Genetic Modification for Wheat Improvement: From Transgenesis to Genome Editing

Copyright © 2019 Nikolai Borisjuk et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.To feed the growing human population, global wheat yields should increase to approximately 5 tonnes per ha from the current 3.3 tonnes by 2050. To reach this goal, existing breeding practices must be complemented with new techniques built upon recent gains from wheat genome sequencing, and the accumulated knowledge of genetic determinants underlying the agricultural traits responsible for crop yield and quality. In this review we primarily focus on the tools and techniques available for accessing gene functions which lead to clear phenotypes in wheat. We provide a view of the development of wheat transformation techniques from a historical perspective, and summarize how techniques have been adapted to obtain gain-of-function phenotypes by gene overexpression, loss-of-function phenotypes by expressing antisense RNAs (RNA interference or RNAi), and most recently the manipulation of gene structure and expression using site-specific nucleases, such as CRISPR/Cas9, for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to increase yield, improve nutritional and health-promoting qualities in wheat, and enhance the crop’s resistance to various biotic and abiotic stresses.Peer Reviewe

A schematic representation of the regulation of abiotic and biotic stress-responsive genes by ERFs and DREBs/CBFs, through three types of stress-responsive <i>cis</i>-acting elements.

<p>A schematic representation of the regulation of abiotic and biotic stress-responsive genes by ERFs and DREBs/CBFs, through three types of stress-responsive <i>cis</i>-acting elements.</p

Expression of <i>TaCor410b</i> and five <i>ERF</i> genes in a variety of wheat tissues in the absence of stress.

<p>Levels of expression were detected by Q-PCR and are shown as normalised transcription levels in arbitrary units (n = 4± SD (P<0.05)).</p

Identification of functional DRE/CRT element in the <i>TdCor410b</i> promoter using transient expression assay.

<p>(A), Identification of functional drought-responsive DRE/CRT elements by 5′ deletion analysis of the <i>TdCor410b</i> promoter, using <i>trans</i>-activation of the <i>GUS</i> reporter gene in a transient expression assay. The full-length <i>TdCor410b</i> promoter and six promoter deletions were linked to the <i>GUS</i> reporter gene and co-transformed <i>via</i> particle bombardment into cell suspension cultures with either pUbi-GFP (negative control) or pUbi-TaDREB3 (transcription activator). A schematic representation of the 5′ terminal deletions of the promoter fused to the <i>GUS</i> gene is shown in the left part of the figure: asterisk (*) denotes the predicted DRE/CRT site. A negative control (basal levels of full-length promoter activity) is shown at the top of the right panel as an empty box. Error bars represent standard deviation (P<0.05) for 3 – 4 independent measurements. (B) Influence of point mutations in the identified functional CRT element on <i>TdCor410b</i> promoter activation, as demonstrated by a transient expression assay. D7 denotes a −263 promoter deletion containing the non-mutated CRT element (positive control), D8 and M5 denote promoter deletions without the CRT element (negative controls), and M1–M4 denote the D7 deletion with different single base pair substitutions to T.</p

Key residues of AP2 domains that underlie selectivity of <i>cis</i>-elements binding, and regulation of the <i>TdCor410b</i> promoter activity.

<p>(A) Multiple sequence alignment of selected AP2 domains using PROMALS3D (44). Representative sequences are coloured according to predicted secondary structures (red: α-helix, blue: β-strand). The black box indicates the boundaries of the AP2 domains. The positions of highly conserved Pro residues in the ERF sequences and of variable non-proline residues in the DREB sequences are highlighted in yellow and green, respectively. The positions of two Pro residues conserved in selected cereal ERF sequences are highlighted in cyan, while the positions of the corresponding Arg residues are highlighted in grey. Consensus of secondary structure elements indicates the position of β-sheets (black arrows) and of an α-helix (purple). The degree of conservation of residues is shown above the sequences by black and brown numbers with a conservation index of 5 and higher. (B) Influence of conserved proline residue substitutions in the AP2 domain of TaERF4a on recognition of the GCC-box. TaDREB3 was used as a negative control and TaERF5a as a positive control of interaction with the GCC-box. Mutation of Pro26 to Arg26 (underlined) has no influence on interaction of the TaERF4a variant with the <i>cis</i>-element. Mutation of Pro42 to Arg42 (underlined and boxed in blue) lead to restoration of interaction and consequent growth of yeast on the selective (-Leu, -His, + 5 mM 3-AT) medium. (C) Regulation of the activity of the <i>TdCor410b</i> promoter and of the artificial promoter with substitution of the CRT element for a tandem of three GCC-boxes by representatives of each isolated ERF subfamily, and variants of TaERF4a with mutations in the AP2 domain. TFs were tested in a transient expression assay in a wheat cell culture. Either pTdCor410b-GUS or 3×GCCbox-GUS constructs were co-bombarded with pUbi-GFP (GFP; negative control), pUbi-TaERF4a (TaERF4a), pUbi-TaERF4a mutated at Pro26 (TaERF4a m1), pUbi-TaERF4a mutated at Pro42 (TaERF4a m2), pUbi-TaERF4a mutated at Pro26 and Pro42 (TaERF4a m1+2), pUbi-TaERF6 (TaERF6), or pUbi-TaERF5a (TaERF5a).</p

Expression of <i>Cor410b</i> and <i>ERF</i> genes in leaves and grain of bread and durum wheat subjected to mechanical wounding.

