Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSGpa1 and Gpg1/Gpg2 in a direct manner, and Gib2 promotes cAMP levels through novel interactions with phosphodiesterase Pde2 and RAS proteins Ras1and Ras2. Using TAP technology, we have identified additional 42 proteins whose putative function range from signal transduction, energy generation,metabolism, and stress response to ribosomal function. Finally, through establishing a protein-protein interactive network, we illustrate that Gib2 adapts ascaffold role to mediate specific protein-protein interactions that drive the formation of various protein complexes. This includes fostering aheterotrimeric complex with Gpa1 and Gpg1/Gpg2, targeting Pde2 (direct) and adenylyl cyclase Cac1 (indirect) to regulate cAMP levels, and likely servingas a conserved ribosomal core protein facilitating fundamental cellular processes to underlie growth and virulence. Our studies reveal the complexity ofthe regulatory network in fungi and advocate Gib2 as a novel target for antifungal therapy.459. Consequences of the loss of transcription factors SreA and HapX on siderophore biosynthesis and iron homeostasis in the perennial ryegrassendophyte, Epichloë festucae. Natasha T. Forester 1,2 , Geoffrey A. Lane 1 , Iain L. Lamont 2 , Linda J. Johnson 1 . 1) Plant Fungal Interactions Team, AgResearchLtd, Palmerston North, New Zealand; 2) Biochemistry Dept, University of Otago, Dunedin, NZ.Siderophores are low molecular weight ferric iron chelators that are made by microorganisms to compete for and to sequester iron, an essentialmicronutrient. Epichloë festucae, a fungal endosymbiont of perennial ryegrass, synthesises two siderophores, epichloënin A and ferricrocin to harvest andutilise iron from its host grass. Work by our group has implicated epichloënin A in the maintenance of symbiosis and is described in another abstract. Toexplore the regulation of siderophore biosynthesis and iron homeostasis processes, we have characterised mutants of two major iron-responsivetranscription factors, SreA and HapX that coordinate cellular responses to iron availability. To evaluate the effect of loss of SreA and HapX on siderophorebiosynthesis, we measured the production of epichloënin A and ferricrocin by LC-MS/MS. Relative to wild type; both siderophores were over-produced inDsreA mycelia grown in the presence of iron, while ferricrocin was produced in excess in DhapX mycelia grown under iron deprived conditions. Irondependentphenotypic deviations from wild type fungal growth were also observed in culture and in planta but neither mutant disrupted the E. festucae -L. perenne association under standard soil conditions. However, under iron-limiting conditions through hydroponic control of symbiotic iron supply, wedemonstrated that DsreA mutants can induce chlorosis in their hosts, indicating that DsreA mutants compete for host iron. In planta, the DsreA fungalhyphae are also markedly increased in girth and lack growth in vascular bundles. In DhapX infected plants grown hydroponically in iron deprivedconditions, we observed inappropriate fungal growth such as highly convoluted and compressed hyphae and elongated fungal structures which hinted atreduced resource conservation by DhapX under growth limiting conditions. Collectively, these results suggest that Epichloë fungi have a tightly regulatediron management system for niche adaptation and actively set limits on iron withdrawal from the host, presumably to prevent competition with its host topromote mutualistic interactions. Mutations that interfere with fungal iron management, either by deregulating siderophore synthesis, can destabilise thefungal-plant association.460. Who is to blame: defining the host responses that lead to ToxA-induced susceptibility. Iovanna Pandelova, Viola Manning, Ashley Chu, LyndaCiuffetti. Botany and Plant Pathology, Oregon State Univ, Corvallis, OR.Pathogenicity by the necrotrophic pathogen of wheat, Pyrenophora tritici-repentis (Ptr), is attributed to the production of host-selective toxins (HSTs).Understanding the mode-of-action of HSTs is essential for a complete characterization of how these pathogenicity factors condition plant diseasesusceptibility. One of the proteinaceous HSTs produced by Ptr, PtrToxA (ToxA), induces necrosis in sensitive cultivars. Several studies suggest that ToxAinteracts with a high affinity receptor, enters mesophyll cells and localizes to chloroplasts. Additionally, ToxA acts as an