Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2009
Publication Date: 4/2/2009
Citation: Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Gabriel, D., Vahling, C.M., Williams, K., Dickerman, A., Sun, Y., Gottwald, T.R. 2009. Complete Genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions. 22(8):1011-1020. Interpretive Summary: Citrus Huanglongbing (greening)is the most devastating disease of citrus in the world and currently threatens the survival of the citrus industry in both Brazil and the U.S., the world's top orange and juice producers. Management of the disease is not only difficult but also expensive and no cure is available for infected trees at the present time. Because of its rapid spread and threat to the citrus industry, efforts are being made to decipher the molecular mechanisms involved in the pathogenicity of the disease. The disease is spread by the Asian citrus psyllid (Diaphorina citri) or the African citrus psyllid (Trioza erytreae) and is associated with low titer, phloem-limited infections by any of three uncultured species of a-Proteobacteria in the genus Candidatus Liberibacter. The species found within the United States is known as 'Ca. L. asiaticus' (Las) and is the only known a-proteobacterium that is both an intracellular plant pathogen and an insect parasite/symbiont. Due to its fastidious nature however, this bacterium has not been cultured in vitro, representing a major obstacle towards advancement in the field. Only 24,477 non-redundant base pairs of sequence have been reported to date and as a result, little progress has been made towards a solution to huanglongbing. In spite of the limitations, we have obtained and annotated a nearly complete genome (>95.0% completion) for Ca. L. asiaticus using multiple displacement amplification and pyrosequencing technologies (454 Life Sciences) on DNA extracted from a single Las-borne Asian citrus psyllid. The draft genome consists of 34 contigs, composed of approximately 1.22 Mb of sequence and has had over 99.9% of accuracy. Annotation of the sequence predicted 1122 coding sequences (CDS) of which 74.15% had homologues with known and putative functions, while 25.85% were hypothetical-conserved open reading frames (ORFs). In addition, 34 pseudogenes were also found. With the aid of the annotated genome, metabolic pathways can be identified and conditions necessary for growth may be ascertained. Virulence factors that allow the pathogen to become established in both its vector and plant hosts may also be determined, thus leading to the development of new control strategies for this devastating disease.
Technical Abstract: Citrus huanglongbing (HLB) is the most destructive disease of citrus worldwide. It is spread by the citrus psyllids (Diaphorina citri and Trioza erytreae), and is associated with low-titer, phloem-limited infections by any of the three uncultured species of a-Proteobacteria: 'Candidatus Liberibacter asiaticus' (Las), 'Ca. L. americanus' and 'Ca. L. africanus'. A nearly complete Las genome was obtained by metagenomics using multiple displacement amplification and pyrosequencing of the DNA extracted from a single psyllid carrying a high titer of Las bacterium. The draft genome of 1.22 Mb assembled into 34 contigs, with an average 37.4% GC content, representing ca. 17X genome coverage and is estimated to be >95.0% complete. Annotation revealed a high number of genes involved in cell motility (4.6%), and active transport in general (92 genes), including 40 ATP binding cassette transport genes, all of which may contribute to virulence. Las appears to have a limited ability for aerobic respiration, a limited defense against reactive oxygen, and is likely auxotrophic for at least 6 amino acids, all of which contribute to its fastidious nature. Consistent with its intracellular nature, Las lacks Type III and Type IV secretion systems as well as typical free living or plant colonizing extracellular degradative enzymes and their cognate Type II main terminal branch components. Las appears to have all Type I secretion system genes needed for both multidrug efflux and repeats in toxin effector secretion. Multiprotein phylogenetic analysis confirmed Ca. L. asiaticus as an early-branching and highly-divergent member of Rhizobiaceae.