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 Ralstonia solanacearum Needs Flp Pili for Virulence on Potato

2012 ◽  
Vol 25 (4) ◽  
pp. 546-556 ◽  
Author(s):  
Charles K. Wairuri ◽  
Jacquie E. van der Waals ◽  
Antoinette van Schalkwyk ◽  
Jacques Theron
Keyword(s):  
Ralstonia Solanacearum ◽  
Type Iv Pili ◽  
Periodontal Pathogen ◽  
Wild Type ◽  
In Planta ◽  
Phytopathogenic Bacterium ◽  
Type Iv ◽  
Plant Pathogenesis

Type IV pili are virulence factors in various bacteria. Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Although type IVa pili have been implicated in the virulence of Ralstonia solanacearum, type IVb pili have not previously been described in this plant pathogen. Here, we report the characterization of two distinct tad loci in the R. solanacearum genome. The tad genes encode functions necessary for biogenesis of the Flp subfamily of type IVb pili initially described for the periodontal pathogen Aggregatibacter actinomycetemcomitans. To determine the role of the tad loci in R. solanacearum virulence, we mutated the tadA2 gene located in the megaplasmid that encodes a predicted NTPase previously reported to function as the energizer for Flp pilus biogenesis. Characterization of the tadA2 mutant revealed that it was not growth impaired in vitro or in planta, produced wild-type levels of exopolysaccharide galactosamine, and exhibited swimming and twitching motility comparable with the wild-type strain. However, the tadA2 mutant was impaired in its ability to cause wilting of potato plants. This is the first report where type IVb pili in a phytopathogenic bacterium contribute significantly to plant pathogenesis.

scholarly journals Mannitol 1-Phosphate Metabolism Is Required for Sporulation in Planta of the Wheat Pathogen Stagonospora nodorum

2005 ◽  
Vol 18 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Peter S. Solomon ◽  
Kar-Chun Tan ◽  
Richard P. Oliver
Keyword(s):  
Insertional Mutagenesis ◽  
Redox Regulation ◽  
Expressed Sequence Tag ◽  
Stagonospora Nodorum ◽  
Resonance Spectroscopy ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Mutant Strains

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wildtype strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.

scholarly journals Isolation and Characterization of Xenorhabdus nematophila Transposon Insertion Mutants Defective in Lipase Activity against Tween

2009 ◽  
Vol 191 (16) ◽  
pp. 5325-5331 ◽  
Author(s):  
Gregory R. Richards ◽  
Eugenio I. Vivas ◽  
Aaron W. Andersen ◽  
Delmarie Rivera-Santos ◽  
Sara Gilmore ◽  
...  
Keyword(s):  
Lipase Activity ◽  
Metabolic Pathways ◽  
Lipase Production ◽  
Xenorhabdus Nematophila ◽  
Transposon Insertion ◽  
Isolation And Characterization ◽  
Transposon Mutants ◽  
Conserved Gene ◽  
Insertion Mutants

ABSTRACT We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

scholarly journals Chromosome-Based Genetic Complementation System for Xylella fastidiosa

2009 ◽  
Vol 75 (6) ◽  
pp. 1679-1687 ◽  
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.

scholarly journals Identification and characterization of Photorhabdus temperata mutants altered in hemolysis and virulence

2016 ◽  
Vol 62 (8) ◽  
pp. 657-667
Author(s):  
Christine Chapman ◽  
Louis S. Tisa
Keyword(s):  
Hemolytic Activity ◽  
Entomopathogenic Nematode ◽  
Rna Turnover ◽  
Transposon Insertion ◽  
Physiological Properties ◽  
Delayed Initiation ◽  
Transposon Mutants ◽  
Photorhabdus Temperata

Photorhabdus temperata is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora and an insect pathogen. This bacterium produces a wide variety of virulence factors and hemolytic activity. The goal of this study was to identify hemolysin-defective mutants and test their virulence. A genetic approach was used to identify mutants with altered hemolytic activity by screening a library of 10 000 P. temperata transposon mutants. Three classes of mutants were identified: (i) defective (no hemolytic activity), (ii) delayed (delayed initiation of hemolytic activity), and (iii) early (early initiation of hemolytic activity). The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis and motility. The hemolysin-defective mutants, P10A-C11, P10A-H12, and P79-B5, had inserts in genes involved in RNA turnover (RNase II and 5′-pentaphospho-5′-adenosine pyrophosphohydrolase) and showed reduced virulence and production of extracellular factors. These data support the role of RNA turnover in insect pathogenesis and other physiological functions.

scholarly journals Phenotypic and Phylogenetic Characterization of Cu Homeostasis among Xylella fastidiosa Strains

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 495
Author(s):  
Qing Ge ◽  
Ranlin Liu ◽  
Paul A. Cobine ◽  
Neha Potnis ◽  
Leonardo De La Fuente
Keyword(s):  
Core Genome ◽  
Xylella Fastidiosa ◽  
Bacterial Pathogen ◽  
Antimicrobial Treatment ◽  
Plant Diseases ◽  
Phylogenetic Characterization ◽  
Cu Homeostasis ◽  
Geographical Locations

