Article

Complete Genome Sequence of Citrus Huanglongbing Bacterium, ‘ Candidatus Liberibacter asiaticus’ Obtained Through Metagenomics

USDA-ARS-USHRL, Fort Pierce, FL 34945, U.S.A.
Molecular Plant-Microbe Interactions (Impact Factor: 3.94). 09/2009; 22(8):1011-20. DOI: 10.1094/MPMI-22-8-1011
Source: PubMed

ABSTRACT

Citrus huanglongbing is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with a low-titer, phloem-limited infection by any of three uncultured species of alpha-Proteobacteria, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. A complete circular 'Ca. L. asiaticus' genome has been obtained by metagenomics, using the DNA extracted from a single 'Ca. L. asiaticus'-infected psyllid. The 1.23-Mb genome has an average 36.5% GC content. Annotation revealed a high percentage of genes involved in both cell motility (4.5%) and active transport in general (8.0%), which may contribute to its virulence. 'Ca. L. asiaticus' appears to have a limited ability for aerobic respiration and is likely auxotrophic for at least five amino acids. Consistent with its intracellular nature, 'Ca. L. asiaticus' lacks type III and type IV secretion systems as well as typical free-living or plant-colonizing extracellular degradative enzymes. 'Ca. L. asiaticus' appears to have all type I secretion system genes needed for both multidrug efflux and toxin effector secretion. Multi-protein phylogenetic analysis confirmed 'Ca. L. asiaticus' as an early-branching and highly divergent member of the family Rhizobiaceae. This is the first genome sequence of an uncultured alpha-proteobacteria that is both an intracellular plant pathogen and insect symbiont.

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    • "influence of the gender of the psyllid on the population levels of CaLas nor was there any obvious pathology associated with high levels of CaLas in the psyllids. This low level of 'super infected' psyllids has been observed previously and a single superinfected psyllid was used to provide the DNA needed to sequence the CaLas genome (Duan et al., 2009). Because the mice were immunized with infected psyllids macerated in buffer, the immune repertoire is also likely to be enriched for proteins expressed by the psyllid in addition to the CaLas proteins. "
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    ABSTRACT: 'Ca. Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.
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    • "Twelve genotypes were identified but 42 out of 62 were identical; only two SNPs were reciprocally fixed between the isolates with no obvious geographic pattern, and the remaining genotypes only differed from one another by one to two SNPs (Furuya et al. 2010). Research on Las genomics has provided researchers with complete sequence data to develop more sensitive molecular markers to study the population biology of the HLB pathogens (Duan et al. 2009;Tyler et al. 2009).Chen et al. (2010)were the first to take advantage of this resource and identified a locus containing a single variable tandem repeat; that is, a microsatellite locus. They investigated the diversity of isolates from Guangdong, China and Florida, USA where the disease was first observed approximately 100 and 15 years ago, respectively. "
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    ABSTRACT: Citrus huanglongbing (HLB) has become a major disease and limiting factor of production in the citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host pathogen interactions, population genetics, and vectoring of the causal agent are discussed.
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    • "L. asiaticus' and 'Ca. L. americanus' genomes have now been fully or partially assembled (Duan et al. 2009; Katoh et al. 2014; Lin et al. 2013a, b; Wulff et al. 2014.; Zheng et al. 2014). Liberibacter crescens, an endophyte of papaya and the only culturable species of this genus, has also been sequenced (Fagen et al. 2014; Leonard et al. 2012). "
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