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.     

Functional characterization of LotP from Liberibacter asiaticus

Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host–pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N‐terminus. Co‐immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP‐interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two‐hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.     

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.     

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。     

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.     

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 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.     

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.     

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.     

Identification and characterization of flavonoids as sialyltransferase inhibitors

Sialyltransferases biosynthesize sialyl-glycoconjugates involved in many biological and pathological processes. We investigated and characterized synthetic flavonoid derivatives as sialyltransferase inhibitors. We first examined 54 compounds by solid-phase enzyme assay using β-galactoside α2,6-sialyltransferase 1 (ST6Gal I) and β-galactoside α2,3-sialyltransferase. Several compounds inhibited sialyltransferase enzyme activity regardless of sialyl-linkage reactions. Among them, two compounds showed inhibitory activity against ST6Gal I with IC₅₀ values less than 10 μM. Three characteristic features of flavonoids were determined by structure-inhibitory activity relationships. First, a double bond between C2-C3 linkages is required for the activity. Second, increasing hydrophilic properties on the B-ring markedly augmented the inhibitory effect. Third, a hydrophobic functional group introduced on the hydroxyl groups of the A-ring enhanced the inhibitory activity. Kinetic analysis using human ST6Gal I indicated a mixed inhibition mechanism of the compounds. In conclusion, the flavonoids identified could be applied for control of cellular expression of sialic acid.     

Effective Antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-Affected Citrus Plants Identified via the Graft-Based Evaluation

Citrus huanglongbing (HLB), caused by three species of fastidious, phloem-limited ‘Candidatus Liberibacter’, is one of the most destructive diseases of citrus worldwide. To date, there is no established cure for this century-old and yet, newly emerging disease. As a potential control strategy for citrus HLB, 31 antibiotics were screened for effectiveness and phytotoxicity using the optimized graft-based screening system with ‘Candidatus Liberibacter asiaticus’ (Las)-infected citrus scions. Actidione and Oxytetracycline were the most phytotoxic to citrus with less than 10% of scions surviving and growing; therefore, this data was not used in additional analyses. Results of principal component (PCA) and hierarchical clustering analyses (HCA) demonstrated that 29 antibiotics were clustered into 3 groups: highly effective, partly effective, and not effective. In spite of different modes of actions, a number of antibiotics such as, Ampicillin, Carbenicillin, Penicillin, Cefalexin, Rifampicin and Sulfadimethoxine were all highly effective in eliminating or suppressing Candidatus Liberibacter asiaticus indicated by both the lowest Las infection rate and titers of the treated scions and inoculated rootstock. The non-effective group, including 11 antibiotics alone with three controls, such as Amikacin, Cinoxacin, Gentamicin, Kasugamycin, Lincomycin, Neomycin, Polymixin B and Tobramycin, did not eliminate or suppress Las in the tested concentrations, resulting in plants with increased titers of Las. The other 12 antibiotics partly eliminated or suppressed Las in the treated and graft-inoculated plants. The effective and non-phytotoxic antibiotics could be potential candidates for control of citrus HLB, either for the rescue of infected citrus germplasm or for restricted field application.     

Lipid-lipid interactions as regulators of carboxylester lipase activity

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.     

黄龙病菌侵染过程中柑橘CsMYB基因克隆及表达分析

MYB类蛋白是一类与植物防卫反应有关的转录因子家族,本研究利用构建的甜橙健株与感染黄龙病的病株差减（SSH）文库,采用RACE技术克隆了一个MYB类基因的cDNA全长序列,命名为CsMYB（GenBank登录号为HQ841074）。柑橘CsMYB基因的cDNA全长为1 306 bp,生物信息学分析显示,该基因包括一个909 bp的完整开放读码框以及一个典型的26 bp poly-A,编码302个氨基酸,分子量为32.97 kD,等电点为8.5。同时,还有MYB类基因的保守特征区域,即在N端有两个典型的MYB DNA结合域：R2和R3。实时荧光定量PCR分析表明,柑橘CsMYB基因受到黄龙病菌侵染后的不同时期表达量不同,伴随着黄龙病病程的变化呈现不同的表达变化。推测CsMYB基因是一个转录因子,可能参与柑橘对黄龙病菌的防御反应过程。     

亚洲柑橘木虱与柑橘黄龙病菌互作的研究进展

亚洲柑橘木虱Diaphorina citri Kuwayama作为柑橘产业重要病害柑橘黄龙病的主要传播媒介,已经成为重点防治对象。该害虫与黄龙病之间的互作一直是相关研究的热点,本文就近年来该领域的研究进展做了一个总结,从亚洲柑橘木虱的获菌与传病机制、病原菌与柑橘木虱之间的互作以及病原菌感染寄主植物后对木虱的影响等方面进行了综述。期望为深入开展黄龙病相关研究、寻找防控新途径提供依据。