Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS466. Vib-1 is required for cellulose utilization in Neurospora crassa. Yi Xiong, Jianping Sun, N. Louise Glass. Department of Plant and Microbial Biology,Univ California, Berkeley, Berkeley, CA.Vib-1 (vegetative incompatibility blocked) encodes a transcriptional regulator that is required for nonself recognition and heterokaryon incompatibility inNeurospora crassa. It is also required for the production of extracellular proteases upon carbon and nitrogen starvation. We found that vib-1 null mutantwas severely defective in utilizing cellulose as a carbon source and this growth defect could be rescued by constitutively expressing clr-2, an essentialtranscription factor for cellulase gene expression. Our transcriptional profiling with RNA-seq showed that most genes that were induced in wild type oncellulose were not induced in the vib-1 null mutant, but were expressed in vib-1 null mutant under non-inducing conditions when clr-2 transcription factorwas constitutively expressed. Our data suggests that vib-1 functions in a signaling pathway upstream of clr-2 for cellulose utilization. By comparing thetranscriptomes of vib-1 and clr1/clr-2 mutants versus wild type in response to different carbon sources including sucrose and cellulosic materials as well ascarbon and nitrogen starvations, we differentiated vib-1 dependent gene expression into several categories. We propose that vib-1 may play an importantrole in carbon starvation signaling ,thus positively regulating preparation for utilization of cellulosic materials.467. The stringency of start codon selection in the filamentous fungus Neurospora crassa. Jiajie Wei 1 , Ying Zhang 1 , Ivaylo P. Ivanov 2 , Matthew S. Sachs 1 .1) Dept Biol, Texas A&M Univ, College Station, TX; 2) BioSciences Institute, University College Cork, Ireland.In eukaryotic cells, initiation may occur from near-cognate codons that differ from AUG by a single nucleotide. The stringency of start codon selectionimpacts the efficiency of initiation at near-cognate codons and the efficiency of initiation at AUG codons in different contexts. We used a codon-optimizedfirefly luciferase reporter initiated with AUG or each of the nine near-cognate codons in preferred context to examine the stringency of start codonselection in the model filamentous fungus Neurospora crassa. In vivo results indicated that the hierarchy of initiation at start codons in N. crassa (AUG >>CUG > GUG > ACG > AUA » UUG > AUU > AUC) is similar to that in human cells. Similar results were obtained by translating mRNAs in a homologous N.crassa in vitro translation system or in rabbit reticulocyte lysate. We next examined the efficiency of initiation at AUG, CUG and UUG codons in differentcontexts in vitro. The preferred context was more important for efficient initiation from near-cognate codons than from AUG. These studies demonstratedthat near-cognate codons are used for initiation in N. crassa. Such events could provide additional coding capacity or have regulatory functions. Analysesof the 5’-leader regions in the N. crassa transcriptome revealed examples of highly conserved near-cognate codons in preferred contexts that could extendthe N-termini of the predicted polypeptides.468. A temperature-dependent complex transcriptional network controls cell shape and virulence in Histoplasma capsulatum. Sinem Beyhan 1 , MatiasGutierrez 1 , Mark Voorhies 1 , Anita Sil 1,2 . 1) Microbiology and Immunology, University of California, San Francisco, San Francisco, CA; 2) Howard HughesMedical Institute, Chevy Chase, MD.Histoplasma capsulatum, which is a respiratory fungal pathogen of humans, is endemic in the United States. Depending on the exposure dose and theimmune status of the host, the infection can lead to mild-respiratory or life-threatening and systemic disease. H. capsulatum has a dimorphic life cycle,switching from an infectious filamentous form in the soil to a pathogenic yeast form in mammalian hosts. This morphological switch, which requires adramatic shift in the gene expression profile of the cells, can be easily recapitulated in the laboratory simply by changing the temperature from roomtemperature to 37°C. We previously identified three regulators, Ryp1, Ryp2 and Ryp3, which are required for the