Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, with CLas+ ACP feeding, as well as studying them in established greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection continues from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo rootstock grafted with non-transgenic rough lemon. Results from 6 and 18 months in the field show transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. A large second planting of Mthionin transgenics went into the ground in April 2019, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants). The next significant data collection for both plantings will be in April 2020; at 2 years for trees from the initial planting and 1 year for the second planting. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are being made a third MThionin planting.The Mthionin construct has also been extensively transformed into Valencia, Ray Ruby and US-942 to provide transgenic material of these critical varieties. The first 36 putative lines are now in soil and are undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′ using adjusted protocols to improve sensitivity and transmission rates (See section 4). . Further detached leaf assays are being run to compare the relative effectiveness between each generation of chimera constructs and to expand the number of lines tested from each. DLA testing has allowed us to identify lines from several constructs with significant effects on CLas transmission and even increased ACP mortalitly. Recent results include up to 95% mortality in ACP after 7 days feeding on detached leaves of the 3rd generation TBL transgenics and 70% for TPK. Lines from promising constructs have been moved forward into greenhouse studies based on DLA results, as noted below.Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All greenhouse experiments are now using an improved protocol to enhance inoculation. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6 month samples are now collected and being analyzed. We are also emphasizing parallel field trials for all phenotyping efforts. Preparations are now being made for a field planting of ~400 `74′ and `188′ transgenics is scheduled for spring 2020. Completed so far are 196 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. All plants will be ready for planting this fall.Fifteen new transformations, totaling over 5000 explants, have been completed to generate Valencia, Ray Ruby, US-942, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Over 100 new putative transgenic lines of 74-Valenica, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion.The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been bud inoculated with HLB+ RL and are undergoing the first post-inoculation analysis. An additional 370 rooted cuttings were propagated for the third round of ACP-inoculations. From which, the first group of 54 plants large enough to use have been inoculated with the higher pressure 21 day protocol. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer and potential therapeutic effects.The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines) and Carrizo/Mthionin (2 lines) have been returned certified clean. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.