Long-read transcriptome profiling reveals UTR-dependent regulation of TCP4 and post-transcriptional responses during TYLCV infection in Nicotiana benthamiana

Long-read sequencing enables accurate reconstruction of full-length transcripts and isoforms, but its application to plant-virus interactions remains limited. Tomato yellow leaf curl virus (TYLCV) is a major DNA virus infecting tobacco and tomato. We used Oxford Nanopore Technologies (ONT) sequencing to characterize transcriptomic responses to TYLCV infection in tobacco. We generated 73.2 million ONT reads from mock- and TYLCV-infected Nicotiana benthamiana plants and identified 87,037 full-length transcripts, including 11,001 previously unannotated transcripts. TYLCV infection induced transcriptional reprogramming, affecting 2,115 transcripts enriched in metabolic processes and plant-pathogen interaction pathways. Long-read sequencing further identified a misannotated leucine-rich repeat gene (LRR), which was strongly induced by TYLCV infection but was dispensable for viral accumulation. Isoform-specific analysis revealed that TCP4 exhibits differential expression of two transcript isoforms with identical coding sequences but distinct 5’ and 3’ untranslated regions (UTRs). Functional assays demonstrated that these UTR differences regulated transcript stability, and silencing TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor TCP4 significantly increased TYLCV accumulation, indicating a positive role in antiviral defense. In parallel, alternative splicing analysis revealed only two events with substantial changes (ΔPSI > 0.3), indicating limited effects of TYLCV on global splicing patterns. Fusion transcript analysis identified condition-specific fusion profiles, with proline-rich protein 4 (PRP4) contributing the largest number of fusion events, and PRP4 silencing increased TYLCV accumulation, suggesting a role in antiviral defense. In addition, we identified 5,611 lncRNAs, among which a TYLCV-induced lncRNA, novel2540.t1 , was associated with increased accumulation of the receptor-like kinase NAK in transient expression assays, suggesting a potential role in regulating NAK transcript abundance. This study provides a high-resolution full-length transcriptome resource for TYLCV-infected tobacco. Our results reveal extensive transcriptional reprogramming but limited changes in transcript structure, and identify TCP4 UTR-dependent regulation as a key antiviral mechanism, together with additional layers of post-transcriptional regulation involving PRP4-associated fusion transcripts and lncRNA-linked modulation of immune-related genes.