Involvement of Mildew Locus O in arbuscular mycorrhizal symbiosis and calcium signalling in Lotus japonicus roots

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are also involved in plant reproduction, root thigmotropism, and arbuscular mycorrhizal (AM) symbiosis. Although MLOs have recently been characterized as Ca2+-permeable channels activated by Rapid Alkalinization Factors (RALF) peptides in plant reproductive cells, their involvement in root Ca2+ signalling during plant-microbe interactions remains unexplored. In this study we investigated the role of MLO4 in the model legume Lotus japonicus, focusing on AM symbiosis. mlo4 insertional mutants exhibited a complex mycorrhization phenotype, showing unaltered AM fungal colonization, but increased phosphate accumulation upon mycorrhization. Ca2+ measurement assays did not highlight significant differences in the cytosolic Ca2+ changes triggered by AM symbiotic signals in mlo4 roots compared to wild-type, but suggested an involvement of LjMLO4 in Ca2+-mediated defence responses. Analyses of LjMLO4 Ca2+-permeable activity via heterologous expression in Escherichia coli strongly supported its function as a Ca2+ channel. Moreover, Ljmlo4 mutants displayed altered Ca2+ responses to RALFs and insensitivity to RALF-induced root growth inhibition, confirming MLO4 participation in RALF signalling. These findings highlight the multifaceted role of MLO4 in L. japonicus, linking it to plant-microbe interactions, Ca2+ signalling, and root development.