MiSeq 16S rDNA amplicon data demonstrated that a single MRE population thrives within each strain. We were successful in "curing" the fungal culture of its endosymbionts and designed experiments to assess impacts of these bacteria on their fungal host. We report on the morphology and phenotypic changes in fungal host biomass and growth when inhabited by MRE. Genome analysis and metatranscriptome data from MRE and their fungal host are expected to provide novel insights on the ecological and evolutionary role of these enigmatic microbes.

**560T Extremotolerant fungi: survivability via symbiosis.** *E. Carr*¹, S. Harris² 1) School of Biological Sciences, University of Nebraska Lincoln, Lincoln, NE; 2) Dept. of Plant pathology, Plant Science Innovation, University of Nebraska Lincoln, Lincoln, NE.

The black yeasts are a group of fungi from the subdivision Pezizomycotina that are known for their extremotolerance. Some of the conditions they can withstand are heat, freezing, desiccation, osmolarity, UV, heavy metals, and in some cases radiation. Unique features of these organisms make them intriguing to study alone, but may also provide insight into the phylogenetically related lichen-forming fungi. Since lichens and these polyextremotolerant fungi share similar resistance features, the black yeasts may enable study of the lichen lifestyle without the challenges of directly working with lichens. In addition to sharing similar traits, black yeasts are also almost always found in the same location as algae, even in desert and polar ecosystems. This raises the intriguing possibility that these organisms interact in a lichen-like manner. We propose that black yeasts and algae indeed interact in a mutualistic fashion that resembles lichens, and that observing these interactions will reveal clues about the nature of the fungal-algal communication that underlies lichen formation. To test our hypothesis we have isolated black yeasts and algae from biological soil crusts and rock surfaces, identified isolates using their ITS sequences, subjected isolates to multiple stressors and nutritional conditions to investigate their phenotypic diversity, and co-cultured the fungi and algae together in a pair-wise manner. Isolates that were taken from a biological soil crust in one location resulted in 24 black yeasts and 43 algal isolates. These have been extensively phenotyped for carbon utilization, nitrogen utilization, temperature resistance, UV resistance, and metal resistance allowing us to observe a moderate range of diversity. Current efforts are underway to integrate the phylogeny and phenotype data, as well as co-culturing the black yeasts with the algal isolates to observe symbiotic tendencies and any phenotypes associated with symbiosis.

**561F SymB and SymC, two membrane associated proteins, are required for Epichloë festucae hyphal cell-cell fusion and maintenance of a mutualistic interaction with Lolium perenne.** K.A. Green¹,², Y Becker¹,³, A Tanaka⁴, D Takemoto⁴, H.L. Fitzsimons¹, S Seiler⁵, H Lalucque⁶, P Silar⁶, B Scott¹,² 1) Institute of Fundamental Science, Massey University, Palmerston North, Manawatu, New Zealand; 2) Bioprotection Research Centre, Massey University, Palmerston North, New Zealand; 3) Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren, Germany; 4) Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan; 5) Freiburg Institute for Advanced Studies, Albert-Ludwigs Universität Freiburg, Freiburg, Germany; 6) Univ Paris Diderot, Sorbonne Paris Cité, Laboratoire des Energies de Demain, Paris, France.

*Epichloë festucae* is a filamentous fungus that forms a mutually beneficial symbiotic association with *Lolium perenne*. The NADPH oxidase complex components *noxA*, *noxR* and *racA*, the transcription factor *proA*, and the cell wall integrity (CWI) MAP kinases, *mkkA* and *mpkA*, are required for mutualistic *E. festucae*-L. *perenne* associations and cell-cell fusion. Homologues of these genes in *Neurospora crassa*, *Sordaria macrospora* and *Podospora anserina* are required for cell-cell fusion and sexual fruiting body maturation. We examined whether *symB* and *symC*, the *E. festucae* homologs of *Podospora anserina* self-signaling genes *IDC2* and *IDC3*, are required for *E. festucae* hyphal fusion and host symbiosis. Deletion mutants of these genes were defective in hyphal cell fusion, formed intra-hyphal hyphae, and had enhanced conidiation. SymB-GFP and SymC-mRFP1 localize to plasma membrane, septa and points of hyphal cell fusion. Plants infected with *ΔsymB* and *ΔsymC* strains were severely stunted. Hyphae of the mutants colonized vascular bundles, were more abundant than wild type in the intercellular spaces and formed intra-hyphal hyphae. Although these phenotypes are identical to those previously observed for cell wall integrity MAP kinase mutants no difference was observed in the basal level of MpkA phosphorylation or its cellular localization in the mutant backgrounds. Both genes contain binding sites for the transcription factor ProA. Collectively these results show that SymB and SymC are key components of a conserved signaling network for *E. festucae* to maintain a mutualistic symbiotic interaction within *L. perenne*.

**562W A novel bacterial endosymbiont related to Mycoavidus cysteinexigens inhabits the mycelium of Mortierella minutissima.** *Gregory Bonito*¹, Alessandro Desiro¹, Jessie Uehling², Natalie Vande Pol¹, Kurt LaButti³, Stephen Mondo³, Kerrie Barry³, Igor Grigoriev³ 1) Michigan State University, East Lansing, USA; 2) Duke University, Durham NC, USA; 3) Joint Genome Institute, Walnut Creek CA, USA.

*Mortierella* are an early diverging genera of soil and plant associated fungi that are known to harbor bacterial endosymbionts. The genome of one of these symbiotic microbes, a *Burkholderia*-related bacterium named *Mycoavidus cysteinexigens*, was recently sequenced from two different *Mortierella elongata* strains isolated in Japan and North America. Here we report on another *Mycoavidus* species that we detected within *Mortierella minutissima*. We sequenced both the fungal host *M. minutissima* and endosymbiont genome with PacBio. The 2.9 MB genome of this bacterium includes 2682 protein coding sequences and is the largest yet for a bacterium belonging to the fungal-associated Glomeribacter-lineage. The genome of this bacterium appears to have type II and type IV secretion systems and many phage capsule and tail protein coding genes. Phylogenetic analyses place this bacterium as sister species to *Mycoavidus cysteinexigens*, within the /glomerobacter lineage. Fungal isolates "cured" of their endobacteria show a difference in fitness when grown under various culture conditions. Transmission studies will be carried out to determine whether bacteria can be acquired horizontally between *Mortierella* species.

**563T The role of SreA-mediated iron regulation in maintaining endophyte-grass symbioses.** Natasha Forester¹, Paul Maclean¹, Geoffrey Lane¹, Iain Lamont², Linda Johnson¹ 1) Forage Improvement, AgResearch Limited, Palmerston North, Manawatu, NZ; 2) University of Otago, Dunedin, New Zealand.

In ascomycetes and basidiomycetes, an iron-responsive GATA-type transcriptional repressor is involved in regulating iron homeostasis. Functional characterisation of an orthologue, SreA, from *Epichloë festucae*, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is critical for symbiotic maintenance. The deletion of sreA (*ΔsreA*) led to the gradual loss of the endophyte from host plants (perennial ryegrass) and confocal microscopy studies identified anomalies in hyphal growth *in planta*, including
misshapen hyphae particularly at branching junctions. SreA also negatively regulates siderophore biosynthesis and high affinity iron uptake
systems by *E. festucae* as shown for other fungi. Preliminary studies indicate that the endophyte tightly controls iron acquisition from the
host apoplast via SreA, as ΔsreA mutants are able to induce premature chlorosis in their host plants during low iron availability.
To understand the influence of iron availability on gene expression and to identify genes that are targets of SreA, we compared the
transcriptomes of plants infected with ΔsreA versus WT (wild-type), and WT-infected plants grown hydroponically under high versus low
iron supply. A disproportionally large number of fungal genes were up-regulated in ΔsreA versus WT, indicating that these genes are likely
direct targets of SreA. Two such highly differentially expressed genes were clustered and putatively encode small secreted proteins (SSPs).
Conserved GATA motifs present in promoter regions, combined with up-regulation of SSP gene expression under low-iron availability,
suggest a novel role for these SSPs in iron metabolism. SreA-responsive genes included genes previously identified as SreA-regulated as
well as a diverse group of genes not obviously associated with iron metabolic processes. Putative siderophore biosynthetic genes not
previously identified from *E. festucae* were also found, including gene links to the ergosterol biosynthetic pathway. Changes in endophyte
secondary metabolite gene expression (for lolitrem and ergot alkaloid biosynthetic genes) with respect to either iron availability or SreA
presence/absence were also observed, highlighting the importance and interconnection of iron availability with fungal growth and secondary
metabolite production.

**564F The Ustilago maydis effector Rsp3 shields hyphae and blocks the anti-fungal activity of a secreted maize protein.** Lay-Sun Ma¹, Lei Wang¹,², Christine Trippel¹, Artemio Mendoza-Mendoza³, Steffen Ullmann⁴, Marino Moretti¹, Stefan Wawra⁵, Stefanie Reissmann¹, Karin Münch¹, Alexander Carsten¹, Bernd Zechmann⁶, Regine Kahmann¹ 1) Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Marburg, Germany ; 2) Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Bad Nauheim, Germany; 3) Bio-Protection Research Centre, Lincoln University, New Zealand; 4) Heinrich-Heine-Universität Düsseldorf, Molecular Microbiology, Düsseldorf, Germany; 5) University of Cologne, Botanical Institute, Cologne, Germany; 6) Center for Microscopy and Imaging, Baylor University, Texas, USA.

The biotrophic fungus *Ustilago maydis* causes smut disease in maize. Hallmarks of the disease are plant tumors and anthocyanin biosynthesis. Here we functionally characterize the secreted effector Rsp3 which contains several repetitive as well as a Cys-rich domain. *rsp3* alleles obtained from field isolates have full biological activity despite of length polymorphisms due to reduced or expanded numbers of certain repeats. *rsp3* is highly expressed during the biotrophic stage and is required for virulence and anthocyanin accumulation. Rsp3-HA can be easily detected in culture supernatants when expressed from a constitutive promoter, but displays highly anomalous migration behavior on SDS-PAGE. During biotrophic growth, Rsp3 decorates the surface of biotrophic hyphae. Using immunoprecipitation and mass spectrometry, we show that Rsp3 interacts with secreted maize DUF26 domain-family proteins. One of these (designated AFP1) was expressed and purified from *Nicotiana benthamiana*. AFP1 was able to bind mannose and showed antifungal activity against the *U. maydis rsp3* deletion mutant but not against a strain over-expressing *rsp3*. This suggests that the Rsp3 effector blocks the antifungal activity of the DUF26-containing maize protein, presumably by inhibiting its binding to the fungal cell wall.

