GENE-EXPRESSION FOLLOWING T-DNA TRANSFER INTO PLANT-CELLS IS APHIDICOLIN-SENSITIVE

A transient assay for gene-expression was used to study the early even ts of T-DNA transfer. Particularly, it was asked if gene expression fo llowing T-DNA transfer required DNA replication in the host cell. A be ta-glucuronidase gene, linked to a CaMV 35S promoter (35S-GUS, enginee red so that it was inactive in Agrobacterium tumefaciens) was introduc ed into Nicotiana plumbaginifolia protoplasts via a disarmed superviru lent strain of Agrobacterium tumefaciens. High beta-glucuronidase acti vity appeared after 3 days of co-cultivation. The activity required th e presence of the vir functions of agrobacteria. The activity was dras tically reduced if the plant cells were treated with aphidicolin, an i nhibitor 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 e xpression if ds-DNA is introduced by electroporation, but is required if ss-DNA is introduced by electroporation, or if DNA is transferred v ia A. tumefaciens. The findings are consistent with a model of T-DNA t ransfer in which ss-DNA molecules, once introduced into plant cells, m ust pass through an aphidicolin sensitive step before they can be tran scribed. The simplest interpretation is that the ss-DNA is replicated by the host cell's aphidicolin-sensitive DNA polymerase before being i ntegrated into the host genome.