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. The first MThionin field trial (45 plants, WT or transgenic Carrizo with rough lemon scions) has shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second MThionin planting (205 total grafted plants of transgenic Hamlin scions, transgenic Carrizo rootstock, or WT/WT controls) is producing encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Leaf samples from the second planting are collected and undergoing CLas quantification. Results from the 2nd generation plantings detailed below also show the included mThionin lines with significantly improved growth and CLas titer numbers compared to WT when grafted to commercial scions. The Mthionin construct has also been extensively transformed into additional varieties; with 10 confirmed transgenic lines of US-942 and 44 putative lines of Valencia and Ray Ruby undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted to screen citrus 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). Testing of all 35s driven Carrizo lines is complete and the analysis of phloem specific and scion-types is well underway. This work has already identified numerous lines with significant effects including increased ACP mortality (up to 95% from TBL, 96% from PKT, 60% from BLT and 70% from TPK) and decreased transmission of CLas into the leaf. Analysis of the ACP endosymbionts show a reduction in titer that may indicate a mode of action for ACP mortality (outlined in Objective 4). The best performing of these lines have been moved forward into greenhouse trials as described below. 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. However, many plants of both treatments remained CLas negative due to low inoculation efficiency. Two follow on greenhouse studies are underway. The first is an improved ACP-inoculated trial with the best performing `74′ and `188′ lines. The second is a larger graft-inoculated study directly comparing the best performing 3rd generation chimeras (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) generation lines. For this, a total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and grafted with CLas+ RL for uniform transmission. In the earlier 6 and 9 month samplings, plants were beginning to test CLas positive and 12 month assessments are underway. An additional two rounds of rooted cuttings (totaling >1600 replicates) have been made from those same lines for paired ACP-inoculated greenhouse and field trials. ACP-inoculations will be done in free flying cages for a 2 month availability window, a protocol developed in a parallel project that has yielded up to a 100% infection rates. Both studies will begin as soon as the plants are large enough. The best performing PKT and LBT lines have also been replicated (>200 plants) for following the same greenhouse to field study pipeline, with graft inoculations as soon as the plants are of sufficient size. Field trials of 2nd generation chimeras (`74′, and `188′) with included MThionin plants are ongoing; with 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) and 70 plants (WT Valencia on transgenic Carrizo) moved to the field in August 2020 and May 2021 respectively. At the 1 year assessment, Hamlin plants with transgenic Carrizo rootstocks showed noticeably reduced CLas titer (in scion midribs) when compared to those on conventional rootstocks. WT Hamlin on `74′ Carrizo had a 10x reduction in CLas titer, WT Hamlin on MThionin a 15x reduction and WT Hamlin on `188′ a 144x reduction. Plants grafted on MThionin-Carrizo also showed better trunk diameter, tree height and canopy volume compared to WT/WT grafts. Because of the high ACP mortality seen in detached leaf assays, field plantings of all chimera are also being surveyed to determine the rates of successful ACP colonization on new flush. An additional planting of transgenic scions (Hamlin) on WT rootstocks is also being prepared to complement these plantings. Eighteen new transformations, totaling over 6200 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. From this effort, over 325 new 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. Transgene expression analysis has confirmed the first 29 of these lines as positive with the remainder still being tested. In addition to the use of the Mthionin and its chimeric variants, new strategies have been implemented in our laboratory to develop HLB resistant citrus. These efforts include the expression of insecticidal peptides (to control ACP) and down-regulation of DMR6 genes (to enhance disease resistance). 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 by detached leaf assay. 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, 27 transgenic Carrizo and Hamlin lines highly expressing Onyx (a code name to protect IP), a peptide with both antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx-Carrizo lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. Strongly performing lines were replicated as rooted cuttings (250 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being further expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 8 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA (for RNA interference) or by Cas9-sgRNA genome editing were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, down-regulated plants could be expected to have heightened immune response. To test this, they were first evaluated for potential Canker resistance which is both a quicker assay and a desirable trait in its own right. After Xanthomonas challenge on detached leaves, both hairpin and gene edited DMR6 lines showed reduced bacterial titers, statistically significant reductions in Canker symptoms and higher expression of some down-stream defense genes compared to WT controls. Several transgenic lines developed no disease symptoms whatsoever. A planting of 190 trees (including WT controls) from the best performing genome edited line (40 trees) and hairpin knockdown lines (150 trees) is being prepared; the plants have been replicated as rooted cuttings and will go into the field once regulatory requirements are met. An amendment to the current BRS permit has been submitted for their addition and is currently under review. As an effort to accelerate development of non-transgenic HLB resistant plants using gene editing, we transformed early flower transgenic plants (carrying FT-Mcherry or FT-scFv gene) with the DMR6 targeting CRISPR construct. Cotyledons from FT-Mcherry line #3 seeds were transformed and 51 putative positive plants are now growing in soil. These plants are already showing strong early flowering phenotypes, but must still be confirmed for presence of the CRISPR transgene. Seeds from 4 more FT-Mcherry lines are now being germinated for additional transformations. 50 additional plants from FT-scFv/CRISPR stacking transformations are also in soil awaiting confirmation. The early flowering trait will greatly decrease the time needed to produce an edited but non-transgenic offspring. A set of 30 confirmed positive plants from this gene stacking effort will be evaluated. Objective 3, ScFv Constructs: ACP inoculated greenhouse studies on 5 scFv lines have 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. The one year assessment is now complete and several scFv lines are showing significantly improved growth characteristics, leaf tissue was collected and is awaiting CLas quantification. An additional 129 rooted cuttings are propagated for follow up plantings with grafted Hamlin scions. A second greenhouse trial testing new lines (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A group of 370 plants for a third greenhouse trial has been propagated with the first 54 plants to reach a suitable size ACP-inoculated using the improved protocol. Plant tissue from both second and third (partial) greenhouse trials has been collected and processed; now awaiting qPCR analysis for CLas quantification. 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. 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 in both whole plant and detached leaf assays. The detached leaf ACP-feeding assay (DLA) 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, Carsonella and Profftella) to better investigate the activity of peptides causing CLas mortality. To further investigate the impact of transgenic or exogenous chimeral application, a DLA protocol to assess changes in ACP inoculability is also being developed. A manuscript detailing the protocol and findings is being prepared. 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 relative to Citrus DNA and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′,`188′, TPK, TBL, DMR6 knockdowns 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), Carrizo/Mthionin (2 lines), Carrizo/’74’ (1 line), Carrizo/’188′ (1 line) and Hamlin/’74’ (1 line) have been returned certified clean. Objective 5, Transgenic Product Characterization: Experiments are also underway to 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 (34 lines) and scFv-TolC (30 lines) have been generated and confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our collaborator for testing.