About the Project
As the protein factory of the cell, the endoplasmic reticulum (ER) underpins the production, folding and quality control of proteins, as well as lipid biosynthesis. It forms a motile and pervasive network of tubules and cisternae that extends throughout the cytoplasm and symplastically across cellular boundaries via plasmodesmata (Hawes et al., 2015). During pathogen infection the demand for de novo protein and lipid biosynthesis increases significantly. This necessitates rapid but highly regulated ER expansion and/or remodelling, together with an enhanced protein folding capacity, as part of a successful host defence strategy.
Since the ER is, therefore, critical to the perception and regulation of adaptive host responses to biotic stress, it is also a prime target for manipulation by the pathogen. Such offensive tactics are primarily orchestrated through the secretion of a diverse arsenal of effector molecules designed to target and manipulate multiple host components and pathways to aid pathogen virulence. To date, however, few ER-targeted phytopathogenic effectors have been fully characterised in terms of identifying their specific host target or mechanism of action (McLellan et al., 2013; Meisrimler et al., 2019).
Using a bioinformatic approach we have recently identified a subset of ER-localised effectors from the economically significant oomycete species Phytophthora infestans, (the causal agent of potato late blight), which share a similar protein topology. We are currently in the process of characterising the host targets of these P. infestans effectors, together with other identified ER-localised effectors from closely related oomycete pathogens, including one which may be involved in ER-chloroplast communication.
We have also performed timeseries imaging experiments to capture the spatial and temporal changes in ER morphology in response to infection with a virulent pathovar of the bacterial phytopathogen, Pseudomonas syringae. Initial analysis has revealed that gross morphological changes are manifested in the ER ~7-9 hours after infection with the bacteria. This is co-incident with initiation of bacterial multiplication and ultimately culminates in the rapid and complete collapse of the ER network by ~10-12 hours. These data firmly place ER remodelling at the forefront of an early, highly conserved and core pathogen virulence strategy.
This multidisciplinary project will focus on the study of i) ER-targeted effectors from multiple pathogen species with different lifestyles and disease strategies; and ii) the changes in ER morphology in response to infection by these pathogens. Specific objectives of the project are:
1. Detailed characterisation of the spatial and temporal remodelling of the ER architecture during biotic stress. This will involve performing in vivo confocal microscopy imaging experiments followed by quantitative image analysis of ER architecture and dynamics using the AnalyzER software developed by one of our collaborators, Prof Mark Fricker (University of Oxford) (Pain et al., 2019).
2. Identification and functional analysis of ER-targeted pathogen effectors. Putative ER effector proteins will initially be identified bioinformatically by applying our in silico screening procedure to the effectoromes of sequenced pathogen species. These candidates, and others, will then be characterised to determine their precise impact on the host.
Ultimately, the project aims to ascertain if it is possible to manipulate ER architecture dynamics at key stages in the infection process in order to improve plant disease resistance. Given the existing global pre- and post-harvest crop losses of ~25% from pests and disease, the development of novel approaches to generate crops with enhanced tolerance to plant pathogens is a key research priority.
BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Plant and Crop Science
Techniques that will be undertaken during the project:
• Confocal microscopy and quantitative image analysis
• Cell biology techniques
• Molecular cloning and generation of novel plant reporter lines
• Whole plant image analysis (eg chlorophyll fluorescence; luciferase and ROS assays).
• Microbiology techniques. Use, manipulation and maintenance of plant pathogens
Typical pattern of working hours:
37.5 hrs per week with flexible working arrangements
McLellan, H. et al. (2013). An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus. PLoS Pathog 9: e1003670.
Meisrimler, C.N. et al. (2019). Multiple downy mildew effectors target the stress‐related NAC transcription factor Ls NAC069 in lettuce. Plant J 99: 1098–1115.
Pain, C. et al. (2019). Quantitative analysis of plant ER architecture and dynamics. Nature Communications: 1–15.
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