Rhynchosporium commune is one of the most destructive pathogens of barley worldwide. It can cause crop yield losses of up to 40% in the UK and decrease in grain quality. Populations of R. commune can change rapidly, defeating new barley resistance (R) genes and fungicides after just a few seasons of their use. Fungicide use is one of the major modes of management of Rhynchosporium and is heavily relied on the agricultural industry. Fungicides that were effective in the past are no longer effective in controlling the disease and many are only effective when used in mixtures. Beyond the currently effective fungicides there is limited new chemistry available so there is a very real need for development in this area. In pathogenic fungi, the cell wall components play a key role in the establishment of pathogenesis. The cell wall forms the outer structure protecting the fungus from the host defence mechanisms. It is involved in initiating the direct contact with the host cells by adhering to their surface. The fungal cell wall also contains important antigens and other compounds modulating host immune responses. R. commune germinated conidia and interaction transcriptome sequencing generated a list of over 30 different cell wall proteins (CWPs) potentially involved in pathogenicity. R. commune genome and interaction transcriptome sequencing provided further information about the extent of CWP families as well as a subset of genes expressed during barley colonisation by R. commune. The use of bioinformatic techniques allowed for the analysis of gene sequences. Putative cell wall associated genes were compared to the sequences from the fungal database via sequence similarity, sequence alignments 15 and conserved domain searches to better understand their function. Phylogenetic analysis also allowed us to understand the evolutionary relationship between R. commune genes and related genes in other organisms. Transcription profiling of R. commune CWPs during the development of infection helped to prioritise them for functional characterisation. Targeted gene disruption unfortunately did not yield mutants but has furthered our understanding of this technique in R. commune for future attempts. Functional complementation was successful however and allowed the uncovering of the function of RSA9. The results show that R. commune RSA9 functions as an allantoicase, an enzyme which breaks down purines as a source of nitrogen when conditions are nitrogen limited. The use of chemical cell wall inhibitors allowed us to better understand the role of carbohydrate cell wall components in R. commune fitness and virulence. Inhibition of cellulose production by DCB showed reduced growth, germination and pathogenicity of R. commune. Similar results were observed when beta-glucan synthesis was impaired; as inhibitor concentration increased, growth and germination of the fungus decreased. The composition of R. commune cell wall was also uncovered during this research. Techniques such as HPLC and FTIR eluded the composition of monosaccharides and polysaccharides respectively. In addition the structure of R. commune cell wall was observed by microscopy, namely TEM. This project revealed some much needed information on the R. commune cell wall and the relation of its components to fitness and virulence during infection of barley.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:646091 |
| Date | January 2014 |
| Creators | Mackenzie, Ashleigh |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=225718 |
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