Pervasive splicing in a plant DNA virus

Viruses maximize their limited coding space through strategies that increase transcript and protein diversity. In mammalian viruses, splicing is a well-established mechanism for proteome expansion, yet in plant viruses it is considered extremely rare and remains poorly characterized. Here, we investigated splicing in the geminivirus tomato yellow leaf curl virus (TYLCV; Begomovirus coheni ), a member of the largest genus of the Geminiviridae family in which splicing events have not previously been described. Analysis of RNA sequencing data from TYLCV-infected plants revealed eight splicing events in viral transcripts arising from both genome strands, most of which harbour canonical splice sites and are predicted to generate novel protein isoforms. Two events, affecting the Rep and CP transcripts, were experimentally validated, confirmed to occur specifically at the RNA level, and shown to depend on the plant spliceosome. Spliced transcripts associate with high-order polysomes, indicating that they are actively translated. Silencing of the spliceosome component SF3B1 reduced viral splicing and limited TYLCV accumulation, indicating a requirement for splicing in full infectivity. Supporting this notion, plants infected with a TYLCV mutant impaired in one of the identified splicing events displayed diminished viral accumulation and attenuated symptoms, demonstrating that viral splicing contributes directly to pathogenicity. Collectively, our results reveal that splicing of viral transcripts is pervasive in TYLCV and promotes infection, suggesting that transcriptome complexity in plant viruses has been substantially underestimated. These findings highlight the need to revisit the transcriptomes and proteomes of plant viruses, particularly DNA viruses replicating in the nucleus, to account for the potential contribution of splicing.