Characterization of a new gene for resistance to wheat powdery mildew on chromosome 1RL of wild rye Secale sylvestre.

KEY MESSAGE: PmSESY, a new wheat powdery mildew resistance gene was characterized and genetically mapped to the terminal region of chromosome 1RL of wild rye Secale sylvestre. The genus Secale is an important resource for wheat improvement. The Secale species are usually considered as non-adapted hosts of Blumeria graminis f. sp. tritici (Bgt) that causes wheat powdery mildew. However, as a wild species of cultivated rye, S. sylvestre is rarely studied. Here, we reported that 25 S. sylvestre accessions were susceptible to isolate BgtYZ01, whereas the other five confer effective resistance to all the tested isolates of Bgt. A population was then constructed by crossing the resistant accession SESY-01 with the susceptible accession SESY-11. Genetic analysis showed that the resistance in SESY-01 was controlled by a single dominant gene, temporarily designated as PmSESY. Subsequently, combining bulked segregant RNA-Seq (BSR-Seq) analysis with molecular analysis, PmSESY was mapped into a 1.88 cM genetic interval in the terminus of the long arm of 1R, which was closely flanked by markers Xss06 and Xss09 with genetic distances of 0.87 cM and 1.01 cM, respectively. Comparative mapping demonstrated that the corresponding physical region of the PmSESY locus was about 3.81 Mb in rye cv. Lo7 genome, where 30 disease resistance-related genes were annotated, including five NLR-type disease resistance genes, three kinase family protein genes, three leucine-rich repeat receptor-like protein kinase genes and so on. This study gives a new insight into S. sylvestre that shows divergence in response to Bgt and reports a new powdery mildew resistance gene that has potential to be used for resistance improvement in wheat.

Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes.

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Baker's Asthma: Is the Ratio of Rye Flour-Specific IgE to Total IgE More Suitable to Predict the Outcome of Challenge Test Than Specific IgE Alone.

Usually the diagnosis of baker's asthma is based on specific inhalation challenge with flours. To a certain extent the concentration of specific IgE to flour predicts the outcome of challenge test in bakers. The aim of this study was to evaluate whether the ratio of specific IgE (sIgE) to total IgE (tIgE) improves challenge test prediction in comparison to sIgE alone. Ninety-five bakers with work-related respiratory symptoms were challenged with rye flour. Total IgE, sIgE, and the sIgE/tIgE ratio were determined. Receiver operator characteristic (ROC) plots including the area under the curve (AUC) were calculated using the challenge test as gold-standard. Total IgE and sIgE concentrations, and their ratio were significantly higher in bakers with a positive challenge test than in those with a negative one (p < 0.0001, p < 0.0001, and p = 0.023, respectively). In ROC analysis, AUC was 0.83 for sIgE alone, 0.79 for tIgE, and 0.64 for the ratio. At optimal cut-offs, tIgE, sIgE, and the ratio reached a positive predicted value (PPV) of 95%, 84% and 77%, respectively. In conclusion, calculating the ratio of rye flour-sIgE to tIgE failed to improve the challenge test prediction in our study group.

Should wheat, barley, rye, and/or gluten be avoided in a 6-food elimination diet?

Eosinophilic esophagitis (EoE), a food antigen-mediated disease, is effectively treated with the dietary elimination of 6 foods commonly associated with food allergies (milk, wheat, egg, soy, tree nuts/peanuts, and fish/shellfish). Because wheat shares homologous proteins (including gluten) with barley and rye and can also be processed with these grains, some clinicians have suggested that barley and rye might also trigger EoE as a result of cross-reaction and/or cross-contamination with wheat. In this article, we discuss the theoretical risks of cross-reactivity and cross-contamination among wheat, barley, and rye proteins (including gluten); assess common practices at EoE treatment centers; and provide recommendations for dietary treatment and future studies of EoE.

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.

Analyzing Genetic Diversity for Virulence and Resistance Phenotypes in Populations of Stem Rust (Puccinia graminis f. sp. secalis) and Winter Rye (Secale cereale).

