Control of Type III-mediated Virulence in Pseudomonas syringae by Cyclic-di-GMP

Ward, Danny (2021) Control of Type III-mediated Virulence in Pseudomonas syringae by Cyclic-di-GMP. Doctoral thesis, University of East Anglia.

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Pseudomonas syringae is a prominent plant pathogen and disease model organism. These bacteria carry out host infection using the type III secretion system (T3SS), which translocates effector proteins into target cells, altering cellular defence mechanisms and metabolism to promote bacterial colonisation. It was previously shown that the secondary signalling molecule cyclic-di-GMP (CdG) binds to the export ATPase complex at the base of the T3SS (HrcN in P. syringae), as well as in closely related homologue proteins. It was hypothesised that this binding interaction plays a role in controlling type III-mediated virulence.
To investigate the CdG:HrcN binding interaction, bacterial strains carrying mutations targeting key predicted CdG binding residues in HrcN were constructed. In vitro analyses of purified HrcN confirmed CdG binding and dodecamerisation for the wildtype. However, a G176A point-mutant of HrcN retained CdG binding but appeared to have compromised CdG-induced downstream oligomerisation. The effect of this mutation on virulence was therefore explored in vivo. Wildtype and mutant hrcN P. syringae pathovar tomato (Pto) DC3000 strains were infiltrated into Arabidopsis thaliana to evaluate disease phenotypes in planta. The G176A hrcN point-mutant exhibited a near-asymptomatic disease phenotype despite having a comparable bacterial load to the WT in A. thaliana Col-0. Disease symptoms returned in immunocompromised A. thaliana lines. The underlying mechanism was then explored. It was shown that a subset of tested effectors (HopAM1, HopAF1, and HopAA1-2) displayed compromised translocation rates for G176A hrcN compared to WT using an effector-CyaA reporter system in Pto. These effector proteins were shown to be important for disease symptom establishment by way of gene over-expression and gene deletion. Candidate interaction targets in the plant host were identified by co-immunoprecipitation.
From this study, first indication that CdG binding to HrcN in Pto may lead to dodecamerisation was shown, and that this interaction is important for full virulence by enabling for efficient translocation of key effector proteins.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Kitty Laine
Date Deposited: 01 Jun 2022 13:02
Last Modified: 01 Jun 2022 13:02

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