Redox regulation of an AP-1-like transcription factor, YapA, in the fungal symbiont Epichloë festucae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
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Date
2013
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Massey University
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Abstract
Reactive oxygen species (ROS) are emerging as important regulators required for
the successful establishment and maintenance of the mutualistic association between
the fungal endophyte Epichloë festucae and its grass host Lolium perenne (perennial
ryegrass). The generation of reactive oxygen species (ROS) by the fungal NADPH
oxidase, NoxA, has previously been shown to regulate hyphal growth of E. festucae in
planta, a result that has led to the hypothesis that fungal-produced ROS are key second
messengers in the symbiosis. However, the highly reactive nature of these molecules
dictates that cells possess efficient redox sensing mechanisms to maintain ROS
homeostasis and prevent oxidative damage to cellular components such as DNA,
lipids and proteins. The Saccharomyces cerevisiae Gpx3-Yap1 and Schizosaccharomyces
pombe Tpx1-Pap1, two-component H2O2 sensors, serve as model redox relays for
coordinating the cellular response to ROS. While proteins related to the Yap1 and Pap1
basic-leucine zipper (bZIP) transcription factors have been identified in a number of
filamentous fungi, the components involved in the upstream regulation remain
unclear. This thesis presents an investigation into the role of the E. festucae Yap1
homologue, YapA, and putative upstream activators GpxC and TpxA, homologues of
Gpx3 and Tpx1, respectively, in responding to ROS. YapA is involved in responding to
ROS generated at the wound site following inoculation into ryegrass seedlings.
However, deletion of yapA did not impair fungal colonisation of the host, indicating
functional redundancy in systems used by E. festucae to sense and respond to plantproduced
ROS. In culture, deletion of E. festucae yapA renders the mutants sensitive to
only a subset of ROS and this sensitivity is influenced by the stage of fungal
development. In contrast to the H2O2-sensitive phenotype widely reported for fungi
lacking the Yap1-like protein, the E. festucae yapA mutant maintains wild-type mycelial
resistance to H2O2 but conidia of the yapA mutant are sensitive to H2O2. Using a
degron-tagged GFP-CL1 as a reporter, we found YapA is required for the expression of
the spore-specific catalase, catA. Moreover, YapA is activated by H2O2, through
disulfide bond formation, independently of both GpxC and TpxA, suggesting a novel
mechanism of regulation exists in E. festucae. This work provides a comprehensive
analysis of the role and regulation of the AP-1 transcription factor pathway in a
filamentous fungal species.
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Keywords
Epichloë, Genetics, Transcription factors