bioactives and toxins such as ochratoxin A). Because of these abilities, they represent a substantial economic interests in pharmaceutical, biotechnology, and bioenergy applications. In a project collaboration with the US Joint Genome Institute and JBEI we are de novo sequencing 300 different species of Aspergillus and establishing an online analysis platform for the scientific community, aspMine. The goal of this project is to develop a targeted tool to expand and improve our knowledge and expertise about this versatile group of fungi. At time of writing, 200 genomes are in various stages of sequencing and a bioinformatic pipeline has been established to analyze and store the data. This project covers a wide range of biologically interesting ideas surrounding the concept of speciation, such as genetic diversity, primary and secondary metabolism and proteome diversity. Complementary to the tools offered by FungiDB and JGI, the aspMine analysis resource offers tools for tracking genes and functions across species, allowing for investigation of shared genes and clusters across the genus as well as species- and clade-specific genes. The online platform also offers comparative analysis of secondary metabolism gene clusters with focus on synteny and functional conservation across species. The aspMine is implemented as a number of web applications created in R shiny, a graphical interface for analysis. The different tools are collected on a webpage which also includes method descriptions and relevant literature. The webpage is available from the beginning of 2016 and will be continually expanded. It is our goal to provide a comprehensive analysis platform for the community for comparative analysis of Aspergillus species.

**PHI-base - the Pathogen-Host Interactions database.** Kim Hammond-Kosack¹, Alayne Cuzick¹, Kim Rutherford², Helder Pedro³, Martin Urban¹
1) Dept of Plant Biology and Crop Sciences, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK; 2) Cambridge Systems Biology and Department of Biochemistry, University of Cambridge, Sanger Building, 80 Tennis Court Road, Cambridge, Cambridgeshire CB2 1GA, UK; 3) The European Molecular Biology Laboratory, The European Bioinformatics Institute, Hinxton, Cambridgeshire, CB10 1SD, UK.

The pathogen-host interactions database PHI-base (www.phi-base.org) is a knowledge database. It contains expertly curated molecular and biological information on genes proven to affect the outcome of pathogen-host interactions reported in peer reviewed research articles. Genes not affecting the disease interaction phenotype are also curated. Viruses are not included. Here we describe a revised PHI-base Version 4 data platform with improved search, filtering and extended data display functions. Also a BLAST search function is now provided. The database links to PHI-Canto, a new multi-species author self-curation tool adapted from PomBase-Canto. The recent release of PHI-base version 4 has an increased data content containing information from >2000 manually curated references. The data provide information on 4460 genes from 264 pathogens tested on 176 hosts in 8046 interactions. Pro- and eukaryotic pathogens are represented in almost equal numbers. Host species belong ~70% to plants and 30% to other species of medical and/or environmental importance. Additional data types included into PHI-base 4 are the direct targets of pathogen effector proteins in experimental and natural host organisms. The different use types and the future directions of PHI-base as a community database are discussed.

Urban et al., (2017) PHI-base: A new interface and further additions for the multi-species pathogen-host interactions database. *Nucleic Acids Research* (database issue Jan 2017)

This work is supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) (BB/I/001077/1, BB/K020056/1). PHI-base receives additional support from the BBSRC as a National Capability (BB/J/004383/1).

**Advances in chemical genetic tools and impact on the research and development of novel crop protection products.** Siân Deller, Helen Carter, Helen Blake, Eileen Scott, Michael Csukai Syngenta, Jealott's Hill, GB.

Chemical control agents are likely to remain a part of the mixture of control strategies open to farmers for the foreseeable future. In order to support the discovery and development of new active ingredients a wide range of chemical genetic tools are utilised and are under constant development. Historically many of these tools used the model organism *Saccharomyces cerevisiae*, but improvements in genomic information and tools make it possible to perform research directly in field-relevant plant pathogens. Some of the wide range of tools used in support of new fungicide discovery will be outlined, including:

- Forward genetic tools, which have long been the mainstay of mode of action diagnosis, provide an unbiased approach for the identification of the molecular target of a chemical inhibitor. If resistant mutants can be generated, the technique can now be applied to any pathogen, providing a reference genome is available and single nucleus cells/spores can be isolated.

- Libraries of engineered model organism strains. Chemistry with lower resistance risk is often associated with the inability to isolate resistant mutants in the plant pathogen of interest. In this case unbiased genetic tools are still of value and model organisms are employed. Haploinsufficiency or overexpression-induced resistance within a yeast library can provide information on proteins or protein families targeted by novel chemistry. Hypotheses can then be based on the mutations seen, and compared to information from additional phenotypes such as biochemical pathway inhibition and microscopic observations.

The improvements in transformation procedures, genetic engineering techniques and availability of multiple selectable markers make it possible to test hypotheses in fungal pathogens of economic importance such as *Zymoseptoria*. Additionally, genes from any species can be tested in a uniform host cell background, for instance, by heterologous expression. Furthermore, these tools can be used to assess the lifetime of a new fungicide product. Mutations induced in the lab or discovered in the field can be assessed for their importance in different genetic backgrounds and an assessment made of the impact on sensitivity and fitness. Advancing molecular biology tools and understanding of phytopathogens are helping to inform and support the development of ever safer and more effective chemical crop protection strategies. How these tools have been used to study Zymoseptoria will be shown.

**Evolutionary and population genetics of culture collections.** K. McCluskey Fungal Genetics Stock Center, Kansas State University, Manhattan, KS.

Living microbe collections are established for a number of reasons including biodiversity, genetics, genomics, industrial and agricultural applications, and in support of patents. The Fungal Genetics Stock Center has holdings in support of multiple areas including genetics, genomics, and biodiversity. Having developed to support researchers using Neurospora and Aspergillus, the FGSC is more of a research resource repository and this is reflected in its narrow taxonomic scope. Similar collections include the Coli Genetic Stock Center, the Bacillus Stock Center, and the Chlamydomonas Center. Other collections, such as the ATCC and the USDA NRRL have much broader
taxonomic scope and are traditional culture collections.

While collections of each type are vulnerable to market effects, the recent change in collection funding by the US National Science Foundation means that collections with narrow focus may not have critical mass sufficient to support curation, quality control, and distribution. As model organisms solve the questions they were developed to address, and as modern questions are directly accessible in key health, industrial, or agriculturally important organisms, the value of the model systems decreases. Overall these effects combine and model organisms and the collections that preserve and distribute them pass into the area of science history. Fungal genetics has seen such areas come and go. Early work with Allomyces, Ustilago, Schiziphyllum, Coprinopsis, and Sordaria generated strains that are held at the FGSC, but are not highly valued in the modern research context.
Saturday, March 18 9:00 AM – 12:00 NOON

Merrill Hall and Chapel

Plenary Session IV: Sensing and Signaling: Perception of the Complex World in which Fungi Thrive or Survive

**Synthetic biology and optogenetics: developing biotechnological solutions and pushing the boundaries between science and art.** L.F. Larrondo Departamento de Genética Molecular y Microbiología, Millennium Nucleus for Fungal Integrative and Synthetic Biology, P. Universidad Católica de Chile.

The filamentous fungus Neurospora crassa has been one of the main models for the study of photobiology, providing great insights on how microorganisms perceive and respond to light. This ascomycete responds specifically to blue light (but not to other wavelengths) through a transcriptional heterocomplex named White Collar Complex (WCC). One of its components, WC-1, possesses a LOV (Light Oxygen Voltage) domain capable of detecting blue light, which promotes a conformational change that leads to dimerization that results in strong transcriptional activation, in a light-intensity dependent manner. In order to design and improve optogenetic switches that can be utilized in other organisms as orthogonal controllers, we have been exploring the dynamics of light responses in this fungus. Thus, through the development of Neurospora-based optogenetic switches we have successfully implemented a blue-light responding transcriptional system in Saccharomyces cerevisiae. Therefore, in yeast, now we can efficiently induce gene expression up to 4000-fold and control biotechnological relevant phenotypes such as flocculation by switching on/off the lights.

On the other hand, we have adopted optogenetic approaches to further delve into Neurospora's circadian and light-responses. In doing so, we were able to genetically program 2D-images in this organism. Thus, we can project a photograph on top of a Neurospora carrying a luciferase reporter under the control of a light responsive promoter and obtain back a bioluminescent pattern mimicking the original image. Thus, we have established a live canvas in which images are genetically processed and reconstituted with real-time dynamics. Such technology not only allows studying light-responses with great resolution, but is also provides a powerful artistic substrate. Remarkably, since the live canvas circuit is integrated in the Neurospora circadian regulatory network, the fungus reproduces on subsequent days -in a circadian manner- the image that it had originally "seen", creating an eidetic (photographic) memory effect. Such phenomenon, based on local discrete phase changes, not only will provide new insights on phase responses, but it also allows for the opportunity to ponder on concepts such as vision and memory. MN-FISB120043, FONDECYT 1131030.

**Fear the titans: when bad yeast get worse.** K. Nielsen Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN.

The human and animal pathogenic yeast *Cryptococcus neoformans* is a major cause of fungal morbidity and mortality worldwide, particularly in immunocompromised individuals. Upon inhalation into the lungs, a subset of *C. neoformans* cells transform into enlarged titan cells. While typical *C. neoformans* cells are 5-7 µm in diameter, the titan cells can be up to 100 µm in diameter. Along with their large size, titan cells also exhibit alterations in their cell wall and capsule structure. These morphological changes impact how the host immune system perceives and responds to the *C. neoformans* infection and ultimately leads to increased disease. In addition to morphological changes, titan cells are also polyploid. Typical *C. neoformans* cells are haploid (1C) but titan cells are tetraploid (4C), octoploid (8C), 16C, etc. with ploidy as high as 312C readily observed. Surprisingly, the polyploid titan cells appear to have a single nucleus and divide by budding to produce haploid or aneuploid daughter cells that can have novel traits such as increased drug resistance and tolerance to various stresses. Current studies aim to understand how *C. neoformans* senses the host environment to trigger titan cell formation as well as the molecular processes underpinning the unique morphological and genomic changes observed in titan cells.

**Developmental consequences of inter-microbial chemical communication.** Nancy Keller¹, Saima Khalid¹, Philipp Wiemann¹, Joshua Baccile², Frank Schroeder², Joseph Spraker¹ 1) University of Wisconsin- Madison; 2) Boyce Thompson Institute - Cornell University.

Filamentous fungi and bacteria are renowned for the production of a diverse array of secondary metabolites (SMs). These natural products are valued for their bioactive properties stemming from their functions in microbial biology, including protection from abiotic and biotic stress and establishment of a secure niche. The rhizosphere provides a rich milieu for fungal/bacterial interactions, one that may be adapted to agricultural practices worldwide. Here we illustrate an unexpected role for SMs in microbial crosstalk among soil-borne pathogens: the bacterium *Ralstonia solanacearum* and the fungi *Aspergillus flavus* and *Fusarium* spp. Ralsolamycin, a lipopeptide inducing chlamydospore formation in fungi (Spraker et al 2016), differentially regulates expression of the bikaverin pathway in *Fusarium fujikuroi* and the imizoquin pathway in *A. flavus*. On one hand, bikaverin specifically accumulates in the chlamydospores and retards *R. solanacearum* proliferation whereas imizoquin, suppressed by ralsolamycin, promotes *Aspergillus* germination via a ROS protective mechanism. We speculate on a role for agricultural in fueling a SMs arms race affecting microbial ecology and disease development.

Spraker JE, Sanchez LM, Lowe TM, Dorrestein PC, Keller NP (2016) *Ralstonia solanacearum* lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues. ISME J. 10(9):2317-30.

**Complex fungal communities in the lung.** Deborah Hogan¹, Elora Demers¹, Amy Biermann¹, Jason Stajich² 1) Dept Microbiology/Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH; 2) Department of Plant Pathology and Microbiology, UC Riverside.

Chronic fungal infections are often recalcitrant to treatment, and the recalcitrance of chronic infections is not well understood. Recent studies in different systems, from bacterial infections to human tumors have found that even within seemingly uniform populations, there is genetic and physiologically-relevant heterogeneity that likely impacts the course of disease and the response to treatment. Heterogeneity within clinical populations of fungal pathogens have not been as well studied. We have identified chronic high burden *C. lusitaniae* lung infections in three unrelated subjects with cystic fibrosis (CF). In all three subjects, *C. lusitaniae* isolates within each population showed high variance in antifungal sensitivity, measured as the minimum inhibitory concentration (MIC), with some isolates stably resistant. In the studies presented here, we will discuss bacterial adaptation to the lung, the repeated selection for genes involved in drug resistance even in
the absence of antifungal therapy, and the roles of antifungal resistance genes in complex populations, microbial communities that contain
bacteria and fungi, and in the host environment.

**Novel interkingdom sensing and signaling in rice blast.** N.I. NAQVI¹, Y KOU², R.N PATKAR³, Y LIU¹, Z QU¹, F YANG¹ 1) Temasek Life Sciences Laboratory, Singapore, SG; 2) China National Rice Research Institute, China; 3) Maharaja Sayajirao Univ. of Baroda, India.

Rice Blast represents a model pathosystem, wherein the causal fungus *Magnaporthe oryzae* perceives and responds to host cues to initiate pathogenic development. Recently, we gained insight into the role of the CFEM motif in surface sensing/response function of the Pth11 receptor, which upon activation utilizes the late endosomal compartment as a scaffold for sustained G-protein/cyclic AMP signaling to induce appressorium formation. Highly regulated redox homeostasis was found to be important for Pth11-mediated appressorium formation in *M. oryzae*. Swapping of the CFEM motifs and *in vitro* binding assays provided further insight into the molecular signatures that are likely recognized by the individual CFEM domains.

Our data suggest that a key step in the appressorium initiation process is cessation of vegetative growth at the germ tube tip, which is achieved through the timely synthesis and action of a novel signaling moiety, Jasmonic acid (JA; fungal oxylipin), in *M. oryzae*. Such intrinsically produced JA is an important signal for appressorium formation and acts in concert with the MAPK cascade in *M. oryzae*. Interestingly, the cAMP signaling itself is necessary for the synthesis of JA in the blast fungus, which subsequently utilizes the ABM monooxygenase to produce a novel metabolite effector ie 12-hydroxy jasmonic acid. Typically, a biotrophic phase precedes necrotrophy during Magnaporthe-Rice interaction and disease development. The blast fungus secretes 12OH-JA into the host tissue and directly inhibits the phytohormone signaling involved in pathogen recognition and defense in rice. We propose a critical role for intrinsic jasmonates in pathogenic differentiation in *M. oryzae*, and a highly intricate chemical communication system during biotrophic interactions in Rice Blast.
Saturday, March 18 2:00 PM – 5:00 PM

Merrill Hall

Signal Transduction and Gene Regulatory Networks

Roles for predicted G protein coupled receptors in plant cell wall sensing in *Neurospora crassa*. K.A. Borkovich, L. Collier, A.

Ghosh, I.E. Cabrera Plant Pathology & Microbiology, University of California, Riverside, CA.

G protein coupled receptors (GPCRs) regulate facets of growth, development and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen *Magnaporthe oryzae*. Largely through the work of undergraduate students in a research-based course at UC Riverside, we have demonstrated that Pth11-related GPCRs are required for normal growth and asexual development on cellulose and other plant-derived carbon sources in the filamentous fungus *Neurospora crassa*. Our work revealed that 3 Pth11-related mutants grew relatively better than wild type on medium containing crystalline cellulose as an alternative to sucrose. We were particularly interested in this result, as this may correlate with sensing of the plant surface for colonization. The most prevalent developmental phenotype for Pth11-related mutants on sucrose was a defect in aerial hyphae height (8 mutants). We are further exploring aerial hyphae height on cellulose and have identified 5 mutants with phenotypes. Our preliminary analysis of conidia production revealed 6 mutants with defects. Based on the cellulose growth and aerial hyphae/conidiation defects, we reasoned that Pth11-related genes may be required for normal aerial hyphae and conidia production on plant-derived carbon sources. Mining of publicly available transcriptional profiling data showed that 14/22 detected Pth11 genes were expressed to higher levels on cellulose than sucrose, including the 3 genes that produced a growth phenotype. These results suggest that the other 19 highly expressed genes may regulate growth on cellulose, but that gene redundancy is masking phenotypes in single mutants. Importantly, these findings also support roles for Pth11-related proteins in sensing plant-derived carbohydrates during growth and development in *N. crassa*.

A role for the velvet protein VE-1 in light sensing and conidiation in Neurospora crassa. María del Mar Gil-Sánchez, Eva M. Luque,

Luis M. Corrochano Department of Genetics, University of Seville, Seville, ES.

The *N. crassa* *ve-1* gene is the homolog of *veA* in *Aspergillus nidulans*. In *A. nidulans* the *VeA* protein forms protein complexes with blue and red photoreceptors and with other regulators to control development and secondary metabolism. The *N. crassa* *ve-1* mutant has defects in aerial hyphal growth and increased conidiation, and we have shown that the *ve-1* mutant has an altered accumulation of carotenoids after light exposure. How does VE-1 regulate pigment biosynthesis and development in *N. crassa*?

We have observed a ten-fold reduction in the sensitivity to light in the *ve-1* mutant, an indication for a role of VE-1 in light sensing. The mutation in *ve-1* results in decreased light-dependent accumulation of the mRNAs of several genes, including the carotenogenesis genes (*al-1*, *al-2*, *al-3*, *cao-2*). However, the absence of any change in the amount of the photoreceptor WC-1 suggests that the reduction of sensitivity to light in the *ve-1* mutant is due to a direct effect of VE-1 on the regulation of transcription. Accordingly, we observed that VE-1 is preferentially located in the nucleus, consistent with a role as a transcriptional regulator.

We have detected the presence of VE-1 during conidial development but the absence of VE-1 in aerial hyphae kept in the dark was puzzling given the presence of *ve-1* mRNA. We have assayed the stability of VE-1 in vegetative mycelia and we have found that the degradation of VE-1 is modulated by the photoreceptor WC-1, and requires the activity of the CSN signalosome and the F-box protein FWD-1. These results suggests that VE-1 is subjected to ubiquitination prior to degradation. We propose that the presence of VE-1 in aerial hyphae and the regulation of its stability by light are key steps in the regulation of conidial development in *N. crassa*.

Signaling through Lrg1, Rho1 and Pkc1 governs *Candida albicans* morphogenesis in response to diverse cues. Jinglin L. Xie¹,

Nora Grahl², Trevor Sless¹, Michelle D. Leach¹,³, Sang H. Kim¹, Deborah A. Hogan², Nicole Robbins¹, Leah Cowen¹ 1) Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; 2) Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, 03755, USA; 3) Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom.

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is *Candida albicans*. *C. albicans* mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of *C. albicans* filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 *C. albicans* homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of *LRG1* or introduction of a *RHO1* mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector *PKC1* results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a *pkc1Δ/pkc1Δ* mutant, adenylate cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in *C. albicans* morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs.

Intracellular pH acts as a second messenger for MAPK signaling in fungi. Tânia Ribeiro Fernandes¹, Antonio Serrano Salces¹,

Teresa Fernández-Acero², David Turrà¹, María Molina², Antonio Di Pietro¹ 1) Department of Genetics, University of Cordoba, Cordoba, Cordoba, ES; 2) Departamento de Microbiología II, Universidad Complutense de Madrid, 28040 Madrid, Spain.

Ambient pH controls fundamental processes in fungi such as growth, development, metabolism and pathogenicity. We previously found
that extracellular pH governs infectious growth in the plant pathogen *Fusarium oxysporum* by reprogramming phosphorylation levels of mitogen-activated protein kinases (MAPKs). The molecular events underlying the pH response are currently unknown. Here we identify intracellular pH (pHi) as a new second messenger regulating MAPK activity in *F. oxysporum*. Using the ratiometric GFP-based pH sensor pHluorin, we found that *F. oxysporum* responds to extracellular alkalinisation and acidification with a transitory shift in pHi. Exogenous application of diethylstilbestrol (DES), a specific inhibitor of the plasma membrane P-type H⁺-ATPase, induced a rapid and sustained decrease of pHi accompanied by rapid and transitory changes in MAPK phosphorylation, supporting the idea that pHi acts as a key switch controlling MAPK activity. To search for fungal proteins involved in pHi-mediated MAPK regulation, we screened a subset of acid-sensitive mutants from the yeast deletion library for loss of DES-triggered MAPK phosphorylation. This identified a number of candidates functioning in conserved cellular processes such as glycosylation, endocytosis or vacuolar ATPase function, many of which have predicted orthologues in Fusarium. Understanding how pHi regulates MAPK signaling may reveal new ways to control fungal growth, development and pathogenicity.

**Investigating how a thermally dimorphic fungal pathogen, *Histoplasma capsulatum*, senses and responds to its host environment.** Lauren Rodriguez, Sarah Gilmore, Sinem Beyhan, Anthony Myint, Mark Voorhies, Anita Sil Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, Ca.

