1) Assessed use of isolated leaf inoculation and small plant destructive sampling: Isolated leaf inoculations with ACP do not readily distinguish between resistant and susceptible citrus selections, but prove useful in assessing CLas-killing transgenics. Within a week, such assays have shown marked reductions in CLas in leaves and in ACP. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin (Mthionin) are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Non-grafted Mthionin are in the field. A propagated group of 200 Mthionin transgenic Carrizo are potted up and grafted with wild type Hamlin to be planted in field next year. More Mthionin Carrizo are propagated and will be used to graft with Valencia and Ray ruby scions for field planting. In greenhouse a group of grafted plants with wt/transgenic and transgenic/wt rookstock and scion combinations were created and subjected to ACP inoculation. The 3-month post inoculation samples were recently collected for titer analysis. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. The 3-month sampling was just completed. Tissue specific constructs of the very promising Mthionin gene have been developed, with 10 Carrizo confirmed. The root specific variant was transformed only into Carrizo, and 17 plantlets were regenerated. A CTV-expression vector for Mthionin was created by Dr. W. Dawson, has been verified, and will be graft inoculated into a range of plants (both infected and healthy for challenging) next quarter. 3) Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 4 below) have been propagated and are in replicated testing. New chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A total of 35 lines of Carrizo with citrus thionin V2-LBP construct, and 20 lines with citrus thionin V1-LBP construct have been generated. A total of 18 independent Carrizo lines, each expressing citrus thionin-EDS and citrus thionin �D2A21 chimeras respectively, were produced with confirmation of high level transgene expressions. A group of Carrizo transgenics expressing two variants of citrus thionin chimera (code 73 and 74) are being tested side-by-side with modified thionin transgenics. These plants were inoculated by ACP no-choice inoculation and reached 3 month mark for the first disease test in July. Using the detached leaf ACP-inoculation assay, it was shown that transgenic Carrizo expressing citrus thionin V1-LBP chimera has significantly less CLas titer after 1 week of ACP feeding than the wild type controls. Psyllid fed on detached leaves of the citrus thionin V1-LBP chimera Carrizo had lower CLas titer in bodies compared to the ones fed on the wide type leaves.. Comparison among individual lines from modified thionin transgenics were conducted using detached leaves and discovered variations in antimicrobial ability between them, suggesting this protocol allows us to screen better performing lines for further tests. His-6 affinity tagged variants of citrus thionin-BPI/LBP expressing constructs have been created with C-terminal and N-terminal thionin orientations. These constructs have been transformed into benthamiana for efficient in plantae production and purification of protein for use in detached tissue assays with multiple lines for each construct confirmed as transgenic and currently undergoing analysis for expression levels. 4) Antibodies (ScFv) to the CLas invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. Two representative constructs, one targeting each gene, have been challenged by CLas + ACP. At all time points measured after inoculation, transgenic plants are showing consistent and statistically significant decreases in bacterial titer (as much as 400x) when measured by qPCR and a much higher incidence of plants with no measurable bacterial DNA amplification. Additional plants representing 21 independent events from all 7 constructs have been replicated as rooted cuttings for ACP challenge of whole plants. A second round of ACP inoculations has been conducted on 150 plants replicated from twelve independent transformation events representing three different ScFv constructs. Additional lines will be inoculated once sufficient mature transgenic material becomes available. Thirty ScFv-transgenic Carrizo plants (10 each from the 3 best performing constructs in greenhouse studies) are being grafted with Ray Ruby scions in parallel with non-transgenic controls for initial field studies. Approximately 120 additional rooted cuttings for a follow up trial are being propagated. 5) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were previously shown to be downregulated in more tolerant �Jackson� compared to susceptible �Marsh� grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in �Hamlin�, �Clementine�, �Carrizo�, rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs targeting citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so far) are ongoing. Carrizo plants carrying these constructs with multiple events each were transferred into larger pots to stimulate growth in early 2018 and subsequent propagations. CRISPR constructs with guide RNA targeting DMR6 and DLO1 were made and transformed into Carrizo, generating 3 and 1 confirmed transgenic line respectively. Mutation at the target locus has been confirmed in the first of these plants after heat shock treatment. The remaining plants are being sequenced to detect mutations and new transformations into Hamlin are in preparation. 6) Budwood from our best performing Mthionin, citrus gene chimeras and ScFv antigen binding fragment expressing transgenics have been sent to DPI for cleanup and then broad field testing.