**565W The Ustilago maydis effector Ten1 interacts with a maize protein phosphatase 2C.** P. Erchinger¹, J. Wu¹,², R. Kahmann¹ 1) Max Planck Institute for Terrestrial Microbiology, Marburg, Germany; 2) Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Science, PR of China.

The fungus *Ustilago maydis* is a pathogen that establishes a biotrophic interaction with *Zea mays*. The interaction with the plant host is largely governed by more than 300 novel, secreted effector proteins, many of which are encoded in gene clusters. The deletion of cluster 10A consisting of 10 effector-encoding genes results in strongly reduced virulence after seedling infection. By generating subdeletions and by complementing the full deletion of cluster 10A with individual genes we could show that *um03744* (termed *ten1*) has a major contribution to virulence. After overexpression in *U. maydis* hyphae, secreted Ten1 protein could be detected in axenic culture supernatant. Through a yeast 2-hybrid screen ZmPP26, a maize protein phosphatase 2C was identified as interaction partner of Ten1. Interaction was further supported by Co-IP experiments after co-expression of Ten1 and ZmPP26 in *Nicotiana benthamiana*. Moreover, ZmPP26 was detected by mass spectrometry after IP of 3xHA-Ten1 from infected maize tissue 52 hours post inoculation. Heterologously expressed ZmPP26 showed strong type 2C-specific phosphatase activity *in vitro*. Current work focuses on the recombinant production of Ten1 to see whether and how the interaction of both proteins alters phosphatase activity of ZmPP26. Furthermore, we are about to identify the interacting domain of Ten1 to be able to test a non-interacting allele in plant infections.

**566T Structure-function analysis of Cmu1, a secreted chorismate mutase in Ustilago maydis.** X. Han¹, J. Schuhmacher², F. Hartwig¹, A. Djamei³, T. Glatter¹, R. Kahmann¹, G. Bange² 1) MPI for Terrestrial Microbiology, Marburg, DE; 2) LOEWE Center for Synthetic Microbiology (Synmikro), Marburg, DE; 3) Gregor Mendel Institute of Molecular Plant Biology, Vienna, AT.

The basidiomycete *Ustilago maydis* is the causal agent for corn smut disease. To successfully colonize the host plant, *U. maydis* secretes a repertoire of around 300 effector proteins to manipulate host processes and suppress plant defense responses. These effector proteins function either in the intercellular space (apoplast) or translocate to plant cells to exert their functions. Previously we have shown that a secreted chorismate mutase Cmu1 is a translocated effector. Cmu1 enters plant cells by a yet unknown mechanism and interferes with salicylic acid biosynthesis to promote fungal infection. To gain more insight into the biological function of Cmu1, we have recently solved its crystal structure by X-ray crystallography using *E. coli* expressed protein. The structure revealed several unique features which might contribute to the function of Cmu1, including a surface-exposed acidic patch, a conserved disulfide bridge and a long loop region situated adjacent to the catalytic site. We have followed up on the relevance of these features for uptake by plant cells, protein stability and allosteric regulation/catalytic activity of Cmu1, respectively. In addition, we have shown that a secreted chorismate mutase from *Sclerotinia sclerotiorum* possessing a chloroplast transit peptide (M. Dickman, personal communication) could complement the virulence phenotype of the *cmu1* mutant. Furthermore, with the help of co-immunoprecipitation followed by mass spectrometry we have identified a secreted maize protein which might be a target of Cmu1 in the apoplast.

**567F Systematic identification of glycoproteins involved in the infection process of Ustilago maydis.** Ismael Moreno-Sanchez, Ramon Barrales, *Jose Ibeas* Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC-Junta de Andalucía, Seville,
Spain.

*Ustilago maydis* has raised as an excellent model for the study of plant-pathogen interactions, and its relation with maize plant is one of the systems in which studies can be tackled from both plant and pathogen perspective (Dean et al., 2012; Djamei & Kahmann, 2012). Protein glycosylation are critical processes in host-pathogen relations. In fact, mutations in essential genes for O-glycosilation (*pmt4*) and N-glycosilation (*gls1* and *gas2*) compromise *U. maydis* virulence on maize, affecting different steps in the infection process (Fernandez-Alvarez et al., 2009; Fernandez-Alvarez et al., 2013). We have now performed a proteome analysis in order to identify cytoplasmatic, secreted and cell wall glycoproteins, produced only when the virulence program is activated by over-expressing the transcription factor Biz1. Up today we have identified by mass spectrometry and MASCOT analysis 48 glycoproteins (33 cytoplasmatic, 11 secreted and 4 from the cell wall), all dependent of the virulence program activation. A systematic characterization of the role of all this glycoproteins in the infection process is currently in progress.

This work was funded by BIO2013-48858-P from MEC Spain.

Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD. (2012). Mol Plant Pathol. 13(7):804.

Djamei A, Kahmann R. (2012). PLoS Pathog. 8(11):e1002955. Fernandez-Alvarez A, Elias-Villalobos A, Ibeas JI. (2009). Plant Cell. 21(10):3397-412.

Fernandez-Alvarez A, Elias-Villalobos A, Jimenez-Martin A, Marin-Menguiano M, Ibeas JI. (2013). Plant Cell.

**568W The comparison of β-1,3-glucanase activity between mycovirus-cured *Trichoderma atroviride* NFCF028 and mycovirus-infected *T. atroviride* NFCF028.** Han-Eul Yang, Song Hee Lee, Suk-Hyun Yun, *Jeesun Chun*, Dae-Hyuk Kim Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea.

Molecular characterization of the most common dsRNA element from a green mold disease-causing *Trichoderma atroviride* indicated that it comprised 8,566 bp and encoded two large open reading frames (ORF1 and 2). The two ORFs were found to overlap by 46 bp with a typical (-1) slippery heptanucelotide sequence. The deduced protein sequences of ORF1 and ORF2 showed significant similarities to those of known mycoviral structural proteins and RNA-dependent RNA polymerases, respectively. Phylogenetic analysis indicated that this dsRNA is a member of a distinct species related to a group of unclassified mycoviruses; therefore, it was named Trichoderma atroviride mycovirus 1 (TaMV1). In order to analyze the effect of TaMV1 infection, β-1,3-glucanase, which is a cell wall-degrading enzyme for controlling plant pathogens and has been associated with the green mold disease by *Trichoderma* spp., was examined. The β-1,3-glucanase activity of TaMV1-cured isolate was higher than that of TaMV1-infected parental *T. atroviride* isolate. In addition, fungal metabolite from the TaMV1-cured isolate was more toxic to other fungal growth, which suggested that the TaMV1 infection resulted in hypovirulence of *T. atroviride*.

**569T Identifying virulence factors in the broad host range necrotrophic pathogen *Sclerotinia sclerotiorum*.** Kassaye Belay², Christopher Misar¹, Mitchell DuFour², Berlin Nelson², Robert Brueggeman², *William Underwood*¹ 1) Sunflower and Plant Biology Research Unit, USDA-ARS, Fargo, ND; 2) Department of Plant Pathology, North Dakota State University, Fargo, ND.

The necrotrophic fungal plant pathogen *Sclerotinia sclerotiorum* exhibits an exceptionally broad host range. This fungus causes significant damage on many agriculturally important broadleaf crops including canola, soybean, common bean, and sunflower. The pathogenicity determinants that allow *S. sclerotiorum* to successfully infect such a wide range of plant species are not well understood and to date, only a few potential effectors or virulence factors have been identified for this pathogen. We have established a diverse collection of 260 *S. sclerotiorum* field isolates collected from numerous host plants primarily in the north central United States. We are currently evaluating the virulence of this isolate collection on multiple plant species and utilizing genotyping-by-sequencing to generate high-density genotypic information for these isolates. Our results thus far indicate a broad range of virulence phenotypes on a single USDA sunflower inbred line and preliminary results indicate that at least some isolates exhibit unique virulence profiles on specific sunflower inbred lines. We are in the process of using this information to identify genetic factors contributing to virulence of this destructive broad host range pathogen.

**570F Tame a virus: a novel defense gene controls virus spread in the cereal pathogen *Fusarium graminearum*.** J. Bormann¹,², C. Heinze¹, M. Mentges¹, A. Brockmann¹, A. Alder¹, C. Blum¹, J. Hartung¹, D. Adpressa³, B. Plitzko³, B. Josephson², S. Loesgen³, M. Freitag², W. Schäfer¹ 1) Molecular Phytopathology, University Hamburg, Hamburg, DE; 2) Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA; 3) Dept. Chemistry, Oregon State University, Corvallis, OR, USA.

The kingdom of the fungi is unique in that defense reactions against pathogens have not been reported yet. Mycoviruses are found in all phyla of the true fungi. Infections are often fatal as they perturb sexual and asexual sporulation, growth, and, if applicable, virulence of the fungal host, a phenomenon known as "hypovirulence". The molecular basis of this effect remains unknown. Here we describe how the plant pathogen *Fusarium graminearum* defends itself against a viral infection in a gene-for-gene manner. We identified the mRNA-binding protein "fungal defense 1" (fd1), as the central facilitator of fungal response to viral infection. The transcription level of *fd1* is markedly reduced in the presence of virus and in strains expressing a viral coat protein acting as a virus-associated molecular pattern. In order to confine virus spread, the fungus triggers *fd1* downregulation, thereby constraining its own vigor and hampering anastomosis formation, a prerequisite for virus transmission within a fungal colony. Deletion of *fd1* causes virus-like symptoms in the absence of virus and abolishes anastomosis formation while constitutive overexpression of *fd1* overrules the cytopathic effects of a virus infection leading to symptomless accumulation of virus particles. Our results extend the recent concepts of virus-host interactions revealing a potentially conserved host defense mechanism reminiscent of immune responses in higher organisms. Metabolomic studies and RNA-seq experiments support the hypothesis of causal connections between *fd1* and mycovirus infection and led to the identification of virus-induced tolerance-associated genes (VITA-genes). Functional characterization of these genes is ongoing and will provide valuable insights into the underlying signaling network for fungal defense. This advance in understanding fungal infection and defense may aid biological pest control approaches using whole mycoviruses or specific viral proteins to prevent future fungal epidemics.
**571W Transposon mutagenesis as a tool to identify genes affecting the pathogenicity of *Talaromyces marneffei***. D. Kaymakci, A. Andrianopoulos School of BioSciences, University of Melbourne, Melbourne, Victoria, AU.

