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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\u202faa). Protein length varied from 130 to 600\u202faa, 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\u202fV genes. RNA-seq and RT-qPCR analyses revealed differential regulation of clade\u202fV members between resistant ( Rpp5) and susceptible (BRS 184) genotypes during Phakopsora pachyrhizi infection. Functional validation by VIGS demonstrated that silencing multiple clade\u202fV 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).<\/jats:p>","DOI":"10.1139\/gen-2025-0125","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T20:02:42Z","timestamp":1785960162000},"page":"1-14","update-policy":"https:\/\/doi.org\/10.1139\/csp-crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterization of the\n                    <i>MLO<\/i>\n                    gene family in\n                    <i>Glycine max<\/i>\n                    and its role in rust infection"],"prefix":"10.1139","volume":"69","author":[{"given":"Matheus Mertz","family":"Ribeiro","sequence":"first","affiliation":[{"name":"Universidade de S\u00e3o Paulo","place":["Ribeir\u00e3o Preto, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liliane Santana","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Federal University of Sergipe (UFS)","place":["Itabaiana, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruna","family":"Barbosa Avelino","sequence":"additional","affiliation":[{"name":"Brazilian Agricultural Research Corporation, National Soybean Research Center (EMBRAPA Soja)","place":["Londrina, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Val\u00e9ria Yukari","family":"Abe","sequence":"additional","affiliation":[{"name":"Brazilian Agricultural Research Coorporation, Embrapa Agrossilvipastoril","place":["Sinop, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fernanda Machado","family":"Castanho","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Genetica e Biologia Molecular","place":["Londrina, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivani de Oliveira Negr\u00e3o","family":"Lopes","sequence":"additional","affiliation":[{"name":"Brazilian Agricultural Research Corporation, National Soybean Research Center (EMBRAPA Soja)","place":["Londrina, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adriana Brombini dos","family":"Santos","sequence":"additional","affiliation":[{"name":"Brazilian Agricultural Research Corporation, National Soybean Research Center (EMBRAPA Soja)","place":["Londrina, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle C. 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