Coker, Timothy L. R. (2016) Infection-site-specific responses of Arabidopsis thaliana to the biotrophic oomycete Hyaloperonospora arabidopsidis. PhD thesis, University of Warwick.

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Abstract

Changes in gene expression form a crucial part of the plant response to infection, and whole-leaf expression profiling has been valuable in our understanding of the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, when studying the interaction between Arabidopsis and the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells, or local and systemic responses of a differing nature may become convoluted.

The aim of this project was to spatially dissect the transcriptional response to Hpa, allowing differentiation of local and more systemic responses. Fluorescence Activated Cell Sorting (FACS), utilising an infection-site-specific fluorescent marker ProDMR6::GFP, was performed in order to isolate Hpa-proximal and Hpa-distal cells from infected seedling samples, and global gene expression measured in these FACS-sorted samples using microarrays. When compared with an uninfected control, 278 transcripts were identified as differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many locally responding genes that were detected for the first time using our sensitive FACS technique.

A portion of locally-responding genes were selected for further study. The promoters of a subset of highly locally induced genes were selected to drive expression of Green Fluorescent Protein (GFP) as a marker of Hpa-contacting cells for further FACS experiments, and to further validate their localised induction. Although some evidence of localised induction was seen in these lines, further investigation is required. We also hypothesised that a number of locally-induced genes would have a functional influence on infection, and tested this through the use of genetic knockouts. Knockouts in 7 of these genes showed altered disease resistance or susceptibility, and the mechanism behind two of these genes was investigated through the use of microarrays.

Overall, the use of FACS to study the Arabidopsis response to Hpa on a spatial scale has allowed identification of new genes with a putative role in the Arabidopsis-Hpa interaction, and contributes to a systems-level understanding of plant-pathogen interactions.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Arabidopsis thaliana -- Disease and pest resistance, Plant-microbe relationships, Oomycetes, Gene expression, Flow cytometry
Official Date:	September 2016
Dates:	Date Event September 2016 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Gifford, Miriam L. ; Beynon, Jim
Sponsors:	Engineering and Physical Sciences Research Council
Format of File:	pdf
Extent:	140 leaves : illustrations, charts
Language:	eng

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