The Tenth International Aspergillus Meeting Asilomar Conference Grounds

s: (*Denotes student presenting poster) Biochemistry and Metabolism 1. Identification of local and cross-chromosomal biosynthetic gene clusters in filamentous fungi using gene expression data. Mikael R. Andersen 1 , Jakob B. Nielsen 1 , Andreas Klitgaard 1 , Lene M. Petersen 1 , Tilde J. Hansen 1 , Lene H. Blicher 1 , Charlotte H. Gottfredsen 2 , Thomas O. Larsen 1 , Kristian F. Nielsen 1 , Uffe H. Mortensen 1 . 1) Department of Systems Biology, Technical University of Denmark, Kgs Lyngby, Denmark; 2) Department of Chemistry, Technical University of Denmark, Kgs Lyngby, Denmark Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodological gene deletions to identify supporting enzymes for key synthases one cluster at a time. In earlier work we presented a method for using a gene expression compendium to accurately predict co-regulated gene clusters in general, and in particular the members of gene clusters for secondary metabolism. A benchmarking of the method in Aspergillus nidulans by comparison to previous gene deletion studies showed the method to be accurate in 13 out of 16 known clusters and nearly accurate for the remaining three. In this work, we have expanded the algorithm to identify cross-chemistry between physically separate gene clusters (super clusters), and validate this both with legacy data and experimentally by prediction and verification of a new supercluster consisting of the non-ribosomal peptide synthetase (NRPS) AN1242 (on chr VIII) and the prenyltransferase AN11080 (on chromosome V) as well as identication of the shared product compound nidulanin A. We also propose further implications of the gene clustering, as our analysis shows that approximately 10 % of the genes seem to be non-randomly (p<0.05) co-regulated with more than two neighboring genes. We have employed A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom. We furthermore present the preliminary analysis of the application of the method to A. niger. *2. N-glycan profiling of Aspergillus nidulans using solid-phase glycan extraction and mass spectrometry. Diana Anyaogu 1 , Shuang Yang 2 , Jakob B. Nielsen 1 , Hui Zhang 2 , Michael Betenbaugh 3 , Uffe Hasbro Mortensen 1 . 1) Department of Systems Biology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; 2) Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21287, United States; 3) Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States Filamentous fungi from the Aspergillus species are widely used as cell factories for the production of chemicals and enzymes, especially Aspergillus niger and Aspergillus oryzae are used as protein producers. Fungi have a high secretion capacity in comparison to other eukaryotic expression systems as algae, yeast and insect cells. The majority of the secreted proteins are glycosylated, thus glycosylation plays an important role in the secretory pathway. Glycosylation is also important in the production of therapeutic proteins as it is involved in protein stability, ligand binding, immunogenicity and serum half-life. Furthermore the efficacy of many therapeutic proteins depends on correct glycosylation. Thus, understanding the glycosylation will enable the directed glycoengineering in Aspergilli to improve protein production. In the present study the Solid-Phase Glycan Extraction (SPGE) method was used to isolate and purify N-glycans from the secretome and whole cell lysates from Aspergillus nidulans. The mass of the glycans was determined using a MALDITOF MS. In addition, A. nidulans strains with mutations in the glycosylation pathway were analyzed and compared to the reference strain. This study shows that some of the mutations had an effect on the N-glycan profile, which shifted the profile towards glycans with a lower mass. The method presented here is thus very efficient for extracting N-glycans and for quantifying the relative abundance of different N-glycans in the secretome and whole cell lysate . *3. Heme regulation in Aspergillus fumigatus. Nicola Beckmann 1 , Ernst R. Werner 2 , Hubertus Haas 1 . 1) Division of Molecular Biology, Biocenter, Innsbruck Medical University, Austria; 2) Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Austria Sufficient iron supply is indispensable for survival of almost all organisms. However, an excess of iron is potentially toxic. In the opportunistic human-pathogenic fungus Aspergillus fumigatus the ability to adapt to iron limitation represents a crucial virulence factor. Iron regulation is tightly interconnected with heme metabolism, as iron-containing heme is an essential cofactor of a variety of cellular processes, e.g. respiration, sterol biosynthesis, oxidative stress detoxification and also reductive iron assimilation. Most knowledge on fungal heme regulation derives from studies in Saccharomyces cerevisiae. A. fumigatus, as well as most other fungal species, lack homologs of key heme regulators found in S. cerevisiae. The goal of this study is to elucidate heme-dependent regulation in A. fumigatus (wt). As a first step, we generated a mutant strain (ΔhemA) lacking the gene encoding aminolevulinic acid synthase (HemA), which catalyzes the committed step in heme biosynthesis. This mutation offers the possibility to control the cellular heme content by supplementation with aminolevulinic acid (ALA). Growth of ΔhemA was blocked at ALA concentrations below 20 μM, but fully restored by addition of 200 μM on solid as well as in liquid media. Supplementation with protoporphyrin IX (PpIX), the iron free heme precursor, and hemin (chloroprotoporphyrin IX iron(III)) supported growth of ΔhemA, proving that A. fumigatus is able to utilize exogenous porphyrins. Nevertheless, A. fumigatus in contrast to several other fungal species is not able to utilize hemin as iron source. Under iron starvation, ALA supplementation led to a tremendous accumulation of PpIX in both ΔhemA and wt, which indicates that HemA represents the major rate limiting step in heme biosynthesis when iron is scarce. In ΔhemA, ALA restriction transcriptionally increased the heme-biosynthetic coproporphyrinogen(III)oxidase and the putative heme receptor CFEM3. Additionally, ALA limitation decreased the resistance of ΔhemA to oxidative stress and the triazole antifungal drug posaconazole, which underlines the crucial role of heme in detoxification and sterol biosynthesis. This work was supported by the Austrian Science Foundation grant FWF P21643-B11 to HH. *4. Key Steps in the Biosynthesis of the Fungal Virulence Factor Gliotoxin. Pranatchareeya Chankhamjon 1 , Daniel H. Scharf 2 , Kirstin Scherlach 1 , Nicole Remme 1 , Andreas Habel 1 , Thorsten Heinek 2 , Martin Roth 3 , Axel A. Brakhage 2 , Christian Hertweck 1 . 1) Biomolecular Chemistry, HKI, Jena, Jena, Germany; 2) Molecular and Applied Microbiology,HKI, Jena, Jena, Germany; 3) Bio Pilot Plant,HKI, Jena, Jena, Germany The prototype of epipolythiodioxopiperazine (ETP) family, gliotoxin, is an infamous virulence factor of the human pathogen Aspergillus fumigatus, notably the leading cause of invasive aspergillosis in the immunocompromised patients. Its toxicity has been attributed to the unusual intramolecular disulfide bridge, which is the functional motif of all ETPs. A number of studies showed that the diketopiperazine core of gliotoxin is assembled by a non-ribosomal peptide synthetase. However, downstream pathway steps have remained elusive, mainly because of the scarcity and instability of pathway in the mediates produced. Here we present the critical role of a specialized glutathione S-transferase (GST), GliG, in the enzymatic sulfurisation and the key step of epidithiol formation by an unprecedented twin carbon-sulfur lyase, GliI. Our studies not only unveil the understanding of key steps in the biosynthesis pathway of an important virulence factor, but also outline a new function of microbial GSTs and gain insights into the formation of organosulfur compounds. 