scholarly journals Transposon Mutagenesis of Xylella fastidiosa by Electroporation of Tn5 Synaptic Complexes

2001 ◽  
Vol 14 (6) ◽  
pp. 701-706 ◽  
Author(s):  
Magalie R. Guilhabert ◽  
Les M. Hoffman ◽  
David A. Mills ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Xylella Fastidiosa ◽  
Insect Vector ◽  
Synaptic Complex ◽  
Tn5 Mutants ◽  
Transposon Mutants ◽  
Plant Pathogenicity ◽  
Synaptic Complexes ◽  
Insertion Sites ◽  
Tn5 Transposase ◽  
Major Impediment

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 × 103 and 4 × 103 Tn5 mutants per μg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

scholarly journals Identification of Xylella fastidiosa Antivirulence Genes: Hemagglutinin Adhesins Contribute to X. fastidiosa Biofilm Maturation and Colonization and Attenuate Virulence

2005 ◽  
Vol 18 (8) ◽  
pp. 856-868 ◽  
Author(s):  
Magalie R. Guilhabert ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Xylella Fastidiosa ◽  
Plant Diseases ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Transposon Tn5 ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
Major Impediment

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.

scholarly journals Site-Directed Mutagenesis Studies of Tn5 Transposase Residues Involved in Synaptic Complex Formation

2007 ◽  
Vol 189 (20) ◽  
pp. 7436-7441 ◽  
Author(s):  
Soheila Vaezeslami ◽  
Rachel Sterling ◽  
William S. Reznikoff
Keyword(s):  
Complex Formation ◽  
Catalytic Reactions ◽  
Site Directed Mutagenesis ◽  
Dna Interactions ◽  
Synaptic Complex ◽  
Biochemical Assays ◽  
Protein Dna Interactions ◽  
End Sequences ◽  
Tn5 Transposase ◽  
Cocrystal Structure

ABSTRACT Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.

scholarly journals Identification of Loci of Pseudomonas syringae pv. actinidiae Involved in Lipolytic Activity and Their Role in Colonization of Kiwifruit Leaves

2017 ◽  
Vol 107 (6) ◽  
pp. 645-653 ◽  
Author(s):  
Hitendra Kumar Patel ◽  
Patrizia Ferrante ◽  
Meng Xianfa ◽  
Sree Gowrinadh Javvadi ◽  
Sujatha Subramoni ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Lipid A ◽  
Lipolytic Activity ◽  
Genetic Screen ◽  
Economic Losses ◽  
In Planta ◽  
Tn5 Transposon ◽  
Tn5 Mutants ◽  
Transposon Mutants

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of P. syringae pv. actinidiae have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity because it is known that mechanisms of regulation, production, and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified P. syringae pv. actinidiae Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate–cysteine ligase, and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of P. syringae pv. actinidiae are presented and discussed.

scholarly journals Assembly of the RAG1/RAG2 Synaptic Complex

2002 ◽  
Vol 22 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Cynthia L. Mundy ◽  
Nadja Patenge ◽  
Adam G. W. Matthews ◽  
Marjorie A. Oettinger
Keyword(s):  
Strand Break ◽  
Synaptic Complex ◽  
Full Complement ◽  
Dna Elements ◽  
Strand Breakage ◽  
Synaptic Complexes ◽  
Recognition Motifs ◽  
Break Formation ◽  
Spacer Dna

ABSTRACT Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.

scholarly journals Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity

2017 ◽  
Vol 30 (11) ◽  
pp. 866-875 ◽  
Author(s):  
R. Caserta ◽  
R. R. Souza-Neto ◽  
M. A. Takita ◽  
S. E. Lindow ◽  
A. A. De Souza
Keyword(s):  
Transgenic Tobacco ◽  
Disease Severity ◽  
Host Plants ◽  
Xylella Fastidiosa ◽  
Ectopic Expression ◽  
Signal Molecules ◽  
Insect Vector ◽  
Expression Of Genes ◽  
Diffusible Signal Factor ◽  
Population Sizes

