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. 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 or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover thetransgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in December and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. Statistical analysis revealed that there was a significant difference in disease both at the second and third ratings. Because of Covid-19 I was unable to rate the plants until late on July 28, 2020 but have not had the time to analyze the data. We have two additional transgenics from plants received from Dr. Vladimir Orbovic. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics are reaching a stage where they can be assayed more thoroughly for resistance. 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 is with Vladimir Orbovic, who has additional transformants. We have screened these via inoculations and so far the ones we have received are susceptibe. More will be available in the future. 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 and were recently rated in late July. I am in the process of analyzing the data. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that have been grafted onto rootstocks. The are in the process of testing this quarter 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. We have grafted the buds and several have developed into branches. We are currently growing these. Unfortunately the control grafts have not developed any branches as of now. Once they are of an appropriate size we will send DNA to Berkeley. Of course this will only occur once we are allowed to conduct research. We have grown the two CRISPR created bs5 Carrizo trees by pinprick inoculation and spray inoculation. For both inoculation methods we saw no clear-cut difference in susceptibility compare to Carrizo rootstock leaves. We have also compared bacterial growth of one of the CRISPR events with Carrizo rootstock leaves and saw no differences in populations. We plan to test the other CRISPR event when the leaves grow outl.