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 ofT-DNA from Agrobacterium cells to suspension-cultured tobaccoBY-2 cells. A modified reporter gene for rß-glucuronidase(GUS) that contained an intron sequence was introduced intothe T-DNA region such that the GUS protein could be synthesizedin plant cells only after transfer of the T-DNA to plant nuclei.When BY-2 cells were co-cultured with Agrobacterium cells thatcontained the modified reporter gene, transient synthesis ofGUS protein was observed between 36 and 48 h after the onsetof co-culture. The level of GUS activity reached a plateau withinas little as 48 h. This temporal profile of GUS activation suggeststhat the transient activity might have been due to expressionof the GUS gene in the T-DNA that had been transferred to theplant nuclei but had not yet been integrated into the plantchromosomes. Levels of transient GUS activity were also examinedwith various vir mutants of Agrobacterium and in a mutant withan altered chromosomal acvB gene, the gene for a protein thathas been postulated to function outside bacterial cells. Duringco-culture with virB, virD2, virD4 and acvB mutants, GUS activityremained at background levels, and the GUS activity in the caseof the virE2 mutant was thirty-fold lower than with the wildtype. On the basis of these results, we discuss the roles ofthese genes during infection by Agrobacterium of plant cells. ⁴Present address: Biochemistry Laboratory, Kanebo Ltd., 5-3-28Kotobuki-cho, Odawara, Kanagawa, 250 Japan