Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS442. Transcriptional regulation of peptidases and nitrogen transporters during the assimilation of organic nitrogen by the ectomycorrhizal fungi Paxillusinvolutus. Firoz Shah 1 , Francois Rineau 2 , Tomas Johansson 1 , Anders Tunlid 1 . 1) Microbial Ecology Group, Department of Biology, Lund University, SE-22362,Lund, Sweden; 2) Centre for Environmental Sciences, Hasselt University, Building D, Agoralaan, 3590 Diepenbeek, Limburg, Belgium.Proteins and amino acids form a major part of the organic nitrogen (N) sources in soils. Though a poorly characterized process, this N is mobilized andbecomes available to plants due to the activity of ectomycorrhizal (ECM) fungi. We have examined the role of ectomycorrhizal extracellular peptidases andamino acid transporters in the degradation, uptake and transfer of various protein sources (BSA, Gliadin and pollen) as well as of plant litter material usingthe ECM model fungus Paxillus involutus. During N-deprived conditions, all substrates induced secretion of peptidase activities. The activity had acidic pHoptimum (2.3-3.0), and it was mainly due to aspartic peptidases and with minor contribution of metallo and serine peptidases. The activity was partly andtemporarily repressed by low concentrations of ammonium (1mg/L). Transcriptional analysis showed that P. involutus expressed a large array of proteinsand enzymes involved in the assimilation of organic N including peptidases, N-transporters and enzymes of the N-metabolism. Extensive in-silico analysisrevealed the presence of genes encoding 312 peptidases, 129 N transporters and 284 enzymes involved in amino acid metabolism. Out of these, 89peptidases and 37 N-transporters and 109 amino acid metabolism enzymes encoding genes were significantly upregulated during organic N assimilation.The genes were encoding a variety of secreted (23) and non-secreted (20) peptidases which were differentially expressed depending on the medium withthe highest expression of the aspartic and metallo peptidases. Apart from the YAAH/ATO family, upregulated genes were found in all the other families oftransporters for amino acids, oligopeptides, ammonium, urea and allantoate/allantoin. The results shows that the expression levels of peptidases andtransporters in P. involutus are coordinately regulated during the assimilation of organic N sources.443. Characterization of genome maintenance components in Neurospora crassa using whole-genome high-throughput approach. Evelina Y. Basenko,Zack Lewis. Department of Microbiology, University of Georgia, Athens, GA.Eukaryotic DNA is packaged into a higher order DNA-protein structure also known as chromatin, which can regulate and impact an array of nuclearprocesses including DNA repair and genome maintenance. Disruptions in genome integrity can lead to serious ailments in humans and also contribute tocancer emergence. DNA repair and genome maintenance have been extensively studied in yeast. We, however, have chosen to investigate genomemaintenance in the filamentous fungus Neurospora crassa. N. crassa possesses a much larger genome than budding yeasts, and it also containsapproximately 1400 genes which are conserved in higher eukaryotes including humans and are absent in yeasts. We utilized the whole-genomeNeurospora knockout library to search for novel regulators of genome maintenance. We screened the knockout library for mutants sensitive to the DNAdamage agent methyl methane sulfonate (MMS). We have further confirmed and investigated additional mutagen sensitivities of confirmed MMSsensitivestrains. For our further studies, we have decided to focus on one of the MMS-sensitive mutants, which contains a deletion of a SNF2-like protein.The whole-genome high-throughput approach is a powerful method to identify novel players of genome maintenance. We have identified several hundredmutants sensitive to DNA damage, that fall within various categories of cellular processes, including but not limited to DNA repair, RNA metabolism, andchromatin maintenance. Our findings and current progress will be reported.444. Circadian regulation and carbon catabolite repression in Neurospora crassa: Two integrated regulatory systems? Rodrigo Díaz-Choque, Luis FLarrondo. Dept Molecular Genetics & Microbiology, Pontificia Universidad Católica de Chile, Santiago, Chile.Circadian clocks are autonomous timers composed of interconnected transcriptional/transcriptional