5. Induction of sclerotia and Aspergillus section Nigri. Jens Frisvad, Lene Petersen, Ellen Lyhne, Thomas Larsen, CMB, Dept Systems Biol, Kgs. Lyngby, Denmark The purpose of this study was to induce sclerotium production in Aspergillus niger and other black Aspergilli. Some species in Aspergillus section Nigri are known for their production of sclerotia, especially A. carbonarius, A. tubingensis (few isolates), A. sclerotioniger, A. sclerotiicarbonarius, A. costaricaensis, A. piperis, A. japonicus, and A. aculeatus. A. heteromorphus was reported in 1955 to produce sclerotia, but this could not be confirmed in later studies. There are also un-confirmed data on sclerotium production in Aspergillus niger, but often isolates reported to produce sclerotia were not A. niger anyway. Induction of sclerotium production in Aspergillus niger is important, since this may help in inducing the perfect state in this important industrial fungus. By screening several media, we were able to develop some media and use some growth conditions that induced sclerotium production in Aspergillus niger and other species hitherto not reported to produce sclerotia. Earlier French beans were suggested as inducers of sclerotium production, but we could not repeat this with any isolate of A. niger. However by using media such as white rice and brown rice or adding different fruits to CYA (Czapek yeast autolysate agar) and incubate at 25 C we were able to induce sclerotium production in certain strains of A. niger. Old strains used for citric acid production, or full genome sequenced strains, were not induced to produce sclerotia, but several fresh strains from different foods did produce abundant sclerotia on the different media, at 25 C, but not 37 C. One older classical citric acid producer from NRRL produced many sclerotia, however. Sclerotium producing isolates also contained aflavinines, confirmed by HPLC-DAD-MS-MS, secondary metabolites only produced in the sclerotia, and detected in A. niger for the first time. Other species, such as A. ibericus, A. neoniger, A. heteromorphus, A. fijiensis, A. luchuensis (formerly A. acidus), A. aculeatinus and A. saccharolyticus could also produce sclerotia on fruit media. The sclerotia contained many sclerotium-specific secondary metabolites. 6. Identification of a gene cluster mediating the biosynthesis of the Aspergillus fumigatus cell wall and secreted polysaccharide, galactosaminogalactan. Fabrice N. Gravelat 1 , Mark J. Lee 1 , Alexander Geller 1 , Dan Chen 2 , Anne Beauvais 3 , Hong Liu 4 , William C. Nierman 2 , Jean-Paul Latge 3 , Thierry Fontaine 3 , Scott G. Filler 4 , Donald C. Sheppard 1 . 1) Microbiology & Immunology Department, McGill University, Montréal, Qc, Canada; 2) J. Craig Venter Institute, Rockville, Maryland, USA; 3) Aspergillus Unit, Institut Pasteur, Paris, France; 4) Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA Aspergillus fumigatus is the most common cause of invasive mold disease in humans. Although adherence of fungal hyphae to host constituents is a critical early step in the pathogenesis of invasive aspergillosis, the molecular mechanisms underlying this process have not been elucidated. Using a forward genetic approach, we identified a glucose epimerase, Uge3, which is required for adherence of hyphae to a wide variety of substrates. Biochemical analyses confirmed that Uge3 is required for the synthesis of the secreted glycan galactosaminogalactan (GAG), which in turn functions as the dominant adhesin of A. fumigatus hyphae and is required for virulence. However, the biochemical and regulatory pathways governing GAG synthesis remain unknown. Using comparative transcriptome analysis, we found that uge3 is found within a cluster of 5 co-regulated genes on chromosome 3. Interestingly, 3 of the 5 proteins (Uge3, Gtb3 and Ega3) encoded by these genes are predicted to contain conserved domains involved in polysaccharide metabolism. The 2 other proteins (Sph3 and Esr3) have no homologs in other organisms. We hypothesized that this cluster of genes may be required for GAG biosynthesis. To test this hypothesis, we constructed deletion mutants of two of the cluster genes: sph3, encoding a cell surface spherulin 4-like protein; and esr3, encoding an extracellular serin-rich protein. Phenotypic analysis of both the Δesr3 and Δsph3 mutant strains confirmed that deletion of these genes resulted in both impaired GAG production and impaired adherence, similar to the phenotype of the Δuge3 mutant strain. Gene deletion for the 2 remaining genes is ongoing. Collectively, these data suggest that the 5 gene cluster identified on chromosome three is likely a carbohydrate biosynthetic cluster required for the synthesis of GAG. Importantly, this is the first description of a gene cluster for the biosynthesis of a cell wall polysaccharide in A. fumigatus, and suggests the possibility that other similar gene clusters may govern the synthesis of glycans in this fungus. The discovery of this cluster, and the subsequent characterization of the role of each of the component elements, may provide insight into the synthesis and function of GAG. *7. Metabolic adaptations in Phytophthora infestans and the role of a phosphagen kinase system in energy metabolism. Meenakshi Kagda, Howard Judelson. Plant Pathology and Microbiology, University of California, Riverside, CA 92521. Nutrient acquisition and metabolic adaptation to host-derived nutrients is an important aspect of pathogen biology. An understanding of the metabolic adaptations made by Phytophthora infestans, an important pathogen of potato and tomato, to optimize nutrient uptake from diverse host tissues and within the microenvironments of the host will lead to a better understanding of host-pathogen relationships. In order to study metabolic adaptations of P. infestans, transcriptional profiling and live cell imaging using promoter-fluorescent protein fusions will be used. Preliminary results demonstrated the differential gene expression of many metabolic genes of P. infestans grown on different natural hosts and that grown on rich media. The next step involves answering the question: Are some metabolic genes expressed in a stage-specific or time-dependent manner? In addition, the role of enzymes involved in energy homeostasis and metabolite channeling are being studied. The roles of two such genes encoding putative creatine kinases are being elucidated using subcellular localization, substrate utilization and loss of function studies. 8. LaeA sleuthing reveals cryptic gene clusters in pathogenic Aspergilli. Nancy Keller 2 , Wenbing Yin 2 , Saori Amaike 2 , Katharyn Affeld 2 , JinWoo Bok 2 , Daniel Schwenk 3 , Dirk Hoffmeister 3 , Joshua Baccile 1 , Ry Forseth 1 , Frank Schroeder 1 1) Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; 2) Department of Plant Pathology, Department of Medical Microbiology and Immunology, and Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA; 3) Department of Pharmaceutical Biology at the Hans‐Knöll‐Institute, Friedrich‐Schiller‐Universität, Beutenbergstraße 11a, 07745 Jena, Germany The human and plant pathogenic Aspergilli, Aspergillus fumigatus and A. flavus, are known to produce a plethora of secondary metabolites. However, most of these metabolites are not yet characterized although their gene clusters are apparent from genomic sequence. In both species, the nuclear protein LaeA regulates the expression of many of these uncharacterized gene clusters. Following leads from laeA mutant microarray data, we created gene deletion and overexpression strains and used 2D NMR-based comparative metabolomic analyses to identify previously undescribed metabolites from both species. In A. fumigatus a tryptophan-derived iron(III)-complex, hexadehydro-astechrome (HAS), was found to be the major product of the cryptic has non-ribosomal peptide synthetase (NRPS) cluster. In A. flavus we show that two separate clusters encode enzymes that produce partially overlapping sets of novel piperazines, pyrazines, and morpholines. These L-tyrosine metabolites are activated by two NRPS-like proteins, LnaA and LnbA. Loss and overexpression of these metabolites impacted fungal development in these species. *9. Characterization of fumiquinazoline biosynthesis in Aspergillus fumigatus. Fang Yun Lim 1 , Brian Ames 2 , Christopher Walsh 2 , Nancy Keller 1 . 1) Medical microbiology and Immunology, University of Wisconsin-Madison, Madison, WI.; 2) Biological chemistry and molecular pharmacology, Harvard Medical School, Boston, MA The fumiquinazolines (FQs) comprise a related, sequentially generated family of bioactive peptidyl alkaloids that are signature metabolites of Aspergillus fumigatus. The FQ framework is built by nonribosomal peptide synthetase (NRPS) machinery with anthranilate as a key non-proteinogenic amino acid building block. Despite being prevalent across the species, its gene cluster has not been characterized. Prior bioinformatic analysis coupled with heterologous expression of the putative A. fumigatus proteins termed here FmqA -FmqD led to the identification of a four-enzymatic process that builds increasingly complex FQ scaffolds. Briefly, FmqA, a trimodular NRPS condenses alanine, tryptophan, and anthranilic acid to form fumiquinazoline F (FQF). The tandem action of a flavoprotein (FmqB) and a monomodular NRPS (FmqC) converts FQF to fumiquinazoline A (FQA). Finally, FmqD, a FAD-dependent oxidoreductase converts FQA to the heptacyclic fumiquinazoline C (FQC). Interestingly, FmqD contains an N-terminus signal peptide predicted for extracellular transport. This study is aimed at providing in vivo validation to the FQ biosynthetic framework and characterizing how cellular localization of FmqD affects production of FQC in A. fumigatus. We found that the conidial metabolite, FQC, is the predominant FQ moiety in two wild type isolates and is selectively accumulated in the conidia. Targeted single gene deletions of FmqA through FmqD coupled with metabolomic profiling of the single biosynthetic gene mutants supported previous biochemical prediction of FQ biosynthesis. Fluorescent microscopy of mutants bearing a C-terminal FmqD-GFP fusion showed that FmqD is localized to the cell wall of the fungus and this localization is abolished when the signal peptide is removed. Future studies will elucidate if cell wall localization of FmqD is crucial for FQC production. 