Advances in genome sequencing has expanded our knowledge of genomic architecture and led to novel gene discovery; however there is a notable lack of tools required for correlating genetic structure with biological function. In an era of escalating global health threatened by pathogenic fungi, correlation of genes to function are essential for identifying unique and essential factors that determine microbial virulence. Studies on disease-causing fungi have revealed that pathogenic potential depends on a complex interplay between the host and pathogen, which is governed by independent *in vivo* cellular pathways. This study utilises a high throughput mutagenesis system coupled with a candidate gene approach to identify genes essential for infection, survival and proliferation of the opportunistic pathogen *Talaromyces marneffei* during human macrophage infection.

**572T Predator Yeasts: genomics and molecular biology of necrotrophic killer yeasts.** Jürgen Wendland¹,², Klara Junker² 1) Bioengineering Sciences, Vrije Universiteit Brussel, Brussel, BE; 2) Carlsberg Research Laboratory, Copenhagen, Denmark.

Yeast of the genus *Saccharomycopsis* can be isolated from diverse habitats around the globe. They exhibit a unique predacious behaviour, which allows them to feed on and kill suitable fungal prey cells. Under nutrient limiting conditions, particularly upon lack of organic sulphur supply, predator yeasts form penetration pegs with which they will penetrate and, consequently, kill their prey. The host range is wide and includes yeasts, such as *Saccharomyces cerevisiae* and *Candida albicans* but also filamentous fungi, e.g. *Ashbya gossypii*. We aim at understanding the biology of predation and characterize molecular pathways and genes required for successful killing of prey cells. To this end we have generated draft genomes of five predator yeasts: *Saccharomycopsis fodiens*, *S. fermentans*, *S. crataegensis*, *S. schoenii* and *Saccharomycopsis* spec. The genome sizes range from 12 Mb to 15 Mb. The genome data, which also include several contigs with telomeric repeats, suggest that loss of genes required for sulphate assimilation is causing methionine auxotrophy within *Saccharomycopsis* species. Interestingly, genomic signatures suggest that *Saccharomycopsis* species are part of the CTG clade, which reassigned the CTG codon from leucine to serine, e.g. also in *C. albicans*. This has guided our molecular approach towards tool development for studying predator yeasts. We have developed synthetic markers, e.g. *SAK1* providing resistance against the antibiotic G418. Selecting for resistance of *S. schoenii* agains 5-Fluoro-Orotic Acid generated a *ura5* mutant. Genome profiling indicated the presence of transposons and of gene families encoding proteins that may play a major role for predacious behaviour. This includes genes encoding proteins for cell-cell adhesion, so called flocculins; genes for cell wall degrading enzymes, e.g. chitinases; and proteases. These and other morphogenesis genes required for penetration peg formation offer excellent target genes to analyze predatory behavior in *Saccharomycopsis*.

**573F Transcriptional response of the opportunistic human pathogen *Mucor circinelloides* to macrophage interaction and phagocytosis.** C. Pérez-Arques, M.I. Navarro-Mendoza, P. Martínez-García, F.E. Nicolás, V. Garre Department of Genetics and Microbiology, University of Murcia, Murcia, Spain.

Mucormycosis is an emerging fungal infection caused by Mucoralean opportunistic human pathogens. Despite the existence of a modern arsenal of antifungal drugs, mortality rates for this infectious disease remain devastatingly high since Mucoreales present an unusual drug resistance. Consequently, a current demand of novel therapeutic targets is triggering the exploration of the genetic basis involved in mucormycosis.

This work aims to identify new virulence factors involved in mucormycosis, using *Mucor circinelloides* as a model due to the great assortment of molecular tools available to manipulate it. Transcriptomic analyses have been performed to compare gene expression in virulent and avirulent strains interacting *in vitro* with mouse macrophages. This comparison revealed a large number of *Mucor* genes (about 20% of the genome) differentially expressed in the interaction with macrophages, which can be clustered in groups according to their expression patterns. Among these differentially expressed genes, a representative sample has been validated by Northern blot, confirming that these expression patterns are triggered in response to macrophage interaction. Interestingly, a small cluster of approximately one hundred genes is differentially expressed in both virulent and avirulent strains in response to the interaction with macrophages, and could contain indispensable genes for fungal defense against phagocytosis. Another remarkable group includes more than five hundred genes that are specifically regulated in the virulent strains in response to the interaction with macrophages, which could represent virulence factors. A deeper analysis of this group of genes revealed an enrichment in functions associated with signal transduction mechanisms, cytoskeleton, cell wall and membrane biogenesis, which is in accordance with an important role in virulence. Thus, to identify essential components in this response, null mutants for ten putative upstream regulators of these signal transduction pathways have been generated and their virulence analyzed in animal host-pathogen interaction models.

This research was funded by Fundación Séneca (19339/PI/14), MECD (FPU14/01983 and FPU14/01832) and MINECO (RYC-2014-15844 and BFU2015-65501-P) co-financed by FEDER.

**574W Effect of cytokinin level manipulation on virulence in the biotrophic fungus *Claviceps purpurea*.** S. Kind¹, J. Hinsch¹, P. Galuszka², P. Tudzynski¹ 1) University of Muenster, Muenster, DE; 2) Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, CZ.

The ergot fungus *Claviceps purpurea* is a biotrophic phytopathogen with a broad host range including economically important crops causing harvest intoxication upon infection. Its infection process is restricted to unfertilized ovaries without causing macroscopic defense symptoms. Thus, sophisticated host manipulation strategies are implicated. The plant hormones cytokinins (CKs) are known to regulate diverse plant cell processes and several plant pathogens alter CK levels during infection. For the first time, we could show that *C. purpurea* synthesizes CKs via two mechanisms and that fungus-derived CKs influence the host-pathogen interaction (Hinsch et al., 2015 and 2016). For a better understanding of the CK biosynthesis and the CK contribution to the plant-fungus interaction we applied different approaches to generate strains with altered CK type composition. The first approach is based on the deletion of the two "LOG" encoding genes which are supposed to be essential for the release of active CKs. We generated both, single and double deletion strains and they were found to be able to produce all types of CKs. Apparently, LOG gene products are dispensable for the formation of CKs. The CK biosynthesis pathway
remains unaffected in the second approach as it is based on heterologous overexpression of CK degrading enzymes from maize (Ckx1) and *Rhodococcus fascians* (FasE). Preliminary results indicate that strains overexpressing *Zmckx1* have strongly increased Ccx-activity and drastically reduced CK levels. Evidently, overexpression of *Zmckx1* resulted in the formation of functional protein. To our knowledge this is the first time a plant gene was successfully expressed in *C. purpurea*. Additionally, the strains showed reduced virulence dependent on the degree of the overexpression. Although characterization of the strains is not completed yet, so far this approach confirms previous findings that fungal-derived CKs are essential for a compatible plant-fungus interaction.

Hinsch et al. (2015) Environ Microbiol: 2935-2951

Hinsch et al. (2016) New Phytol: 980-992

**575T *A Setosphaeria turcica* secondary metabolite effector prompts a resistance response on *Ht1* maize. *B. Gillian Turgeon*¹, Dongliang Wu¹, Santiago Mideros¹, Zeran Lin¹, Rebecca Nelson¹,² 1) Section of Plant Pathology & Plant-Microbe Biology, SIPS, Cornell University, Ithaca, NY; 2) Section of Plant Breeding, SIPS, Cornell University, Ithaca, NY.**

*Setosphaeria turcica* is the causal agent of Northern Leaf Blight of maize, one of the most significant maize foliar diseases. *S. turcica* is a hemibiotrophic pathogen and several races have been described based on the symptoms they cause on a panel of maize lines carrying the 'major resistance' genes, *Ht1*, *Ht2*, *Ht3*, and *HtN*. By definition, maize carrying *e.g.*, *Ht1* is susceptible to *S. turcica* race 1. Our recent evidence suggests that a gene encoding a hybrid polyketide synthase:nonribosomal peptide synthetase (PKS:NRPS) enzyme for biosynthesis of a secondary metabolite in *S. turcica* race 23N sequenced strain 28A is critically important for the specific resistance interaction of this race on *Ht1* maize. Thus, mechanism of action is similar to the (hemi)biotrophic microbial effector protein-plant resistance protein model, but different in that a secondary metabolite, not a protein, acts as a fungal effector. Genotyping by sequencing and analysis of >200 progeny of a *S. turcica* race 23N by race 1 cross and concomitant phenotyping on *Ht1* maize identified a SNP, in the same PKS:NPS gene, that segregates perfectly with susceptible/resistant phenotypes. Co-inoculations with wild-type (resistance reaction) and mutant (susceptible reaction) strains in 1:1, 1:3, and 1:5 ratios complement the mutant phenotype. The *S. turcica* PKS:NPS gene is highly expressed *in planta* in the resistant interaction. On the maize side, qRT-PCR of maize inoculated with *S. turcica* wild type race 23N and the mutant demonstrate that both salicylic acid (SA) and jasmonic acid (JA) signaling are involved. Thus, a fungal small molecule associates in a specific manner with a plant resistance protein to prompt resistance.

