2011 Annual Report 1a.Objectives (from AD-416) To dissect the disease complex of citrus Huanglongbing (HLB) in Florida from the following five angles:. 1)to complete Las genome sequence and to conduct comparative genomics of the Liberibacter species;. 2)to validate Koch’s postulates by culturing the Las bacterium in vitro and development of artificial inoculation methods;. 3)to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development;. 4)to confirm if Las bacteria are seed-transmissible and their role in HLB development; and. 5)to monitor Las population dynamics in citrus and periwinkle plants after chemical and heat treatment. 1b.Approach (from AD-416) Through molecular biology techniques, including state-of-the-art genomic sequencing, microarrays, and library construction, we will finish sequencing the Las genome, perform comparative genomics, seed transmission studies and microbial ecology analyses. Using a combination of long established and newly developed microbiology techniques, we will continue to pursue the culturing of Las. Greenhouse and lab experiments will be conducted to monitor Las population dynamics and when appropriate for the progress of other objectives. 3.Progress Report This project is related to inhouse objective 1: Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens. This is the last year of this project. A complete circular genome of Candidatus Liberibacter asiaticus was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb. All bacterial artificial chromosome (BAC) clones of Las were sequenced, and some new sequences will be added to the published genome. We have characterized the adenosine triphosphate (ATP) translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based "drug" to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, confirming only one operon responsible for zinc uptake. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 cells/ml were obtained within 48 hrs based on quantitative polymerase chain reaction (qPCR) estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 22.00 (16S rDNA-based RT-PCR) in liquid media. We are looking into factors affecting further growth.