ACTIVITY OF THE AGROBACTERIUM T-DNA TRANSFER MACHINERY IS AFFECTED BY VIRB GENE-PRODUCTS

The oriT (origin of transfer) sequence and mob (mobilization) genes of plasmid RSF1010 can functionally replace transfer DNA (T-DNA) borders to generate an RSF1010 intermediate transferable to plants through activities of the tumor-inducing (Ti)-plasmid virulence (vir) genes of Agrobacterium tumefaciens. Because the Ti plasmid virB gene products are hypothesized to form a membrane-localized T-DNA transport apparatus, we investigated whether specific virB genes were needed for RSF1010 transfer. Here we report that transformation of Nicotiana tabacum leaf explants by an RSF1010-derivative plasmid (pJW323) requires the essential virulence genes virB9, virB10, and virB11, consistent with the hypothesis that both the T-DNA and RSF1010 transfer intermediates utilize the same transport machinery. Further, while pJW323 is transferred into plant cells by Agrobacterium strains harboring both pJW323 and pTiA6, the initiation of crown gall tumors (i.e., T-DNA transfer) is greatly suppressed. Coordinate overexpression of the virB9, virB10, and virB11 gene products relieves pJW323-mediated oncogenic suppression and restores tumorigenicity, but does not increase the transfer frequency of pJW323 into plant cells. We propose that the virB9, virB10, and virB11 gene products function coordinately and stoichiometrically to enhance DNA transfer in a fashion specific for the T-DNA intermediate.