The replication-associated protein (RepA) of Maize streak virus interacts in yeast with retinoblastoma-related protein (RBR), the negative regulator of cell-cycle progression. This may allow geminiviruses to subvert cell-cycle control to provide an environment that is suitable for viral DNA replication. To determine the importance of this interaction for MSV infection, the RBR-binding motif, LxCxE, was mutated to IxCxE or LxCxK. Whilst RBR binding in yeast could not be detected for the LxCxK mutant, the IxCxE protein retained limited binding activity. Both mutants were able to replicate in maize cultures and to infect maize plants. However, whereas the wild-type virus invaded mesophyll cells of mature leaves, the LxCxK mutant was restricted to the vasculature, which is invaded prior to leaf maturity. Mature leaves contain high levels of RBR and it is suggested that the MSV RepA-RBR interaction is essential only in tissues with high levels of active RBR.