feedback loops. They are thought to confer aselective advantage to individuals by temporally coordinating several processes and contributing to cellular homeostasis. In Neurospora crassa, a modelorganism in circadian studies, ~20%; of its genes are under circadian control and interestingly; many of them are related to metabolism. Indeed, the ideaof a crosstalk between metabolism and the circadian clock has become stronger in the last years, and several examples have been obtained in mammaliansystems. However, we still don’t know in Neurospora the actual influence of circadian regulation in its physiology and its real impact in the “real-world”.Moreover, the different transcriptional regulators linking time-of-day information and the expression of genes involved in metabolically relevantprocesses, like Carbon-Catabolite Repression (CCR) or cellulose degradation remain largely unknown. Thus, we are analyzing glucose repression in acircadian context, using N. crassa as a model. We hypothesize that there is an intimate crosstalk between both regulatory systems over the expression ofseveral rhythmic CCR-controlled genes. We are using gene expression assays and a codon-optimized luciferase transcriptional reporter, to evaluate therole of the transcription factor CRE-1 (carbon catabolite regulation-1), a conserved metabolic regulator, in this potential cross-talk. Also, CRE-1 is a crucialtranscription factor involved in several important cellular processes as cellulose degradation and catabolic repression. Further, we are studying how bothinputs are integrated to control the expression of target genes. Our results suggest CRE-1 as a link between both pathways, as it appears to be importantfor both CCR and circadian control of target genes. In addition, we describe for the first time In Neurospora the presence of a functional clock in cellulose(Avicel) -containing media. This observation strengthens the hypothesis that a circadian clock may regulate the expression of several cellulase-encodinggenes, having a real impact in such a physiologically relevant process.445. Opposing activities of the HCHC and DMM complexes maintain proper DNA methylation in Neurospora crassa. Shinji Honda 1,2 , Eun Yu 1 , Eric Selker 1 .1) University of Oregon, Institute of Molecular Biology, Eugene, OR; 2) University of Fukui, Life Science Unite, Fukui, Japan.Proper regulation of heterochromatin and DNA methylation is critical for the normal function of cells. We show that heterochromatin and DNAmethylation are faithfully controlled in Neurospora by opposing activities of the silencing complex HCHC and the anti-silencing complex DMM. Theworkings of these two complexes were investigated. HCHC consists of four proteins, the two chromo domain proteins HP1 and CDP-2, the histonedeacetylase HDA-1 and the AT-hook motif protein CHAP. We found that histone deacetylase activity is critical for HCHC function but the H3K9me3 bindingactivity of the CDP-2 chromo domain is not. Instead, CDP-2 serves as an essential bridge between HP1 and HDA-1. CHAP interacts directly with HDA-1,binds in a methylation-independent way to the A:T-rich DNA that forms the cores of methylated regions and is important for stable association of HDA-1with chromatin. HCHC is involved in the spreading of DNA methylation in dmm mutants. The DMM complex consists of a presumed histone demethylase,DMM-1, plus DMM-2, which is characterized by a fungal-specific Zn(II) 2Cys 6 DNA-binding domain (“Zn-Cys”). We found that DMM-2 strongly binds to DNAfrom euchromatin/heterochromatin junctions, thereby promoting the stable association of DMM-1 at the edge of heterochromatin domains to preventaberrant spreading of DNA methylation.446. The transcription factor FL is phosphorylated and interacts with a trehalose related protein in Neurospora crassa. Carmen Ruger-Herreros 1 , GencerSancar 2 , Michael Brunner 2 , Luis M. Corrochano 1 . 1) Departamento de Genetica, Universidad de Sevilla, Spain; 2) BZH, Universität Heidelberg, Germany.Several environmental cues, including light, promote a developmental transition in Neurospora crassa that leads to the formation of conidia. Conidiationis controlled by FLUFFY (FL), a zinc finger transcription factor. Light activates the transcription of fl through the transient binding of the WC complex to thefl promoter. Light also activates the transcription of several conidiation genes in Aspergillus nidulans, and their Neurospora homologs have been identified230