10. Engineering Cyclic Peptide Biosynthesis in Poisonous Mushrooms. Hong Luo, John S. Scott Craig, Robert M. Sgambelluri, Sung-Yong Hong, Jonathan D. Walton. Department of Energy Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, United States Ninety percent of fatal mushroom poisonings are caused by alpha-amanitin and related bicyclic peptides found in some species of Amanita, Galerina, Lepiota, and Conocybe. We showed that the amatoxins (mainly amanitins) and related phallotoxins are synthesized on ribosomes in A. bisporigera and the unrelated mushroom G. marginata. The primary gene products are short (34-35 amino acid) proproteins that are initially processed by a dedicated prolyl oligopeptidase. A genome survey sequence of A. bisporigera suggested that it has a repertoire of over 40 cyclic peptides, all produced on a single biosynthetic scaffold. Members of this extended gene family are characterized by conserved upstream and downstream amino acid sequences, including two invariant proline residues, flanking a six to ten-amino acid “hypervariable” region that encodes the amino acids found in the mature toxins (or predicted toxins). The evidence indicates that A. bisporigera has evolved a combinatorial strategy that could in principle biosynthesize billions of small cyclic peptides. In order to study the other steps in amanitin biosynthesis, and to engineer novel cyclic peptides, we have developed a transformation strategy for the amanitin-producing mushroom G. marginata. This first transformation method uses Agrobacterium-mediated transformation followed by hygromycin selection. Taking advantage of this platform, we are introducing artificial toxin genes that are deliberately designed to provide insights into the pathway. The synthetic genes include those that encode the cyclic octapeptide beta-amanitin, the heptapeptides phalloidin and phallacidin, examples of the toxin gene family known from A. bisporigera but not G. marginata, and randomly generated artificial sequences. Currently, thousands of transformants have been generated through an efficient pipeline and the transformants are being analyzed for production of the expected products. If successful, the novel peptides will be screened in a number of assays including RNA polymerase (the site of action of alpha-amanitin), membrane ion channels, pathogenic bacteria, and cancer cell lines. *11. Genomic and metabolomic analysis of polyketide production in Ascocoryne sarcoides. Ross C. Mann 1,2* , Scott W. Mattner 2 , Simone J. Rochfort 1,2 , Ian J. Porter 1,2 , German C. Spangenberg 1,2 . 1) Department of Primary Industries, Biosciences Research Division, Bundoora, Victoria, Australia; 2) La Trobe University, Bundoora, Victoria, Australia. Polyketides are an important group of secondary metabolites from fungi that exhibit a wide range of structural diversity and function, with many reported to be involved in protection (eg. pigments) and defense (eg. antibiotics). The structural diversity of polyketides is largely attributed to the polyketide synthases that catalyse their synthesis, due to their exceptional assemblage and combinatorial power. An isolate of Ascocoryne sarcoides was found to produce four linear polyketides of similar structure, and these exhibited bioactivity against the Gram positive bacteria Bacillus subtilis. Structural characterisation of one of the polyketides via HREIMS and NMR indicated a new metabolite. The polyketide exhibited varying levels of reduction along the central carbon chain, which is indicative of biosynthesis via a highly reducing iterative polyketide synthase. The genome of the isolate of A.sarcoides was sequenced, assembled and annotated, which enabled genome mining for polyketide synthases. A total of three polyketide synthases were identified, which contained domains associated with highly reducing iterative polyketide synthases. Genetic diversity studies were conducted to ascertain relatedness to functional polyketide synthases of fungi (eg. lovastatin, fumonisin, T-toxin). The gene clusters surrounding the polyketide synthases were also profiled to ascertain genetic mechanisms involved in polyketide production (ie. transporters, regulators, etc). This study has used advances in metabolomics and genomics to conduct an in depth analysis of polyketides from A. sarcoides, and the genes that regulate their production. Future studies will look to functionally characterise these genes, and determine their potential application in the agricultural and biofuel sectors. *12. Genome mining to identify gene clusters involved in monoand sesquiterpene production in the bioactive endophytic fungus, Nodulisporium sp. Ross C. Mann 1,2* , Scott W. Mattner 2 , Simone J. Rochfort 1,2 , Ian J. Porter 1,2 , German C. Spangenberg 1,2 . 1) Department of Primary Industries, Biosciences Research Division, Bundoora, Victoria, Australia; 2) La Trobe University, Bundoora, Victoria, Australia. Endophytic fungi are an emerging tool in agriculture, in part, due to their ability to protect their host plant against biotic stresses. The secondary metabolism of endophytic fungi is central to their unique role in defence of their host. Endophytic isolates of a Nodulisporium sp. were found to produce a suite of volatile secondary metabolites, bioactive against a range of agricultural pathogens (fungi and bacteria), pests (insects) and weeds. Profiling the volatile metabolome of isolates of Nodulisporium sp. identified 52 metabolites, with the majority belonging to the mono(eg. eucalyptol) and sesqui(eg. βElemene) terpene structural class. Terpenes are important defense metabolites of plants and have been researched extensively, along with the terpene synthase genes that regulate their production. However, only a limited amount of research has been conducted on fungal terpenes and their biosynthesis (eg. mycotoxins), while no monoterpene synthases have been identified from fungal species. Terpene synthases are unique in their function, in that they can synthesise multiple terpene structures from the one enzyme, and this potentially offers an endophyte a diverse arsenal against pathogens and pests. The genomes of six isolates of Nodulisporium sp. were sequenced, assembled and annotated, and the genes involved in terpene production identified through genome mining. A total of eight terpene synthases were identified, which is disproportionately high compared to other sequenced fungal species. The gene clusters surrounding the terpene synthases were profiled to ascertain genetic mechanisms involved in terpene production (ie. transporters, regulators, etc). Comparative genomic studies were also employed to evaluate genetic diversity across the terpene synthase gene clusters of the six isolates. This metabolomic and genomic study provides new information about metabolite diversity of terpenes in endophytic fungi and the genes regulating their production. This knowledge is providing the foundation for future functional analysis of this important gene class, and also genetic information for the creation of designer endophytes to improve disease resistance of the host plant. 13. Platforms for secondary metabolite analysis in filamentous fungi. Uffe H. Mortensen 1 , Jakob B. Nielsen 1 , Diana C. Anyaogu 1 , Dorte K. Holm 1 , Lene M. Petersen 1 , Morten T. Nielsen 1 , Mikael S. Joergensen 1,2 , Kristian F. Nielsen 1 , Pia F. Johannesen 2 , Dominique A. Skovlund 2 , Thomas O. Larsen 1 . 