<p>(A) Expression of <i>TaCor410b</i> and <i>TaERF</i> genes in leaves of bread wheat plants following wounding. Levels of expression, detected by Q-PCR, are shown as normalised transcription levels in arbitrary units. (B) Expression of <i>TdCor410b</i> and <i>TdERF6</i> following wounding in leaves of durum wheat plants at flowering. (C) Expression of <i>TdCor410b</i> and <i>TdERF6</i> wheat grains following wounding, with the wounding being applied at 8–15 days after pollination. Values are means (± SD (P<0.05)) of 3 measurements.</p

Wheat TFs isolated in Y1H screens and their properties.

<p>(A) An unrooted radial phylogenetic tree of AP2-domain containing TFs from monocotyledonous and dicotyledonous plant species. Amino acid sequences of 32 proteins were aligned with ProMals3D (44) and branch lengths were drawn to scale. Grey shading indicates distinct branches of ERF and DREB TFs. Two-letter prefixes in the sequence identifiers indicate species of origin (Ta  =  <i>Triticum aestivum</i>; Hv  =  <i>Hordeum vulgare</i>; Os  =  <i>Oriza sativa</i>; Gm  =  <i>Glycine max</i>; At  =  <i>Arabidopsis thaliana</i>; Bj  =  <i>Brassica juncea</i>; Gh  =  <i>Gossypium hirsutum</i>; Nt  =  <i>Nicotiana tabacum</i>; Ns  =  <i>Nicotiana sylvestris</i>; Ca  =  <i>Capsicum annuum</i>). Protein accession numbers are specified in the Materials and Methods. TFs isolated in this work are shown in bold. The <i>Arabidopsis</i> AtERF1 TF was used for construction of 3D models of the AP2 domains of TaERF4a, TaERF5a and TaDREB3, and is shown in bold and underlined. (B) Specificity of recognition of known stress-responsive <i>cis</i>-elements by ERF and DREB TFs detected <i>via</i> a Y1H assay. Growth of yeast on selective medium (-Leu, -His,+5 mM 3-AT) indicates protein-DNA interaction. The <i>cis</i>-element CAATGATTG of the HD-Zip class II TF was used as a negative control. (C) Demonstration of activator properties using ERFs in a Y1H assay. The presence of their own activation domains in the representatives from each subfamily of ERFs supports the activation of the yeast genes and consequent growth of yeast on the selective (-Leu, -Trp, -His, -Ade) medium. (D) Regulation of <i>TdCor410b</i> promoter activity by representatives of each isolated subfamily of ERFs. TFs were tested in a transient expression assay in a wheat cell culture. The pTdCor410b-GUS construct was co-bombarded with pUbi-GFP (GFP; negative control), pUbi-TaERF4a (TaERF4a), pUbi-TaERF4a mutated in the ERF-associated amphiphilic repression (EAR) motif (TaERF4a m), pUbi-TaERF6 (TaERF6), and pUbi-TaERF5a (TaERF5a), and GUS expression in the cultures was quantified (n = 4±SD (P<0.05)).</p

Molecular models of AP2 domains in complex with <i>cis</i>-elements.

<p>(A) Molecular surface morphologies of the AP2 domains of AtERF1, TaERF4a, TaERF5a and TaDREB3 TFs in complex with <i>cis</i>-elements. White, blue and red patches on protein surfaces indicate electro-neutral, electropositive and electronegative patches; the charged patched are contoured at ±5 kT/e. Double-stranded DNA sequences of the <i>cis</i>-elements (GCCGCC/GGCGGC, GCCGAC/GTCGGC and ACCGAC/GTCGGT) are indicated by sticks, where the coding and complementary strands are shown in green and yellow atomic colours, respectively. (B) Molecular folds of the AP2 domains of AtERF1, TaERF4a, TaERF5a and TaDREB3 TFs in complex with <i>cis</i>-elements. Ribbon representations show the disposition of secondary structure elements, where anti-parallel strands carry amino acid residues that mediate contacts between individual <i>cis</i>-elements and the AP2 domains. The ribbons are coloured in green (AtERF1), cyan (TaERF4a), yellow (TaERF5a) and magenta (TaDREB3). The black arrows point to the NH₂-termini of the AP2 domains. The coding strands of <i>cis</i>-elements GCCGCC, GCGGAC and ACCGAC are shown as stick models and are coloured in atomic colours. The interacting residues in the AP2 domains are also shown as sticks, and are coloured in green (AtERF1), cyan (TaERF4a), yellow (TaERF5a) and magenta (TaDREB3). Distances of ≥3.4 Å between the contacting residues (Arg and Trp) and <i>cis</i>-elements are indicated by dotted lines. The positions of respective Pro or Gly residues, adjacent to the contacting Arg residues, are also indicated. The interplay of these residues within the structures suggested that structural rigidity or flexibility could impact upon selectivity of binding of individual <i>cis</i>-elements.</p