The goal of this project is to understand the biology of HLB by identifying key host components and processes involved in disease development. We use secreted proteins (also called effectors) from the causative agent, Candidatus Liberibacter asiaticus (CLas), as molecular probes because they have been considered key virulence proteins of bacterial pathogens. Our previous research using bioinformatic and experimental approaches identified four CLas effectors that are highly expressed in infected trees. We will isolate the direct citrus targets of these effectors, which will reveal important information of HLB pathogenesis. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first two quarters of this project, we cloned the four CLas effector genes into the Y2H bait vector, transformed them into the yeast strain AH109, and confirmed that the effectors are highly expressed in the yeast without self activation activities. Therefore, these constructs are appropriate for Y2H screens. In the third quarter (Year 1) of this project, our main efforts include: 1) Construct citrus cDNA libraries that will be used for Y2H screening. We collected RNA samples from asymptomatic and symptomatic tissues of HLB-infected sweet orange leaves. These RNA samples were mixed with RNA extracted from healthy tissues to ensure that we would be able to cover as many genes as possible. The RNA has been sent out for cDNA library construction by a company. We have been working with the company to normalize the library in order to minimize the influence from the over-representative transcripts of highly expressed, housekeeping genes, which may bias the screening later on. 2) Make gene expression constructs that produce fusion proteins with each CLas effector gene tagged to a gene encoding the yellow fluorescence protein (YFP). These fusion proteins have been transformed to plant cells to determine the localizations of these effectors in plants using microscope.