Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

Plant species-specific recognition of long and short ß-1,3-linked glucans is mediated by different receptor systems.

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying ß-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different ß-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) ß-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long ß-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short ß-1,3-glucans. Hydrolysis of the ß-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain ß-glucans. Moreover, in contrast to the recognition of short ß-1,3-glucans in A. thaliana, perception of long ß-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that ß-glucan recognition may be mediated by multiple ß-glucan receptor systems.

Stripe rust induced defence mechanisms in the leaves of contrasting barley genotypes (Hordeum vulgare L.) at the seedling stage.

Puccinia striiformis f. sp. hordei, the causal organism of stripe rust in barley poses serious threats to its production. The present study examined the seedling response and changes in antioxidant defence system along with NADPH oxidase, hydrogen peroxide, and lipid peroxidation marker-malondialdehyde (MDA) in the four barley genotypes namely Jyoti, RD2900, RD2901, and RD2552 in response to M and G-races of stripe rust pathogen. Disease reaction showed Jyoti as susceptible genotype, RD2901 and RD2552 as resistant, whereas RD2900 behaved differentially to both the races. M-race which is predominant was found to be more virulent than G-race of barley stripe rust pathogen. RD2901 showed an increase in activities of NADPH oxidase, catalase, peroxidase, and enzymes of ascorbate-glutathione pathway along with ascorbate and glutathione pool on inoculation with M-race, which was accompanied by the decrease in hydrogen peroxide and MDA contents. Jyoti, on the other hand, showed an increase in peroxidase and glutathione-S-transferase activities only which were unable to maintain redox homeostasis. The scrutiny of data indicated an increase in ASA/DHA ratio on infection in all the genotypes irrespective of their behaviour towards the races. However, GSH/GSSG ratio significantly declined in Jyoti and increased or remained unaffected in the resistant genotypes which suggested that GSH/GSSG might be playing a vital role in imparting tolerance against stripe rust. Further, correlation studies also revealed that leaf damage was positively correlated with H2O2 and MDA contents.

Distinct branches of the N-end rule pathway modulate the plant immune response.

The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

A Conserved Puccinia striiformis Protein Interacts with Wheat NPR1 and Reduces Induction of Pathogenesis-Related Genes in Response to Pathogens.

In Arabidopsis, NPR1 is a key transcriptional coregulator of systemic acquired resistance. Upon pathogen challenge, NPR1 translocates from the cytoplasm to the nucleus, in which it interacts with TGA-bZIP transcription factors to activate the expression of several pathogenesis-related (PR) genes. In a screen of a yeast two-hybrid library from wheat leaves infected with Puccinia striiformis f. sp. tritici, we identified a conserved rust protein that interacts with wheat NPR1 and named it PNPi (for Puccinia NPR1 interactor). PNPi interacts with the NPR1/NIM1-like domain of NPR1 via its C-terminal DPBB_1 domain. Using bimolecular fluorescence complementation assays, we detected the interaction between PNPi and wheat NPR1 in the nucleus of Nicotiana benthamiana protoplasts. A yeast three-hybrid assay showed that PNPi interaction with NPR1 competes with the interaction between wheat NPR1 and TGA2.2. In barley transgenic lines overexpressing PNPi, we observed reduced induction of multiple PR genes in the region adjacent to Pseudomonas syringae pv. tomato DC3000 infection. Based on these results, we hypothesize that PNPi has a role in manipulating wheat defense response via its interactions with NPR1.

The fungal-specific ß-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants.

ß-glucans are well-known modulators of the immune system in mammals but little is known about ß-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific ß-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress ß-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of ß-glucan in plants.

An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew.

