Carmen V. Ozuna

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

Diversification of the celiac disease α‐gliadin complex in wheat: a 33‐mer peptide with six overlapping epitopes, evolved following polyploidization

The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides.

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α-, γ-, ω-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ- and α-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω-gliadins and γ-gliadins, and three of these also silenced α-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α- and ω-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.

Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9

Summary Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.

Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the Triticeae : cereal domestication and breeding contributed to decrease the content of gliadins and gluten

Wheat proteins are important for the physico-chemical properties of bread-dough and contribute to the protein intake in the human diet. In certain individuals, an immunological reactivity of the gluten protein family is strongly implicated in the etiology of celiac disease (CD) and non-celiac wheat sensitivity (NCWS). There is evidence that gluten-related disorders have increased in frequency in recent years. Gluten proteins were characterized and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) while the occurrence of CD immunogenic epitopes was searched in the gliadin sequences of Triticeae within the NCBI database. We have observed a tendency toward low content of gliadins in cultivated species compared to that of the wild ancestors in all Triticeae members. Regarding the glutenin subunits, there was no clear trend, but levels tended to be higher in cultivated species. Thousand-kernel weight is higher for domesticated and cultivated species. Quantification of DQ2- and DQ8-restricted epitopes in gliadin sequences showed a great variability in the number of CD epitopes per species and genome. A higher frequency of immunnogenic epitopes was found to be associated with genomes of the DD, BBAADD, and RR type. Durum wheats tend to have a lower content of gluten and CD immunogenic epitopes. Cultivated barley could be an alternative cereal with low immunogenic epitopes and low gluten. The results reported in this study suggest that domestication and breeding have contributed to a decrease in the content of gliadins and total gluten in the Triticeae species over time.

Genetic Transformation of Wheat: Advances in the Transformation Method and Applications for Obtaining Lines with Improved Bread-Making Quality and Low Toxicity in Relation to Celiac Disease

Wheat is one of the most important crops and is counted among the “big three” cereal crops (rice, wheat and maize), with an annual world production of around 680 million tonnes in 2009. Wheat is also one of the main sources of calories and proteins in the human diet. However, in spite of its global importance, wheat has been one of the last crops being transformed and it was not until 1992 when Vasil et al. (1992) obtained the first fertile transgenic plant of wheat. Nowadays, wheat transformation still presents more difficulties than transformation of other cereals, such as rice and maize, with lower transformation efficiencies and greater genotype dependence (Shewry & Jones, 2005). Particle bombardment is the most widely used method for genetic transformation of wheat, presenting higher transformation efficiencies than Agrobacterium-mediated transformation (Lazzeri & Jones, 2009). However, particle bombardment causes physical damage to the scutellar tissues used for transformation, negatively affecting the embryogenesis, in vitro regeneration of the explants and therefore the transformation efficiency. Osmotic treatment is thought to offer protection to bombarded material by minimising cytoplasm leakage from target cells (Vain et al., 1993), so it is of great importance to optimise the duration and moment of application of the osmotic treatment to the explants. Among the applications of genetic transformation, gene over-expression and posttranscriptional gene silencing (PTGS) are two strategies successfully used to enhance the wheat quality. In particular, the baking quality of wheat, largely determined by the high molecular weight glutenin subunits (HMW-GS), is one of the most important targets for genetic transformation. Transgenic wheat lines expressing additional copies of the 1Ax1, 1Dx5, 1Dy10 HMW-GS genes were obtained by particle bombardment by Leon et al. (2009) (Fig. 1 A). In addition, new lines combining the three transgenic events were obtained by conventional crossing (Leon et al., 2010) (Fig. 1 B). Therefore, a set of