Dataset Information

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

ABSTRACT: Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/β-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

SUBMITTER: Yu Z

PROVIDER: S-EPMC10955484 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Yu Zitong Z Yunusbaev Ural U Fritz Allan A Tilley Michael M Akhunova Alina A Trick Harold H Akhunov Eduard E

Plant biotechnology journal 20231117 4

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. Th ...[more]

PMID: 37975410

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Project description:Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development.A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences.GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream.The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

Project description:BackgroundThe unique properties of wheat flour primarily depend on gluten, which is the most important source of protein for human being. gamma-Gliadins have been considered to be the most ancient of the wheat gluten family. The complex family structure of gamma-gliadins complicates the determination of their function. Moreover, gamma-gliadins contain several sets of celiac disease epitopes. However, no systematic research has been conducted yet.ResultsA total of 170 gamma-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences are putatively functional. The ORF lengths of these sequences range from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity of gamma-gliadins. The repeat motif P(Q/L/S/T/I/V/R/A)F(S/Y/V/Q/I/C/L)P(R/L/S/T/H/C/Y)Q1-2(P(S/L/T/A/F/H)QQ)1-2is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that gamma-gliadins are highly diverse. Phylogenic analyses indicate that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classified gamma-gliadins into two types based on the length of repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids in gamma-gliadins, and those gamma-gliadins from subgroup SG-10 and SG-12 and gamma-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that gamma-gliadins with a pattern of C9 and gamma-gliadins with a short repetitive domain almost lack any epitopes.Conclusiongamma-Gliadin sequences in wheat and closely related Aegilops species are diverse. Each group/subgroup contributes differently to nutritional quality and epitope content. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, it is possible to breed wheat varieties, the gamma-gliadins of which are less, even non-toxic and more nutritional.

Dataset Information

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Publications

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Similar Datasets

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