Proteome Analysis of Soybean Root Apoplast Combined with AlphaFold Prediction Reveal Macrophomina phaseolina Infection Strategies and Potential Targets for Engineering Resistance

Macrophomina phaseolina (Tassi) Goid. is a hemibiotrophic 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. Therefore, the identification of mechanistic targets is crucial for improving resistance against the pathogen. 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 dynamics of soybean root apoplast during M. phaseolina infection. We have detected several secreted proteins of M. phaseolina and differential regulation of soybean-secreted proteins in root apoplast during infections. Glycome analysis and callose deposition assays have revealed changes in soybean root cell wall compositions and potential polysaccharide targets of M. phaseolina. AlphaFold 2 (AF2) analysis was instrumental in revealing several interesting sequence-unrelated structurally similar (SUSS) effectors and effectors with novel structural folds secreted by M. phaseolina. Structured-guided engineering of protease-inhibitor complexes is emerging as an important strategy to engineer resistance in plants against pathogens. AlphaFold Multimer (AFM) analysis of candidate-secreted proteins from soybean and M. phaseolina has predicted cysteine and serine protease-inhibitor complexes with high confidence. We have validated these interactions using molecular dynamics (MD) and competitive activity-based protein profiling (ABPP) approaches. Therefore, our work provides insights into Soybean-M. phaseolina interactions in the root apoplast and unveil potential candidates for engineering resistance.