoding the transmembrane receptor protein TREM2 is a risk gene of Alzheimer's disease (AD), and the impairment of TREM2 functions in microglia due to mutations in TREM2 may significantly increase the risk of AD by promoting AD pathologies. However, how the expression of TREM2 is regulated and the transcription factors required for TREM2 expression are largely unknown. By luciferase assay, DNA pull-down, and in silico predictions, we identified Yin Yang 1(YY1) as a binding protein of the minimal promoter of the TREM2 gene, and the binding was further confirmed by EMSA and DNA pull-down assay. shRNA-mediated YY1 silencing significantly reduced the activity of the TREM2 minimal promoter and TREM2 protein levels in the microglial cell line BV2 and the neuroblastoma Neuro2A. Furthermore, we found that the levels of TREM2 and YY1 were both downregulated in lipopolysaccharide-treated BV2 cells and in the brain of AD model mice. These results demonstrated that YY1 plays a crucial role in the regulation of TREM2 expression. Our study suggests that microglial YY1 could be targeted to maintain TREM2 expression for AD prevention and therapy.

Effects of Alpinae Oxyphyllae Fructus on microglial polarization in a LPS-induced BV2 cells model of neuroinflammation via TREM2.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the important traditional Chinese medicines, Alpinia oxyphylla could warm and tonify the kidney and spleen. It has been used as anti-salivation, anti-diarrhea in various diseases. In recent years, many studies have reported the significant effect of Alpinia oxyphylla on improving cognitive ability, anti oxidative stress and protecting neurons. AIMS OF THE STUDY: In this paper, we studied whether AE and its main active components could improve M1 and M2 polarization, inhibit neuroinflammation through triggering receptor expressed on myeloid cells 2 (TREM2), and exert anti-inflammatory effects. MATERIALS AND METHODS: In this paper, the concentrations of inflammatory cytokines such as NO, TNF-α, IL-10 were assessed using detection kits respectively. Arg-1 and Iba-1, as polarized markers of M1 and M2, were detected by Immunofluorescence staining. CD86 and CD206 were tested by flow cytometry as surface markers of M1 and M2. Furthermore, RT-PCR was performed to determinate TNF-α, IL-10, Arg-1, and Iba-1. Western blot was used to test the activation of PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. TREM2 siRNA treatment further verified the action target of Chrysin, the main active ingredient of Alpinia oxyphylla. Molecular docking study was performed to investigate the binding mode between Chrysin and the human TREM2. RESULTS: We found that AE could promote the phenotypic transformation of microglia from M1 to M2, and similar effects of Chrysin were observed. Furthermore, downregulation of TREM2 blocked the anti-neuroinflammation of Chrysin, and inhibited the shift of M1 phenotype to M2 phenotype. Additionally, TREM2-siRNA suppressed the effects of Chrysin on PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. CONCLUSIONS: Our findings indicated that AE could improve the polarization response of microglia. TREM2 plays a vital role in the microglial repolarization effects of Chrysin through PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways regulated by neuroinflammation.

Exon 2-mediated deletion of Trem2 does not worsen metabolic function in diet-induced obese mice.

Triggering receptor expressed on myeloid cells 2 (Trem2) is highly expressed on myeloid cells and is involved in cellular lipid homeostasis and inflammatory processes. Trem2 deletion in mice (Trem2-/- ) evokes adipose tissue dysfunction, but its role in worsening obesity-induced metabolic dysfunction has not been resolved. Here we aimed to determine the causal role of Trem2 in regulating glucose homeostasis and insulin sensitivity in mice. Nine-week-old male and female littermate wild-type (WT) and Trem2-/- mice were fed a low- or high-fat diet for 18 weeks and phenotyped for metabolic function. Diet-induced weight gain was similar between genotypes, irrespective of sex. Consistent with previous reports, we find that loss of Trem2 causes massive adipocyte hypertrophy and an attenuation in the lipid-associated macrophage transcriptional response to obesity. In contrast to published data, we find that loss of Trem2 does not worsen metabolic function in obese mice. No differences in intraperitoneal glucose tolerance (ipGTT), oral GTT or mixed meal substrate control, including postprandial glucose, non-esterified fatty acids, insulin or triglycerides, were found between WT and Trem2-/- animals. Similarly, no phenotypic differences existed when animals were challenged with stressors on metabolic demand (i.e. acute exercise or environmental temperature modulation). Collectively, we report a disassociation between adipose tissue remodelling caused by loss of Trem2 and whole-body metabolic homeostasis in obese mice. The complementary nature of experiments conducted gives credence to the conclusion that loss of Trem2 is unlikely to worsen glucose homeostasis in mice.

