A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans , induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid c lones mapping to the right of the known oncogenic T-region of pTiChry5 conf erred Amadori opine production on tumors induced by the nopaline strain C58 . Sequence analysis of DNA held in common among these cosmids identified tw o 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5, These proba ble border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (T-R), that confer s production of the Amadori opines. The oncogenic T-left region (T-L) was l ocated precisely by identifying and sequencing the likely border repeats de fining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir he lper plasmid derived from pTiChry5, still contains all of TR and the leftmo st 9 kb of T-L. Consistent with this hypothesis, transgenic Arabidopsis tha liana plants selected for with a marker encoded by a binary plasmid followi ng transformation with KYRT1 coinherited production of the Amadori opines a t high frequency. All opine-positive transgenic plants also contained T-R-D NA, while those plants that lacked T-R-DNA failed to produce the opines. Mo reover, A. thaliana infected with KYRT1 in which an nptII gene driven by th e 35S prometer of Cauliflower mosaic virus was inserted directly into tbe v ir helper plasmid yielded kanamycin-resistant transformants at a low but de tectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was con structed from pTiChry5 by two rounds of sacB-mediated selection for deletio n events. This plasmid, called pKPSF2, lacks both of the known T-regions an d their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mo bilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.