1) DTU Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark; 2) Department 463, Fungal Gene Technology, Novozymes, Denmark We are developing versatile methods that allows for rapid and simple genetic manipulation of filamentous fungi. Currently, we use our methods for elucidation of pathways for secondary metabolite production in a number of different species. The platform includes simple systems for gene targeting and defined expression platforms for pathway reconstitution. Alternatively, if few or no genetic tools are available for the fungus, we use AMA1 based plasmids for transformation. All DNA handling prior to fungal transformation is based on assembly by efficient USER cloning that allows for many DNA fragments to be merged in a single cloning step. Examples of pathway reconstitution will be presented including functional transfer of the entire geodin producing gene cluster from Aspergillus terreus into A. nidulans. In an attempt to map the first intermediates of polyketide pathways in a fungal species, we have individually expressed all PKS genes from A. niger as a starting point for pathway elucidation. Using this approach we identified a PKS gene responsible for production of 6MSA. Next, we individually deleted all genes in the corresponding gene cluster in A. niger to further map the pathway. Theses analyses suggest that 6-MSA is a precursor of Yanuthone D/E. In a similar study, we have identified a related PKS gene in A. aculeatus that also produces 6-MSA when expressed in A. nidulans. The corresponding gene cluster in A. aculeatus contains a gene encoding a transcription factor. Using our AMA1 based expression system, this gene has been overexpressed in A.aculeatus. As a result a new 6-MSA based compound has been identified. Lastly, using a knockin/knock-out platform in Trichoderma reesei we use the same principles to uncover a gene cluster that is responsible for a very complex family of sorbicillinoids. 14. Characterization of the 3-methyl orsellinic acid gene cluster in Aspergillus nidulans. Jakob B. Nielsen 1 , Marie L. Klejnstrup 1 , Paiman K. Jamal 1 , Dorte K. Holm 1 , Michael L. Nielsen 1 , Anna M. Kabat 2 , Charlotte H. Gotfredsen 3 , Thomas O. Larsen 1 , Uffe H. Mortensen 1 . 1) Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark; 2) Center for Systems Microbiology, Department of Systems Biology, Technical University of Denmark; 3) Department of Chemistry, Technical University of Denmark With the aim of mapping the polyketome of Aspergillus nidulans we have made a library of strains, which individually overexpress PKS genes from an ectopic locus. A screen of this collection on different media demonstrated that overexpression of AN6448 (pkbA) leads to increased production of 3-methyl orsellinic acid. An inspection of the DNA sequence surrounding this gene uncovered a putative gene cluster including a gene, AN6446 (pkbR), with homology to transcription factors. Based on this observation, we decided to overexpress pkbR. A qRT-PCR analysis of this strain was used to delineate the borders of the gene cluster as well as to stimulate formation of cichorine, cichorinic acid, nidulol and a novel cichonidulol dimer, just to name a few of the products that we have linked to this gene cluster. Subsequent deletion of all genes in the cluster has allowed us to propose a comprehensive model for the biosynthetic pathway of this cluster. 15. Analyzing the impact of compartmentalization on organic acid production in Aspergillus niger. Matthias G. Steiger 1,2* , Marzena L. Blumhoff 1,2,3 , Diethard Mattanovich 1,2 , Michael Sauer 1,2 . 1) Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 11, 1190 Vienna, Austria; 2) University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 18, 1190 Vienna, Austria; 3) University of Applied Sciences FHCampus Vienna, School of Bioengineering, Muthgasse 86, 1190 Vienna, Austria. sAspergillus niger is a well-established host organism for the production of carboxylic acids. Acids like citric, gluconic and oxalic acids can already be produced by A. niger and high titers are obtained. The formation of carboxylic acids involves the shuttling of intermediate metabolites between different intracellular compartments and utilizes different enzymatic capabilities of the respective compartment. The knowledge about the involved shuttling mechanisms and the localization of the necessary enzymes is still fragmentary. Using fluorescence microscopy, it is possible to characterize the intracellular localization of GFP tagged proteins and hence mitochondrial leader sequences can be functionally tested. In order to analyze the influence of the compartmentalization on the organic acid production, we have chosen itaconic acid as a target substance. Itaconic acid, which was selected by the US Department of Energy as one of the 12 building block chemicals for the industrial biotechnology, is currently produced by A. terreus. Heterologous expression of the A. terreus cadA gene also enables the formation of itaconic acid in A. niger although only low titers are obtained. We set out to characterize the influence of the compartmentalization on the productivity and re-engineered the enzymatic cascade by flipping the enzymatic activities of the cis-aconitic acid decarboxylase and aconitase between the mitochondrion and the cytosol. We will present new leader sequences for mitochondrial targeting in A. niger alongside with results about the positive impact of the enzymatic re-localization on the itaconic acid production. 16. Increased production of fatty acids and triglycerides in Aspergillus oryzae by modifying fatty acid metabolism. Koichi Tamano 1 , Kenneth Bruno 2 , Tomoko Ishii 1 , Sue Karagiosis 2 , David Culley 2 , Shuang Deng 2 , James Collet 2 , Myco Umemura 1 , Hideaki Koike 1 , Scott Baker 2 , Masayuki Machida 1 . 1) National Institute of Advanced Industrial Science and Technology (AIST); 2) Pacific Northwest National Laboratory (PNNL) Biofuels are attractive substitutes for petroleum based fuels. Biofuels are considered they do not contribute to global warming in the sense they are carbon-neutral and do not increase carbons on the globe. Hydrocarbons that are synthesized by microorganisms have potential of being used as biofuels or the source compounds. In the hydrocarbon compounds synthesized by A. oryzae, fatty acids and triglycerides are the source compounds of biodiesel that is fatty acid methyl ester. We have increased the production by modifying fatty acid metabolism with genetic engineering in A. oryzae. Firstly, enhanced-expression strategy was used for the increase. For four enzyme genes related to the synthesis of palmitic acid [C16:0-fatty acid], the individual enhanced-expression mutants were made. And the fatty acids and triglycerides in cytosol were assayed by enzyme assay kits, respectively. As a result, both fatty acids and triglycerides were most synthesized in the enhanced-expression mutant of fatty acid synthase gene at 2.1-fold and 2.2-fold more than the wild-type strain, respectively. Secondly, gene disruption strategy was used for the increase. Disruptants of several enzyme genes related to long-chain fatty acid synthesis were made individually. And one of them showed drastic increase in fatty acid synthesis. In the future, further increase in the synthesis is expected by utilizing genetic engineering in A. oryzae. 17. Molecular genetic characterization of secondary metabolism pathways in Asperillus species. Clay Wang 1 , Yiming Chiang 1 , Nancy Keller 3 , Kenneth Bruno 4 , Scott Baker 4 , Chun jun Guo 1 , James Sanchez 1 , Benjamin Knox 4 , Alexandra Soukup 3 , Jin Woo Bok 3 , Manmeet Ahuja 2 , Ruth Entwistle 2 , Liz Oakley 2 , Shu-lin Chang 1 , Hsu-Hua Yeh 1 , Mike Praseuth 1 , Berl Oakley 2 . 1) Pharma Sci & Chemistry, Univ Southern California, Los Angeles, CA.; 2) Department of Molecular Biosciences, University of Kansas; 3) Department of Medical Microbiology and Immunology and Department of Bacteriology, University of Wisconsin Madison; 4) Pacific Northwest National Laboratory Advances in next generation DNA sequencing have provided a large number of fungal genome sequences in public databases. Within these genomes are large numbers of cryptic secondary metabolism pathways. Data will be presented where we use a comparative genomics approaches to identify the products of these cryptic pathways. Next we use a gene knock out approach to create mutants followed by isolation and characterization of intermediates and shunt products. Using this approach we have been able to identify the products of a meroterpenoid pathway in A. terreus. Cell Biology and Development 18. Aspergillus nidulans SNXA HRB1 is an SR/RRM family protein that rescues defects in the CDC2/CYCLINB pathway. Steven James 1 , Travis Banta 2 , James Barra 1 , Clifford Coile 2 , Ryan Day 2 , Cheshil Dixit 2 , Steven Eastlack 2 , Anh Giang 2 , Yulon Huff 2 , Julie Kobie 1 , Faustin Mwambutsa 2 , Mimi Nguyen 2 , Amanda Orzechowski 1 , Kristin Shingler 1 , Sarah Lea Anglin 2 . 