Abstract
Fusarium oxysporum-induced root rot severely threatens global soybean production, yet limited understanding of resistance mechanisms constrains breeding progress. This study conducted comparative transcriptomic analysis between highly resistant (Xiaoheiqi) and susceptible (L83-4752) soybean accessions following pathogen inoculation across four time points (8-17 days post-infection). RNA-seq analysis identified 1496 differentially expressed genes following pathogen challenge. KEGG pathway enrichment analysis revealed significant enrichment in MAPK signaling pathway (12 genes) and plant-pathogen interaction pathway (13 genes). Eight genes co-occurred in both pathways, with GmCML (Glyma.10G178400) exhibiting the most dramatic differential expression among these candidates. This gene encodes a 151-amino acid calmodulin-like protein showing 185-fold higher expression in resistant plants at 17 days post-inoculation, confirmed by qRT-PCR validation. Functional validation through transgenic hairy root overexpression demonstrated that GmCML significantly enhanced disease resistance by coordinately activating antioxidant defense systems. Overexpression of GmCML in transgenic soybean enhanced resistance to F. oxysporum by modulating the activity of antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT) and the accumulation of osmoregulatory substances (proline and soluble sugars). Population genetic analysis of 295 diverse soybean accessions revealed three GmCML haplotypes based on promoter region polymorphisms. Two favorable variants (Hap2 and Hap3) conferred significantly lower disease indices and exhibited evidence of positive selection during domestication, indicating evolutionary importance in disease resistance. This research provides the first comprehensive characterization of GmCML's role in soybean-Fusarium interactions, establishing this calmodulin-like protein as a regulatory hub linking calcium signaling to coordinated defense responses. The identified natural variants and functional mechanisms offer validated targets for both marker-assisted breeding and genetic engineering approaches to enhance soybean disease resistance.