Active Oxygen-producing and -scavenging Enzymes in Pepper Leaves Infected with Xanthomonas campestris pv. vesicatoria

Lipoxygenase (LOX), peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities were determined in pepper (Capsicum annuum) leaves infected with Xanthomonas campestris pv. uesicatoria, from 3 to 15 days after inoculation, before symptom appearance and during the development of the disease. Strong Stimulation of LOX and POX activities was observed in infected leaves at an advanced stage of the disease (12–15 days after inoculation), when bacterial water-soaked spots and a slight chlorosis of the inoculated leaf areas were evident and a decrease in chlorophyll content of infected tissue was detected. The infection also induced a significant increase in CAT activity at the day 9 and a decrease in SOD and CAT activities at the day 12. On the basis of the changes observed, uncontrolled production of active oxygen species at advanced stages of infection is hypothesized.     

野生型油菜黄单胞菌的过氧化氢释放特性

采用外源过氧化氢和油菜组织处理油菜黄单胞菌野生型(XpW)和烷基过氧化物还原酶亚基C突变型(Xp1),检测了各体系中过氧化氢的释放情况,并测定了油菜黄单胞菌野生型的最大过氧化氢耐受浓度范围,以明确植物-病原菌互作过程中是否具有病原细菌源的过氧化氢产生.结果显示:(1)过氧化氢处理3.5 h后,对外源过氧化氢的清除率相对于0.5 h时XpW为100%,Xp1为-26%,说明野生型对培养体系中过氧化氢的清除率高于突变型;油菜组织处理后,体系中产生和积累的过氧化氢情况是:XpW为3.5 h时比0.5 h时高2.105倍,Xp1为3.5 h时比0.5 h时低25.2%,说明野生型培养体系中产生和积累的过氧化氢量高于突变型.(2)野生型的最大过氧化氢耐受浓度范围为1.715 08×105～2.450 11×105 μmol/L,远远高于油菜组织处理XpW菌液时体系中最大检测到的过氧化氢浓度,说明本实验中油菜组织处理XpW菌液过程中产生的过氧化氢的浓度不能够杀灭XpW.由此推测,油菜组织处理体系中野生型油菜黄单胞菌能够产生部分的过氧化氢,其过氧化氢的产生与烷基过氧化物还原酶亚基C相关,支持植物-病原细菌相互作用过程中可能有细菌源的过氧化氢产生的观点.     

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.     

Transaldolase exhibits a protective role against menadione toxicity in Xanthomonas campestris pv. phaseoli

A talA gene encoded transaldolase, a rate-limiting enzyme in the non-oxidative branch of the pentose-phosphate pathway, was cloned from Xanthomonas campestris pv. phaseoli. talA located in a region of the bacterial genome rich in genes involved in oxidative stress protection and regulation. TalA from X. campestris pv. phaseoli showed a high degree of homology to many previously reported transaldolases from both prokaryotic and eukaryotic sources. The expression of X. campestris pv. phaseoli talA was high at log-phase of growth, then declined at stationary phase, and could not be induced by oxidants. A talA mutant constructed by insertional inactivation did not possess any detectable transaldolase activity. Lack of a functional talA gene did not affect bacterial growth in a rich medium containing glucose or sucrose as a carbon source. However, the talA knockout mutant showed increased sensitivity to the superoxide generator menadione, but not to other oxidants. This increased menadione sensitivity phenotype could be complemented by expression of talA in a plasmid vector. The data demonstrated a novel and essential role of transaldolase in protection against menadione toxicity in X. campestris.     

Methods for recovery and immunodetection of Xanthomonas campestris pv. phaseoli in navy bean seed

Non-selective enrichment procedures were evaluated for recovery of Xanthomonas campestris pv. phaseoli (fuscans strain) from artificially inoculated navy bean seed. A marked increase in recovery of the pathogen was obtained when the mixtures (bacterium plus bean seed) were suspended in Pseudomonas Agar F medium at 28°C for 48 h. Detection of this pathogen by indirect immunofluorescence microscopy (IF) and indirect enzyme-linked immunosorbent assay (ELISA) with a specific monoclonal antibody was compared. The IF system was not only more sensitive but also more reliable than ELISA for detection of the pathogen. The method is particularly useful for evaluation of the common bacterial blight status of seedlots before planting out.     

PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli

A PCR-based method was developed for the specific detection of Xanthomonas campestris pv. phaseoli var. fuscans from plant material. Primers Xf1 and Xf2, based on a sequence conserved amplified region (SCAR) derived from RAPD PCR analysis of X. c. pv. phaseoli var. fuscans , amplified a DNA fragment of 450 bp from all such isolates. In contrast, no amplification product was obtained from any X. c. pv. phaseoli isolates, or from any other DNAs tested. As few as 10 cells of X. c . pv. phaseoli var. fuscans (equivalent to about 100 fg DNA) could be detected in vitro . In planta , following an initial inoculation of as little as one cell, an amplification product was generated after only 2 d of incubation, allowing highly sensitive detection 10 d before disease symptoms were observed. Moreover, the failure to amplify DNA from X. c . pv. phaseoli isolates shows that these primers provide a rapid, improved method to differentiate these two varieties using PCR.     

Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Antibacterial activity of metabolite produced by Paenibacillus polymyxa strain HKA-15 against Xanthomonas campestris pv. phaseoli

An antibacterial metabolite extracted from Paenibacillus polymyxa HKA-15 showed strong inhibition against Xanthomonas campestris pv. phaseoli strains CP-1-1 and M-5. Minimum inhibitory concentration (MIC) of crude extract against strains CP-1-1 and M-5 was found to be 1.7 mg/ml and 1.52 mg/ml, respectively. In UV-Vis range, the absorption peak of crude extract was maximum at 240 nm. The compound is resilience to wide range of temperature, pH, surfactants and organic solvents. The complete loss of activity was observed when crude metabolite was treated with pepsin (400 unit/ml). Characterization of crude metabolite suggested its hydrophobic and peptide nature. Inhibition of Xanthomonas campestris pv. phaseoli by peptide like metabolite produced by Paenibacillus polymyxa strain HKA-15 under in vitro conditions showed ecological and biotechnological potential of strain HKA-15 to control common blight disease in beans.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae

Xanthomonas phaseoli pv. manihotis (Xpm) and X. cassavae (Xc) are two bacterial pathogens attacking cassava. Cassava bacterial blight (CBB) is a systemic disease caused by Xpm, which might have dramatic effects on plant growth and crop production. Cassava bacterial necrosis is a nonvascular disease caused by Xc with foliar symptoms similar to CBB, but its impacts on the plant vigour and the crop are limited. In this review, we describe the epidemiology and ecology of the two pathogens, the impacts and management of the diseases, and the main research achievements for each pathosystem. Because Xc data are sparse, our main focus is on Xpm and CBB.     

Differential Esterase Expression in Leaves of Manihot esculenta Crantz Infected with Xanthomonas axonopodis pv. manihotis

The polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases in plants and to show a differential expression of esterases as markers of pathogenesis in cassava plants (Manihot esculenta Crantz). The characterization of alpha- and beta-esterases from leaves of M. esculenta by the PAGE system was possible using an extraction solution containing two phenol-complexing agents (PVP-40 and sodium metabisulfite), three antioxidant agents (EDTA, beta-mercaptoethanol, and DTT), and one quinone reducer (ascorbic acid). Fourteen esterase isozymes were detected in young unexpanded leaves of M. esculenta cultivars. The inhibition pattern of alpha- and beta-esterases of M. esculenta showed that Est-9 is an arylesterase, and in the unexpanded leaves of the M. esculenta plants infected with Xanthomonas axonopodis pv. manihotis, the Est-7 beta-esterase showed the characteristic staining of an alpha/beta-esterase. This diffrential expression of Est-7 isozyme in young unexpanded leaves of cassava plants can be used as a marker of pathogenesis after infection with X. axonopodis pv. manihotis.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.     

Systemic Migration and Establishment of Xanthomonas campestris pv. pruni in Plum Leaves and Twigs

The systemic migration of Xanthomonas campestris pv. pruni (Xcp) through vascular bundles of leaves and twigs of plum was investigated. A rifampicin-resistant strain of Xcp was inoculated into leaves located midway from the tip of new green twigs of Golden King plum trees in a glasshouse. High numbers of the pathogen were recovered 4 and 8 weeks after inoculation from sections of uninoculated and symptomless veins, petioles, and twig tissue. Symptoms of bacterial spot developedwithin 8 weeks on main and secondary veins of uninoculated leaves located as far as 13 cm from the inoculated leaf on the same twig. Weekly isolation indicated the constant presence of Xcp in apparently unaffected shields of twig tissue obtained from a naturally infected Golden King orchard. Xcp apparently enters plum twigs through veins of infected leaves and migrates systemically through twigs to leaves.     

十字花科黑腐病菌hrpG基因原核表达

在十字花科黑腐病菌(Xcc)中,hrp基因对寄主的致病性和非寄主的超敏反应中起核心作用,而hrpG对整个hrp基因簇起调控作用.HrpG为OmpR家族的双组分系统感受调控蛋白,含有两个结构域,分别是N端Response_reg和C端Trans reg_C.本研究利用表达载体pQE-30 Xa,成功构建了HrpG的表达重组子,在E.coli M15 [pREP4]中进行诱导表达.通过调节诱导温度、IPTG浓度和诱导时间最终确定在温度为20℃,IPTG浓度为0.8 mmol/L,诱导表达4 h.hrpG基因在宿主细胞E.coli M15获得高效可溶性表达.目前尚未有可溶性HrpG蛋白获得成功表达的报导,本研究中获得HrpG蛋白在大肠杆菌获得大量可溶性的表达,将为in vitro研究HrpG的生理活性,特异的结合位点和调控功能研究打下良好基础.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.