Microrchidia (MORC)
proteins comprise a family of
proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type
ATPase and an S5-fold. In plants, MORC
proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC
proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC
proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley
RNA Pol III-dependent
U3 small nuclear RNA (
snRNA) promoter to drive expression of the synthetic
single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%-100%), which constitutes an important improvement of the gene-editing technology in cereals. Corroborating and extending earlier findings, SpCas9-edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased
disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of
transposable elements (
TEs), substantiating that plant MORC
proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.