EARLY TRANSCRIPTION OF AGROBACTERIUM T-DNA GENES IN TOBACCO AND MAIZE

We developed a sensitive procedure to investigate the kinetics of tran scription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant susp ension culture cells. The procedure uses a reverse transcriptase-polym erase 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 a ttenuated 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. Howev er, expression of gusA transcripts in tobacco infected by this virD2 m utant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a m utant virD2 allele from which the omega region had been deleted result ed in similar transient expression of gusA mRNA. These data indicate t hat the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that th e omega domain of VirD2 may be required for efficient integration of T -DNA into the plant genome. The finding that the initial kinetics of g usA gene expression in maize cells are similar to those shown in infec ted tobacco cells but that the presence of gusA mRNA in maize is highl y transient suggests that the block to maize transformation involves T -DNA integration and not T-DNA entry into the cell or nuclear targetin g.