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-talks occur 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 significant changes in soybean root cell wall compositions and potential polysaccharide targets of M. phaseolina . AlphaFold 2 (AF2) analysis has revealed several interesting sequence-unrelated structurally similar (SUSS) effectors and effectors with novel structural folds secreted by M. phaseolina . Furthermore, AlphaFold Multimer (AFM) analysis has revealed protease-inhibitor interactions displayed by candidate-secreted proteins from soybean and M. phaseolina . 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.