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DNA microarray and mutational analysis to identify virulence genes in Xylella fastidiosa
2004
The development of successful management and control strategies of Pierces disease of grape requires the identification of virulence and pathogenicity genes and determining how they functions to control the disease development process. Based on the presumption that biofilm formation is a major pathogenicity factor of Xylella and that it may play a major role in the disease causing process, we have been studying the factors-genetic and environmental that affect biofilm formation by Xylella fastidiosa. We have identified that, Bovine serum albumen, a component of PW medium specifically inhibits biofilm formation in X. fastidiosa and that this inhibition is BSA concentration dependent. Because of its effect on the biofilm formation in vitro, we are studying the expression profiles of X. fastidiosa genes in the presence and absence of BSA in the media. We have also identified a global regulatory gene, rsmA (rsm = regulator of secondary metabolism) that control biofilm. An rsmA-deficient strain of X. fastidiosa forms more biofilm in vitro than the wild type. In a preliminary nylon membrane DNA macroarray experiment using about a 100 select candidate pathogenicity genes, we have determined an increased expression of 15 genes in the mutant when compared to the wild type parent. We are now using full genome microarrays of Xylella fastidiosa to catalogue the genes whose expressions are controlled by either rsmA or BSA. The results from these ongoing analyses using both approaches should help us catalogue X. fastidiosa genes which may be involved in pathogenicity and biofilm formation. Subsequent genetic analysis of the genes to be identified should give us some understanding of not only how pathogenicity is regulated in this bacterium but also how to tackle the problems posed by Pierces disease.
Site-directed gene disruption in Xylella fastidiosa
FEMS Microbiology Letters, 2002
As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa
Protein Expression and Purification, 2004
In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37°C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.
Genome and pathogenicity of Xylella fastidiosa
2001
The complete sequencing of the The complete sequencing of the Xylella fastidiosa Xylella fastidiosa genome was the first project of this kind genome was the first project of this kind to focus on a plant pathogen. The choice of this bacteria was tightly associated with its to focus on a plant pathogen. The choice of this bacteria was tightly associated with its importance as the causal importance as the causal agent of citrus variegated chlorosis (CVC). Adopting a sequencing agent of citrus variegated chlorosis (CVC). Adopting a sequencing strategy based on shotgun and cosmids, the project allowed a 10 fold coverage of the strategy based on shotgun and cosmids, the project allowed a 10 fold coverage of the genome. The specific mechanisms of pathogenicity are not genome. The specific mechanisms of pathogenicity are not yet clear while the annotation of yet clear while the annotation of the genome took in consideration several hypothesis. Based on the current pathogenicity the genome took in consideration several hypothesis. Based on the current pathogenicity hypothesis, the genes were sorted into different categories with special focus on hypothesis, the genes were sorted into different categories with special focus on those those associated with attachment to bacterial cells and different hosts, as well as genes related associated with attachment to bacterial cells and different hosts, as well as genes related to the adaptation capacity to the xylem conditions. Genes previously found only in animal to the adaptation capacity to the xylem conditions. Genes previously found only in animal pathogens were also pathogens were also observed in observed in X. fastidiosa X. fastidiosa, suggesting that they could share some of , suggesting that they could share some of the pathogenicity mechanisms. the pathogenicity mechanisms.
FEMS Microbiology Letters, 2003
Xylella fastidiosa is the causal agent of economically important plant diseases, including citrus variegated chlorosis and Pierce's disease. Hitherto, there has been no information on the molecular mechanisms controlling X. fastidiosa^plant interactions. To determine whether predicted open reading frames (ORFs) encoding putative pathogenicity-related factors were expressed by X. fastidiosa 9a5c cells grown at low (LCD) and high cell density (HCD) conditions in liquid modified PW medium, reverse Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed. Our results indicated that ORFs XF2344, XF2369, XF1851 and XF0125, encoding putative Fur, GumC, a serine-protease and RsmA, respectively, were significantly suppressed at HCD conditions. In contrast, ORF XF1115, encoding putative RpfF, was significantly induced at HCD conditions. Expressions of ORFs XF2367, XF2362 and XF0290, encoding putative GumD, GumJ and RpfA, respectively, were detected only at HCD conditions, whereas expression of ORF XF0287, encoding putative RpfB was detected only at LCD conditions. Bioassays with an Agrobacterium traG: :lacZ reporter system indicated that X. fastidiosa does not synthesize N-acyl-homoserine lactones, whereas bioassays with a diffusible signal factor (DSF)-responsive Xanthomonas campestris pv. campestris mutant indicate that X. fastidiosa synthesizes a molecule similar to DSF in modified PW medium. Our data also suggest that the synthesis of the DSF-like molecule and fastidian gum by X. fastidiosa is affected by cell density in vitro.
Molecular Plant-Microbe Interactions, 2012
In Xylella fastidiosa the fatty acid signal molecule diffusible signaling factor (DSF) is produced and sensed by components of the regulation of pathogenicity factors (rpf) cluster; lack of DSF production in RpfF mutants results in a nonvector-transmissible phenotype yet cells are hypervirulent to grape. rpfB has not been characterized in Xylella fastidiosa, although its homolog has been suggested to be required for DSF synthesis in Xanthomonas campestris pv. campestris. We show that RpfB is involved in DSF processing in both Xylella fastidiosa and Xanthomonas campestris, affecting the profile of DSF-like fatty acids observed in thin-layer chromatography. Although three fatty acids whose production is dependent on RpfF were detected in Xylella fastidiosa and Xanthomonas campestris wild-type strains, their respective rpfB mutants accumulated primarily one chemical species. Although no quantifiable effect of rpfB on plant colonization by Xylella fastidiosa was found, insect colonization and transmission was reduced. Thus, RpfB apparently is involved in DSF processing, and like Xanthomonas campestris, Xylella fastidiosa also produces multiple DSF molecules. It is possible that Xylella fastidiosa coordinates host vector and plant colonization by varying the proportions of different forms of DSF signals via RpfB.
Molecular Plant-Microbe Interactions, 2003
Using real time quantitative polymerase chain reaction, we verified that the colonization of FP cells was more efficient for both hosts. The sequence of the complete X. fastidiosa genome allowed the construction of a DNA microarray that was used to investigate the total changes in gene expression associated with the FP condition. Most genes found to be induced in the FP condition were associated with adhesion and probably with adaptation to the host environment. This report represents the first study of the transcriptome of this pathogen, which has recently gained more importance, since the genome of several strains has been either partially or entirely sequenced.