Replication modes of Maize streak virus mutants lacking RepA or the RepA–pRBR interaction motif

• Replication of MSV RepA mutants was investigated by qPCR and 2D gel electrophoresis.
• RepA–pRBR binding is not essential for RCR or RDR replication modes.
• However, RepA–pRBR binding does enhance efficiency of both replication modes.
• Absence of RepA strongly influences viral titres and replicative intermediates.
• MSV RepA is necessary for efficient RCR and ssDNA production.

The plant-infecting mastreviruses (family Geminiviridae) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize streak virus (MSV) mutants that either fail to express RepA (RepA−), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA− viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA–pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication.