Xanthomonas albilineans

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Philippe Rott - One of the best experts on this subject based on the ideXlab platform.

  • Identification of pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by Transposon Mutagenesis
    2020
    Co-Authors: Philippe Rott, Monique Royer, Gary C Marlow, Laura Fleites, Dean W Gabriel
    Abstract:

    Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In addition to the previously identified albicidin biosynthesis gene ciuster mutations, 33 new loci were identified in which insertions were correlated with reduced pathogenicity. These insertions affected genes predicted to encode proteins involved in a variety of functions, inciuding exopolysaccharide and lipopolysaccharide biosynthesis, fatty acid biosynthesis, regulatory and cell signaling, and secretion systems. Several of these have been associated with virulence in other bacterial plant pathogens that invade the xylem. However, sorne loci were identified that are predicted to encode previously unrecognized and apparently essential pathogenicity factors, at least for sugarcane leaf scald, inciuding an OmpA family outer membrane protein. Five independent TnS insertions in OmpA locus XALc_0557 of X albilineans strain XaFL07-1 produced no or very few leaf symptoms. These mutants produced albicidin in vitro and were able to multiply in sugarcane leaf tissue to levels similar to the wild-type strain, but did not efficiently colonize the sugarcane stalk. These ompA mutants were also affected in growth rate, motility and biofilm formation in vitro. (Resume d'auteur)

  • Towards identification of genes involved in pathogenicity of Xanthomonas albilineans, the pathogen causing sugarcane leaf scald
    2020
    Co-Authors: Patrice Champoiseau, Jeanheinrich Daugrois, Isabelle Pieretti, Monique Royer, Stephane Cociancich, Philippe Rott
    Abstract:

    The hrp or avr genes found in most plant pathogenic bacteria were not found in Xanthomonas albilineans, the causal agent of sugarcane leaf scald. This pathogen produces, however, a pathotoxin called albicidin that is involved in disease symptom development. Recently, all genes involved in albicidin biosynthesis were cloned and sequenced. Variation in these genes was, however, not correlated with variation in pathogenicity of X. albilineans. In this study, we attempted to identify new pathogenicity genes of X. albilineans using several approaches and 19 strains of the pathogen differing in disease severity and stalk colonization in Guadeloupe. The in vitro production of albicidin varied among strains of X. albilineans, but all strains showed the same RFLP (restriction fragment length polymorphism) pattern with albicidin biosynthetic genes and the same Spel PFGE (pulsed-field gel electrophoresis) pattern. In contrast, variation among strains was found by AFLP (amplified fragment length polymorphism) with 16 selective primer combinations, after enzymatic digestion of total genomic 'DNA with Sacl and Mspl. No relationship between this genetic variation and variation in pathogenicity was, however, identified. A phylogenetic study with housekeeping genes ihfA and efp suggested that X. albilineans is on an evolutionary road between the Xanthomonas campestris group and Xylella fastidiosa, another vascular plant pathogen that does not possess hrp or avr genes. Sequencing of the whole genome of X. albilineans, in progress at Genoscope/France, will be the next step in unravelling pathogenicity of this sugarcane pathogen. (Texte integral)

  • Towards identification of pathogenicity genes specific to Xanthomonas albilineans strains closely associated with sugarcane leaf disease outbreaks
    2020
    Co-Authors: Isabelle Pieretti, Philippe Rott, Monique Royer, Stephane Cociancich, Sandrine Duplan, Dean W Gabriel
    Abstract:

    Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, one of the major diseases of sugarcane. Interestingly, this pathogen lacks both the xanthan gum genes cluster and a type III secretion system (T3SS) of the Hrp1 and Hrp2 injectisome families (1). X. albilineans produces a unique and specific toxin, albicidin, which is a powerful DNA gyrase inhibitor. Consequently, albicidin blocks chloroplasts differentiation, resulting in leaf scald symptoms. High genetic and pathogenic variability exists among strains of X. albilineans, and 10 genetic groups were identified by pulsed field gel electrophoresis (PFGE). All strains involved in sugarcane leaf scald disease outbreaks since the late 1980s belong to the same genetic group called PFGE-B, whereas the strains isolated previously belong to other groups, especially to group PFGE-A. These two groups were also revealed by multilocus sequence analysis (MLSA) using seven housekeeping genes (groEL, dnaK, gyrB, atpD, efp, recA and glnA). The complete genome sequence of strain GPE PC73 belonging to PFGE-B is now available (1). To better understand the genetic differences between the PFGE-A and PFGE-B strains, Suppression Subtractive Hybridization (SSH) analysis was performed to compare the genomes of XaFL07-1 (PFGE-B) and Xa23R1 (PFGE-A), both isolated in Florida. SSH is a method used to identify DNA fragments that are uniquely found in one strain when compared with another, closely related bacterial strain (2,3). We enriched a library of unique DNA sequences from strain XaFL07-1 (tester strain), using Xa23R1 DNA as the driver strain. A total of 188 XaFL07-1-specific clones were generated and sequenced. Sequences were all compared against the GPE PC73 genome and against the GenBank non redundant database (NCBI). Initial screening focused on 12 genes with potential pathogenicity function and for which SSH data were confirmed by PCR and Southern blot hybridization. These included a DNA methyltransferase, a chemotaxis protein, a permease, a CRISPR protein, and an Rhs protein. However, after further distribution studies of these genes among strains of X. albilineans representing the genetic diversity observed in this pathogen, only one gene encoding a DNA (cytosine-5)-methyltransferase was found to be specific to PFGE-B strains. In GPE PC73, this gene is localized in a 53 kb chromosome region of phage origin that includes also two other DNA methyltransferases. SSH often results in identification of phage DNA (3); DNA methyltransferases are known to play a role in genetic regulation by modifying the binding of DNA polymerases on promoters. Additional PCR and Southern blot screening of all SSH clones, including genes with unknown functions, could also lead to the identification of other pathogenicity genes specific to PFGE-B strains of X. albilineans. Knockout mutagenesis of the DNA methyltransferases and of other SSH candidate genes will be performed to investigate the role of these genes in pathogenicity of Xanthomonas albilineans strains closely associated with sugarcane leaf scald disease outbreaks. (Texte integral)

  • identification and characterization of Xanthomonas albilineans causing sugarcane leaf scald in china using multilocus sequence analysis
    Plant Pathology, 2019
    Co-Authors: Mbuya Sylvain Ntambo, Philippe Rott, Monique Royer, Jianyu Meng, Huili Zhang
    Abstract:

    Xanthomonas albilineans is the causal agent of leaf scald, a disease that can cause considerable damage to sugarcane industries. This study analysed the phylogenetic relationship of 14 samples of X. albilineans from China and 13 reference strains retrieved from the GenBank database by multilocus sequence analysis (MLSA). To reach this goal, five housekeeping genes of X. albilineans were amplified from diseased leaves and sequenced: gyrB, abc, rpoD, atpD and glnA. Based on the concatenated sequence of these genes (4473 nt), the 14 samples of X. albilineans from China had 99.9–100% sequence identity with one another and with five strains of the pathogen from the French West Indies and the USA (Florida). The 27 samples or strains of X. albilineans were distributed in two distinct clades in the MLSA‐based phylogenetic tree. Clade 1 was formed by four strains of the pathogen from Fiji, Papua New Guinea and the USA. All the other strains from worldwide locations, including the 14 samples from China, were grouped in clade 2. This latter clade included all strains of the pathogen that were associated with outbreaks of leaf scald that have occurred over the last two decades, especially in the Caribbean islands and the USA. The very low diversity of X. albilineans in four Chinese provinces suggests recent spread of a single strain (from genetic group PFGE‐B) of the leaf scald pathogen within China.

  • Comparison of loop-mediated isothermal amplification, polymerase chain reaction, and selective isolation assays for detection of Xanthomonas albilineans from sugarcane
    Tropical Plant Pathology, 2018
    Co-Authors: Vanessa Duarte Dias, Isabelle Pieretti, Emmanuel Fernandez, Marcos G. Cunha, Martha Hincapie, Philippe Roumagnac, Jack C. Comstock, Philippe Rott
    Abstract:

    A loop-mediated isothermal amplification (LAMP) assay was developed and compared to polymerase chain reaction (PCR), nested PCR, and selective isolation assays for detection of Xanthomonas albilineans , the causal agent of sugarcane leaf scald. The pathogen was isolated on selective medium from 44 out of 45 (98%) samples taken from symptomatic stalks, and from 44 out of 70 (63%) samples from asymptomatic stalks that were collected from plots with symptomatic stalks. Forty-two (93%), 41 (91%), and 42 (93%) symptomatic samples tested positive by LAMP, PCR and nested PCR, respectively. The pathogen was detected in 19 (27%), 8 (11%), and 25 (36%) of the 70 asymptomatic samples by LAMP, PCR and nested PCR, respectively. Symptomatic stalks were mainly, but not always, associated with high populations of the pathogen (10^7–10^9 CFU/ml), and asymptomatic stalks with low populations (

Carlos Vicente - One of the best experts on this subject based on the ideXlab platform.

