Non-technical paragraph: The introduction of DNA into plants (plant transformation to generate genetically modified or transgenic plants) has become a core technology for basic plant research and the agricultural biotechnology industry. Agrobacterium-mediated plant genetic transformation is the most commonly used method to generate transgenic plants. The bacterium Agrobacterium can transfer large DNA-molecules (T-DNA) capable of encoding numerous genes sufficient to, e.g., encode a novel biosynthetic pathway. Thus, Agrobacterium-mediated transformation is a key tool for synthetic biology. Although the Agrobacterium host range is broad, many agronomic important species or specific cultivars remain recalcitrant to transformation. In addition, T-DNA integrates randomly into the plant genome. Consequently, T-DNA may disrupt genes important for plant development and productivity. Random T-DNA integration often occurs in genomic regions that silence encoded transgenes, leading to unpredictable and unstable transgene expression. It is therefore important to develop novel technologies to increase transformation efficiency of a broader range of crop species and agronomic important varieties, and to insert T-DNA into defined locations of any plant genome. This project will develop novel technologies to broaden the plant host range of Agrobacterium, and to direct the integration of T-DNA to specific plant chromosomal regions pre-selected by the scientist. In addition, this project will develop novel technologies to deliver genes to plants efficiently without subsequent integration into the plant genome. This latter technology is important for delivering plant genome engineering reagents without maintaining these reagents after they have accomplished their tasks.
Technical paragraph: This project proposes tool development for the plant research community to help enable functional genomics for a broad spectrum of species. Because Agrobacterium-mediated transformation is the preferred DNA transfer approach, one goal of this project is to increase the efficiency of transformation by debilitating plant defense responses to Agrobacterium. This will be accomplished by engineering Agrobacterium strains that can secrete Type III effectors and suppress plant defense responses to enhance transformation. Because controlled T-DNA integration and predictable transgene expression is important for synthetic biology, a second goal is to build a system to facilitate effective and precise DNA integration into plant genomes. This system will be developed using CRISPR/Cas9 to generate breaks in specific tomato genomic sequences that will "trap" T-DNA. These integration sites will be chosen to maximize the probability of stable transgene expression through numerous plant generations and under field conditions. The expression of targeted and randomly integrated transgenes will be assessed. Agrobacterium is also used to deliver genome engineering reagents to plants. However, integration of these reagents is undesirable, and they usually are segregated out of the engineered plants. This is difficult for vegetative-propagated species. The third goal of this project is to develop Agrobacterium strains that can efficiently deliver but not integrate T-DNA encoding genome editing reagents, or will secrete these reagents through a Type III secretion system. This will be accomplished by altering Agrobacterium VirD2, the protein that leads T-DNA from the bacterium into the plant and which is important for T-DNA integration.
