Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS415. Artificial miRNA constructs for Phytophthora sojae transformation. Stephanie R. Bollmann 1 , Felipe D. Arredondo 1 , Noah Fahlgren 2 , James C.Carrington 2 , Niklaus J. Grünwald 3 , Brett M. Tyler 1 . 1) Center for Genome Research and Biocomputing, and Department of Botany and Plant Pathology,Oregon State University, Corvallis, OR; 2) Donald Danforth Plant Science Center, St. Louis, MO; 3) Horticultural Crops Research Laboratory, USDAAgricultural Research Service, Corvallis, OR.Phytophthora, a genus of fungal-like oomycetes, contains some of the most devastating plant pathogens, causing multi-billion dollar damage to crops,ornamental plants, and natural environments. The genomes of five Phytophthora species, including the soybean pathogen P. sojae, have recently beensequenced, with many more species soon to be completed. Gene regulation by small RNA pathways is highly conserved among eukaryotes, although littleis known about small RNA pathways in the Stramenopile kingdom. Two Dicer homologs, DCL1 and DCL2, and one RDR homolog were cloned andannotated from P. sojae, and gene expression analysis revealed only minor changes in transcript levels among different lifestages and infection timepoints.At this point, the role of the two oomycete Dicer homologs are only speculated. This study aims to down-regulate DCL1 and DCL2 expression in order toanalyze the contribution of each homolog to small RNA biogenesis. Traditional RNAi, such as overexpression of RNA complementary to a target mRNAtranscript, has been used to knockdown gene expression in Phytophthora, although the effect is most often short-lived. Dicer homologs are involved in theRNAi pathway, therefore this method may not be effective, especially for the homolog involved in the siRNA pathway. Artificial miRNAs, designed fromendogenous miRNAs, have recently been used to target transcripts such as these. We designed artificial miRNA constructs based on the conservedPhytophthora miRNA found in P. sojae, targeting both DCL1 and DCL2 as well as the effector Avr1k, the histidine biosynthesis enzyme HISG, and GFP forcontrols. Analysis of transformants is currently underway.416. RNAi-dependent epimutations evolve antifungal drug resistance in the zygomycete fungal pathogen Mucor. Silvia Calo Varela 1 , Cecelia Shertz 1 ,Robert J Bastidas 1 , Soo Chan Lee 1 , Piotr Mieczkowski 2 , Joshua A Garnek 1 , Rosa Ruiz-Vazquez 3 , Santiago Torres-Martinez 3 , Maria E Cardenas 1 , JosephHeitman 1 . 1) Molecular Genetics and Microbiol, DUKE University Medical Center, Durham, NC; 2) High Throughput Sequencing Facility, CCGS, UNC,Chapel Hill, NC; 3) Department of Molecular Genetics and Microbiology, University of Murcia, Murcia, Spain.Microorganisms evolve via a panoply of mechanisms spanning aneuploidy, sexual/parasexual reproduction, mutators, Hsp90, and even prions.Mechanisms that may seem detrimental can be repurposed to generate diversity. The pathogenic fungus Mucor circinelloides grows as a hyphaeaerobically, but as a yeast in anaerobic conditions or in the presence of the immunosuppressive drug FK506. FKBP12 is a protein folding enzyme conservedthroughout eukaryotes that interacts with FK506 and mediates antifungal activity of this drug. The FK506-FKBP12 complex inhibits the proteinphosphatase calcineurin and thereby blocks hyphal growth of M. circinelloides. Continued exposure to FK506 yields resistant isolates, which exhibit hyphalgrowth emerging from the yeast colony. Some isolates harbor a variety of mutations in the fkbA gene that encodes FKBP12. However, other isolatesharbor no mutations in the fkbA gene. These unusual epimutant isolates also revert frequently within several generations of vegetative growth in drugfreemedia and are restored to wild-type (yeast growth in the presence of FK506). Northern and Western analyses revealed a loss of fkbA mRNA andFKBP12 protein in the epimutants. High-throughput sequencing and Northern blot also detected sRNA generated from fkbA in the epimutant strains,revealing a new role for RNAi in the development of transient, reversible resistance to an antifungal drug treatment. RNAi could be triggered via dsRNAproduction from an overlap in the 3’ regions of the mRNA of fkbA and its neighboring gene patA, which encodes a putative polyamine transporter. Ourresults reveal a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity in fungi.417. Heterochromatic marks are involved in the repression of plant-regulated secondary metabolism in Epichloë festucae and for symbiotic interactionwith the host perennial ryegrass. Tetsuya Chujo, Barry Scott. Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.The fungal endophyte Epichloë festucae systemically colonizes perennial ryegrass (Lolium perenne), and produces a range of secondary metabolites toprotect the host plant. The alkaloid peramine provides protection against insect herbivory. Protection against mammalian herbivory is afforded by theproduction of alkaloids, such as ergot alkaloids and lolitrems. Using the E. festucae-perennial ryegrass symbiotic association as a model experimentalsystem we have shown that gene clusters for the synthesis of these bioprotective metabolites are all preferentially and highly expressed in planta, but notexpressed in culture. Recent work showed that disruption of genes encoding either heterochromatin protein-1 (HepA) or the H3K9 methyltransferase(ClrD) in Aspergillus nidulans resulted in enhanced expression of secondary metabolite gene clusters, demonstrating that heterochromatic marks areinvolved in the repression of these clusters. Thus, we hypothesized that plant-regulated E. festucae secondary metabolite gene clusters have a repressivechromatin structure in culture, and chromatin remodeling is an important component for activation of these gene clusters in planta. To test thishypothesis we have deleted the hepA and clrD homologues from E. festucae by targeted gene replacement. Deletion of hepA resulted in a slight reductionin culture radial growth whereas deletion of clrD resulted in a severe reduction. Western blot analysis revealed that the level of H3K9 tri-methylation(H3K9me3) is dramatically decreased in DclrD mutants. Expression levels of ltmG & ltmM (cluster 1) and ltmP & ltmF (cluster 2), as measured by qRT-PCR,increased in both the DhepA and DclrD mutants grown in a defined medium. Introduction of a wild-type allele of either hepA or clrD complemented DhepAor DclrD mutant phenotypes, respectively. In addition, the DhepA mutant has a dramatic host interaction phenotype, inducing severe stunting andpremature senescence of the ryegrass host. On the other hand, DclrD mutant is an infection mutant. These results strongly suggest that heterochromaticmarks regulate both secondary metabolite gene expression and the mutualistic symbiotic interaction of E. festucae with its host perennial ryegrass.418. Cellulose Degradation Regulator 2 Induces Expression of a Conserved Core of Genes for Plant Cell Wall Saccharification in Neurospora crassa andAspergillus nidulans. Samuel T. Coradetti, Yi Xiong, N Louise Glass. Department of Plant and Micorbial Biology, University of California, Berkeley, CA.To better understand mechanisms of cellulase gene regulation and genome-wide gene regulation enabling robust enzyme secretion, we studied theconservation of gene regulation by cellulose degradation regulator 2 (CLR-2) in Neurospora crassa and Aspergillus nidulans. Misexpression of CLR-2 undernormally repressive and non-inducing culture conditions was sufficient for cellulases secretion in N. crassa, but not A. nidulans. We used RNAseq to mapthe trascriptome in wild-type, deletion and mis-expression strains of both species. We identified a cohort of conserved enzymes with conserved sequenceand CLR-2 dependent regulation across evolutionarily divergent ascomycetes, which represent a core of essential enzymes for degradation of complexcellulosic substrates. We also identified non-conserved CLR-2 regulated genes in each species, which may have function specific to a particular substrate orniche. These data suggest that manipulation of CLR-2 has significant potential for improved cellulase production from industrial production strains.419. The transcriptional repressor CRE-1 regulates glycogen metabolism in Neurospora crassa. Fernanda B. Cupertino, Stela Virgilio, Fernanda Z. Freitas,Thiago S. Candido, Maria Célia Bertolini. Instituto de Quimica,UNESP, Araraquara, São Paulo, Brazil.In Neurospora crassa the RCO-1 co-repressor, an orthologue of the yeast Tup1, has been identified as a protein involved in glycogen metabolismregulation in a screening of a transcription factor knocked-out strains set. The Saccharomyces cerevisiae Tup1 protein participates in the Tup-1-Ssn627th Fungal Genetics Conference | 223