  • Glycoproteins of sugarcane plants facilitate the infectivity of Ustilago scitaminea and Xanthomonas albilineans, two sugar- cane pathogens
    2020
    Co-Authors: María Blanch, Carlos Vicente
    Abstract:

    Sugarcane plants produce heterofructans composed of homofructan domains consisting of β-1,2-fructofuranoside chains which intercalate variable-length segments of polygalactitol. Sometimes, these heterofructans appear as the glycosidic moiety of glycoproteins. They are produced as a response to mechanical injuries and pathogen infections. These glycoproteins act as a defense mechanism against smut (Ustilago scitaminea) by inducing homotypic adhesion and by inhibiting teliospore germination. When smut teliospores are cultured on glycoproteins produced by resistant cultivars of sugarcane plants, the actin capping, which occurs before teliospore germination, is efficiently inhibited. Then, cell polarization is not achieved and the growth of germinative tube is completely inhibited. However, inoculation of sensitive cultivars with smut teliospores induce glycoprotein fractions that promote teliospore polarity after binding to their cell wall ligand, and are different from those obtained from resistant plants. On the other hand, leaf scald, a bacterial-vascular disease of sugar cane, has Xanthomonas albilineans as casual organism. The pathogen is confined mainly to the leaf and stalk vascular bundles, which are often partly or completely occluded with a gum-like substance, identified as a xanthan-like polysaccharide. This xanthan-like polysaccharide produced consists of a basal tetramer that is repeated to form the macromolecule. This basal tetrasaccharide is composed by two molecules of glucose, one mannose rest and a final glucuronic acid. Since xanthans contain glucuronic acid, the ability of these bacteria to produce an active UDP glucose dehydrogenase (the enzyme that produces UDP glucuronic acid from UDPG) is often seen as a virulence factor. Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose but the enzyme activity rapidly decays after hydrolysis of the enzyme by bacterial proteases. Thus, X. albilineans axenically cultured did not secrete xanthans to liquid media but the use of inoculated sugarcane tissues for producing and characterizing xanthans is absolutely required. Glycoproteins from sugarcane, the natural host of the bacterium, also assures the production of the active enzyme by inhibiting bacterial proteases.

  • Analysis of the xanthans, produced by Xanthomonas albilineans, by capillary electrophoresis: Identification of the product of reaction of an UDP-glucose dehydrogenase
    Analytical chemistry an Indian journal, 2020
    Co-Authors: MarÃÂía Blanch, María-estrella Legaz, Carlos Vicente
    Abstract:

    Leaf scald, a bacterial-vascular disease of sugar cane, has Xanthomonas albilineans as casual organism. The organism may invade the parenchyma between the bundles and produces reddened pockets of gum, identified as a xanthan. This xanthan consists of a basal tetramer composed by two molecules of glucose, one mannose rest and a final glucuronic acid that is highly repeated to formthe macromolecule. The occurrence of glucuronate rest in the polysaccharide requires the action of bacterial UDP-glucose dehydrogenase. In order to verify that the obtained protein is a true UDP-glucose dehydrogenase, the identification of theUDP-glucuronic acid like only product of reaction was necessary. The detection of the UDP-glucuronic acid has been carried out by Capillary Electrophoresis.

