Plant genetic transformation is a fascinating process by which the Agrobacterium transforms its host by delivering a well-defined fraction of its own genome (designated transfer DNA or T-DNA) as a single-stranded (ss) DNA molecule (designated T-strand) into the host cell. The T-DNA integrates into the host genome and is expressed there. Many of the biological mechanisms that govern the transformation process remain poorly understood and our understanding of the mechanisms by which T-DNA molecules integrate into the host-cell genome is still sketchy. The T-DNA molecule does not code for the machinery needed for its transport and integration into the plant genome. Thus, the T-DNA sequence can potentially be replaced by any other sequence of interest without affecting the transformation and integration process, which is likely to be governed largely by plant proteins and machineries. T-DNA molecules were shown to be capturable by genomic double-strand breaks (DSBs), which led to the suggestion that DSBs may act as ‘hot spots’ for T-DNA integration. Data from my laboratory suggest that a double-stranded (ds) intermediate (dsT-DNA) may serve as a dominant substrate in the integration process. The mechanism by which T-strands are complemented to dsT-DNAs is still unknown and to the best of our knowledge, have never been studied and we have only recently begun to reveal the molecular mechanisms by which dsT-DNA molecules integrate into genomic DSBs. My hypothesis is that dsT-DNA intermediate molecules and genomic DSBs play a significant role in T-DNA integration and in my interdisciplinary proposal I plan to combine the use of functional assays, biochemical studies, genetic approaches and imaging methods to (i) unveil the molecular mechanism of T-strand complementation to dsT-DNA and (ii) study the role played by DSBs in capturing dsT-DNA molecules.
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