Early Transcription of Agrobacterium T-DNA Genes in Tobacco and Maize

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded B-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, virDllvirD2, 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 Cterminal 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 o region from VirDP 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. lnfection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the o 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 w 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 maire 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.