Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSThursday, March 14 3:00 PM–6:00 PMMerrill HallCool Tools for Fungal BiologyCo-chairs: Miguel Penalva and Kevin McCluskeyThe Environmental Molecular Sciences Laboratory molecular analysis capabilities for fungal biology. S. E. Baker. Environmental Molecular SciencesLaboratory, Pacific Northwest Natl Lab, Richland, WA.Tools for analysis of classical and reverse genetic mutants play an important role in fungal biology research. The Environmental Molecular SciencesLaboratory (EMSL) at the Pacific Northwest National Laboratory is a US Department of Energy national user facility. EMSL develops and utilizes cuttingedge mass spectrometry, NMR, imaging and computational capabilities to accelerate research in a number of areas. We have used EMSL’s massspectrometry capabilities to characterize glycosylation of secreted proteins of Aspergillus niger. In addition, we have explored the use of laser ablation andnano-DESI mass spectrometry for spatial localization of molecules associated with Trichoderma reesei mycelium. Finally, spores from wildtype and albinostrains of Aspergillus carbonarius were characterized using helium ion microscopy. As a national user facility, the EMSL is open to the fungal biologycommunity through a competitive, peer-reviewed proposal process.Development and utilization of arrayed mutant sets for yeasts and filamentous fungi. Aric E Wiest, Kevin McCluskey. Fungal Genetics Stock Center,Kansas City, MO.Advancements in high throughput functional genomics has allowed the generation of vastly increasing numbers of strains carrying single gene deletions.For some organisms these include mutations distributed across the genome. The FGSC has generated or acquired sets of arrayed mutants for severaldifferent yeast or filamentous fungal species including Neurospora crassa, Magnaporthe grisea, Cryptococcus neoformans, Candida albicans, Aspergilusnidulans, and Pichia pastoris. These arrayed sets allow rapid screening for desired traits across a broad number of gene deletions. Details of construction,replication and manipulation of these arrayed sets will be presented. Custom arraying, construction of functional sets, and cryopreservation will also bediscussed.Sequencing-based solutions to identify and characterize fungal developmental genes. Minou Nowrousian, Ines Teichert, Gabriele Wolff, Ulrich Kück.Dept. of General & Molecular Botany, Ruhr University Bochum, Bochum, Germany.During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of spores. We areusing a combination of classical genetics, next generation sequencing, molecular and microscopic methods to learn more about this differentiation processin the model organism Sordaria macrospora, and here we present data on the identification/characterization of transcription factors and signalingmolecules that are involved in development. Whole genome sequencing of mutant pro44 was used to identify the mutation that causes sterility in themutant strain. For Illumina/Solexa sequencing, pooled DNA from progeny of crosses of the mutant with the wild type was used, and we were able topinpoint the causative mutation in the mutant strain through bioinformatics analysis. pro44 carries a mutation in a GATA-type transcription factor, andfertility can be restored by transformation with the wild-type allele. In a second approach, we used laser microdissection to isolate young fruiting bodies(protoperithecia) of the wild type and mutant pro1 that carries a deletion of another transcription factor gene essential for sexual development. Linearamplification of RNA from microdissected protoperithecia yielded enough material for RNA-seq analysis. A comparison with total mycelium revealedsignificant differences in in gene expression between protoperithecia and non-reproductive mycelia. Among the genes strongly up-regulated inprotoperithecia were the pheromone precursor genes ppg1 and ppg2. This was confirmed by fluorescence microscopy of egfp expression under control ofppg1 regulatory sequences. In protoperithecia, many genes are under control of the transcription factor PRO1; thus, by combining laser microdissectionand RNA-seq, we can now perform genome-wide analyses of genes that are dependent on a development-specific transcription factor for correctexpression in a defined developmental structure in fungi. Among the genes that are dependent on PRO1 for correct expression in protoperithecia is pro44,which is among the 500 most strongly expressed genes in wild-type, but not pro1 protoperitheica. In summary, our data indicate that PRO1 and PRO44 aremembers of a transcription factor network that regulates gene expression and cell differentiation in developing fruiting bodies.Aspergillus nidulans as an experimental system to identify novel cell wall growth and maintenance genes through identification of anti-fungal drugresistance mutations. Xiaoxiao Sean He, Shengnan Jill Li, Susan Kaminskyj. Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.Systemic fungal infections are estimated to contribute to ~10% of hospital deaths. Systemic fungal infections are most dangerous for the young, the old,and the already sick, since their immune systems are less vigorous. Most antifungal drugs in current clinical use target ergosterol (polyenes) or theergosterol biosynthetic pathway (azoles and allylamines). Drugs against beta-glucan synthesis (echinocandins) are effective against aspergillosis andcandidaisis. The use of compounds that target fungal enzymes inevitably leads to the development and natural selection of drug resistant fungal strains.Not only are the anti-fungal drugs in current clinical use losing efficacy in some situations, but in addition the high level of conservation between animaland fungal physiology leaves relatively few relevant targets to explore. However, it is likely that for any drug-enzyme combination there will be relativelyfew mutations that could increase drug resistance while still maintaining enzyme function. We are using Aspergillus nidulans as an experimental modelsystem to assess the number and identity of mutations that lead to drug resistance. As proof of concept, we grew wild type A. nidulans on replicate platescontaining a sub-lethal concentration of Calcofluor. These developed fast-growing sectors beginning at ~ 5 d (70 rounds of mitosis). Preliminary resultsshow that many of these sectors harboured heritable, single-gene mutations. To date, mutated genes that confer robust, heritable resistance to Calcofluorthat were identified by next generation sequencing have roles in cell wall synthesis, cell wall integrity regulation, or drug detoxification. We suggest thisstrategy will be useful for predicting genetically-mediated anti fungal resistance adaptation and help us to be ahead in the drug-resistance arms race.27th Fungal Genetics Conference | 47