The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

柑橘黄龙病菌内参基因的筛选与评估

【背景】柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一,主要由候选韧皮部杆菌属亚洲种("Candidatus Liberibacter asiaticus",CLas)引起。CLas全基因组测序已经完成,因而该病原菌的基因表达研究和功能验证得以进行。【目的】筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性。【方法】基于基因功能类别,利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16Sr RNA基因作为参照基因)。结合Ct值标准差和geNorm、NormFinder、RefFinder软件,评价内参基因的表达稳定性。【结果】在感染黄龙病不同时期和不同品种的植物寄主样本中,14对引物表现出较强的特异性和稳定性。内参基因稳定性排名:ftsZgyrArpoB1ftsAsecAgapzapEgmk2rpoDsecYrpoOftsWgmk1recA,根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估。以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因LasΔ5313的表达水平,所得的表达模式相同。【结论】柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定,在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参。本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础。     

Diversity of "Candidatus Liberibacter asiaticus," based on the omp gene sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of "Ca. Liberibacter asiaticus" isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species "Ca. Liberibacter asiaticus," even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating "Ca. Liberibacter asiaticus" isolates.     

Predictive sequence analysis of the Candidatus Liberibacter asiaticus proteome

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a parasitic gram-negative bacterium that is closely associated with Huanglongbing (HLB), a worldwide citrus disease. Given the difficulty in culturing the bacterium and thus in its experimental characterization, computational analyses of the whole Ca. L. asiaticus proteome can provide much needed insights into the mechanisms of the disease and guide the development of treatment strategies. In this study, we applied state-of-the-art sequence analysis tools to every Ca. L. asiaticus protein. Our results are available as a public website at http://prodata.swmed.edu/liberibacter_asiaticus/. In particular, we manually curated the results to predict the subcellular localization, spatial structure and function of all Ca. L. asiaticus proteins (http://prodata.swmed.edu/liberibacter_asiaticus/curated/). This extensive information should facilitate the study of Ca. L. asiaticus proteome function and its relationship to disease. Pilot studies based on the information from our website have revealed several potential virulence factors, discussed herein.     

Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves

Huanglongbing is one of the most devastating diseases of citrus (Citrus spp.). One management tactic against huanglongbing is aggressive management of the vector, the Asian citrus psyllid (Diaphorina citri Kuwayama), with insecticide applications. However, D. citri in abandoned groves are not controlled and therefore pose a risk of reinfestation for nearby commercial citrus. These abandoned groves could serve as a reservoir for the vector, as well as a source of the presumed causal agent for huanglongbing in Florida, Candidatus Liberibacter asiaticus (Las). The current study was conducted to determine the degree to which Las is present in abandoned Florida citrus groves and to compare relative inoculum levels in nearby managed and abandoned groves during times of the year when D. citri are abundant (June, July, and August). In addition, the movement of Las by dispersing D. citri adults from inner and edge rows of abandoned grove plots to the corresponding rows of managed plots was quantified during the same 3 mo. The results of the current study confirmed the presence of Las in both D. citri and plant tissue in abandoned groves at statistically equivalent levels to those in nearby managed groves. The mean number of D. citri adults dispersing from abandoned to managed grove plots ranged from 7.25 +/- 1.70 to 70.25 +/- 21.25 per 4-d intervals. Of those, the mean number of dispersing D. citri adults that were carrying the Las pathogen ranged from 1.00 +/- 0.58 to 1.50 +/- 0.50. Our results indicate that abandoned citrus groves are a significant source of Ca. Las and that dispersing D. citri move this pathogen into nearby managed groves.     

Infection and distribution of Candidatus Liberibacter asiaticus in citrus plants and psyllid vectors at the cellular level

Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus-growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri, in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri. To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus-growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP-225, OMP-333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP-47 and OMP-225 displayed specificity to the D. citri vector. The application of OMP-225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri. The application of OMP-333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.     

Differentiation of "Candidatus Liberibacter asiaticus" isolates by variable-number tandem-repeat analysis

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of "Candidatus Liberibacter asiaticus." The Nei's measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.     

Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design

Candidatus Liberibacter asiaticus is the causal agent of Huanglongbing (HLB) disease of citrus. Current management practices have not been able to control HLB and stop the spread of HLB. The current study is focused on screening small molecule inhibitors against SecA protein of Ca. L. asiaticus. Homology modeling, structure based virtual screening and molecular docking methods have been used to find the novel inhibitory compounds against SecA activity at ATP binding region. At 20 μm 17 compounds showed >50% inhibition and four compounds had more than 65% inhibition. The most active compound has IC₅₀ value of 2.5 μM. The differences between the activities of the compounds are explained by their inter-molecular interactions at ATP binding site.     

Genetic Diversity of Candidatus Liberibacter asiaticus Based on Two Hypervariable Effector Genes in Thailand

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA _I and lasA _II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA _I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA _II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.     

Diversity and characterization of Candidatus Liberibacter asiaticus strains causing huanglongbing disease in Iran,based on two prophage loci

Huanglongbing (HLB), also known as citrus greening, is a destructive disease of citrus; it is considered a newly emerging disease which has spread to the Middle East and North Africa (MENA). In Iran, the disease was first found in 2009. In this study, two hypervariable prophage and phage‐related loci, bacteriophage repressor protein C1 (CLIBASIA_ 01645 locus) and prophage terminase gene (CLIBASIA_05610 locus), were used to determine the diversity and characterization of Candidatus Liberibacter asiaticus ( CLas) strains associated with HLB samples. Analyses of the CLIBASIA_01645 locus, characteristic of variable tandem repeat numbers (VTRNs), revealed the homogeneity of Iranian CLas isolates: However, this result showed two distinct genotypes (TRN < 10 and TRN > 10) of CLas in Iran. This is the first report documenting the presence of two differentially distributed genotypes of CLas in Iran. Sequence analysis of prophage terminase revealed the presence of two putative prophages (prophage I and prophage II) in the genome of CLas isolates of Iran. Frequency analysis of these two prophages by specific loci revealed the association between prophages populations, the development HLB symptoms and CLas genotypes and their interactions with another obligate symbiontic, HLB phytoplasma.     

Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.     

Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae)

The purpose of this investigation was to evaluate acquisition and inoculation (together, transmission) efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with citrus huanglongbing (HLB) by the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae). In laboratory studies, nymphs reared on Las infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60 to 100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Las-infected plants. Similar rates of pathogen acquisition by psyllids after nymphal and adult feeding were observed in the field. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4 to 10%, whereas groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Las-positive adult psyllids, determined using quantitative real-time polymerase chain reaction, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long time period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Las by D. citri did not result in Las-infected plants after >1 yr of incubation after inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs.