Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like α-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T₀ generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines.

Plant diseases are globally causing substantial losses in staple crop production, undermining the urgent goal of a 60% increase needed to meet the food demand, a task made more challenging by the climate changes. Main consequences concern the reduction of food amount and quality. Crop diseases also compromise food safety due to the presence of pesticides and/or toxins. Nowadays, biotechnology represents our best resource both for protecting crop yield and for a science-based increased sustainability in agriculture. Over the last decades, agricultural biotechnologies have made important progress based on the diffusion of new, fast and efficient technologies, offering a broad spectrum of options for understanding plant molecular mechanisms and breeding. This knowledge is accelerating the identification of key resistance traits to be rapidly and efficiently transferred and applied in crop breeding programs. This review gathers examples of how disease resistance may be implemented in cereals by exploiting a combination of basic research derived knowledge with fast and precise genetic engineering techniques. Priming and/or boosting the immune system in crops represent a sustainable, rapid and effective way to save part of the global harvest currently lost to diseases and to prevent food contamination.

Among wheat gluten proteins, the α-type gliadins are the major responsible for celiac disease, an autoimmune disorder that affects about 1% of the world population. In fact, these proteins contain several toxic and immunogenic epitopes that trigger the onset of the disease. The α-type gliadins are a multigene family, encoded by genes located at the complex Gli-2 loci. Here, three bread wheat deletion lines (Gli-A2, Gli-D2 and Gli-A2/Gli-D2) at the Gli-2 loci were generated by the introgression in the bread wheat cultivar Pegaso of natural mutations, detected in different bread wheat cultivars. The molecular characterization of these lines allowed the isolation of 49 unique expressed genes coding α-type gliadins, that were assigned to each of the three Gli-2 loci. The number and the amount of α-type gliadin transcripts were drastically reduced in the deletion lines. In particular, the line Gli-A2/Gli-D2 contained only 12 active α-type gliadin genes (−75.6% respect to the cv. Pegaso) and a minor level of transcripts (−80% compared to cv. Pegaso). Compensatory pleiotropic effects were observed in the two other classes of gliadins (ω- and γ-gliadins) either at gene expression or protein levels. Although the comparative analysis of the deduced amino acid sequences highlighted the typical structural features of α-type gliadin proteins, substantial differences were displayed among the 49 proteins for the presence of toxic and immunogenic epitopes. The deletion line Gli-A2/Gli-D2 did not contain the 33-mer peptide, one of the major epitopes triggering the celiac disease, representing an interesting material to develop less “toxic” wheat varieties.

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker’s asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Durum wheat milling is a key process step to improve the quality and safety of final products. The aim of this study was to characterize three bran-enriched milling fractions (i.e., F250, G230 and G250), obtained from three durum wheat grain samples, by using an innovative micronization and air-classification technology. Milling fractions were characterized for main standard quality parameters and for alveographic properties, starch composition and content, phenolic acids, antioxidant activity and ATIs. Results showed that yield recovery, ash content and particle size distributions were influenced either by the operating conditions (230 or 250) or by the grain samples. While total starch content was lower in the micronized sample and air-classified fractions, the P/L ratio increased in air-classified fractions as compared to semolina. Six main individual phenolic acids were identified through HPLC-DAD analysis (i.e., ferulic acid, vanillic acid, p-coumaric acid, sinapic acid, syringic and p-hydroxybenzoic acids). Compared to semolina, higher contents of all individual phenolic components were found in all bran-enriched fractions. The highest rise of TPAs occurred in the F250 fraction, which was maintained in the derived pasta. Moreover, bran-enriched fractions showed significant reductions of ATIs content versus semolina. Overall, our data suggest the potential health benefits of F250, G230 and G250 and support their use to make durum-based foods.

Climate change and the need to feed an increasing population undermines food production and safety, representing the reasons behind the development of a new agriculture that is much more sustainable, productive and accessible worldwide. Genome editing and, in particular, clustered regularly interspaced palindromic repeats/CRISPR-associated protein (CRISPR/Cas) tools will play a major role in plant breeding to address these concerns. CRISPR/Cas includes a series of genome editing tools relying on the recognition and cleavage of target DNA/RNA sequences to introduce specific mutations.