Detection of Xylella fastidiosa from resistant and susceptible grapevine by tissue sectioning and membrane entrapment immunofluorescence.
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Immunofluorescence detection was performed by tissue sectioning and membrane entrapment of Xylella fastidiosa from the inoculated hybrid selection F8909-08 (Vitis rupestris A. de Serres x V. arizonica/candicans b43-17; resistant) and Chardonnay (susceptible). In both techniques, tissue sections and bacteria-trapped polycarbonate membranes were incubated with specific polyclonal IgG and stained with fluorescein isothiocyanate (FITC)-conjugated IgG from rabbits to X. fastidiosa cells. The stained preparations were observed by fluorescence microscopy. Rapid identification of the bacteria within 3 weeks post inoculation (wpi) was possible in thin cross sections of the petioles, which allowed penetration of the specific antibody. Examination of the bacteria over time was also possible, and allowed observation of bacterial multiplication and invasion of xylem vessels. The membrane entrapment technique was able to isolate bacteria at low concentrations in infected but asymptomatic plants. (+info)
Upstream migration of Xylella fastidiosa via pilus-driven twitching motility.
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Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient. (+info)
Multilocus simple sequence repeat markers for differentiating strains and evaluating genetic diversity of Xylella fastidiosa.
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A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics. (+info)
Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidios and colonization and attenuate virulence.
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Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels. (+info)
HTself: self-self based statistical test for low replication microarray studies.
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Different statistical methods have been used to classify a gene as differentially expressed in microarray experiments. They usually require a number of experimental observations to be adequately applied. However, many microarray experiments are constrained to low replication designs for different reasons, from financial restrictions to scarcely available RNA samples. Although performed in a high-throughput framework, there are few experimental replicas for each gene to allow the use of traditional or state-of-art statistical methods. In this work, we present a web-based bioinformatics tool that deals with real-life problems concerning low replication experiments. It uses an empirically derived criterion to classify a gene as differentially expressed by combining two widely accepted ideas in microarray analysis: self-self experiments to derive intensity-dependent cutoffs and non-parametric estimation techniques. To help laboratories without a bioinformatics infrastructure, we implemented the tool in a user-friendly website (http://blasto.iq.usp.br/~rvencio/HTself). (+info)
Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity.
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Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member. (+info)
SpotWhatR: a user-friendly microarray data analysis system.
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SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between "single experiment" and "batch processing" versions. (+info)
Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa.
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Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively. (+info)