Secondary metabolites in the Parastagonospora nodorum-wheat pathosystem
Date
2016
Authors
Muria Gonzalez, Mariano Jordi
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Abstract
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.
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Keywords
Parastagonospora nodorum, secondary metabolites, mass spectrometry, LC-MS, GC-MS, volatile organic compounds, VOCs, sesquiterpenes
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