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Detection and visualization of an exopolysaccharide produced by Xylella fastidiosa in vitro and in planta.

Department of Plant Pathology, University of California, Davis, Davis, CA 95616, USA.
Applied and Environmental Microbiology (Impact Factor: 3.68). 12/2007; 73(22):7252-8. DOI: 10.1128/AEM.00895-07
Source: PubMed

ABSTRACT Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.

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    ABSTRACT: Exopolysaccharides (EPS) synthesized by plant pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared to the wild type. Furthermore, GC-MS chromatography showed that both mutant strains did not produce oligosaccharides. Biologically the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects, they were very poorly transmitted by and retained within vectors. Gene expression profile indicates up regulation of genes implicated in cell-to-cell signaling and adhesins while down-regulation in genes required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in Xylella fastidiosa interactions with both plants and insects.
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    ABSTRACT: Xylella fastidiosa is a Gram-negative, xylem-limited bacterium that causes a lethal disease of grapevine called Pierce's disease. Lipopolysaccharide (LPS) comprises approximately 75% of the outer membrane of Gram-negative bacteria, and because it is largely displayed on the cell surface, it mediates interactions between the bacterial cell and its surrounding environment. LPS is comprised of a conserved lipid A-core oligosaccharide component and a variable O-antigen portion. By targeting a key O-antigen biosynthetic gene, we demonstrate the contribution of the rhamnose-rich O-antigen to surface attachment, cell-cell aggregation and biofilm maturation: critical steps for successful infection of the host xylem tissue. Moreover, we have demonstrated that a fully formed O-antigen moiety is an important virulence factor for Pierce's disease development in grape and that depletion of the O-antigen compromises its ability to colonize the host. It has long been speculated that cell surface polysaccharides play a role in X. fastidiosa virulence and this study confirms that LPS is a major virulence factor for this important agricultural pathogen.
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    ABSTRACT: Eal is an EAL-domain protein in X. fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP-domain proteins are implicated in the hydrolysis of the secondary messenger cyclic di-GMP. Cell density dependent communication mediated by Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both will affect virulence, was thus addressed. eal was induced by sub-inhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hyper-motility, reduced biofilm formation and hyper-virulence to grape. Consistent with that, the rpfF mutant was more susceptible to tobramycin than the wild type strain. Therefore, we propose that both cell-cell communication and antibiotic stress apparently can lead to similar modulations of cyclic di-GMP in X. fastidiosa resulting in similar phenotypes. However, the effect of cell density is dominant to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.
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