Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSstudy the role of genes encoding proteins of the wall and secretome, and the differences existing in the behavior of the diploid and haploid, taking intoconsideration that only the first one is pathogenic to the natural host.382. Expression of the Trichophyton rubrum ace2 and pacC genes during degradation of keratinized substrates. Larissa Silva, Nalu Peres, GabrielaPersinoti, Elza Lang, Vanderci Oliveira, Antonio Rossi, Nilce Martinez-Rossi. University of Sao Paulo, Ribeirão Preto/SP/Brazil, Sao Paulo, Brazil.Trichophyton rubrum is a pathogenic, cosmopolitan and anthropophilic fungus that infect keratinized tissues, mainly skin and nails. The genomes ofseveral dermatophytes, including T. rubrum, were sequenced by the Broad Institute/NIH, enabling studies on the regulation of the expression of genesrelated to several cellular processes. The transcription factor (TF) Ace2 participates of a network of genes called RAM (Regulation of Ace2 activity andcellular morphogenesis), involved in the regulation of morphogenesis, cell division, and development of conidiophores. The TF PacC regulates thetranscription of genes in response to extracellular pH and also genes related to the biosynthesis of cell wall, suggesting a crosstalk between these twopathways. Therefore, the aim of this study was to analyze the expression profile of the pacC and ace2 genes in different nutritional sources (nail and skinex vivo infections) to understand the regulation of these TF during pathogenesis and development. In silico analyses of the putative promoter regions ofthe pacC and ace2 genes revealed the presence of the DNA binding motifs of both TFs, suggesting a possible cross- and co-regulation of these TFsexpression in T. rubrum. Gene expression analyses during growth in keratinized tissues suggested an opposite expression profile in nail interaction assays,the ace2 gene was up-regulated and pacC was down-regulated. Moreover, in ex vivo skin infective both genes presented a similar expression level. Theseresults suggest a different gene expression modulation of ace2 and pacC according to the nutrient source and possibly the infection site. Moreover, thisevaluation provides a better comprehension of the involvement of both pathways in regulating a variety of cellular processes that enable cell viabilityduring infection of keratinized tissues.383. Control and Function of Two Fatty Acid Regulators in Neurospora crassa. Erin L. Bredeweg 1 , Fei Yang 2 , Kristina Smith 1 , Rigzin Dekhang 2 , JillianEmerson 3 , Jay Dunlap 3 , Deborah Bell-Pedersen 2 , Matthew Sachs 2 , Michael Freitag 1 . 1) Program for Molecular and Cellular Biology, Department ofBiochemistry and Biophysics, and Center for Genome Research and Biocomputing (CGRB), Oregon State University, Corvallis, OR 97331; 2) Department ofBiology and Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX; 3) Department of Genetics, Geisel School ofMedicine at Dartmouth, Hanover, NH.The filamentous saprobe Neurospora crassa is an excellent model for describing the behavior of transcriptional regulators. We describe the genomewidebehavior of two Fatty Acid Regulators (FAR) proteins, transcription factors that modulate the response of N. crassa to the presence of fatty acids. Weused ChIP-seq to find the localization of FAR-1 and FAR-2 under nutrient conditions targeting short and long chain fatty acid carbon sources, with sucroseas a control. Bioinformatic analyses describe variant binding sites for FAR-1 and FAR-2, with overlap in about a third of all target regions. Functions underthe control of ChIP-seq targets were further examined by phenotypic assays for siderophore production, oxidative stress, and linear growth. We foundreduced siderophore production, and increased vulnerability to oxidative stress in far-1 mutants, but not far-2 mutants. Linear growth showed a carbonspecificreduced growth rates for far-2, as well as Tween-20 sensitivity and conidiation defects for far-1. RNA-seq identified numerous differentiallyregulated transcripts under different growth conditions and in the single or double mutants. Many of these transcripts are part of the gene set identifiedby ChIP-seq, and many were affected by the absence of one or both FARs. Our analyses identified groups of co-regulated proteins not previously identifiedas affected by FAR transcription factors, in addition to those involved in the control of the core cellular machinery for energy production by beta-oxidation.384. Characterization of genomic targets for the Neurospora crassa hypothetical transcription factor NCU04390 by ChIP-sequencing. R. Gonçalves 1 , E.Bredeweg 2 , M. Freitag 2 , M. C. Bertolini 1 . 1) Instituto de Química, UNESP, Araraquara, São Paulo, Brazil; 2) Department of Biochemistry and Biophysics, OSU,Corvallis, OR, USA.The mechanisms by which glycogen content is controlled in microorganisms are intricate, involving co-regulation of many proteins. In Neurospora crassa,glycogen reaches maximal levels at the end of the exponential growth phase, however under heat shock, glycogen content and transcription of theglycogen synthase gene (gsn) rapidly decrease. In a previous analysis, the NCU04390 KO strain showed a drastic increase in glycogen levels and upregulationof the gsn gene after heat shock when compared to the wild-type strain, suggesting that the NCU04390 gene product is involved in theregulation of glycogen metabolism. Because the product of this ORF is annotated as a hypothetical transcription factor (TF) with an N-terminal zinc-fingerand a central fungal-specific TF domain, chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) was expected to revealgenes that are directly regulated by the NCU04390 gene product. First, GFP tag was fused to the 3'-end of the ORF NCU04390 by gene replacement. ChIPwas performed with NCU04390-GFP at 30ºC and 45ºC with antibodies against the GFP tag. ChIP-libraries were sequenced on a HiSeq2000 (Illumina/Solexa)genome analyzer and data from 45ºC experiment revealed that most of the genes regulated by the transcription factor encode hypothetical proteins.However genes encoding proteins with known functions, such as proteins involved in carbon metabolism and transporters were also identified. Amongthese genes, it is important to mention the glycogen debranching enzyme coding gene (ORF NCU00743), which participates in the glycogen degradation.The His::4390 recombinant protein was produced in E. coli, partially purified by IMAC and used in EMSA experiments to validate the result found in theChIP-Seq assay. The results showed specific binding of the recombinant His::4390 in the NCU00743 promoter, suggesting that the transcription factormight regulate glycogen metabolism under heat stress through the gene encoding the debranching enzyme. Data from EMSA validation analysis for morepeaks found in ChIP-seq will be presented. Supported by FAPESP, CNPq, CAPES and US NIH.385. The KMT6 Histone H3 K27 Methyltransferase Regulates Expression of Secondary Metabolites and Development in Fusarium graminearum. KristinaM. Smith, Lanelle R. Connolly, Michael Freitag. Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon StateUniversity, Corvallis, OR 97331.The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation ofsecondary metabolite gene clusters remains largely a mystery. By ChIP-sequencing we found that regions of the F. graminearum genome with secondarymetabolite clusters are enriched for a histone modification, trimethylated histone H3 lysine 27 (H3K27me3), associated with gene silencing. Thismodification was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. H3K27me3 and di- ortrimethylated H3K4 (H3K4me2/3), modifications associated with gene activity, are found in mutually exclusive regions of the genome. To betterunderstand the role of H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z).The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-sequencing, displays growth defects, is sterile, and produces mycotoxins underconditions where they are not generated in wildtype (WT) strains. RNA-sequencing showed that genes modified by H3K27me3 are most often silent, asabout 75% of the 4,449 silent genes are enriched for H3K27me3. Surprisingly, we found 22% of the 8,855 expressed genes enriched for H3K27me3. Asubset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6 (1,780 genes), and an overlapping set of genes showed increased214