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

Hoffie, Robert Eric; Otto, Ingrid; Perović, Dragan; Budhagatapalli, Nagaveni; Habekuß, Antje; Ordon, Frank; Kumlehn, Jochen

doi:10.3389/fgeed.2021.784233

Cited by 27 publications

(15 citation statements)

References 24 publications

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“…The proportion of mutants among primary transgenic plants was 0%-57%, which is on par with previously published results in targeted mutagenesis with cas9 in barley (see Koeppel et al (2019), Table 1). The comparatively high proportion of homozygous mutants in M 1 using the cultivar 'Igri' indicated that cas9 and gRNA could induce mutations prior to spontaneous genome duplication, which is a frequent occurrence during (haploid) microspore-derived plant regeneration (see also Hoffie et al (2021)). However, for the same reason, T-DNA-free individuals occurred rather rarely among Cas endonucleaseinduced mutants with an 'Igri' background.…”

Section: Discussionmentioning

confidence: 99%

“…Since only EIF4E ‐mediated resistance is present in current winter barley cultivars, there is an urgent need for new resistance mechanisms in winter barley breeding material. The full knockout of HvEIF4E renders barley resistant to bymovirus infection, as described for other plants, but it is associated with a substantial yield penalty (Hoffie et al ., 2021 ). In the landrace HOR1363 (formerly PI1963) and the Russian cultivar ‘Russia 57’, rym11 , a locus associated with resistance to all known strains of BaYMV and BaMMV, was described (Nissan‐Azzouz et al ., 2005 ; Sedláček and Mařík, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

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Novel resistance to the BymovirusBaMMVestablished by targeted mutagenesis of thePDIL5‐1susceptibility gene in barley

Hoffie

Perović

Habekuß

et al. 2022

Plant Biotechnology Journal

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The Potyviridae are the largest family of plant-pathogenic viruses. Members of this family are the soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5-1 (PDIL5-1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5-1-based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time-consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR-associated endonuclease technology to induce site-directed mutations similar to those described for PDIL5-1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene-free homozygous M 2 mutants were produced in spring barley. A variety of mutants carrying novel PDIL5-1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in-frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus-resistant mutants showed no adverse effects in terms of growth and yield.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Novel resistance to the BymovirusBaMMVestablished by targeted mutagenesis of thePDIL5‐1susceptibility gene in barley

Hoffie

Perović

Habekuß

et al. 2022

Plant Biotechnology Journal

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“…Considering all the evidence, it seems that the gene HORVU.MOREX.r3.6HG0573610 is the most likely candidate for BaMMV resistance encoded by rym15 . Functional analysis of the two candidate genes, for example by gene editing ( Hoffie et al, 2021 ) will likely lead to cloning of the causal gene for rym15 .…”

Section: Discussionmentioning

confidence: 99%

High-Resolution Mapping of Barley mild mosaic virus Resistance Gene rym15

et al. 2022

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Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), which are transmitted by the soil-borne plasmodiophorid Polymyxa graminis, cause high yield losses in barley. In previous studies, the recessive BaMMV resistance gene rym15, derived from the Japanese landrace Chikurin Ibaraki 1, was mapped on chromosome 6HS of Hordeum vulgare. In this study, 423 F4 segmental recombinant inbred lines (RILs) were developed from crosses of Chikurin Ibaraki 1 with two BaMMV-susceptible cultivars, Igri (139 RILs) and Uschi (284 RILs). A set of 32 competitive allele-specific PCR (KASP) assays, designed using single nucleotide polymorphisms (SNPs) from the barley 50 K Illumina Infinium iSelect SNP chip, genotyping by sequencing (GBS) and whole-genome sequencing (WGS), was used as a backbone for construction of two high-resolution maps. Using this approach, the target locus was narrowed down to 0.161 cM and 0.036 cM in the Igri × Chikurin Ibaraki 1 (I × C) and Chikurin Ibaraki 1 × Uschi (C × U) populations, respectively. Corresponding physical intervals of 11.3 Mbp and 0.281 Mbp were calculated for I × C and C × U, respectively, according to the Morex v3 genome sequence. In the 0.281 Mbp target region, six high confidence (HC) and two low confidence (LC) genes were identified. Genome assemblies of BaMMV-susceptible cultivars Igri and Golden Promise from the barley pan-genome, and a HiFi assembly of Chikurin Ibaraki 1 together with re-sequencing data for the six HC and two LC genes in susceptible parental cultivar Uschi revealed functional SNPs between resistant and susceptible genotypes only in two of the HC genes. These SNPs are the most promising candidates for the development of functional markers and the two genes represent promising candidates for functional analysis.