Following the detection of pathogen cognate effectors, plant Nod-like receptors (NLRs) trigger isolate-specific immunity that is generally associated with cell death. The regulation of NLR stability is important to ensure effective immunity. In barley (Hordeum vulgare), the allelic Mildew locus A (MLA) receptors mediate isolate-specific disease resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Currently, how MLA stability is controlled remains unknown. Here, we identified an MLA-interacting RING-type E3 ligase, MIR1, that interacts with several MLAs. We showed that the carboxyl-terminal TPR domain of MIR1 mediates the interaction with the coiled-coil domain-containing region of functional MLAs, such as MLA1, MLA6, and MLA10, but not with that of the nonfunctional MLA18-1. MIR1 can ubiquitinate the amino-terminal region of MLAs in vitro and promotes the proteasomal degradation of MLAs in vitro and in planta. Both proteasome inhibitor treatment and virus-induced gene silencing-mediated MIR1 silencing significantly increased MLA abundance in barley transgenic lines. Furthermore, overexpression of MIR1 specifically compromised MLA-mediated disease resistance in barley, while coexpression of MIR1 and MLA10 attenuated MLA10-induced cell death signaling in Nicotiana benthamiana Together, our data reveal a mechanism for the control of the stability of MLA immune receptors and for the attenuation of MLA-triggered defense signaling by a RING-type E3 ligase via the ubiquitin proteasome system.

Coeliac disease: immunogenicity studies of barley hordein and rye secalin-derived peptides.

Coeliac disease (CD) is an inflammatory disorder of the small intestine. It includes aberrant adaptive immunity with presentation of CD toxic gluten peptides by HLA-DQ2 or DQ8 molecules to gluten-sensitive T cells. A ω-gliadin/C-hordein peptide (QPFPQPEQPFPW) and a rye-derived secalin peptide (QPFPQPQQPIPQ) were proposed to be toxic in CD, as they yielded positive responses when assessed with peripheral blood T-cell clones derived from individuals with CD. We sought to assess the immunogenicity of the candidate peptides using gluten-sensitive T-cell lines obtained from CD small intestinal biopsies. We also sought to investigate the potential cross-reactivity of wheat gluten-sensitive T-cell lines with peptic-tryptic digested barley hordein (PTH) and rye secalin (PTS). Synthesised candidate peptides were deamidated with tissue transglutaminase (tTG). Gluten-sensitive T-cell lines were generated by culturing small intestinal biopsies from CD patients with peptic-tryptic gluten (PTG), PTH or PTS, along with autologous PBMCs for antigen presentation. The stimulation indices were determined by measuring the relative cellular proliferation via incorporation of 3 H-thymidine. The majority of T-cell lines reacted to the peptides studied. There was also cross-reactivity between wheat gluten-sensitive T-cell lines and the hordein, gliadin and secalin peptides. PTH, PTS, barley hordein and rye secalin-derived CD antigen-sensitive T-cell lines showed positive stimulation with PTG. ω-gliadin/C-hordein peptide and rye-derived peptide are immunogenic to gluten-sensitive T-cell lines and potentially present in wheat, rye and barley. Additional CD toxic peptides may be shared.

The dynamics of cereal cyst nematode infection differ between susceptible and resistant barley cultivars and lead to changes in (1,3;1,4)-ß-glucan levels and HvCslF gene transcript abundance.

Heterodera avenae (cereal cyst nematode, CCN) infects the roots of barley (Hordeum vulgare) forming syncytial feeding sites. In resistant host plants, relatively few females develop to maturity. Little is known about the physiological and biochemical changes induced during CCN infection. Responses to CCN infection were investigated in resistant (Rha2) and susceptible barley cultivars through histological, compositional and transcriptional analysis. Two phases were identified that influence CCN viability, including feeding site establishment and subsequent cyst maturation. Syncytial development progressed faster in the resistant cultivar Chebec than in the susceptible cultivar Skiff, and was accompanied by changes in cell wall polysaccharide abundance, particularly (1,3;1,4)-ß-glucan. Transcriptional profiling identified several glycosyl transferase genes, including CELLULOSE SYNTHASE-LIKE F10 (HvCslF10), which may contribute to differences in polysaccharide abundance between resistant and susceptible cultivars. In barley, Rha2-mediated CCN resistance drives rapid deterioration of CCN feeding sites, specific changes in cell wall-related transcript abundance and changes in cell wall composition. During H. avenae infection, (1,3;1,4)-ß-glucan may influence CCN feeding site development by limiting solute flow, similar to (1,3)-ß-glucan during dicot cyst nematode infections. Dynamic transcriptional changes in uncharacterized HvCslF genes, possibly involved in (1,3;1,4)-ß-glucan synthesis, suggest a role for these genes in the CCN infection process.

