This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants plus the addition of a new strategy using gene editing. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. We are in the process of characterizing if different TAL effector variants activate the transgene in order to determine if variants of TAL effectors are effectively targeted. In order to do this TAL variants we are in the process of inoculating into leaves of the transgenic plant, JJ5, and 5′-RACE will be used to identify transcription starts. We will be able to determine if several unique TAL effectors are able to activate transcription. As for developing a different transgenic with ProBs314EBE:avrGf2, we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. However, there were issues with the construct. Therefore a new construct was made. This construct was recently sent to Vladimir Orbovic, who is in the process of creating additional transformants. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. We will rate these in the next few weeks. We will be conducting futher greenhouse assays on ROS activity to determine if other EFR transgenics may have more potential for resistance to citrus canker than the two currently in the field. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process, but that was negative for the gene. The latter was to serve as the negative control as it had undergone the transformation process. We have grafted the buds and several have developed into branches. We are currently growing these. In the past few months neither of the branches that were created by crispr were actively growing. Recently both of the branches for the transgenic trees have begun growing and we plan on conducting further population experiments in order to confirm previous results on the the level of resistance.