SGER: Homology-Based Gene Targeting in Arabidopsis

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Dissection of biochemical functions and developmental pathways by mutating genes is central to functional genomics. Genes can be mutated by nucleotide sequence changes, insertions or deletions. Insertional mutagenesis with T-DNA and transposons has become the most widely used method for mutating plant genes. By contrast, homology-dependent gene targeting, a powerful technique that permits precise changes to be made in either coding or regulatory sequences, has not yet become a useful technique in plants. A number of factors have been identified that may contribute to the lack of gene targeting success or which can be optimized to enhance the frequency and increase the ease of detecting homology-based recombinational events in plants. This information has been used to design a new gene targeting strategy for plants. This project utilizes a high-risk strategy, however if successful, it will lead to the rapid development of a practicable system for homologous gene targeting in plants, which will have a very high pay-off in plant research. The strategy for plant gene targeting seeks to 1) enhance the probability of identifying homology-based interactions by detecting both gene conversion and recombination, 2) minimize illegitimate recombination associated with both naked DNA and T-DNA transformation, 3) make use of the observation that double-stranded DNA breaks stimulate recombination in plants and 4) enrich for plants in which the recombination donor molecule has been released from the chromosome. Because evidence has already been obtained indicating that all of the sub-components of the present strategy work in plants, putting the whole system together and testing it is the next step. The results of these experiments will provide the preliminary data for subsequent research to study molecular mechanisms in plant recombination and to identify favorable genetic backgrounds for gene targeting.