**576F A widely conserved type 2 Glycosyl Transferase is essential for fungal pathogenesis of plants through enabling hyphal growth across solid surfaces. Robert King¹, Martin Urban¹, Rebecca Lauder², Sreedhar Kilaru³, Gero Steinberg³, Kim Hammond-Kosack¹, Jason Rudd¹ 1) Wheat Pathogenomics Team, Rothamsted Research, Harpenden, GB; 2) BioImaging unit, Rothamsted Research, Harpenden, GB; 3) BioSciences Dept, Exeter University, Exeter, GB.**

Fungal spores of plant pathogens must differentiate hyphal filaments in order to grow across and through plant surfaces to initiate infection. Failure to undergo this transition would in almost all cases result in a loss of virulence. From a T-DNA mutagenesis screen performed on the wheat leaf pathogen *Zymoseptoria tritici*, we recovered a mutant which was unable to correctly differentiate and extend hyphae on low nutrient agar, despite retaining full ability to (1) undergo 'yeast-like' blastic conidiogenesis on solid rich nutrient agar, (2) grow hyphae in liquid culture, and (3) generate aerial mycelium. The mutant also failed to form hyphae on wheat leaf surfaces resulting in a complete loss of virulence. The affected gene was identified and functionally validated through complementation and independent targeted deletion. It encoded a putative type 2 Glycosyl Transferase (*ZtGT2*) widely conserved throughout the fungal kingdom. The orthologous gene from the taxonomically un-related wheat ear-infecting fungus *Fusarium graminearum* was also functionally characterised. *ZtGT2* transcripts accumulated in conditions which stimulate hyphal growth of the wild-type strain and the protein was present only in the cell wall fraction. Global RNAseq gene expression analysis comparing *Zt?GT2* versus the wild-type strain growing in liquid culture, and during early leaf infection, highlighted a rapid growth arrest of *Zt?GT2* on the leaf surface, but a comparable early transcriptional adaptation, suggesting leaf surface sensing was largely unaffected. Comparison of fungal transcriptomes in liquid culture revealed an unexpected up-regulation of numerous candidate and functionally validated secreted effector proteins in *Zt?GT2* including the chitin binding LysM domain effector *Zt3LysM*. These data highlight the essential role of a widely conserved *GT2* in regulating both common and variable aspects required for fungal pathogenesis of plants, and suggest it may represent a viable target for future widespread control of plant diseases.

**577W RNAi-based functional genomics identifies new virulence determinants in mucormycosis. M.I. Navarro-Mendoza¹, T.A. Trieu¹,², C. Pérez-Arques¹, M. Sanchis³, J. Capilla³, P. Navarro-Rodríguez³, L. López-Fernández³, S. Torres-Martínez¹, V. Garre¹, R. Ruiz-Vázquez¹, F.E. Nicolás¹ 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 *mcplD*, 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.
**578T Unconventional recombination in the mating type locus of heterothallic apple canker pathogen *Valsa mali*.** Z. Yin, X. Ke, Z. Li, X. Gao, L. Huang State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, China.

Sexual reproduction in filamentous ascomycetes is controlled by mating type (*MAT*) locus, including two idiomorphs *MAT1-1* and *MAT1-2*. Understanding the *MAT* locus can provide clues for unveiling the sexual development and virulence factors for fungal pathogens. The genus *Valsa* (Sordariomycetes, Diaporthales) contains many tree pathogens responsible for destructive canker diseases. Sexual stage of these ascomycetes is occasionally observed in nature and no *MAT* locus has been reported up till now. Thus, we identified the *MAT* locus of apple canker pathogen *V. mali*, which causes extensive damage and even death to trees. *V. mali* is heterothallic that each isolate carries either *MAT1-1* or *MAT1-2* idiomorph. However, the *MAT* structure is distinct from that of many other heterothallic fungi in Sordariomycetes. Two flanking genes *COX13* and *APN2* were cooperated into the *MAT* locus, possibly by intra-chromosomal rearrangement. After the acquisition of foreign genes, unequal recombination occurred between *MAT1-1/2* idiomorphs, resulting in a reverse insertion in *MAT1-2* idiomorph. Evolutionary analysis showed that these three complete *MAT1-1-2*, *COX13* and *APN2* genes in this region diverged independently due to different selection pressure. Additionally, the unbalanced distribution (*MAT1-1/2*, 3:1) of mating-type idiomorphs was revealed by PCR detection of 24 isolates from different geographic regions. The unconventional remodeling in *MAT* structure and unbalanced *MAT* ratio probably limits the regular sexual reproduction. These results provide insight into the evolution of the mating systems in Sordariomycetes.

**579F Developing bi-parental fungal population and mapping of the genetic locus conditioning Ptr ToxC production in *Pyrenophora tritici-repentis*.** G. K. Kariyawasam, Z. Liu Department of Plant Pathology, North Dakota State University, Fargo, ND 58108, USA.

*Pyrenophora tritici-repentis* (*Ptr*) is a necrotrophic plant pathogen that causes tan spot disease on both bread and durum wheat worldwide. The fungal pathogen is known to produce at least three necrotrophic effectors including Ptr ToxA, Ptr ToxB, and Ptr ToxC as important virulence factors. Both Ptr ToxA and Ptr ToxB are a protein and the fungal genes encoding them have been cloned. However, Ptr ToxC is a non-ionic, polar, and low molecular mass molecule and the gene(s) conditioning its production remain unknown. Because Ptr is a homothallic fungus, it precludes the development of segregating fungal population for genetic analysis. In this work, we created heterothallic fungal strains by deleting one of the *MAT1* genes in isolate 86-124 (race 2, non Ptr ToxC-producer) and AR crossB10 (new race, Ptr ToxC-producer), and developed a bi-parental population by crossing them. The developed population consisted of 112 progenies and phenotyping of all progenies on Ptr ToxC differential lines suggested a single major gene conferring Ptr ToxC production in this population. The whole population is being genotyped using SSR and SNP markers. The obtained genotypic and phenotypic data will be used to map the genetic locus conditioning Ptr ToxC production. This work will help us to gain a better understanding of the disease system and aid in the breeding efforts to increase tan spot resistance.

**580W Fatal attraction: nematophagous fungus *A. oligospora* mimics olfactory cues of sex and food to lure nematodes.** Y. Hsueh¹, M. Gronquist³, E. Schwarz⁴, R. Nath², F. Schroeder⁵, P. W. Sternberg² 1) IMB, Academia Sinica, Taipei, TW; 2) Howard Hughes Medical Institute and Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; 3) Department of Chemistry, State University of New York at Fredonia, Fredonia, NY 14063, USA; 4) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA; 5) Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.

To study the molecular basis for predator-prey coevolution, we investigated how *Caenorhabditis elegans* responds to the predatory fungus *Arthrobotrys oligospora*. *C. elegans* and other nematodes were attracted to volatile compounds produced by *A. oligospora*. Gas-chromatographic mass-spectral analyses of *A. oligospora*-derived volatile metabolites identified several odors mimicking food cues attractive to nematodes. One compound, methyl 3-methyl-2-butenoate (MMB) additionally triggered strong sex- and stage-specific attraction in several *Caenorhabditis* species; furthermore, when MMB is present, it interferes with nematode mating, suggesting that MMB might mimic sex pheromone in *Caenorhabditis* species. Forward genetic screening suggests that multiple receptors are involved in sensing MMB. Response to fungal odors involves the olfactory neuron AWCs. Single-cell RNA-seq revealed the GPCRs expressed in AWC. We propose that *A. oligospora* likely evolved the means to use olfactory mimicry to attract its nematode prey through the olfactory neurons in *C. elegans* and related species.

**581T A Gpr1 receptor interactor protein from the Sur7-family is involved in cell wall stability and stress response in the mycoparasite *Trichoderma atroviride*.** Lea Atanasova¹, Theresa Radebner², Sabine Gruber¹, Alexander Lichius¹, Gerhard Schütz³, David Kreil⁴, Susanne Zeilinger¹ 1) Institute of Microbiology, University of Innsbruck, Innsbruck, Austria; 2) Institute of Chemical Engineering, TU Wien, Vienna, Austria; 3) Institute of Applied Physics, TU Wien, Vienna, Austria; 4) Chair of Bioinformatics, University of Natural Resources and Life Sciences, Vienna, Austria.

The ability of fungi to feed on other fungi (necrotrophic hyperparasitism or mycoparasitism) - a trait prominent within the genus *Trichoderma* - represents a promising alternative to hazardous chemical fungicides for plant diseases control. We previously showed that the 7-transmembrane receptor Gpr1 is involved in prey sensing and regulation of mycoparasitism-related processes in *Trichoderma atroviride*. Based on transcriptome profiling and membrane-based yeast two-hybrid screening we identified several candidates among which a protein of the fungal-specific Sur7 superfamily was recognized to be transcriptionally regulated by Gpr1 as well as being a putative interactor of this receptor. Additionally, EGFP labeled Gpr1 and Sur7 proteins showed similar, partly overlapping localization patterns in *T. atroviride* hyphae. Sur7 family proteins are typical residents of membrane microdomains that exhibit a composition, structure, and biological function distinct from the surrounding membrane. In yeasts such Sur7 containing membrane compartment of Can1(MCC)/eisosome domains were recently discovered and represent a novel type of membrane domain that is important for plasma membrane organization, sphingolipid homeostasis, and cell wall morphogenesis. However, for filamentous fungi the function of Sur7 proteins is less known. We show that in the mycoparasite *T. atroviride* the deletion of the *sur7-15* gene causes significantly reduced antagonistic response against living prey fungi, whereas its overexpression triggers full overgrowth and killing of the prey. Chitinase and chitin synthase-encoding genes
were significantly upregulated in *Δsur7* implying substantial cell-wall stress response. In accordance to that, degenerated resistance to cell-
wall intercalation and activation of a specific self-response chitinase and actin in mutant's self-confrontation underlined the important role of
*sur7-15* in the maintenance of the *T. atroviride* cell-wall integrity. Moreover, a regulatory impact of *sur7* on genes from the putative
MCC/eisosome domain and its involvement in the *T. atroviride* cell membrane stability and osmotic tolerance was also indicated.
Morphologically the *sur7* mutants showed a distinct phenotype with slower germination and enhanced hyphal and germ-tube branching.

