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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

Screening of Pseudomonas syringae pv. phaseolicola 1448A transposon disruption mutants.Tn mutant colonies (48) were inoculated onto; A. standard agar plates and changes in colony morphology recorded after 72hr, B. soft agar plates and reduction in swarming ability recorded after 72hr. Selected mutants are highlighted with red arrows. WT, wild type.
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pone.0137355.g001: Screening of Pseudomonas syringae pv. phaseolicola 1448A transposon disruption mutants.Tn mutant colonies (48) were inoculated onto; A. standard agar plates and changes in colony morphology recorded after 72hr, B. soft agar plates and reduction in swarming ability recorded after 72hr. Selected mutants are highlighted with red arrows. WT, wild type.

Mentions: Tn mutant libraries (960 disruption mutants each) were made for Pph strains 1448A (causes disease on CW and TG) and 1302A (causes disease on CW and HR on TG) to screen for mutants that exhibited phenotypic changes. Firstly, colonies were screened for changes in colony morphology which may reflect changes in cell wall structure or motility that are important for colonisation of the plant. Pph1448A::Tn and 1302A::Tn libraries were replica plated (three replicates) onto KB plates, incubated at 25°C for 72 hr and visually compared to their equivalent WT that was also included on each 48 colony plate (Fig 1A). A total of 15 colonies for 1302A::Tn and 42 colonies for 1448A::Tn were observed as being consistently different, that is, either larger or smaller, to their WT equivalents (Table 2). The morphology of selected mutants was also confirmed by subsequently plating out the mutants individually: see Fig 2 for examples.


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)

Screening of Pseudomonas syringae pv. phaseolicola 1448A transposon disruption mutants.Tn mutant colonies (48) were inoculated onto; A. standard agar plates and changes in colony morphology recorded after 72hr, B. soft agar plates and reduction in swarming ability recorded after 72hr. Selected mutants are highlighted with red arrows. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137355.g001: Screening of Pseudomonas syringae pv. phaseolicola 1448A transposon disruption mutants.Tn mutant colonies (48) were inoculated onto; A. standard agar plates and changes in colony morphology recorded after 72hr, B. soft agar plates and reduction in swarming ability recorded after 72hr. Selected mutants are highlighted with red arrows. WT, wild type.
Mentions: Tn mutant libraries (960 disruption mutants each) were made for Pph strains 1448A (causes disease on CW and TG) and 1302A (causes disease on CW and HR on TG) to screen for mutants that exhibited phenotypic changes. Firstly, colonies were screened for changes in colony morphology which may reflect changes in cell wall structure or motility that are important for colonisation of the plant. Pph1448A::Tn and 1302A::Tn libraries were replica plated (three replicates) onto KB plates, incubated at 25°C for 72 hr and visually compared to their equivalent WT that was also included on each 48 colony plate (Fig 1A). A total of 15 colonies for 1302A::Tn and 42 colonies for 1448A::Tn were observed as being consistently different, that is, either larger or smaller, to their WT equivalents (Table 2). The morphology of selected mutants was also confirmed by subsequently plating out the mutants individually: see Fig 2 for examples.

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