The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection and develop improved and long term HLB management strategies. Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varietiesPotential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding protein) and pilin assembly genes were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We have tested their receptors in Tobacco and citrus. Specifically, we are identifying the receptors in HLB susceptible variety Valencia sweet orange and HLB resistant variety Poncirus and HLB tolerant variety Sugar Belle. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation using pull-down assay or co-immunoprecipitation assays. We also hypothesized that Las outer membrane proteins might directly induce plant immune response in the phloem sieve elements because Las lives in the phloem. 21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid (Y2H) system and surface plasmon resonance (SPR) assay. Two outer membrane proteins showed positive interactions with citrus proteins based on Y2H assays. We are further confirming the interactions using GST pull-down assaysIn addition, multiple Las PAMPs have been tested for their effects in inducing plant defense against Las in the greenhouse and at least four different Las PAMPs showed significant effect in inducing plant immunity. We are testing whether those Las PAMPs can inhibit Las titers after foliar spray in the greenhouse. We have conducted RNA-seq analyses of Poncirus and sweet orange and we currently analyzing the data. We are testing the control effects of different PAMPs against HLB. Three PAMPs showed strong activity in inducing plant defenses. We have identified 21 genes that might contribute to the tolerance of Poncirus to HLB. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are transgenically expressing putative receptors or targets (identified in Poncirus) of Las PAMPs in Valencia sweet orange or Duncan grapefruit. They are driven by 35S promoter and phloem specific promoter AtSuc2. We will conduct Las inoculation via grafting or psyllid transmission once the transgenic plants are about one year old. For those identified receptors or targets, we are sequencing the promoter regions in Valencia, Sugar Belle, and Poncirus to compare their differences. If the native promoter of Poncirus is strong enough, we will use Poncirus promoter to drive the expression of PAMP receptors or other target genes to avoid concerns about 35S promoter or AtSUC2 promoter. We are also driving the expression of one defense inducing gene using a pathogen-inducing promoter. Several plants expressing the constructs were generated. Testing of those plants showed that they resonded to canker. We will test whether they are resistant to HLB. Right now, we are propagating to more plants for testing. Objective 3. Investigate the roles of effectors in HLB disease developmentWe have completed screening of 30 putative Las effectors and 4 of them repressed plant defense. We are screening another 20 putative Las effectors and 3 more effectors that suppress plant defense. We have completed Y2H for the four defense-suppressing effectors and identified their targets in Valencia sweet orange. Confirmation of the targets is ongoing using coimmunoprecipitation and BiFC assays. Meanwhile, we have conducted CTV-mediated gene silencing of 15 putative HLB susceptibility genes in collaboration with Dawson lab. Sweet orange plants carrying the CTV constructs were inoculated with Las via grafting. Interestingly, gene silencing of one of the putative HLB susceptible genes led to significant HLB tolerance. The plants showed mild HLB symptoms, similar growth as non-inoculated plants whereas the growth of control plants was significantly reduced and showed severe HLB symptoms. We are characterizing the putative mechanism of the HLB S gene. We are conducting genome editing of the identified HLB S gene of Valencia sweet orange and Duncan grapefruit to generate HLB resistant or tolerant citrus. In addition, we also overexpressed the HLB S gene in Valencia sweet orange to further understand the mechanism and will inoculate them with Las once they are one year old. We will continue to test other targets of putative effector genes. In addition, we hypothesized the effectors might induce plant defense in Poncirus and Sugar Belle. We are conducting Y2H to identify putative targets of effectors in Poncirus and Sugar Belle. We have conducted RNA-seq analyses of Sugar Belle. The data is under analyses. One manuscript entitled Citrus CsACD2 is a target of Candidatus Liberibacter asiaticus in Huanglongbing disease has been accepted by Plant Physiology. We are investigating the binding sites of CsACD2 with SDE15. We have tested the effect of effectors in suppressing plant immune responses caused by PAMPs. We have identified another important effector. In total, six promising HLB susceptibility genes were identified.