  • sugarcane glycoproteins may act as signals for the production of xanthan in the plant associated bacterium Xanthomonas albilineans
    Plant Signaling & Behavior, 2011
    Co-Authors: María-estrella Legaz, María Blanch, Blanca Fontaniella, Dolores Pinon, Yolanda Blanco, Mariateresa Solas, Rocio Santiago, Carlos Vicente
    Abstract:

    Visual symptoms of leaf scald necrosis in sugarcane (Saccharum officinarum) leaves develop in parallel to the accumulation of a fibrous material invading exocellular spaces and both xylem and phloem. These fibers are produced and secreted by the plant-associated bacterium Xanthomonas albilineans. Electron microscopy and specific staining methods for polysaccharides reveal the polysaccharidic nature of this material. These polysaccharides are not present in healthy leaves or in those from diseased plants without visual symptoms of leaf scald. Bacteria in several leaf tissues have been detected by immunogold labelling. The bacterial polysaccharide is not produced in axenic culture but it is actively synthesized when the microbes invade the host plant. This finding may be due to the production of plant glycoproteins after bacteria infection, which inhibit microbial proteases. In summary, our data are consistent with the existence of a positive feedback loop in which plant-produced glycoproteins act as a cell...

  • gluconacetobacter diazotrophicus a sugarcane endophyte inhibits xanthan production by sugarcane invading Xanthomonas albilineans
    Journal of Plant Interactions, 2010
    Co-Authors: Yolanda Blanco, María-estrella Legaz, Carlos Vicente
    Abstract:

    Abstract Gluconacetobacter diazotrophicus secretes to the culture medium a lysozyme, active against Xanthomonas albilineans. High molecular mass glycoproteins, obtained from sugarcane stalks, initially inhibit the secretion of the enzyme from bacteria to the culture medium, but it is actively secreted later. Alternatively, the production of these plant glycoproteins decreases when G. diazotrophicus is experimentally inoculated into sugarcane stalk segments. Xanthan production is achieved by the pathogen X. albilineans infecting sugarcane stalks but it was nullified by G. diazotrophicus when the endosymbiont is growing in plant tissues together the pathogen.

  • purification and properties of an unusual udp glucose dehydrogenase nadph dependent from Xanthomonas albilineans
    Microbiological Research, 2008
    Co-Authors: María Blanch, María-estrella Legaz, Carlos Vicente
    Abstract:

    Summary Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K m for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

Robert G Birch - One of the best experts on this subject based on the ideXlab platform.

  • A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans.
    Microbiology (Reading England), 2020
    Co-Authors: Guozhong Huang, L Zhang, Robert G Birch
    Abstract:

    Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted M:(r) 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS-NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus-acyl-CoA ligase (AL)-acyl carrier protein (ACP)-beta-ketoacyl synthase (KS)-beta-ketoacyl reductase (KR)-ACP-ACP-KS-peptidyl carrier protein (PCP)-condensation (C)-adenylation-PCP-C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS-NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.

  • functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans
    Applied Microbiology and Biotechnology, 2010
    Co-Authors: Saeed M Hashimi, Robert G Birch
    Abstract:

    Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.

  • dna gyrase from the albicidin producer Xanthomonas albilineans has multiple antibiotic resistance and unusual enzymatic properties
    Antimicrobial Agents and Chemotherapy, 2008
    Co-Authors: Saeed M Hashimi, Guozhong Huang, Anthony Maxwell, Robert G Birch
    Abstract:

    The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase. The X. albilineans gyrA and gyrB genes are not clustered with previously described albicidin biosynthesis and self-protection genes. The GyrA and GyrB products differ from Escherichia coli homologues through several insertions and through changes in several amino acid residues implicated in quinolone and coumarin resistance. Reconstituted X. albilineans DNA gyrase showed 20- to 25-fold-higher resistance than E. coli DNA gyrase to albicidin and ciprofloxacin and 8-fold-higher resistance to novobiocin in the supercoiling assay. The X. albilineans DNA gyrase is unusual in showing a high degree of distributive supercoiling and little DNA relaxation activity. X. albilineans GyrA (XaA) forms a functional gyrase heterotetramer with E. coli GyrB (EcB) and can account for albicidin and quinolone resistance and low levels of relaxation activity. XaB probably contributes to both coumarin resistance and the distributive supercoiling pattern. Although XaB shows fewer apparent changes relative to EcB, the EcA·XaB hybrid relaxed DNA in the presence or absence of ATP and was unable to supercoil. A fuller understanding of structural differences between albicidin-sensitive and -resistant gyrases may provide new clues into features of the enzyme amenable to interference by novel antibiotics.

