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, as well as studying them in established greenhouse and field trials. 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 has continued for the two Mthionin field plantings. The first plantings of transgenic or control Carrizo grafted with non-transgenic rough lemon scions (45 total plants) have shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second planting of transgenic Carrizo with WT Hamlin scions, transgenic Hamlin on non-transgenic Carrizo and WT/WT controls (205 total plants) have shown very encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Scheduled assessments for both plantings have been prioritized under pandemic conditions, allowing the 36 month field assessment of the first planting and 24 month assessment for the second planting to be completed. Leaf samples associated with these assessments have also 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 56 putative lines from these transformations are in soil and undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted for lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, ’73’, and ‘188’ (as well as scFv-InvA, scFv-TolC, Topaz and Onyx). esting of all 35s driven 3rd generation Carrizo lines is complete and the analysis of phloem specific and scion-type lines is well underway. This work has already 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 have been moved forward to greenhouse trials. Initial ACP-inoculated greenhouse trials on 8 lines of citrus Thionin-LBP 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 due to low inoculation efficiency. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using a new protocol to improve inoculation rates. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6-12 month tissue samples are now collected and processed, awaiting qPCR analysis. A large greenhouse study is 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+ RL to ensure high transmission. The first leaf collection will be collected in March. An additional ~1200 rooted cuttings have been made from those same lines for matching ACP-inoculated greenhouse and field trials. An earlier field planting of 1st and 2nd generation lines (~400 plants of Mthionin, `74′, and `188′) is also underway. The first 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) went into the soil in August 2020, a second set of 70 WT Valencia on transgenic Carrizo are ready to be moved to the field this season and the remaining 200 transgenic Hamlin on WT root-stocks are being grafted. 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 325 new putative transgenic lines from 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: ACP inoculated greenhouse studies on 5 scFv lines has been completed with transgenics showing significantly reduced CLas titer (up to 250x reduction) and a significantly higher incidence of no CLas rDNA amplification in roots and leaves compared to WT. These lines have been grafted with WT Ray Ruby scions and are undergoing field trials at Picos farm. Their first assessment will be in March 2021. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. A second greenhouse trial (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 ACP-inoculated using the improved protocol. Tissue from both trials for testing CLas titer 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 screened through this assay have since been confirmed to reduce CLas titer by foliar application to grapefruit trees in tests performed by CRADA partners. Citron, Hamlin and Valencia trees have been selected and blocked for trunk application trials using 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 exogenous applications. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; adjusting feeding period and ACP numbers. We have also expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Profftella, and Carsonella) to better investigate the activity of peptides causing CLas mortality. 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′,`188′, TPK, TBL 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 and its chimeric variants, new strategies have been implemented in our Laboratory to fight HLB. These efforts include the expression of insecticidal peptides to control ACP (CLas vector) and 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 (SCAmpP-3) promoters were evaluated for their ability to kill ACP. From these, 12 lines (4 event of each genotype) showed significant ACP mortality and were selected to move up in the screening pipeline for HLB/ACP tolerance. 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. The 5 Onyx lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These strongly performing lines were replicated as rooted cuttings (55 Onyx and 131 Topaz plants) that will enter greenhouse trials as soon as the plants are of appropriate size. The available Onyx transgenic material is being expanded with 40 Hamlin and Carrizo plants transformed with the phloem specific version and 33 Ray Ruby and Valencia plants with the constitutive version undergoing expression analysis. Down regulated DMR6 Carrizo transgenic citrus, either by expression of specific hairpin RNA or by specific Cas9-sgRNA were generated, cloned, and are ready to be assessed. Since DMR6 is a broad immune suppressor, downregulated transgenic plants will be first evaluated for Canker resistance as a quicker assay. For that, clones from 5 selected lines are being inoculated with Xanthomonas citri and data is to be collected soon. As an effort to accelerate the development of non-transgenic HLB resistant plants through gene editing, we transformed early flower transgenic plants (carrying FT-SCFV gene) with the DMR6 targeting CRISPR construct. A set of 30 plants resulted from this gene stacking effort will be evaluated for the presence of both genes. Objective 5, Transgenic product Characterization: Experiments are also underway track the movement and distribution of transgene products using antibodies and affinity tagged protein variants. 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 and/or Flag tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), PKT (20 lines), scFv-InvA (22 lines) and scFv-TolC (18 lines) have been generated and expression confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing