Transient gene expression in plant cells mediated by Agrobacterium tumefaciens: Application for the analysis of virulence loci

A simple system is described for detection of the transfer of T-DNA from Agrobacterium cells to suspension-cultured tobacco BY-2 cells. A modified reporter gene for β-glucuronidase (GUS) that contained an intron sequence was introduced into the T-DNA region such that the GUS protein could be synthesized in plant cells only after transfer of the T-DNA to plant nuclei. When BY-2 cells were co-cultured with Agrobacterium cells that contained the modified reporter gene, transient synthesis of GUS protein was observed between 36 and 48 h after the onset of co-culture. The level of GUS activity reached a plateau within as little as 48 h. This temporal profile of GUS activation suggests that the transient activity might have been due to expression of the GUS gene in the T-DNA that had been transferred to the plant nuclei but had not yet been integrated into the plant chromosomes. Levels of transient GUS activity were also examined with various vir mutants of Agrobacterium and in a mutant with an altered chromosomal acvB gene, the gene for a protein that has been postulated to function outside bacterial cells. During co-culture with virB, virD2, virD4 and acvB mutants, GUS activity remained at background levels, and the GUS activity in the case of the virE2 mutant was thirty-fold lower than with the wild type. On the basis of these results, we discuss the roles of these genes during infection by Agrobacterium of plant cells.