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The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

Manoharan B, Neale HC, Hancock JT, Jackson RW, Arnold DL - PLoS ONE (2015)

Bottom Line: We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation.Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified.This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Bioscience, Faculty of Health and Applied Sciences, University of the West of England, Frenchay Campus, Bristol, BS16 1QY, United Kingdom.

ABSTRACT
The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

No MeSH data available.


Related in: MedlinePlus

Insertion points of transposon hits on a map of the circular chromosome of Pseudomonas syringae pv. phasiolicola 1448A.Outer two circles show positions of protein-coding genes on the plus and minus strands. Red dashes, Pph 1448A Tn hits (56); blue dashes, Pph 1302A Tn hits (41). The genome map was generated by using the DNAplotter [28].
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pone.0137355.g004: Insertion points of transposon hits on a map of the circular chromosome of Pseudomonas syringae pv. phasiolicola 1448A.Outer two circles show positions of protein-coding genes on the plus and minus strands. Red dashes, Pph 1448A Tn hits (56); blue dashes, Pph 1302A Tn hits (41). The genome map was generated by using the DNAplotter [28].

Mentions: From the phenotypic screens of the Tn mutant libraries, 160 mutants were selected as showing differences to the WT strain. The 160 selected mutants consisted of 106 individual Tn mutants as some mutants were selected in multiple screens. DNA sequences were obtained from 104 of the 106 mutants (we failed on multiple occasions to obtain sequence from two mutants) and the insertion points of the Tn in the P. syringae 1448A genome were determined (S2 Table). The Tn hits were plotted on to a genome map of Pph 1448A using DNAPlotter (Fig 4). The map of the Tn hits shows a diverse distribution of gene knockouts around the genome, with a Tn rich area around 3,900,000 bp, which corresponds to the flagella gene cluster.


The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

Manoharan B, Neale HC, Hancock JT, Jackson RW, Arnold DL - PLoS ONE (2015)

Insertion points of transposon hits on a map of the circular chromosome of Pseudomonas syringae pv. phasiolicola 1448A.Outer two circles show positions of protein-coding genes on the plus and minus strands. Red dashes, Pph 1448A Tn hits (56); blue dashes, Pph 1302A Tn hits (41). The genome map was generated by using the DNAplotter [28].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556710&req=5

pone.0137355.g004: Insertion points of transposon hits on a map of the circular chromosome of Pseudomonas syringae pv. phasiolicola 1448A.Outer two circles show positions of protein-coding genes on the plus and minus strands. Red dashes, Pph 1448A Tn hits (56); blue dashes, Pph 1302A Tn hits (41). The genome map was generated by using the DNAplotter [28].
Mentions: From the phenotypic screens of the Tn mutant libraries, 160 mutants were selected as showing differences to the WT strain. The 160 selected mutants consisted of 106 individual Tn mutants as some mutants were selected in multiple screens. DNA sequences were obtained from 104 of the 106 mutants (we failed on multiple occasions to obtain sequence from two mutants) and the insertion points of the Tn in the P. syringae 1448A genome were determined (S2 Table). The Tn hits were plotted on to a genome map of Pph 1448A using DNAPlotter (Fig 4). The map of the Tn hits shows a diverse distribution of gene knockouts around the genome, with a Tn rich area around 3,900,000 bp, which corresponds to the flagella gene cluster.

Bottom Line: We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation.Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified.This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Bioscience, Faculty of Health and Applied Sciences, University of the West of England, Frenchay Campus, Bristol, BS16 1QY, United Kingdom.

ABSTRACT
The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

No MeSH data available.


Related in: MedlinePlus