Am. Chaudhury et al., GENE-EXPRESSION FOLLOWING T-DNA TRANSFER INTO PLANT-CELLS IS APHIDICOLIN-SENSITIVE, Australian journal of plant physiology, 21(2), 1994, pp. 125-131
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.