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Title: Quantum pathogen evolution by integron-mediated effector capture
Author: Payne, Joseph David
ISNI:       0000 0004 6497 0241
Awarding Body: University of the West of England
Current Institution: University of the West of England, Bristol
Date of Award: 2017
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Plant pathogenic Pseudomonads are responsible for the loss of millions of pounds in crop revenue each year. They export effector molecules via the type three secretion system into the plants’ cells in order to elicit disease. If the plant has the corresponding resistance genes to detect the type three effector molecule then the plant will mount an immune response called the plant hypersensitive response (HR). Type three effector molecules can also supress the plants’ immune response including pathogen associated molecular pattern triggered immunity and effector triggered immunity. Pseudomonads can evade HR by potentially gaining different effector molecules using mobile DNA elements. Integrons are one such type of element. Integrons are elements that allow bacteria to acquire and store genes from the environment particularly during times of stress. They also allow differential expression of the captured genes dependent on the environmental conditions. Integron-like elements (ILEs) within Pseudomonas syringae pathovars and other Pseudomonads can be identified by using conserved genes such as the xerC integrase and the UV damage repair gene rulB. RulB encodes a DNA polymerase V which appears to be a hotspot for ILE insertion. Using the rulAB operon, the xerC gene and the ILE insertion junction, rulB-xerC, it was possible to identify a number of ILEs. The screening of 164 plant pathogenic Pseudomonas strains revealed new ILEs from 21 strains all containing at least one type three effector molecule. The screening also revealed that the xerC integrase was conserved across multiple ILEs within plant pathogens. Expression studies of the ILE integrase genes, type three effector genes and the disrupted rulB gene showed that the genes on both ILEs present in P. syringae pv. pisi 203 and pv. syringae 3023 are upregulated in times of cellular stress and DNA damage. This led to the conclusion that ILEs may be more active when the bacteria was in need of exogenous genes to overcome the cellular stress. The ILE may also be excised following DNA damage to restore full rulB functionality. It was identified that rulB was a hotspot for ILE insertion but it was not known why the ILEs choose this site or if any other genes were required for ILE insertion. Cloned versions of the rulAB operon from the pWW0 plasmid found in Pseudomonas putida PaW340 showed that only rulAB was required for P. fluorescens ILE insertion but rulAB must be intact. P. syringae ILEs were also tested but did not show any insertion. Due to ILEs inserting into and disrupting rulB their effect on UV tolerance was tested. A range of strains containing an intact rulB gene were tested alongside the ILE containing strains with increasing amounts of UVB irradiation applied. The results showed very minor differences in growth rates between the two groups with only one UVB irradiation amount of 60 seconds causing a significant difference in growth rate at the 95% confidence interval between the two groups of strains. This research has contributed to the understanding of ILEs in phytopathogenic bacteria. It has also increased our understanding of the mechanisms of ILE gene expression, the mechanism surrounding ILE excision and insertion and the effect of ILEs on bacterial growth in high UV environments.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pseudomonas ; pathogen ; effector ; integron ; evolution ; Pseudomonas syringae