Activity of T-DNA borders in plant cell transformation by mini-T plasmids.
By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.
D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010935:Plant Diseases; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics
D004269:DNA, Bacterial; C036483:T-DNA; D004247:DNA; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII