Subfamily-level Comparative Transcriptomics of Key Immune Regulators in Plants and Suspension Cells Reveals Novel Rice Blast-resistance Genes

Plants activate pattern-triggered immunity (PTI) through key immune components, including pattern recognition receptors (PRRs), receptor-like cytoplasmic kinases (RLCKs), and transcription factors (TFs), to combat pathogens. However, a comprehensive transcriptional overview of these immune regulators at the subfamily level during biotic stress in rice is currently lacking. The aims of this study were to characterize the expression profiles of OsPRRs, OsRLCKs, and OsTFs and establish a robust pipeline for selecting novel candidate genes involved in plant immunity. We identified differentially expressed genes (DEGs) within these families using transcriptomic data from both rice plants infected with Magnaporthe oryzae infection and rice suspension cell treated with chitin treatment. Our analysis revealed the transcriptional regulation of well-known immune-related subfamilies of OsPRRs, OsRLCKs, and OsTFs, such as RLK-LRR-XII and RLCK-VII, and identified several novel subfamilies with high proportions of DEGs that may contribute to pathogen perception and plant defense. We demonstrated that selecting candidates from overlapping DEGs between plant and suspension cell systems is an effective strategy for screening genes involved in rice immunity. Using this pipeline, novel immune regulators were identified, and their functions were confirmed. Two RLCKs, i.e., OsRLCK298 and OsBSR1, act as positive regulators of immunity against rice blast fungus, whereas two transcription factors, i.e., OsERF65 and OsERF96.2, act as negative regulators. This study provides a valuable transcriptomic resource and establishes a validated pipeline for gene discovery that could be applied to other stress responses and in other plant species. Overall design: RNA-seq profiling of wild rice plants and their knockout mutants when sprayed with Magnaporthe oryzae and harvested at 48 hpi