1) Dept. Biology, Gettysburg College, Gettysburg, PA; 2) Dept. Biology, Millsaps College, Jackson, MS Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated. To further characterize this regulation in Aspergillus nidulans, we conducted a screen for extragenic suppressors of nimX2 cdc2 that resulted in the identification of the cold-sensitive, G1-arresting snxA1 mutation. Our data show that snxA1 suppresses defects in regulators of the G2/M transition, including nimX2 cdc2 , nimE6 cyclinB , and nimT23 cdc25 , but does not suppress the G1/S-arresting nimE10 cyclinB mutation or any of four S phase mutations. Furthermore, the snxA1 mutation or deletion of snxA alter localization patterns of NIME CYCLINB at the restrictive temperatures for snxA1 and nimX2, supporting a role for SNXA in cell cycle control. snxA encodes the A. nidulans ortholog of Saccharomyces cerevisiae Hrb1/Gbp2, nonessential shuttling mRNA binding proteins belonging to the SR (Serine-Arginine Rich) and RRM (RNA Recognition Motif) protein family. snxA hrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and overexpression of gDNAs or of alternatively spliced snxA cDNAs rescues snxA1 mutant phenotypes. SNXA HRB1 is predominantly nuclear, but is not retained in the nucleus during the partiallyclosed mitosis of A. nidulans. We further demonstrate that the snxA1 mutation does not suppress nimX2 by altering NIMX2 CDC2 /NIME CYCLINB kinase activity, suggesting that the effects of SNXA1 on NIMX2 CDC2 /NIME CYCLINB may be due to altered localization of NIME CYCLINB . These data suggest a novel role in G2/M regulation for this SR/RRM family member. This work was supported by the Mississippi INBRE funded by grants from the National Center for Research Resources (5P20RR016476-11) and the National Institute of General Medical Sciences (8 P20 GM103476-11) from the National Institutes of Health. 19. The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. Oezguer Bayram 1* , Oezlem Sarikaya Bayram 1 , Yasar Luqman Ahmed 2 , Jun-Ichi Maruyama 1,4 , Oliver Valerius 1 , Silvio Rizzoli 3 , Ralf Ficner 2 , Stefan Irniger 1 , Gerhard Braus 1 . 1) Institute of Microbiology & Genetics, Department of Molecular Microbiology and Genetics, Georg-August-Universität, Grisebachstr. 8, D 37077 Goettingen, Germany; 2) Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Georg-August-Universität, Goettingen; 3) European Neuroscience Institute, Deutsche Forschungsgemeinschaft Center for Molecular Physiology of the Brain/Excellence Cluster 171, 37077 Göttingen; 4) Department of Biotechnology, The University of Tokyo, Tokyo, Japan. The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog but similar to yeast the entire MAPK module interacts with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3 but even the entire MAPK module complex of four physically interacting proteins can migrate from plasma membrane to nuclear envelope. *20. Two methyltransferase protein complexes control fungal development and secondary metabolite production. Oezlem Sarikaya Bayram 1 , Oezguer Bayram 1 , Jong-Hwa Kim 2 , Keon-Sang Chae 3 , Dong-Min Han 4 , Kap-Hoon Han 2 , Gerhard Braus 1 . 1) Institute of Microbiology & Genetics, Dept. of Molecular Microbiology and Genetics, Georg August University, Grisebachstr. 8, D 37077 Goettingen, Germany; 2) Department of Pharmaceutical Engineering, Woosuk University, Wanju, 565-701, Korea; 3) Division of Biological Sciences, Chonbuk National University, Jeonju, 561-756, Korea; 4) Division of Life Sciences, Wonkwang University, Iksan, 570-749, Korea Coordination of development and secondary metabolism of the filamentous fungus Aspergillus nidulans requires the trimeric velvet complex consisting of VelB-VeA and the putative methyltransferase LaeA. We discovered a second trimeric protein complex for the same control mechanism consisting of an unusual zinc finger domain protein and even two subunits containing canonical methyltransferase domains. In contrast to velvet, which is assembled in the nucleus, the novel trimeric protein complex is formed at the plasma membrane. Functional green fluorescent protein fusions revealed that both methyltransferases are released from the membrane-bound zinc finger domain and migrate to the nucleus. The dimeric nuclear methyltransferase complex physically interacts with chromatin factors as heterochromatin protein and has an impact on the expression of asexual or sexual developmental genes as well as secondary metabolite gene clusters. Consistently, deletions of the corresponding genes result in defects in light response. Our results support that a trimeric membrane complex initiates a signalling pathway which is mediated by two methyltransferases which transduce the signal to nuclear chromatin and affect gene expression. The interplay between the novel methyltransferase complex and the velvet complex remains to be elucidated.70-749, Korea *21. Functional analysis of sterol transporter in filamentous fungus Aspergillus nidulans. Nicole Bühler, R. Fischer, N. Takeshita. Microbiology, Karlsruhe Institut of Technology, Karlsruhe, Germany A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip provides the delivery of proteins and lipids to the tip and is essential for cell wall and cell membrane extension at the tip. Apical sterol-rich plasma membrane domains (SRDs), which can be viewed using the sterol-binding fluorescent dye filipin, are gaining attention for their important roles in polarized growth of filamentous fungi. Although the importance of SRDs is becoming clear, their exact roles and formation mechanisms remain rather unclear. Transport of sterol to hyphal tips is thought to be important for the SRDs organization. Oxysterol binding proteins, which are conserved from yeast to human and involved in vesicular trafficking, signalling, lipid metabolism and non-vesicular sterol transport. Saccharomyces cerevisiae has seven oxysterol binding protein homologues (OSH1-7). Their subcellular distributions are regulated respectively. The OSH proteins are thought to function as a sterol transporter between closely located membranes independently of vesicle transport. In the filamentous fungus Aspergillus nidulans, we found five OSH genes. To investigate their functions for the polarized growth and SRDs organization, their localization are analyzed by GFP tagging. The gene-deletion strains are constructed and analyzed. Their expression levels are analyzed via qRT-PCR. *22. Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger. Maria A. Burggraaf 1,2 , Benjamin M. Nitsche 1,2 , Gerda Lamers 1 , Vera Meyer 2,3 , Arthur F.J. Ram 1,2 . 1) Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden, The Netherlands; 2) Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands; 3) Institute of Biotechnology, Applied and Molecular Microbiology, Berlin University of Technology, Berlin, Germany The filamentous fungus Aspergillus niger is an important and versatile cell factory commonly exploited for the industrialscale production of a wide range of enzymes and organic acids. Although numerous studies have been conducted aiming at improving our knowledge of degradative cellular activities that determine product yields in A. niger including secretion of proteases and the unfolded protein response, there is a catabolic pathway that has yet not been studied in this industrially exploited fungus, namely Autophagy. Autophagy is a well conserved catabolic process constitutively active in eukaryotes that is involved in cellular homeostasis by targeting of cytoplasmic content and organelles to vacuoles. Autophagy is strongly induced by limitation of nutrients including carbon, nitrogen and oxygen and is clearly associated with cell death. We previously demonstrated that the accumulation of empty hyphal compartments and secondary regrowth in carbon starved submerged batch cultures of A. niger were accompanied by a joint transcriptional induction of autophagy genes. In this study we examined the role of autophagy by deleting the atg1, atg8 and atg17 orthologues in A. niger and phenotypically analyzing the deletion strains in surface and submerged cultures. Our results indicate that atg1 and atg8 are essential for efficient autophagy whereas deletion of atg17 has little to no effect on autophagy. Depending on the stressor, autophagy deficiency renders A. niger both more resistant and more sensitive to oxidative stress. Fluorescence microscopy showed that mitochondrial turnover upon carbon depletion in submerged cultures is severely blocked in autophagy impaired mutants. Furthermore, automated image analysis demonstrated that autophagy promotes survival in maintained carbon starved cultures of A. niger. Taken together, our results suggest that besides its function in nutrient recycling, autophagy plays important roles in physiological adaptation by organelle turnover and protection against cell death upon carbon depletion in submerged cultures. *23. Pheromone-induced G2 cell cycle arrest in Ustilago maydis requires inhibitory phosphorylation of Cdk1. Sónia M. Castanheira, José Perez-Martín. Centro Nacional de Biotecnología. CSIC. Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain. Ustilago maydis is a dimorphic basidiomycete that infects maize. In this fungus virulence and sexual development are intricately interconnected. Induction of pathogenicity program requires that two haploid compatible cells fuse and form an infective filament after pheromone signaling. The pheromone signal is transmitted by a well-known MAPK cascade. Interestingly, Saccharomyces cerevisiae and Ustilago maydis use a similar MAPK cascade to respond to sexual pheromone and in both cases a morphogenetic response is provided (shmoo and conjugative hypha, respectively). However, while S. cerevisiae arrests its cell cycle in G1 in response to pheromone, U. maydis does this by arresting at G2. The mechanisms and physiological reasons involved in the distinct cell cycle response to pheromone in U. maydis are largely unknown. In this communication we will introduce our attempts to characterize the molecular mechanisms behind pheromone-induced cell cycle arrest in U. maydis .Our results have indicated that inhibitory phosphorylation of Cdk1 is part of the mechanism of the pheromone-induced G2 cell cycle arrest. This inhibitory phosphorylation depends on the essential kinase Wee1. We analyzed the transcriptional pattern of cell cycle related genes in response to overactivation of pheromone pathway (using a constitutively activated allele of fuz7, the MAPKK of the cascade) and found that two main G2/M regulators -Hsl1, a kinase involved in downregulation of Wee1 and Clb2, the mitotic cyclinwere downregulated at transcriptional level. Using chimeric promoter fusions we found that transcriptional downregulation was not as important for pheromone-induced cell cycle arrest as expected and we are analyzing other possible regulatory options such as stability or subcellular localization of these regulators. 24. The arrestin-like protein ArtA is essential for ubiquitylation and endocytosis of the UapA transporter in response to both broad-range and specific signals. George Diallinas, Mayia Karachaliou, Sotiris Amillis, Minos Evangelinos, Alexandros Kokotos. Faculty of Biology, University of Athens, Athens, Greece We investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. All arrestin null mutants are viable showing wild-type growth and morphology, except one which is affected in conidiospore production, but several have modified profiles in respect to N or C source utilization and drug sensitivity. A single arrestin, ArtA, is essential for HulARsp5-dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Genetic analysis further showed that residues 545-561 of the UapA C-tail, which includes a critical di-acidic motif, is required for efficient UapA endocytosis. Mutational analysis of ArtA shows that the N-terminal region (2-123) and both PY elements are essential for its function. ArtA undergoes HulA-dependent ubiquitination at residue Lys343 and this modification is critical for the efficiency of UapA ubiquitination and endocytosis, especially in response to ammonium. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or L-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates. 25. Unique protein domains regulate Aspergillus fumigatus RasA localization and signaling during invasive growth. Rachel V. Lovingood 1 , Praveen R. Juvvadi 2 , William J. Steinbach 2 , Jarrod R. Fortwendel 1 . 1) Microbiology and Immunology, University of South Alabama, Mobile AL, USA; 2) Pediatric Infectious Diseases, Duke University, Durham NC, USA Invasive pulmonary aspergillosis (IPA) is propagated by inhalation of A. fumigatus spores that germinate and invade the lung tissue in search of nutrients. We have shown that the A. fumigatus RasA GTPase protein is necessary for hyphal morphogenesis, cell wall integrity, and virulence during IPA. Our previous studies focused on conserved protein domains regulating RasA localization and signaling. These studies revealed the requirement for plasma membrane (PM)-localized Ras for proper signaling and regulation of A. fumigatus growth and virulence. Therefore, mechanisms controlling Ras localization are of interest in designing novel antifungal Ras inhibitors. Although Ras pathways may represent valid antifungal targets, the importance of fungal-specific Ras protein domains to Ras function in fungal pathogenesis remains unexplored. To address this important knowledge gap, we identified fungal-specific Ras protein domains by comparing fungal Ras sequences to their human counterpart, H-ras. We hypothesized that such domains could serve as targetable areas to selectively inhibit the fungal Ras protein. This analysis revealed two areas of significant divergence with H-ras: i) the Invariant Arginine Domain (IRD), a novel domain conserved in the RasA homologs of every available fungal genome but not present in H-ras and ii) an extended hypervariable region (HVR). Truncation analysis of the HVR identified a serine-rich region that is necessary for localization to the PM and for RasA signaling during hyphal morphogenesis. *26. Colletotrichum orbiculare Bub2-Bfa1 complex, a spindle position checkpoint (SPOC) component in Saccharomyces cerevisiae, is involved in proper progression of cell cycle. Fumi Fukada 1 , Ayumu Sakaguchi 2 , Yasuyuki Kubo 1 . 1) Laboratory of Plant Pathology, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan; 2) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan Colletotrichum orbiculare is an ascomycete fungus that causes anthracnose of cucumber. In Saccharomyces cerevisiae, the orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. A surveillance mechanism named spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-daughter polarity axis. BUB2 is a component of SPOC and constitutes the main switch for the mitotic exit network (MEN) signaling. We identified and named this homolog as CoBUB2 in C. orbiculare and generated gene knock-out mutants. First, we observed morphogenesis and pathogenesis of the cobub2 mutants. The cobub2 mutants formed abnormal appressoria and penetration hyphae on model substrates, and the cobub2 mutants also showed attenuate pathogenesis to cucumber leaves. Second, we observed mitosis based on mitotic spindle behavior and nuclear DAPI staining during appressorium development. In the wild type, mitosis occurred in appressorium developing conidia after 4h incubation, whereas interestingly, in the cobub2 mutants, mitosis occurred in pre-germinated conidia after 2h incubation. After development of appressorium, in some germlings the daughter nucleus was delivered from conidia to appressoria, and the others perform second round of mitosis in appressorium developing conidia after 4h incubation. Third, we evaluated the timing of S phase and M phase during appressorium development in wild type and the cobub2 mutants by cell cycle specific inhibitors. In the cobub2 mutants, it was shown that the transition period from G1 phase to S phase accelerated about 2h than that of the wild type. Last, in S. cerevisiae, Bub2 forms GTPase activating protein (GAP) complex with Bfa1, and Bub2-Bfa1 GAP complex constitutes SPOC. Then we named homolog of BFA1 as CoBFA1 in C. orbiculare and generated cobfa1 mutants. From observation of nuclear division, the cobfa1 mutants showed similar behavior of nuclear division to the cobub2 mutants. Therefore, it is assumed that CoBub2 forms GAP complex with CoBfa1, however, CoBub2-CoBfa1 GAP complex has different function from that in S. cerevisiae maintaining G1 phase duration or setting up the proper time of S phase. Interestingly, mutational analysis of the IRD produced a properly localized yet non-functional RasA protein. However, activation of the IRD RasA mutant was not altered suggesting a role for the IRD during interactions of RasA with downstream effectors. Further characterization of the IRD and HVR, and the protein interactions to which they contribute, will reveal fungal-specific aspects of Ras function and may define a new paradigm for Ras signal transduction in fungal organisms. *27. The exocyst complex is necessary for secretion of effector proteins during plant infection by Magnaporthe oryzae. Yogesh ogesh K. Gupta 1 , Martha Giraldo 2 , Yasin Dagdas 1 , Barbara Valent 2 , Nicholas J. Talbot 1 . 