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation.

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)-a RAGE ligand-was also reduced by SE treatment in wild-type Ager+/+ mice as well as in cultured peritoneal macrophages from Ager+/+ mice but not in Ager-/- mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.

Effects of electroacupuncture on DNA methylation of the TREM2 gene in senescence-accelerated mouse prone 8 mice.

OBJECTIVE: To explore the mechanism by which electroacupuncture (EA) upregulates triggering receptor expressed on myeloid cells 2 (TREM2) protein in the hippocampus of Alzheimer's disease (AD) model animals from the perspective of TREM2 DNA methylation. METHODS: In total, 24 eight-month-old senescence-accelerated mouse prone 8 (SAMP8) mice were divided into an (untreated) AD group (n = 8), donepezil group (receiving donepezil treatment, n = 8) or EA group (receiving an EA intervention, n = 8). A healthy control group comprising 8-month-old senescence-accelerated mouse resistant 1 (SAMR1) mice (n = 8) was also included. Western blotting, bisulfite sequencing, and oxidative bisulfite sequencing were applied to test the relative expression of TREM2 protein and the methylation levels of the TREM2 gene. RESULTS: EA significantly upregulated the relative expression of TREM2 protein (p < 0.01), downregulated the 5-methylcytosine level (p < 0.01) and upregulated the 5-hydroxymethylcytosine level (p < 0.05) in the hippocampus. CONCLUSION: Downregulation of 5-methylcytosine levels and upregulation of 5-hydroxymethylcytosine levels in the TREM2 gene might be the mechanism by which EA promotes the expression of TREM2 protein.

Epimedii Folium and Curculiginis Rhizoma ameliorate lipopolysaccharides-induced cognitive impairment by regulating the TREM2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Neuroinflammation induced by microglia is closely related to a variety of neurodegenerative diseases including Alzheimer's disease (AD). Previous study has found that aqueous extract of Epimedii Folium and Curculiginis Rhizoma (EX) had anti-inflammatory effect on AD by activating the NLRP3 inflammasome and inhibiting NF-κB/MAPK pathway. However, whether the anti-neuroinflammatory effect of EX is related to microglia or not remains unclear. AIM OF THE STUDY: The present study aimed to investigate the protective effect of EX on cognitive impairment induced by LPS and explore the underlying mechanism of EX. MATERIALS AND METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was performed to qualify the major components of EX, EX in the serum and cerebrospinal fluid. To evaluate the anti-inflammatory effects of EX in vivo, the mice were orally administrated with EX (2.34, 4.68 g kg-1•d-1) for 28 days before cotreatment with LPS (1 mg kg-1•d-1, i.p.). The leaning and memory abilities of mice were examined by Morris water maze test. The expression of inflammatory related proteins and the activation of microglia were detected by ELISA, immunofluorescence, real-time PCR and Western blotting. RESULTS: HPLC-MS analysis confirmed and quantified 9 components in EX, 5 components in the serum and 4 components in the cerebrospinal fluid. In a LPS-induced neuroinflammatory mouse model, EX was found to exert anti-inflammatory activity by reducing the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), regulating the expression of different phenotypes of microglia, and increasing the expression of proteins related with TREM2 in the hippocampus tissue. Moreover, LPS-induced microglia activation was markedly attenuated in the hippocampus. CONCLUSIONS: These findings demonstrate that EX exerts anti-neuroinflammatory effects via reducing the production of inflammatory mediators, regulating the conversion of microglia and activating the proteins related with TREM2. EX might become a novel herb pairs to treat neuroinflammatory diseases.

