Bacterial pathogens rely on the secreted “effector” proteins as essential virulence factors to cause disease. The HLB-associated bacterium Candidatus Liberibacter asiaticus (Las) possesses the Sec secretion system, which is a general protein secretion machinery that delivers effectors from the pathogen cells into the phloem of infected plants. Our research interests have been primarily in the Sec-delivered effectors (SDEs) produced by Las and our long-term goals are: 1) using SDEs as detection markers for Las diagnosis; 2) using SDEs as molecular probes to understand HLB pathogenesis. Previously, we identified ~30 SDEs from Las through bioinformatic analysis of the Psy62 genome; these SDEs were then analyzed on their gene expression levels in infected citrus trees. Results from these analyses allowed us to focus on three SDEs, which are highly expressed in infected tissues of various citrus varieties. Building on these previous results, this project aims to characterize the citrus targets of these three SDEs. Our central hypothesis is that SDEs contribute to HLB development by manipulating their host target(s) in citrus. Understanding how SDEs modulate citrus physiology and immunity will provide important mechanistic insight into HLB pathogenesis. This knowledge will then facilitate the development of sustainable control strategies. We proposed to pursue three objectives at the beginning of this project and we are happy to report that all of these objectives have been successfully accomplished. 1) Identify citrus proteins associating with three selected Las effectors using yeast two hybrid screens. We used the yeast two hybrid (Y2H) screening approach and identified the citrus targets of each of the three SDEs. For this purpose, we constructed a normalized citrus cDNA library containing more than 3 millions of primary clones using HLB-infected RNA samples. This library was then subjected to a next generation sequencing-based screening using each SDE as the bait. 2) Confirm the effector-host target interactions using a series of in vitro and in vivo assays. We confirmed the SDE-citrus target interaction using a series of biochemical assays including targeted Y2H and in vitro co-immunoprecipitation. We first analyzed the potential SDE targets identified from the Y2H screening for promising candidates that are: 1) differentially expressed in HLB-infected citrus; and 2) potentially contributing to immunity and/or HLB symptom development. The full-length cDNA of these genes were then cloned in various plasmid vectors for experiments to confirm their interaction with the SDEs. In average, we examined the interaction of each SDE with ~10 candidate targets. Through this process, we further focus our research on one SDE, which specifically interacts with a class of enzymes that have known function in plant defense. Intriguingly, these targets are also manipulated by effectors produced by other bacterial and fungal pathogens to promote virulence. 3) Design control strategies aiming to enhance the resistance/tolerance of citrus to HLB based on effector activities and the functions of their targets. We are designing chemical treatment strategies based on the enzymatic activity of the citrus targets that we identified for one SDE. This work will continue beyond the funded period of this project.