Thermally dimorphic fungal pathogens utilize temperature as a signal to distinguish between growth in the environment and growth within a mammalian host. Growth at 37°C triggers alterations in morphology and gene expression. We study *Histoplasma capsulatum*, which exists in a multicellular hyphal phase in the soil that transitions to a unicellular yeast phase after inhalation by a mammalian host. Here we describe the identification of insertion mutants that are locked in the yeast phase independent of temperature. These mutants are either defective in sensing temperature or transducing the temperature signal. One insertion mutation disrupts the regulation of the *MSB2* gene, whose ortholog encodes a putative osmosensor in *Saccharomyces cerevisiae*. Gene expression profiling of the *MSB2* mutant revealed that, unlike wild-type cells, the transcription factor Efg1 fails to be induced at room temperature in the yeast-locked mutant. Since Efg1 promotes filamentation in *C. albicans*, we explored the role of Efg1 in filamentation in *H. capsulatum*. We determined that overexpression of Efg1 is sufficient to promote filamentation even at 37°C. Taken together, these data suggest that Msb2 may be required for Efg1 induction to promote filamentation in response to temperature. We are currently characterizing the role of *MSB2* in temperature response pathways in *H. capsulatum*.

**Functional analyses of the RNAi pathway required for sexual development in the cereal pathogen *Fusarium graminearum*.** Da-Woon Kim, Hee-Kyoung Kim, Sung-Hwan Yun Dept Med Biotech, Soonchunhyang Univ, Asan, Chungnam, KR.

*Fusarium graminearum*, the causal agent of Fusarium head blight in cereal crops, produces sexual progeny (ascospore) as an important overwintering and dissemination strategy for completing the disease cycle. This homothallic ascomycetous species carries two opposite mating-type (MAT) loci in a single nucleus to control sexual development. Recently, we have identified putative target genes or regulatory pathways controlled by the MAT proteins, among which a RNA interference (RNAi) pathway has been suggested to control a late stage of sexual development. Using several molecular strategies, we have determined the functions of several genes involved in the putative RNAi pathway such as *FgSAD-1*, *FgSAD-3*, *FgDCL-1*, *FgDCL-2*, *FgRecQ-2*, *FgQIP*, *FgSMS-2*, *FgQDE-3*, *FgQDE-2* and *FgQDE-1*. In particular, we have focused on the role of *FgSMS-2* encoding an Argonaute-like protein, which is a part of the RNA-induced silencing complex for specific cleavage of target mRNAs, in *F. graminearum*. Both gene deletion- and gene overexpression-strains of *FgSMS-2* were defective in ascospore/asci maturation. A GFP-tagging analysis showed that FgSMS-2 was specifically localized on perinuclear regions inside the immature asci. A BiFC analysis revealed that FgSMS-2 was able to bind to a Dicer-like protein, FgDCL-1 in cytoplasmic region during the early stage of sexual development. However, three isoforms of the AGO2 protein from *Drosophila melanogaster* were not able to complement the defects caused by *FgSMS-2* in *F. graminearum*, indicating an evolutionary divergence of the AGO-2 like proteins between these two eukaryotes. In addition, we identified a putative target gene (TAG1) of FgSMS-2, which showed an unknown protein similarity. The overexpression of TAG1 led at least 12 h-delay of ascospore formation in *F. graminearum*. Overall, it is likely that the RNAi pathway plays an important role during the sexual development in *F. graminearum*, particularly the Argonaute-like protein, FgSMS-2 protein, controls a set of mRNAs that might be unnecessary during the meiotic event in *F. graminearum*.

**A conserved regulator of meiosis has been rewired to control chlamydosporulation in the fungal pathogen of humans *Candida albicans*.** A. Hernandez-Cervantes¹, S. Znaidi¹,², V. Basso¹, J. Ropars¹, N. Sertour¹, D. Sullivan³, G. Moran³, F. Vincent⁴, F. Dalle⁴, M.E. Bougnoux¹, S. Bachellier-Bassi¹, C. d'Enfer¹ 1) Fungal Biology and Pathogenicity, Institut Pasteur, INRA, Paris, FR; 2) Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique, Institut Pasteur de Tunis, Tunis, TUN; 3) Trinity College, Dublin, IRL; 4) UMR 1347, Université de Bourgogne, Dijon, FR.

Chlamydospores are large, spherical, thick-walled cells produced by several fungi, including the fungal pathogens of humans *Candida albicans* and *Candida dubliniensis*. Little is known regarding the function of these cells and the pathway controlling chlamydosporulation. While investigating the function of the transcription factor Rme1 in the regulation of morphogenesis in *C. albicans*, we have observed that it binds and positively regulates genes that have been previously associated to chlamydospore formation (1). Consistently, inactivation of *RME1* abolished chlamydospore formation in *C. albicans* and *C. dubliniensis* while its over-expression resulted in profuse chlamydosporulation. *RME1* over-expression could also bypass the requirement for other chlamydospore formation regulators such as Efg1 and Hog1. Finally, the expression levels of *RME1* and its targets were highly correlated to the varying ability of *C. albicans* clinical isolates to produce chlamydospores. Thus, Rme1 plays a central role in the control of chlamydosporulation in *C. albicans* and *C. dubliniensis*.

Consistent with this role, *RME1* expression increased in the course of chlamydosporulation. Chromatin-immunoprecipitation experiments, experiments using conditional *RME1* expression and gene fusion experiments with a luciferase reporter allowed to demonstrate that *RME1* expression is subject to a dual regulation whereby (i) elevation of *RME1* mRNA levels in response to chlamydospore-inducing conditions is independent of Rme1 and (ii) sustained expression of *RME1* during chlamydosporulation is subject to autoregulation. Taken together, our results establish Rme1 as a central regulator of the chlamydospore developmental pathway in *C. albicans*. Surprisingly, in *S. cerevisiae* and other yeasts, Rme1 plays an important role in the control of meiosis, and thus ascospore formation. Therefore, our
results indicate that Rme1 can be used alternatively to control sexual or asexual development in Saccharomycotina.

(1) Palige et al. PLoS One 8:e61940, 2013

**Subcellular dynamics of the MAP kinase MAK-2 correlate with its activity during germling fusion, thereby mediating a conserved cell-cell signaling mechanism.** *A. Serrano*¹, *M. Weichert*¹, *R. Marschall*², *J. Illgen*¹, *U. Brandt*¹, *N. Thieme*¹, *A. Lichius*³, *N. Read*³, *P. Tudzynski*², *A. Fleissner*¹
1) Department of Genetics, TU Braunschweig, Braunschweig, DE; 2) Institute of Molecular Biology and Biotechnology of Fungi, Schlossplatz 8, Münster, DE; 3) Institute of Inflammation and Repair, University of Manchester, CTF Building 46, UK.

Cell-cell fusion is essential for the development of most eukaryotic organisms. However, the molecular basis of this process is poorly understood. In *Neurospora crassa*, fusion occurs between germinating vegetative spores so-called germlings. This process employs an unusual mode of communication, in which the two fusion partners coordinately alternate between signal sending and receiving in a "cell dialog". It includes the alternating membrane recruitment of the SO protein (signal sending) and the MAP kinase MAK-2 (signal receiving). To analyze the relationship between the localization and activity of MAK-2, we permanently tethered the kinase to the plasma membrane by using a CaaX motif. This mislocalization resulted in a fusion-defective phenotype in the *Δmak-2* background, indicating that the dynamics of MAK-2 are essential for its function. Western blot analyses revealed a hyper-phosphorylation of the membrane-bound MAK-2 by its upstream MAP kinases. In addition, membrane tethering of activated MAK-2 in WT background disrupts the dynamic localization of SO and reduces the number of tropic interactions.

Similar to *N. crassa*, the grey mold *Botrytis cinerea* undergo cell fusion during colony formation. Localization of the MAK-2 homolog, BMP-1, shows a comparable subcellular dynamic localization during *B. cinerea* cell interactions. Surprisingly, we found that *N. crassa* and *B. cinerea* germlings undergo mutual attraction and establish physical contact, although complete fusion events were not detected.

Together, these data suggest that the "cell dialog" mode of communication is conserved in filamentous ascomycete fungi and that divergent fungal species share a common language
Saturday, March 18 2:00 PM – 5:00 PM

Chapel

Fungal Multicellular Development

ChIP-Seq analysis identifies components of the cell wall integrity, NADPH oxidase and pheromone signaling integrity pathways as target genes of the fungal developmental regulator PRO1. U. Kück, S. Steffens, K. Becker, S. Krevet, I. Teichert Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE.

Fruiting body formation is a fundamental step during the life cycle of filamentous fungi that requires tight spatiotemporal regulation. Working with the model fungus *Sordaria macrospora*, we have previously demonstrated that this major process in the sexual life cycle is controlled by the Zn(II)₂Cys₆ zinc finger transcription factor PRO1.

Here, we further investigated the genome-wide regulatory network controlled by PRO1 by employing chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to identify binding sites for PRO1 (Steffens et al. 2016). We identified several target regions that occur in the promoter regions of genes encoding components of diverse signaling pathways. Furthermore, we identified a conserved DNA-binding motif that is bound specifically by PRO1 in vitro. In addition, PRO1 controls in vivo the expression of a DsRed reporter gene under the control of the esdC target gene promoter. Our ChIP-seq data suggest that PRO1 also controls target genes previously shown to be involved in regulating the pathways controlling cell wall integrity, NADPH oxidase and pheromone signaling. Our data point to PRO1 acting as a master regulator of genes for signaling components that comprise a developmental cascade controlling fruiting body formation.

Steffens E, Becker K, Krevet S, Teichert I, Kück U (2016) Mol Microbiol (in press)

Findings on the origin and genetic background of fungal multicellularity. K. Krizsan¹, B. Balint², A. Prasanna N.¹, B. Kiss¹, E. Almasi¹, Gy. Sipos³, I. Nagy¹, L. Nagy G.² 1) Biochemistry Inst, BRC, Szeged, Hungary; 2) Seqomics Ltd, 6782 Mórahalom, Hungary; 3) Institute of Silviculture and Forest Protection, University of West-Hungary, 9400 Sopron, Hungary.

The independent emergence of complex fruiting bodies was a major step in fungal evolution. Despite decades-long interest in fruiting body development, its genetic bases and the origins of the involved genes are still incompletely known. Here, we set out to identify structural and regulator genes underlying the development of 3 mushroom-forming fungi and to infer a core set of developmentally regulated genes required to build an agaricoid fruiting body. We obtained expression profiles for equivalent developmental stages (vegetative mycelium, early and late primordium, young and mature fruiting body) and tissue types (cap, stipe, lamellae) of *Coprinopsis cinerea* AmutBmut, *Schizophyllum commune* H4-8, *Armillaria ostoyae* C-18 using RNA-Seq. Our results demonstrate that fruiting body formation requires a drastic reorganization of gene expression patterns, with the most dramatic changes observed at the transition from vegetative mycelium to primordia and from young to mature fruiting bodies. Based on global expression profiles in both *Coprinopsis* and *Armillaria*, early primordia grouped with samples derived from stipes, while cap and gill tissues comprised a distinct similarity group, suggesting that two separate developmental pathways exist during the formation of agaricoid fruiting body types. Mining of the genomes of related mushroom-forming fungi indicated a high conservation for most structural genes across the Agaricomycetes, whereas, regulators of gene expression showed more restricted taxonomic distributions. These results will contribute to understanding the genetic background and conserved and lineage-specific mechanisms of fruiting body formation in the Agaricomycetes.

IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus

*Podospora anserina*. Herve Lalucque¹, Fabienne Malagnac¹,², Kimberly Green³, Valérie Gautier¹, Pierre Grognet¹,², Laetitia Chan Ho Tong¹,⁴, Barry Scott³, Philippe Silar¹ 1) Univ Paris Diderot, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, Case courrier 7040 Lamarck, 75205 Paris, France; 2) Present adress : Institut de Biologie Intégrative de la Cellule, UMR 9198 CEA CNRS Univ Paris Sud, 91405 Orsay, France; 3) Institute of Fundamental Sciences, Massey University, Palmerston North, 4442, New Zealand; 4) Present adress : Institut Français du Pétrole Energies Nouvelles 1-4 Avenue du Bois Préau, 92852 Rueil-Malmaison, France.

Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus *Podospora anserina*. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.

Origins of novel sexual development phenotypes in the evolution of the transcriptome in five fungi. Jeffrey P. Townsend¹, Zheng Wang¹, Kayla Stefanko², Caitlyn Cubba², Frances Trail² 1) Yale University, New Haven, CT; 2) Michigan State University, East Lansing, MI.

Changes in gene expression have been hypothesized to play an important role in the evolution of divergent morphologies. To test this hypothesis, we examined developmental differences in fruiting body morphology of filamentous fungi, culturing five species in a common garden environment and profiling genome-wide gene expression at five stages. We reconstructed ancestral gene expression, identifying genes with the largest evolved increases in gene expression across development. Conducting knockouts and performing phenotypic analysis in two divergent species typically demonstrated altered fruiting body development in the species that had evolved increased expression. Our evolutionary approach to finding relevant genes proved far more efficient than other gene deletion studies targeting whole genomes or gene families. Combining gene expression measurements with knockout phenotypes facilitated the refinement of Bayesian models of the gene network underlying fruiting body development, regulation of which is one of the least understood processes of multicellular development.
**Role of blue light for fruiting body induction and development in basidiomycetous fungi.** *Y. Sakamoto*¹, *H. Muraguchi*² 1) Department of Bioresource Sciences, Iwate Biotechnology Research Center, Kitakami, JP; 2) Department of Biotechnology, Akita Prefectural University, Akita, JP.

Light is one of crucial environmental factor for sexual development in fungi. Especially basidomycetous fungi form lager fruiting bodies compared with other filamentous fungi. Therefore relationship between light and fruiting body development in basidiomycetous fungi has been investigated, but molecular mechanism of fruiting body development under light has not been well understood yet compared with ascomycetous fungi, like *Neurospora crassa*. Several basidiomycetous species can form fruiting body under complete darkness. In *Flammulina velutipes* and *Coprinopsis cinerea*, fruiting bodies can be formed under dark, but cap can not mature under darkness, called pinhead fruiting body, or dark stipe. Light can induce cap development on pinhead fruiting bodies by light stimulation in *F. velutipes*. In *Lentinula edodes*, fruiting body formed under dark is whiter compared with normal fruiting body formed under light, and light can stimulate pigmentation toward white fruiting bodies formed under dark. These suggest that light stimulates fruiting body morphogenesis in basidiomycetous fungi. Blue light receptors were identified in *C. cinerea*, dst-1 and dst-2, from dark stipe mutant. The dst-1 encodes WC-1, a homologue of White Collar-1 in *N. crassa*, and dst-2 encodes a protein that has putative flavin adenine dinucleotide (FAD)-binding-4 domain. Homologue of WC-2, a partner of WC-1, in basidiomycetous fungi was first found in *L. edodes*, and disruption of WC-2 in *C. cinerea* resulted dark stipe.

Light also promotes fruiting body induction in several basidomycetous fungi. In *C. cinerea* AmutBmut strain #326, hyphal knot, initial of fruiting body, can be induced concurrently on concentric circle like fairy ring by blue light stimulation. Concurrent hyphal knot induction can be induced in WC-2 mutant. This suggests that WC-1/WC-2 complex will not be involved in hyphal knot induction in *C. cinerea*. Gene expression after blue light stimulation for concurrent hyphal knot induction will be discussed.

**RNA editing: posttranscriptional control of multicellular development in *Sordaria macrospora*.** *I. Teichert*, T. Dahlmann, U. Kück, M. Nowrousian Ruhr-University Bochum, Allgemeine und Molekulare Botanik, Bochum, DE.

The filamentous ascomycete *Sordaria macrospora* has a long history as a model system for studying fungal fruiting body formation. *S. macrospora* generates mature perithecia within seven days without a mating partner and does not produce any conidia, allowing easy observation of sexual structures. The genetic analysis of sterile mutants has enabled the identification of many developmental factors, such as kinases and phosphatases, divers signaling proteins, and transcription factors [1, 2]. Further, transcriptomic analysis of mutants and different developmental stages revealed a large number of potential regulators of fruiting body formation [3, 4]. Studying *S. macrospora* RNA-seq data in more detail, we detected an increase of putative A-to-I RNA editing events in wild type protoperithecia compared to sexually developing mycelia. These data are consistent with a recent study in *Fusarium graminearum*, which identified fruiting body-specific A-to-I RNA editing [5]. Strikingly, the above-mentioned increase of putative editing events in *S. macrospora* wild type was not observed in RNA-seq data of protoperithecia from sterile mutants pro1 and Dnox1, lacking genes for a transcription factor and an NADPH oxidase, respectively. These data indicate that A-to-I RNA editing is linked to fruiting body formation in *S. macrospora*. Putative editing sites in five genes were verified from independent cDNA samples by Sanger sequencing. The same sites were not edited in cDNA from sterile mutant pro1. The effect of RNA editing on protein function is under investigation.

[1] Teichert et al. 2014 Adv Genet 87:199

[2] Teichert et al. 2014 PLoS Genet 10:e1004582

[3] Teichert et al. 2012 BMC Genomics 13:511

[4] Dirschnabel et al. 2014 Genetics 196:729

[5] Liu et al. 2016 Genome Res 26:499

**Cag1, one of the Tup1 paralogues in *Coprinopsis cinerea*, is required for gill formation in fruiting body development.** *H. Muraguchi*, R. Masuda, N. Iguchi, K. Tsukuta, T. Nagoshi, K. Kemuriyama Dept Biotechnology, Akita Prefectural Univ, Akita, JP.

The pileus (cap) of the fruiting body in homobasidiomycete fungi bears the hymenium, a layer of cells that includes the basidia where nuclear fusion, meiosis and sporulation occur. *Coprinopsis cinerea* is a model system for studying fruiting body development. The hymenium of *C. cinerea* forms at the surface of the gills in the pileus. We have mutagenized a homokaryotic fruiting strain, #326, and isolated a mutant that fails to enlarge the cap tissue on the primordial shaft in fruiting. Genetic analysis of this mutant, *cap-growthless*, indicated that the mutant phenotype was brought about by a single gene, designated as *cag1*. The *cag1* locus was mapped on chromosome IX by linkage analysis using RAPD markers that were mapped to each chromosome. The *cag1* gene was identified by transformation experiments using BAC DNAs and their subclones derived from chromosome IX, and found to encode a homologue of *Saccharomyces cerevisiae* Tup1. The *C. cinerea* genome contains another Tup1 homologue called *Cc.tupA*. We examined whether the expression levels of *cag1* and *Cc.tupA* are developmentally regulated during fruiting. In the vegetative mycelium, *Cc.tupA* were expressed more than *cag1*. In contrast, in the growing pileus, the *cag1* expression levels were higher than those of *Cc.tupA*. To examine changes in the spatial and temporal expression of Cag1 and Cc.TupA in the pileus, we reciprocally fused fluorescent tags, i.e. EGFP and mCherry, to these proteins. Compared with Cc.TupA, Cag1 was preferentially expressed in the gill trama tissue cells, suggesting that the function of Cag1 is required for gill trama tissue differentiation and maintenance. Because it is known that *S. cerevisiae* Tup1 forms homotetramer, we examined whether Cag1 interacts with itself and with Cc.TupA using yeast two-hybrid (Y2H) system. Y2H analysis reveals that Cag1 can interact with itself and with Cc.TupA through their N-terminal regions. Like Tup1, which interacts with Cyc8, the N-terminal region of Cag1 also interacts with the N-terminal region of Cc.Cyc8, which contains tetratricopeptide repeats. Y2H analysis and co-localization of Cag1 and Cc.TupA suggested that Cag1 interacts with Cc.TupA in the nuclei of certain cells.

**Feeding developing button mushrooms.** *A. Sonnenberg*, J. Baars Plant Breeding, Wageningen University and Research Centre, Wageningen, NL.

The number and sizes of fruiting bodies of saprotrophic fungi heavily depend on the amount and quality of substrate available for these fungi. Is thus not surprising that substrate for the production of edible mushrooms is one of the main research areas to improve yield and quality and as a result the profitability of commercial production systems. For button mushrooms (*Agaricus bisporus*), next to substrate, a
nutritionally poor casing layer on top of the substrate is a prerequisite to induce the vegetative mycelium to form fruiting initials. The quality of this layer also has a prominent influence on the quality of mushrooms. Quality is reduced when mushrooms mature, i.e. when the cap is opening and sporulation starts. Previous research on button mushrooms has shown that there is a signalling between the top layer where fruiting bodies are formed and the bottom layer where substrate is degraded and where nutrients are transported to the developing mushrooms. The induction of fruiting changes the physiology and gene expression of the vegetative mycelium in the compost towards feeding the developing mushrooms. Changing the nutritional quality and the total amount of nutrients in the substrate layer influences the yield and quality but not always in a logical way. Many observations have been done in the past in mainly empirical and applied research that have generated some understanding.

