T-DNA border sequences required for crown gall tumorigenesis.

Abstract

Similar 23-base-pair (bp) direct repeats occur at the ends of two adjacent but noncontiguous T-DNAs, TL and TR (left and right T-DNA), in the tumor-inducing plasmid pTiA6NC. Thus, three border repeats lie right and one lies left of TL, which carries the genes needed for tumor maintenance. To determine whether T-DNA transfer and integration (subsequently called T-DNA transmission) require sequences in addition to the 23-bp border repeat, we constructed a deletion removing the three potential TL right borders (the TL right border and both TR borders). Since this deletion severely attenuated virulence, we reintroduced restriction fragments containing the TL right border repeat at a new location to the right of TL and tested their ability to restore virulence. Fragments that carried the border repeat flanked by at least 67 bp of wild-type Ti plasmid sequences on the left and 1035 bp on the right restored virulence completely. Smaller fragments restored virulence significantly but not fully, even though the border repeat remained intact. Therefore, T-region sequences flanking the border repeat in the fully active fragments stimulated T-DNA integration. Fragments that restored virulence fully when inserted in the wild-type orientation stimulated virulence only slightly in the opposite orientation. Thus, the right border sequence promotes T-DNA transfer and integration best in one direction.