Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSChem. Soc, 2003).586. Response of Alternaria brassicicola to the antifungal activity of isothiocyanates. Benoit Calmes, Jérôme Dumur, Thomas Guillemette, Nelly Bataillésimoneau,Phillippe Simoneau. Institut de Recherche en Horticulture et Semences UMR1345, Angers, France.Alternaria brassicicola is the causative agent of Brassicaceae black spot disease. This necrotrophic fungus causes glucosinolates degradation by plantmyrosinase during infection. Isothiocyanates (ITCs), the major breakdown compounds, have been shown to be exert some toxicity to various A.brassicicola isolates. We showed, by application of specific fluorescent probes, that ITCs cause ROS production, disrupt the mitochondrial membranepotential and trigger apoptosis in A. brassicicola cells. The generation of an oxidative stress following ITCs application was confirmed by the fact that KOmutants deficient for the transcription factors AP1 and SKN7 (both being involved in the oxidative-stress response) are hypersensitive to ITCs and havedecreased aggressiveness on glucosinolates accumulating host plants. Despite this host defense system, A. brassicicola is still able to complete itsinfectious cycle, indicating the existence of strategies to cope with this oxidative stress. Hypersensitivity to Al-ITC, Bz-ITC or Ph-ITC was observed in KOmutants deficient for distinct glutathione-S-transferases. Some of them were differentially expressed following exposure to ITCs and exhibited hightransferase activity with ITCs as substrate. The polyol mannitol has been proposed to act as an antioxidant agent and protect fungal cells by quenching ROSproduced by hosts in response to attack. We isolated the genes encoding the MPD and MDH enzymes, two essential enzymes of the mannitol metabolismin A. brassicicola, and used targeted gene disruption to create single and double mutants for each gene. Only mutants unable to accumulate mannitol inhyphae and conidia were sensitive to ITCs. Our results supported the involvement of fungal GST and mannitol metabolism in ITC-derived oxidative stress.GST participates in ITCs detoxification mechanisms and mannitol accumulation in protection against ROS. They highlight their importance with respect tothe ability of A. brassicicola to efficiently accomplish its pathogen life cycle despite exposure to plant-derived antifungal metabolites.587. Redox regulation of an AP-1-like transcription factor, YapA, in the fungal symbiont Epichloë festucae. Gemma M. Cartwright, Barry Scott, YvonneBecker. Molec Biosci, Massey Univ, Palmerston Nth, New Zealand.Reactive oxygen species (ROS) are emerging as important regulators required for the successful establishment and maintenance of the mutualisticassociation between the fungal endophyte Epichloë festucae and its grass host Lolium perenne. The generation of reactive oxygen species (ROS) by thefungal NADPH oxidase, NoxA has previously been shown to regulate hyphal growth of E. festucae in planta; a result that has led to the hypothesis thatfungal-produced ROS are key second messengers in the symbiosis. However, the highly reactive nature of these molecules dictates that cells possessefficient sensing mechanisms to maintain ROS homeostasis and prevent oxidative damage to cellular components. The Saccharomyces cerevisiae Gpx3-Yap1 and Schizosaccharomyces pombe Tpx1-Pap1, two-component H 2O 2 sensors, serve as model redox relays for coordinating the cellular response toROS. 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 study investigated the role of the E. festucae Yap1 homologue, YapA, andputative upstream activators GpxC and TpxA, homologues of Gpx3 and Tpx1, respectively, in responding to ROS. YapA is involved in responding to ROSgenerated at the wound site following inoculation into ryegrass seedlings. However, deletion of yapA did not impair host colonization indicatingredundancy in systems used by E. festucae to sense and respond to plant-produced ROS. In culture, deletion of E. festucae yapA, renders the mutantssensitive to only a subset of ROS and this sensitivity is influenced by the stage of fungal development. In contrast to the H 2O 2-sensitive phenotype widelyreported for fungi lacking the Yap1-like protein, the E. festucae yapA mutant maintains wild-type mycelial resistance to H 2O 2 but conidia of the yapAmutant are very sensitive to H 2O 2. Using a degron-tagged GFP-CL1 as a reporter, we found YapA is required for the expression of the spore specificcatalase, catA. Moreover, YapA is activated by H 2O 2 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.27th Fungal Genetics Conference | 265