Transcriptomic and alternative splicing reprogramming by low-dose gamma irradiation in tomato under ToBRFV infection

Tomato brown rugose fruit virus (ToBRFV) is a rapidly spreading pathogen that threatens global tomato production by overcoming established genetic resistance mechanisms. Conventional seed disinfection strategies often fail to eliminate the virus, necessitating the exploration of alternative antiviral approaches. In this study, we examined the effects of low-dose gamma irradiation (15 Gy) on transcriptomic reprogramming and alternative splicing (AS) in tomato seedlings infected with ToBRFV. Through RNA sequencing (RNA-Seq), distinct AS patterns were identified between irradiated and non-irradiated plants, with significant enrichment in exon skipping and alternative splice site usage in the gamma-treated group. High-throughput RNA sequencing data were analyzed using CLC Genomics Workbench, custom Python scripts, and functional enrichment tools including the STRING database and KEGG REST API. These AS events were non-randomly distributed across the genome, with hotspots located in defense-related loci. Six genes were identified that were both differentially expressed and alternatively spliced (DE-ASGs), including kinases, lipases, and auxin response factors, suggesting a dual-layered regulatory response. Functional enrichment analysis revealed that gamma-induced AS genes were significantly involved in plant–pathogen interactions, MAPK signaling, and hormonal response pathways. Furthermore, predicted miRNA–target interactions indicated sly-miR6024 and sly-miR5303 as central regulators of alternatively spliced transcripts. These findings underscore the role of AS as a key component of gamma-induced antiviral defense and point to a synergistic regulatory network involving transcriptional modulation, alternative splicing, and miRNA-mediated control. This study provides novel insights into radiation-induced resistance and transcriptomic plasticity, offering a foundation for developing stress-resilient tomato cultivars. • 15 Gy gamma irradiation reshapes AS in ToBRFV-infected tomato seedlings. • Exon skipping & splice site shifts enriched in defense-linked genomic regions. • DE-ASGs found: kinases, lipases, ARFs imply dual transcriptional-splicing control. • Gamma-induced AS genes linked to plant–pathogen, MAPK, and hormone pathways. • sly-miR6024 and sly-miR5303 predicted as master regulators of spliced transcripts.