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. We have identified multiple receptors for the aforementioned PAMPs. 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 were identified using Yeast 2 hybrid (Y2H) system. Two outer membrane proteins showed positive interactions with citrus proteins based on Y2H assays which were confirmed using GST pull-down assaysIn addition, multiple PAMPs have been tested for their effects in inducing plant defense against Las in the greenhouse and at least four different PAMPs showed significant effect in manipulating plant immunity. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant Sugar Belle mandarin in winter, spring, summer and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both varieties, spring had the highest number of DEGs. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, ROS, and PR genes in both varieties. The immune responses of both varieties to CLas result in oxidative stress with induced expression of RBOH genes and suppressed expression of many genes encoding antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase, thioredoxins and glutaredoxins. HLB-positive Sugar Belle trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, Sugar Belle showed higher expression of genes involved in phloem regeneration that might contribute to its tolerance to HLB. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.We have the control effects of different PAMPs against HLB. Three PAMPs showed strong activity in inducing plant defenses. We analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJLas interacts with FlgB but not with FliE. Ectopic expression of flaALas in A. tumefaciens mutants restored the swimming motility of .flaA mutant and .flaAD mutant, but not that of the null mutant .flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.The type IVc tight adherence (Tad) pilus locus is a putative PAMP encoded by Las. The Tad loci are conserved among members of Rhizobiaceae, including ‘Ca. L. asiaticus’ and Agrobacterium spp. Ectopic expression of the ‘Ca. L. asiaticus’ cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of ‘Ca. L. asiaticus’ was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ‘Ca. L. asiaticus’ Tad pilus-encoding genes and ‘Ca. L. asiaticus’ pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ‘Ca. L. asiaticus’ VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the ß-galactosidase reporter gene. Comparative expression analysis confirmed that ‘Ca. L. asiaticus’ visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ‘Ca. L. asiaticus’ visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ‘Ca. L. asiaticus’ and shed light on the involvement of VisNR in regulation of the Tad locus. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe have transgenically expressed 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 have made 6 constructs to express PAMP receptors individually or in combinations. Three overexpression lines have been generated. 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 have completed Y2H for the four defense-suppressing effectors and identified their targets in Valencia sweet orange. 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. 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. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.