Limits...
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

In vitro growth of Pseudomonas syringae pv. phaseolicola transposon mutants.Transposon mutants and wild type (WT) strains were inoculated in LB broth with shaking for 24 hr and OD600 recorded. Phenotypic screens: Sw, swarming reduction; S, small colony; L, large colony; Bf, biofilm formation. 14-, Pph 1448A; 13-, Pph 1302A. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556710&req=5

pone.0137355.g005: In vitro growth of Pseudomonas syringae pv. phaseolicola transposon mutants.Transposon mutants and wild type (WT) strains were inoculated in LB broth with shaking for 24 hr and OD600 recorded. Phenotypic screens: Sw, swarming reduction; S, small colony; L, large colony; Bf, biofilm formation. 14-, Pph 1448A; 13-, Pph 1302A. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.

Mentions: A more manageable number of Tn mutants (22) were selected for further investigation (Table 4), based on representation of each phenotypic screen, changes in their response on inoculated bean pods and some mutants with gene knockouts in known virulence factors. We first tested the in vitro growth of the mutants compared to the WT to determine whether the mutations fundamentally altered bacterial growth and to provide the foundation for in planta population growth analysis. The mutants were grown in liquid culture (LB broth) and their OD600 was measured after 24 hours (Fig 5). We observed that a number of the small colony mutants grew to a similar density to the WT indicating that small colony phenotype was not exclusively due to altered growth rate. However, several mutants were significantly different to the WT. Mutants 14–4.44, 14–5.32 and 14–7.09 which have gene disruptions annotated as carA, carB and pyrB all showed very low growth in liquid culture. Genes carA, carB and pyrB are involved in pyrimidine biosynthesis [38, 39]. Mutant 14–7.80 has a disruption in pyrD that is annotated as a dihydroorotate dehydrogenase, which is also involved in pyrimidine biosynthesis, but this does not appear to have affected in vitro growth as much as disruptions in the other genes.


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)

In vitro growth of Pseudomonas syringae pv. phaseolicola transposon mutants.Transposon mutants and wild type (WT) strains were inoculated in LB broth with shaking for 24 hr and OD600 recorded. Phenotypic screens: Sw, swarming reduction; S, small colony; L, large colony; Bf, biofilm formation. 14-, Pph 1448A; 13-, Pph 1302A. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137355.g005: In vitro growth of Pseudomonas syringae pv. phaseolicola transposon mutants.Transposon mutants and wild type (WT) strains were inoculated in LB broth with shaking for 24 hr and OD600 recorded. Phenotypic screens: Sw, swarming reduction; S, small colony; L, large colony; Bf, biofilm formation. 14-, Pph 1448A; 13-, Pph 1302A. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
Mentions: A more manageable number of Tn mutants (22) were selected for further investigation (Table 4), based on representation of each phenotypic screen, changes in their response on inoculated bean pods and some mutants with gene knockouts in known virulence factors. We first tested the in vitro growth of the mutants compared to the WT to determine whether the mutations fundamentally altered bacterial growth and to provide the foundation for in planta population growth analysis. The mutants were grown in liquid culture (LB broth) and their OD600 was measured after 24 hours (Fig 5). We observed that a number of the small colony mutants grew to a similar density to the WT indicating that small colony phenotype was not exclusively due to altered growth rate. However, several mutants were significantly different to the WT. Mutants 14–4.44, 14–5.32 and 14–7.09 which have gene disruptions annotated as carA, carB and pyrB all showed very low growth in liquid culture. Genes carA, carB and pyrB are involved in pyrimidine biosynthesis [38, 39]. Mutant 14–7.80 has a disruption in pyrD that is annotated as a dihydroorotate dehydrogenase, which is also involved in pyrimidine biosynthesis, but this does not appear to have affected in vitro growth as much as disruptions in the other genes.

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