Bacteria-triggered systemic immunity in barley is associated with WRKY and ETHYLENE RESPONSIVE FACTORs but not with salicylic acid.

Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.

The compromised recognition of turnip crinkle virus1 subfamily of microrchidia ATPases regulates disease resistance in barley to biotrophic and necrotrophic pathogens.

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

[Celiac disease--the chameleon among the food intolerances]. / Zöliakie. Das Chamäleon unter den Nahrungsmittelintoleranzen.

Celiac disease is an autoimmune disorder resulting from gluten intolerance and is based on a genetically predisposition. Symptoms occur upon exposure to prolamin from wheat, rye, barley and related grain. The pathogenesis of celiac disease has not yet been sufficiently elucidated but is being considered as an autoimmune process. At its core are the deamidation of prolamin fragments, the building of specific antibodies and the activation of cytotoxic T-cells. The immunological inflammatory process is accompanied by structural damages of the enterocytes (villous atrophy, colonization and crypt hyperplasia). The symptoms and their extent depend on the type of the celiac disease; classic and non-classic forms are being distinguished (atypical, oligosymptomatic, latent and silent celiac disease). Characteristics of the classic presentation are malabsorption syndrome and intestinal symptoms such as mushy diarrhea and abdominal distension. The diagnosis of celiac disease is based on four pillars: Anamnesis and clinical presentation, serological evidence of coeliac specific antibodies (IgA-t-TG; IgA-EmA), small intestine biopsy and improvement of symptoms after institution of a gluten-free diet. The basis of the therapy is a lifelong gluten-free diet, i. e. wheat, rye, barley, spelt, green-core, faro-wheat, kamuth and conventional oats as well as food items obtained therefrom. Small amounts of up to 50 mg gluten per day are usually tolerated by most patients; amounts of > or = 100 mg/day lead mostly to symptoms. Gluten-free foods contain < or = 20 ppm or 20 mg/kg (Sign: symbol of the 'crossed ear' or label 'gluten-free'). At the beginning of the therapy the fat and lactose intake may need to be reduced; also the supplementation of single micronutrients (fat-soluble vitamins, folic acid, B12, iron, and calcium) may be required. Alternative therapies are being developed but have not yet been clinically tested.

Barley γ3-hordein: glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat.

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.

Significant differences in coeliac immunotoxicity of barley varieties.

SCOPE: The only treatment available for coeliac disease (CD) is a strict diet in which the intake of wheat, barley, rye, or oats is avoided. Barley is a major cereal crop, grown mainly for its use in brewing, and it has high nutritional value. The identification of varieties with a reduced toxicity profile may contribute to improve the diet, the quality of life and health of CD patients. METHODS AND RESULTS: Searching for harmless barleys, we investigated accessions of malting and wild barley, used for developing new cultivated cereals. The CD toxicity profile of barleys was screened using G12 antibody and cell proliferation and IFN-γ release from peripheral blood mononuclear cells and intestinal biopsies from CD patients. We found a direct correlation between the reactivity with G12 and the immunogenicity of the different barleys. CONCLUSION: The malting barleys were less immunogenic, with reduced levels of toxic gluten, and were possibly less harmful to CD patients. Our findings could raise the prospect of breeding barley species with low levels of harmful gluten, and the attractive goal of developing nontoxic barley cultivars, always taking into account the Codex standard for foods for special dietary use for persons intolerant to gluten.

Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance.

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.

Defense gene expression is potentiated in transgenic barley expressing antifungal peptide Metchnikowin throughout powdery mildew challenge.

