Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSRedox regulation of an AP-1-like transcription factor, YapA, in the fungal symbiont Epichloë festucae. Gemma M. Cartwright, Barry Scott, Yvonne Becker.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.Interaction between phenolic and oxidant signaling in Cochliobolus heterostrophus. Benjamin A Horwitz 1 , Samer Shalaby 1 , Olga Larkov 1 , MordechaiRonen 2 , Sophie Lev 3 . 1) Department of Biology, Technion - IIT, Haifa, Israel; 2) Department of Plant Science, Tel Aviv University, Ramat Aviv, Israel; 3)Centre for Infectious Diseases and Microbiology, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia.The transcription factor ChAP1 is an ortholog of yeast YAP1 in the maize pathogen Cochliobolus heterostrophus. ChAP1 migrates to the nucleus uponexposure to oxidative stress, inducing antioxidant genes such as thioredoxin and glutathione reductase [1]. ChAP1 also localizes to nuclei on contact withthe leaf and during invasive growth. Though reactive oxygen species are encountered on the host, ChAP1 nuclear retention can occur without oxidativestress. One of the signals responsible is provided by phenolic compounds [1-3]. Using a genetically-encoded ratiometric reporter of the redox state, weshowed that leaf extract and phenolics, despite their antioxidant properties, promote nuclear accumulation of ChAP1. To study this dual role of ChAP1 weidentified genes expressed in response to phenolics. Intradiol dioxygenase CCHD1 is rapidly upregulated, independent of ChAP1 [2]. Coumaric acid causedrapid and simultaneous upregulation of most of the b-ketoadipate pathway genes. Deletion of CCHD1 provided genetic evidence that protocatechuic acidis an intermediate in catabolism of many aromatics [3]. The activity of a structure series showed complementary requirements for upregulation of CCHD1and ChAP1 nuclear retention. The ability to metabolize a compound and ChAP1 nuclear retention are inversely correlated. To find additional genesinduced by phenolics, microarrays designed from the predicted coding sequences of the C. heterostrophus genome [4] were hybridized to probes madefrom RNA of cultures exposed to coumaric acid, or controls. Expression of about 90 genes from different pathways primarily for metabolism, for example,the b-ketoadipate, quinic acid and shikimic acid pathways, as well as transporters from different families was altered in response to coumaric acid. Theability to respond to phenolics and detoxify or metabolize them via the b-ketoadipate pathway confers an advantage to plant pathogens, and explains thepresence of at least two response pathways detecting these compounds. [1] Lev et al. (2005) Eukaryot. Cell 4:443-454; [2] Shanmugam et al. (2010) Cell.Microbiol. 12:1421-1434; [3] Shalaby et al. (2012) MPMI 25: 931-940; [4] Ohm et al. (2012) PLoS Pathog 8: e1003037. Supported in part by the IsraelScience Foundation. We thank Michal Levin and Itai Yanai for help with microarray hybridization.74