  • a dha14 drug efflux gene from Xanthomonas albilineans confers high level albicidin antibiotic resistance in escherichia coli
    Journal of Applied Microbiology, 2006
    Co-Authors: J M Bostock, L Zhang, Guozhong Huang, Saeed M Hashimi, Robert G Birch
    Abstract:

    Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

  • A DHA14 drug efflux gene from Xanthomonas albilineans confers high‐level albicidin antibiotic resistance in Escherichia coli
    Journal of Applied Microbiology, 2006
    Co-Authors: J M Bostock, L Zhang, Guozhong Huang, Saeed M Hashimi, Robert G Birch
    Abstract:

    Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Dean W Gabriel - One of the best experts on this subject based on the ideXlab platform.

  • Towards identification of pathogenicity genes specific to Xanthomonas albilineans strains closely associated with sugarcane leaf disease outbreaks
    2020
    Co-Authors: Isabelle Pieretti, Philippe Rott, Monique Royer, Stephane Cociancich, Sandrine Duplan, Dean W Gabriel
    Abstract:

    Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, one of the major diseases of sugarcane. Interestingly, this pathogen lacks both the xanthan gum genes cluster and a type III secretion system (T3SS) of the Hrp1 and Hrp2 injectisome families (1). X. albilineans produces a unique and specific toxin, albicidin, which is a powerful DNA gyrase inhibitor. Consequently, albicidin blocks chloroplasts differentiation, resulting in leaf scald symptoms. High genetic and pathogenic variability exists among strains of X. albilineans, and 10 genetic groups were identified by pulsed field gel electrophoresis (PFGE). All strains involved in sugarcane leaf scald disease outbreaks since the late 1980s belong to the same genetic group called PFGE-B, whereas the strains isolated previously belong to other groups, especially to group PFGE-A. These two groups were also revealed by multilocus sequence analysis (MLSA) using seven housekeeping genes (groEL, dnaK, gyrB, atpD, efp, recA and glnA). The complete genome sequence of strain GPE PC73 belonging to PFGE-B is now available (1). To better understand the genetic differences between the PFGE-A and PFGE-B strains, Suppression Subtractive Hybridization (SSH) analysis was performed to compare the genomes of XaFL07-1 (PFGE-B) and Xa23R1 (PFGE-A), both isolated in Florida. SSH is a method used to identify DNA fragments that are uniquely found in one strain when compared with another, closely related bacterial strain (2,3). We enriched a library of unique DNA sequences from strain XaFL07-1 (tester strain), using Xa23R1 DNA as the driver strain. A total of 188 XaFL07-1-specific clones were generated and sequenced. Sequences were all compared against the GPE PC73 genome and against the GenBank non redundant database (NCBI). Initial screening focused on 12 genes with potential pathogenicity function and for which SSH data were confirmed by PCR and Southern blot hybridization. These included a DNA methyltransferase, a chemotaxis protein, a permease, a CRISPR protein, and an Rhs protein. However, after further distribution studies of these genes among strains of X. albilineans representing the genetic diversity observed in this pathogen, only one gene encoding a DNA (cytosine-5)-methyltransferase was found to be specific to PFGE-B strains. In GPE PC73, this gene is localized in a 53 kb chromosome region of phage origin that includes also two other DNA methyltransferases. SSH often results in identification of phage DNA (3); DNA methyltransferases are known to play a role in genetic regulation by modifying the binding of DNA polymerases on promoters. Additional PCR and Southern blot screening of all SSH clones, including genes with unknown functions, could also lead to the identification of other pathogenicity genes specific to PFGE-B strains of X. albilineans. Knockout mutagenesis of the DNA methyltransferases and of other SSH candidate genes will be performed to investigate the role of these genes in pathogenicity of Xanthomonas albilineans strains closely associated with sugarcane leaf scald disease outbreaks. (Texte integral)

  • Identification of pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by Transposon Mutagenesis
    2020
    Co-Authors: Philippe Rott, Monique Royer, Gary C Marlow, Laura Fleites, Dean W Gabriel
    Abstract:

    Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In addition to the previously identified albicidin biosynthesis gene ciuster mutations, 33 new loci were identified in which insertions were correlated with reduced pathogenicity. These insertions affected genes predicted to encode proteins involved in a variety of functions, inciuding exopolysaccharide and lipopolysaccharide biosynthesis, fatty acid biosynthesis, regulatory and cell signaling, and secretion systems. Several of these have been associated with virulence in other bacterial plant pathogens that invade the xylem. However, sorne loci were identified that are predicted to encode previously unrecognized and apparently essential pathogenicity factors, at least for sugarcane leaf scald, inciuding an OmpA family outer membrane protein. Five independent TnS insertions in OmpA locus XALc_0557 of X albilineans strain XaFL07-1 produced no or very few leaf symptoms. These mutants produced albicidin in vitro and were able to multiply in sugarcane leaf tissue to levels similar to the wild-type strain, but did not efficiently colonize the sugarcane stalk. These ompA mutants were also affected in growth rate, motility and biofilm formation in vitro. (Resume d'auteur)

  • surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves
    Molecular Plant Pathology, 2016
    Co-Authors: Imene Mensi, Jeanheinrich Daugrois, Dean W Gabriel, Isabelle Pieretti, Laura Fleites, Daniel Gargani, Julie Noell, Francois Bonnot, Philippe Rott
    Abstract:

    Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue-cultured plantlets grown in vitro. Six mutants of strain XaFL07-1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly-β-hydroxybutyrate than the wild-type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non-ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild-type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves. (Resume d'auteur)

  • Xanthomonas albilineans ompa1 appears to be functionally modular and both the omc and c like domains are necessary for leaf scald disease of sugarcane
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Laura Fleites, Philippe Rott, Imene Mensi, Shujian Zhang, Daniel Gargani, Dean W Gabriel
    Abstract:

    Several EZ-Tn5 insertions in gene locus XALc_0557 (OmpA1) of the sugarcane leaf scald pathogen Xanthomonas albilineans XaFL07-1 were previously found to strongly affect pathogenicity and endophytic stalk colonization. XALc_0557 has a predicted OmpA N-terminal outer membrane channel (OMC) domain and an OmpA C-like domain. Further analysis of mutant M468, with an EZ-Tn5 insertion in the upstream OMC domain coding region, revealed impaired epiphytic and endophytic leaf survival, impaired resistance to sodium dodecyl sulfate (SDS), structural defects in the outer membrane (OM), and hyperproduction of OM vesicles. Cloned full-length XALc_0557 complemented M468 for all phenotypes tested, including pathogenicity, resistance to SDS, and ability to survive both endophytically and epiphytically. Another construct, pCT47.3, which expressed only the C-like domain of XALc_0557, restored resistance to SDS in M468 but failed to complement any other mutant phenotype, indicating that the C-like domain functioned independe...

  • the rpfcg two component system negatively regulates the colonization of sugar cane stalks by Xanthomonas albilineans
    Microbiology, 2013
    Co-Authors: Philippe Rott, Laura Fleites, Imene Mensi, Lauren Sheppard, Jeanheinrich Daugrois, Dean W Gabriel
    Abstract:

    The genome of Xanthomonas albilineans, the causal agent of sugar cane leaf scald, carries a gene cluster encoding a predicted quorum sensing system that is highly related to the diffusible signalling factor (DSF) systems of the plant pathogens Xylella fastidiosa and Xanthomonas campestris. In these latter pathogens, a cluster of regulation of pathogenicity factors (rpf) genes encodes the DSF system and is involved in control of various cellular processes. Mutation of Xanthomonas albilineans rpfF, encoding a predicted DSF synthase, in Florida strain XaFL07-1 resulted in a small reduction of disease severity (DS). Single-knockout mutations of rpfC and rpfG (encoding a predicted DSF sensor and regulator, respectively) had no effect on DS or swimming motility of the pathogen. However, capacity of the pathogen to cause disease was slightly reduced and swimming motility was severely affected when rpfG and rpfC were both deleted. Similar results were obtained when the entire rpfGCF region was deleted. Surprisingly, when the pathogen was mutated in rpfG or rpfC (single or double mutations) it was able to colonize sugar cane spatially more efficiently than the wild-type. Mutation in rpfF alone did not affect the degree of spatial invasion. We conclude that the DSF signal contributes to symptom expression but not to invasion of sugar cane stalks by Xanthomonas albilineans strain XaFL07-1, which is mainly controlled by the RpfCG two-component system.