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

scholarly journals Aphrophoridae Role in Xylella fastidiosa subsp. pauca ST53 Invasion in Southern Italy

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1035
Author(s):  
Ugo Picciotti ◽  
Nada Lahbib ◽  
Valdete Sefa ◽  
Francesco Porcelli ◽  
Francesca Garganese
Keyword(s):  
Pest Management ◽  
Plant Pathogen ◽  
Southern Italy ◽  
Xylella Fastidiosa ◽  
Xylem Sap ◽  
Disease Spread ◽  
Insect Vector ◽  
Transmission Control ◽  
Olive Quick Decline Syndrome ◽  
Olive Trees

The Philaenus spumarius L. (Hemiptera Aphrophoridae) is a xylem-sap feeder vector that acquires Xylella fastidiosa subsp. pauca ST53 during feeding on infected plants. The bacterium is the plant pathogen responsible for olive quick decline syndrome that has decimated olive trees in Southern Italy. Damage originates mainly from the insect vector attitude that multiplies the pathogen potentialities propagating Xf in time and space. The principal action to manage insect-borne pathogens and to contain the disease spread consists in vector and transmission control. The analysis of an innovative and sustainable integrated pest management quantitative strategy that targets the vector and the infection by combining chemical and physical control means demonstrates that it is possible to stop the Xylella invasion. This review updates the available topics addressing vectors’ identification, bionomics, infection management, and induced disease by Xylella invasion to discuss major available tools to mitigate the damage consequent to the disease.

scholarly journals Rationally designed mariner vectors to allow functional genomic analysis of Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-site sequencing (TraDIS)

2018 ◽  
Author(s):  
Janine T Bossé ◽  
Yanwen Li ◽  
Leon G. Leanse ◽  
Liqing Zhou ◽  
Roy R Chaudhuri ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Insertion Site ◽  
Genomic Analysis ◽  
Actinobacillus Pleuropneumoniae ◽  
Transposase Gene ◽  
Functional Genomic Analysis ◽  
High Throughput Method ◽  
Transposon Mutants ◽  
Insertion Sites ◽  
First Time

AbstractTransposon Directed Insertion Sequencing (TraDIS) is a high-throughput method for mapping insertion sites in large libraries of transposon mutants. The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide. In this study, we generated two novel mariner vectors, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), in order to facilitate TraDIS identification of conditionally essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a near random distribution of insertions around the respective chromosomes. A preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, that are required for growth under anaerobic conditions.

scholarly journals Identification of New Candidate Pathogenicity Factors in the Xylem-Invading Pathogen Xanthomonas albilineans by Transposon Mutagenesis

2011 ◽  
Vol 24 (5) ◽  
pp. 594-605 ◽  
Author(s):  
Philippe Rott ◽  
Laura Fleites ◽  
Gary Marlow ◽  
Monique Royer ◽  
Dean W. Gabriel
Keyword(s):  
Outer Membrane ◽  
Disease Symptoms ◽  
Pathogenicity Factors ◽  
Membrane Fusion Protein ◽  
Tn5 Mutants ◽  
Insertion Sites ◽  
Outer Membrane Transporters ◽  
Genomic Dna Sequence ◽  
Xanthomonas Albilineans ◽  
Leaf Symptoms

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane ‘CP80-1743’ was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.

scholarly journals A Highly Efficient Transposon Mutagenesis System for the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

2001 ◽  
Vol 14 (11) ◽  
pp. 1312-1318 ◽  
Author(s):  
Oliver Kirchner ◽  
Karl-Heinz Gartemann ◽  
Eva-Maria Zellermann ◽  
Rudolf Eichenlaub ◽  
Annette Burger
Keyword(s):  
Transposon Mutagenesis ◽  
Saturation Mutagenesis ◽  
Clavibacter Michiganensis ◽  
Insertion Element ◽  
Suicide Vector ◽  
Clavibacter Michiganensis Subsp ◽  
Transposon Mutants ◽  
Insertion Sites ◽  
Tomato Pathogen ◽  
Electroporation Efficiency

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

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