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“…By contrast, editing of other S genes can impair plant growth and lead to developmental defects. For example, knockout variants of the barley host factor gene eIF4E ( Eukaryotic Translation Initiation Factor 4E ) show decreased total grain yield per plant ( Hoffie et al., 2021 ). The loss of function of eIF4E or its isoforms eIF(iso)4E leads to dwarfism or the loss of fertility in Arabidopsis, tobacco, tomato, and melon ( Combe et al., 2005 ; Mazier et al., 2011 ; Callot and Gallois, 2014 ; Patrick et al., 2014 ; Gauffier et al., 2016 ; Pechar et al., 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Mutation of barley HvPDIL5-1 improves resistance to yellow mosaic virus disease without growth or yield penalties

Cheng

Kan

et al. 2022

Front. Plant Sci.

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The soil-borne yellow mosaic virus disease, which is caused by the bymoviruses barley yellow mosaic virus (BaYMV) and/or barley mild mosaic virus (BaMMV), seriously threatens winter barley production in Europe and East Asia. Both viruses are transmitted by the soil-borne plasmodiophorid Polymyxa graminis and are difficult to eliminate through chemical or physical measures in the field, making breeding for resistant cultivars the optimal strategy for disease control. The resistance locus rym1/11 was cloned encoding the host factor gene Protein Disulfide Isomerase Like 5-1 (PDIL5-1), whose loss-of-function variants confer broad-spectrum resistance to multiple strains of BaMMV/BaYMV. Most resistance-conferring variants have been identified in six-rowed barley landraces/historic cultivars, and their introgression into modern two-rowed malting cultivars is difficult because PDIL5-1 is located in a peri-centromeric region with suppressed recombination. In this study, we used CRISPR/Cas9 genome editing to modify PDIL5-1 in the BaYMV/BaMMV-susceptible elite malting barley cv. ‘Golden Promise’ and obtained the mutants pdil5-1-a and pdil5-1-b. PDIL5-1 in the pdil5-1-a mutant encodes a protein lacking a cysteine residue, and pdil5-1-b contains a protein-coding frameshift. Both mutants were completely resistant to BaYMV. The knockout mutant pdil5-1-b showed complete BaMMV resistance, while pdil5-1-a showed decreased viral accumulation but no disease symptoms if compared to ‘Golden Promise’. Both PDIL5-1 edited lines, as well as the previously produced EMS-induced pdil5-1 mutant ‘10253-1-5’ in the elite malting barley cv. ‘Barke’ background, displayed no growth or yield penalties in garden experiments or bymovirus-free field trials. Line ‘10253-1-5’ showed improved resistance and yield performance compared to the wild-type and its sibling line when grown in infectious fields. Therefore, genome editing of the host factor gene PDIL5-1 could facilitate the breeding of barley varieties with resistance to bymoviruses.

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

Cited by 27 publications

References 24 publications

Novel resistance to the BymovirusBaMMVestablished by targeted mutagenesis of thePDIL5‐1susceptibility gene in barley

Novel resistance to the BymovirusBaMMVestablished by targeted mutagenesis of thePDIL5‐1susceptibility gene in barley

High-Resolution Mapping of Barley mild mosaic virus Resistance Gene rym15

Mutation of barley HvPDIL5-1 improves resistance to yellow mosaic virus disease without growth or yield penalties

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