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  • Analysis of the xanthans, produced by Xanthomonas albilineans, by capillary electrophoresis: Identification of the product of reaction of an UDP-glucose dehydrogenase
    Analytical chemistry an Indian journal, 2020
    Co-Authors: MarÃÂía Blanch, María-estrella Legaz, Carlos Vicente
    Abstract:

    Leaf scald, a bacterial-vascular disease of sugar cane, has Xanthomonas albilineans as casual organism. The organism may invade the parenchyma between the bundles and produces reddened pockets of gum, identified as a xanthan. This xanthan consists of a basal tetramer composed by two molecules of glucose, one mannose rest and a final glucuronic acid that is highly repeated to formthe macromolecule. The occurrence of glucuronate rest in the polysaccharide requires the action of bacterial UDP-glucose dehydrogenase. In order to verify that the obtained protein is a true UDP-glucose dehydrogenase, the identification of theUDP-glucuronic acid like only product of reaction was necessary. The detection of the UDP-glucuronic acid has been carried out by Capillary Electrophoresis.

  • sugarcane glycoproteins may act as signals for the production of xanthan in the plant associated bacterium Xanthomonas albilineans
    Plant Signaling & Behavior, 2011
    Co-Authors: María-estrella Legaz, María Blanch, Blanca Fontaniella, Dolores Pinon, Yolanda Blanco, Mariateresa Solas, Rocio Santiago, Carlos Vicente
    Abstract:

    Visual symptoms of leaf scald necrosis in sugarcane (Saccharum officinarum) leaves develop in parallel to the accumulation of a fibrous material invading exocellular spaces and both xylem and phloem. These fibers are produced and secreted by the plant-associated bacterium Xanthomonas albilineans. Electron microscopy and specific staining methods for polysaccharides reveal the polysaccharidic nature of this material. These polysaccharides are not present in healthy leaves or in those from diseased plants without visual symptoms of leaf scald. Bacteria in several leaf tissues have been detected by immunogold labelling. The bacterial polysaccharide is not produced in axenic culture but it is actively synthesized when the microbes invade the host plant. This finding may be due to the production of plant glycoproteins after bacteria infection, which inhibit microbial proteases. In summary, our data are consistent with the existence of a positive feedback loop in which plant-produced glycoproteins act as a cell...

  • gluconacetobacter diazotrophicus a sugarcane endophyte inhibits xanthan production by sugarcane invading Xanthomonas albilineans
    Journal of Plant Interactions, 2010
    Co-Authors: Yolanda Blanco, María-estrella Legaz, Carlos Vicente
    Abstract:

    Abstract Gluconacetobacter diazotrophicus secretes to the culture medium a lysozyme, active against Xanthomonas albilineans. High molecular mass glycoproteins, obtained from sugarcane stalks, initially inhibit the secretion of the enzyme from bacteria to the culture medium, but it is actively secreted later. Alternatively, the production of these plant glycoproteins decreases when G. diazotrophicus is experimentally inoculated into sugarcane stalk segments. Xanthan production is achieved by the pathogen X. albilineans infecting sugarcane stalks but it was nullified by G. diazotrophicus when the endosymbiont is growing in plant tissues together the pathogen.

  • purification and properties of an unusual udp glucose dehydrogenase nadph dependent from Xanthomonas albilineans
    Microbiological Research, 2008
    Co-Authors: María Blanch, María-estrella Legaz, Carlos Vicente
    Abstract:

    Summary Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K m for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

  • Effect of leaf scald (Xanthomonas albilineans) on polyamine and phenolic acid metabolism of two sugarcane cultivars
    European Journal of Plant Pathology, 2007
    Co-Authors: Blanca Fontaniella, Carlos Vicente, Roberto Armas, María-estrella Legaz
    Abstract:

    Polyamine and phenolic acid levels as well as activities of some enzymes of their biosynthetic metabolism were examined in two sugarcane ( Saccharum officinarum ) cultivars differing in susceptibility to leaf scald, a disease caused by the bacterium, Xanthomonas albilineans. Juice obtained from both infected cultivars showed significantly increased levels of free putrescine and ornithine decarboxylase activity. However, the pathogen induced different changes in the two cultivars in subsequent metabolic steps. Whereas acid insoluble conjugated spermidine completely disappeared from the highly susceptible cv. C 439-52, an increase in acid insoluble conjugated polyamines was observed in the moderately susceptible cv. L 55-5. Phenolic acid metabolism also differed in the two cultivars. Since total phenolic acid content and phenylalanine ammonium lyase activity was greater in both cultivars after infection, distribution of phenolic acids between free or conjugated forms diverted into different pathways. The level of susceptibility of the two cultivars is discussed in terms of changes in these compounds.