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. We have tested the transgenic Duncan grapefruit containing the Bs3-executor transgene for resistance to an array of strains representing a worldwide collection.� All of the strains were shown to elicit a hypersensitive reaction when infiltrated with a bacterial suspension of� Xanthomona citri�into young leaves of the transgenic citrus plant but a susceptible reaction when infiltrated into non-transgenic susceptible Duncan grapefruit leaves. We are continuing further experiments to confirm the resistance in the greenhouse.� Furthermore, we will quantify bacterial populations in transgenic and susceptible trees to demonstrate if the transgene is activated without the TAL effector that is predicted to activate this gene.� We are also confirming activation of the transgene using a realtime protocol to determine if the transgene is activated properly. During the past three months we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contained 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.� The construct was recently sent to Dr. Vladimir Orbovic, who is in the process of transforming grapefruit and sweet orange. 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 are in the process of planting in the field at Fort Pierce in collaboration with Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic that contain the Bs3-executor transgene.� One of the putatve transgenics has a growth defect and in all likelihood will be of no use in future experiments. The other putative transgenic tree which appears to contain the gene may be useful for future testing.��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 are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have additional transgenics from plants received from Dr. Vladimir Orbovic that need to be tested for the presence of EFR.� �Objective 3. To determine if�bs5-generated transgenic Carrizo plants are resistant to�X. citri�and to generate transgenic grapefruit carrying�bs5. Currently the trees are being grown and then will be grafted onto rootstocks for further testing.