Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Abstract

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation (Ds), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep+) and non-replicating (rep-) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep+) or mutant, inactive derivatives of these genes (rep-). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds-vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 x 105-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.