1) School of Biosciences, University of Exeter, EX4 4QD, UK; 2) Department of Plant Pathology, Kansas State University, Manhattan, Kansas, USA Magnaporthe oryzae is a devastating plant pathogenic fungus, which causes blast disease in a broad range of cereals and grasses. A specialized infection structure called the appressorium breaches the leaf cuticle and subsequently the fungus colonizes host epidermal cells. Colonization of host tissue is facilitated by small secreted proteins called effectors, that suppress plant immunity responses and may also mediate invasive growth. Some of these effectors have been shown to localize at the appressorium pore prior to plant infection, at the tips of primary invasive hyphae and in a specialized plant-derived, membrane-rich structure called the Biotrophic Interfacial Complex (BIC). However the underlying mechanism controlling polarized secretion is not well defined in M. oryzae. The exocyst is an octameric protein complex (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) that appears to be evolutionary conserved in fungi and to play a crucial role in vesicle tethering to the plasma-membrane. The exocyst plays an important role in polarized exocytosis and interacts with various signaling pathways at the apex of fungal cells. We are currently characterizing components of exocyst complex during infection related development of M. oryzae. We have shown that the exocyst localizes to hyphal tips as in other fungi during hyphal growth in culture. Interestingly, exocyst components also localize around the appressorium pore, which suggests the pore is an active site for secretion at the point of plant infection. We have recently shown that organization of the appressorium pore requires a hetero polymeric septin network and we show here that localization of the exocyst at the appressorium pore is septin dependent. The exocyst is furthermore involved in secretion of symplastic (host cell-delivered) effectors but not apoplastic effectors. Targeted gene deletion of exocyst components Exo70 and Sec5 causes significant virulence defects because of impaired secretion. We will present new information on the role of the exocyst during invasive growth of M. oryzae. 28. A highly conserved sequence motif is required for PkcA localization to septation sites and protein function in Aspergillus nidulans. Loretta Jackson-Hayes 1 , Terry Hill 1 , Darlene Loprete 1 , Claire DelBove 1 , Omolola Dawodu 2 , Jordan Henley 3 , Ashley Poullard 3 , Justin Shapiro 1 . 1) Rhodes College, Memphis, TN 38112; 2) Rust College, Holly Springs, MS 38635; 3) Tougaloo College, Tougaloo, MS 39174 Many proteins with diverse functions contribute to cell wall synthesis in polarized growth and septation. Some of these proteins play similar roles at tips and septa, while others are exclusively involved in one process or the other. In Aspergillus nidulans, wild type protein kinase C (PkcA) localizes to growing hyphal tips and septation sites, and a role for PkcA in cell wall synthesis is supported by the inability of PkcA mutant strains to exhibit resistance to cell wall perturbing agents. PkcA localization to septation sites is dynamic. Upon initiation of septum formation PkcA is organized as a ring at periphery of the septation site. The ring constricts in synchrony with the actin/myosin contractile ring and dissipates when septa are fully matured. To determine which domains are important for septum site localization, green fluorescent protein tagged, domain-deleted versions of PkcA were constructed. The domains that are vital to A. nidulans maintenance of cell wall integrity were separately identified by growing the domain deleted stains in the presence of the cell wall stressor calcofluor white. We have determined that the localization signal and the domain responsible for resistance to calcofluor white are distinct. The PkcA septation site localization signal is found within a region having homology with C2 domains of PKC proteins found in other organisms. Observations of both Nand Cterminal truncations support the conclusion that the PkcA septation site localization signal lies within the final 20 amino acids of the C2 domain. Removal of these amino acids causes PkcA mislocalization to the cytoplasm. Furthermore, removal of the localization signal renders the resulting truncated proteins less able to complement calcofluor white hypersensitivity in a strain carrying a mutation in its PkcA gene, highlighting the requirement of proper localization for this aspect of PkcA function. 29. Beyond green mining: analysis of fungal cytochemistry using gold nanoparticles. Fatemeh Farazkhorasani 1 , Martin Prusinkiewicz 2 , Kathleen M Gough 1 , Susan GW Kaminskyj 2 . 1) Chemistry, University of Manitoba, Winnipeg, Canada; 2) Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Cells including fungal hyphae and other microorganisms, as well as fungal growth medium including both complex and defined composition, can reduce solutions of HAuCl4 to elemental gold nanoparticles (AuNPs). As described in 2012 Analyst 137:4934-42, we have shown that AuNPs formed by growing fungal hyphae can be used as analytical substrates for surface-enhanced Raman scattering (SERS) spectroscopic analysis. These SERS spectra are in the same energy range as our Fourier-transform infrared (FTIR) spectroscopic studies that provided information about cell composition. However, SERS is orders of magnitude more sensitive, and analysis is limited to cell components within a few nanometers of the AuNP. Our current interest is the fungal cell wall, which forms a porous interface between the cell and its environment. Cell wall chemistry is intrinsically related to cell-environment interactions, particularly for pathogenesis. The fungal wall is about 25 % of fungal dry weight, and its synthesis and maintenance is estimated to require ~25 % of the fungal genome. Fungal walls are ~ 80 % carbohydrate. Minor structural differences in carbohydrate bonding can cause profound changes in their metabolism, which complicates analysis. Preliminary studies described in the Analyst paper showed that SERS-active AuNPs can be generated by living hyphae. Higher Au concentrations produced larger AuNPs within and on the hypha, but in addition were lethal within 30 min. Lower Au concentrations produced clusters of smaller AuNPs on the cell wall surface, and were not lethal. These were also SERS-active. We are using SERS to probe the wall composition of engineered mutants in the Aspergillus galactofuranose biosynthesis pathway, which plays key roles in fungal growth and drug resistance. We expect the combination of fungal genetic engineering and high sensitivity/high spatial-resolution chemical analysis will provide novel information about fungal growth and infectivity. 30. Aspergillus nidulans as an experimental system to identify novel cell wall growth and maintenance genes through identification of anti-fungal drug resistance 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 the ergosterol biosynthetic pathway (azoles and allylamines). Drugs against beta-glucan synthesis (echinocandins) are effective against aspergillosis and candidaisis. 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 animal and fungal physiology leaves relatively few relevant targets to explore. However, it is likely that for any drug-enzyme combination there will be relatively few mutations that could increase drug resistance while still maintaining enzyme function. We are using Aspergillus nidulans as an experimental model system 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 plates containing a sub-lethal concentration of Calcofluor. These developed fast-growing sectors beginning at ~ 5 d (70 rounds of mitosis). Preliminary results show that many of these sectors harboured heritable, single-gene mutations. To date, mutated genes that confer robust, heritable resistance to Calcofluor that were identified by next generation sequencing have roles in cell wall synthesis, cell wall integrity regulation, or drug detoxification. We suggest this strategy will be useful for predicting genetically-mediated anti-fungal resistance adaptation and help us to be ahead in the drug-resistance arms race. 31. Aspergillus nidulans cell walls lacking galactofuranose are more susceptible to glucan degrading enzymes. Biplab Paul 1 , Tanya Dahms 1 , Susan Kaminskyj 2 . 