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: 3.67). 12/2007; 73(22):7252-8. DOI: 10.1128/AEM.00895-07
Source: PubMed


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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    • ", 2005 ) and the primary occluding structures in leaf veins ( Mollenhauer and Hopkins , 1976 ) . However , some reports have described a vessel - filling accumulation of gels in veins ( Fry and Milholland , 1990b ) and petioles ( Stevenson et al . , 2004a ) of infected grapevines . These authors suggested that these gels were of bacterial origin ( Roper et al . , 2007a ) due to their close association with X . fastidiosa cells . It is im - portant to note that the organs investigated in these studies ( i . e . grape leaf petioles , veins , and young stems ) contain mostly or only primary xylem tissues ."
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    ABSTRACT: Vascular occlusions are common structural modifications made by many plant species in response to pathogen infection. However, the functional role(s) of occlusions in host plant disease resistance/susceptibility remains controversial. This study focuses on vascular occlusions that form in stem secondary xylem of grapevines infected with Pierce's disease (PD) and the impact of occlusions on the hosts' water transport and the systemic spread of the causal bacterium Xylella fastidiosa in infected vines. Tyloses are the predominant type of occlusion that forms in grapevine genotypes with differing PD resistances. Tyloses form throughout PD-susceptible grapevines with over 60% of the vessels in transverse sections of all examined internodes becoming fully blocked. In sharp contrast, tylose development was mainly limited to a few internodes close to the point of inoculation in PD-resistant grapevines, impacting only 20% or less of the vessels. The extensive vessel blockage in PD-susceptible grapevines was correlated to an over 90% decrease in stem hydraulic conductivity, compared to an approximately 30% reduction in the stems of PD-resistant vines. Despite the systemic spread of X. fastidiosa in PD-susceptible grapevines, the pathogen colonized only 15% or less of the vessels in any internode and occurred in relatively small numbers, amounts much too small to directly block the vessels. Therefore we concluded that the extensive formation of vascular occlusions in PD-susceptible grapevines does not prevent the pathogen's systemic spread in them, but may significantly suppress the vines' water conduction, contributing to PD symptom development and the vines' eventual death.
    Full-text · Article · Jan 2013 · Plant physiology
    • "Interestingly , gene neighbourhood analysis suggested that xhpT participates in the regulation of EPS synthesis, because of its location directly upstream of the gum operon, which encodes proteins putatively involved in the biosynthesis of the EPS polymer, fastidian gum (da Silva et al., 2001). This polymer is a major component of the X. fastidiosa biofilm matrix in vitro and in planta (Roper et al., 2007b). This observation prompted us to investigate the role of XhpT as a regulator of EPS synthesis, as well as of phenotypes associated with other steps involved in biofilm formation. "
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    ABSTRACT: Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.
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    ABSTRACT: This project focuses on the lipopolysaccharide (LPS) component of the outer membrane of Xylella fastidiosa (Xf). In particular, we are investigating if the O-antigen portion of this molecule contributes to Xf surface attachment and biofilm formation. More importantly, by targeting genes involved in O-antigen biosynthesis, we will determine if LPS is an important virulence factor for Xf infection of grape. Additionally, we will determine if LPS contributes to the high level of host specificity observed for this pathogen. LAYPERSON SUMMARY Xylella fastidiosa (Xf) is a bacterium that has the ability to infect many different plant species. In some plants, this bacterium can cause serious disease. In grapevine, this disease is known as Pierce's disease (PD) and has caused millions of dollars of damage to the California grape industry alone. Xf species also infect other economically important crops such as almond, oleander and citrus. Interestingly, while all Xf isolates belong to the same group or species, some isolates can cause disease in one host but not another. For example, oleander strains cannot cause disease in grapevine and vice versa. One major goal of this project is to understand the bacterial mechanisms that dictate this high level of host specificity. We are focusing on a key component of the bacterial cell membrane, called lipopolysaccharide (LPS), and how certain parts of this molecule may be important in dictating host specificity. We are also investigating how the LPS molecule is related to bacterial virulence and other key aspects of the disease process, like attachment to the plant cell wall. This molecule makes up more than 70% of the bacterial membrane and if LPS does prove to be an important factor during Xf plant infection, its abundance in the bacterial cell membrane makes it a logical target for disease control. Furthermore, antimicrobial compounds that weaken the LPS molecule generally make the bacterium more sensitive to other stresses. Therefore, compounds targeted towards LPS synthesis could increase the efficacy of other anti-Xylella compounds currently being developed when both are used in conjunction.
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