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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

Complementation of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.Genes identified through transposon (Tn) insertion were cloned into a broad host range vector and transformed into their respective mutant strain (TnC). An empty vector was also transformed into the strains to use as a control (TnE). A number of tests were carried out with these strains: A.in vitro growth in LB broth after 16hrs; B.in planta growth in bean cultivar Canadian Wonder after 2 days; C. colony size after 2 days incubation, shown at the same magnification; D. swarming ability in soft agar after 5 days incubation, shown at the same magnification. 14-, Pph 1448A; 13- Pph 1302A; CHP, conserved hypothetical protein. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
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pone.0137355.g006: Complementation of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.Genes identified through transposon (Tn) insertion were cloned into a broad host range vector and transformed into their respective mutant strain (TnC). An empty vector was also transformed into the strains to use as a control (TnE). A number of tests were carried out with these strains: A.in vitro growth in LB broth after 16hrs; B.in planta growth in bean cultivar Canadian Wonder after 2 days; C. colony size after 2 days incubation, shown at the same magnification; D. swarming ability in soft agar after 5 days incubation, shown at the same magnification. 14-, Pph 1448A; 13- Pph 1302A; CHP, conserved hypothetical protein. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.

Mentions: To validate our approach and confirm that the phenotypes observed from the mutants were due to the mutated genes, complementation experiments were undertaken. We focused on four mutants that showed significant differences to WT growth in planta and represented a selection of the phenotypes we had identified. For example one of these, the 1302A::fliO mutant, showed enhanced growth in planta despite still causing an HR. Each gene (open reading frame plus some flanking sequence) was amplified from the WT strain and cloned into broad host range vector pBBR1MCS-5 before electroporation into the mutant strain. An empty vector was also used as a control in all mutants tested. Complementation to WT phenotypes (or near WT) was observed for all four mutants tested, while the empty vector did not affect the mutant phenotypes, confirming that the gene mutated by the Tn was responsible for the change in phenotype (Fig 6; S3 Table).


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)

Complementation of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.Genes identified through transposon (Tn) insertion were cloned into a broad host range vector and transformed into their respective mutant strain (TnC). An empty vector was also transformed into the strains to use as a control (TnE). A number of tests were carried out with these strains: A.in vitro growth in LB broth after 16hrs; B.in planta growth in bean cultivar Canadian Wonder after 2 days; C. colony size after 2 days incubation, shown at the same magnification; D. swarming ability in soft agar after 5 days incubation, shown at the same magnification. 14-, Pph 1448A; 13- Pph 1302A; CHP, conserved hypothetical protein. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137355.g006: Complementation of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.Genes identified through transposon (Tn) insertion were cloned into a broad host range vector and transformed into their respective mutant strain (TnC). An empty vector was also transformed into the strains to use as a control (TnE). A number of tests were carried out with these strains: A.in vitro growth in LB broth after 16hrs; B.in planta growth in bean cultivar Canadian Wonder after 2 days; C. colony size after 2 days incubation, shown at the same magnification; D. swarming ability in soft agar after 5 days incubation, shown at the same magnification. 14-, Pph 1448A; 13- Pph 1302A; CHP, conserved hypothetical protein. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
Mentions: To validate our approach and confirm that the phenotypes observed from the mutants were due to the mutated genes, complementation experiments were undertaken. We focused on four mutants that showed significant differences to WT growth in planta and represented a selection of the phenotypes we had identified. For example one of these, the 1302A::fliO mutant, showed enhanced growth in planta despite still causing an HR. Each gene (open reading frame plus some flanking sequence) was amplified from the WT strain and cloned into broad host range vector pBBR1MCS-5 before electroporation into the mutant strain. An empty vector was also used as a control in all mutants tested. Complementation to WT phenotypes (or near WT) was observed for all four mutants tested, while the empty vector did not affect the mutant phenotypes, confirming that the gene mutated by the Tn was responsible for the change in phenotype (Fig 6; S3 Table).

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