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 are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. 67 plants representing 13 independent events and wild types (4-5 replicates each) were transferred into larger pots and are getting ready for field planting and resistance evaluation. The remaining plants were used for bud-inoculation tests as transgenic root stock and/or scion. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. Another 805 rooted cuttings were created at the end of 2017 from 18 events for future experimentations. Tissue specific constructs of the very promising Mthionin gene have been developed. The phloem-specific variant has been transformed into Carrizo and is currently being transformed into other citrus types including Hamlin and Ray Ruby. The root specific variant is being transformed only into Carrizo. 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. Using the detached leaf ACP-inoculation assay, it was shown than transgenic Carrizo expressing citrus thionin V1-LBP chimera have significantly less CLas titer after 1 week of ACP feeding than the wild type controls. 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 six and nine months post inoculation, transgenic plants are showing consistent and significant decreases in bacterial titer (300x) as 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. 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 6) Budwood from our best performing Mthionin transgenics and citrus gene chimeras have been sent to DPI for cleanup and then broad field testing.