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 (Fv/Fm) 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.