The goal of this project is to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins secreted by the causative agent Candidatus Liberibacter asiaticus (CLas) into the phloem. One of the most important virulence mechanisms of bacterial pathogens is their protein secretion systems, which secrete proteins (called ‘effectors’) to manipulate the hosts. CLas possesses the general Sec secretion system that secretes effectors carrying a specific N-terminal secretion signal. Sec effectors have been demonstrated to be important for pathogenicity of insect-transmitted, phloem-limited bacteria. Therefore, it is likely that Sec effectors of CLas also contribute to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are using these effectors as molecular probes to identify key components in HLB. We aim to find the citrus targets of these CLas effectors, which may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In this report period, we have completed the Y2H screening. For each effector (i.e. bait), more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the baits. This practice eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with each bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. We are still doing in-depth analysis of these data, but have already seen interesting citrus proteins that are potentially targeted by the CLas effectors. For example, one effector targets several transcription factors in citrus. These transcription factors may directly regulate the expression of genes that contribute to HLB susceptibility and/or symptom development. Indeed, one transcription factor targeted by this effector contains a “WRKY” domain, which is well-known for its regulatory role in plant defense. Interestingly, our microscopy analysis also shows that this effector is located in the nucleus of plant cells. These data suggest that CLas effectors may directly manipulate gene expression in citrus.