Temporal proteomic profiling via 4D-DIA reveals early defense mechanisms and core resistance determinants in soybean against Phakopsora pachyrhizi

Asian soybean rust, caused by Phakopsora pachyrhizi, is a devastating fungal disease threatening global soybean production, particularly in tropical regions where chemical control is increasingly unsustainable. This study employed cutting-edge 4D-DIA proteomics to investigate molecular defense mechanisms in resistant (SX6907) and susceptible (Tianlong 1) soybean cultivars during early infection (12 hpi and 3 dpi). We identified 12,852 proteins, with 1,510 differentially expressed proteins (DEPs) revealing genotype-specific responses. Resistant plants exhibited sustained upregulation of immune receptors (CRKs, LRR-RLKs), MAPK signaling components, and cell wall reinforcement proteins (peroxidases, XTHs), alongside dynamic modulation of calcium signaling and ROS homeostasis. These patterns suggest key pathways enriched in resistance may include phenylpropanoid biosynthesis, isoflavonoid production, and ER stress responses, while susceptible plants showed suppression of photosynthesis and defense pathways. Weighted Protein Co-expression Network Analysis(WPCNA) highlighted co-expression modules linked to resistance, potentially including NLR-mediated effector-triggered immunity. Crucially, DIR proteins and organelle-specific defense hubs (e.g., chloroplasts, nuclei) were implicated in rust resistance. Validation by qPCR confirmed concordance for 84% of tested DEPs. Our findings provide a protein-level blueprint of soybean rust resistance, identifying candidate targets for marker-assisted breeding and genetic engineering to develop durable resistant varieties, reducing reliance on fungicides.