Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci

Background - A-gliadins form a multigene protein family encoded by multiple ¿-gliadin (Gli-2) genes at three genomic loci, Gli-A2, Gli-B2 and Gli-D2, respectively located on the homoeologous wheat chromosomes 6AS, 6BS, and 6DS. These proteins contain a number of important celiac disease (CD)-immunogenic domains. The ¿-gliadins expressed from the Gli-B2 locus harbour fewer conserved CD-epitopes than those from Gli-A2, whereas the Gli-D2 gliadins have the highest CD-immunogenic potential. In order to detect differences in the highly CD-immunogenic ¿-gliadin fraction we determined the relative expression level from the homoeologous Gli-2 loci in various tetraploid and hexaploid wheat genotypes by using a quantitative pyrosequencing method and by analyzing expressed sequence tag (EST) sequences. Results - We detected large differences in relative expression levels of ¿-gliadin genes from the three homoeologous loci among wheat genotypes, both as relative numbers of expressed sequence tag (EST) sequences from specific varieties and when using a quantitative pyrosequencing assay specific for Gli-A2 genes. The relative Gli-A2 expression level in a tetraploid durum wheat cultivar ('Probstdorfer Pandur') was 41%. In genotypes derived from landraces, the Gli-A2 frequency varied between 12% and 58%. In some advanced hexaploid bread wheat cultivars the genes from locus Gli-B2 were hardly expressed (e.g., less than 5% in 'Lavett') but in others they made up more than 40% (e.g., in 'Baldus'). Conclusion - Here, we have shown that large differences exist in relative expression levels of ¿-gliadins from the homoeologous Gli-2 loci among wheat genotypes. Since the homoelogous genes differ in the amount of conserved CD-epitopes, screening for differential expression from the homoeologous Gli-2 loci can be employed for the pre-selection of wheat varieties in the search for varieties with very low CD-immunogenic potential. Pyrosequencing is a method that can be employed for such a 'gene family-specific quantitative transcriptome profiling

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing

BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

<p>Abstract</p> <p>Background</p> <p>Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (<it>Triticum aestivum</it>) (AABBDD).</p> <p>Results</p> <p>The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of <it>Triticum aestivum </it>cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties.</p> <p>Conclusion</p> <p>The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.</p

Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation

<p>Abstract</p> <p>Background</p> <p>Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains.</p> <p>Results</p> <p>A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (<it>T. aestivum</it>, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, <it>Gli-A1</it>, <it>Gli-B1</it> or <it>Gli-D1</it>. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus <it>Gli-D1</it>. Transcripts from each locus differed in CD epitope content and composition. The <it>Gli-D1</it> transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the <it>Gli-A1</it> transcripts (8.6) and the <it>Gli-B1</it> transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce <it>in vitro</it> proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone.</p> <p>Conclusions</p> <p>Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).</p