A transient assay for gene-expression was used to study the early events of T-DNA transfer. Particularly, it was asked if gene expression following T-DNA transfer required DNA replication in the host cell. A β-glucuronidase gene, linked to a CaMV 35S promoter (35S-GUS, engineered so that it was inactive in Agrobacterium tumefaciens) was introduced into Nicotiana plumbaginifolia protoplasts via a disarmed supervirulent strain of Agrobacterium tumefaciens. High β-glucuronidase activity appeared after 3 days of co-cultivation. The activity required the presence of the vir functions of agrobacteria. The activity was drastically reduced if the plant cells were treated with aphidicolin, an inhibitor of DNA replication in eukaryotic cells. While double-stranded (ds) 35S-GUS DNA, introduced by electroporation, showed undiminished expression in the presence of aphidicolin, gene expression from single-stranded (ss) 35S-GUS DNA was inhibited by aphidicolin. These results suggest that DNA replication in host cells is not required for gene expression if ds-DNA is introduced by electroporation, but is required if ss-DNA is introduced by electroporation, or if DNA is transferred via A. tumefaciens. The findings are consistent with a model of T-DNA transfer in which ss-DNA molecules, once introduced into plant cells, must pass through an aphidicolin sensitive step before they can be transcribed. The simplest interpretation is that the ss-DNA is replicated by the host cell's aphidicolin-sensitive DNA polymerase before being integrated into the host genome.
Construction and screening of subtracted cDNA libraries from limited populations of plant cells: a comparative analysis of gene expression between maize egg cells and central cells
