Objective 1. Characterize Rep splicing events during infection using next-generation sequencing and molecular approaches.
Using a combination of next-generation sequencing (short- and long-read RNA-sequencing) we could confirm eight splicing events in TYLCV transcripts, four of them affecting Rep transcripts. We further confirmed these Rep splicing events by RT-PCR and Sanger sequencing of cDNA from infected samples. In addition, following this approach, we were also able to identify a fifth splicing event affecting Rep transcript. Curiously, we observed that four of the splicing events were similar and were targeting the central region of the transcript; at the protein level, these four splicing events would lead to the removal of the central oligomerization domain of Rep, without affecting the C-terminal helicase domain. Importantly, through a collaboration with Dr. Catharina Merchante from the IHSM-CSIC-UMA (Malaga, Spain), we were able to confirm the translation of Rep spliced variants by polysome profiling.
In parallel, we generated two mutants of TYLCV in which the splicing of the oligomerization domain of Rep was impaired by the mutation of the splice sites, without affecting Rep underlying amino acid sequence. We confirm that a functional Rep full-length protein was produced in the mutants and then tested their ability to replicate and accumulate in local and systemic infections. While replication in local infections (leaf patch assays) was not significantly impacted in the mutant compared to TYLCV WT virus, systemic accumulation in both tomato (natural host) and Nicotiana benthamiana (model host) was significantly impaired in both mutants, suggesting that the splicing of Rep is required for the completion of the viral cycle and viral fitness (Pott et al., 2025, 2026).
Objective 2. Determine the functional roles of Rep spliced variants, including their impact on viral replication, transcriptional regulation, protein localization, and host factors interactions.
We first tested the potential role of Rep spliced variants on viral replication, as this is the main known function of Rep proteins. None of the spliced variants were able to initate replication in complementation assays or in a virus-free reporter system, suggesting that they are not involved in this process, at least not by themselves. As in natural infections, both Rep full length and Rep spliced variants co-exist, we co-expressed Rep spliced variants together with TYLCV infectious clones in leaf patch assays. Surprisingly, we found that Rep spliced variants have a dominant negative role on viral replication. This prompted us to test whether Rep spliced variants would be involved in the negative regulation of Rep expression, another known function of Rep, required for the completion of the viral cycle. Importantly, we found out that the four spliced variants lacking the oligomerization domain indeed are stronger repressors of Rep promoter activity (compared to Rep full length) and that the mutants impaired in Rep splicing fail to repress Rep expression. Furthermore, we confirmed the ability of Rep spliced variants to bind to Rep promoter region in TYLCV intergenic region, as does Rep full length (Pott et al., 2026).
In parallel, we performed TurboID-proximity labelling to decipher the proxiome of two Rep spliced variants; we selected one of the spliced variants lacking the oligomerization domain (Rep306 for its length in amino acid) and the fifth one lacking part of the N-terminal DNA-binding domain (while maintaining inctact the central and C-terminal domains; Rep328). Interestingly, we found that Rep306 interacts with several members of transcriptional repressors belonging to the TOPLESS/LEUNIG family, which could explain the mechanism by which this spliced variant repress Rep promoter activity (unpublished results). Rep328 shows a nucleo-cytoplasmatic subcellular localization compared to Rep full length which accumulates in the nuclei of the infected cells; interestingly, we found many cytosolic host factors in its proxiome; their potential role during the viral infection will require further testing.
Objective 3. Assess the broader relevance of Rep splicing across CRESS viruses, establishing whether this mechanism is conserved and functionally significant beyond geminiviruses.
As Rep is conserved among different geminiviruses and other related ssDNA viruses, we wondered whether the Rep transcripts from other viruses will also undergo alternative splicing. To test this hypothesis, we followed several strategies: 1) splicing prediction using bioinformatic tools, 2) confirmation of splicing by RNA-seq and/or RT-PCR and Sanger sequencing, 3) data mining. Together, our results suggest that splicing may be prevalent among geminiviruses and other related viral families. Many of these splicing events target the central region of Rep transcript, with several cases leading to a similar domain organization than the spliced variants of Rep lacking the oligomerization domain but with intact C-terminal.