yeast-phase growth. ryp1, ryp2 and ryp3mutants are unable to respond to change in temperature and grow constitutively in the filamentous form even at 37°C. Ryp1 belongs to a conservedfamily of fungal proteins that regulate cellular differentiation in response to environmental signals. The best-studied member of this family of proteins isWor1, which is a master regulator of white-to-opaque switching in Candida albicans. Ryp2 and Ryp3 are orthologous to VosA and VelB, respectively, whichare developmental regulators in Aspergillus nidulans. In this study, using transcriptional profiling and chromatin immunoprecipitation (ChIP) experiments,we explored complementary and unique roles of Ryp1, Ryp2, and Ryp3 in regulating yeast-phase growth. Our results reveal that Ryp1, Ryp2 and Ryp3physically interact and associate with DNA throughout the genome. Additionally, we identified a fourth transcription factor, Ryp4, which is a direct targetof Ryp1, Ryp2 and Ryp3, as a novel regulator of yeast-phase growth in H. capsulatum. Further transcriptional profiling and ChIP experiments show thatRyp4 regulates and associates with the upstream regions of a subset of Ryp1, Ryp2, and Ryp3 targets, which are involved in morphology and virulence in H.capsulatum. Finally, we identified two distinct cis-regulatory elements that are utilized by Ryp1 or the Ryp2/Ryp3 complex to facilitate gene expression.Our results reveal a tightly regulated and interwoven transcriptional network that controls the ability of a pathogenic fungus to cause disease in responseto host temperature.469. Evolutionary analysis of Dicer proteins: a preliminary analysis to study of microRNAs in the mushroom, Coprinopsis cinerea. Xuanjin Cheng, HoiShan Kwan. Life Sciences, The Chinese University of Hong Kong, New Territory, Hong Kong.Coprinopsis cinerea is a mushroom of limited edible value and is extensively used as a model organism to study the development of homobasidiomycetefungi. Unraveling the molecular basis of the fungus developmental processes would contribute to evolutionary studies and lead to improvement in thebreeding and cultivation of edible or medical homobasidiomycete mushrooms. MicroRNA (miRNA) is a group of endogenous non-coding regulatory RNAsof ~22 nt that regulate gene expression in various biological processes such as cell differentiation, development regulation and heterochromatinformation. Dicer is a key enzyme involved in the biogenesis of miRNAs and is highly conserved through eukaryotes. A miRNA-like RNA cannot be defined asa miRNA unless a Dicer (or Dicer-like) protein is found participating in its biogenesis. There are three Dicer homologs (CC1G_00230, CC1G_03181,CC1G_13988) identified in the C. cinerea genome. In order to gain an insight into the roles of Dicer proteins in C. cinerea and to investigate whether Diceris involved in miRNA biogenesis, we employed a comprehensive phylogenetic analysis of the Dicer protein family in all of the three kingdoms underEukaryota - animal, plant and fungus - and highlighted the results of Dicer homologs in C. cinerea. We showed that Dicer genes duplicated and diversifiedindependently in early animal, plant and fungus evolution, coincident with the origins of multicellularity. Besides, identified a group of Dicer homologs thatare specific to mushroom-forming fungi. We also showed that changes in one of the Dicer domains, the double-stranded RNA binding domain (dsRBD),alone may lead to diversification of Dicer proteins. As a whole, we revealed a dynamic picture in which the evolution of Dicer proteins has drivenelaboration of parallel RNAi functional pathways in the animal, plant and fungus kingdoms.470. Effect of the trp1 gene on transformation frequencies in Coprinopsis cinerea. Bastian Doernte, Ursula Kües. Molecular Wood Biotechnology andTechnical Mycology, University of Goettingen, Germany.Genetic transformation of the basidiomycete Coprinopsis cinerea has first been described by Binninger et al. in 1987 (1). For the transfer of geneticmaterial, chromosomal integrative vectors are used, which contain a selectable marker gene and/or a gene of interest. During transformation the geneticmaterial integrates at ectopic sites into the host chromosomes. Binninger et al. (1987) created the vector pCc1001, by cloning a 6.5 kb PstI genomic236