The barley ERF protein HvRAF enhances multi-stress resilience in rice via modulation of stress-responsive gene expression programs

Transcription factor (TF)-based engineering offers an efficient approach to improve multi-stress resilience in crops. The barley HvRAF gene encodes an ethylene-responsive factor (ERF)-type TF previously implicated in both abiotic and biotic stress responses. To test its cross-species functionality, HvRAF was overexpressed in rice ( Oryza sativa ) under the constitutive GOS2 promoter. Transgenic lines (GOS2:: HvRAF ) exhibited significantly higher photosystem II efficiency ( F v / F m ) and survival rates than non-transgenic controls under drought and high salinity, indicating enhanced maintenance of photosynthetic performance and cellular homeostasis. They also showed reduced lesion lengths after infection with Xanthomonas oryzae pv. oryzae , demonstrating enhanced basal resistance to bacterial blight. Transcriptome profiling identified 250 up- and 132 down-regulated genes, with up-regulated sets associated with stress-related functions including ROS scavenging, heat-shock response, and accumulation of ABA- and SA-responsive proteins. Promoter analyses revealed multiple cis -elements (GCC-box, DRE/CRT, ABRE, and W-box), suggesting coordinated regulation through ERF, bZIP, and WRKY factors. Quantitative RT-PCR validated constitutive and hormone-enhanced expression of SalT , HSP20 , ISC5 , and PR genes in transgenic plants. Together, these findings indicate that HvRAF protein modulates stress-responsive gene expression programs, thereby conferring broad-spectrum tolerance to abiotic and biotic challenges. The results highlight HvRAF as a promising regulatory gene for TF-based molecular breeding of multi-stress-resilient rice cultivars.