Evidence and opportunities for developing non-transgenic genome edited crops using site-directed nuclease 1 approach.

The innovations and progress in genome editing/new breeding technologies have revolutionized research in the field of functional genomics and crop improvement. This revolution has expanded the horizons of agricultural research, presenting fresh possibilities for creating novel plant varieties equipped with desired traits that can effectively combat the challenges posed by climate change. However, the regulation and social acceptance of genome-edited crops still remain as major barriers. Only a few countries considered the site-directed nuclease 1 (SDN1) approach-based genome-edited plants under less or no regulation. Hence, the present review aims to comprise information on the research work conducted using SDN1 in crops by various genome editing tools. It also elucidates the promising candidate genes that can be used for editing and has listed the studies on non-transgenic crops developed through SDN1 either by Agrobacterium-mediated transformation or by ribo nucleoprotein (RNP) complex. The review also hoards the existing regulatory landscape of genome editing and provides an overview of globally commercialized genome-edited crops. These compilations will enable confidence in researchers and policymakers, across the globe, to recognize the full potential of this technology and reconsider the regulatory aspects associated with genome-edited crops. Furthermore, this compilation serves as a valuable resource for researchers embarking on the development of customized non-transgenic crops through the utilization of SDN1.

Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Targeted Knockout of Eukaryotic Translation Initiation Factor 4E Confers Bymovirus Resistance in Winter Barley.

The Eukaryotic Translation Initiation Factor 4E (EIF4E) is a well-known susceptibility factor for potyvirus infections in many plant species. The barley yellow mosaic virus disease, caused by the bymoviruses Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), can lead to yield losses of up to 50% in winter barley. In autumn, the roots of young barley plants are infected by the soil-borne plasmodiophoraceous parasite Polymyxa graminis L. that serves as viral vector. Upon viral establishment and systemic spreading into the upper parts of the plants, yellow mosaics occur as first symptoms on leaves. In the further course of plant development, the disease entails leaf necrosis and increased susceptibility to frost damage. Thanks to the rym4 and rym5 allelic variants of the HvEIF4E gene, more than two thirds of current European winter barley cultivars are resistant to BaYMV and BaMMV. However, several strains of BaYMV and BaMMV have already overcome rym4- and rym5-mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the EIF4E gene by Cas9 endonuclease in BaMMV/BaYMV-susceptible winter barley cv. "Igri". Small insertions were generated, resulting in a shift of the translational reading frame, thereby causing the loss-of-function of EIF4E. The mutations occurred in the homozygous state already in the primary mutants. Their progeny proved invariably homozygous and fully resistant to mechanical inoculation with BaMMV. EIF4E knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.

Engineering Smut Resistance in Maize by Site-Directed Mutagenesis of LIPOXYGENASE 3.

Biotic stresses caused by microbial pathogens impair crop yield and quality if not restricted by expensive and often ecologically problematic pesticides. For a sustainable agriculture of tomorrow, breeding or engineering of pathogen-resistant crop varieties is therefore a major cornerstone. Maize is one of the four most important cereal crops in the world. The biotrophic fungal pathogen Ustilago maydis causes galls on all aerial parts of the maize plant. Biotrophic pathogens like U. maydis co-evolved with their host plant and depend during their life cycle on successful manipulation of the host's cellular machinery. Therefore, removing or altering plant susceptibility genes is an effective and usually durable way to obtain resistance in plants. Transcriptional time course experiments in U. maydis-infected maize revealed numerous maize genes being upregulated upon establishment of biotrophy. Among these genes is the maize LIPOXYGENASE 3 (LOX3) previously shown to be a susceptibility factor for other fungal genera as well. Aiming to engineer durable resistance in maize against U. maydis and possibly other pathogens, we took a Cas endonuclease technology approach to generate loss of function mutations in LOX3. lox3 maize mutant plants react with an enhanced PAMP-triggered ROS burst implicating an enhanced defense response. Based on visual assessment of disease symptoms and quantification of relative fungal biomass, homozygous lox3 mutant plants exposed to U. maydis show significantly decreased susceptibility. U. maydis infection assays using a transposon mutant lox3 maize line further substantiated that LOX3 is a susceptibility factor for this important maize pathogen.

Kmasker plants - a tool for assessing complex sequence space in plant species.

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain Gene in albostrians Barley.

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley (Hordeum vulgare) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvCMF7 is a homolog of the Arabidopsis (Arabidopsis thaliana) CONSTANS, CO-like, and TOC1 (CCT) Motif transcription factor gene CHLOROPLAST IMPORT APPARATUS2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization.

A simple test for the cleavage activity of customized endonucleases in plants.

BACKGROUND: Although customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants. RESULTS: A simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations. CONCLUSIONS: A convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN's functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.

Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.

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.