AbstractThe molecular interaction between Rhynchosporium commune and its host barley was studied to gain a better understanding of the pathogen during infection and provide further characterisation of resistance in barley, using a combination of bioinformatics, transcript expression analysis, proteomics and confocal microscopy.
Expression analysis of potential effector sequences identified novel candidate effectors Rc_10934, Rc_2091 and Rc_2835 which showed the highest abundance during the biotrophic infection. A further two novel candidates Rc07_03591 and Rc07_02334 and a LysM containing protein (RcLysM3) were identified using a proteomic analysis of infected plant apoplast.
Further apoplastic analysis revealed some of the most abundant proteins that are present in R. commune’s infection toolkit. Cell wall degrading enzymes (CWDEs), virulence factors and proteins involved in detoxification were all highlighted as some of the main key players of pathogenesis.
A large family of LysM domain containing proteins was later identified in the R. commune genome. Expression profiling revealed the upregulation of some of the transcripts during infection, indicating a potential role in pathogenesis, whereas others were expressed in vitro indicating potential functions for the proteins in fungal growth and development. RcLysM3 containing 3 LysM domains and sharing similarities with the well-known C. fulvum Ecp6 effector was selected for further characterisation. Bioinformatics predictions showed a high affinity for chitin binding which was confirmed in vitro. Binding analysis revealed that it can also bind chitosan but not plant cell wall polysaccharides, indicating that it is potentially involved in the evasion of plant immune responses. The presence of the effector was also identified in the apoplast of infected barley leaves using a proteomic approach.
R. commune strain expressing GFP was used to characterise differences in pathogen growth and colony morphology in response to different genetic backgrounds of barley using lines carrying the Rrs3 (Abyssinian), Rrs4 (CI11549) and Rrs13 (BC line 30) genes and barley landraces with uncharacterised resistance.
Rrs1 resistance was further analysed using comparative proteomics to identify proteins differentially expressed in resistant and susceptible cultivars. Pathogenesis related proteins - chitinase, glucanase and thaumatin-like protease, were identified in the barley apoplastic fluid and were shown to be upregulated during infection. In addition, serine carboxypeptidase and purple acid phosphatase proteins were identified that were novel to the barley resistance interaction but have been identified in other incompatible interactions as defence related proteins.
The final chapter of this thesis is dedicated to the analysis of asymptomatic growth of R. commune on the model dicotyledonous plant N. benthamiana and analysis of effector transcription during growth on a non-host. R. commune growth was shown to be confined to the leaf surface, with no evidence of plant cell deterioration in transgenic N. benthamiana plants expressing an mRFP-tagged plasma membrane protein. This system could be used for further research into non-host interactions and provides insights into the growth of R. commune on alternative plant species.
|Date of Award||2016|
|Sponsors||HGCA & The James Hutton Institute|
|Supervisor||Paul Birch (Supervisor), Anna O. Avrova (Supervisor) & Adrian C. Newton (Supervisor)|