The α-Gliadins in Bread Wheat: Effect of Nitrogen Treatment on the Expression of the Major Celiac Disease Immunogenic Complex in Two RNAi Low-Gliadin Lines

Abstract

Celiac Disease (CD) is an autoimmune disorder that affects approximately 1% of the worldwide population. The α-gliadins of wheat contain the 33-mer peptide, the most active peptide in CD both in adults and pediatric patients. In this study, we have characterized the variants and expression profile of an α-gliadins amplicon, harboring the 33-mer peptide, in two low-gliadin RNAi wheat lines, under two different Nitrogen (N) treatments. We estimated that the amplicon expands 45 different α-gliadin variants with high variability due to length, randomly distributed SNPs, and the presence of encoded CD epitopes. Expression of this amplicon is reduced in both RNAi lines in comparison to the wild type. High N treatment significantly increases transcripts of the amplicon in the wild type, but not in the transgenic lines. Classification of α-gliadin variants, considering the number of epitopes, revealed that amplicon variants containing the full complement of 33-mer peptide were affected by N treatment, increasing their expression when N was increased. Line D793 provided higher and more stable silencing through different N fertilization regimes, expressing fewer CD epitopes than D783. Results of this study are important for better understanding of RNAi α-gliadin silencing in response to N treatments, and for undertaking new strategies by RNAi or CRISPR/Cas toward obtaining new varieties suitable for people suffering gluten intolerances.

Unraveling the celiac disease-related immunogenic complexes in a set of wheat and tritordeum genotypes: implications for low-gluten precision breeding in cereal crops

“…We report 27.6% and 22.0% of pseudogenes for the alpha-and gamma-gliadin amplicons, and many of them presented the mutation of the glutamine into a stop codon, mainly in the alpha-gliadins. However, the number of pseudogenes could be underestimated because the amplicon does not cover the entire sequence of both gliadins genes as reported by Sańchez-Leoń et al (2021). In fact, there was previously described 45% of pseudogenes in alpha-gliadins, while the gamma-gliadin-genes presented 21% of them, close to our results for this family (Huo et al, 2018a;Huo et al, 2018b).…”

“…We report 27.6% and 22.0% of pseudogenes for the alpha- and gamma-gliadin amplicons, and many of them presented the mutation of the glutamine into a stop codon, mainly in the alpha-gliadins. However, the number of pseudogenes could be underestimated because the amplicon does not cover the entire sequence of both gliadins genes as reported by Sánchez-León et al. (2021) .…”

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

“…Moreover, the exact copy number of these genes and their specific chromosome distribution are still uncertain. For Chinese Spring and BobWhite, 47 and 45 α-gliadin genes have been characterized, respectively [ 20 , 21 ]. This high variability found in WT lines along with the similarity of these sequences makes the analysis of InDels very complex.…”

“…On the other hand, CRISPR/Cas mutation analysis is extremely challenging because of the nature of α-gliadin genes which comprise approximately 100 copies of genes and pseudogenes disposed in tandem at the Gli-2 loci of chromosomes 6A, 6B, and 6D [ 19 ]. Recently, 47 α-gliadin genes were characterized for the Chinese Spring variety by Huo et al [ 20 ], whereas 45 different α-gliadin variants were identified for the BobWhite variety using amplicon sequencing [ 21 ]. The repetitive DNA composition of these regions along with the duplication of genes and their tandem organization hinders the genomic sequence assembly and subsequent InDels analysis.…”

Polymerase chain reaction: a powerful analytical tool in the field of food safety

“…It is worth emphasizing that in the PCR test, it is possible to use several pairs of primers to detect multiple targets in a single reaction (so-called multiplex PCR), thus reducing costs and time. The PCR method was successfully implemented for the detection of gliadin which is a major component of wheat gluten and is responsible for most of the harmful effects on the health of sensitive people (Sánchez-León et al 2021). PCR method using a pair of primers specific for the gluten-coding DNA sequence revealed the presence of gluten in guaranteed gluten-free products but also in naturally glutenfree foods.…”