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

Effect of six weeks 1000 mg/day vitamin C supplementation and healthy training in elderly women on genes expression associated with the immune response - a randomized controlled trial.

BACKGROUND: In this study, we investigated the effects of supplementation and exercise on the expression of genes associated with inflammation like CCL2, CRP, IL1, IL6, IL10 mRNA in elderly women. METHODS: Twenty four participants divided randomly into two groups were subjected to 6 weeks of the same health training program (three times per week). SUP group (supplemented, n = 12, mean age 72.8 ± 5.26 years and mean body mass 68.1 ± 8.3 kg) received 1000 mg of Vitamin C/day during the training period, while CON group (control, n = 12, mean age 72.4 ± 5.5 years and body mass 67.7 ± 7.5 kg) received placebo. RESULTS: No significant changes in IL-1, IL-6, IL-10 and CRP mRNA were observed within and between groups. However, there was a clear tendency of a decrease in IL-6 (two-way ANOVA, significant between investigated time points) and an increase in IL-10 mRNA noted in the supplemented group. A significant decrease in CCL2 mRNA was observed only in the CON group (from 2^0.2 to 2^0.1, p = 0.01). CONCLUSIONS: It can be concluded, that 6 weeks of supplementation and exercise was too short to obtain significant changes in gene expression in leukocytes, but supplementation of 1000 mg vitamin C positively affected IL-6 and IL-10 expression - which are key changes in the adaptation to training. However, changes in body mass, IL1 and CCL2 were positive in CON group. It is possible that Vitamin C during 6 weeks of supplementation could have different effects on the expression of individual genes involved in the immune response. TRIAL REGISTRATION: Retrospectively registered.

Geniposide Enhances Macrophage Autophagy through Downregulation of TREM2 in Atherosclerosis.

Macrophage autophagy defect is closely related to the progression of atherosclerosis (AS) and is regulated by the triggering receptor expressed on myeloid cell 2 (TREM2). TREM2 is a key factor in the development of Alzheimer's disease (AD), the deficiency of which leads to anomalous autophagy in microglia. However, the role of TREM2 in the autophagy of plaque macrophages is still unclear. Geniposide (GP) can inhibit AS progression and enhance macrophage autophagy, although the underlying mechanisms remain unknown. We found that high-fat diet (HFD) feeding significantly increased TREM2 levels and inhibited autophagy in the macrophages of ApoE[Formula: see text] mice. TREM2 overexpression in RAW264.7 macrophages decreased autophagy via activation of mTOR signaling. GP inhibited the progression of AS in ApoE[Formula: see text] mice, reinforced macrophage autophagy, and downregulated TREM2 by inhibiting mTOR signaling. Taken together, augmenting the autophagy levels in plaque macrophages by inhibiting the TREM2/mTOR axis can potentially impede atherosclerotic progression. The promising therapeutic effects of GP seen in this study should be validated in future trials, and the underlying mechanisms have to be elucidated in greater detail.

Microglia TREM2: A Potential Role in the Mechanism of Action of Electroacupuncture in an Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is one of the most serious public health concerns facing the world. Its characteristic feature is neuroinflammation due to microglial activation. Electroacupuncture is one of the therapies employed to improve the condition of patients with AD, although its mechanism of action is still to be determined. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor that is involved in regulating neuroinflammation in AD. In this study, we applied senescence-accelerated mouse-prone 8 mice as the AD animal model, used the Morris water maze, and applied hematoxylin and eosin staining, immunofluorescence double staining, and Western blotting, to explore the effects and potential mechanisms of action of electroacupuncture. In summary, this study suggested that electroacupuncture treatment could improve the learning and memory abilities (p < 0.05) and protect neurons. These effects result from acupuncture could upregulate TREM2 expression in the hippocampus (p < 0.01), which was essential for the anti-inflammatory effects in the AD animal model. However, further studies are needed to conclusively demonstrate the mechanism of action of electroacupuncture in AD.

Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages.

Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due to their immunomodulatory properties. The overall immunomodulatory response of hMSCs resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages (Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into improved Candida albicans phagocytosis activity of macrophages. Taken together, these results demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated immunomodulation with increased CD163 expression and decreased gene expression of matrix metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.

Association between hereditary predisposition to common cancers and congenital multimalformations.

In a previous article we reported that mutations favoring cancer at adulthood seemed to improve fertility and limit miscarriages. Because spontaneous abortion may result from anomalies in embryo, we questioned if an increased frequency of congenital malformation could be evidenced among cancer-prone families. Oncogenetics database (≈193 000 members) of the comprehensive cancer center Jean Perrin was crossed with regional registry of congenital malformations (≈10 000). Among children born between 1986 and 2011, 176 children with malformation matched in both databases. In breast/ovaries cancer-prone families, the risk for malformations was multiplied by 2.4 [1.2-4.5] in case of a BRCA1 mutation. Frequencies of malformation in BRCA2 and MMR mutated families were similar to families without a cancer syndrome. In comparison to malformations concerning a unique anatomical system, multimalformations were significantly more frequent in case of BRCA or MMR mutations: compared to families without cancer syndrome, the risk of multimalformations was multiplied by 4.1 [0.8-21.7] for cancer-prone families but with no known deleterious mutation, by 6.9 [1.2-38.6] in families with a known mutation but an unknown parental mutational status and by 10.4 [2.3-46.0] when one parent carried the familial mutation. No association with the type of anatomical system was found, nor with multiple births. These results suggest that BRCA and MMR genes play an important role in human embryogenesis and that if their function is lowered because of heterozygote mutations, congenital malformations are either more likely (BRCA1 mutations) and/or more susceptible to concern several anatomical systems.

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b-/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor.

Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.

Wake-promoting effects of ONO-4127Na, a prostaglandin DP1 receptor antagonist, in hypocretin/orexin deficient narcoleptic mice.

Prostaglandin (PG)D2 is an endogenous sleep substance, and a series of animal studies reported that PGD2 or PGD2 receptor (DP1) agonists promote sleep, while DP1 antagonists promote wakefulness. This suggests the possibility of use of PG DP1 antagonists as wake-promoting compounds. We therefore evaluated the wake-promoting effects of ONO-4127Na, a DP1 antagonist, in a mouse model of narcolepsy (i.e., orexin/ataxin-3 transgenic mice) and compared those to effects of modafinil. ONO-4127Na perfused in the basal forebrain (BF) area potently promoted wakefulness in both wild type and narcoleptic mice, and the wake-promoting effects of ONO-4127Na at 2.93 × 10(-4) M roughly corresponded to those of modafinil at 100 mg/kg (p.o.). The wake promoting effects of ONO-4127Na was observed both during light and dark periods, and much larger effects were seen during the light period when mice slept most of the time. ONO-4127Na, when perfused in the hypothalamic area, had no effects on sleep. We further demonstrated that wake-promoting effects of ONO-4127Na were abolished in DP1 KO mice, confirming that the wake-promoting effect of ONO-4127Na is mediated by blockade of the PG DP1 receptors located in the BF area. ONO-4127Na reduced DREM, an EEG/EMG assessment of behavioral cataplexy in narcoleptic mice, suggesting that ONO-4127Na is likely to have anticataplectic effects. DP1 antagonists may be a new class of compounds for the treatment of narcolepsy-cataplexy, and further studies are warranted.