Network-based analysis of crucial genes for salt tolerance in rice

Rice responds to salt stress by modulating a vast array of genes integrated into a sophisticated regulatory network. This complexity makes it challenging to identify the key genes and the specific alleles that confer tolerance. We used time-course expression analysis to profile gene and miRNA expression associated with salt tolerance in Pokkali, a salt-tolerant rice variety. We established a framework for studying transcription factor-target(mRNA/miRNA) interactions using gene co-expression and machine-learning models. Moreover, we developed a hypergeometric distribution-based method to elucidate the interactions of salt stress-related miRNA-targets. Using these approaches, we established co-expression (GCN) and gene regulatory networks (GRN) based on co-expression and TF/miRNA-target interactions. Hub genes with high connectivity in our networks were enriched for previously reported salt tolerance genes, a finding largely supported by subsequent haplotype analysis of 374 rice accessions. Finally, we functionally validated three crucial hub genes, OsCAF1B, OsADR3 and Ospdr9, by demonstrating their roles in salt tolerance using their knockout mutants. The crucial genes, haplotypes and networks for salt tolerance identified in this study (resource available at https://cbi.njau.edu.cn/RiceSALTnet) provide a foundation for breeding rice cultivars with enhanced salt tolerance.