Alternative splicing expands the functional portfolio of a plant virus to control the viral cycle

Viruses frequently have limited coding space, yet efficiently manipulate complex hosts by deploying intricate, often poorly understood strategies. Geminiviruses, devastating plant pathogens with small single-stranded (ss) DNA genomes, replicate in the nucleus aided by the viral replication-associated protein (Rep), the most conserved protein within this family and related ssDNA viruses. Rep initiates viral DNA replication and represses its own promoter to regulate the infection cycle. The molecular mechanisms enabling this dual functionality are so far unknown. Here, we show that the geminivirus tomato yellow leaf curl virus (TYLCV) exploits the host spliceosome to produce novel Rep splice variants. Splicing generates Rep isoforms lacking the central oligomerization domain that cannot initiate replication but strongly repress the Rep promoter. Conversely, Rep mutants deficient in splicing promote replication but fail to repress transcription. Reduced Rep splicing interferes with the viral gene expression hierarchy and decreases infectivity. Our findings therefore reveal a previously unrecognized viral strategy in which alternative splicing produces functionally specialized viral protein isoforms, providing a mechanistic explanation for the dual role of Rep in viral replication and gene regulation. Splicing events with potentially similar functional consequences in related viruses suggest that this strategy may have convergently evolved across diverse viral lineages infecting different domains of life.