1) Dept Chemistry and Biochemistry, Univ Regina, Regina, SK, Canada; 2) Dept Biology, Univ Saskatchewan, Saskatoon, SK, Canada The cell wall of Aspergillus is a dynamic organ, consisting of a semi-permeable network of mannoprotein, and alphaand beta-glucans. These components are remodeled as fungal cell grows and responds to its environments. By weight, fungal walls are estimated to be 35-45% alpha-(1,3)-glucan, 20-35% beta-(1,3)-glucan, 20-25% galactomannan, 7-15% chitin (beta-1,4-glucan), and 4% beta-(1,6)-glucan. Evidence from literature sources suggest that the Aspergillus wall 'core' is chitin and galactomannan linked to beta-1, 6and beta-1, 6-glucan. Galactofuranose (Gal-f) appears to play a central role in Aspergillus cell wall maturation. Previously, we showed that Gal-f biosynthesis is important for wild type chemical, physical, structural properties of the A. nidulans cell wall. We propose that the lack of Gal-f disrupts the proper packing of cell wall components, giving rise to more disordered surface subunits and so to greater deformability. Here, we show results from an investigation of the susceptibility of Aspergillus Gal-f biosynthesis deletion strains to glucan degrading enzyme using atomic force microscopy. Topographic images of glucanaseand laminarinase-treated wildtype strains suggest that glucan is at least one component of the cell surface subunits. Strains that lacked Gal-f were more susceptible to beta-1,3-glucanase. *32. Identification of novel genes involved in induction of appressorium development triggered by plant-derived signals in Colletotrichum orbiculare. Sayo Kodama 1 , Ayumu Sakaguchi 2 , Yasuyuki Kubo 1 . 1) Laboratory of Plant Pathology, Graduate School of Life and Environmental Science, Kyoto Prefectural University, Kyoto, Japan; 2) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan Many plant pathogenic fungi initiate infection of host leaves by the germination of conidia and differentiation of appressoria at the tip of germ tubes. These morphological changes are triggered by various external signals such as physical or chemical signals from the plant surface. In our previous study, cucumber anthracnose fungus Colletotrichum orbiculare CoKEL2, a Schizosaccharomyces pombe tea1 homologue, encoding a kelch repeat protein was identified. The cokel2 mutants formed abnormal appressoria on glass slides, and those appressoria were defective in penetration hyphae development into cellulose membranes, an artificial model substrate for fungal infection. In contrast, the cokel2 mutants formed normal appressoria on the host cucumber plant and retained penetration ability. Moreover, when conidia were incubated in the presence of exudates from cucumber cotyledon, normal appressorium formation on the artificial substrate by the cokel2 mutants was restored. These results suggest that CoKEL2 is essential for normal morphogenesis of appressoria and that there is a bypass pathway that transduces plant-derived signals for appressorium formation independent of CoKEL2. These plant-derived signaling pathways for appressorium formation have not been characterized in fungal pathogens including C. orbiculare. To determine specific components of the plant-derived signaling pathway that leads to appressorium formation, we screened six cokel2 double mutants that formed abnormal appressoria not only on artificial substrates but also on the host plant surface. Furthermore, reintroduction of CoKEL2 into those cokel2 double mutants restored normal appressorium formation on artificial substrates, suggesting that cokel2 double mutants have defects in CoKEL2-independent and plant-derived specific signaling pathway for appressorium formation. We identified and characterized candidate mutated genes by whole genome sequencing of the six cokel2 double mutants. To define the involvement of those candidate mutated genes in appressorium formation, we observed the phenotypes of candidate geneΔ single mutants, cokel2Δ candidate geneΔ double mutants, and complementation strains. As expected, candidate geneΔ mutants in cokel2Δ back ground showed same phenotypes as those of screened cokel2 double mutants. 33. Distinctive Mitotic Localization of a Novel Suppressor of nimA1 Provides New Insight into NIMA Function. Jennifer R. Larson, Stephen A. Osmani. Department of Molecular Genetics, the Ohio State University, Columbus, OH. The NIMA kinase is an essential regulator of mitotic events in Aspergillus nidulans. Not only is NIMA essential for initiating mitosis its overexpression can prematurely induce mitotic events including DNA condensation and nuclear pore complex (NPC) disassembly in A. nidulans and human cells. One of the key roles for NIMA at the onset of mitosis is its regulation of NPCs. A previous study aimed at identifying suppressors of the temperature-sensitive nimA1 allele isolated two NPC proteins, which were named SONA and SONB for Suppressors Of NimA1. Although NIMA is essential for mitotic entry there is also evidence that NIMA and conserved related kinases have functions later in mitosis and in the DNA damage response. To further characterize the roles of NIMA we designed a genetic screen to isolate additional suppressors of nimA1 that also cause conditional temperature-dependent DNA damage sensitivity. Our expectation was the identification of additional genes involved in NIMA regulation and in the DNA damage response. Here we describe one such gene, which we have named sonC. SonC contains a unique Zn(II)Cys6 binuclear DNA binding domain, which is highly conserved among the Ascomycota. Deletion of sonC results in swollen, ungerminated spores, suggesting it is essential for a core growth process. As expected for a DNA binding protein, SonC localizes to nuclei during interphase. Interestingly, dual fluorescence imaging of SonC with histone H1 during mitosis revealed that a portion of SonC localizes with histone H1 along a distinct projection of chromatin that juts away from the main, condensed chromatin mass, which we hypothesize may be the NOR. Supporting this hypothesis, the region of DNA that likely forms the projection is cradled by the nucleolus prior to mitosis as seen by colocalization studies of SonC with the nucleolar protein Bop1. As mitosis proceeds, the H1 histones are evicted from the middle region of this projection but not at its distal end. This indicates that the chromatin in this region of the genome is altered during mitotic progression and we are testing the idea that SonC might be important for NOR condensation and/or nucleolar disassembly during its mitotic segregation. Because SonC was identified as a suppressor of NIMA we propose that NIMA may have a function in regulating nucleolar disassembly during mitosis. 34. The GATA-type transcription factor NsdD is a key regulator of conidiation and secondary metabolism in Aspergillus. Mi-Kyung Lee 1 , Nak-Jung Kwon 1 , Im-Soon Lee 2 , Jae-Hyuk Yu 1 . 1) Bacteriology, University of Wisconsin Madison, MADISON, WI, USA; 2) Department of Biological Sciences, Konkuk University, Seoul, Republic of Korea Asexual development (conidiation for higher fungi) is the most common reproductive mode of many fungi; yet, its regulatory mechanisms remain to be understood. In this study, we carried out a multi-copy based genetic screen in the absence of the repressor of conidiation sfgA, which is designed to identify a new set of negative regulator(s) of conidiation. Among over 100,000 colonies, 45 transformants showing altered conidiation were isolated, of which 10 defined the nsdD gene (AN3152), a key activator of sexual fruiting. The others have defined AN7507, AN2009, AN1652, AN5833 and AN9141. A series of verification, genetic and mycotoxin analyses revealed that only NsdD is a true negative regulator of brlA (an essential activator of conidiation) and conidiation, and that NsdD acts downstream of fluG and flbA~E, but upstream of brlA. The removal of NsdD was sufficient to cause hyper-active conidiation even in liquid submerged culture, as well as early and prolonged activation of brlA, suggesting that NsdD is indeed a key repressor of brlA and conidiation. Moreover, the deletion of nsdD results in hyper-active conidiation and altered production of mycotoxins in the opportunistic human pathogen Aspergillus fumigatus and the aflatoxin-producing human/plant pathogen Aspergillus flavus. Importantly, we have discovered that nsdD encodes two differentially expressed mRNAs and polypeptides (β and α). Finally, the subsequent transient promoter analysis using the brlA promoter::luciferase fusion constructs have revealed that NsdD negatively regulates the brlAβ promoter activity. In summary, NsdD is a key negative regulator of conidiation acting direct upstream of brlA in A. nidulans, and couples conidiation and mycotoxin biosynthesis in Aspergilli. *35. Stability of a G protein alpha subunit in genetic backgrounds lacking the G beta subunit or a cytosolic guanine nucleotide exchange factor. Alexander V. Michkov, Katherine A. Borkovich. Plant Pathology and Microbiology, University of California, Riverside,