Xylella fastidiosa is a bacterial pathogen causing severe diseases and asymptomatic colonization in more than 600 plants worldwide. Copper (Cu) is a widely used antimicrobial treatment for various plant diseases, including those affecting X. fastidiosa hosts. Cu homeostasis among X. fastidiosa strains from different geographical locations and host species has not been characterized. Here, we assessed minimum inhibitory concentration (MIC) of Cu for 54 X. fastidiosa strains. We observed strain-level variation in MIC values within each subspecies. We hypothesized that these differences could be explained by sequence variation in Cu homeostasis genes. Phylogenies based on copA, copB, copL, and cutC were created using 74 genomes (including 43 strains used in vitro) of X. fastidiosa, showing that the phylogenetic clustering of Cu homeostasis associated with clustering was based on core genome phylogenies, rather than on pattern of MIC. No association was found among Cu MIC, subspecies classification, and host and location of isolation, probably due to uneven and limited group of strains whose genomes are available. Further analysis focused on a subgroup of isolates from Georgia’s vineyards that shared similar Cu-related phenotypes. Further research is needed to better understand the distribution of Cu homeostasis for this pathogen.

scholarly journals Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12405
Author(s):  
Viet Tru Nguyen ◽  
Nanami Sakata ◽  
Giyu Usuki ◽  
Takako Ishiga ◽  
Yoshiteru Hashimoto ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Bacterial Blight ◽  
Mutational Analysis ◽  
Expression Profiles ◽  
Critical Role ◽  
Wild Type ◽  
In Planta ◽  
Bacterial Populations ◽  
Transposon Mutants ◽  
Soybean Leaves

Pseudomonas savastanoi pv. glycinea (Psg) causes bacterial blight of soybean. To identify candidate virulence factors, transposon-mediated mutational analysis of Psg was carried out. We syringe-inoculated soybean leaves with Psg transposon mutants and identified 28 mutants which showed reduced virulence from 1,000 mutants screened. Next, we spray-inoculated soybean leaves with these mutants and demonstrated that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. Expression profiles comparison between the Psg wild-type (WT) and algU mutant in HSC broth revealed that expression of coronatine (COR)-related genes (including cmaA and corR) were down-regulated in the algU mutant compared with Psg WT. Moreover, we also showed that COR production were reduced in the algU mutant compared with WT. We also demonstrated that algD, which is related to alginate biosynthesis, showed reduced expression and biofilm formation was significantly suppressed in the algU mutant. Furthermore, hrpL also showed less expression in the algU mutant. These results indicate that AlgU plays a critical role in promoting Psg pathogenesis by regulating multiple virulence factors.

scholarly journals Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

2011 ◽  
Vol 24 (10) ◽  
pp. 1198-1206 ◽  
Author(s):  
Luciana Cursino ◽  
Cheryl D. Galvani ◽  
Dusit Athinuwat ◽  
Paulo A. Zaini ◽  
Yaxin Li ◽  
...  
Keyword(s):  
Xylella Fastidiosa ◽  
Severe Disease ◽  
Type Iv Pili ◽  
Plant Diseases ◽  
Twitching Motility ◽  
In Planta ◽  
Disease Manifestation ◽  
Phytopathogenic Bacterium ◽  
Type Iv ◽  
Genes Encoding

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

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 Virulence of Pseudomonas syringae pv. tomato DC3000 Is Influenced by the Catabolite Repression Control Protein Crc

2017 ◽  
Vol 30 (4) ◽  
pp. 283-294 ◽  
Author(s):  
Suma Chakravarthy ◽  
Bronwyn G. Butcher ◽  
Yingyu Liu ◽  
Katherine D’Amico ◽  
Matthew Coster ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Rna Binding ◽  
Plant Diseases ◽  
Wild Type ◽  
In Planta ◽  
Growth And Survival ◽  
Control Protein

Pseudomonas syringae infects diverse plant species and is widely used as a model system in the study of effector function and the molecular basis of plant diseases. Although the relationship between bacterial metabolism, nutrient acquisition, and virulence has attracted increasing attention in bacterial pathology, it is largely unexplored in P. syringae. The Crc (catabolite repression control) protein is a putative RNA-binding protein that regulates carbon metabolism as well as a number of other factors in the pseudomonads. Here, we show that deletion of crc increased bacterial swarming motility and biofilm formation. The crc mutant showed reduced growth and symptoms in Arabidopsis and tomato when compared with the wild-type strain. We have evidence that the crc mutant shows delayed hypersensitive response (HR) when infiltrated into Nicotiana benthamiana and tobacco. Interestingly, the crc mutant was more susceptible to hydrogen peroxide, suggesting that, in planta, the mutant may be sensitive to reactive oxygen species generated during pathogen-associated molecular pattern–triggered immunity (PTI). Indeed, HR was further delayed when PTI-induced tissues were challenged with the crc mutant. The crc mutant did not elicit an altered PTI response in plants compared with the wild-type strain. We conclude that Crc plays an important role in growth and survival during infection.

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