Identification and validation of hub genes associated with biotic and abiotic stresses by modular gene co-expression analysis in Oryza sativa L

Rice, a staple food consumed by half of the world's population, is severely affected by the combined impact of abiotic and biotic stresses, with the former causing increased susceptibility of the plant to pathogens. Four microarray datasets for drought, salinity, tungro virus, and blast pathogen were retrieved from the Gene Expression Omnibus database. A modular gene co-expression (mGCE) analysis was conducted, followed by gene set enrichment analysis to evaluate the upregulation of module activity across different stress conditions. Over-representation analysis was conducted to determine the functional association of each gene module with stress-related processes and pathways. The protein-protein interaction network of mGCE hub genes was constructed, and the Maximal Clique Centrality (MCC) algorithm was applied to enhance precision in identifying key genes. Finally, genes implicated in both abiotic and biotic stress responses were validated using RT-qPCR. A total of 11, 12, 46, and 14 modules containing 85, 106, 253, and 143 hub genes were detected in drought, salinity, tungro virus, and blast. Modular genes in drought were primarily enriched in response to heat stimulus and water deprivation, while salinity-related genes were enriched in response to external stimuli. For the tungro virus and blast pathogen, enrichment was mainly observed in the defence and stress responses. Interestingly, RPS5, PKG, HSP90, HSP70, and MCM were consistently present in abiotic and biotic stresses. The DEG analysis revealed the upregulation of MCM under the tungro virus and downregulation under blast and drought in resistant rice, indicating its role in viral resistance. HSP70 showed no changes, while HSP90 was upregulated in susceptible rice during blast and drought. PKG increased during drought but decreased in japonica rice under salinity. RPS5 was highly upregulated during blast in both resistant and susceptible rice. The RT-qPCR analysis showed that all five hub genes were upregulated in all treatments, indicating their role in stress responses and potential for crop improvement.