Preventing crown gall disease using oncogene silencing or VirE1-expressing transgenics.

摘要

Crown gall disease is caused by the ubiquitous soil bacterium Agrobacterium tumefaciens which transfers a portion if DNA (T-DNA) into the plant cell. Preventing infection by using the biocontrol strain Agrobacterium radiobacter K84 is currently the only defense for crown gall. Two different resistance strategies were examined in this work. The first was the use of VirE1-expressing transgenic Arabidopsis thaliana . VirE1 is the secretory chaperone of VirE2. VirE2 is exported into plant cells during infection and is essential for tumorigenesis. Once in plant cells VirE2, a single-stranded DNA binding protein, protects the single-stranded T-strand and also helps localize it to the nucleus. Since the interaction of VirE1 and VirE2 is favored over a VirE2 and T-strand interaction we hypothesized that plant expressed VirE1 would prevent VirE2 from binding the T-strand. One plant line exhibited 3.5-fold fewer tumors than wild-type plants. Some amino acid residues of VirE1 needed for interaction with VirE2 have also been identified in this work. This research provides further information about the VirE1-VirE2 interaction that may be used in future constructs for VirE1-mediated crown gall resistance. The other resistance strategy examined in this work is gene silencing of the A. tumefaciens oncogenes iaaM and ipt. These oncogenes are part of the T-DNA and once in the cells express auxin and cytokinin which cause gall growth. By generating constructs that made double-stranded RNA and creating transgenic apple tissue that expressed these constructs, sequence specific degradation of the target mRNA was initiated. Six apple lines were completely resistant to crown gall disease using gene silencing. These methods may be further improved and even combined to eventually create crown gall resistant transgenics for commercial production.