We intend to give an overview on research done in the past and more recently on the cultivation of button mushrooms (*Agaricus bisporus*) and evaluate what this has learned us so far. We intend to focus also on the type of research is needed to generate the knowledge necessary to understand the production system and to use this to either improve the present cultivation system or even to develop an entirely new one.
Saturday, March 18 2:00 PM – 5:00 PM

Fred Farr Forum

Synthetic Biology, Fungal Tools, and Fungi as Tools

**JGI fungal single cell genomics pipeline.** Doina Ciobanu¹, Alicia Clum¹, Asaf Salamov¹, William Andreopoulos¹, Steven Ahrendt¹,²,
Alisha Quandt³, Igor Grigoriev¹,², Timothy James³, Jan-Fang Cheng¹ 1) Genomics, Joint Genome Institute, Walnut Creek, CA; 2) Plant and
Microbial Biology, University of California, Berkeley, CA; 3) Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

We developed a single cell genomics pipeline for noncultured fungal species and used it for sequencing seven early diverging fungi:
* *Blyttiomyces helicus* (Chytridiomycota), *Caulochytrium protostelioides* (Chytridiomycota), *Rozella allomycis* (Cryptomycota), *Syncephalis pseudoplumigaleata* (Zoopagomycotina), *Thamnocephalis sphaerospora* (Zoopagomycotina), *Piptocephalis cylindrospora* (Zoopagomycotina), *Dimargaris cristalligena* (Kickxellomycotina), and one Dikarya: *Metschnikowia bicuspidata* (Ascomycota). The established pipeline for fungal single-cell genome recovery consists of seven steps: environmental sample collection and analysis, single cell isolation, single cell lysis and whole genome amplification, single cell amplified genome (SCAG) quality screening, best SCAG genome deep sequencing and assembly and finally best SCAG or combined SCAG (C-SCAG) annotation and functional analysis. Each of these steps can have an impact on the quality of the resulting genome and annotation. We validated each step of the pipeline using a set of quality check criteria such as start genome amplification (SGA), fold genome amplification (FGA), random twenty-mer uniqueness (RTU), genome GC content, assembled genome size plus a set of metrics for genome assembly quality and evaluated each using criteria for its predictability power in relationship to genome completeness, which was estimated using CEGMA (CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. G. Parra, K.Bradnam, I.Korf. Bioinformatics, V23, Iss 9, pp1061-1067). We examined all the factors that have a critical impact for fungal single cell genome recovery, the most important of which is amplification bias, which can be characterized by the combination of SGA, FGA, RTU and genome GC content. We offer a suite of protocols for a large scale workflow that can be applied to a wider range of organisms than fungi.

**A global co-expression network approach for connecting genes to specialized metabolic pathways.** Jennifer Wisecaver, Abigail Lind, Antonis Rokas Vanderbilt University, Nashville, TN.

Both fungi and plants produce a tremendous diversity of specialized metabolites (SMs) to interact with and manage their environment. A major challenge hindering efforts to tap this seemingly boundless source of pharmacopeia is the identification of SM pathways and their constituent genes. To address this challenge, much attention has been paid to fungal SM gene clusters, but the number of SM pathways in fungi that are non-clustered or only partially clustered is completely unknown. The genome of *Aspergillus fumigatus*, an opportunistic human pathogen and one of the leading causes of fungal-related human deaths, provides a stark example; 21 of its 37 SM gene clusters are missing either transporters, transcription factors or both, suggesting these genes may be located elsewhere in the genome. Given the well-established observation that the genes comprising a SM pathway are co-regulated in response to specific environmental conditions, we hypothesized that genes from a given SM pathway would form tight associations (modules) with each other in gene co-expression networks, facilitating their identification even without knowledge of the genome sequence. To evaluate this hypothesis, we first used 10 global co-expression datasets—each a meta-analysis of hundreds to thousands of expression experiments—across eight plant model organisms to identify hundreds of modules of co-expressed genes for each species. In support of our hypothesis, 15.3-52.6% of modules contained two or more known SM biosynthetic genes (e.g., cytochrome P450s, terpene synthases, and polyketide synthases), and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways in these species, including all those known to form SM gene clusters. For each clustered SM pathway, the module analysis expanded the known genetic repertoire of the pathway by identifying additional unclustered enzymatic genes as well as unclustered genes involved in pathway regulation and product transport. We are currently expanding our network analysis to fungal taxa including *Aspergillus* and submit that global gene co-expression is a rich, but largely untapped, data source for discovering the genetic basis and architecture of natural products.

**Universal expression system for eukaryotic microorganisms.** A. Rantasalo, C. Landowski, J. Kuivanen, J. Jäntti, D. Mojzita VTT Technical Research Centre of Finland Ltd.

We have developed a novel orthogonal expression system that functions in a wide spectrum of eukaryotic microorganisms. The expression system is based upon a synthetic transcription factor (sTF) that regulates expression of the target gene via a sTF-dependent promoter. The sTF expression is driven by an engineered, universal core promoter that provides a low, but sufficient expression level of the sTF. The sTF expression is constitutive and thus present in the cells in all growth conditions and in all developmental and growth stages. The sTF-dependent promoter regulating the expression of the target gene also contains a similar type of universal core promoter functional in diverse species. This core promoter can be further modified by inserting synthetic repressor (sR) binding sites, through which an additional expression control is gained upon expression of the sR. The combination of multiple sTF-binding sites and the core promoters (including the repressible versions) enables specific adjustment over a wide range of target gene expression levels, from very low to very high. This expression system provides robust, stable, and tuneable expression levels of the target genes in a broad spectrum of host organisms. Further it simplifies the genetic tools needed for the construction of new protein expression hosts, including those with undeveloped know-how. The method for selecting the universal core promoters, constructions of the expression systems, and demonstrations of their performance in diverse hosts, including yeasts and filamentous fungi, will be presented.

**A CRISPR/Cas9 based toolbox for efficient genome editing of filamentous fungi.** U.H. Mortensen, C. S. Nødvig, Z.D. Jarczynska, M.L. Nielsen, F. H. Kirchner, J.B. Hoof Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, DK.

Large numbers of fungi are currently being fully sequenced and will contribute dramatically to our understanding of fungal biology. However, the fact that gene targeting is inefficient in most fungal species hampers exploitation of the genome sequences. This problem has been significantly reduced after efficient CRISPR/Cas9 gene editing has been introduced in several different fungal species. A challenge of the CRISPR/Cas9 system is to deliver gRNAs to form the Cas9 ribonuclease. Several approaches have been presented in the literature including methods where gRNAs are co-transformed into the cells along with the gene editing DNA substrates, methods where the gRNA is
produced by RNA polymerase III, and methods where the gRNA is liberated by ribozymes from a larger transcript produced by RNA
polymerase II. Since the different methods have different advantages/disadvantages, we envision that they may work with different
efficiencies in different fungal species. We have therefore developed a flexible CRISPR/Cas9 toolbox adapted for filamentous fungi to
facilitate genome editing. Our toolbox includes bio-bricks containing e.g. different genetic markers and polymerase promoter types allowing
for a rapid and efficient vector assembly and bricks that allow for quick insertion of new genes into strong expression sites for heterologous
expression. Moreover, it includes bricks to facilitate trouble shooting including a *cas9-RFP* reporter gene to evaluate Cas9 levels in new
hosts and a system allowing the efficiency of individual gRNA species to be tested in vivo. Using our toolbox, we have successfully edited
the genomes of more than 10 species and used it to make a different range of genetic alterations including site specific mutations by using
oligonucleotides as repair templates and deletions. In this way we have linked secondary metabolites to genes in species that have not
previously been genetically engineered. For strains where we plan to do extensive gene targeting, we typically use CRISPR to mutate the
*pyrG* gene, hence, producing a marker than can be selected/counter selected. Next, we mutate a gene in the NHEJ pathway to produce a
strain where gene targeting is very efficient. We will show how this strategy can be used to investigate the biosynthetic pathway of gene
clusters. Lastly, we will show how markers and mutations in NHEJ genes can be easily reverted to wild-type if a wild-type background is
desirable in the subsequent analyses.

**Synthetic biology as powerful discovery tool to understand and rewire the cell factory Aspergillus niger.** V. Meyer Applied and Molecular Microbiology, TU Berlin, Berlin.

Synthetic Biology interrogates the complexity of biological systems on a molecular level. It takes a constructive approach to design novel
biological parts and circuits with non-natural function and to reengineer biological networks. Obviously, systems biology and synthetic
biology disciplines – systems analysis and design – ideally complement each other and a multitude of methodological crosslinks exist.
Our focus is to pioneer genetic tools and molecular gene switches for the industrial strain *Aspergillus niger* and to explore its capabilities as
producer of secreted proteins and natural products. By engineering titratable and tightly regulated conditional mono- and polycistronic gene
expression systems and targeting them to specific genomic loci, we rewire the metabolism of *A. niger*. On the one hand, this enables us to
improve this cell factory as expression host for proteins and secondary metabolites with product yields high enough for commercial
exploitation. On the other hand, this approach helps us to unravel the function of complex gene networks *in vivo*.

**Measuring intracellular pH as a well-controlled physiological parameter responding to extracellular cues.** G. Smits Dept Molecular Biol, SILS, Univ Amsterdam, Amsterdam, NL.

In baker's yeast, intracellular pH can be monitored in a time-resolved, organelle specific manner in living and growing cells, using the
genetically encoded pH responsive GFP variant pHLuorin. We assessed intracellular pH, and found that it dynamically responds to changes
in the cellular environment, and cytoplasmic pH can vary between 4.5 and 7.4. In Candida glabrata, pH responds to cues that are
associated with pathogenicity. Because pH also changes in response to the influx of weak organic acids, such as those used as food
preservatives or produced as industrial precursors using yeast or filamentous fungi. We have established online intracellular pH
measurement as a valid proxy measurement of acid influx, allowing the establishment of kinetic parameters.
In a genome-wide analysis using the yeast deletion collection, we identified genes responsible for pH homeostasis. We established that
intracellular pH is a second messenger, controlling yeast's maximal cell division rate.

**Engineering of *Ustilago maydis* for efficient organic acid production.** E. Geiser¹,², S.K. Przybilla¹, M. Bölker³, T. den Hartog⁴, L.M. Blank¹, N. Wierckx¹ ¹) Institute of Applied Microbiology (iAMB), RWTH Aachen University, Aachen, DE; ²) BioSC, c/o Forschungszentrum Jülich, Jülich, DE; ³) Department of Biology, Philipps-University Marburg, Marburg, DE; ⁴) Institute for Technical and Macromolecular Chemistry (ITMC), RWTH Aachen University, Aachen, DE.

The family of Ustilaginaceae contains promising biocatalysts for a variety of industrially relevant chemicals. One promising product is itaconic acid, a platform chemical for the production of pharmaceuticals, adhesives and polymers. We recently characterized the itaconate biosynthesis pathway and related gene cluster in *U. maydis* MB215 (1). The *U. maydis* itaconate gene cluster is remarkably similar to that of *Aspergillus terreus* (2). However, in contrast to the known biosynthesis pathway of *A. terreus*, itaconate production in *Ustilago* proceeds via the unusual intermediate *trans*-aconitate. Two additional genes in the cluster, *cyp3* (cytochrome P450 monooxygenase) and *rdo1* (ring-cleaving dioxygenase) are not directly involved in itaconate production. Instead, *Cyp3* has a role in the further oxidation of itaconate into 2-hydroxyparaconitate (3). We will present the application of this new biochemical knowledge to enhance the overall product titer and yield up to 4.5-fold, but also to control product specificity.

**References**

1. Geiser et al. 2016. *Ustilago maydis* produces itaconic acid via the unusual intermediate *trans*-aconitate. *Microbial Biotechnology* **9**:116-126

2. Li et al. 2011. A clone-based transcriptomics approach for the identification of genes relevant for itaconic acid production in *Aspergillus*. *Fungal Genetics and Biology* **48**:602-611

3. Geiser et al. 2016. Genetic and biochemical insights into the itaconate pathway of *Ustilago maydis* enable enhanced production. *Metabolic Engineering* **38**:427-435

**Kluyveromyces marxianus as a robust systems and synthetic biology platform.** R. Estrela¹,³, P. Cernak¹,³, C. Zhang¹,³, V. Yu¹,³, J. Cate¹,²,³ ¹) Molecular and Cell Biology Department, UC Berkeley, Berkeley, CA; ²) Department of Chemistry, UC Berkeley, Berkeley, CA; ³) Energy Biosciences Institute, Berkeley, CA.

Among "non-standard" yeast, there is increasing interest in studying *Kluyveromyces marxianus* (*Km*) given its combination of industry-
appealing traits: broad range of carbon source utilization, ability to grow at temperatures up to 52°C and ferment at 45°C and one of the
highest growth rates among eukaryotes. It is not surprising, however, that most of the studies published on *Km* up to date exploits its
general biotechnological potential rather than studying the basic biology underlying its interesting traits. The lack of genetic tools has
hampered in-depth genetics and biochemistry focused studies in *Km* and in many other non-model organisms. Our goal is to tackle this
problem and develop *K. marxianus* into a robust platform for systems biology studies and synthetic biology applications in industrial
microbiology. We also established CRISPR-Cas9 genome editing in *Km* and used this system to engineer several strains to use in classical genetics. We have been using these synthetic biology tools to probe the genetic basis for thermotolerance in *Km* and also to use *Km* to produce value-added chemicals. We are successfully using CRISPR-Cas9 to knockout genes of interest and identify ones that might have an important role in thermotolerance. Our synthetic biology tools are also being used to explore the potential of *Km* to produce renewable fuels and chemicals.
Saturday, March 18 2:00 PM – 5:00 PM

Kiln

## Chromosome Dynamics

**What drives speciation? Examination into the evolutionary events of more than 100 Aspergillus species.** J.L. Nybo¹, T.C. Vesth¹, S. Theobald¹, I. Kjaerboelling¹, J.C. Frisvad¹, T.O. Larsen¹, R. Riley², A. Salamov², I.V. Grigoriev², S.E. Baker³, M.R. Andersen¹ 1) Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, DK; 2) Joint Genome Institute, Walnut Creek, CA, USA; 3) Joint Bioenergy Institute, Berkeley, CA, USA.

The study of speciation - how new species arise, diverge and remain separate, has a central role in evolutionary biology. Partly because it embraces so many disciplines, including population genetics, behavioral sciences, comparative genomics, evolutionary biology, biodiversity, biogeography and ecology. It also remains one of the most fascinating questions in evolution.

Speciation is nearly impossible to study and in most cases, we know very little about the genetic basis of species formation. But in this project we aim to identify evolutionary events that can drive speciation, such as gene duplications, creations and losses, and horizontal gene transfers between closely or distantly related species within the genus of the filamentous fungi Aspergillus. This diverse genus holds species relevant to both plant and human pathology, food biotechnology, enzyme and bulk chemical production, model organisms, and it even contains some extremophiles.

To identify these events, we have developed a homologous protein prediction software that has been used to generate a high-resolution pan-genomic map. From where, we have identified genes specific to species, clades and core that allows for guilt-by-association-based mapping of genotype-to-phenotype.

Our results illustrate a highly diverse genus where 500-2000 genes are unique to each species. These genes are predominantly within regulation or compound biosynthesis, supporting the notion of natural selection. A conservative estimate of the number of protein families shared by all Aspergillus species is surprisingly low, only about 2600 core families, suggesting high environmental adaptation within this genus.

**Transitions between tetrapolar and bipolar fungal mating type driven by chromosomal translocations involving intercentromeric recombination.** Sheng Sun¹, Vikas Yadav², R. Blake Billmyre¹, Christina A. Cuomo³, Minou Nowrousian⁴, Jean-Luc Souciet⁵, Teun Boekhout⁶, Betina Porcel⁷, Patrick Wincker⁷, Joshua A. Granek¹, Liuyang Wang¹, Kaustuv Sanyal², Joseph Heitman¹ 1) Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC; 2) Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India; 3) Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; 4) Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum, Germany; 5) Université de Strasbourg, CNRS UMR7156, Strasbourg, 67000, France; 6) CBS-KNAW Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands; Institute of Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, The Netherlands; 7) Commissariat à l'Energie Atomique (CEA), Institut de Génomique (IG), Genoscope, Evry, France, Université d'Evry, UMR 8030, Evry, France, Centre National de Recherche Scientifique (CNRS), UMR 8030, Evry, France.

Species within the human pathogenic *Cryptococcus* species complex are major threats to public health, causing more than one million infections globally each year. *Cryptococcus amylolentus* is the most closely known related species of the pathogenic *Cryptococcus* species complex, and it is non-pathogenic. Additionally, while pathogenic *Cryptococcus* species have bipolar mating systems with a single large *MAT* locus that represents a derived state in Basidiomycetes, *C. amylolentus* has a tetrapolar mating system with two *MAT* loci (*P/R* and *HD*) located on different chromosomes. Thus, studying *C. amylolentus* could shed light on the origin and evolution of pathogenesis, as well as the transition from tetrapolar to bipolar mating systems in the pathogenic *Cryptococcus* species. In this study, we sequenced, assembled, and annotated the genomes of two *C. amylolentus* isolates, CBS6039 and CBS6273, which are interfertile. Genome comparison between the two *C. amylolentus* isolates identified the boundaries and the complete gene contents of the *P/R* and *HD* loci. Also, bioinformatics and ChIP-seq analyses showed that *C. amylolentus* has regional centromeres that are enriched with species-specific transposable and repetitive elements, similar to the centromeric structures in the pathogenic *Cryptococcus* species. Additionally, we found that while neither of the *P/R* and *HD* loci in *C. amylolentus* is physically linked to its centromere, both *MAT* loci showed centromere linkage in meiosis, suggesting the presence of recombination repressors and/or epistatic gene interactions in the inter *MAT*-CEN regions. Furthermore, genomic comparison between *C. amylolentus* and pathogenic *Cryptococcus* species provided evidence that chromosomal rearrangements mediated by intercentromeric recombination have occurred after the two lineages split from their common ancestor. We propose a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by repetitive elements located within the centromeric regions and shared between chromosomes. This translocation brought the *P/R* and *HD* loci onto the same chromosome, and was followed by chromosomal rearrangements that resulted in the two *MAT* loci becoming physically linked and eventually fused to form the single contiguous *MAT* locus that is now extant in the pathogenic *Cryptococcus* species.

**Genome plasticity impacts adaptive genome evolution in the vascular wilt pathogen Verticillium.** M.F. Seidl, L. Faino, D.E. Cook, M. Kramer, X. Shi-Kunne, G.C.M. van den Berg, B.P.H.J. Thomma Laboratory of Phytopathology, Wageningen University & Research, Wageningen, NL.

Genome plasticity enables organisms to adapt to environmental changes and to occupy novel niches. This is established by mechanisms ranging from single-nucleotide polymorphisms to large-scale chromosomal variations, all of which contribute to differences in chromosomal size, organization and gene content. While these mechanisms operate in all organisms, they are particularly relevant for plant pathogens that engage in a co-evolutionary arms race with their hosts. Plant pathogens secrete so-called effectors that contribute to host colonization and counteract host immunity. Effector genes often cluster in highly plastic, transposon-rich genomic regions. However, mechanistic understanding of the evolution of these plastic genomic regions remains scarce. We study these molecular mechanisms in the fungal genus
*Verticillium* that contains economically and ecologically important plant pathogens, among which *Verticillium dahliae* is the most notorious pathogen that causes vascular wilt disease on >200 plant species. Using long-read sequencing technology, we completely assembled two *V. dahliae* strains. By comparative genomics, we established that transposable elements play important roles in shaping the genome of *V. dahliae*. Plastic genomic regions in *V. dahliae* that contain all known effectors evolve by extensive genomic rearrangements that are mediated by erroneous double-strand breaks, often over transposons. Extensive genomic rearrangements are not only restricted to *V. dahliae*, but also occur in related *Verticillium* species. Furthermore, recent segmental duplications are enhanced in the plastic regions. These regions, in contrast to the core genome, are also enriched in active transposons that further contribute to local plasticity. In fungi, transposons are located in tightly condensed chromatin, so called heterochromatin, that is supposed to suppress transposon activity and repress structural variations. In contrast, many fungal pathogens have highly plastic transposon-rich regions. Therefore, research into chromatin opens new avenues to link genome organization, genome plasticity and adaptive genome evolution in fungal pathogens.

**The adaptive potential of *Candida albicans* in response to *in vivo* stress.** G. Cromie¹, E. Jeffery¹, S. Filler², J. Berman³, A. Dudley¹, A. Forche⁴ 1) PNRI, Seattle, WA USA; 2) UCLA, Los Angeles, CA USA; 3) Tel-Aviv University, Tel-Aviv, Israel; 4) Bowdoin College, Brunswick, ME USA.

