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THE PLANT CELL, Vol 8, Issue 5 873-886, Copyright © 1996 by American Society of Plant Biologists
Early Transcription of Agrobacterium T-DNA Genes in Tobacco and Maize
S. B. Narasimhulu, Xb. Deng, R. Sarria and S. B. Gelvin
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
We developed a sensitive procedure to investigate the kinetics of
transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded
[beta]-glucuronidase gusA (uidA) gene soon after infection of plant
suspension culture cells. The procedure uses a reverse
transcriptase-polymerase chain reaction and enables detection of gusA
transcripts within 18 to 24 hr after cocultivation of the bacteria with
either tobacco or maize cells. Detection of gusA transcripts depended
absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4
within the bacterium. Mutations in virC and virE resulted in delayed and
highly attenuated expression of the gusA gene. A nonpolar transposon
insertion into the C-terminal coding region of virD2 resulted in only
slightly decreased production of gusA mRNA, although this insertion
resulted in the loss of the nuclear localization sequence and the important
[omega] region from VirD2 protein and rendered the bacterium avirulent.
However, expression of gusA transcripts in tobacco infected by this virD2
mutant was more transient than in cells infected by a wild-type strain.
Infection of tobacco cells with an Agrobacterium strain harboring a mutant
virD2 allele from which the [omega] region had been deleted resulted in
similar transient expression of gusA mRNA. These data indicate that the
C-terminal nuclear localization signal of the VirD2 protein is not
essential for nuclear uptake of T-DNA and further suggest that the [omega]
domain of VirD2 may be required for efficient integration of T-DNA into the
plant genome. The finding that the initial kinetics of gusA gene expression
in maize cells are similar to those shown in infected tobacco cells but
that the presence of gusA mRNA in maize is highly transient suggests that
the block to maize transformation involves T-DNA integration and not T-DNA
entry into the cell or nuclear targeting.
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