**582F Ménage à trois: fluorescent rhizosphere pseudomonads pursue combined strategies to control polarity and growth of pathogenic Verticillium fungi and their plant hosts.** K. Nesemann¹, S. A. Braus-Stromeyer¹, *R. Harting*¹, H. Kusch¹, A. Hoefer¹, C. E. Stanley², M. Stöckli³, M. Landesfeind¹, A. Kaever¹, A. Ambrosio¹, C. Timpner¹, K. Hoff⁴, T. Pena-Centeno⁴, I. Bulla⁴, M. Starke⁴, A. J. deMello², M. Künzler³, M. Aebi³, G. H. Braus¹ 1) Institute of Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Georg-August-Universität Göttingen, DE; 2) Institute of Chemical and Bioengineering, ETH Zürich, CH; 3) Institute of Microbiology, ETH Zürich, CH; 4) Institute of Mathematics and Computer Science, Ernst-Moritz-Arndt-Universität Greifswald, DE.

The genus Verticillium includes several root-inhabiting fungi, which can colonize plants as endophytes or phytopathogens. The impact of
genes from rhizosphere Pseudomonas species for secreted metabolites including phenazines or the genes controlled by the bacterial
GacA/GacS two component regulatory system on fungal growth was investigated. *Pseudomonas fluorescens* from the rhizosphere of the
rapeseed *Brassica napus* cannot synthesize phenazine or 2,4-diacetylphloroglucinol mycotoxins, but caused similar growth inhibitions
towards the rapeseed pathogen *Verticillium longisporum* or *V. dahliae* infecting tomato as a different host. The fungal secondary
metabolism control genes *Lae1* or *Csn5* are dispensable for the fungal-bacteria interaction. Bacterial co-cultivations with different strains
supports media-dependent bacterial strategies to inhibit fungal growth during agar or liquid co-cultivation. Phenazines caused Verticillium
growth inhibition on high glucose, but not on pectin/amino acid agar. Co-cultivation with *P. protegens* in liquid pectin/amino acid medium
alters the relative fungal transcripts ratio including 16% up-regulated genes for mitochondrial activity, detoxification or melanine biosynthesis
and about half decreased gene fractions for protein synthesis or pectin degradation. Bacterial-fungal interactions at the single cell level
were probed in microfluid devices in liquid channels with pectin/amino acid medium. These confrontations with physical constraints revealed
that rhizosphere Pseudomonads can inhibit 80% of fungal hyphal growth independently of phenazine and 2,4-diacetylphloroglucinol by
complex combinations of metabolites. 90% of *V. longisporum* hyphal tips exhibited strong polarity defects with a 'curly' phenotype. Co-
infection experiments of host plants with fungi and bacteria revealed that only the *gacA*-controlled Pseudomonas regulatory network
protects plants against Verticillium induced pathogenicity.

Population and Evolutionary Genetics

**583W Discovery of a novel azole-resistance mutation in Aspergillus fumigatus and the possible role of sexual reproduction in its evolution.** J. Zhang¹, Bas Zwaan¹, E Snelders¹, S Schoustra¹, K Dijk², J Meis³,⁴, F Hagen³,⁴, E Kuijper⁵, G Kampinga⁶, J Zoll⁴,⁷, W Melchers⁴,⁷, P Verweij⁴,⁷, A Debets¹ 1) Laboratory of Genetics, Wageningen University, Wageningen, NL; 2) Department of Medical Microbiology, Vu University Medical Centre, Amsterdam, The Netherlands; 3) Department of Medical Microbiology and Infectious Diseases, CWZ Hospital, Nijmegen, The Netherlands; 4) Expert Centre in Mycology Radboudumc/CWZ (EMRC); 5) Department of Medical Microbiology, Leiden University Medical Centre, Leiden, The Netherlands; 6) Department of Medical Microbiology, University Medical Centre Groningen, Groningen, The Netherlands; 7) Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.

We investigated two compost-heaps, one containing azoles and one without azoles, for the presence of azole-resistant and azole-
sensitive *A. fumigatus* isolates. The azole-free compost yielded 98% (49/50) sensitive and 2% (1/50) azole resistant isolates, whereas the
azole-containing compost yielded 8% (4/45) sensitive and 92% (41/45) resistant isolates. From the latter, 84% (37/45) were of the highly
resistant TR46 Y121F/T289A genotype and 8% (4/45) had a novel pan-triazole-resistance harbouring a triple tandem repeat: TR46³/Y121F/
M172I/T289A/G448S. The stark contrast between the two composts indicates that azole-containing compost is a hot spot for the
development and maintenance of azole resistance. Subsequent screening of the clinical *A. fumigatus* collection from the Dutch national
surveillance programme indicated that this resistance mechanism was already present in 2012, and is now found in all participating medical
centres. We were able to recover this novel TR46³ mutation among the sexual progeny in the lab, from a cross between two TR46 isolates of
opposite mating type that were from the same compost. This strongly indicates a role of sex in the emergence of this novel azole-
resistance mechanism in *A. fumigatus*. Furthermore, we provide further evidence indicating the potential occurrence of sexual reproduction
in compost, but this still needs further confirmation. Our findings alarm the fast development of the azole resistance issue in *A. fumigatus*
and further indicate the role of sexual reproduction of *A. fumigatus* in the development of azole resistance.

**Key words:** Aspergillus fumigatus; novel new mutation; compost; azole resistance; hot spot; sexual reproduction; ascospores; conidiospores

**584T The role of lifecycle in azole-resistance development in Aspergillus fumigatus.** *E. Snelders*¹, J. Zhang¹, A.J.M. Debets¹, S.E. Schoustra¹, W.J.G. Melchers², B.J. Zwaan¹, P.E. Verweij² 1) Laboratory of Genetics, Wageningen University, Wageningen, NL; 2) Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, NL.

*Aspergillus fumigatus* is a ubiquitous fungus that plays an important role in carbon and nitrogen recycling in nature by degrading organic biomass. It is also a fungus that can cause several diseases in humans ranging from allergic conditions to acute invasive aspergillosis. To survive and thrive, *A. fumigatus* needs to rapidly adapt to these environments that often entail various challenges. Genetic adaptations by mutation and recombination can be defined as the acquisition of heritable modifications in an organism through natural selection, which enable it to survive and reproduce in the prevailing or new environment. One example of this adaptive process is the development of azole resistance. Triazoles have become the cornerstone of medical treatment of aspergillus-related diseases. Although triazole resistance can develop during patient therapy, another route of resistance selection in *A. fumigatus* can be through exposure to azole fungicides in the environment. Using the case of triazole resistance development, we show that the process of reproduction, sexual, parasexual, or asexual,
is crucial for the adaptive potential of *A. fumigatus*. In this study we describe the different life cycles in *A. fumigatus* and their characteristics and probability of occurring in the human host or in the environment.

**585F Pivotal role for the alternatively spliced, spliceosomal twin intron in one-bp intron drift.** *E. Fekete*¹, *M. Flipphi*¹, *N. Ág*¹, *N. Kavalecz*¹, *G.C. Cerqueira*², *C. Scazzocchio*³, *L. Karaffa*¹ 1) Department of Biochemical Engineering, University of Debrecen, Debrecen, HU; 2) Broad Institute of MIT & Harvard, Cambridge MA, USA; 3) Dept. of Microbiology, Imperial College London, UK.

In the primary transcript of nuclear genes, coding sequences – exons – usually alternate with non-coding sequences – introns. The latter are removed and former are joined by means of splicing to create the mRNA ORF that translates into the functional peptide product. In the study of the control of fungal gene expression, the ubiquitous splicing process is largely ignored. In the evolution of genic intron-exon structure, introns are gained at new positions or lost from extant positions or they move to a new position. The latter "intron drift" or "intron slide" leads to discordant introns in ortholog genes.

Previously we described stwintrons (spliceosomal twin introns) in filamentous ascomycota (*). These are complex intervening sequences in which a canonical "internal" intron interrupts one of the three conserved domains of a canonical "external" intron and consequently, they can only be removed with two subsequent splicing reactions. A stwintron in which the donor of the external intron is interrupted between the first and second bp – [D1,2] – can also be removed alternatively if the acceptor of the other external intron is interrupted between the penultimate and ultimate bp – [A2,3] – where the resultant mRNA is identical for both splicing routes.

Here we present a new stwintron, the first uncovered in *Aspergillus nidulans*. Ortholog genes occur in 4 Pezizomycotina classes. Most encompass either a [D1,2] or an alternatively spliced [D1,2] /[A2,3] stwintron at the same position. However, 9 species harbor there a discordant, canonical intron that conforms a one-bp intron drift: 7 have a phase-2 intron and appear to miss the [D1,2] internal intron while two carry a phase-0 intron and they appear to lack the [A2,3] internal intron instead. This is one of the few instances of intron drift where its mechanism could be elucidated.

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.

**586W Sexual fertility in Aspergillus flavus: understanding genetic exchange and phenotypic inheritance through analysis of F1 progeny.** *R.M. Gell*, I. Carbone Center for Integrated Fungal Research, Program of Genetics, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA.

The carcinogenic mycotoxin aflatoxin is a constant threat and economic burden to corn and oil seed crops grown within the United States and globally. Aflatoxin is produced by species in *Aspergillus* section *Flavi*, primarily *Aspergillus flavus*. Within the US and Africa, aflatoxin contamination is controlled through the high-density application of non-aflatoxin producing *A. flavus* strains. Though previously thought of as only asexual, *A. flavus* has recently been found to undergo sexual reproduction both in laboratory crosses and in the field. During the mating process, the sclerotium, a survival structure, of one strain acts as the female parent providing both the mitochondria and a matrix for the ascocarps and progeny to grow, while a spore or propagule from a second compatible strain fertilizes as the male. The fertility of mating pairs is highly variable and strongly influenced by the directionality of the cross but little is known about the genetic basis of fertility and how it is regulated. We are examining crosses that exhibit high fertility in one direction, but low fertility when male and female parents are reversed. Genome wide data using double digest Restriction Associated DNA sequencing was obtained for the progeny of these biased crosses. These data are being used to explore genome-wide recombination and as markers for mapping genomic regions that influence phenotypes, such as bias in fertility. By understanding this variation, we create opportunities to utilize strain fertility in the selection of biological control agents and increase our understanding of genetic exchange within populations of *A. flavus*.

