Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS312. Integrated transcriptional profiling and analysis for identification of Cryptococcus neoformans genes regulated during human cryptococcalmeningitis. Y. Chen 1 , J. Tenor 1 , D. Toffaletti 1 , A. Litvintseva 2 , T. Mitchell 1 , J. Perfect 1 . 1) Duke University School of Medicine, Durham, NC; 2) Centers forDisease Control and Prevention, Atlanta, GA.Background: Cryptococcus neoformans is an opportunistic fungal pathogen that is the major cause of fungal meningitis in immunocompromisedindividuals worldwide. Accurate and comprehensive de novo transcriptome profiling of C. neoformans in the human host may allow a betterunderstanding of how it survives and produces disease. Methods and Results: To identify genes, whose expression is differentially regulated under in vivoand in vitro conditions, we selected two strains of C. neoformans var. grubii (serotype A), which were isolated from the cerebrospinal fluid (CSF) of twoAIDS patients from Uganda and the United States. Multilocus sequence typing (MLST) showed that one strain was from VNI clade and one strain from VNII.Next-generation sequencing (RNA-Seq) was used to determine transcriptional profiles of these strains under three conditions: fungal cells were directlytaken from CSF of the patients; fungal cells were grown in YPD at 37°C until the stationary phase; fungal cells that reached the stationary phase in YPDwere exposed to sterile human CSF for 9 hours. The sequencing results showed that there was no major difference in sequencing quality andcontaminations between in vivo and in vitro samples. Hierarchical clustering analysis revealed that the samples treated with same environment have moresimilarity in transcriptional profile. Comparative analysis of the expression pattern shows that 144 genes up-regulated in CSF when compared to YPD and87 genes were up-regulated in vivo compared to YPD and 39 genes overlapped between the CSF and in vivo condition. Some of the overlapping genes inCSF and in vivo have been reported to be related to the virulence composite of C. neoformans, such as Rim101 and ENA P-type ATPase 1. Furthermore, wesearched for the 100 most divergent expressed genes between the two strains. Gene Ontology (GO) term enrichment analysis showed an enrichment ofGO terms in transporter activity between the strains. Conclusion: We provide the first transcriptome profiling of C. neoformans taken directly from the CSFof two human patients. The comparisons between in vivo and in vitro samples helped us to identify a group of genes that may be important for surviving,adapting and proliferating of C. neoformans in the CSF of the human host.313. Structural and functional characterization of microRNA-like RNAs in the penicillin producing fungus Penicillium chrysogenum. Tim Dahlmann,Minou Nowrousian, Ulrich Kück. Christian Doppler Laboratory for Fungal Biotechnology, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum,Deutschland, tim.dahlmann@rub.de.MicroRNAs are endogenous RNAs with a size of about 22 nt and post-transcriptionally regulate gene expression in metazoan and plants. MicroRNAs arederived from RNA hairpin precursors, which are usually transcribed by RNA polymerase II. Recent studies on small RNA binding components of the RNAinducedsilencing complex (RISC) show the existence of microRNA-like RNAs (milRNAs) in Neurospora crassa, which give a first hint of a post-transcriptionalregulatory mechanism based on microRNAs in fungi [1]. So far only little is known about microRNA-like molecules in other fungi, especially about their rolein fungal development and gene regulation.To investigate the occurrence of milRNAs and their involvement in gene regulation in the penicillin producing fungus Penicillium chrysogenum, weperformed predictions of putative microRNAs. Therefore small RNAs (19 - 50 nt) representing different growing conditions and developmental stages,were used for RNA next generation sequencing. The calculation of putative microRNA precursors was performed with the program miRDeep [2], and isbased on the distribution of RNA sequence reads in afore predicted RNA hairpin molecules. By this approach, we were able to identify structures, whichshow the typical characteristics of microRNA precursors. To confirm the in silico predictions, transcript analyses were performed. These analyses supportthe existence of small RNAs and their precursors and show various expression pattern of the putative milRNAs under different growing conditions. Toinvestigate the regulatory role of the identified milRNAs, strains lacking or overexpressing milRNAs were generated. In addition, we have constructedartificial microRNAs to investigate their use as molecular genetic tools to mediate gene specific RNA interference (RNAi). The results of this study provideevidence for milRNAs in P. chrysogenum and indicate a milRNA based silencing mechanism in this fungus.