Molecular characterisation of the rhynchosporium commune interaction with barley (Phd)

Abstract

The interaction between Rhynchosporium commune and its host barley were studied to gain a better understanding of how the pathogen infects its host and to 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 domain containing protein (RcLysM3) were identified using a proteomic analysis of infected plant apoplast.  Functional assays were used to characterise one of the LysM domain containing proteins indicating its potential involvement in the evasion of plant immune responses.  Further analysis of the apoplast revealed some of the most abundant molecules 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 key players of pathogenesis.

A R. commune strain expressing green fluorescent 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. This study also identified R. commune strains recognised by barley genotypes containing Rrs3 (Abyssinian), Rrs4 (CI11549), and Rrs13 (BC Line30) resistance genes as well as two super virulent strains that overcome these resistances.

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.

Asymptomatic growth of R. commune on the model dicotyledonous plant Nicotiana benthamiana was shown to be confined to the leaf surface making it a good model for characterisation of non-host interactions.