ANALYSIS OF THE TRANSFER OF THE TI-PLASMID FROM AGROBACTERIUM TUMEFACIENS TO PETUNIA CELLS
Abstract
The causative agent of crown gall, a neoplastic transformation of dicotyledonous plants, is the tumor inducing (Ti)-plasmid harbored by virulent strains of Agrobacterium tumefaciens. A portion of the Ti-plasmid, known as the transferred (T)-DNA, is stably integrated and transcribed in the plant nuclear DNA (9, 33, 79, 82). This thesis is concerned with the development and use of a procedure designed to investigate the initial transfer of the nucleic acid from the bacterium to the plant cell. The method involves the infection of Petunia protoplasts with the virulent Agrobacterium tumefaciens strain A348, or the avirulent strain A136. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the T-DNA may be transferred to the plant cell rapidly (within 2-6 hours) after the bacteria bind to the cell wall. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Another topic covered in this thesis is the determination of the T-DNA organization found in an uncloned octopine-type tumor line derived by co-cultivation. Southern blot analysis (69), has shown that the T-DNA arrangement of this tumor is similar to the organization found in established crown gall lines (71). A third aspect of crown gall tumorigenesis discussed involves the transfer role of the repeated sequences flanking the T-DNA. Petunia protoplasts were infected with an avirulent strain of Agrobacterium, WR 3095, harboring a mutated Ti-plasmid in which only the left repeat of the T-DNA remains. Southern analysis of the DNA isolated from infected plant cells suggested that the repeats which have been deleted are not required for transfer. The final topic described in this thesis concerns the investigation of the mechanism of transfer of the T-DNA to the plant cell. These results suggested that the T-region may excise from the Ti-plasmid in the bacterium. This smaller molecule may be the transfer intermediate.
Degree
Ph.D.
Subject Area
Molecular biology
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