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.
Highlights
Pseudomonas syringae is a Gram-negative plant pathogenic bacterial species with multiple pathovars that cause a number of diseases of a wide range of plants, but can exist out of the PLOS ONE | DOI:10.1371/journal.pone.0137355 September 1, 2015In Vitro Transposon Screen of Bacteria for Plant Colonisation Genes host plant in non-agricultural environments including rivers and snow pack [1]
Pph1448A::Tn and 1302A::Tn libraries were replica plated onto KB plates, incubated at 25°C for 72 hr and visually compared to their equivalent wild type (WT) that was included on each 48 colony plate (Fig 1A)
The mutant libraries as above were screened for strains exhibiting altered ability to swarm on soft agar, to identify genes potentially involved in spreading motility, which has been shown to be important for virulence on the plant [25]
Summary
Pseudomonas syringae is a Gram-negative plant pathogenic bacterial species with multiple pathovars that cause a number of diseases of a wide range of plants, but can exist out of the PLOS ONE | DOI:10.1371/journal.pone.0137355 September 1, 2015In Vitro Transposon Screen of Bacteria for Plant Colonisation Genes host plant in non-agricultural environments including rivers and snow pack [1]. Pseudomonas syringae is a Gram-negative plant pathogenic bacterial species with multiple pathovars that cause a number of diseases of a wide range of plants, but can exist out of the PLOS ONE | DOI:10.1371/journal.pone.0137355. Pph is typically associated with causing disease lesions in the leaf of bean. Like most P. syringae strains, Pph usually exhibits an epiphytic lifecycle phase, existing on leaf surfaces [4, 5]. The pathogen will rapidly colonise the plant tissue, usually making polysaccharides and forming biofilms [7]. The pathogen spreads through the apoplast to colonise uninfected tissue. The spreading lesions will cause bacteria to remerge onto the external surfaces of the plants where they can be dispersed
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