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

Abstract

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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“…Pathogens often exploit susceptible plant genes to facilitate their infection ( Van Schie and Takken, 2014 ). KOs have been used on a variety of susceptible genes to increase disease tolerance in apple ( Pompili et al., 2020 ), barley ( Hoffie et al., 2022 ), canola with the susceptibility gene only in the A genome ( Pröbsting et al., 2020 ), cassava ( Gomez et al., 2019 ), corn ( Liu et al., 2022 ), cucumber ( Chandrasekaran et al., 2016 ), rice ( Zhou et al., 2018 ), tomato ( Nekrasov et al., 2017 ), and watermelon ( Zhang et al., 2020a ) ( Table 1 ). Increased resistance to wheat stem rust Ug99 was conferred into wheat ( T. aestivum ) by introducing resistance genes from either einkorn wheat ( T. monococcum ) ( Chen et al., 2018 ) or durham wheat T. turgidum ( Zhang et al., 2017b ), both of which are in the wheat gene pool.…”