To understand the adaptive potential of *C. albicans* to its host, we characterized the genomes of ~1000 isolates recovered after single passage through mice from an oropharyngeal Candidiasis model (OPC) and a systemic model of Candidiasis (BSI). We used flow cytometry to determine plody and ddRADseq to assess whole genome karyotypes. Our analysis revealed high levels of aneuploidy and loss of heterozygosity (LOH) among isolates from both models. There was no significant difference in the overall frequencies of aneuploidy and LOH. However, chromosome (Chr) 6 trisomy was significantly higher in the OPC population. Intriguingly, Chr6 is particularly enriched for genes from multiple virulence gene families. Segmental aneuploidies and LOH occurred at similar frequencies in both model. Interestingly, all but one aneuploidy breakpoint (at the rDNA locus) were unique to each model. The majority of aneuploidy and LOH breakpoints were found near repetitive DNA and tRNAs. In addition, many LOH breakpoints were located near proposed origins of replication. This finding suggests that *in vivo* environments may trigger replication stress-induced genome instability, which could be due partially to the resolution of arrested replication forks. In contrast to similar overall frequencies of genome changes in the two models, genetic diversity within each mouse host was significantly higher for the OPC model, suggesting that the oral cavity represents a much more dynamic and diverse niche.

**Processing of replication forks by homologous recombination factors to avoid mitotic abnormalities.** A. Ait-saada, A. Da Silva, K. Fréon, S.A.E. Lambert Institut Curie, CNRS, University Paris Sud, Paris-Saclay, University PSL Research University, UMR3348 F-91405, Orsay, France.

The completion of eukaryotic DNA replication requires the sequential activation of replication origins and the merging of converging forks. The completion of DNA replication is continuously threatened by a broad spectrum of unavoidable replication fork barriers (RFBs) that are caused by intrinsic chromosomal features (such as DNA sequence and chromatin), endogenous stress linked to cellular metabolism (such as transcription) and environmental factors including DNA damage (1). RFBs interrupt replication fork elongation, often causing multiple temporary pauses to a single replisome and occasionally causing terminal fork arrest. To avoid these perturbations creating chromosomal aberrations additional replication-based pathways have evolved to ensure the completion of DNA replication and thus help maintain genome stability. Terminally-arrested forks can be reactivated by DNA repair pathways such as homologous recombination (HR), and maintaining replication fidelity relies on close links between the replication machinery and HR (2). To investigate the consequences of terminally arrested forks on genome stability, we have developed conditional and genetically encoded RFBs which allow the block of replication forks at specific loci, in the fission yeast S. pombe (3 and 4). Our recent data on understanding how dysfunctional forks are processed and restarted by the HR pathway will be presented and discussed. More precisely, our data reveal a new role for Rad51-mediated fork-protection that ensures the rescue of arrested forks by an incoming converging fork to avoid sister chromatid bridging at site of replication stress.

1. Lambert and Carr. Impediments to replication fork movement: stabilisation, reactivation and genome instability. Chromosoma. 2013; 122(1-2):33-45.

2. Carr and Lambert. Replication Stress-Induced Genome Instability: The Dark Side of Replication Maintenance by Homologous Recombination. J Mol Biol. 2013; 425(23):4733-44.

3. Lambert et al. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange. Mol. Cell. 2010 13;39(3):346-59.

4. Iraqui et al. Recovery of arrested replication forks by homologous recombination is error-prone. PLoS Genetic. 2012 Oct;8(10):e1002976.

**The role of histone-methyltransferases KMT1 and KMT6 in genome and chromatin organization of the wheat pathogen Zymoseptoria tritici.** M. Moeller¹,², K. Schotanus¹,²,⁴, M. Freitag³, Eva H. Stukenbrock¹,² 1) Christian-Albrechts-University, Kiel, Germany; 2) Max Planck Institute for Evolutionary Biology, Plön, Germany; 3) Oregon State University, Corvallis, OR, United States of America; 4) Duke University Medical Center, Durham, NC, United States of America.

*Zymoseptoria tritici* is a plant pathogenic fungus specialized to infect wheat (*Triticum aestivum*). The genome of the sequenced reference isolate comprises 21 chromosomes of which eight are accessory chromosomes. These chromosomes are highly instable during meiosis and mitosis, transcriptionally repressed and show enrichment of repetitive elements and of heterochromatic histone marks. To elucidate the role of heterochromatin-associated histone modifications on genome stability and transcriptional regulation in *Z. tritici*, we created deletion mutants of the methyltransferases KMT6 and KMT1 that are responsible for H3K27me3 and H3K9me3, respectively. We combined experimental evolution, genetic and high-resolution microscopic analyses to follow the impact of these deletions on chromosome and genome stability. We used ChIP-seq, whole genome sequencing and RNA-seq to compare changes in chromatin and genome structure and differences in gene expression between mutant and wild type strains. Analyses of genome and ChIP-seq data from the Δ*kmt1* mutants reveal dramatic chromatin reorganization, genome rearrangements and formation of “neochromosomes”. Transcriptome analysis revealed transposable element activation. The Δ*kmt6* mutant, however, displays an increased stability of the accessory chromosomes compared to
the wild type under normal growth conditions *in vitro*. Based on these results we conclude a strong impact of H3K9me3 in chromatin and genome organization and an important role of H3K27me3 for the stability of accessory chromosomes.

**Epigenetic suppression of transposons co-opted as global regulator of virulence gene expression in a fungal phytopathogen.** *Adam P. Taranto*, Megan C. McDonald, Peter S. Solomon Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, Australia.

Many fungal plant-pathogens display a rapid transcriptomic switch in gene expression upon infection of a susceptible host. We have explored the role of epigenetic suppression in regulation of this shift towards virulence in the wheat-pathogen *Parastagonospora nodorum*. We have characterised the methylome of *P. nodorum* via whole genome bisulfite sequencing and assessed the contribution of DNA-methylation to gene expression by RNA-seq. Genomic regions subject to epigenetic control were enriched in transposable elements, effector-like genes, and putatively horizontally acquired genes. Key necrotrophic effectors, *ToxA*, *Tox1* and *Tox3* occur in close proximity to transposons. Molecular disruption of epigenetic silencing mechanisms active in these regions (H3K9me3, H3K27me3, and 5mC DNA methylation) allowed for partial recapitulation of the early-infection transcriptomic profile of *P. nodorum*. Similar epigenetic reprogramming of pathogen gene expression was achieved through application of chemical inhibitors of epigenetic pathways.

**Dynamics of accessory chromosomes in *Fusarium oxysporum*.** *M. Rep*, P. van Dam, P.M. Houterman, H.C. van der Does, S. Shahi, S. Schmidt, I. Vlaardingerbroek, S. Widinugraheni, J. Li, M. Constantin, N. Tintor, M. de Sain, B.V. Chellappan, B.J.C. Cornelissen, L. Fokkens Molecular Plant Pathology, University of Amsterdam, Amsterdam, NL.

Genomes of the *Fusarium oxysporum* (Fo) species complex contain eleven 'core' chromosomes and a variable number of 'accessory' chromosomes. The three smallest of the core chromosomes are different from the eight larger ones in that they have a higher SNP frequency between strains, a higher fraction of *in planta*-induced genes and, similar to accessory chromosomes and subtelomeric regions, a high incidence of histone H3 lysine 27 trimethylation. They do not, however, have the very high repeat-content characteristic of accessory chromosomes. Surprisingly, one of the three small core chromosomes is dispensable for saprophytic growth and pathogenicity. Also among the accessory chromosomes some stand out: in plant-pathogenic strains of Fo, effector genes are present on specific accessory chromosomes that we call 'pathogenicity chromosomes'. Expression of the effector genes is very low *in vitro* and highly induced *in planta*. We have shown horizontal transfer of two pathogenicity chromosomes, one for tomato- and one for cucurbit-infection, in both cases conferring disease-causing ability to the recipient strain. SMRT sequencing has allowed us to assemble repeat-rich pathogenicity chromosomes and identify centromeres in all chromosomes. Comparison of pathogenicity chromosomes between strains reveals a high rate of intra-chromosomal recombination. In several cases, we could localize recombination to repetitive elements, suggesting that the repeat-rich nature of accessory chromosomes is one factor underlying their highly dynamic nature.
Saturday, March 18 2:00 PM – 5:00 PM

Heather

## Early-diverging Fungi

**Evolutionary genomics of the zygomycete fungi.** J.W. Spatafora¹, Y. Chang¹, J.E. Stajich², ZyGoLife Research Consortium 1) Dept Botany & Plant Pathology, Oregon State Univ, Corvallis, OR; 2) Department of Plant Pathology and Microbiology and Institute for Integrative Genome Biology, University of California, Riverside, Riverside CA.

Zygomycetes comprise two separate phyla, Zoopagomycota and Mucoromycota, of Kingdom fungi. They represent early diverging lineages of nonflagellated fungi that arose after the zoosporic lineages of chytrids (Chytridiomycota and Blastocladiomycota) and prior to Dikyara (Ascomycota and Basidiomycota). As such, they are thought to represent lineages that colonized early terrestrial landscapes and associated with some of the earliest forms of terrestrial life. Zoopagomycota is the earliest diverging lineage and includes three subphyla, Entomophthoromycotina, Kickxellomycotina and Zoopagomycotina, which are primarily parasites and pathogens of animals, mycoparasites, and soil saprobes. Mucoromycota is sister group to Dikarya and includes Glomeromycotina, Mortierellomycotina and Mucoromycotina, which are primarily symbionts of plants (e.g., arbuscular mycorrhizae, root endophytes), saprobes of plant material, and soil fungi, although animal pathogens and mycoparasites are known. Although nonflagellated, Zoopagomycota possess a centriole-like organelle, while Mucoromycota possesses a spindle pole body typical of nonflagellated fungi. Based on this foundation of ecology and cell biology, phylogenomic analyses of these taxa stand to provide critical insights into the evolution of fungi and the origin of fungal associations and interactions with other terrestrial life forms. Here we will emphasize the use of genome scale data to resolve evolutionary relationships, and comparative genomics to understand transitions associated with the loss of flagellum and evolution of nutritional modes reliant on animal, fungal and plant-based carbon sources among early diverging fungi.

**The genome of an uncultured nematode-destroying fungus and its role in resolving the zygomycete tree of life.** K.R. Amses, T.Y. James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

Phylogenomic analyses of the artificial Zygomycota s.l. show support for its reorganization into two segregate phyla, each containing three subphyla. Of these six subphyla of zygomycetes, many are represented by only one or a few draft genomes, leading to some ambiguity in analyses. The sampling of more taxa from these poorly represented groups will bolster the strength of phylogenomic hypotheses formed at these deep nodes, strengthening the lines delineating the proposed taxonomic groupings. The Zoopagomycotina, a subphylum comprised of obligate parasites of other zygomycetes and small soil animals, is represented by a single draft genome in the most recent analyses. Establishment of axenic cultures has proven a central obstacle to the genome-enabled study of these cryptic soil fungi as acquisition of sufficient material for traditional whole genome sequencing is difficult. Single cell genomics approaches offer a solution to these complications, allowing for the sequencing and assembly of whole genome data sets from miniscule amounts of input material summing to only one or a few cells. Leveraging these techniques in order to address the underrepresentation of Zoopagomycotina in phylogenomic analyses, we have sequenced the genome of *Stylopage hadra*, a cryptic but ubiquitous fungus that captures and consumes nematodes in natural and agricultural soils. Our single cell genomics approach generated four sequencing libraries with an average cumulative scaffold size of 22.81 Mbp and genome completeness scores of 56.65%, suggesting a *S. hadra* genome size of approximately 40 Mbp. Preliminary phylogenetic analyses based on concatenated RPB1 and RPB2 sequences support *S. hadra's* placement in the Zoopagomycotina. Sequencing libraries contain a putative endosymbiont of *S. hadra* belonging to the beta-proteobacterian genus *Glomeribacter* with cumulative scaffold sizes indicating a genome size of approximately 1.80 Mbp. Known from the cytoplasm of arbuscular mycorrhizal fungi and species of *Mortierella*, species of *Glomeribacter* comprise a group of opportunistic soil-borne endosymbionts that tend to reduce the growth rates of their associated fungi, indicating a weakly parasitic interaction.

**E2F and SBF transcription factors in early-diverging fungi bind overlapping and unique cis-regulatory sites.** A.M. Augustus¹,², E.M. Medina¹,², R. Gordan²,³,⁴, N.E. Buchler¹,² 1) Department of Biology, Duke University, Durham, NC; 2) Center for Genomic & Computational Biology, Duke University, Durham, NC; 3) Department of Biostatistics & Bioinformatics, Duke University, Durham, NC; 4) Department of Computer Science, Duke University, Durham, NC.

Although cell cycle control is a conserved and essential process, some core animal and fungal cell cycle regulators are not homologous (e.g. E2F and SBF). Using eukaryote-wide phylogenetic analyses, we have shown that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor, a protein with homology to the KiIA-N domain of DNA viruses. Ancestral SBF may have hijacked cell cycle control by binding cis-regulatory elements targeted by E2F and activated transcription of G1/S genes to drive cell proliferation. Cell cycle evolution in the fungal ancestor proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in early-diverging fungi such as *Spizellomyces punctatus* (a Chytrid). The hybrid network had parallel E2F and SBF pathways that regulate similar target genes, thus providing redundant control which enabled the eventual loss of E2F and its replacement by the SBF pathway in the ancestor of Dikarya.

To address the question of redundancy and specificity of E2F and SBF, we measured the binding specificity of E2F and SBF in chytrids and zygomycetes using a high-throughput protein-binding microarray (PBM) assay. PBM assays measure, in a single experiment, the binding of recombinant proteins to tens of thousands of synthetic DNA sequences, guaranteed to cover all possible 10-bp DNA sequences in a maximally compact representation. Our data show that chytrid and zygomycete E2F and SBF have nearly identical DNA-binding specificity to their human and yeast counterparts. We further show that E2F and SBF can bind a common set of motifs, which supports the hijacking hypothesis and binding redundancy between E2F and SBF. Last, we show that there are specific motifs that can be bound only by E2F or only by SBF. This suggests that certain classes of genes could be under E2F-only or SBF-only control, which may explain why both transcription factors are still maintained in some species, such as *Spizellomycyes punctatus*.
**Pervasive adenine N6-methylation of active genes in fungi.** S.J. Mondo¹, R.O. Dannebaum¹, R.C. Kuo¹, A. Gryganskyi², J. Magnuson³, T.Y. James⁴, M.A. O'Malley⁵, J.E. Stajich⁶, J.W. Spatafora⁷, A. Visel¹, I.V. Grigoriev¹ 1) Fungal Genomics, Joint Genome Institute, Walnut Creek, CA; 2) L. F. Lambert Spawn Co, Coatesville, PA; 3) Pacific Northwest National Laboratory, Richland, WA; 4) Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI; 5) Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA; 6) Department of Plant Pathology and Microbiology, University of California, Riverside, California; 7) Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.

Epigenetics plays a critical role in regulation and expression of genetic information. Many important epigenetic marks are encoded as DNA-based modifications, for example 5-methylcytosine (5mC) or N6-methyldeoxyadenine (6mA). 6mA is a non-canonical eukaryotic DNA base modification reported genome-wide in only four species: an alga, *Chlamydomonas reinhardtii* and three animals, *Drosophila melanogaster*, *Caenorhabditis elegans* and *Mus musculus*. However, the genome-wide abundance of 6mA in these organisms ranges from low (0.4% of all A's in *C. reinhardtii*) to extremely low (0.001%-0.07% in *D. melanogaster*). Despite low levels, 6mA is critical for proper development, as it plays an important role in regulation of gene expression. Regrettably, exploration of 6mA thus far has been limited to single model eukaryotes and therefore its relevance in many major branches of Eukaryota is still unknown. Here we conduct the first kingdom-wide exploration of both 6mA and 5mC, where we interrogate the Fungi, an ancient and extremely diverse eukaryotic lineage. We found heavy utilization of 6mA in early-diverging fungi where up to 2.8% of all adenines were methylated, vastly exceeding the levels observed in other eukaryotes and more derived fungi, whereas 5mC was primarily in the CpG context and restricted to repeats. Interesting, with one exception 6mA and 5mC were mutually exclusive (i.e. lineages with high 6mA had no 5mC and vice versa). 6mA occurred symmetrically at ApT dinucleotides, where it concentrated in dense 6mA 'islands' at gene promoters. Islands were positioned nearby promoter thymine-blocks and ≈95% of methylated genes were expressed. Our results demonstrate the importance of 6mA as a broadly conserved epigenomic mark in eukaryotes as well as identify key components involved in positioning of genomic 6mA. Furthermore, our results implicate 6mA as an epigenomic mark transmissible across nuclear division and suggest its involvement in nucleosome organization. We anticipate that our study will provide a foundation for deeper exploration of 6mA not only in fungi, but across all Eukaryota.

**Bacterial endosymbionts as a tool to unravel the basic biology of Mucoromycotina.** Olga Lastovetsky¹, Stephen Mondo², Maria Gaspar³, Colin Barber², Susan Henry³, *Teresa Pawlowska*² 1) Graduate Field of Microbiology, Cornell University, Ithaca, NY 14853; 2) School of Integrative Plant Science, Plant Pathology & Plant Microbe-Biology, Cornell University, Ithaca, NY 14853; 3) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.

Despite their ecological significance and formative role in unraveling the principles of fungal reproduction, Mucoromycotina remain one of the least understood lineages of filamentous fungi. This knowledge gap is largely caused by limited genetic tractability that bedevils these organisms. To overcome this impediment, we rely on the *Rhizopus microsporus-Burkholderia* symbiosis, in which the endobacteria can be manipulated and used as a tool to perturb various processed in the fungus. Through transcriptional profiling, pharmacological manipulation, identification of genes that display altered expression patterns due to endobacteria, and mapping them to well-characterized regulatory and metabolic networks in other fungi, we identified candidate genes responsible for regulation of several processes in Mucoromycotina, including reproductive biology, lipid metabolism, and defense against antagonistic bacteria. These findings create a foundation for functional characterization of key biological processes in Mucoromycotina.

**Resolving the Mortierellaceae phylogeny through Multi-Locus Sequence Typing (MLST) and phylogenomics.** Natalie Vande Pol¹, Jason Stajich², Kerry O'Donnell³, Alessandro Desiro¹, Gregory Bonito¹ 1) Michigan State University, East Lansing, MI; 2) University of California at Riverside, Riverside, CA; 3) Mycotoxin Prevention & Applied Microbiology Research Unit, NCAUR-ARS-USDA, Peoria, IL.

The Mortierellaceae (Mortierellomycotina) are a diverse family of fungi that are of evolutionary and ecological relevance. They are the closest lineage to the arbuscular mycorrhizae (Glomeromycotina) and include some of the first species to evolve fruiting body production. The Mortierellaceae are estimated to contain at least 100 species classified within six polyphyletic genera that cannot be resolved with ribosomal markers. With advances in DNA sequencing technology, it is now feasible to generate sequence data from many loci in parallel (MLST), or to perform low-coverage genome (LCG) sequencing to identify phylogenetically informative loci. In collaboration with the ZyGo Life consortium and the Joint Genome Institute, we sequenced 68 LCGs representing 50 unique species of Mortierellaceae. From these, we identified 400 informative loci and used RaxML to build a concatenated tree. The resulting phylogeny has very strong bootstrap support and a very different structure from existing ribosomal trees. In parallel, we analyzed three *de novo* sequenced *Mortierella* genomes and extracted 13 informative loci. For each locus, we designed PCR primers for multiplexed PCR amplification across 333 Mortierellaceae isolates, which included the 68 LCG isolates. We will discuss the strengths and limitations of these two approaches.

**An Omics-based approach to understanding Host-Pathogen interactions during Mucormycosis.** V.M. Bruno, T.N. Watkins, M.C. Chibucos, S. Soliman, T. Ghebremarimam, A.S. Ibrahim Dept of Microbiology & Immunology, University of Maryland Sch of Med, Baltimore, MD.

Mucormycosis is a deadly invasive infection caused by several fungal organisms belonging to the subphylum Mucormycotina, order Mucorales. The major risk factors include uncontrolled diabetes mellitus that results in hyperglycemia and ketoacidosis (DKA), other forms of acidosis, treatment with corticosteroids, solid organ or bone marrow transplantation, neutropenia, trauma and burns (e.g., wounded soldiers in Iraq and Afghanistan), malignant haematological disorders and deferoxamine therapy in patients receiving haemodialysis. The infection is generally acquired by inhalation of spores that are ubiquitous in nature and cause either rhino-orbital (almost exclusively in DKA patients) or pulmonary (mainly in neutropenic leukemic patients) disease. There are currently no vaccines and only two antifungal agents approved by the USA FDA to treat this disease. In the absence of surgical removal of the infected focus (such as excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative. Even when surgical debridement is combined with high-dose antifungal therapy, the mortality associated with mucormycosis is >50%. In patients with prolonged neutropenia or disseminated disease, mortality is 90-100%. The high mortality rate, limited options for therapy and the extreme morbidity of highly disfiguring surgical therapy make it imperative to look for alternative strategies to treat and prevent mucormycosis. In this work, we have undertaken an unbiased, systematic approach to identify and characterize fungal and host factors that contribute to disease progression with the goal of developing novel therapeutic targets.
**RNAi-based functional genomics identifies new virulence determinants in mucormycosis.** *M.I. Navarro-Mendoza*¹, *T.A. Trieu*¹,², *C. Pérez-Arques*¹, *M. Sanchis*³, *J. Capilla*³, *P. Navarro-Rodríguez*³, *L. López-Fernández*³, *S. Torres-Martínez*¹, *V. Garre*¹, *R. Ruiz-Vázquez*¹, *F.E. Nicolás*¹
1) Department of Genetics and Microbiology, University of Murcia, Murcia, Spain; 2) Current address: Department of Genetics, Faculty of Biology, Hanoi National University of Education, Hanoi, Vietnam; 3) Microbiology Unit, University Rovira i Virgili, IISPV. Tarragona, Spain.

