Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSThe role of NADPH oxidases in Neurospora crassa cell fusion. Nallely Cano-Dominguez 1 , Ernestina Casto-Longoria 1 , Jesus Aguirre 2 . 1) Departamento deMicrobiologia, CICESE, Ensenada, Baja California, Mexico; 2) Departamento de Biologia Celular y Desarrollo. Instituto de Fisiologia Celular UNAM, MexicoCity, D.F. Mexico.Hansberg and Aguirre proposed that reactive oxygen species (ROS) play essential roles in cell differentiation in microorganisms. ROS are generatedmainly during mitochondrial electron transport and by the action of certain enzymes. The NADPH oxidases (NOX) are enzymes that catalyze the productionof superoxide by transferring electrons from NADPH to oxygen. Neurospora crassa contains the NADPH oxidases NOX-1 and NOX-2 and a commonregulatory subunit NOR-1. NOX-2 is essential for ascospore germination, while NOX-1 is required for sexual and asexual development, polar growth andcell fusion. NOR-1 is essential for all these NOX functions. We have found that a functional NOR-1::GFP fusion is localized throughout the cytoplasm,enriched at the hyphal tip and sometimes in aggregates. This suggests that the functional NOX complexes are probably not localized at the plasmamembrane. Up to now NOX function in fungi has been evaluated in mutants that completely lack NOX proteins. We generated nox-1 alleles that result inNOX-1 proteins carrying substitutions of proline 382 by histidine or cysteine 542 by arginine, which affect NADPH-binding. Equivalent mutations inphagocytic Nox2/gp91phox do not affect protein stability but completely lack oxidase activity. P382H and C542R mutants did not produce sexual fruitingbodies and showed a decreased growth and differentiation of aerial mycelia, without affecting production of conida. These results indicate that sexualdevelopment depends on ROS production by NOX-1, whereas during asexual differentiation NOX-1 plays an important role independently of its catalyticactivity. Dnox-1, Dnor-1, P382H NOX-1 and C542R NOX-1 mutants were all able to produce some conidial anastomosis tubes (CATs) but they were unableto complete cell-cell fusion. All these mutants are also impaired in vegetative hyphae-hyphae fusion, which might explain the growth defects in Dnox-1 andDnor-1 strains. CATs production is delayed in the presence of antioxidant N- acetyl cystein (NAC) and Dsod-1 strains show an increase in CATs fusions. Theresults suggest that some ROS may be implicated in signaling CATs homing and vegetative fusion.Peroxiredoxins in ROS responses -Why evolve peroxidases that are inactivated by peroxides? Alison M. Day, Jonathon D Brown, Sarah R Taylor, JonathanD Rand, Brian A Morgan, Elizabeth A Veal. Inst Cell & Molecular Biosciences, Newcastle Univ, Newcastle Tyne, United Kingdom.Peroxiredoxins (Prx) are extremely abundant antioxidant enzymes with important roles in protecting against oxidative stress, ageing and cancer.Thethioredoxin peroxidase activity of eukaryotic typical 2-Cys Prx detoxifies hydrogen peroxide but, enigmatically, is highly sensitive to inactivation byperoxide-induced hyperoxidation of a catalytic cysteine residue. It has been proposed that hyperoxidation might allow hydrogen peroxide to act as a signaland/or promote an alternative activity of Prx as a chaperone [1, 2]. However, any advantage to be gained by inhibiting the thioredoxin peroxidase activityand preventing Prx from removing peroxides under oxidative stress conditions has remained obscure. The fission yeast Schizosaccharomyces pombecontains a single 2-Cys Prx, Tpx1. Our previous work has established that, counterintuitively, Tpx1 is vital for adaptive transcriptional responses tohydrogen peroxide due to essential roles in the hydrogen peroxide-induced activation of the p38/JNK/Hog1-related MAPK Sty1 and AP-1-like transcriptionfactor Pap1 [3, 4]. In seeking to understand why the thioredoxin peroxidase activity of Tpx1 should be important for Pap1 activation, we have identifiedthat Tpx1 is the major cellular substrate for thioredoxin. Accordingly, in hydrogen peroxide-treated cells, Tpx1 competitively inhibits the activity ofthioredoxin towards other substrates, including Pap1, and the methionine sulphoxide reductase A, Mxr1. Consequently, we show that the oxidativeinactivation of the thioredoxin peroxidase activity of Tpx1 is important to maintain active Mxr1, repair oxidative protein damage and maintain cell viabilityfollowing exposure to toxic levels of hydrogen peroxide [5]. Based on these discoveries in yeast, we propose that an important function for the reversiblehyperoxidation of eukaryotic 2-Cys Prx is to regulate thioredoxin and thus thioredoxin-mediated signalling and repair processes. I will present further datasupporting this conclusion and discuss its implications for hydrogen peroxide signal transduction.NADPH oxidases regulate septin-mediated cytoskeletal re-modeling during plant infection by the rice blast fungus Magnaporthe oryzae. Lauren S.Ryder 1 , Yasin F. Dagdas 1 , Thomas A. Mentlak 1 , Michael J Kershaw 1 , Martin Schuster 1 , Christopher R Thornton 1 , Jisheng Chen 2 , Zonghua Wang 2 , Nicholas JTalbot 1 . 1) Dept Biosciences, Univ Exeter, Exeter, United Kingdom; 2) Fujian agricultural university.NADPH oxidases (Nox) are flavoenzymes that function by transferring electrons across biological membranes to catalyze reduction of molecular oxygento superoxide. In animal cells, Nox enzymes are implicated in cell proliferation, cell signalling and apoptosis, while in plants Nox are necessary forprogrammed cell death, the response to environmental stresses, pathogen infection, and polarised growth of root hairs. In filamentous fungi, Nox arenecessary for cellular differentiation during sexual reproduction and for developmental processes that involve transitions from non-polarised to polarisedcell growth, such as tissue invasion by mutualistic and pathogenic fungi, and fungal virulence. The underlying function of Nox enzymes in these diversedevelopmental processes remains unclear. The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, whichuses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientationof the dynamic F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2-NoxR complex spatiallyorganises a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration pegemergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessaryfor penetration hypha elongation. Organisation of F-actin and septins in appressoria are disrupted by application of anti-oxidants, while latrunculinmediateddepolymerisation of appressorial F-actin is competitively inhibited by reactive oxygen species (ROS), providing evidence that regulated synthesisof ROS by fungal NADPH oxidases directly controls septin and F-actin dynamics.27th Fungal Genetics Conference | 73