Stem rust (Puccinia graminis f. sp. secalis) leads to considerable yield losses in rye-growing areas with continental climate, from Eastern Germany to Siberia. For implementing resistance breeding, it is of utmost importance to (i) analyze the diversity of stem rust populations in terms of pathotypes (= virulence combinations) and (ii) identify resistance sources in winter rye populations. We analyzed 323 single-uredinial isolates mainly collected from German rye-growing areas across 3 years for their avirulence/virulence on 15 rye inbred differentials. Out of these, 226 pathotypes were detected and only 56 pathotypes occurred more than once. This high diversity was confirmed by a Simpson index of 1.0, a high Shannon index (5.27), and an evenness index of 0.97. In parallel, we investigated stem rust resistance among and within 121 heterogeneous rye populations originating mainly from Russia, Poland, Austria, and the United States across 3 to 15 environments (location-year combinations). While German rye populations had an average stem rust severity of 49.7%, 23 nonadapted populations were significantly (P < 0.01) more resistant with a stem rust severity ranging from 3 to 40%. Out of these, two modern Russian breeding populations and two old Austrian landraces were the best harboring 32 to 70% fully resistant plants across 8 to 10 environments. These populations with the lowest disease severity in adult-plant stage in the field also displayed resistance in leaf segment tests. In conclusion, stem rust populations are highly diverse and the majority of resistances in rye populations seems to be race specific.

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew.

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology-based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single-cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3- and Pm8-like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co-evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.

Testing safety of germinated rye sourdough in a celiac disease model based on the adoptive transfer of prolamin-primed memory T cells into lymphopenic mice.

UNLABELLED: The current treatment for celiac disease is strict gluten-free diet. Technical processing may render gluten-containing foods safe for consumption by celiac patients, but so far in vivo safety testing can only be performed on patients. We modified a celiac disease mouse model to test antigenicity and inflammatory effects of germinated rye sourdough, a food product characterized by extensive prolamin hydrolysis. Lymphopenic Rag1-/- or nude mice were injected with splenic CD4+CD62L-CD44high-memory T cells from gliadin- or secalin-immunized wild-type donor mice. We found that: 1) Rag1-/- recipients challenged with wheat or rye gluten lost more body weight and developed more severe histological duodenitis than mice on gluten-free diet. This correlated with increased secretion of IFNγ, IL-2, and IL-17 by secalin-restimulated splenocytes. 2) In vitro gluten testing using competitive R5 ELISA demonstrated extensive degradation of the gluten R5 epitope in germinated rye sourdough. 3) However, in nude recipients challenged with germinated rye sourdough (vs. native rye sourdough), serum anti-secalin IgG/CD4+ T helper 1-associated IgG2c titers were only reduced, but not eliminated. In addition, there were no reductions in body weight loss, histological duodenitis, or T cell cytokine secretion in Rag1-/- recipients challenged accordingly. IN CONCLUSION: 1) prolamin-primed CD4+CD62L-CD44high-memory T cells induce gluten-sensitive enteropathy in Rag1-/- mice. 2) Hydrolysis of secalins in germinated rye sourdough remains incomplete. Secalin peptides retain B and T cell stimulatory capacity and remain harmful to the intestinal mucosa in this celiac disease model. 3) Current antibody-based prolamin detection methods may fail to detect antigenic gluten fragments in processed cereal food products.

Serum autoantibodies directed against transglutaminase-2 have a low avidity compared with alloantibodies against gliadin in coeliac disease.

Coeliac disease is characterized by intolerance to gliadin and related gluten components present in wheat, barley and rye. Coeliac disease patients harbour antibodies directed against alloantigens such as gliadin, but also against the autoantigen transglutaminase-2 (TG2). The type and quality of antibody responses provides insight into the underlying immune activation processes. Therefore, in this study we have analysed the avidity of the antibody response directed against the autoantigen TG2 and compared this with antibody responses against the alloantigens gliadin and Escherichia coli. We observed that the immunoglobulin (Ig)A autoantibody response directed against TG2 is of low avidity compared with the IgA response against the alloantigens gliadin and E. coli in the same patients; the same was true for IgG, both in IgA-deficient and in -sufficient coeliac patients. The observed avidities appear not to be related to disease stage, antibody levels, age or duration of exposure to gluten. In conclusion, in coeliac disease there is a clear difference in avidity of the antibody responses directed against the auto- and alloantigens, indicating different regulation or site of initiation of these responses.

Diagnostic Accuracy of Specific IgE Against Wheat and Rye in Flour-Induced Occupational Asthma.