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. In this work, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of *Mucor circinelloides* silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of *mcpID*, which encodes a Phospholipase D, and *mcmyo5*, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of *M. circinelloides*. Knock-out mutants for those genes showed reduced virulence in both *Galleria mellonella* and *Mus musculus* models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in *M. circinelloides* that could represent promising targets for future antifungal therapies.

This research was funded by Fundación Séneca (19339/PI/14), MECD (FPU14/01832 and FPU14/01983) and MINECO (RYC-2014-15844 and BFU2015-65501-P) co-financed by FEDER.
Saturday, March 18 2:00 PM – 5:00 PM

Nautilus

Disease Epidemiology

**Azole resistance in Aspergillus fumigatus: the silent tsunami.** *Paul Verweij*¹,² 1) Radboud University Medical Center, Nijmegen, NL; 2) Center of Expertise in Mycology Radboudumc/CWZ.

*Aspergillus fumigatus* is an important opportunistic fungus that causes a spectrum of diseases in humans ranging from allergic syndromes to acute invasive disease. Since nearly two decades acquired resistance to medical azoles has emerged and the application of azole compounds in the environment appears to play an important role in resistance selection. Resistance mutations associated with the Cyp51A-gene continue to emerge, which confer a pan-azole resistant phenotype. As *A. fumigatus* is not a phytopathogen, growth of the fungus in organic waste, such as compost, which contains azole fungicide residues appears to be a condition that facilitates the selection of resistance mutations. Although these mutations confer resistance to azole fungicides, the isolates are cross resistant to medical triazoles due to molecule similarity. Over the years three environmental mutations have been identified consisting of a tandem repeat in the gene promoter region often combined with point mutations in the Cyp51A-gene (TR₃₄/L98H; TR₅₃; TR₄₆/Y121F/T289A). Recently another two mutations were found in patients and the environment, indicating that the current situation is not sustainable and unless measures are taken the class of azoles may be lost for treatment of fungal diseases.

The clinical significance of azole resistance is substantial in patients at risk of invasive aspergillosis. A recent Dutch study showed that within a five-year cohort of patients with invasive aspergillosis the mortality rate was 36% in patients infected with an azole-susceptible isolate, compared with 86% in those with azole-resistant aspergillosis. Early diagnosis of resistance is difficult and delay of treatment with an effective drug contributes to the poor outcome. Furthermore, due to lack of effective alternative agents, certain manifestations of invasive aspergillosis, such as central nervous system aspergillosis, are virtually untreatable. In the Netherlands very high resistance rates of 25% to 32% have been reported in critically-ill patients and those with hematological malignancy, prompting changes in initial treatment strategies. Unlike antibacterial resistance, there appears to be little awareness of resistance in *A. fumigatus* and associated problems in the management of aspergillus diseases. This silent tsunami will continue to spread unless research is initiated that investigates the dynamics of environmental resistance selection and enables effective measures to be taken.

**Genome-wide genetic dissection of trait variation between species.** Carly Weiss¹, Rylee Hackley¹,², Jeremy Roop¹, Jeffrey Skerker³, *Rachel Brem*¹,² 1) Department of Plant and Microbial Biology, UC Berkeley, Berkeley, CA; 2) Buck Institute for Research on Aging, Novato, CA; 3) Energy Biosciences Institute, UC Berkeley, Berkeley, CA.

Geneticists since Mendel have sought to explain how and why traits vary among wild individuals. The industry standard toward this end is to test for DNA sequence variants that correlate with phenotype across a population, but such methods cannot be applied across reproductive barriers. To fill this analysis gap, we developed a high-throughput, interspecific version of the reciprocal hemizygote test. In a viable hybrid formed from the mating of two species, we introduce disrupting mutations at each allele of each gene in turn via transposon mutagenesis, and we quantify phenotypes of the resulting hemizygote mutants in a pooled format via Tn-seq. As a testbed for our approach, we focused on thermotolerance in yeasts. At 39°C, *Saccharomyces cerevisiae* grows faster and accumulates more biomass than its sister species *S. paradoxus*, to a degree far exceeding the variation among isolates of each species. Culture and sequencing of *S. cerevisiae* × *S. paradoxus* hemizygotes revealed a battery of loci at which the species' alleles conferred differences in thermotolerance. Few of these genes had known roles in resistance to heat shock or other stresses. At most mapped loci, the *S. cerevisiae* allele was associated with improved thermotolerance relative to the *S. paradoxus* allele. Independent transgenesis experiments verified the benefit at high temperature of *S. cerevisiae* versions of the housekeeping genes *AFG2*, *CEP3*, and *NIP100*. Together, these results elucidate the complex genetics of *S. cerevisiae* thermotolerance, and they pioneer a genetic mapping method that will be applicable across the tree of life.

**Population structure and drivers of genetic diversity in Candida glabrata.** *E. Shor*¹, K. Healey¹, C. Jimenez-Ortigosa¹, S. Lockhart², V. Loparev², D. Perlin¹ 1) Public Health Research Institute, Rutgers University, Newark, NJ; 2) Centers for Disease Control, Atlanta, GA.

The prevalence of *Candida glabrata* infections has been rising for several decades, and it now accounts for approximately 25% of all Candida bloodstream infections in the U.S. and can in some settings predominate as the principal bloodstream fungal pathogen. Furthermore, *C. glabrata* exhibits elevated intrinsic resistance to triazoles and readily acquires resistance to echinocandins. It also becoming increasingly apparent that *C. glabrata* has a highly genetically diverse population structure. However, how this diversity arises and whether it contributes to the virulence and drug resistance of *C. glabrata* is unknown. A multi-locus sequence typing (MLST) scheme has thus far identified over 80 different *C. glabrata* sequence types (STs); however, our preliminary analysis indicates that this number is likely much higher. We also found that different STs carry different alleles of DNA repair gene *MSH2*, some of which cause elevated rates of drug resistance *in vitro*, indicating that different STs have different propensity towards mutability and acquiring drug resistant gene variants. *C. glabrata* also displays a high degree of variability at the level of chromosomal architecture. For instance, it has been reported that clinical *C. glabrata* isolates show great diversity in terms of chromosomal number and structure, displaying a high number of chromosomal rearrangements relative to the reference laboratory strain. This observation has led to the hypothesis that emergence of new chromosomes is a virulence mechanism in *C. glabrata* and may underlie emergence of drug resistance. However, we find that strains of the same ST have similar chromosomal patterns, suggesting that specific chromosomal configurations may pre-exist emergence of virulence and may be a feature of commensal *C. glabrata* populations. To identify new determinants of virulence and/or drug resistance in *C. glabrata*, we are performing optical mapping and long read genome sequencing (PacBio) of several STs that are prevalent in the U.S. We are also investigating the importance of the DNA replication checkpoint in facilitating chromosomal rearrangements in *C. glabrata*.

**Epidemiology of Cryptococcus spp. using comparative genomics.** *D.M. Engelthaler*¹, C.C. Roe¹, G.R. Thompson³, W. Meyer², S. Lockhart⁴ 1) TGen North, Translational Genomics Research, Flagstaff, AZ; 2) University of Sydney; 3) University of California Davis; 4) Centers for Disease Control and Prevention.

**Background:** Fungi present a number of problems for molecular epidemiology due to their reproductive, morphologic, ecologic, and
genomic complexities. While numerous methods of genotyping can infer genetic relationships among isolates, they provide limited empirical estimates of genetic relatedness among strains. The combination of advances in whole genome sequencing and bioinformatics analyses along with the recent emergences of *Cryptococcus* in previously non-endemic regions has allowed for the use of advanced genomic analyses to understand the dispersal and epidemiology of *Cryptococcus* and other environmental fungi. Prototypical examples are the emergence of novel populations of *C. gattii* in the temperate North American Pacific Northwest, and the recent genomic understanding of distinct lineages Southeastern U.S.

**Methods:** Using Illumina next generation sequencing technology, we sequenced >250 *C. gattii* isolates, representing the known global diversity, and applied several bioinformatics approaches to establish genomic and genetic relatedness and distance. These analyses also included genomes from sequenced isolates of *C. neoformans* var. *neoformans* and *C. n. var. grubii*.

**Results:** WGS-based analysis was able to confirm outbreak sources, elucidate the population structure of the various subtypes and establish phylogeographic understanding of the various species and subtypes. These analyses describe the highly diversity of global populations of major subtypes and established likely source of dispersal events (e.g., VGII emanating out of South America); additionally they also established recent emerging subtypes as being completely clonal but with continuing their local evolution in newly endemic regions. Numerous epidemiologically significant gene content differences were identifiable between and among subtypes, including genes potentially related to habitat adaptation, virulence and clinical differentiation. Various statistical analyses have established likely age estimates for dispersal and emergence events.

**Discussion:** The next generation of molecular epidemiology of fungal pathogens must include WGS analyses. The use of WGS and appropriate bioinformatics enables investigations to be informed with empirical knowledge of strain and subtype relatedness rather than reliance on epidemiological inference based on genetic similarity. Additionally, the continued use of WGS in epidemiology allows for the creation of public archives of sequence data for future research and investigations, further improving overall biological, public health and clinical understanding. More specific, the findings here provide greater understanding of dispersal, emergence and epidemiology of *Cryptococcus* species.

**Genomic diversity of *Malassezia* yeasts: implications for epidemiology and the clinic.** Teun Boekhout¹,², Anna Kolecka¹, Bart Theelen¹, Claudia Cafarchia³, Roberta Latti³, Thomas Dawson⁴, the Malassezia Genome Consortium 1) CBS Fungal Biodiversity Centre, Utrecht, NL; 2) Institute of Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, NL; 3) Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, 70010 Valenzano, Bari, Italy; 4) A*STAR Institute of Medical Biology (IMB), Singapore.

*Malassezia* is a genus of lipid-dependent basidiomycetous yeast species belonging to Ustilaginomycotina. *Malassezia* spp. are a major component of the human skin mycobiome and may be implicated in several skin disorders, such as pityriasis versicolor, seborrheic dermatitis and atopic dermatitis. Some species may also causes sepsis, especially in neonates. Here we present results from a comparative genomics study of all species of the genus with the aim to contribute to our understanding of the biodiversity, disease potential and genetic processes such as hybridization.

**Methods.**

The genomes of all species were sequenced by Illumina technology, and for some species more than one isolate using Illumina and Pacific BioSciences technology. Bioinformatics analysis included de novo assembly, annotation and gene family analysis. Additional analysis also included MultiLocus sequencing, MALDI-TOF MS and Amplified Fragment length Polymorphism (AFLP) applied to a greater set of isolates, including outbreak isolates.

**Results.**

*Malassezia* have among the smallest free-living eukaryotic genomes, ranging between 7-9 Mb, with some exceptions having ca. 14Mb genomes. Three phylogenetic clusters can be discerned, largely confirming previous insight obtained by the analysis of rDNA. The compact genomes differ from those of related Ustilaginomycotina by major gene loss and limited gene gain processes. Interestingly, the larger genomes all belonged to the *M. furfur* complex with three distinct clades, with one representing potential aneuploids as hypothesized based on previous MLST, AFLP and PFGE data. Strains belonging to one of the potential *M. furfur* parental clades seem to be more prevalent in causing invasive infection. Detailed comparative genomics analysis of the *M. furfur* strains may shed light on the molecular mechanisms. In addition, MLST revealed a significant genetic heterogeneity of outbreak isolates suggesting that patients can be infected by multiple isolates.

**Conclusions:** All 17 hitherto described species of *Malassezia* belong to three major clades that differ in gene loss/gain patterns. The genomes of all species are small, except for those isolates that seem to represent hybrids in the *M. furfur* complex. Invasive isolates of *M. furfur* mainly belong to a specific parental group but also show considerable diversity. It remains to be seen how this relates to the *Malassezia* diversity present on skin of neonates, parents and hospital staff.

**Emergence of bat white-nose syndrome in North America.** D. Blehert US Geological Survey, National Wildlife Health Center, Madison, WI.

White-nose syndrome (WNS) is an emergent wildlife disease that has spread rapidly and caused unprecedented mortality among bats of eastern North America. Since first detected in New York during winter of 2006-2007, WNS has spread to 29 US states and five Canadian provinces, and the disease is estimated to have killed over five million bats. Prior to emergence of WNS, such massive population declines of mammalian species due to an infectious disease were unprecedented. White-nose syndrome is caused by the psychrophilic fungus *Pseudogymnoascus destructans*. The fungus grows optimally at temperatures consistent with bat hibernation (approximately 2 to 12°C), causing a lethal skin infection that disrupts hibernation physiology and behavior. Additionally, the fungus persists throughout the year in soil of underground bat hibernation sites, including during summer months, establishing environmental reservoirs of the pathogen. A growing body of evidence suggests that *P. destructans* was introduced to North America, likely from Eurasia where bats seem to co-exist with the fungus, suggesting that WNS in North America is the result of a novel pathogen introduced into a population of naïve host species.
Naturally occurring mismatch repair mutants mediate rapid phenotypic change and drug resistance in the Pacific Northwest

**Cryptococcus gattii** outbreak. R. Blake Billmyre, Shelly Clancey, Shelby Priest, Joseph Heitman Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC.

Pathogenic microbes confront a constant evolutionary conflict between the pressure to maintain genome stability and the need to adapt in response to mounting external stresses. Over the past fifteen years, an ongoing outbreak of the human fungal pathogen *Cryptococcus deuterogattii* has occurred in the Pacific Northwest of the United States and Canada. Whole genome resequencing of outbreak strains identified one lineage of the outbreak that harbors a nonsense mutation in *MSH2* and exhibits a hypermutator phenotype. Genetic analysis of progeny as well as independent deletions of *MSH2* demonstrate linkage of the hypermutator phenotype to *MSH2* mutation. This defect in mismatch repair destabilizes homopolymer runs throughout the *Cryptococcus* genome, resulting in inactivation of genes with coding homopolymer runs. As a result, resistance to the clinically-used immunosuppressive drugs FK506 and rapamycin is rapidly generated, as well as resistance to 5-fluorocytosine (5-FC), a frontline drug often used to treat Cryptococcal infections and for which spontaneous drug resistance occurs rapidly, necessitating use only in combination with amphotericin B. One environmental isolate with an *MSH2* mutation even exhibited an unselected FK506/rapamycin drug resistance phenotype attributable to a homopolymer run shift. Competition experiments demonstrate this hypermutator state is deleterious in rich growth conditions, but allows more rapid adaptation to stressful conditions. Ongoing work has identified strains with elevated mutation rate present throughout the population of the sister species of *Cryptococcus neoformans* and representing multiple independent origins of hypermutation. Hypermutator states may represent a general mechanism by which fungi adapt to changing environmental conditions or drug challenges.

Latest insights in the epidemiology and diversity of *Fusarium oxysporum* f.sp. cubense, the causal agent of Panama disease in

banana. Gert H.J. Kema¹,³, Fernando Garcia²,³, Nadia Ordóñez¹,³, Maricar Salacinas¹,³, Michael F Seidl¹, Bart P.H.J. Thomma¹, Harold JG Meijer³ 1) Wageningen University and Research, Laboratory for Phytopathology; 2) Wageningen University and Research, Laboratory for Plant Breeding; 3) Wageningen Plant Research.

Panama disease or Fusarium wilt of banana draws global attention. The currently developing epidemic of the so-called Tropical Race 4 (TR4) is caused by a single clone of *Fusarium oxysporum* f.sp. cubense (Foc); vegetative compatibility group 01213. It is reminiscent of the previous epidemic that wiped out “Gros Michel” bananas in Central America, which pushed the banana industry into bankruptcy. The epidemic was eventually quenched by cultivating “Cavendish” bananas, which are resistant to the so-called Foc Race 1 strains that caused the epidemic in “Gros Michel”. The industry revived and thrives by the success of “Cavendish” that has developed into a global monoculture. The emergence of TR4 caused havoc and wipes out “Cavendish” plantations in South East Asia, from where the disease now has spread into the Near and Middle East and Africaª. Banana production in many regions is at stake and there are no sustainable solutions available. Our research focuses on the international complexity and addresses mostly genetic diversity in host and pathogen as well as epidemiological aspects embedded in multidisciplinary programs. We have used genotyping by sequencing technologies to describe global and regional diversity in the causal agent Foc and have phenotyped hundreds of banana accessions with various Foc genotypes. Methods to rapidly detect - particularly TR4 - and manage the disease have been developed to slow down the epidemic. This provides the necessary time for developing durable solutions that also contribute to break the hegemony of the global “Cavendish” monoculture by introducing a diversified panel of banana cultivars. The latest developments will be presented and discussed.

<footnote>ª Ordonez Román, N.I., Seidl, M.F., Waalwijk, C., Drenth, A., Kilian, A., Thomma, B.P.H.J., Ploetz, R.C., and Kema, G.H.J., 2015. Worse comes to worst: Bananas and Panama disease - when plant and pathogen clones meet. PLoS Pathogens, DOI: 10.1371/journal.ppat.1005197.</footnote>
Saturday, March 18 2:00 PM – 5:00 PM

Scripps

Cell Walls and Polysaccharides

**Aspergillus** biofilm exopolysaccharide – from virulence factor to therapeutic target. *Don Sheppard*¹,² 1) Microbiology and Immunology, McGill University, Montreal; 2) Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal.

*Aspergillus fumigatus* is the most common cause of invasive mold infection in humans. Reverse-genetic studies from our group have discovered that *A. fumigatus* virulence is enhanced by the production of galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that is required for fungal biofilm formation, adherence to and invasion of host cells as well as evasion of host immune defenses.

Using molecular and biochemical approaches we have elucidated the biosynthetic pathways governing the synthesis of GAG. These studies have revealed parallels between *Aspergillus* and bacterial exopolysaccharides, most notably the requirement for post-synthetic deacetylation, which renders the final polymer cationic and able to mediate adhesion. Within the GAG biosynthetic complex, we have identified two enzymes, Sph3 and Ega3, which contain glycoside hydrolase domains that can degrade pre-formed GAG. Recombinant hydrolase domains from both of these proteins are able to disrupt pre-formed biofilms of *A. fumigatus* at nanomolar concentrations. Treatment of *A. fumigatus* with recombinant hydrolases increases susceptibility to antifungal agents, and prevents invasion and damage of pulmonary epithelial cells in vitro. Recombinant Sph3 is non-toxic when administered intratracheally to mice, and treatment of neutropenic mice with Sph3 dramatically attenuates virulence.

Collectively these studies have revealed that GAG is a key virulence factor of the pathogenic fungus *A. fumigatus*, and reveal a promising new therapeutic strategy to target this virulence factor.

**Some like it on the rocks: recurring stresses select for rock-inhabiting fungi with manifold protective pigments.** *Nicole Knabe*¹,
Romy Breitenbach¹,², Polina Dementyeva¹, Felix Heeger³, Camila Mazzoni³, Anna A. Gorbushina¹,² 1) Federal Institute for Materials Research and Testing (BAM), Department Materials and the Environment, Berlin, Germany; 2) Freie Universität Berlin, Department of Biology, Chemistry and Pharmacy & Department of Earth Sciences, Berlin, Germany; 3) Berlin Center for Genomics in Biodiversity Research, Berlin, Germany.

Black ascomycetous microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in deserts as well as ubiquitous in other wide-spread terrestrial ecosystems including man-made materials such as solar panels. The ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance. Also, applied research is necessary to find ways of preventing MCF from colonising and degrading solar panels and historic monuments.

Carotenoids and melanin are amongst the protective pigments that contribute to the robustness of MCF. We studied their role in stress resistance of the non-pathogenic rock-inhabiting fungus *Knufia petricola* (Chaetothyriales) strain A95. Several knock-out mutants have been produced in melanin- (A95ΔSDH and A95ΔPKS) as well carotenoid- (A95ΔPDG) synthesis and a double mutant (A95ΔPKS/ΔPDG) also exists. Disruption of melanin synthesis affects not only oxidative-stress resistance but also the extracellular polysaccharide (EPS) matrix and lipid composition of the cell membrane. Exposure to ≤ 30 mM H₂O₂ did not affect the growth rate of A95ΔPKS as compared to wild type cells. Almost twice as much water-soluble EPS was present in cultures of the A95ΔPKS mutant compared to the wild type and A95ΔPDG strains. Comparative gene expression analyses of the wild type and the A95ΔPKS mutant identified genes regulated under oxidative stress conditions. These data will help elucidate mechanisms of cell wall maturation and oxidative stress defence strategies.

