Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens.
Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.
Veluthambi
K
K
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Ream
W
W
Gelvin
S B
SB
D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D015247:Deoxyribonuclease HindIII; D005798:Genes, Bacterial; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000382:microbiology*; D010957:Plasmids; D014176:Protein Biosynthesis; D011523:Protoplasts; D012231:Rhizobium / Q000235:genetics* / Q000502:physiology; D014774:Virulence
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII