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 Mthionin field plantings. Results from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo root-stock grafted with non-transgenic rough lemon 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 root-stock (108 plants) and WT/WT controls (16 plants). Scheduled assessments for both field plantings is being prioritized under current covid-19 pandemic conditions; the 24 month field assessment of the first planting and 12 month assessment for the second planting are completed. Leaf samples from both populations have been collected and are being processed for Clas quantification. Additional grafts of WT Hamlin and Ray Ruby scions to Mthionin root-stock were made and are included in the ongoing chimera planting discussed in Objective 2. 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 51 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, TBL, BLT, LBP/’74’, `73′, and `188′ (as well as scFv-InvA, scFv-TolC) 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. We have completed DLA testing of all 35s and many phloem specific (ScampP) promoter driven 3rd generation Carrizo lines. These assays have identified numerous lines with significant effects on CLas transmission and increased ACP mortality (up to 95% from TBL and >70% from TPK). The best performing lines are now in 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 ACP inoculated greenhouse experiments are now using an improved protocol using a combination of smaller plants, more aggressive trimming and close observation to safely extend the caged ACP infestation time from 7 days to 21 without harming the plants. Additional greenhouse studies are also being prepared in parallel using bud inoculations. 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 tissue samples are now collected and processed, awaiting qPCR analysis. A large additional greenhouse study is now underway to directly compare the best performing 3rd generation chimera (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) lines. A total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and bud inoculated with CLas+ Rough Lemon. The same lines are now being replicated for additional greenhouse and field trials. This is part of our efforts to emphasize parallel field trials for all phenotyping efforts. Towards this goal, a field planting of ~400 `74′, `188′ and Mthionin transgenics is underway. The first 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) went into the soil in August 2020 and will be undergoing their first assessment by February 2021. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT root-stocks are being made to complete the planting. Eighteen new transformations, totaling over 6200 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Over 275 new putative transgenic lines including 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 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 are now in the ground at the Picos farm location undergoing field trials. They are scheduled to receive their first assessment in March 2021. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. A second round of greenhouse trials (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A third set of 370 plants for greenhouse trials has been propagated with the first 54 plants to reach a suitable size already inoculated using the new ACP inoculation protocol. Tissue for testing CLas titer from both sets of plants has been collected and processed; now awaiting qPCR analysis. 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. Several peptides variants screened through this assay have now shown significant ability to reduce CLas titer by foliar application to grapefruit trees in initial testing- conducted by CRADA partners. Hamlin and Valencia trees have been selected and blocked for trunk application trials with these peptides. Transgenic Nicotiana benthamiana plants expressing His-6 tagged variants of the chimeras TBL, TPK, PKT and LBP have also been generated to produce sufficient protein extracts for use in trunk application trials. 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. In order to better investigate the effects of peptides producing ACP mortality, we have expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Profftella, and Carsonella) in addition to CLas. 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. In addition to the use of the AMP Mthionin, its variants and chimeric proteins, new strategies have been implemented in our Laboratory to fight HLB, including the evaluation of insecticidal peptides to control the ACP (CLas vector), as well as the downregulation of the DMR6 genes to enhance defense responses against HLB disease. 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific promoter (SCAmpP-3) were evaluated for their ability to kill ACP and 12 lines (4 event of each genotype) were selected to move up in the screening pipeline for HLB/ACP tolerance, since they showed significant ACP mortality. Also 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA, and 5 lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These high performing lines from Topaz and Onyx have now been replicated as rooted cuttings for greenhouse trials. Approximately 400 cuttings were made from these transgenic lines in September and October, and around 40% are already rooted and producing new shoots. Onyx has been introduced also in Hamlin, Ray Ruby and Valencia, under SCAmpP-3 promoter. A set of 30 new putative transgenic events are well acclimatized and are undergoing transgene expression analysis. Newly regenerated transformed plants have being transfer to soil.Down regulated DMR6 Carrizo transgenic citrus, either by expression of specific hairpin RNA or by specific Cas9-sgRNA were generated and are ready to be assessed for HLB resistance by grafting with infected scion. Objective 5, Transgenic product Characterization: CLas+ RL have been grafted as scions onto MThionin expressing Carrizo as a platform to test peptide movement and effects across the graft union. 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. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing. Experiments are also underway to use these plants to track the movement and distribution of transgene products as are parallel efforts with antibody based approaches.