Transfer of the T-DNA from Agrobacterium Tumefaciens to Plant Cells

Abstract

We have devised a technique to follow the transfer of the A. tumefaciens Ti-plasmid from the bacterium to the plant cell. This assay, based on co-cultivation procedure of Marton et al. (Nature 277:129,-131, 1979), does not depend upon the expression of the T-DNA but rather assays the physical presence of the T-DNA in the infected plant cells. Numerous controls have indicated that we are, on Southern blots, detecting T-DNA sequences within the plant cell and not merely T-DNA sequences from contaminating bacteria. We have also shown that the DNA is transferred to the plant at a bacterial/plant interface and not by transformation of naked Ti-plasmid DNA. Our results indicate that multiple copies, on average, of the T-DNA can be transferred to the plant cells within hours of the initiation of co-cultivation, but most of this DNA is rapidly degraded in the plant cell. Rearrangements in the T-DNA have occasionally been seen. Regions of the Ti-plasmid far from the T-region have sometimes been detected in the plant by a sensitive dot blot analysis. Preliminary evidence suggests that deletion of the right border repeat from the Ti-plasmid (which results in an extremely attenuated plant response with respect to virulence) does not markedly decrease the transfer efficiency of the mutant Ti-plasmid to the plant.