Detection and Visualization of an Exopolysaccharide Produced by Xylella fastidiosa In Vitro and In Planta

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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Further In Vitro Assessment and Mid-Term Evaluation of Control Strategy of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy)

Pathogens ◽

10.3390/pathogens10010085 ◽

2021 ◽

Vol 10 (1) ◽

pp. 85

Author(s):

Giuseppe Tatulli ◽

Vanessa Modesti ◽

Nicoletta Pucci ◽

Valeria Scala ◽

Alessia L’Aurora ◽

...

Keyword(s):

Control Strategy ◽

Xylella Fastidiosa ◽

In Planta ◽

Bacterial Concentration ◽

Infected Area ◽

Olive Groves ◽

Term Evaluation ◽

In Vitro Assessment ◽

Olive Trees

During recent years; Xylella fastidiosa subsp. pauca (Xfp) has spread in Salento causing relevant damage to the olive groves. Measures to contain the spreading of the pathogen include the monitoring of the areas bordering the so-called “infected” zone and the tree eradication in case of positive detection. In order to provide a control strategy aimed to maintain the tree productivity in the infected areas, we further evaluated the in vitro and in planta mid-term effectiveness of a zinc-copper-citric acid biocomplex. The compound showed an in vitro bactericidal activity and inhibited the biofilm formation in representative strains of X. fastidiosa subspecies, including Xfp isolated in Apulia from olive trees. The field mid-term evaluation of the control strategy assessed by quantitative real-time PCR in 41 trees of two olive groves of the “infected” area revealed a low concentration of Xfp over the seasons upon the regular spraying of the biocomplex over 3 or 4 consecutive years. In particular, the bacterial concentration lowered in July and October with respect to March, after six consecutive treatments. The trend was not affected by the cultivar and it was similar either in the Xfp-sensitive cultivars Ogliarola salentina and Cellina di Nardò or in the Xfp-resistant Leccino. Moreover, the scoring of the number of wilted twigs over the seasons confirmed the trend. The efficacy of the treatment in the management of olive groves subjected to a high pathogen pressure is highlighted by the yielded a good oil production

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Expression of Pathogenicity-Related Genes of Xylella fastidiosa In Vitro and In Planta

Current Microbiology ◽

10.1007/s00284-004-4447-8 ◽

2005 ◽

Vol 50 (4) ◽

pp. 223-228 ◽

Cited By ~ 28

Author(s):

Alessandra A. de Souza ◽

Marco A. Takita ◽

Eridan O. Pereira ◽

Helvécio D. Coletta-Filho ◽

Marcos A. Machado

Keyword(s):

Xylella Fastidiosa ◽

In Planta

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection

Plant Disease ◽

10.1094/pdis-08-19-1748-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 724-730 ◽

Cited By ~ 2

Author(s):

Qing Ge ◽

Paul A. Cobine ◽

Leonardo De La Fuente

Keyword(s):

Biofilm Formation ◽

Inductively Coupled Plasma ◽

Tap Water ◽

Xylella Fastidiosa ◽

In Vitro Study ◽

Optical Emission ◽

In Planta ◽

Inductively Coupled ◽

High Concentrations

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

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Expression of the gum Operon Directing Xanthan Biosynthesis in Xanthomonas campestris and Its Regulation In Planta

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.6.768 ◽

2001 ◽

Vol 14 (6) ◽

pp. 768-774 ◽

Cited By ~ 51

Author(s):

Adrian A. Vojnov ◽

Holly Slater ◽

Michael J. Daniels ◽

J. Maxwell Dow

Keyword(s):

Xanthomonas Campestris ◽

Carbon Sources ◽

Regulatory System ◽

Extracellular Polysaccharide ◽

In Planta ◽

Glucuronidase Activity ◽

Gum Gene Cluster ◽

Diffusible Signal Factor ◽

Turnip Leaves

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of β-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.

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Immunochemical Analysis of UMP Kinase fromEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.3.833-840.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 833-840 ◽

Cited By ~ 17

Author(s):

Stéphanie Landais ◽

Pierre Gounon ◽

Christine Laurent-Winter ◽

Jean-Claude Mazié ◽

Antoine Danchin ◽

...

