Characterization of the MLO gene family in Glycine max and its role in rust infection

The MLO gene family plays a critical role in plant–pathogen interactions, regulating susceptibility and resistance to fungal diseases. In this study, we performed a genome-wide characterization of the MLO genes in soybean ( Glycine max), integrating structural analysis, phylogenetic classification, expression profiling, functional validation by virus-induced gene silencing (VIGS), and SNP mining. We identified 40 MLO genes, including a novel member ( GmMLO25) with an unusually small MLO domain (93 aa). Protein length varied from 130 to 600 aa, with differences in the number of transmembrane domains and conserved motifs, including calmodulin-binding domains (CaMBDs). Analysis of SNPs in coding sequences revealed missense substitutions affecting conserved motifs and transmembrane regions, particularly in clade V genes. RNA-seq and RT-qPCR analyses revealed differential regulation of clade V members between resistant ( Rpp5) and susceptible (BRS 184) genotypes during Phakopsora pachyrhizi infection. Functional validation by VIGS demonstrated that silencing multiple clade V genes did not reduce the number of uredinia but drastically reduced fungal sporulation. This study identified GmMLO38 as a functional susceptibility gene in soybean and as a prime target for gene editing or RNAi-based strategies to enhance resistance to Asian soybean rust (ASR).