Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.     

XacFhaB adhesin,an important Xanthomonas citri ssp. citri virulence factor,is recognized as a pathogen‐associated molecular pattern

Adhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non‐fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753‐residue‐long protein with a predicted β‐helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino‐terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen‐associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non‐host plants, with a stronger activation by the carboxyl‐terminal region. Furthermore, pre‐infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.     

Injection–Infiltration of Attached Grapefruit with Xanthomonas citri subsp. citri to Evaluate Seasonal Population Dynamics in Citrus Canker Lesions

To study population dynamics of Xanthomonas citri subsp. citri (Xcc) in citrus canker lesions on fruit, a needle‐free injector was used for infiltration of bacterial inoculum into fruit in situ on mature ‘Ruby Red’ grapefruit (Citrus paradisi Macf.) trees in Florida. Inoculations of Xcc at 10⁵ colony‐forming units (cfu) per ml were conducted in 2012 and 2013 on attached fruit varying from 15 mm to 90 mm in diameter. Inoculations were repeated every 2–3 weeks until the fruit were no longer injectable. On fruit less than 40 mm in size, erumpent lesions formed within 2 weeks of inoculation and expanded 1–9 mm in diameter from 30 to 120 days postinoculation (dpi). Xcc populations in lesions were 6–8 log cfu per lesion at 30 dpi and maintained this population up to 90 dpi. By 120 dpi, Xcc populations declined 1–3 log units as rainfall and temperature decreased in September–October. Xcc populations declined to ~3 log cfu per lesion after 120 dpi in November 2012 and 2013, whereas the population resurged to 5 log cfu per lesion after 180 dpi in January–February 2014.     

柑橘溃疡病菌II型分泌通道基因xpsD突变体的鉴定及功能分析

【目的】鉴定柑橘溃疡病菌胞外水解酶减弱突变体Mxac56-20的Tn5插入位点,及其在柑橘上的致病力。【方法】采用质粒拯救方法获得Tn5旁侧序列,与基因组信息比对后明确突变体的插入位点;构建功能互补载体对突变体进行功能互补,检测互补菌株胞外蛋白水解酶、纤维素酶和淀粉酶的恢复情况;在寄主植物柑橘上观察致病力变化。【结果】Mxac56-20的Tn5插入位点是II型分泌系统xpsD基因,所构建的互补载体使突变体的胞外水解酶活性和致病力得到恢复。【结论】柑橘溃疡病菌xpsD基因的突变,导致胞外水解酶活性降低,在寄主上的致病力减弱,说明柑橘溃疡病菌的II型分泌系统在与寄主互作过程中起到致病因子的作用。     

Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence

The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole.     

利用激光共聚焦扫描显微镜对溃疡病菌侵染柑橘叶片的实时观察

柑橘溃疡病对柑橘产业造成了巨大损失,而研究柑橘与溃疡病菌的互作关系以及柑橘的感病和抗病性均需要观察溃疡病菌在柑橘寄主中的侵染和定殖过程。激光共聚焦扫描显微镜不仅可以观察活细胞,活组织的动态代谢过程,而且可以获得三维图像,对于病原菌在柑橘植物组织内的繁殖和致病机制研究具有重要意义。但是,选择适宜的植物材料和制片方法对激光共聚焦扫描显微镜的观察效果影响很大。本文对激光共聚焦扫描显微镜所观察的材料在其处理和观察方法上加以改进,获得了质量更好的图片和实验结果,也使得实验更为方便快捷。激光共聚焦扫描显微观察还在瞬时表达分析中得到应用,提高了柑橘瞬时表达分析的效果。通过将切片和压片相结合观察到溃疡病菌在不同时间点对柑橘叶片的侵染情况,而通过3D建模能观察到柑橘叶片不同组织层面中的病菌数量和病菌位置,为研究溃疡病菌在叶片中的定殖方式和入侵数量提供了前期基础。     

The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein-protein interaction

The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant-pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N- and C- termini and a structurally well defined core composed of three beta-strands and two small alpha-helices, which pack against each other forming a two-layer alpha/beta scaffold. This protein fold resembles the domains of the beta-lactamase inhibitory protein BLIP, involved in protein-protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein-protein interactions required during X. citri infection.     

Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker Disease

Twenty-four strains of Xanthomonas axonopodis pv. citri ( Xac ), the causal agent of bacterial canker of citrus, isolated from Mexican lime ( Citrus aurantifolia ) and lemon ( Citrus limon ) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using Eco RI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A.     

HrpM is involved in glucan biosynthesis,biofilm formation and pathogenicity in Xanthomonas citri ssp. citri

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm‐deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ‐Tn5 <KAN‐2> Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ‐Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β‐1,2‐glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H ₂ O ₂ in comparison with the wild‐type strain. All defective phenotypes were restored to wild‐type levels by complementation with the plasmid pBBR1‐MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.     

Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.