BACKGROUND: Assessment of IgE-mediated sensitization to flour allergens is widely used to investigate flour-induced occupational asthma. The diagnostic efficiency of detecting specific IgE antibodies (sIgEs) against wheat and rye flour, however, has not been thoroughly compared with other diagnostic procedures. OBJECTIVE: We sought to evaluate the diagnostic accuracy of sIgE against wheat and rye compared with specific inhalation challenge (SIC) with flour as the reference standard. METHODS: This retrospective multicenter study included 264 subjects who completed an SIC with flour in eight tertiary centers, of whom 205 subjects showed a positive SIC result. RESULTS: Compared with SIC, sIgE levels of 0.35 kUA/L or greater against wheat and rye provided similar sensitivities (84% to 85%, respectively), specificities (71% to 78%), positive predictive values (91% to 93%), and negative predictive values (56% to 61%). Increasing the threshold sIgE value to 5.10 kUA/L for wheat and to 6.20 kUA/L for rye provided a specificity of 95% or greater and further enhanced the positive predictive value to 98%. Among subjects with a positive SIC, those who failed to demonstrate sIgE against wheat and rye (n = 26) had significantly lower total serum IgE level and blood and sputum eosinophil counts and a lesser increase in postchallenge FeNO compared with subjects with a detectable sIgE. CONCLUSION: High levels of sIgE against wheat and/or rye flour strongly support a diagnosis of flour-induced occupational asthma without the need to perform an SIC. The absence of detectable sIgE against wheat and rye in subjects with a positive SIC seems to be associated with lower levels of TH2 biomarkers.

[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.

Characterization of wheat: Secale africanum introgression lines reveals evolutionary aspects of chromosome 1R in rye.

Wild Secale species, Secale africanum Stapf., serve as a valuable source for increasing the diversity of cultivated rye (Secale cereale L.) and provide novel genes for wheat improvement. New wheat - S. africanum chromosome 1R(afr) addition, 1R(afr)(1D) substitution, 1BL.1R(afr)S and 1DS.1R(afr)L translocation, and 1R(afr)L monotelocentric addition lines were identified by chromosome banding and in situ hybridization. Disease resistance screening revealed that chromosome 1R(afr)S carries resistance gene(s) to new stripe rust races. Twenty-nine molecular markers were localized on S. africanum chromosome 1R(afr) by the wheat - S. africanum introgression lines. Twenty markers can also identically amplify other reported wheat - S. cereale chromosome 1R derivative lines, indicating that there is high conservation between the wild and cultivated Secale chromosome 1R. Nine markers displayed polymorphic amplification between S. africanum and S. cereale chromosome 1R(afr) derivatives. The comparison of the nucleotide sequences of these polymorphic markers suggested that gene duplication and sequence divergence may have occurred among Secale species during its evolution and domestication.

Maternal antenatal peanut consumption and peanut and rye sensitization in the offspring at adolescence.

BACKGROUND: There is considerable controversy whether maternal peanut ingestion during pregnancy might influence sensitization in later life. Objective To examine whether maternal peanut ingestion during pregnancy might increase sensitization in the offspring. METHODS: A population-based longitudinal cohort study with 16 years follow-up was conducted (N=373). Subjects were recruited at birth as part of an infant health study. Maternal antenatal peanut consumption was documented at birth and peanut and rye sensitization were determined by measurement of serum-specific IgE at age 16. RESULTS: Peanut sensitization was common (14%). In the entire cohort (n=310), there was no association between antenatal peanut ingestion and peanut sensitization (P=0.17). However, there was a strong association between antenatal peanut ingestion and decreased risk of rye sensitization and peanut sensitization in those (n=201) without a family history (FH) of asthma (Rye OR 0.30, 95% CI 0.14-0.63, P=0.001 and Peanut OR 0.18, 95% CI 0.04-0.78, P=0.02). There was an increased risk of rye sensitization in those (n=108) with a FH of asthma and antenatal peanut ingestion (Rye OR 2.69, 95% CI 1.11-6.51 P=0.03). It was considered that these sensitizations were likely to be related to the presence of IgE antibodies to cross-reacting carbohydrate epitopes common to rye and peanut allergens. CONCLUSIONS AND CLINICAL RELEVANCE: Antenatal peanut ingestion may influence the development of IgE antibody to cross-reacting carbohydrate epitopes in later life. Genetic factors may modify this association.

[Establishment of wheat-rye addition lines and De Novo powdery mildew resistance gene from chromosome 5R].

The F5 plants derived from octoploid triticale × common wheat were investigated by FISH methods using repetitive DNA sequences pAS1 and pSc119.2 as probes. The disease resistance of these plants was also screened and evaluated in the field. The 1R, 2R, 3R, 4R, 5R, 6R and 7R monosomic addition lines and 1R and 2R disomic addition lines were found. The occurrence frequencies of chromosomes 1R and 4R addition lines were higher than that of chromosomes 2R, 3R, 5R, 6R, and 7R addition lines in the high generation screened. The 5R and 6R monosomic addition lines were immune to powdery mildew. The chromosome 5R in this study might carry new powdery mildew resistance gene(s). In addition, the preferential elimination of chromosome 4B was observed in several plants.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

Discovery of a Novel Leaf Rust (Puccinia recondita) Resistance Gene in Rye (Secale cereale L.) Using Association Genomics.

Leaf rust constitutes one of the most important foliar diseases in rye (Secale cereale L.). To discover new sources of resistance, we phenotyped 180 lines belonging to a less well-characterized Gülzow germplasm at three field trial locations in Denmark and Northern Germany in 2018 and 2019. We observed lines with high leaf rust resistance efficacy at all locations in both years. A genome-wide association study using 261,406 informative single-nucleotide polymorphisms revealed two genomic regions associated with resistance on chromosome arms 1RS and 7RS, respectively. The most resistance-associated marker on chromosome arm 1RS physically co-localized with molecular markers delimiting Pr3. In the reference genomes Lo7 and Weining, the genomic region associated with resistance on chromosome arm 7RS contained a large number of nucleotide-binding leucine-rich repeat (NLR) genes. Residing in close proximity to the most resistance-associated marker, we identified a cluster of NLRs exhibiting close protein sequence similarity with the wheat leaf rust Lr1 gene situated on chromosome arm 5DL in wheat, which is syntenic to chromosome arm 7RS in rye. Due to the close proximity to the most resistance-associated marker, our findings suggest that the considered leaf rust R gene, provisionally denoted Pr6, could be a Lr1 ortholog in rye.

Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals.

Allergen specific CD4+ T cell clones generated from allergic individuals have been shown to produce increased levels of the cytokine interleukin 4 (IL-4), compared to allergen specific clones generated from nonallergic individuals. This difference between CD4+ T cells from allergic and nonallergic individuals with regard to cytokine production in response to allergen is thought to be responsible for the development of allergic disease with increased IgE synthesis in atopic individuals. We examined the production of IL-4 in subjects with allergic rhinitis and in allergic individuals treated with allergen immunotherapy, a treatment which involves the subcutaneous administration of increasing doses of allergen and which is highly effective and beneficial for individuals with severe allergic rhinitis. We demonstrated that the quantity of IL-4 produced by allergen specific memory CD4+ T cells from allergic individuals could be considerably reduced by in vivo treatment with allergen (allergen immunotherapy). Immunotherapy reduced IL-4 production by allergen specific CD4+ T cells to levels observed with T cells from nonallergic subjects, or to levels induced with nonallergic antigens such as tetanus toxoid. In most cases the levels of IL-4 produced were inversely related to the length of time on immunotherapy. These observations indicate that immunotherapy accomplishes its clinical effects by reducing IL-4 synthesis in allergen specific CD4+ T cells. In addition, these observations indicate that the cytokine profiles of memory CD4+ T cells can indeed be altered by in vivo therapies. Thus, the cytokine profiles of memory CD4+ T cells are mutable, and are not fixed as had been suggested by studies of murine CD4+ memory T cells. Finally, treatment of allergic diseases with allergen immunotherapy may be a model for other diseases which may require therapies that alter inappropriate cytokine profiles of memory CD4+ T cells.

A novel approach to produce phage single domain antibody fragments for the detection of gluten in foods.

In this study, we demonstrated the feasibility of isolating recombinant phage-antibodies against gluten from a non-immunized library of human single-domain antibodies (dAbs). Phage display technology enabled the selection of affinity probes by successive rounds of biopanning against a biotinylated synthetic peptide comprising repetitive immunogenic gluten motifs. The analysis of a wide representation of heterologous plant species corroborated that two of the isolated clones were specific to wheat, barley and rye proteins. The phage antibody selected as the most appropriate clone for the detection of gluten in foods (dAb8E-phage) was further applied in an indirect ELISA to the analysis of 50 commercial food samples. Although the limit of detection achieved did not improve those of current immunoassays, the proposed methodology could provide promising new pathways for the generation of recombinant antibodies that allow a comprehensive determination of gluten in foods, whilst replacing the need for animal immunization.