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.