Agrobacterium, a fascinating microorganism that can genetically modify its plant hosts, represents an important research subject for plant biology and a major biotechnological tool for agriculture. Despite four decades of research, critical aspects of the Agrobacterium-mediated transformation process remain obscure. One of the most intriguing, yet least understood, aspects is how the bacterium utilizes its protein effectors, which it transfers with the Transferred (T-)-DNA, into the host cell, to "colonize" the host genome; prepare it for T-DNA invasion at additional integration sites, recruit bacterial and host DNA repair machinery, and repress host defenses to assure optimal expression of the T-DNA. This project aims to provide these missing pieces of information by investigating functional interactions between Agrobacterium effector proteins and their host cell partners. The broader importance of the resulting data lies in the enhancement of our basic understanding of plant-bacterium interactions and Agrobacterium pathogenicity, translation of the developed models to a broader range of bacterium-host interactions, and in providing a scientific foundation for the design of safer and more efficient genetic transformation procedures that benefit science, agriculture and the society. This research will serve as a platform for teaching undergraduate and community college science and introducing young scientists into the fields of experimental plant microbiology and biology.<br/><br/>Agrobacterium has been considered a unique prokaryote that encodes a protein machinery for DNA transfer and integration into eukaryotic genomes. This dogma is challenged by recent discoveries that other diverse bacterial species, also encode functional DNA transfer machineries. Enhancing fundamental knowledge about genetic transformation with Agrobacterium, therefore, will advance understanding of plant-pathogen interactions and improve Agrobacterium as a gene transfer tool for agriculture, but also will inform translation to a broad range of eukaryotic host-prokaryotic pathogen interactions. This project's goals are to understand how Agrobacterium creates double-stranded DNA breaks (DSBs), which are used as preferred integration sites, how the T-DNA molecule and the appropriate cellular DNA repair factors are recruited to DSBs, and how Agrobacterium overcomes host defenses that can silence T-DNA expression. Aim 1 examines the mechanism by which Agrobacterium enriches DSBs in the plant genomic DNA. A hypothesis will be tested that a bacterial virulence (Vir) effector protein (e.g., VirE3) interacts with a host S1-type endonuclease to initiate generation of DSBs. Aim 2 examines the mechanism by which Agrobacterium targets the T-DNA to the DSB and recruits the host DNA polymerase to integrate the T-DNA and repair the DSB. A hypothesis will be tested that a bacterial Vir effector associated with the T-DNA (e.g., VirE2) interacts with a host DSB-bound factor (e.g., a BRCA-like protein) to direct the T-DNA to DSB, and another T-DNA-associated Vir effector (e.g., VirD2) recruits a host DNA polymerase (e.g., Pol theta) to the same DSB. Aim 3 will examine how Agrobacterium suppresses the host defensive RNA silencing to facilitate expression of T-DNA. A hypothesis will be tested that Agrobacterium utilizes one of its F-box protein effectors to target the components of the host RNA silencing machinery to proteasomal degradation via the SCF pathway.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.