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 (BaYMV) and (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 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 and allelic variants of the 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 and -mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the 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 . 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. knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.