Gene disruption by homologous recombination in the Xylella fastidiosa citrus variegated chlorosis strain.
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ABSTRACT:
SUBMITTER: Gaurivaud P
PROVIDER: S-EPMC124079 | biostudies-literature | 2002 Sep
REPOSITORIES: biostudies-literature
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Gene disruption by homologous recombination in the Xylella fastidiosa citrus variegated chlorosis strain.
Gaurivaud Patrice P Souza Leonardo C A LC Virgílio Andrea C D AC Mariano Anelise G AG Palma Renê R RR Monteiro Patrícia B PB
Applied and environmental microbiology 20020901 9
Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for beta-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to beta-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. ...[more]
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Project description:Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.
| S-EPMC142809 | biostudies-literature
Project description:Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants.
| S-EPMC4771316 | biostudies-literature
Project description:A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.
| S-EPMC3843690 | biostudies-literature
Project description:High rates of homologous recombination (HR) in the bacterial plant pathogen Xylella fastidiosa have been previously detected. This study aimed to determine the extent and explore the ecological significance of HR in the genomes of recombinants experimentally generated by natural transformation and wild-type isolates. Both sets of strains displayed widespread HR and similar average size of recombined fragments consisting of random events (2-10 kb) of inter- and intrasubspecific recombination. A significantly higher proportion and greater lengths (>10 kb, maximum 31.5 kb) of recombined fragments were observed in subsp. morus and in strains isolated in Europe from intercepted coffee plants shipped from the Americas. Such highly recombinant strains pose a serious risk of emergence of novel variants, as genetically distinct and formerly geographically isolated genotypes are brought in close proximity by global trade. Recently recombined regions in wild-type strains included genes involved in regulation and signaling, host colonization, nutrient acquisition, and host evasion, all fundamental traits for X. fastidiosa ecology. Identification of four recombinant loci shared between wild-type and experimentally generated recombinants suggests potential hotspots of recombination in this naturally competent pathogen. These findings provide insights into evolutionary forces possibly affecting the adaptive potential to colonize the host environments of X. fastidiosa.
| S-EPMC6776109 | biostudies-literature
Project description:BackgroundThe xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains - which is particularly important for CVC-associated strains.ResultsThis paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria.ConclusionResults from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10% increase in our current knowledge of the South American Xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. Surprisingly, this list of novel elements include sequences previously believed to be unique to North American strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct Xf strains.
| S-EPMC2262912 | biostudies-literature
Project description:Xylella fastidiosa is an economically important bacterial plant pathogen. With insights gained from 72 genomes, this study investigated differences among the three main subspecies, which have allopatric origins: X. fastidiosa subsp. fastidiosa, multiplex, and pauca The origin of recombinogenic X. fastidiosa subsp. morus and sandyi was also assessed. The evolutionary rate of the 622 genes of the species core genome was estimated at the scale of an X. fastidiosa subsp. pauca subclade (7.62 × 10-7 substitutions per site per year), which was subsequently used to estimate divergence time for the subspecies and introduction events. The study characterized genes present in the accessory genome of each of the three subspecies and investigated the core genome to detect genes potentially under positive selection. Recombination is recognized to be the major driver of diversity in X. fastidiosa, potentially facilitating shifts to novel plant hosts. The relative effect of recombination in comparison to point mutation was calculated (r/m = 2.259). Evidence of recombination was uncovered in the core genome alignment; X. fastidiosa subsp. fastidiosa in the United States was less prone to recombination, with an average of 3.22 of the 622 core genes identified as recombining regions, whereas a specific clade of X. fastidiosa subsp. multiplex was found to have on average 9.60 recombining genes, 93.2% of which originated from X. fastidiosa subsp. fastidiosa Interestingly, for X. fastidiosa subsp. morus, which was initially thought to be the outcome of genome-wide recombination between X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, intersubspecies homologous recombination levels reached 15.30% in the core genome. Finally, there is evidence of X. fastidiosa subsp. pauca strains from citrus containing genetic elements acquired from strains infecting coffee plants as well as genetic elements from both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex In summary, our data provide new insights into the evolution and epidemiology of this plant pathogen.IMPORTANCEXylella fastidiosa is an important vector-borne plant pathogen. We used a set of 72 genomes that constitutes the largest assembled data set for this bacterial species so far to investigate genetic relationships and the impact of recombination on phylogenetic clades and to compare genome content at the subspecies level, and we used a molecular dating approach to infer the evolutionary rate of X. fastidiosa The results demonstrate that recombination is important in shaping the genomes of X. fastidiosa and that each of the main subspecies is under different selective pressures. We hope insights from this study will improve our understanding of X. fastidiosa evolution and biology.
| S-EPMC6581164 | biostudies-literature
Project description:With the aid of a biochip, carrying representative sequences from approximately 2200 sequences from the genome of isolate 9a5c from X. fastidiosa (Xf), microarray-based comparisons have been performed with 6 different Xf isolates obtained from citrus plants (Table 1). Four of these isolates (56a, 9.12c, 187b, and 36f) were obtained from CVC-affected trees and are representatives of the most prevalent Xf haplotypes found in sweet orange orchards across the state of São Paulo, while isolate CV21 was obtained from a non-symptomatic tree from the same region. Isolate Fb7, on the other hand, was obtained from a sweet orange tree that displayed symptoms of “Pecosita”, a disease similar to CVC that occurs in some citrus-growing regions of Argentina. Keywords: Comparative Genomic Hybridization
2007-07-18 | GSE8493 | GEO
Project description:Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.
| S-EPMC3591940 | biostudies-literature
Project description:Proteins containing PDZ domains are involved in a large number of biological functions, including protein scaffolding, organization of ion channels, and signal transduction. We recently identified a novel PDZ domain-containing protein, PDZK1, that is selectively expressed in normal tissues, where it is associated and colocalized with MAP17, a small 17-kDa membrane-associated protein; cMOAT, an organic anion transporter implicated in multidrug resistance; and the type IIa Na/Pi cotransporter. The protein cluster formed by PDZK1, MAP17, and cMOAT is upregulated in a significant number of human carcinomas originating in the colon, breast, lung, and kidney. In order to better define the function of PDZK1 in the protein cluster and its potential role in the organization of ion channels, we generated a PDZK1 knockout mouse. While PDZK1-deficient mice developed normally, did not display any gross phenotypic abnormalities, and were fecund, lack of PDZK1 resulted in modulation of expression of selective ion channels in the kidney, as well as increased serum cholesterol levels. However, no significant redistribution of proteins known to interact with PDZK1, such as MAP17, cMOAT, and the type IIa Na/Pi cotransporter, was observed. The absence of a more significant phenotype in PDZK1-deficient mice may be due to functional compensation by other PDZ domain-containing proteins, which could be instrumental in determining the location of interacting proteins such as ion channels and other membrane-associated proteins in defined areas of the plasma membrane.
| S-EPMC141144 | biostudies-literature
Project description:Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM(2) and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.
| S-EPMC3036931 | biostudies-literature