Proteome Analysis of Soybean Root Apoplast Combined With AlphaFold Prediction Reveals Macrophomina Phaseolina Infection Strategies and Potential Targets for Engineering Resistance

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes charcoal rot (CR) disease in various legumes, including soybean. To date, no reliable resistance gene sources have been identified in soybean or other legumes to combat M. phaseolina . The apoplast is a critical region where intense molecular cross‐talk occurs between plants and pathogens, and the outcome of their interactions is determined in this compartment. Here, we employed label‐free quantitative (LFQ) proteomics to investigate the changes in soybean root apoplast during M. phaseolina infection. We have detected several secreted proteins of M. phaseolina and differential accumulation of soybean‐secreted proteins during infections. Glycome analysis and callose deposition assays have revealed changes in soybean root cell wall compositions during M. phaseolina infection. AlphaFold 2 (AF2) analysis was instrumental in revealing several sequence‐unrelated structurally similar (SUSS) effectors and effectors with novel structural folds secreted by M. phaseolina . AlphaFold multimer (AFM) analysis of candidate‐secreted proteins from soybean and M. phaseolina has predicted cysteine and serine protease‐inhibitor complexes. We have validated these interactions using molecular dynamics (MD), inhibition assays and competitive activity‐based protein profiling (ABPP) approaches. Therefore, our work provides insights into Soybean‐ M. phaseolina interactions in the root apoplast and potential candidates for engineering resistance.