Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSCellular development integrating primary and induced secondary metabolism in the filamentous fungus Fusarium graminearum. Jon Menke 1 , JakobWeber 2 , Karen Broz 3 , H. Corby Kistler 1,3* . 1) Department of Plant Pathology, University of Minnesota, St. Paul, USA; 2) Molekulare Phytopathologie,Universität Hamburg, Germany; 3) USDA ARS Cereal Disease Laboratory, St. Paul, MN, USA.Several species of the filamentous fungus Fusarium colonize plants and produce toxic small molecules that contaminate agricultural products, renderingthem unsuitable for consumption. Among the most destructive of these species is F. graminearum, which causes disease in wheat and barley and oftencontaminates the grain with harmful trichothecene mycotoxins. Induction of these secondary metabolites occurs during plant infection or in culture inresponse to chemical signals. Here we report that trichothecene biosynthesis involves a complex developmental process that includes dynamic changes incell morphology and the biogenesis of novel subcellular structures. Two cytochrome P-450 oxygenases (Tri4p and Tri1p) involved in early and late steps intrichothecene biosynthesis were tagged with fluorescent proteins and shown to co-localize to vesicles we call “toxisomes.” Toxisomes, the inferred site oftrichothecene biosynthesis, dynamically interact with motile vesicles containing a predicted major facilitator superfamily protein (Tri12p) previouslyimplicated in trichothecene export and tolerance. The immediate isoprenoid precursor of trichothecenes is the primary metabolite farnesylpyrophosphate. When cultures are shifted from non-inducing to trichothecene inducing conditions, changes occur in the localization of the isoprenoidbiosynthetic enzyme HMG CoA reductase. Initially localized in the cellular endomembrane system, HMG CoA reductase increasingly is targeted totoxisomes. Metabolic pathways of primary and secondary metabolism thus may be coordinated and co-localized under conditions when trichothecenesynthesis occurs.LaeA sleuthing reveals cryptic gene clusters in pathogenic Aspergilli. Nancy Keller 2 , Wenbing Yin 2 , Saori Amaike 2 , Katharyn Affeld 2 , JinWoo Bok 2 , DanielSchwenk 3 , Dirk Hoffmeister 3 , Joshua Baccile 1 , Ry Forseth 1 , Frank Schroeder 1 . 1) Boyce Thompson Institute and Department of Chemistry and ChemicalBiology, Cornell University, Ithaca, NY 14853, USA; 2) Department of Plant Pathology, Department of Medical Microbiology and Immunology, andDepartment of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA; 3) Department of Pharmaceutical Biology at the Hans-Knöll-Institute, Friedrich-Schiller-Universität, Beutenbergstrabe 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 nuclearprotein LaeA regulates the expression of many of these uncharacterized gene clusters. Following leads from laeA mutant microarray data, we created genedeletion and overexpression strains and used 2D NMR-based comparative metabolomic analyses to identify previously undescribed metabolites from bothspecies. In A. fumigatus a tryptophan-derived iron(III)-complex, hexadehydro-astechrome (HAS), was found to be the major product of the cryptic has nonribosomalpeptide synthetase (NRPS) cluster. In A. flavus we show that two separate clusters encode enzymes that produce partially overlapping sets ofnovel piperazines, pyrazines, and morpholines. These L-tyrosine metabolites are activated by two NRPS-like proteins, LnaA and LnbA. Loss andoverexpression of these metabolites impacted fungal development in these species.The KMT6 Histone H3 K27 Methyltransferase Regulates Expression of Secondary Metabolites and Development in Fusarium graminearum. Kristina M.Smith, Lanelle R. Connolly, Michael Freitag. Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon StateUniversity, Corvallis, OR 97331.The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation ofsecondary metabolite gene clusters remains largely a mystery. By ChIP-sequencing we found that regions of the F. graminearum genome with secondarymetabolite clusters are enriched for a histone modification, trimethylated histone H3 lysine 27 (H3K27me3), associated with gene silencing. Thismodification was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. H3K27me3 and di- ortrimethylated H3K4 (H3K4me2/3), modifications associated with gene activity, are found in mutually exclusive regions of the genome. To betterunderstand the role of H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z).The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-sequencing, displays growth defects, is sterile, and produces mycotoxins underconditions where they are not generated in wildtype (WT) strains. RNA-sequencing showed that genes modified by H3K27me3 are most often silent, asabout 75% of the 4,449 silent genes are enriched for H3K27me3. Surprisingly, we found 22% of the 8,855 expressed genes enriched for H3K27me3. Asubset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6 (1,780 genes), and an overlapping set of genes showed increasedexpression. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3. In these cases absenceof H3K27me3 is insufficient for expression, which suggests a requirement for additional factors for gene expression. Taken together, we show that absenceof H3K27me3 allows expression of 14% of all annotated genes, resulting in derepression of predominantly secondary metabolite pathways and otherspecies-specific functions, including potentially secreted pathogenicity factors. This study provides the framework for novel targeted strategies to controlthe “cryptic genome” and specifically secondary metabolite expression.88