Keyword(s):

Monoclonal Antibody ◽

Polyclonal Antibodies ◽

Intact Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Membranes ◽

E Coli ◽

Specific Localization ◽

Ump Kinase ◽

Carboxy Terminal

ABSTRACT Mono- and polyclonal antibodies directed against UMP kinase fromEscherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in wholeE. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

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Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection

Phytopathology ◽

10.1094/phyto-02-16-0097-r ◽

2016 ◽

Vol 106 (8) ◽

pp. 928-936 ◽

Cited By ~ 6

Author(s):

Lindsey P. Burbank ◽

Drake C. Stenger

Keyword(s):

Copy Number ◽

Xylella Fastidiosa ◽

Expression Vectors ◽

Broad Host Range ◽

In Planta ◽

Plasmid Vectors ◽

Antibiotic Selection ◽

Shuttle Vectors ◽

Range Vector

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

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Chromosome-Based Genetic Complementation System for Xylella fastidiosa

Applied and Environmental Microbiology ◽

10.1128/aem.00024-09 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1679-1687 ◽

Cited By ~ 36

Author(s):

Ayumi Matsumoto ◽

Glenn M. Young ◽

Michele M. Igo

Keyword(s):

Dna Sequences ◽

Xylella Fastidiosa ◽

Cell Physiology ◽

Multiple Cloning Site ◽

Wild Type ◽

In Planta ◽

Catalase Peroxidase ◽

The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

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Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Molecules ◽

10.3390/molecules25112622 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2622

Author(s):

Aliah Zannierah Mohsin ◽

Rashidah Sukor ◽

Jinap Selamat ◽

Anis Shobirin Meor Hussin ◽

Intan Hakimah Ismail ◽

...

Keyword(s):

Binding Affinity ◽

Analytical Methods ◽

Polyclonal Antibodies ◽

Sequence Similarity ◽

High Specificity ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

High Sequence Similarity ◽

High Affinity

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

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Detection of a Single-Copy Plasmid, pXFSL21, in Xylella fastidiosa Strain Stag’s Leap with Two Toxin-Antitoxin Systems Using Next-Generation Sequencing

Phytopathology ◽

10.1094/phyto-07-18-0249-fi ◽

2019 ◽

Vol 109 (2) ◽

pp. 240-247 ◽

Cited By ~ 4

Author(s):

Christopher Van Horn ◽

Fengnian Wu ◽

Zheng Zheng ◽

Zehan Dai ◽

Jianchi Chen

Keyword(s):

Next Generation Sequencing ◽

Xylella Fastidiosa ◽

Bacterial Genome ◽

Illumina Miseq ◽

Single Copy ◽

Next Generation ◽

In Planta ◽

Term Survival ◽

Generation Sequencing

Plasmids are important genetic elements contributing to bacterial evolution and environmental adaptation. Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing economically devastating diseases such as Pierce’s disease (PD) of grapevine. In this study, the plasmid status of a highly virulent PD strain, Stag’s Leap, originally isolated from Napa Valley, CA, was studied using sequencing and bioinformatics tools. DNA samples extracted from a pure culture in periwinkle wilt medium (in vitro DNA) and a PD-symptomatic grapevine artificially inoculated in the greenhouse (in planta DNA) were subject to next-generation sequencing (NGS) analyses (Illumina MiSeq or HiSeq). Sequence analyses and polymerase chain reaction experiments revealed the presence of a circular plasmid, pXFSL21, of 21,665 bp. This plasmid existed as a single copy per bacterial genome under both in vitro and in planta conditions. Two toxin-antitoxin (T-A) systems (ydcD-ydcE and higB-higA) were detected in pXFSL21, a possible mechanism for the long-term survival of this single-copy plasmid in the bacterial population. BLAST searches against the GenBank database (version 222) detected homologs of the two T-A systems in chromosomes or plasmids of some X. fastidiosa strains. However, double T-A systems were found only in pXFSL21. pXFSL21 was not found in other known PD strains and, therefore, could serve as a molecular marker for strain Stag’s Leap monitoring and tracking. The NGS-based technique outlined in this article provides an effective tool for identifying single- or low-copy-number plasmids in fastidious prokaryotes.

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