**587T The adaptive potential of *Candida albicans* in response to *in vivo* stress.** *G. Cromie*¹, *E. Jeffery*¹, *S. Filler*², *J. Berman*³, *A. Dudley*¹, *A. Forche*⁴ 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 ploidy 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.

**588F Transitions between tetrapolar and bipolar fungal mating type driven by chromosomal translocations involving intercentromeric recombination.** *Sheng Sun*¹, *Vikas Yadav*², *R. Blake Billmyre*¹, *Christina A. Cuomo*³, *Minou Nowrousian*⁴, *Jean-Luc Souciet*⁵, *Teun Boekhout*⁶, *Betina Porcel*⁷, *Patrick Wincker*⁷, *Joshua A. Granek*¹, *Liuyang Wang*¹, *Kaustuv Sanyal*², *Joseph Heitman*¹ 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; 7) Commissariat à l'Energie Atomique (CEA), Institut de Génomique (IG), Genoscope, Evry, France, Université d'Evry, UMR 8030, Evry, France, Centre National de

<footnote>(*) Ág et al. (2015) Fungal Genet Biol 85:7 & Flipphi et al. (2013) Fungal Genet Biol 57:48.</footnote>
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.

**589W** 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.

**590T** A genetic comparison of *Exserohilum turcicum* isolates from maize and sorghum based on microsatellite haplotypes. A. Nieuwoudt¹, MP. Human¹, M. Craven², BG Crampton¹ 1) Plant and Soil Sciences, FABI, University of Pretoria, Pretoria, Gauteng, ZA; 2) Agricultural Research Council-Grain Crops, Potchefstroom, 2520, South Africa.

*Exserohilum turcicum* is the causal agent of northern leaf blight (NLB), a devastating foliar disease of both maize and sorghum in South Africa. Although *E. turcicum* is considered to cause disease in both crops, host specificity has been observed between isolates obtained from either maize or sorghum. The genetic differences underlying host specialization must still be determined. Population studies of *E. turcicum* isolated from maize has been conducted in Europe, America, Kenya, Northern China and South Africa, but studies comparing sorghum and maize isolates only refer to a few sorghum isolates. Therefore, a population genetics study was performed on *E. turcicum* isolates from both maize and sorghum to compare haplotypes of isolates from the different crops in KwaZulu-Natal (2014) and Mpumalanga (2015) in South Africa. Twelve microsatellite markers were used to screen 61 maize isolates and 64 sorghum isolates. No shared haplotypes were detected between the maize and sorghum isolates, but two shared haplotypes were identified between the maize isolates from the two locations. Principal coordinate analysis between maize and sorghum isolates showed no distinct separation between the maize and sorghum populations, since there is overlap in the central axis between the isolates from the different crops. Analysis of molecular variance indicated that isolates from the same host in the two locations are more similar than isolates from different hosts in the same location. Mating type distribution and indices of association gave no indication that random mating is taking place within the populations. However, haplotype diversities were too high to be associated with strictly clonal populations suggesting that a mixed reproductive life cycle is present. There is high genetic diversity in *E. turcicum* isolates from both maize and sorghum, evidence that *E. turcicum* follows a mixed reproductive strategy and there are indications for some differentiation between isolates based on the plant host from which isolates were collected.

**591F** The impact of heavy metal contamination on particular ectomycorrhizal fungi and associated plant hosts. Jessica Velez¹, Christopher Schadt² 1) University of Tennessee Knoxville, Knoxville, TN; 2) Oak Ridge National Laboratory.

Agriculture has become a multifaceted industry as the production of biofuels and the need for both greater productivity and sustainability grows in importance. However, there is a finite amount of land available, and a portion of this land is unusable due to the presence of major pollutants, such as heavy metal contaminants. As the demand for agricultural land rises, the viability of crops within soils that were previously considered unusable has become more appealing. There is a possibility that with modifications to the rhizosphere, a plant species may be able to survive in conditions that would otherwise be toxic to the plant, increasing overall land availability for use in agriculture. This would allow the planting of biofuel crops within fields that may not be optimized for the growth of crops intended for
consumption due to soil contamination, thereby avoiding the "food-for-fuel" tradeoff that has driven agronomic policy concerns in the corn-based ethanol industry. This research will aim to lead to a greater understanding of the exchange that occurs within a plant-fungal system and determine if there is an increase in the overall hardiness of the plant when the fungal species are present and how this varies with the genetic makeup of the symbionts. Looking to the future, this research will impact the direction of biofuel crop production as knowledge of the plant-fungal relationship increases, as well as potentially open new land for agricultural development.

**592W Unusual evolutionary mechanisms to escape Effector-Triggered-Immunity in the fungal phytopathogen *Leptosphaeria maculans*.** Clémence Plissonneau, *Thierry Rouxel*, Marie-Hélène Balesdent Plant Pathology, INRA-Bioger, Thiverval-Grignon, FR. *Leptosphaeria maculans* is the fungus responsible for the stem canker disease of oilseed rape (*Brassica napus*). *AvrLm3* and *AvrLm7*, two avirulence effector genes of *L. maculans*, are involved in an unusual relationship: the presence of *AvrLm7* suppresses the *Rlm3*-mediated resistance of *AvrLm3*. Following the large-scale cropping of *Rlm7* cultivars in Europe, the on-going breakdown of the *Rlm7* resistance and the concomitant inactivation of *AvrLm7* was accompanied by the resurgence of isolates expressing the *AvrLm3* phenotype. Here, we evaluated adaptation mechanisms to escape *Rlm3*-mediated ETI by assessing *AvrLm3* polymorphism in a collection of 236 *L. maculans* isolates. In isolates virulent towards both *Rlm3* and *Rlm7* (a3a7), the loss of the *Rlm3*-mediated resistance response was due to two distinct mechanisms. First, when *AvrLm7* was inactivated (deletion or inactivating mutations), amino acid substitutions in *AvrLm3* generated virulent isoforms of the protein. Secondly, point mutations in *AvrLm7* could maintain the masking of the *AvrLm3* phenotype while also allowing escape from the *Rlm7* recognition. This virulence mechanism was found in 56.4% of the a3a7 isolates, and these contained an avirulent allele of *AvrLm3*. Signatures of positive selection were observed in *AvrLm3* and, in spite of the telomeric location of *AvrLm3*, no a3a7 isolates exhibited deletion events or inactivating mutations in *AvrLm3* as commonly observed for other avirulence genes of *L. maculans*. The complex evolutionary mechanisms enabling *L. maculans* to escape *Rlm3*-mediated resistance while preserving *AvrLm3* sequence integrity, along with observed reduced aggressiveness of isolates silenced for *AvrLm3*, serves to emphasize importance of this effector in pathogenicity towards *B. napus*.

**593T Divergent evolution of two populations within a lineage of the hybrid fungal oilseed rape pathogen *Verticillium longisporum*.** Jasper R.L. Depotter¹,², Michael F. Seidl¹, Grardy C.M. van den Berg¹, Bart P.H.J. Thomma¹, Thomas A. Wood² 1) Laboratory of Phytopathology, Wageningen University and Research; 2) Department of Crops and Agronomy, National Institute of Agricultural Botany.

Population genetic structures represent evolutionary trajectories of organisms while they adapt to differential environmental conditions. Here, pathogen populations are additionally shaped by their host, as co-evolution between pathogen and host can lead to corresponding genetic co-structuring. Verticillium stem striping is a relatively new disease of oilseed rape that was mainly observed in continental Europe, but has recently expanded to other countries including the UK. Verticillium stem striping is caused by the hybrid fungal species *Verticillium longisporum* that originates from at least three separate hybridization events. Strains from the hybridization event between *Verticillium* species A1 and D1 are predominantly responsible for Verticillium stem striping. In this study, the population structure of *V. longisporum* lineage A1/D1 was investigated. Multi-locus genotype analysis revealed a hitherto un-described dichotomy that correlates with the geographic origin of the isolates. The genetic clusters are provisionally called "A1/D1 West" and "A1/D1 East" according to their relative location in Europe. Whereas A1/D1 East is the dominant population in Germany and Sweden, where Verticillium stem striping already occurs since the 1960s, the distribution of A1/D1 West reaches further than Europe, with isolates found in the USA and Japan. Genome comparison between representatives of the A1/D1 West and East clusters confirmed their mutual origin, excluding putative distinctiveness through separate hybridizations. An A1/D1 West population caused the sudden rise of Verticillium stem striping in the UK. Remarkably, the genetic diversity of the UK isolates was higher than that of the whole A1/D1 East cluster. Conceivably, the lower genetic variation within A1/D1 East indicates a founder effect, where A1/D1 West is the original population and A1/D1 East a founder population. A1/D1 East may have been able to establish by its initial capacity to cause Verticillium stem striping disease on oilseed rape, whereas A1/D1 West, until recently, occurred only on alternative hosts.

**594F Exploring hybridization in fungi: a lesson from the forest pathogens *Heterobasidion spp.*** M.M. Garbelotto¹, F. Sillo², L. Giordano²,³, P. Gonthier² 1) ESPM, University of California Berkeley, 54 Mulford Hall, 94720 Berkeley (CA), USA; 2) DISAFA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco (TO), Italy; 3) AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco (TO), Italy.

