# Rescue of tomato yellow leaf curl virus mutants harboring heterologous iterons through in planta evolution

## TL;DR

This study shows that tomato yellow leaf curl virus can evolve in plants to overcome replication repression caused by foreign iteron sequences.

## Contribution

The study reveals that iterons act as repressors of replication and are overcome through in planta evolution, not by restoring original sequences.

## Key findings

- Tomato yellow leaf curl virus mutants with foreign iterons replicated locally but had systemic movement defects.
- De novo mutations rescued systemic movement without restoring original iteron sequences.
- Iterons likely compete with host transcription factors and cognate Rep for binding, influencing replication.

## Abstract

The single-stranded, circular DNA genomes of geminiviruses contain iterated motifs of five to six nucleotides, known as iterons, upstream of the replication protein (Rep) coding region. Iterons were previously found to interact with cognate Rep in a sequence-specific manner, and the iteron-Rep interaction was needed for viral DNA replication. Nonetheless, iterons of closely related viruses often have different sequences, suggesting diversifying selection. To identify selection pressures driving iteron diversification, we constructed tomato yellow leaf curl virus (isolate SH2) mutants in which the iteron motifs were replaced with those of closely related tobacco curly shoot virus (isolate Y35). All mutants replicated in inoculated leaves of Nicotiana benthamiana, but some failed to spread systemically. However, the systemic movement defects were mostly rescued by de novo mutations. Intriguingly, these de novo mutations did not restore the iterons to SH2 sequences. Rather, they likely enabled viral escape from repression exerted by the heterogeneous Y35 iterons in the absence of a matching Rep. These results suggest that iterons probably act as sites of competitive binding by host-encoded transcription factors (TFs) and the cognate Rep. We further speculate that iteron-TF binding commences as soon as viral genomes enter cell nuclei, committing genome copies to Rep mRNA transcription and protein translation, but also blocking them from replication. Conversely, iteron-Rep binding would be possible only after Rep is produced and likely repels TFs from some genome copies, permitting replication initiation. Testing this model through future research should clarify the intricate evolutionary interplays between geminiviruses and their crop hosts and inform novel management strategies.

Geminiviruses are important crop pathogens worldwide for which effective control measures are lacking due to an incomplete understanding of their evolutionary dynamics in infected plants. The current study focuses on a class of short sequence repeats in geminiviral genomic DNA, known as iterons, located immediately upstream of the viral gene encoding replication protein (Rep). Iterons are interesting because, although their positions and repeat patterns are conserved across all geminiviruses, their sequence identities are highly diverse. Our investigations revealed that, contrary to previous reports, the sequence identity of iterons is non-essential for tomato yellow leaf curl virus replication. Rather, they are repressors of replication, and this repression is overcome by their binding with cognate Rep. Future investigations will likely unveil novel targets for more effective management of crop diseases caused by geminiviruses.