elicitor of defense responses byincreasing production of phenolic compounds and by the up-regulation of genes involved in jasmonic acid and ethylene production pathways. After ToxAtreatment and incubation in constant light, there is a decrease in photosystem (PS) I and II transcripts observable already at 9 and 14 hours post infiltration(hpi), which is followed by the drastic reduction in levels of both PSI- and PSII-complex proteins. It is proposed that photosystem dysfunction leads to lightdependentaccumulation of reactive oxygen species (ROS) and the development of necrosis. To better understand the role of ROS, photosynthesis anddefense responses in necrosis development induced by ToxA, plants were incubated in light (presence of ROS) or in dark (absence of ROS). In order todetermine the impact of ToxA on gene regulation and to establish when early changes in protein content of PS complexes occur, both biochemical andmicroarray analyses were performed. Some defense-related genes are up-regulated in ToxA-treated leaves incubated in the dark (ToxA/dark), althoughthe number of probesets was considerably less compared to ToxA-treated leaves incubated in light (ToxA/light). Furthermore, ethylene biosynthesis genes,that play a role in symptom development in ToxA/light treatrment are not significantly up-regulated in ToxA/dark-treated leaves. Finally, only a smallfraction of PSI- and II-related and chlorophyll a/b-binding genes are down-regulated in ToxA/dark compared to ToxA/light treatment. These data suggestthat only certain defense-related pathways are involved in ToxA-induced necrosis development, and help to identify those genes whose differentialregulation by ToxA is light and/or ROS-dependent.461. RNA silencing of pacC increases aflR transcript levels under alkaline pH conditions in Aspergillus flavus. Benesh M Somai, Kyle W van der Holst, EssaSuleman. Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, 6031, Eastern Cape, South Africa.Aspergillus flavus produces aflatoxin B1 which is an important hepatocarcinogen, especially amongst the developing third world countries which have alarge number of poor, rural, subsistence communities with little access to fungicides. The master regulator of aflatoxin production is aflR which, in turn,appears to be negatively regulated by pacC. However, until now, there were never any direct measurements of the relative aflR/pacC transcript ratiosproduced under aflatoxin conducive and non-conducive conditions. In the current study, pacC was down-regulated in two transformants by a syntheticpacCRNAi construct under the control of a thiamine inducible promoter. Expression of pacC and aflR transcripts was then measured via RT-qPCR incultures grown under alkaline or acid conditions. At pH 4, between pacCRNAi inducing and repressing conditions, an aflR/pacC transcript ratio of 1.09relative to the reference gene was obtained indicating the production of an equal abundance of aflR and pacC mRNA. It is generally accepted that at acidicpH the majority of pacC mRNA is unprocessed, remains untranslated and non-functional thereby being incapable of repressing AFLR protein production.This stimulates aflatoxin production at acidic pH. Between pH 8 and pH 4, when pacCRNAi was suppressed, the aflR/pacC ratio was 0.2 indicating that pacCproduction was higher than that of aflR. aflR transcript levels were reduced between 76% and 80% therefore explaining the normal lack of aflatoxindetection at pH 8. Between pH 8 and pH 4, when pacCRNAi was induced, the aflR/pacC ratio was between 1.77 and 13.21 indicating that at alkaline pH,suppression of pacC allowed a large increase in AFLR which stimulated aflatoxin production. It is concluded that pacC is produced at acidic pH, but remainslargely non-functional. Furthermore, at pH 8, aflR production decreases only by about 80% and therefore it is possible that the remaining 20% oftranscripts still stimulates aflatoxin production. Finally, via RNAi silencing it is conclusively proved that pacC negatively regulates aflR production at pH 8.462. High-throughput prediction and functional validation of promoter motifs regulating gene expression in spore and infection stages of Phytophthorainfestans. H. Judelson, S. Roy, M Kagda. Dept of Plant Pathology and Microbiology, University of California, Riverside, CA.Most filamentous pathogens have complex life cycles in which gene expression networks orchestrate the formation of cells specialized for dissemination234