Hybridization is recognized as one of the major force to rapid adaptive evolution in fungi. However, the evolutionary potentials of hybrids are still poorly understood. The American forest pathogens *Heterobasidion irregulare* is currently hybridizing with the Eurasian sister species *H. annosum* in Italy. By using this model system, the aims of this work were I) clarifying the role of the mitochondrial genome in heterokaryotic hybrids, II) identifying introgressed genes from the invasive *H. irregulare* into the native *H. annosum*, III) estimating the pathogenic and saprobic fitness of hybrids. To reach these aims, artificial heterokaryotic hybrids were generated *in vitro* and their pathogenic and saprobic capabilities were assessed through pine seedling inoculations and growth experiments under controlled conditions. A target transcriptomic analysis through RT-qPCR was also performed. In addition, whole genomes of 9 natural homokaryotic hybrids collected in the sympatric area in Italy were sequenced and compared with that of pure genotypes of the two species. In the saprobic assay, the heterokaryotic hybrids carrying only one mitochondrial genome perfectly mirror the respective parental donor of mitochondria, suggesting a role for the mitochondrion in saprobicity. RT-qPCR results support this hypothesis, since during saprobic processes heterokaryotic hybrids regulate their genes similarly to the respective parental donor of mitochondrial genome. The comparative genomic analysis in natural hybrids allowed to identify and quantify introgressed genomic regions in each genotype. Recurrent introgressed genes from the *H. irregulare* to *H. annosum* seem to be associated to saprobic processes and to secretory Golgi-related pathways. This work provides insights on the genomics of hybridization, towards a broader understanding of the evolutionary trajectories of hybrid fungal pathogens.
**595W Evidence of birth and death evolution in the α-pheromone precursor gene of *Fusarium circinatum*.** O.O. Adenigba¹, B.D. Wingfield², T.A. Duong², N.A. van der Merwe², M.J. Wingfield¹, E.T. Steenkamp¹ 1) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; 2) Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa.

Ascomycetes encode two classes of pheromones (α and a), which are expressed in a mating-type specific manner to initiate the process of sexual reproduction. Pheromone genes encode precursor proteins that undergo proteolytic processing to release tandem repeats of the respective pheromone peptides. We hypothesized that the evolution of these genes and their repeats are driven by birth-and-death and concerted evolution, as has been shown for multi-gene families. To test this hypothesis and to determine the mode of selection experienced by these genes, we utilized the α-pheromone precursor gene in the pine pitch canker pathogen, *Fusarium circinatum*. We amplified, sequenced and analysed the α-pheromone precursor gene in 69 isolates of *F. circinatum*. The aligned sequences of the repeats were used to construct a neighbour joining tree and to infer dN/dS ratios to investigate modes of selection. Among the 69 isolates, we identified 13 haplotypes, each with a unique combination of variants of the α-peptide repeat at the DNA level. The total number of tandem α-peptide repeats per haplotype ranged from 10 to 14, possibly due to the birth and loss of repeats. Phylogenetic relationships further indicated that repeats grouped according to repeat variant and not according to haplotype. All dN/dS ratios were also less than 1, suggesting that this gene and its repeats are subjected to negative selection. Collectively, our findings showed evidence of birth and death evolution and strong purifying selection acting on the α-pheromone precursor gene of *F. circinatum*. These findings could imply a need for increased dosage of a specific pheromone during sexual reproduction, while the occurrence of variant repeats suggested that new α-peptides with new or similar functions might evolve.

**596T Fitness landscape driven by the interaction between gene mutation and aneuploidy.** H-J. Tsai, A. Nelliat, R. Li Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD.

Aneuploidy, an abnormal genome state with gain or loss of chromosomes, has detrimental effects to cellular physiology due to dramatic changes in gene copy number. This unbalanced state of genome content leads to genome instability and developmental defects. Interestingly, aneuploidy also confers phenotypic variation in fungal populations to gain selective advantages for stress adaptation. For example, gain of chromosome 5 in pathogenic yeast *Candida abnicans* is highly associated with fluconazole resistance. Moreover, aneuploidy is widespread and well tolerated in wild yeasts to drive rapid phenotypic evolution as an adaptive strategy. The paradoxical nature between the beneficial and detrimental roles of aneuploidy in organismal fitness remain unclear. Here, we investigate how aneuploidy rewires genetic network to change population fitness by performing a genetic screen in a heterogeneous aneuploid population in *Saccharomyces cerevisiae*. We took advantage of triploid meiosis to generate a karyotypically heterogeneous aneuploid population and unbiasedly tested the growth impacts of aneuploidy. In parallel, we applied this strategy to aneuploid populations carrying a single gene mutation in each population. Thus, we were able to observe the fitness impact from the interaction between gene mutation and aneuploidy. Intriguingly, despite cell growth being compromised due to the negative effects of gene mutation or aneuploidy, majority of aneuploid populations carrying a gene mutation exhibited comparable fitness to euploids. To further examine this growth phenotype, we utilized a high-throughput, quantitative microscopy assay to measure the fitness distribution of microcolonies and revealed high cell-to-cell heterogeneity in aneuploid populations. Under the absence of stress in the environment, a wide range of growth capabilities existed in aneuploid populations, and few of them showed advantageous growth to euploid cells. The results suggest that aneuploidy may confer phenotypic variation due to the preservation of inner heterogeneity and could show high adaptability when stress presents.

In summary, we have mimicked the naturally occurring karyotypic complexity in a diverse aneuploid population to elucidate fungal adaptive evolution. Most importantly, our genetic screen and quantitative fitness analysis of the heterogeneous aneuploid populations could reveal detailed molecular trajectory in aneuploidy during evolution.

**597F Multilevel selection in *Neurospora tetrasperma*.** C. Meunier, S. Hosseini, Z. Maryush, H. Johannesson Systematic Biology, Evolution Biology Centre, Uppsala, SE.

Heterokaryosis, the coexistence of genetically divergent nuclei in the same mycelium, is a common trait in fungi. In the filamentous ascomycete *Neurospora tetrasperma*, a shift in the mating system has led to predominant heterokaryosis during the life cycle: both mating-type nuclei (*mat A* and *mat a*) are packaged together in conidia and ascospores- however, monokaryotic, haploid individuals can still be found. Such a shift can be considered as an evolutionary transition to higher complexity. During this transition, a potential threat to heterokaryons is discordant selection, when selection operating at level of haploid nuclei acts counter to selection operating at the level of the heterokaryon. Furthermore, a crucial step in the transition is the export of fitness heritability from monokaryon-level to heterokaryon-level, potentially leading to division of labor among nuclear types. In our study, we investigated pros and cons of heterokaryosis in *Neurospora tetrasperma*, studying nuclear ratios and contrasting fitness traits between the heterokaryotic mycelium and its two genetically divergent, totipotent and free ranging nuclear types. We showed that the ratio of the two nuclear types varies within a mycelium and verified that the ratio is homogeneous, but that it can deviate from a diploid-like 1:1-ratio. When measuring fitness traits of the mycelium and its component nuclear genotypes, we found that nuclear types have different fitness optima during the life cycle and that the phenotype of the heterokaryotic mycelium is reflecting the nuclear ratio in an additive manner. Thus, our data indicate subfunctionalization of the nuclear types for mycelial function, a form of division of labor. Our results also hint towards variation in nuclear ratio resulting in phenotypic flexibility of the mycelium. However, the occurrence of seemingly selfish nuclei, enjoying better replication and transmission than sister nuclei, yet being detrimental to the heterokaryotic organism, points towards ongoing discordant selection.

**598W Can meiotic drive drive speciation?** A.A. Vogan, Jesper Svedberg, Sandra L. Ament-Velásquez, Hanna Johannesson Systematic Biology, Uppsala University, Uppsala, SE.

Selfish elements can bias their own transmission from one generation to the next in a process referred to as meiotic drive. The driving element will be passed to the majority of offspring, thereby increasing in frequency over generations. The drive can occur regardless of its effect on fitness and may be a detriment to the organism as a whole. This intragenomic conflict could lead to strong selection against individuals carrying the element and promote reproductive isolation between so called killer and sensitive strains.
In ascomycetous fungi, meiotic drive manifests itself as a spore killing phenomenon. Briefly, ascospores carrying a killer element will induce the death of ascospores in the same ascus that do not carry the element. The killing is assumed to have a large cost to fitness, as a cross between killer and sensitive strains would result in the death of roughly half of all sexual progeny. We have examined 4 different spore killers in both *Neurospora* and *Podospora*. All of these examples of drive show patterns that suggest they may promote reproductive isolation between killer and sensitive strains.

In *N. sitophila*, we have indications that two separate defense mechanisms have evolved to protect against drive. Both Tahitian and European populations are polymorphic for spore killing. In Tahiti, a resistance locus has evolved that allows a number of sensitive spores to escape killing. In Europe, no such resistance has been observed. However, sensitive strains appear to be a distinct lineage from killer strains. This suggests that reproductive isolation may be a viable alternative to resistance as a way to protect against spore killing.

In *N. intermedia*, there are also indications of reproductive isolation between killer and sensitive strains. Crosses between certain populations produce little to no viable progeny when one parent carries a killer element, but produce moderate levels of viable progeny when both parents are sensitive. This pattern is also observed when crosses are conducted between *N. intermedia* and its sister species, *N. metzenbergii*.

The final example is from a population of *P. anserina* in the Netherlands, where the investigated spore killer element appears to be nearly fixed. A small number of individuals can be found that do not have this killer element and again, these isolates appear to be a distinct lineage from the other Dutch strains.

We are currently performing experiments to confirm the link between meiotic drive and speciation.

**599T Impact of promoter evolution on gene expression levels in Zymoseptoria tritici.** C.J. Eschenbrenner¹,², A. Kumar¹, J. Haueisen¹,², E.H. Stukenbrock¹,² 1) Botanical Institute, University of Kiel, Kiel, Germany; 2) Max Planck Institute of Evolutionary Biology, Plön, Germany.