Transgenesis of antimicrobial peptides (AMPs) from different origins has emerged as an option for improvement of crop disease resistance since proof-of-concept for their activities against microbial phytopathogens is provided, persistently. Nevertheless, a more systematic approach based on knowledge of AMPs modes of action including elucidation of their cellular targets and possible impact on immune system considerably improves and diversifies the armory against harmful plant diseases. In present study, the impact of Metchnikowin (Mtk) expression in barley in terms of modulating different immune pathways was investigated. Monitoring of transcript abundance of different genes involved in key immune pathways of SAR, ISR, and redox milieu during interaction of Mtk barley with biotrophic Blumeria graminis f. sp. hordei (Bgh) demonstrated that several immune responses are modulated in Mtk transgenic plants. Present findings substantiate the higher activation of SAR pathway as well as ISR pathway in transgenic plants. Regarding susceptibility factors, nonetheless MLO gene is expressed more in Mtk plants and should lead to an increased cellular accessibility to Bgh, its impact is presumably overwhelmed by other mechanism(s) so that the plants show more resistance when challenging with Bgh. On the other hand, no obvious difference was observed between expression level of Bax inhibitor-1 (BI-1) in transgenic and wild type plants, which could be an indicative of its neutrality in resistance/susceptibility of transgenic plants to Bgh. The provided evidence on the involved pathways in Mtk-induced resistance improves our knowledge concerning impacts of AMPs expressed in diverse plant species on immune system of relevant transgenic plants.

Barley leaf transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora indica-mediated systemic induced resistance to powdery mildew.

Colonization of barley roots with the basidiomycete fungus Piriformospora indica (Sebacinales) induces systemic resistance against the biotrophic leaf pathogen Blumeria graminis f. sp. hordei (B. graminis). To identify genes involved in this mycorrhiza-induced systemic resistance, we compared the leaf transcriptome of P. indica-colonized and noncolonized barley plants 12, 24, and 96 h after challenge with a virulent race of B. graminis. The leaf pathogen induced specific gene sets (e.g., LRR receptor kinases and WRKY transcription factors) at 12 h postinoculation (hpi) (prepenetration phase) and vesicle-localized gene products 24 hpi (haustorium establishment). Metabolic analysis revealed a progressing shift of steady state contents of the intermediates glucose-1-phosphate, uridinediphosphate-glucose, and phosphoenolpyruvate 24 and 96 hpi, indicating that B. graminis shifts central carbohydrate metabolism in favor of sucrose biosynthesis. Both B. graminis and P. indica increased glutamine and alanine contents, whereas substrates for starch and nitrogen assimilation (adenosinediphosphate- glucose and oxoglutarate) decreased. In plants that were more B. graminis resistant due to P. indica root colonization, 22 transcripts, including those of pathogenesis-related genes and genes encoding heat-shock proteins, were differentially expressed ?twofold in leaves after B. graminis inoculation compared with non-mycorrhized plants. Detailed expression analysis revealed a faster induction after B. graminis inoculation between 8 and 16 hpi, suggesting that priming of these genes is an important mechanism of P. indica-induced systemic disease resistance.

Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.

Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

Barley-derived beta-D-glucan induces immunostimulation via a dectin-1-mediated pathway.

Barley-derived beta-glucan, a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose polymers, binds to dectin-1. However, whether it can trigger signal transduction via dectin-1 remains unclear. In this study, we used a reporter gene assay to determine whether barley-derived beta-d-glucan can activate NF-kappaB via dectin-1-mediated signaling when dectin-1 is cotransfected with Syk, CARD9, and Bcl10 in 293T cells. We found that barley-derived beta-D-glucan can activate NF-kappaB leading to cytokine production when dectin-1, Syk, CARD9, and Bcl10 are coexpressed in the cells. We also found that barley-derived beta-D-glucan can induce the phosphorylation of Syk and production of IL-6 in thioglycolate-elicited peritoneal macrophages. These results indicated that the immunostimulatory effects of barley-derived beta-d-glucan might be exerted, at least in part, via dectin-1.

Nuclear actions in innate immune signaling.

Innate immunity in plants and animals is mediated through pattern recognition receptors, which were thought to initiate signaling in the cytoplasm to activate defense pathways. Shen et al. (2006) and Burch-Smith et al. (2007) now provide compelling evidence that certain plant disease resistance proteins, which detect specific pathogenic effectors, act in the nucleus to trigger downstream signaling and defense pathways.