Binci, F.; Guarneri, G.; Somoza, S. C.; Vascon, F.; Capparotto, A.; Di Nuzzo, E.; Rago, G.; Cendron, L.; Navazio, L.; Giovannetti, M.

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent studies have revealed that MLO proteins can act as Ca2+-permeable channels activated by Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca2+-mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus, focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in arbuscular mycorrhizal (AM) fungus-colonized root cells, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting independently of AM symbiosis but in a Ca2+-dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca2+ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca2+ signalling. Together, these findings identify LjMLO4 as a key molecular hub between peptide signalling, Ca2+ transport and root system architecture, highlighting MLO integration of developmental, nutrition and symbiotic cues.