Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications

Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is <i>mildew resistance locus o</i> (<i>mlo</i>)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of <i>mlo</i> mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel <i>mlo</i> mutant lines and evaluated their resistance to causal fungus <i>Blumeria hordei</i>. We further examined if <i>mlo</i> knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant <i>mlo</i> lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced <i>B. hordei</i> colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating <i>mlo</i>-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited <i>mlo</i>-associated effects on root microbiome diversity and relative abundance of microbial taxa.