Proteomic signatures uncover phenotypic plasticity of susceptible and resistant genotypes by wall remodelers in rice blast

Synergistic action of shared and conserved extracellular matrix (ECM) signaling network determines the fate of pathogen attack and triggers innate immunity in plants. Rice blast, caused by Magnaporthe oryzae adversely affects rice productivity. As a major communication interface, ECM senses external perturbation and provides selective biochemical barrier to segregate cellular organization and signaling pathways from external environment Extensive research has shed light on how plant immune components are activated, regulated and function in response to environmental perturbation. However, two key questions remain largely unresolved: (i) how does ECM dynamics govern susceptibility and disease resistance, (ii) what are the components that underpin these phenomena? Rice cultivars were inoculated with Magnaporthe oryzae, scanning electron microscopy and biochemical analysis including, malondialdehyde production, electrolyte leakage, was performed. ECM proteins was isolated by mechanical disruption, CaCl2 extraction, followed by purity assessment using transmission electron microscopy and enzyme assays. To develop disease and immune responsive ECM proteome, TMT-10Plex labeling, trypsin digestion and reverse phase fractionation was performed. LC-MS/MS of 12 fractions was carried out using Orbitrap Fusion Tribrid interfaced with Easy-nLC II nanoflow. Data were analyzed in MaxQuant v.1.6.0.1 using concatenated Oryza sativa and Magnaporthe oryzae protein database. ECM cellulose, pectin and lignin was determined. Real-time PCR of putative markers was performed. Protein-protein interaction network was developed to identify regulatory disease/immune regulatory hubs. To understand ECM regulated genotype-phenotype plasticity in blast disease, we temporally profiled two contrasting rice genotypes in disease- and immune-state. Morpho-histological, biochemical and electron microscopy analyses revealed that increased necrotic lesions accompanied by electrolyte leakage governs disease-state. Wall carbohydrate quantification showed that changes in pectin level was more significant in blast-susceptible (BS) compared to blast-resistant (BR) cultivar. Temporally-resolved quantitative disease- and immune-responsive ECM proteomes identified 308 and 334 proteins, respectively involved in wall remodeling and integrity, signaling and disease/immune response. Pairwise comparisons between time and treatment, mRNA expression, diseasome- and immunome-networks revealed novel blast-related functional modules. We observed accumulation of wall deformation- and homeostasis-related components in BS cultivar, whereas deregulation of pattern-triggered immunity, wall remodeler and calcium signaling components were observed in BR cultivar. Data demonstrated accumulation of α-galactosidase and phosphatase were associated with disease-state, while ROS, induction of Lysin motif proteins, CAZymes and extracellular Ca-receptor protein govern immune-state.