CRISPR/Cas9-mediated knockout of MsMLO1 confers enhanced powdery mildew resistance in alfalfa

Powdery mildew, a disease primarily caused by Erysiphe pisi, is one of the most devastating foliar diseases of alfalfa (Medicago sativa L.), a globally important forage crop. The MLO gene is a well-known susceptibility factor for powdery mildew in many plant species, but its role in alfalfa remains unclear. In this study, we used the CRISPR/Cas9 system to edit MsMLO1 in alfalfa cultivar 'Aohan'. We designed an sgRNA based on a conserved target site in the exon region of this gene, and introduced the genome-editing vector into alfalfa via Agrobacterium tumefaciens-mediated transformation. Among the regenerated plants, we obtained a homozygous mutant (Msmlo1-3) with all four haplotypes edited, as well as two heterozygous mutants (Msmlo1-1 and Msmlo1-2). In disease-resistance assays, the homozygous mutant exhibited significantly enhanced resistance to E. pisi, with markedly reduced hyphal growth and conidial density compared to wild-type (WT) plants, whereas heterozygous mutants displayed no obvious resistance. Importantly, the Msmlo1-3 mutant showed no detectable growth penalties or altered agricultural traits grown in a greenhouse for six months. Transcriptome analysis revealed that the MsMLO1 mutation affects a broad gene regulatory network, including genes encoding transcription factors (MYB, AP2/ERF, WRKY) and genes involved in phyto hormone signaling, cell wall remodeling, secondary metabolism, and reactive oxygen species pathways. Therefore CRISPR/Cas9-mediated knockout of MsMLO1 conferred robust and durable powdery mildew resistance without compromising normal plant growth, highlighting the potential of MsMLO1 as a target for breeding disease-resistant in alfalfa.