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 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. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltratin. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These trangenics will be grafted onto rootstock once they reach the appropriate size. 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 is in the process of screening putative grapefruit and sweet orange transformants. We hope to have additional transgenics later this year. We have also grafted our lone transgenic plant onto two rootstocks (812 and Sour Orange) and planted these in late March in the field at Fort Pierce in collaboration Dr. Ed Stover. 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 our tow most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March. We have identified additional transgenics from plants received from Dr. Vladimir Orbovicthat that will be graftered onto rootstocks in the near future.