1. Please state project objectives and what work was done this quarter to address them: Knocking out disease susceptibility genes (S genes) has resulted in broad disease resistance in multiple crops. The bottleneck to using CRISPR for engineering HLB resistance in citrus has been the lack of suitable and validated gene targets. Identifying suitable gene targets has been the most urgent task for achieving HLB resistance through gene editing. We are the first group in the world that have knocked out two S genes, DMR6 and SWEET1, in HLB-susceptible `Duncan’ grapefruit and produced dmr6 and sweet1 mutants. The objectives of this project are to evaluate the resistance of four `Duncan’ mutants to HLB after graft inoculation (Objective 1a) and exposure to infected Asian citrus psyllids (ACP) (Objective 1b,) and to assess potential side effects of these mutations on citrus plant growth and morphology (Objective 2). The overall goal of this project is to determine the effectiveness of editing DMR6 and SWEET1 for engineering HLB resistance in citrus. Experiment 1 is being conducted to achieve Objective 1a. Four `Duncan’ mutants and one control wildtype line were grafted onto HLB-free sour orange rootstock in two batches. The first batch of clonal plants were graft-inoculated with CLas-positive buds or CLas-free buds (mock inoculation) on June 17, 2023, resulting in 29 graft-inoculated plants and 10 mock-inoculated plants. These plants have been grown in a temperature-controlled greenhouse. Data on plant height, trunk diameter below and above graft union, HLB symptoms, and CLas titer were collected on June 6 (before inoculation), September 21, (3 months post inoculation), and December 21, 2023 (6 months post inoculation). The Ct value of these mutants and wildtype was approximately 40, indicating undetectable CLas in these inoculated plants. This result was unexpected, and it suggests that the graft inoculation did not perform as expected, even though the CLas inoculum source plants continued to test positive, with a Ct values of 27 to 28. Consequently, these plants were re-inoculated in December 2023; tender shoots were fed upon by 10 female and 10 male hot Asian citrus psyllids (ACP) per plant for 10 days, then the ACPs were collected and analyzed for CLas titer. On average, 63% of these ACPs tested CLas-positive. These re-inoculated plants will be tested for CLas titer in March 2024 (3 months post re-inoculation). The second batch of clonal plants (total 34, with 4 to 8 per mutant) were propagated onto sour orange on July 24, 2023. These plants have grown to a height of 16 to 49 inches; when new shoots become available in spring 2024, we will use hot ACP to inoculate them. Experiment 2 and Experiment 3 are being conducted for Objective 1b and 2, respectively. These experiments require 20 clonal plants of similar stem diameter for each mutant or wildtype line. To produce the required clonal plants, 50 to 75 cuttings were taken from each mutant stock plant and stuck into potting mix in early July 2023. The cuttings were rooted under an intermittent misting system in a secure greenhouse for 3 months. For each mutant, six to 37 cuttings have rooted and produced new shoots. To produce additional clonal plants for Objective 1b and 2, another batch of cuttings were taken on November 3, 2023. These cuttings were rooted in Oasis Rootcubes. Now, enough numbers of rooted cuttings have been produced for all four mutants and wildtype `Duncan’. These cuttings have been potted and are being promoted to grow and produce tender shoots for ACP-based inoculation and plant morphological comparison. 2. Please state what work is anticipated for next quarter: Objective 1a (Experiment 1) -1st batch of inoculated plants: This batch of plants was re-inoculated in December 2023 by exposing them to infected ACP. All plants are growing well in a secure air-conditioned greenhouse. Data on plant height, trunk diameter, HLB symptom will be collected in March 2024. Leaves from each plant will be sampled for DNA isolation and qPCR analysis to quantify CLas titer 3 months post re-inoculation. Objective 1a (Experiment 1) – 2nd batch of clonal plants: This batch of plants is growing well and will be inoculated with CLas by 10 days’ feeding of hot ACP. Our initial plan was to inoculate this group of plants in January 2024, but the infected ACP colonies were ruined by invading ants. Our collaborating entomology group is raising new hot ACPs, which are expected to be ready for us in March 2024. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 1b (Experiment 2): Rooted cuttings are being pushed to grow rapidly for ACP inoculation in April 2024, as described above. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 2 (Experiment 3): Rooted cuttings will be potted up in containers in 4 weeks and grown in a secure greenhouse at CREC. Plant growth and leaf and shoot morphology of mutants will be monitored closely and compared with the wildtype to determine potential side effects from the edited S genes. 3. Please state budget status (underspend or overspend, and why): The total spendings by far add to $15,301.04, about 15.4% of the total budget. This is below the expected spending for the second quarter of the project. The primary reason was the difficulty we experienced in propagating the mutants and the wildtype to produce clonal plants for Objective 1b and 2 during the hot summer and fall months. Without enough numbers of clonal plants, the planned CLas inoculation, DNA extraction, qRT-PCR, and horticultural experiments had to be postponed to the third quarter of the project.