**Fungal surface amyloids function in host-microbe interactions.** *Melissa Garcia-Sherman*¹, Safraz Hamid², Desmond Jackson¹,
James Thomas¹, Peter Lipke¹ 1) Dept of Biology, Brooklyn College CUNY, Brooklyn, NY; 2) Dept of Biology, Princeton University, Princeton, NJ.

Previously we have shown that Als5p, a member of *Candida albicans* cell wall adhesins, contains amyloid forming sequences which are conserved in other Als family members. These sequences are critical for amyloid fibril formation by soluble peptides and for the intact Als5 protein. The Als5p amyloid sequence is also critical for cell adhesion and biofilm formation (Garcia *et al*, PLoS ONE e17632). I will present data that demonstrate an amyloid forming peptide that contains a sequence of Als5p and binds to the surface of non-albicans species of *Candida*. Additionally, *Candida tropicalis* aggregates stained positive with the amyloid binding dye thioflavin T, and an Als5p amyloid-inhibiting peptide blocked cell aggregation. The amyloid inhibiting peptide also blocked *C. albicans*, *C. tropicalis*, and *Candida parapsilosis* adhesion to monolayers of FaDu epithelial-derived cells, monolayers. These data demonstrates a functional consequence of amyloid inhibition and is the first evidence that amyloid-like interactions function in adhesion to human cells by opportunistic fungi. I will also present data that show that fungal surface amyloids are present in autopsy tissue sections from patients inflicted with several fungal infections. The fungi present in these patients test positive with amyloid binding dyes, such as thioflavin T, thioflavin S, and Congo red. In the infected tissue and *in vitro*, amyloid-expressing fungi were coated with human serum amyloid P component (SAP), a amyloid-binding, pattern recognition receptor with anti-inflammatory function.

Supported by R01GM098616

**Comparative analysis of the function of α-1,3-glucan synthases, AgsA and AgsB, in Aspergillus nidulans.** *K. Miyazawa*¹, A. Yoshimi², S. Yano³, S. Kasahara⁴, F. Hasegawa², K. Abe¹,² 1) Grad. Sch. Agric. Sci., Tohoku Univ., Sendai, JP; 2) NICHe, Tohoku Univ., Sendai, JP; 3) Yamagata Univ., Yonezawa, JP; 4) Miyagi Univ., Sendai, JP.

Although α-1,3-glucan (AG) is one of major polysaccharides in the cell wall of *Aspergillus* species, the biological function of AG remains unclear, except for the role as a virulence factor in some pathogenic fungi. Previously, we carried out functional analysis of two α-1,3-glucan synthase (AGS) genes (i.e. *agsA* and *agsB*) in the model filamentous fungus *Aspergillus nidulans*. The *agsB*? strain lost most cell wall AG, suggesting that a main AGS in this fungus is *agsB*. Interestingly, the hyphae of the *agsB*? strain was dispersed under liquid culture
conditions, whereas the wild-type strain formed hyphal pellets under same culture conditions. These results suggest that AG has the role as an adhesive factor for hyphal cells. On the other hand, because the expression of *agsA* gene was scarcely detected under normal growth conditions and the *agsA*? strain did not show phenotypic defects, the role of *agsA* remains unclear. In this study, in order to investigate the roles of AgsA and AgsB in cell wall AG synthesis, we comparatively analyzed cell wall polysaccharides synthesized by AgsA and AgsB. First, we constructed *agsA* or *agsB* gene overexpression (O/E) strain by replacing the promoter region of *agsA* or *agsB* with *tet1* promoter under the genetic background of the other AGS gene disruption, and confirmed the high expression of either of the AGS genes in the O/E strains. The O/E of *agsA* restored the growth characteristics of the *agsB*? strain under liquid culture conditions: the O/E *agsA* strain formed the hyphal pellets. This suggests that the *agsA* gene encodes a functional AGS. To elucidate the differences of cell wall structure between these two strains, we performed the alkaline-fractionation of cell wall and analyzed the sugar composition of the fractions. The carbohydrate analyses revealed that the sugar compositions of the AS2 fraction were similar in these two strains, but the texture of the AS2 fraction derived from these two strains was markedly different from each other, suggesting that the detailed chemical structure of AG obtained from the O/E *agsA* differs from that of AG derived from the O/E *agsB* strains.

**Mannan molecular sub-structures control nanoscale glucan exposure in *Candida*.** Matthew S. Graus¹,², Michael J. Wester³, David L. Williams⁴, Michael D. Kruppa⁴, Douglas W. Lowman⁴,⁵, Jesse M. Young¹, Harry C. Pappas², Keith A. Lidke⁶, *Aaron K. Neumann*¹ ¹) Department of Pathology, University of New Mexico, Albuquerque, NM 87131; ²) Department of Nanoscience & Microsystems Engineering, University of New Mexico, Albuquerque, NM 87131; ³) Department of Mathematics and Statistics, University of New Mexico, Albuquerque, NM 87131; ⁴) Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37684; ⁵) AppRidge International, LLC, Telford, TN 37690; ⁶) Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM 87131.

N-linked mannans (N-mannans) in the cell wall of *Candida albicans* are thought to mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Lateral cell wall exposures of glucan on *C. albicans* are predominantly single receptor-ligand interaction sites and are restricted to nanoscale geometries. *Candida* species exhibit a range of basal glucan exposures and their mannan also vary in size and complexity at the molecular level. We used super resolution fluorescence imaging and a series of protein mannosylation mutants in *C. albicans* and *C. glabrata* to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in *C. albicans* and *C. glabrata*, respectively. Furthermore, a *C. albicans* clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and exhibited similar changes to nanoscopic glucan exposure geometry. We conclude that acid labile N-mannan controls glucan exposure geometry at the nanoscale. Furthermore, variations in glucan nanoexposure characteristics are clinically relevant and are likely to impact the nature of the pathogenic surface presented to innate immunocytes at dimensions relevant to receptor engagement, aggregation and signaling.

**Regulation of hyphal guidance by Pxl1 and Cst20 in *C. albicans*.** M.C. Almeida¹, E.M. Morrison¹, J. Craven², N.A.R. Gow¹, A.C. Brand¹ ¹) MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, Aberdeen, GB; ²) Department of Molecular Biology and Biotechnology, The Krebs Institute, University of Sheffield, Sheffield, UK.

*Candida albicans* lives as a commensal yeast in humans but can cause life-threatening bloodstream infections in susceptible patient groups. The formation of invasive hyphae is a characteristic virulence trait of *C. albicans* and we have shown that the penetration of host tissue depends on the ability of hyphae to steer as they grow. We are investigating the regulatory mechanisms of how hyphae sense guidance cues in the environment and translate these signals to re-orient the direction of growth. We have identified two types of 'steering-locked' mutants and the proteins associated with them. A wavy hyphal phenotype is generated by deletion of Pxl1, a Paxillin-like homologue, and its putative regulatory kinase, Ptk2 (homologue of Focal Adhesion Kinase). In contrast, truncation of Cst20 (Cst20<sup>1-549</sup>) or hyper-activation of the small GTPase, Rho1, cause hyphae to grow uni-directionally. Both the wavy and unidirectional phenotypes correlate with an inability to respond normally to directional cues. Pxl1-GFP localises to bud tip and bud neck in yeast and to the growing tip in hyphae, but not to the septa, suggesting that Pxl1 is involved in the transport or localisation of a specific subset of cell-wall remodelling proteins. RFP-Ptk2 localises throughout the cytoplasm. Although the wavy and unidirectional phenotypes are strikingly different, the Pxl1 and Cst20<sup>1-549</sup> strains exhibit other phenotypes in common. In yeast, TEM analysis showed both mutants have cell separation defects. In hyphae, they exhibit aberrant intracellular structures. These shared phenotypes suggest that Pxl1 and Cst20 are involved in the same pathway that governs cell growth. We hypothesise that Cst20, a homolog of PAK that negatively regulates Paxillin in humans, may have a similar function in *C. albicans*, suggesting that constant turnover of the Pxl1 complex is required for hyphal steering responses.

**Functional genomic analysis reveals fungal modulators of host immune responses.** Teresa O'Meara, Kwamaa Duah, Leah Cowen Molecular Genetics, University of Toronto, Toronto, Ontario, CA.

*Candida albicans* is a leading human fungal pathogen that causes life-threatening systemic infections. The first line of defense against this pathogen is the innate immune response. Macrophages readily engulf *C. albicans*, and the engulfed *C. albicans* cells subsequently undergo a morphogenetic switch from yeast to a filamentous growth. This change in morphology is temporally coupled to the induction of macrophage pyroptosis, an inflammatory host cell death program mediated via activation of NLRP3 and caspase-1. Recently, we discovered that it is the remodeling of the fungal cell wall in response to the macrophage environment that exposes the trigger of host cell death, not the filament itself. Further, we found that heat-killed previously phagocytized *C. albicans* cells could drive macrophage lysis, unlike their counterparts that had not been phagocytized prior to killing, suggesting that this fungal cell wall remodeling is sufficient to drive macrophage lysis, and that the remodeling occurs specifically in response to the macrophage phagosome. However, the specific trigger and mechanisms involved remain enigmatic.

To address this question, we developed a high-content imaging platform to monitor interactions between *C. albicans* and macrophages, and have leveraged this platform to identify *C. albicans* mutants that are defective in pyroptosis. Using murine macrophages expressing an ASC-fluorescent protein (FP) reporter, we quantified ASC-FP paranuclear specks, which are indicative of NLRP3 inflammasome activation. Our primary screen allows us to simultaneously quantify not only pyroptosis, but also *C. albicans* filamentation within macrophages. Genes required for filamentation in the macrophage cannot be predicted *a priori* given that distinct genes regulate filamentation in response to
distinct cues. Our initial analysis of 96 filamentation-competent conditional expression strains that enable transcriptional repression of cell wall genes identified 34 genes that are important for activation of pyroptosis. This included genes that encode glucan biogenesis enzymes, mannosyltransferases, and GPI-anchored proteins, and their potential regulators. Together, we have identified novel circuitry regulating fungal induction of host immune cell death.

**Capsule synthesis and regulation in the fungal pathogen *Cryptococcus neoformans*.** T.L. Doering¹, L.X. Li¹, S.R. Gish¹, E.J. Maier²,³, M.R. Brent²,³,⁴ 1) Department of Molecular Microbiology, Washington University Medical School, Saint Louis, MO; 2) Department of Computer Science & Engineering, Washington University, Saint Louis, MO; 3) Center for Genome Sciences, Washington University Medical School, Saint Louis, MO; 4) Department of Genetics, Washington University Medical School, Saint Louis, MO.

*Cryptococcus neoformans* is a pathogenic fungus that kills hundreds of thousands of people a year, mainly immunocompromised individuals in developing areas of the world. This yeast bears a protective polysaccharide capsule that is required for virulence; capsule polysaccharides are also shed from the cell and impair the host immune response to infection. Capsule thickness varies dramatically with environmental conditions, becoming significantly greater during infection. We are dissecting this regulation, with the dual goals of understanding how environmental signals are integrated to determine capsule size and defining the downstream capsule synthetic machinery.

We have developed computational approaches¹ to map the transcriptional regulatory network of *C. neoformans* and model interactions between transcription factors (TFs) and their target genes.²,³ We used these network maps to predict which TFs were mostly likely to impact capsule regulation and test our predictions by deleting the corresponding genes. The resulting mutants produced abnormal capsules, validating our predictions.³ Many also showed reduced virulence, in one case completely altering the course of the disease.³,⁴ We further used our network maps to examine the target genes of capsule-implicated TFs and understand how these regulators influence capsule thickness. We are continuing to probe capsule regulation and investigate the roles of the affected gene products, with particular interest in proteins involved in the glycan synthetic pathways that produce capsule.

1 Haynes, B. C. et al. Mapping functional transcription factor networks from gene expression data. *Genome Res* **23**, 1319-1328 (2013).

2 Haynes, B. C. et al. Toward an integrated model of capsule regulation in *Cryptococcus neoformans*. *PLoS Pathog* **7**, e1002411 (2011).

3 Maier, E. J. et al. Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation. *Genome Res* **25**, 690-700 (2015).

4 Gish, S. R. et al. Computational Analysis Reveals a Key Regulator of Cryptococcal Virulence and Determinant of Host Response. *mBio* **7** (2016).

**Biochemistry and Metabolism**

**1W A global co-expression network approach for connecting genes to specialized metabolic pathways.** Jennifer Wisecaver, Abigail Lind, Antonis Rokas Vanderbilt University, Nashville, TN.

Both fungi and plants produce a tremendous diversity of specialized metabolites (SMs) to interact with and manage their environment. A major challenge hindering efforts to tap this seemingly boundless source of pharmacopeia is the identification of SM pathways and their constituent genes. To address this challenge, much attention has been paid to fungal SM gene clusters, but the number of SM pathways in fungi that are non-clustered or only partially clustered is completely unknown. The genome of *Aspergillus fumigatus*, an opportunistic human pathogen and one of the leading causes of fungal-related human deaths, provides a stark example; 21 of its 37 SM gene clusters are missing either transporters, transcription factors or both, suggesting these genes may be located elsewhere in the genome. Given the well-established observation that the genes comprising a SM pathway are co-regulated in response to specific environmental conditions, we hypothesized that genes from a given SM pathway would form tight associations (modules) with each other in gene co-expression networks, facilitating their identification even without knowledge of the genome sequence. To evaluate this hypothesis, we first used 10 global co-expression datasets—each a meta-analysis of hundreds to thousands of expression experiments—across eight plant model organisms to identify hundreds of modules of co-expressed genes for each species. In support of our hypothesis, 15.3-52.6% of modules contained two or more known SM biosynthetic genes (e.g., cytochrome P450s, terpene synthases, and polyketide synthases), and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways in these species, including all those known to form SM gene clusters. For each clustered SM pathway, the module analysis expanded the known genetic repertoire of the pathway by identifying additional unclustered enzymatic genes as well as unclustered genes involved in pathway regulation and product transport. We are currently expanding our network analysis to fungal taxa including *Aspergillus* and submit that global gene co-expression is a rich, but largely untapped, data source for discovering the genetic basis and architecture of natural products.

**2T A novel integral membrane protein (Imp1) mediates TOR signaling in *Magnaporthe oryzae* and is required for deterministic (non-random) appressorium formation and biotrophic growth in rice cells.** G. Sun¹, R. Wilson¹,² 1) Department of Plant Pathology, University of Nebraska Lincoln, Lincoln, NE; 2) Redox Biology Center, University of Nebraska–Lincoln, Lincoln, Nebraska.

The devastating rice blast fungus *Magnaporthe oryzae* accesses rice plants by forming appressorial cells that disrupt the rice leave cuticle using internal turgor acting on a thin penetration peg. We recently reported that Target of Rapamycin (TOR), a conserved regulator of cell growth in response to nutrient availability, drives appressorium formation when inactivated on the nutrient-free leaf surface and, when activated, promotes biotrophic growth in rice cells following host penetration. We sought to identify and characterize additional components of this critical signaling pathway and here we report that a novel integral membrane protein, Imp1, mediates TOR signaling to ensure normal appressoria formation and in planta development by *M. oryzae*. We discovered *IMP1* by using Agrobacterium tumefaciens-mediated mutagenesis to select for rapamycin resistant *M. oryzae* strains. Following targeted deletion of *IMP1* by homologous gene recombination, we found that the resulting *Δimp1* mutant strain was impaired for appressorium formation. Appressorium formation in *Δimp1* deletants was stochastic such that some germinating spores of *Δimp1* produced appressoria, but most formed undifferentiated swellings at germ tube tips. These latter germinating spores demonstrated increased mitosis and loss of autophagy, consistent with constitutive TOR activation. Appressorium formation rates in *Δimp1* deletants were not remediated by rapamycin (or cAMP) treatment, suggesting Imp1 is required either for TOR inactivation or transduction of the inactive TOR signaling cascade. Those spores that formed appressoria could penetrate leaf cuticles but elaborated morphologically abnormal invasive hyphae that failed to conduct cell-to-cell movement in rice cells.
Imp1 localized to surface of vacuoles and is likely involved in autophagosome-vacuole fusion and vacuole function in response to TOR activity status. We thus propose that Imp1 is a downstream component of the TOR signaling pathway such that in response to TOR inactivation, Imp1 mediates autophagosome-vacuole fusion resulting in autophagy and, in germinating spores, mitotic arrest and deterministic appressorium development. Our results thus reveal new insights on both the mediation of TOR signaling and the molecular mechanisms underlying appressorium formation by the rice blast fungus *M. oryzae*.

**3F** A trehalose-regulatory subunit moonlights to regulate cell wall homeostasis through modulation of chitin synthase activity in *Aspergillus fumigatus*. Arsa Thammahong, Alayna Caffrey, Sourabh Dhingra, Josh Obar, Robert Cramer Microbiology and Immunology, Dartmouth College, Hanover, NH.

*Purpose:* Trehalose biosynthesis is a metabolic pathway found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for human and plant fungal pathogen virulence. Loss of canonical trehalose biosynthesis genes in the human pathogen *Aspergillus fumigatus* alters virulence and cell wall integrity through undefined mechanisms. Here we characterize additional genes, herein called *tsIA* and *tsIB*, which encode proteins that contain similar protein domains as *OrlA* (a trehalose-6-phosphate phosphatase), but lack critical catalytic residues for phosphatase activity.

*Methods:* We utilized a genetics approach to generate null mutants of *tsIA* and *tsIB*. To observe the phenotypes of these mutants, we used trehalose assays and cell wall perturbing agents. Furthermore, LC-MS/MS and co-immunoprecipitation were performed to define protein-protein interactions of *TsIA*. To further characterize the phenotype of *tsIA* null mutant, chitin synthase activity assay and spinning-disk confocal microscope were used to study the chitin content and the localization of CsmA. A chemotherapeutic murine model was utilized to study the host-pathogen interaction.

*Results:* Loss of *tsIA* reduced trehalose content in both conidia and mycelia, impaired cell wall integrity, and significantly altered cell wall structure. Unexpectedly, immunoprecipitation assays coupled with LC-MS/MS revealed a protein interaction between *TsIA* and *CsmA*, a type V chitin synthase enzyme. *TsIA* regulates not only chitin synthase activity but also *CsmA* localization. Loss of *TsIA* directly affected the host immune response to *A. fumigatus* characterized by an increase in murine mortality likely due to enhanced immune cell recruitment.

*Conclusion:* Our data provide a mechanistic model whereby proteins in the trehalose pathway play critical roles in fungal cell wall homeostasis that alters fungal-host interactions. Future studies are underway to elucidate the mechanism(s) through which *OrlA*, *TsIA*, *TsIB*, and their interacting partners control fungal cell wall homeostasis and virulence.

**4W Disruption of mgr2 gene in Aspergillus fumigatus alters redox balance, cell wall structure, spore germination and susceptibility to macrophage killing.** E.S. Santos¹, L.L.L. Balico¹, A.C. Silva², L.O. Sousa¹, S.A. Uyemura¹ 1) FCFRP - USP, Ribeirão Preto, BR; 2) FMRP - USP, Ribeirão Preto, BR.

*Aspergillus fumigatus* is a filamentous saprophytic fungus and a major pathogen in immunosuppressed patients. mgr2 (mitochondrial genome maintenance protein) gene encodes a protein of 108 amino acids whose functions have not yet been elucidated. In humans, ROMO1 (MGR2 orthologous) regulates ROS balance, proliferation, cell growth and death, and mitochondrial fission/fusion. In *S. cerevisiae*, MGR2 is part of TIM23 complex, a translocase of the mitochondrial inner membrane. The aim of this study was to understand MGR2 function in *A. fumigatus* by generating a knockout mutant (*Δmgr2*) and to phenotypically characterize the mutant in comparison to wild type strain (KU80) and to *Δmgr2* complemented with mgr2 gene (*Δmgr2::mgr2*'). Our results show that MGR2 deletion increases spore germination and ROS formation (analyzed by carbonylated protein assay), alters cell wall composition as observed by confocal microscopy after Congo Red and Calcofluor White staining, and alters sensitivity to enzymatic cell wall degradation. Interestingly, *Δmgr2* also showed a lower susceptibility to macrophage killing of conidia. New studies are necessary to further understand MGR2 function in *A. fumigatus*.

**5T Mechanism for Subcellular Compartmentalization of Melanization in Aspergillus fumigatus.** Srijana Upadhyay, Xinping Xu, Xiaorong Lin Dept Biol, Texas A&M University, College Station, TX.

Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously we discovered that *Aspergillus fumigatus* compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered "atypical" secretory proteins. Here, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, forming protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. This post-translational lipid modification correlates the endosomal localization of all early melanin enzymes. Furthermore, treatment with palmitoylation inhibitor not only drastically reduced the palmitoylation of the PKS enzyme *Alb1*, but also weakened its endosomal localization. Intriguingly, bioinformatic analyses predict palmitoylation a common mechanism for potential membrane association of other polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in *A. fumigatus*. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling while post-translational lipid modifications help recruit the "atypical" enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

**6F Copper-responsive isocyanide biosynthetic cluster in Aspergillus fumigatus.** F. Lim¹, J. Baccile², T. Won², P. Wiemann¹, A. Lind³, A. Rokas³, F. Schroeder², N. Keller¹ 1) University of Wisconsin-Madison, Madison, WI, U.S.A; 2) Cornell University, Ithaca, NY, U.S.A; 3) Vanderbilt University, Nashville, TN, U.S.A.

Isocyanide-containing natural products are of immense interest in the biological and chemical milieu owing to its broad range of bioactivity, which is attributed by the highly reactive isocyano- functional groups tethered to structurally diverse carbon scaffolds. In contrast to the fast-growing list of naturally-occurring isocyanides, knowledge on the biosynthetic machineries that give rise to such unique chemistry is still in its infancy with only four characterized biosynthetic gene clusters in bacteria, and unprecedented in eukaryotes. A hallmark function for these isocyanide natural products is their capability for metal coordination, which is shown to impart the bioactivites for many of these naturally-occurring isocyanides and from an ecological perspective, crucial to the pathogenesis of the entomopathogenic bacterium,
*Xenorhabdus nematophila* (by disabling the innate immune defense via inhibition of the cuproenzyme, phenoloxidase). Here we report on the identification of four isonitrile synthases (INS) in the genome of the human opportunistic pathogen, *Aspergillus fumigatus* and the discovery of a novel copper-responsive INS-NRPS-like hybrid enzyme (CrmA) involved in the synthesis of xanthocillin analogues, the first reported biosynthetic pathway dedicated to this family of naturally-occurring isocyanides. This work also elucidates the regulatory circuitry that bridges cellular metal homeostasis and fungal development.

**7W Biosynthesis of dihydrolysergic acid by heterologous expression of Claviceps spp. genes in Neosartorya fumigata.** Stephanie Arnold, Paige Bragg, *Daniel Panaccione* West Virginia University, Morgantown, WV.

Ergot alkaloids derived from lysergic acid (LA) and dihydrolysergic acid (DHLA) can be used to treat dementia, migraines, hyperprolactinemia, and other disorders. LA and DHLA are structurally similar fungal secondary metabolites, but they have different activities related to cognitive function and other clinical applications. LA has been produced by genetic modification of the model fungus *Neosartorya fumigata* (synonym *Aspergillus fumigatus*), but the biosynthetic pathway to DHLA has not been established. Previous studies showed that the enzyme CloA from the LA-producing fungus *Epichloë typhina* x *festucae* oxidized the substrate agroclavine to LA. We transformed *cloA* from *E. typhina* x *festucae* into a *N. fumigata* strain that accumulates festuclavine, a precursor to DHLA, but it failed to oxidize festuclavine to DHLA. We hypothesized that CloA from *Claviceps africana*, a DHLA-producing fungus, would oxidize festuclavine to DHLA. A genomic clone of *C. africana* cloA was not processed correctly in *N. fumigata*, so coding sequence only versions of *cloA* alleles from *C. africana* and closely related *C. gigantea* were synthesized and expressed in the festuclavine-accumulating mutant background. HPLC and mass spectrometry analyses demonstrated that transformants expressing CloA from either of these *Claviceps* spp. produced DHLA. Substrate feeding studies indicated interesting differences in specificity of CloA from LA producing and DHLA producing fungi. Our results show that production of DHLA requires specialized alleles of two ergot alkaloid pathway genes compared to those found in the LA pathway.

**8T Role of the urea cycle in the synthesis of nitric oxide.** *D. Canovas*¹,², A.T. Marcos¹, J.F. Marcos³, T. Schinko², J. Strauss² 1) Department of Genetics, University of Sevilla, Sevilla, Spain; 2) Division of Microbial Genetics and Pathogen Interactions, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Sciences Vienna, Austria; 3) Department of Food Science, Institute of Agrochemistry and Food Technology (IATA), Valencia, Spain.

Nitric oxide (NO), and its role in signalling, has been extensively studied in mammals and to some extent in plants. However, little is known about the role of NO in fungi and how it is synthesized in these organisms. Recently, we reported that NO production in *A. nidulans* is coupled to conidiation and requires a functional nitrate reductase (NR) gene (*niaD*) that is upregulated under these conditions even in the presence of the repressing nitrogen source ammonium. NO levels influence the balance between conidiation and sexual reproduction. Here we report that NO levels are also modulated by light, a general environmental cue and a regulator of fungal development. The light-dependent modulation of nitric oxide levels involves NO catabolism by the mitochondrial flavohemoglobin *fhbB*, and *agaA*, an arginase that controls the intracellular concentration of the NO precursor arginine. Addition of arginine to the cultures provokes a transient increase of the production of NO. However, analogues of arginine did not affect the production of NO. Mutants in the urea cycle genes show differences in NO levels compared to the wild type strain. Taken together our findings indicate that light-dependent developmental processes in *A. nidulans* interfere with nitric oxide metabolism which – in addition to nitrate reduction - is modulated by enzymes of the urea cycle.

**Reference:**

Marcos AT, Ramos MS, Marcos JF, Carmona L, Strauss J, Cánovas D. Nitric oxide synthesis by nitrate reductase is regulated during development in *Aspergillus*. Mol Microbiol (2016) 99:15-33.

**9F COP9 signalosome and Cand mediated Cullin-RING ligase control and Aspergillus nidulans development.** A. M. Koehler, G. H. Braus Molekulare Mikrobiologie und Genetik, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany.

Cullin RING ligases (CRLs) are the specificity factors which mark target substrates for degradation by the ubiquitin 26S proteasome system. CRLs are activated by linkage of the ubiquitin-like modifier NeddH/Nedd8 to a conserved lysine residue within the cullin core module of this enzyme complex. *Aspergillus nidulans* posseses approximately 70 F-box proteins as receptors for different substrates, which are linked through the adaptor SkpA to cullins. Fungal development requires the controlled degradation of various substrates and therefore exchanges of F-box proteins within CRLs. This exchange depends on the inactivation of CRLs by removing NeddH and subsequent reassembly. At the molecular level this process is catalysed by the two deneddylases COP9 signalosome and DenA [1, 2], and the Cand complex [3]. In fungi a seven-subunit pre-COP9 signalosome is first assembled and activated by the integration of the eighth CsnE NeddH-specific isopeptidase subunit [4]. Cand binds to deneddylated cullins at the SkpA binding and the neddylation site and its function is a prerequisite for CRL reassembly. Defects in COP9 or Cand subunits result in impaired fungal development and a dysregulated secondary metabolism. Cand represents a single polypeptide in numerous filamentous fungi and mammals, but is split in *A. nidulans* into at least two polypeptides (34kDa CandAN blocks neddylation site; 114 kDa CandAC blocks SkpA binding site), which form a cullin binding heterodimeric complex and are presumably the result of a genomic rearrangement event [3]. The gene locus of the two *A. nidulans* cand genes was analysed in more detail and revealed that the fungal Cand complex consists apparently of even three proteins, which all are required for fungal development and coordinated secondary metabolism. The interplay between Cand subunits and the two deneddylases as well as their molecular function and consequences for fungal differentiation are presented.

[1] Christmann M et al., 2013. PLoS Genet. 9, e1003275.

[2] Schinke J et al., 2016. PLoS Genetics 12, e1005949.

[3] Helmstaedt K et al., 2011. Mol Biol Cell. 22, 153-164.

[4] Beckmann EA et al., 2015. Mol Microbiol 97: 110-124.
**10W Growth-phase sterigmatocystin formation on lactose is mediated via low specific growth rates in Aspergillus nidulans. L.**

Karaffa<fnref n="1" />, Z. Németh<fnref n="1" />, Á.P. Molnár<fnref n="1" />, B. Fejes<fnref n="1" />, L. Novák<fnref n="2" />, N.P. Keller³,⁴, E. Fekete<fnref n="1" />

1) Department of Biochemical Engineering, University of Debrecen, Debrecen, HU; 2) Department of Physical Chemistry, Faculty of Science and Technology, University of Debrecen, HU; 3) Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, USA; 4) Department of Bacteriology, University of Wisconsin, Madison, USA.

Seed contamination with polyketide mycotoxins such as aflatoxin (AF) and sterigmatocystin (ST) produced by *Aspergillus spp.*, is an agricultural, economic, and medical issue worldwide. ST is the penultimate intermediate in the biosynthesis of AF, and in several fungi including the model fungus *A. nidulans*, it is the end product of the AF pathway. This biosynthetic pathway is well-characterized in *A. nidulans*, but many of the regulatory aspects related to the carbon source available for the fungus are still enigmatic. This is particularly true for the heterodisaccharide lactose (milk sugar; 1,4-O-beta-D-galactopyranosyl-D-glucose), inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates including the customary D-glucose. Here, kinetic data from well-controlled single-carbon substrate submerged fermentations revealed that on D-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when the majority of the carbon source is still available. Maximal biomass-specified ST production in lactose medium was significantly higher than on D-glucose. These data suggested that ST formation may either be mediated by a carbon catabolite regulatory mechanism prominent on D-glucose, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by constant-mass chemostat-type continuous fermentations on D-glucose as a sole carbon source at two different dilution rates (D = 0.090 h⁻<fnref n="1" /> and D = 0.020 h⁻<fnref n="1" />), representing a state of carbon catabolite repression and derepression, respectively. ST production under such conditions negatively correlated with the dilution rate, i.e., no ST formed at high growth rate, while low growth rate led to the formation of 0.4 mg L⁻<fnref n="1" /> ST. Essentially identical results were obtained with a CreA mutant strain, indicating that CreA does not regulate the formation of ST during growth on D-glucose. We concluded that low specific growth rates may be the primary cause of sustained, mid-growth ST formation on the slowly assimilating lactose in *A. nidulans*, and that carbon utilization rates likely play a general regulatory role during biosynthesis.

This research was supported by the EU and co-financed by the European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008.

**11T Discovery of novel aromatic processing enzymes from ferulic acid-tolerant *Aspergillus niger* evolution mutant.** *April J. M.*

Liwanag<fnref n="1" />,<fnref n="2" />, Adiphol Dilokpimol<fnref n="1" />,<fnref n="2" />, Isabelle Benoit-Gelber <fnref n="1" />,<fnref n="2" />, Ronald de Vries<fnref n="1" />,<fnref n="2" />

1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, NL; 2) Fungal Molecular Physiology, Utrecht University, Utrecht, NL.

The biological degradation of lignin generates a complex mixture of aromatic compounds in the environments, but the metabolic pathways by which fungi may utilize these are poorly understood. A putative aromatic pathway has been described in *Aspergillus japonicus*<fnref n="1" />.

However, most enzymes involved in aromatic conversions have not been identified, despite the potential in using such enzymes for the conversion of lignin-derived aromatic compounds.

*Aspergillus niger* is widely used for industrial applications (e.g., enzyme and metabolite production) and is a model organism for strain improvement. Benzoate-para-hydroxylase (BphA, EC 1.14.13.12.) is the only aromatic processing enzyme that has been characterized so far in *A. niger*, which converts hydroxybenzoate into protocatechuate<fnref n="2" />. To identify enzymes involved in the aromatic metabolism of fungi and strains capable of efficient aromatic conversion, we have developed an *A. niger* adaptive evolution mutant by growing *A. niger* N402 on incrementally increasing concentrations of ferulic acid. This mutant has a growth threshold on 0.1% ferulic acid, in the absence of any sugar source, and it tolerates a 10 fold-higher concentration of ferulic acid compared to the parental strain.

Transcriptomics was performed to investigate the difference in the expression level of genes between the mutant and parental strain grown on ferulic acid. The results revealed candidate genes encoding enzymes that are potentially involved in aromatic metabolism. The selected genes were then analyzed by qPCR and cloned for biochemical characterization of the corresponding enzymes.

**12F Analysis of aspirochlorine (ACL) productivity and ACL cluster sequence in Aspergillus oryzae strain.** *S. Ryota*<fnref n="1" />, *O. Tami*<fnref n="1" />,
*U. Miyuki*<fnref n="1" />, *O. Ken*<fnref n="1" />, *I. Kazuhiro*<fnref n="1" />,<fnref n="2" />

1) Nstional Research Institution of Brewing, Hiroshima, Hiroshima, Japan; 2) Graduate School of Advanced Science of Matter, Hiroshima, Hiroshima, Japan.

*Aspergillus oryzae* is GRAS (generally recognized as safe) and various type of strains are used in the industry. The *A. oryzae* genome sequence analysis conducted in 2005, revealed the presence of many conserved secondary metabolism clusters in *A. oryzae*. In these studies, a cluster similar to the gliotoxin synthesis cluster was identified in the *A. oryzae* genome. Recently, the product of this cluster was identified as aspirochlorine (ACL), and this cluster was designated as the ACL cluster. ACL is antibiotics A30641, which inhibits fungal protein synthesis and represses the growth of *Candida albicans*. Although, some *A. oryzae* strains are known to produce ACL, the ACL productivity in several *A. oryzae* strains remain unclear. In this study, we investigated the physiological and molecular biological features of ACL productivity among different *A. oryzae* strains. First, we investigated ACL productivity in 3 different media (WATM, CYA, YES) in agar plate condition and rice koji condition by using *A. oryzae* RIB40. We observed that the amount of ACL was different in each plate culture medium. However, in rice koji conditions, RIB40 did not produce ACL even after 8 days of cultivation. We then examined the ACL production of 13 *A. oryzae* strains, one from each of the 13 phylogenetic clusters of *A. oryzae*. We examined the ACL productivity of these strains in YES agar medium, as RIB40 showed the maximum ACL production; ACL productivities among these strains were considerably different. In particular, RIB301, RIB430 and RIB1172 did not produce ACL. Furthermore, we examined the ACL production by these strains in rice koji conditions and found that none of the strains produced ACL. This difference in ACL productivity likely be based on the difference between the ACL cluster genes sequences. Hence, we examined the genome sequence of ACL clusters in these 13 strains. The ACL cluster was found to be conserved in all the 13 strains and many mutations were found in each strain. Some strain-specific mutations were also observed; in particular, in the strains that did not produce ACL.

In this study, it is clear that ACL productivity is affected by nutrient conditions. Interestingly, in the rice koji condition, no strains produced

<footnote n="1">¹ O. Milstein *et al.* (1983) *Arch. Microbiol.*, 135, 147-154.</footnote>

<footnote n="2">² CC. Reddy & CS. Vaidyanathan. (1975) *Biochim. Biophys. Acta.*, 1, 46-57.</footnote>
ACL, and it supposed that Japanese sake does not contain ACL. Hence, we investigated the ACL concentration in Japanese sake. We
collected 11 types of sake from 20 prefectures, and did not find ACL.

**13W Characterization of small proteins secreted by lignolytic fungi.** N. Valette¹,², Eric Gelhaye¹,², Mélanie Morel-Rouhier¹,² 1) UMR 1136 Tree-Microbe interactions, Lorraine University, Vandoeuvre les Nancy, FR; 2) INRA, Interactions Arbres–Microorganismes, UMR1136, F-54280 Champenoux, France.

Saprophytic fungi play an important biological and ecological role in forest ecosystems. Indeed, thanks to many extracellular enzymes, they are able to degrade all wood components, a key step for carbon recycling. During this process, wood releases various molecules, called extractives, which can be toxic for fungal cells. This is the reason why these microorganisms have developed extensive detoxification systems. Interestingly, a transcriptomic analysis revealed that numerous up-regulated genes involved in extractive response in *Phanerochaete chrysosporium* are still of unknown function. Among them, one group called small secreted proteins (SSP) has been highlighted. A SSP from *P. chrysosporium* has been produced as a recombinant protein in *Escherichia coli* and purified for functional characterization. Our results show that this SSP has several interesting structural features. Indeed, it can form a kind of hydrogel and forms fibers depending on pH. Moreover, the protein is highly stable being insensitive to temperature, denaturating agents or reductants. Since it can interact with wood extractives, this protein can be involved in the process by which fungi protect themselves against toxic molecules.

**14T Powdery mildew conidiophore initiation but not spore fitness is correlated with host plant lipid precursor availability.** A. McRae*¹, J. Jaenisch*¹, M.Y. Lee¹, C. Hirai¹, L. Silva², T. Northen², M.C. Wildermuth¹ 1) Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA; 2) Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Walnut Creek, CA.**

Powdery mildews are widespread plant pathogens that infect agriculturally important crops. These obligate biotrophs depend on host
metabolism for nutrients to complete their life cycle. Powdery mildews grow epiphytically and develop specialized feeding structures called
haustoria in the epidermal cells of its host. These feeding structures assimilate nutrients to fuel the metabolically demanding process of
asexual reproduction. Asexual reproductive structures (conidiophores) consist of chains of newly formed spores which contain many lipid
bodies. Others previously demonstrated that lipid storage bodies in germinating spores decrease over time, suggesting these energy rich
molecules are consumed to support development prepenetration in *Blumeria graminis* f sp hordei (Bgh). cDNA microarray analysis
of Bgh showed lipid degradation pathways are upregulated prepenetration, consistent with this function (Plant Cell 17:2107-
22). *Golovinomyces orontii* MGH1 infects the model plant, *Arabidopsis thaliana*. Infection site transcriptomics revealed that select metabolic
genes/pathways are up regulated at 5 days post inoculation (dpi), concurrent with the production of asexual reproductive structures. This
includes genes involved in the pyruvate dehydrogenase (PDH) bypass. The PDH bypass can facilitate increased production of Acetyl-CoA,
which can be utilized as precursors for lipid biosynthesis. ¹³C-ethanol feeding to detached infected leaves to provide labelled lipid precursor
via the host PDH bypass showed isotopic enrichment in plant lipids and fungal spore lipids. Furthermore, powdery mildew colonies on plant
mutants in PDH bypass genes have reduced numbers of conidiophores 5 dpi. However, the number of newly formed spores per
conidiophore remains unchanged. The lipid content of spores was assessed using flow cytometry with Nile Red and BODIPY 505/515 lipid
stains. Flow cytometry analysis found the spores from powdery mildew grown on these plants versus wild-type plants have the same lipid
content. Furthermore, these two sets of spores also exhibit similar germination rates. Taken together, this suggests that not only does the
induced host PDH bypass provide lipid precursors for fungal spore formation, but that a PDH bypass-derived lipid precursor acts as signal
to regulate conidiophore formation while maintaining the fitness of those spores that are produced.

*presenting authors

**15F Powdery Mildew Manipulation of Plant Host Metabolism Fuels Asexual Reproduction.** Mi Yeon Lee¹, Chihiro Hirai¹, Johan Jaenisch¹, Amanda McRae¹, Leslie Silva², Trent Northen², *Mary Wildermuth*¹ 1) University of California, Berkeley, CA; 2) Lawrence Berkeley National Laboratory, Walnut Creek, CA.

Powdery mildew fungi are obligate biotrophs that alter plant cellular architecture and metabolism to acquire their nutrients from the plant, while limiting plant defense. Using site-specific analyses, we found that the powdery mildew *Golovinomyces orontii* induces endoreduplication in plant mesophyll cells underlying the fungal feeding structure concomitant with fungal proliferation (PNAS 107:460-5). Multiple rounds of DNA replication without mitosis result in enhanced cell ploidy with ~16-fold elevated DNA content. Increased endoploidy has long been associated with enhanced metabolism, but the detailed mechanisms underlying this relationship are not well understood. We identified a conserved eukaryotic transcription factor and plant-specific factors that control the extent of induced ploidy and found the extent of fungal proliferation correlates with DNA ploidy levels (MPMI 26:537-45). This suggests ploidy-dependent enhanced host metabolism is needed to support the metabolically demanding proliferation phase of the powdery mildew life cycle. Using transcriptome, genetic, and biochemical analyses we have identified specific ploidy-dependent alterations in Arabidopsis primary metabolism that are utilized by the powdery mildew to fuel its asexual reproduction. Specifically, use of the plant pyruvate dehydrogenase (PDH) bypass which can increase Acetyl-CoA and precursors for lipid synthesis is correlated with the number of fungal asexual reproductive structures. However, the number of new spores per reproductive structure remains unchanged. Powdery mildew spores contain lipid bodies which appear to be utilized to fuel germination and pre-penetration events (Plant Cell 17:2107). 13C-ethanol feeding to detached infected leaves to provide labelled lipid precursor via the plant PDH bypass showed isotopic enrichment in plant lipids and fungal spore lipids. FACS analysis of lipid stained spores from powdery mildew grown on PDH bypass mutant plants versus wild-type plants show they have similar lipid content. Furthermore, these two sets of spores also exhibit similar germination rates. Taken together, this suggests that not only does the induced ploidy-associated use of the host PDH bypass provide lipid precursors for fungal spore formation, but that a PDH bypass-derived lipid precursor acts as signal to regulate conidiophore formation while maintaining the fitness of spores that are produced.

