Effectors are essential virulence factors in microbial pathogens. The HLB-associated bacterium Candidatus Liberibacter asiaticus (Las) is known to encode the Sec secretion system, which is predicted to deliver effector proteins into plant phloem. Our previous research identified four Sec-secreted proteins from Las. The goal of this project is to characterize the targets of these effectors in citrus. This research will provide important knowledge on the basic biology of HLB pathogenesis and facilitate the development of control strategies. Our research has been focusing on four Las effectors whose expression can be consistently detected from various citrus varieties that are infected by Las. We hypothesized that their targets in citrus contribute to HLB development. The main approach we are using to identify the effector targets is yeast two hybrid (Y2H) screening. In the past two years of this project, we accomplished the following experiments: 1) expression analysis of the Las effectors; 2) cloning and expression of the effector genes in yeast; 3) construction of a normalized citrus cDNA library with more than 3 millions of primary clones using HLB-infected RNA samples; 4) Illumina sequencing-based Y2H screening using each of the four Las effectors as the bait and sequencing data analysis; 5) subcellular localization analysis of the Las effectors. During this report period, we focused on experimental confirmation of effector targets. From Y2H screening, multiple potential targets were identified for each effector. We performed extensive literature search and prioritized our effort on candidates with potential roles in plant defense or HLB symptom development. These candidates were re-cloned from citrus cDNA individually and examined for their interaction with the corresponding effector by targeted Y2H. Due to the large amount of work, we have further focused on two effectors, which exhibit significantly higher expression (10-40 folds) in HLB-infected citrus tissues than in psyllids. So far, we examined a total of over 20 potential interacting proteins of these two effectors. Our results strongly suggest that each of these effectors specifically interacts with a class of citrus proteins in yeast. Importantly, these two citrus plant families have been reported to play a role in plant defense and they are also well-known virulence targets of other bacterial and fungal pathogens. Furthermore, one of the protein family has been reported to be present in the vascular system. We have made exciting progress in the project. Our on-going effort includes: 1) further confirm the physical interaction of the effectors and their targets using in vitro and in vivo co-immunoprecipitation assays; 2) characterize the target proteins as a family to understand how they interact with the effectors.