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, in the tumor-inducing plasmid pTiA6NC. Thus, three border repeats lie right and one lies left of TL (the left T-DNA) which carried 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 195 bp on the right restored virulence completely. Smaller fragments and border repeat oligonucleotides 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.