**16W Resistance to anilinopyrimidine fungicides in field populations of *Botrytis cinerea* is caused by independent mutations in two nuclear-encoded mitochondrial proteins.** A. Mosbach¹, D. Edel¹, S. Widdison², A. Farmer³, R. Dietrich⁴, A. Corran², G. Scalliet¹ 1) Syngenta Crop Protection AG, 4332 Stein, Switzerland; 2) Syngenta Jealott's Hill Int. Research Cen, Bracknell RG42 6EY, UK; 3) National Center for Genome Resources, Santa Fe NM 87505, USA; 4) Syngenta Crop Protection LLC, Research Triangle Park, USA.

Anilinopyrimidine (AP) fungicides were first introduced in Europe more than 20 years ago and still display a good efficacy for the control of
a range of ascomycetes in various crops worldwide. Although resistant field isolates occurring at low to moderate frequency in most of these pathogens are known for many years, the underlying resistance mechanisms have not been deciphered.
The grey mold *Botrytis cinerea* is a high risk pathogen for the development of fungicide resistance, and isolates showing reduced sensitivity to APs are frequently found in populations from grapes, strawberries and other crops. To characterize the resistance mechanisms in *B. cinerea*, two complementary approaches were undertaken: The induction of resistance-conferring mutations by *in vitro* random mutagenesis followed by next generation sequencing, and the characterization of mapping populations from crosses between resistant field isolates and reference strains. These approaches led to the identification of nine different nuclear genes conferring resistance, all coding for proteins associated with the mitochondria. Such diversity of possible fungicide resistance mechanisms is so far unique.
Some of these genes have been reported to play roles in mitochondrial protein maturation and quality control, or the defense against oxidative damage. One of the nine genes was found both *in vitro* and in the field, another one exclusively in nature. Based on our monitoring, these two genes account for all of the resistance observed in the field. By targeted gene knock-out we could demonstrate that some of the resistance-conferring mutations most likely lead to a partial loss of function of the encoded proteins, as the deletion mutants displayed increased tolerance to cyprodinil. These results, in addition to AP treatment-related metabolomics and transcriptomics information enable us to progress our understanding of the mode of action for this class of fungicides.

**17T The fungal cell wall as an antifungal drug target.** Carol Munro, Louise Walker, Sami Alawfi, Giuseppe Buda de Cesare, Chibuike Ibe MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, GB.

The cell wall of the pathogenic yeast *Candida albicans* is a dynamic organelle, primarily composed of chitin, β-1,3-glucan, β-1,6-glucan and mannoproteins. The polysaccharides give the wall its mechanical strength and the mannoproteins include important virulence factors as well as carbohydrate active enzymes that contribute to maintaining cellular integrity. The majority of cell wall components are fungal-specific and attractive targets for the development of much needed therapies. This potential has already been realised by the development of the echinocandins; antifungal drugs that target the cell wall by inhibiting β-1,3-glucan synthesis. The echinocandins generally provide effective therapy but sporadic resistant *Candida* isolates have emerged through acquisition of point mutations in the *FKS* target genes. *C. albicans*, and other fungal species, respond to sub-MIC echinocandins by up-regulating chitin biosynthesis and modulating the cell wall glycoproteome. The changes are brought about by activation of cell wall salvage mechanisms that involve PKC and calcium/calcineurin signalling. Modifications to the cell wall such as elevated chitin reduce susceptibility to echinocandins *in vitro*, as well as in infection models. Several predicted carbohydrate-active (CAZy) enzymes involved in modulating and cross-linking chitin and glucan (Phr1, Phr2, Pga4, Crh11, Utr2) are among the cell wall proteins (CWPs) positively regulated in response to cell wall damage. Overexpression or deletion of specific CWP genes results in altered caspofungin susceptibility, paradoxical growth and influences host-pathogen interactions. Changes in environmental conditions and the application of cell envelope stresses can substantially change the cell wall, which should be seen as a moving target.

**18F Managing Plant Diseases Through Disruption of Fungal Nicotinamide Adenine Dinucleotide Cycle.** Shay Covo, Daniel Waiger, Vinay Bary, Nuria Vital Plant pathology and Microbiology, Hebrew University, Rehovot, IL.

Nicotinamide adenine dinucleotide (NAD) is a common and important metabolite in eukaryotic cells. NAD is crucial for proper redox balance and it is a cofactor of DNA repair enzymes and histone deacetylases from the sirtuins superfamily. During histone deacetylation, sirtuins convert NAD to nicotinamide (NAM). It was previously shown that NAM can reduce fungal growth in mice infected with *Candida albicans* due to inhibition of the ascomycete-specific sirtuin HST4. Here we tested the ability of NAM and other compounds in the NAD pathway to restrict the growth of fungal plant pathogens. NAM was able to inhibit both hyphal growth and conidial germination of *Fusarium oxysporum* and *Botrytis cinerea* in vitro (IC50 ~ 4 mM). NAM was able to reduce significantly fungal growth on slices of tomatoes. We are looking for ways to reduce the effective dose of NAM to manage plant disease. One direction is to inhibit the enzyme that further metabolites NAM in the cell. PNC1 is a fungal-specific nictinamidase that was shown to be inhibited in vitro by nicotinaldehyde (NA). We were able to show that NA inhibits the growth of *Fusarium* and *Botrytis* using even lower concentrations than NAM. Surprisingly, as determined by RNAseq the mode of action of NAM and NA is completely different. Genes that are upregulated due to NAM exposure are mainly transcription factors as expected from histone deacetylase inhibitor. Some of the GO terms enriched for genes that are downregulated by NAM belong to the redox balance. In contrast, genes that are up-regulated by NA belong to the redox- balance. These results suggested to us that while NAM inhibits chromatin biology NA disrupts NAD biosynthesis. This is in agreement of the role of PNC1 in NAD salvage biosynthesis pathway. Indeed we were able to show that NA toxicity can be bypassed by addition of niacin the next compound in NAD biosynthesis cycle. Our preliminary results show that NA indeed reduces NAD cellular concentrations. Finally, we were able to show that NA is synthetic lethal with Boscalid a SDHI. We propose that by developing better chemistry to inhibit PNC1 and HST4 a novel class of fungicides that have a broad effect on cellular functions will be obtained.

**19W Elucidating the structural basis of effector induced susceptibility in the *Parastagonospora nodorum* - wheat interaction.** M.A Outram¹, X Zhao¹, S Breen², P.S Solomon², B Kobe¹, S.J Williams¹,² 1) School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AU; 2) Research School of Biology, Australian National University, Canberra, ACT, AU.

*Parastagonospora nodorum*, the causal agent of Septoria Nodorum Blotch, is a major necrotrophic fungal pathogen of wheat worldwide. *P. nodorum* secretes small cysteine-rich proteinaceous effectors (ToxA, Tox1 and Tox3) that interact with corresponding dominant host sensitivity gene products, promoting cell death and rendering the plant susceptible to disease. Whilst it is now understood that these effectors are required for disease, there is little understanding of their function. Recently Tox3 from *P. nodorum* has been shown by yeast two-hybrid and co-immunoprecipitation assays to interact with basic and acidic wheat pathogenesis-related 1 (PR1) proteins. We have developed a protein expression and purification system that produces Tox3 and wheat PR1 proteins and the recombinant Tox3 protein was determined to be functional based on wheat infiltration experiments. We present here the first crystal structure of an acidic wheat PR1 protein and have obtained crystals of Tox3. These data provide a platform to understand the molecular details of the Tox3 and wheat PR1 interaction.
**20T Biosynthesis and detoxification of trichosetin is regulated by two cluster-specific transcription factors in Fusarium fujikuroi.** S. Janevska¹, B. Arndt², L. Baumann¹, L. Apken¹, H.-U. Humpf², B. Tudzynski¹ 1) Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, DE; 2) Institute of Food Chemistry, University of Muenster, Muenster, DE.

The PKS-NRPS-derived tetramic acid equisetin and its *N*-desmethyl derivative trichosetin exhibit significant toxic properties against a variety of organisms, including plants and bacteria (methicillin-resistant *Staphylococcus aureus*). The first equisetin biosynthetic gene cluster was described for *Fusarium heterosporum* (Kakule *et al.*, 2013), a species distantly related to the notorious rice pathogen *Fusarium fujikuroi*.

Here we present the identification and activation of a homologous but silent gene cluster in *F. fujikuroi*. Bioinformatic analysis revealed that this cluster does not harbor the equisetin *N*-methyltransferase gene *eqxD* and accordingly, trichosetin was isolated as final product. Indeed, heterologous expression of *F. heterosporum* *eqxD* in a trichosetin-producing *F. fujikuroi* strain resulted in the accumulation of equisetin. Constitutive overexpression of one of the two cluster-specific transcription factor (TF) genes, designated *TF22*, led to induced expression of the three biosynthetic cluster genes, including the PKS-NRPS key gene. Accumulation of trichosetin strongly inhibited growth of the producing fungus. Therefore, we adapted the inducible bacterial-fungal hybrid promoter system Tet-on (Meyer *et al.*, 2011) for *F. fujikuroi*, to achieve a controlled overproduction of trichosetin and a functional characterization of each cluster gene. In contrast, overexpression of the second cluster-specific TF gene, designated *TF23*, did not activate the expression of the other cluster genes. Instead, *TF23* was induced by the final product trichosetin itself via a yet unknown mechanism. TF23 in turn was shown to be essential for expression of the cluster gene *MFS-T*, encoding a transporter of the major facilitator superfamily. Our evidence suggests that TF23 and MFS-T contribute to detoxification of trichosetin and thus, self-protection of the producing fungus.

Kakule TB *et al.* (2013) ACS Chem Biol 8: 1549–57

Meyer V *et al.* (2011) Appl Environ Microbiol 77: 2975-83

**21F Cell wall biosynthesis in Fusarium graminearum.** Rohan Lowe¹, Neil Shirley², Alan Little², Mark Bleackley¹, Vincent Bulone², Marilyn Anderson¹ 1) Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia; 2) ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, SA, Australia.

Fungal cell walls contain several polysaccharides that are not encountered in plants and/or animals and thus represent an ideal target for control of fungal diseases. However, a better understanding of cell wall structure and biosynthesis in pathogenic fungi is needed for the rational design of new efficient cell wall inhibitors. Fungal cell walls have been understudied during the past decades due to the general assumption that they are generally homogeneous in structure throughout the kingdom Fungi. Instead, the fungal cell wall is a highly dynamic structure whose composition constantly changes as a response to the environment and developmental cues. Using the cereal pathogen Fusarium graminearum as our model fungus, genes encoding carbohydrate active enzymes (CAZy) were tracked by RNA-Seq during growth in a corn stalk rot time course and in vitro. In total, 516 CAZy genes were identified in the Fusarium genome and 494 were detected as expressed in either in planta or in vitro. Twelve of these genes were identified as potentially encoding chitin synthases. Eight were expressed both in vitro and in the corn stalk, with differential expression levels observed as infection progressed. An analytical carbohydrate analysis was performed to determine the basal *F. graminearum* cell wall composition during in vitro growth. In addition to major wall components, subtle structural features in cell walls can play an essential role in wall stability and be targeted by specific inhibitors for disease control. Future work will aim to deploy specific defensin plant innate immunity proteins to disrupt the biosynthesis of fungal cell wall carbohydrates, such as chitin.

**22W Brown rot fungi do the 'two-step' to deploy an ephemeral reactive oxygen species (ROS) pretreatment in wood.** J. Schilling¹, J. Zhang¹, G. Presley¹, C. Hunt², K. Hammel²,³, E. Panisko⁴, M. Figueroa¹ 1) University of Minnesota, Saint Paul, MN; 2) U.S. Forest Products Laboratory, Madison, WI; 3) Unviersity of Wisconsin, Madison, WI; 4) Pacific Northwest National Laboratory, Richland, WA.

We recently showed that brown rot wood-degrading fungi differentially express genes for lignocellulose oxidation (LOX) ahead of those associated with traditional cell wall hydrolysis, including glycoside hydrolases (GHs). This window of oxidative upregulation was very brief when colonizing wood (<48 hrs). Here, I will present the thin wafer system being used to map these fine-scale temporal dynamics, and share the results of two studies harnessing this set-up.

For the first study, we have been able to address the question 'What cues the gene expression transition from oxidative to hydrolytic during brown rot?' To do this, we have matched one effort to quantify soluble wood carbon fractions, spatially, as the model brown rot fungus colonizes and degrades wood with another effort to test carbon source regulation of gene expression. This paired effort, using qPCR to target genes that we previously showed via whole transcriptome profiling are differentially-expressed, indicates that key soluble sugars play a dual role in repression of LOX and simultaneous upregulation of GHs. Notably, cellulase production in brown rot fungi that has long been characterized as 'constitutive' given the lack of glucose repression we demonstrated instead is inducible but not repressed, later. This has been overlooked given the brief window prior to induction of GHs, lost in the noise of whole-block decay trials. Concerning the transition, coordinating regulation is a logical approach to segregating pretreatment from saccharification, given that ROS generated from oxidative pathways will denature enzymes. This also offers insight into the control of this unique mechanism, one that consolidates harsh oxidative pretreatments with enzymatic hydrolysis without requiring separate 'reactor' volumes.

Second, we have begun to address the question 'How representative is the staggered two-step mechanism in *P. placenta* of other brown rot fungi, and how does it compare with its white rot ancestors?' To do this, we have use whole transcriptome sequencing (RNAseq) to overlay wafer data among two brown rot species (*P. placenta* one of them) from distinct clades and two white rot species. Data has revealed surprisingly more similarities in mechanisms than expected, but some key distinctions in the analyses that are ongoing. These similarities and distinctions will help target genes unique to the brown rot mechanism as well as those common among these important carbon-cycling decomposers.
**23T Mapping of defective in silencing (dis) mutants in Fusarium graminearum by bulk segregant analysis and high-throughput sequencing.** Kristina Smith, Lanelle Connolly, Xiao Lan Chang, Brett Pierce, Corinne Fargo, Brian Josephson, Zackary Bango, Madison Esposito, Michael Freitag Dept. of Biochem & Biophysics, Oregon State University, Corvallis, OR.

Polycomb Group (PcG) proteins generate facultative heterochromatin in some fungi by trimethylating histone H3 lysine 27 (H3K27me3). Members of the conserved Polycomb Repressive Complex 2 (PRC2) include the H3K27 methyltransferase, KMT6, and binding partners SUZ12, EED, and CAF1-3 (MSL1). Loss of *kmt6*, *eed*, or *suz12* leads to complete loss of H3K27me3 accompanied by developmental defects and novel or increased expression of ~25% of all genes. Most genes silenced by PcG have unknown functions but are predicted to be involved in secondary metabolism and pathogenicity. To uncover suppressors of H3K27me3 silencing, and identify functional equivalents of PRC1, the complex that may initiate PRC2 action and also binds H3K27me3 in animals but does not exist in fungi, we developed a forward genetics approach utilizing UV mutagenesis. We inserted a *neo* reporter gene into the *pks2* region, which is reliably silenced by H3K27me3 in WT but de-repressed in the *kmt6* mutant. WT strains with *neo* at the heterochromatic locus were UV-irradiated, and mutants were selected on G418-containing medium. Dozens of primary mutants, which we call *defective in silencing (dis)*, fall into several classes with distinct growth phenotypes and global gene expression patterns evidenced by transcriptome sequencing. Bulk segregants from *dis* mutants backcrossed to mapping strains with *mat* deletions to force outcrossing were sequenced and analyzed with the Genome Analysis Tool Kit to identify SNPs, insertions and deletions. The first mutant analyzed, *dis1*, carries a truncated *eed* gene, and completely phenocopies the deletion mutant we constructed. Here we will report on additional components of the PcG silencing system.

**24F Roles of polyamine metabolism in appressorium function.** Raquel Rocha, Richard Wilson Plant Pathology, University of Nebraska - Lincoln, Lincoln, NE.

Polyamines are a group of ubiquitous and essential metabolites found in all living organisms. In fungi, the most abundant molecules - putrescine, spermidine, and spermine - have roles in conidiation and spore germination in *Aspergillus*, *Mucor*, and *Phycomyces* species. Little is known about how polyamine metabolism affects the development of fungal infection structures. Here, we report that a spermine synthase - encoded by *SMT1* - is required by the rice blast fungus *Magnaporthe oryzae* to generate spermine from spermidine and is essential for developing functional infection cells (appressoria) on rice leaf surfaces. *M. oryzae*, the most important pathogen of cultivated rice, elaborates appressoria at the tips of germ tubes emerging from spores on the leaf surface. The melanin-rich wall of the appressorium allows solutes such as glycerol to accumulate and generate enormous internal turgor pressure. This pressure is translated into mechanical force acting on the penetration peg emerging from a pore at the base of the appressorium, forcing it through the plant cuticle. In contrast, incipient cytorrhysis experiments determined that the appressoria of *Δsmt1* mutant strains generated turgor pressures that were 1.8 higher than wild type (WT), but no penetration pegs were observed breaching rice leaf cuticles by confocal microscopy. These developmental defects were not due to misregulated lipid mobilization or cell cycle progression, which occurred like WT in the *Dsmt1* mutant strains. Rather, by calculating the ratio of appressoria plasmolysis to cytorrhysis following PEG treatment, we determined that appressorial cell wall porosity was increased 3.5 in *Δsmt1* mutant strains compared to WT. We conclude that polyamine metabolism in *M. oryzae* appressoria, like in *Arabidopsis*, might control cell wall porosity. Perturbing porosity in *Δsmt1* appressoria could affect turgor and/or the rigidity of the cell wall. In turn, these structural defects might affect the polarized growth of the penetration peg from the appressorial pore. Ongoing studies of *Dsmt1* will, compared to WT, examine the structure of the appressorial cell wall, examine the pore at the base of the appressorium, and determine the status of penetration peg formation. Taken together, our results reveal novel roles for polyamine metabolism in appressorium function that enrich our understanding of the metabolic pathways intrinsic to rice cell colonization by *M. oryzae*.

**25W Production and composition characteristics of *Tremella fuciformis* exopolysaccharide.** Ji Young Kang¹, Jeesun Chun²,
Baek Rock Oh¹ 1) Industrial Microbiology and Bioprocess Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup-si, 580-185, Republic of Korea; 2) Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea.

An edible mushroom *Tremella fuciformis* is known to produce exopolysaccharides. Yeast-like cells (blastospore) were isolated from the fruiting body of the mushroom, and designated to the strain *T. fuciformis* TFC6. The exopolysaccharides of the strain are most highly produced in submerged culture at 4 culture day (2g/l). Moreover, TFCUV-5 obtained by UV mutagenesis to increase the exopolysaccharide productivity shown that the exopolysaccharide production reached 3.6g/l in flask culture which is an increase of 80% compared with the corresponding values of wild type TFC6. The optimum culture condition for maximum exopolysaccharide productivity was 25°C and pH7. The exopolysaccharides derived by the strain TFCUV-5 contained sucrose, glucose, xylose, mannose and glucuronic acid about 17.1%, 1.3%, 22%, 40.3% and 19.3%, respectively, so called a glucuronoxylomannan. The mutant enhanced the exopolysaccharide production might be applicable to several filed such as cosmetic and dietary supplements industries.

**26T Expression data integration in an *Aspergillus niger* genome-scale metabolic model.** M. V. Aguilar-Pontes¹, E. McDonnell², K. Strasser², D. Fulton², A. Tsang², R. P. de Vries¹ 1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Utrecht University, Utrecht, NL; 2) CSFG-Concordia University, Montreal, Canada.

Filamentous fungi include important species used in industrial applications. One of the main representatives is *Aspergillus niger*, an industrial workhorse used for enzyme and metabolite production. In order for *A. niger* to achieve its full potential as a cell factory, deeper knowledge of its metabolism is needed. We propose a new metabolic network based on a manually curated annotation of the *Aspergillus niger* genome, including literature references, as well as information from curated and automated protein, metabolite and reaction databases and RNA-seq data. Reconstruction of the model and manual curation of the network based on different expression data sets was performed using Pathway Tools. In order to assess and improve our model, a comparison with previously published genome scale models was performed (Andersen et al., 2008; Lu et al., 2016). Experimental results will be used to validate predictions of essential genes involved in growth as well as mutant behavior.

Our aim is to create a model that will give us new insights into the carbon metabolic pathways of *A. niger* and obtain leads to improve industrial processes. This model will also enable us and other researchers to study carbon utilization by fungi in more detail.