[1] Lee HC et al. (2010) Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Molecular Cell 38:803-814[2] Friedländer MR et al. (2008) Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol 26:407-415.314. Metatranscriptomic analysis of ectomycorrhizal root clusters in Pinus taeda: new methodologies for assessing functional gene expression in situ.H.-L. Liao 1 , Y. Chen 2 , T. D. Bruns 3 , K. G. Peay 4 , J. W. Taylor 3 , S. Branco 3 , J. M. Talbot 4 , R. Vilgalys 1 . 1) Department of Biology Duke University, Durham, NC; 2)School of Medicine, Duke University, Durham, NC; 3) Department of Plant and Microbial Biology, UC-Berkeley, Berkeley, CA; 4) Department of Biology,Stanford University, Stanford, CA.A highly diverse community of ectomycorrhizal (ECM) fungi are known to associate with members of the genus Pinus. Less is known about how diversefungal communities affect functional diversity within ECM roots. Here we present an optimized method for metatranscriptomic analysis of the ECM-pineroot interaction in a natural system. RNA was purified using a CTAB method from individual ECM root clusters collected at varying spatial scales across thedistribution range of P. taeda, and sequenced using Illumina HiSeq technology. About 35 million qualified reads were obtained. Sequences were initiallyassembled using reference based mapping (Bowtie) to sort the reads that represent rRNA from fungal and bacterial species. Reads from divergent regions(D1-D2) of fungal LSU rRNA were used to identify dominant ECM and other fungal community members. Subsequently, P. taeda genes and functionalgenes of dominant fungal species were sorted using public cDNA databases. The Trinity package was used for de novo assembly of un-mapped reads(mostly fungal genes). Blastx and Go packages were used for gene annotation. A typical ECM root cluster was found 45% P. taeda genes, 3% fungal rRNA,0.05% bacterial 16S rRNA, 30% fungal functional genes, 10% unknown sequences, and 12% unassembled reads. Analysis of D1-D2 LSU sequencesconfirmed that a single ECM fungal species usually dominates individual root clusters. De novo assemblies of fungal genes yielded 120 thousand contigsfrom 10 million reads representing 90 thousand unique genes with highly similarity to known ECM fungi. Functional analysis revealed that most of thetranscripts recovered were involved with translation, protein degradation, heat shock, superoxide metabolism, electron transfer, signaling, and C/Nmetabolism. Highly expressed transcripts recovered from Piloderma, which was abundant in our samples, included genes encoding a wide array ofmetabolic enzymes: chitosanase, phosphatase, glutamine synthetase, terpene synthases, b-glucanase; transporters for P+ and oligopeptides; cell signaling:calmodulin, cAMP-regulated phosphoprotein (Igo1); C/N related genes: lectin, cross-pathway control (cpc1); as well as several genes with unknownfunction. Future studies will seek to address how ECM metatranscriptomes change in response to different Pinus hosts and across different spatial scales.315. Transcriptomic response of Neurospora crassa germinating conidia to chitosan in sub-lethal dose. Federico Lopez-Moya 1 , David Kowbel 2 , N. LouiseGlass 2 , Luis Vicente Lopez-Llorca 1 . 1) Laboratory of Plant Pathology, Multidisciplinary Institute for Environment Studies (MIES) Ramón Margalef. Universityof Alicante, Alicante, SPAIN; 2) Department of Plant and Microbial Biology, University of California, Berkeley CA, 94720-3120 USA.Chitosan is a natural polymer able to permeabilize Neurospora crassa membranes, in an energy dependent manner. Plasma membrane permeabilisationby chitosan depends on membrane fluidity, with FFA unsaturated membrane fungi (N. crassa) being chitosan sensitive, the plasma membrane fluidity is an27th Fungal Genetics Conference | 197