Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover.