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).