This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. 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. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. 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. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. As stated in a previous report, the transgenic Duncan grapefruit containing the Bs3-executor transgene, designated JJ5, shows a high level of resistance to an array of strains representing a worldwide collection. Furthermore, using real time PCR, we have validated that the gene is activated by one or more TAL effectors and that there is minimal activation without these genes. Buds from the original transgenic tree were grafted onto two rootstocks (812 and Sour Orange) and planted in late March in the field at Fort Pierce in collaboration Dr. Ed Stover. Citrus canker has developed on plants in the field and the trees were rated for disease in September and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both, and an additional one which has since been identified, responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltration. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. All three transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics were grafted onto rootstock and are in various stages (i.e, some of buds have broken and the shoots are developing while others are still dormant). During the past three months 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 that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was sent to Vladimir Orbovic, who has developed 45 putative grapefruit and sweet orange transformants. We have screened these via PCR and there was one weak positive. The transgenic was grown and inoculated with Xcc and found to be susceptible. 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. They were planted in the field in late March. There was some citrus canker on the trees, although they were not uniformly infected. In September the trees were rated for disease severity and the transgenics carrying EFR had considerably more disease than the susceptible wild-type Duncan grapefruit. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that that have been grafted onto rootstocks. These will be tested for ROS activity and for EFR gene expression. 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, serves as budwood that had undergone the transformation process but that was negative for the transgene. This will serve as a negative control.