Secondary metabolites in the Parastagonospora nodorum-wheat pathosystem
Parastagonospora nodorum is a fungal pathogen and the causal agent of Septoria nodorum blotch (SNB), a disease of wheat responsible for significant yield losses around the globe. This pathogen can be easily cultured under laboratory conditions and genetically manipulated following standard procedures. Furthermore, its genome has been sequenced and several “omics” resources are available making P. nodorum a model organism to study necrotrophic fungal pathogens. It is now well recognised that this pathogen relies on the production of proteinaceous toxins (effectors) to drive the infection process. There are though many other facets of the SNB disease that are poorly understood compared to the effectors. Such is the case for the P. nodorum secondary metabolites (SMs). Bioinformatic analysis of the P. nodorum genome has revealed that it contains around 40 genes responsible for biosynthesising the core structure of different SMs which provides this fungus the capacity to produce a vast array of small molecule diversity. However, the potential of P. nodorum to produce SMs contrast with the relatively low number of identified compounds. In this thesis, I embarked upon the task of looking for P. nodorum SMs involved in the interactions occurring within the plant-pathogen system. The first experimental chapter presents an assessment of the in vitro capacity of P. nodorum to produce SMs utilising liquid chromatography-mass spectrometry (LC-MS). The data obtained from these analyses demonstrated the high capacity of P. nodorum to produce an array of metabolites. The spectrometric data was compiled in a spectral library of unknown SMs which was used in the second chapter to hunt for ecologically relevant SMs. The production of these substances was studied in vitro, during the interaction of P. nodorum and a possible antagonist, the wheat pathogen Zymoseptoria tritici; and in planta, during the infection of wheat by P. nodorum and during the co-infection of P. nodorum and Z. tritici. The results confirmed the presence of P. nodorum SMs and their possible involvement in the P. nodorum-wheat interaction and fungal antagonism. The third experimental chapter continues the assessment of SMs in P. nodorum biotic interactions by focusing on the volatile organic compounds (VOCs) using solid phase micro-extractions coupled to gas chromatography-mass spectrometry (SPME-GC-MS). It was found that P. nodorum produces phytotoxic and self-inhibitory VOCs that also iv exhibited mild antibacterial activities. Furthermore, several sesquiterpenes were detected within the VOCs mixture. Since previous bioinformatics survey on P. nodorum revealed the presence of three sesquiterpene synthases genes, Sts01, Sts02 and Sts03, a reverse genetics approach was then exploited to link these genes to their metabolic products. Sts01 and Sts02 were found to be the biosynthetic genes of the detected sesquiterpenes. The isolation of the metabolic products of Sts01 and Sts02 was achieved by expresion in a heterologous host. Finally, using spectrometric and spectroscopic techniques the eudesma-4,11-diene and β-elemene were identified as the main products of Sts02, α-cyperone as a downstream transformation of the eudesmadiene, and the acora-4,9-diene as the major product of Sts01. This constitutes the first report of terpenes isolated from P. nodorum.
Parastagonospora nodorum, secondary metabolites, mass spectrometry, LC-MS, GC-MS, volatile organic compounds, VOCs, sesquiterpenes
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