Triticeae cereals are among the most important crop plants grown worldwide and being used for animal feed, food and beverages. Although breeding efforts evolved over the last ten thousand years our ...today's crop plants, biotechnological methods would help to speed up the process and incorporate traits impossible by conventional breeding. The main research topics were related to cover the future demand on our agricultural practices to supply sufficient food for a growing world population. Target traits are resistances against viral and fungal diseases, improvement of water and nitrogen use efficiency, to tackle plant architecture, both below and aboveground and to develop varieties that could grow on dry or salty locations. Other applications are considering accumulation of useful compounds or decreasing allergenicity. This review will summarize methods to generate the material including a section how genome engineering using gRNA/Cas (CRISPR/Cas) technology could further improve the methodology and will give an overview about recent and future applications.
Protein disulfide isomerases (PDIs) catalyze the correct folding of proteins and prevent the aggregation of unfolded or partially folded precursors. Whereas suppression of members of the PDI gene ...family can delay replication of several human and animal viruses (e.g., HIV), their role in interactions with plant viruses is largely unknown. Here, using a positional cloning strategy we identified variants of PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (HvPDIL5-1) as the cause of naturally occurring resistance to multiple strains of Bymoviruses. The role of wild-type HvPDIL5-1 in conferring susceptibility was confirmed by targeting induced local lesions in genomes for induced mutant alleles, transgene-induced complementation, and allelism tests using different natural resistance alleles. The geographical distribution of natural genetic variants of HvPDIL5-1 revealed the origin of resistance conferring alleles in domesticated barley in Eastern Asia. Higher sequence diversity was correlated with areas with increased pathogen diversity suggesting adaptive selection for bymovirus resistance. HvPDIL5-1 homologs are highly conserved across species of the plant and animal kingdoms implying that orthologs of HvPDIL5-1 or other closely related members of the PDI gene family may be potential susceptibility factors to viruses in other eukaryotic species.
Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can ...be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.
The angiosperm embryo and endosperm are limited in space because they grow insidematernal seed tissues. The elimination of cell layers of the maternal seed coat by programmedcell death (PCD) could ...provide space and nutrition to the filial organs.
Using the barley (Hordeumvulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains.
The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains.
The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals.
Genome editing strategies in barley (
) typically rely on
-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are ...genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express
. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in
-transgenic barley. Somatic and heritable editing in the
gene (
) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.
Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host’s cell walls by ...mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops.
To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi).
The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes.
The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.
Summary
Exploring genes with impact on yield‐related phenotypes is the preceding step to accomplishing crop improvements while facing a growing world population. A genome‐wide association scan on ...leaf blade area (LA) in a worldwide spring barley collection (Hordeum vulgare L.), including 125 two‐ and 93 six‐rowed accessions, identified a gene encoding the homeobox transcription factor, Six‐rowed spike 1 (VRS1). VRS1 was previously described as a key domestication gene affecting spike development. Its mutation converts two‐rowed (wild‐type VRS1, only central fertile spikelets) into six‐rowed spikes (mutant vrs1, fully developed fertile central and lateral spikelets). Phenotypic analyses of mutant and wild‐type leaves revealed that mutants had an increased leaf width with more longitudinal veins. The observed significant increase of LA and leaf nitrogen (%) during pre‐anthesis development in vrs1 mutants also implies a link between wider leaf and grain number, which was validated from the association of vrs1 locus with wider leaf and grain number. Histological and gene expression analyses indicated that VRS1 might influence the size of leaf primordia by affecting cell proliferation of leaf primordial cells. This finding was supported by the transcriptome analysis of mutant and wild‐type leaf primordia where in the mutant transcriptional activation of genes related to cell proliferation was detectable. Here we show that VRS1 has an independent role on barley leaf development which might influence the grain number.
Significance Statement
By applying a genome‐wide association study on leaf blade area (LA) in two‐ and six‐rowed spring barley (Hordeum vulgare L.), homeobox transcription factor six‐rowed spike 1 (VRS1) was identified. Mutant studies, histological and gene expression analyses suggest that VRS1 influences the size of leaf primordia resulting in wider leaves with more longitudinal veins.
Prime editing, developed by Anzalone et al., brings genome editing to a new level, because this approach allows introduction of all mutation types, including insertions, deletions, and all putative ...12 types of base-to-base conversions. Previously tested in human cells, this technique has been adapted for use in plants by Lin et al.
Cereal grains contribute substantially to the human diet. The maternal plant provides the carbohydrate and nitrogen sources deposited in the endosperm, but the basis for their spatial allocation ...during the grain filling process is obscure. Here, vacuolar processing enzymes have been shown to both mediate programmed cell death (PCD) in the maternal tissues of a barley grain and influence the delivery of assimilate to the endosperm. The proposed centrality of PCD has implications for cereal crop improvement.
DNA double-strand break (DSB) repair mechanisms differ in their requirements for a homologous repair template and in the accuracy of the result. We aimed to quantify the outcome of repair of a single ...targeted DSB in somatic cells of young barley (Hordeum vulgare) plants. Amplicon sequencing of three reporter constructs revealed 47 to 58% of reads as repaired via nonhomologous end-joining (NHEJ) with deletions and/or small (1 to 3 bp) insertions. Alternative NHEJ revealed 2 to 5 bp microhomology (15.7% of cases) or new replication-mediated short duplications at sealed breaks. Although deletions outweigh insertions in barley, this bias was less pronounced and deleted sequences were shorter than in Arabidopsis thaliana. Between 17 and 33% of reads likely represent restoration of the original sequence. Depending on the construct, 20 to 33% of reads arose via gene conversion (homologous recombination). Remarkably, <1 to >8% of reads apparently display synthesis-dependent strand annealing linked with NHEJ, inserting 4 to 61 bp, mostly originating from the surrounding of breakpoints. Positional coincidence of >81% of sister chromatid exchanges with target loci is unprecedented for higher eukaryotes and indicates that most repair events for staggered DSBs, at least in barley, involve the sister chromatid and occur during S or G2 phase of the cell cycle.
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