In a previous study, we compared transcriptome data from three isolates of the fungal wheat pathogen *Zymoseptoria tritici* during wheat infection. We used three isolates that cause the same level of disease symptoms on the susceptible wheat cultivar Obelisk. While the three isolates are confronted with the same host immune system and show the same ability to grow and reproduce in Obelisk wheat, we found that up to 20% of all shared genes are differentially expressed between isolates during host colonization. We concluded that isolates of *Z. tritici* use different transcriptional programs to successfully invade the same host. In this study, we address the underlying genetic and genomic basis of transcriptional variation in *Z. tritici* and focus on evolution in promoter sequences. For this purpose, we created *de novo* assemblies for 26 genomes of *Z. tritici* from several locations worldwide (Australia, Denmark, France, Germany and Iran). In addition, we used the Dutch isolate IPO323 as reference for all analyses. Genome assemblies of the 26 isolates were used to generate a multiple genome alignment with the *tba* program package. The alignment was further processed and filtered using the program *maffilter*. After filtering we retained a genome alignment of 26 Mb comprising only alignment blocks with sequence data for all 26 genomes. The filtered alignment was then used to identify 741,269 single nucleotide polymorphisms (SNPs). From the multiple genome alignments we extracted 500 bp sequences upstream of each aligned coding sequences and used the SNP data to compute a range of parameters for each promoter including SNP densities, Tajima's D, and the nucleotide diversity computed as Tajima's Pi. Genome-wide correlation analyses show a strong negative correlation between gene expression and genetic variation in promoters. Thus as expected, highly expressed genes have more conserved promoter sequences. We also find that genetic variation in promoters varies greatly according to the chromosomal context. Promoters in gene dense regions are more conserved than promoters in gene sparse regions in agreement with a stronger effect on background selection in gene rich regions.

**600F 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.

**601W An experimental approach to studying the role of Horizontal Gene Transfer in shaping fungal secondary metabolism.** P.E. de Reus, K.F.N. Nielsen, R.J.N. Frandsen Biotechnology and Biomedicine, Technical University Denmark, Copenhagen, DK.

The *in-silico* discovery rate of putative secondary metabolite gene clusters in filamentous fungi is on a surge. With it come increasingly many reports of Horizontal Gene Transfer (HGT) of these gene clusters. Such events are inferred from discrepancies in the synteny, nucleotide composition or taxonomical distribution of the gene clusters in question. Clustered genes have been reported to undergo HGT more often than unclustered genes, for which various hypotheses can be offered. This 'genetic sharing economy' is very interesting from an evolutionary perspective, and yet *in-silico* studies leave fundamental questions about the functional implications of HGT unanswered.

Our study takes an experimental approach to studying the role of HGT in shaping fungal secondary metabolism, focusing on the immediate impact of a whole-cluster HGT event on the recipient. Four naphto-y-pyrone gene clusters - two confirmed and two putative - were selected from four different native fungal hosts (*Fusarium graminearum*, *Fusarium fujikuroi*, *Trichophyton rubrum*, and *Aspergillus eucalypticola*).
Each cluster was transferred to the same locus in host organism *Aspergillus nidulans* using a one-step PCR-based *in-vivo* recombination method. Alongside the clusters, a second library was created in which the cluster pathway-specific transcription factors were overexpressed in a second locus. In a third library, the transcription factors were overexpressed in strains lacking the cluster.

Transcriptional activity of the clusters was investigated using RT-qPCR to address splicing and trans-regulatory interactions as two of the potential barriers to the functional expression of HGT gene clusters in the new host. Metabolic profiling of the libraries was done by HPLC-UV/Vis-High Resolution MS to screen for production of pathway products and intermediates, as well as other novel metabolites arising from cross-chemistry or cross-regulation.

This case study, on a small scale, demonstrates the added value of *in-vivo* experimental work to complement *in-silico* findings and hypotheses, offering additional insights into the role of HGT in fungal secondary metabolism, as well as providing an additional tool for cluster validation.

**602T Mating type loci suggest ancestral heterothallism and convergent evolution towards homothallism in *Ophiostomatales* lineages.** T.A. Duong¹, M.J. Wingfield¹, Z.W. de Beer², B.D. Wingfield¹ 1) Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, ZA; 2) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, ZA.

The order *Ophiostomatales* (Ascomycota: Sordariomycetes) is a large group of fungi with diverse biological and ecological adaptations including the ability to cause disease in animals and plants. The order includes ten genera including more than 300 species. In many of these species, especially those residing in *Leptographium*, *Raffaelea* and *Sporothrix*, sexual structures are unknown and they have been presumed to be asexual. However, several recent studies have shown that many are actually heterothallic fungi most likely with cryptic sexual cycles. The mating type loci have also been fully characterized for a number of species and these all have a heterothallic mating system. The aim of this study was to characterize the mating type locus in selected homothallic species in the *Ophiostomatales*, and to compare this with the locus in heterothallic species. The overall aim was to gain insight into the evolution of the mating type locus in the *Ophiostomatales*. The genomes of several homothallic species were sequenced and their mating type loci characterized. Comparisons of the mating type loci in homothallic and heterothallic species belonging to five different genera in the *Ophiostomatales*, we were able to show that heterothallism is the ancestral state for the order. Furthermore, that convergent evolution has given rise to a small number of homothallic species residing in distantly related lineages. This is the first study to fully characterize the mating type loci of homothallic members of the *Ophiostomatales* and it has improved our understanding of the evolution of the mating systems in this interesting and important group of fungi.

**603F Evolutionary genomics of Spore Killing in *Podospora anserina*.** S.L. Ament-Velasquez¹, E. Bastiaans¹,², A. Vogan¹, J. Svedberg¹, A.J.M. Debets², S.J. Saupe³, H. Johannesson¹ 1) Systematic Biology, Department of Organismal Biology, Uppsala University, Norbyvagen 18D, 752 36 Uppsala, Sweden; 2) Laboratory of Genetics, Wageningen University, Droevendaalsesteeg, Wageningen, The Netherlands; 3) Institut de Biochimie et de Genetique Cellulaire, UMR 5095 CNRS, Universite de Bordeaux 2, France.

Mendel's law of equal segregation states that during meiosis each allele has the same probability of being transmitted to the offspring. However, some selfish genetic elements are known to "cheat" and promote their own transmission at the expense of the other allele. These *meiotic drivers* introduce intragenomic conflict within the host organism, which has potential implications for the evolution of genome architecture, population structure, and speciation. In ascomycete fungi, meiotic drivers are known as "spore killers" since half of the progeny (the ones with the killer locus) induce the death of the other half during ascus development. The pseudohomothallic fungus *Podospora anserina* exhibits a particularly complex case of spore killing, where up to six different coexisting spore killers (Psk's) have been described in a natural population sampled in the Netherlands. When confronted in matings, these Dutch Psk's can either kill or not kill other Psk in a hierarchical pattern. Although a novel type of gene (*spok*) conferring spore-killing abilities has been described in French strains, nothing is known on the genetic basis of Dutch Psk's or their effects on the organism fitness. In this context, we used Illumina Hi-Seq (paired-end) technology to sequence the entire Dutch collection (123 strains) plus two French strains. We further selected nine representatives of all Psk and sensitive strains and obtained chromosome-size genome assemblies using PacBio sequencing technology. We discovered that different *spok* paralogs segregate in a ~150kb translocation block located in either arm of chromosome 5 or in chromosome 3 of *P. anserina*, depending on the killer phenotype. The block has been subjected to internal rearrangements and deletions of *spok* genes. Moreover, although the gene *spok2* (placed outside of the block) is nearly fixed in the population, a few samples were found to be truly naive, having no *spok* genes at all. Preliminary population genomic analyses suggest that these naive strains are differentiated from the rest of the Dutch strains. To reconcile our findings with the observed killing hierarchy, we propose the existence of an unidentified killer locus unrelated to the *spok* genes. Future research will focus on functional verification of the *spok* genes for spore killing, fitness effects of carrying the *spok* block and its implications on the evolutionary dynamics of spore killing in *P. anserina*.

**604W AmanitaBASE: population genomics to elucidate the intersection of mating systems, genome organization, and invasion.** M. Holly Elmore¹, Jaqueline Hess², Anne Pringle³ 1) Organismic & Evolutionary Biology, Harvard University GSAS, Cambridge, MA; 2) University of Vienna, Vienna, AT; 3) Botany and Bacteriology, University of Wisconsin-Madison, Madison, WI.

We have sequenced and are assembling and annotating 99 *Amanita* genomes to enable *AmanitaBASE*, a massive comparative genomics effort in this emerging system. In addition to previously sequenced genomes from *A. brunescens*, *A. muscaria* var. *guessowii*, *A. polypyramis*, *A. thiersii*, and *A. inopinata*, *AmanitaBASE* will boast 70 specimens of *A. phalloides* from two invasive populations in California, 11 *A. phalloides* specimens from their native range in Portugal, 3 specimens of *A. thiersii*, a saprotrophic *Amanita*, and 11 *Amanita* specimens from across Europe provided by Kew Gardens. The 70 specimens from invasive *A. phalloides* populations in California were collected at three different timepoints over 10 years, which will give us insight into population dynamics. Each sequenced mushroom was meticulously mapped and photographed before being collected and detailed ecological notes were takne for each collection site. *Amanita* have a tetrapolar mating system, where compatible mates must have different alleles at both the homeodomain (HD) locus and the pheromone/receptor (PR) locus. The mating loci extend over several kb of repressed recombination and are expanding in a manner similar
to the evolution of sex chromosomes. Whole genomes will enable an analysis of linkage disequilibrium and the comparison of genomes
from the same location and timepoints will enable a detailed study of population-level variation in the architecture of mating types.

Comparison of genomes from different populations and timepoints can shed light on the evolution of mating type genes, including the origin
of new HD and PR alleles.

Comparing native to invasive populations may reveal genomic changes in response to invasion. *A. thiersii* fits the more classic paradigm of a low diversity invasive population that spreads rapidly, whereas *A. phalloides*, which in California appears to be very genetically diverse, is spreading more slowly over a smaller area. This may be explained by the fact that *A. thiersii* is a free-living saprotroph while *A. phalloides* must find compatible mycorrhizal hosts. *A. thiersii*'s near clonality may be related to anomalies in the mating loci that were found in the first sequenced *A. thiersii* genome. Multiple genomes from these populations will allow the study the intersection of mating systems, symbiosis, and range expansion in unprecedented resolution.

**605T** The important root rot pathogen *Thielaviopsis basicola* shown to be heterothallic. W.J. Nel¹, T.A. Duong², B.D. Wingfield², M.J. Wingfield¹, A. Hammerbacher¹, Z.W. de Beer¹ 1) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, Gauteng, ZA; 2) Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, Gauteng, ZA.

The ascomycete fungus, *Thielaviopsis basicola* (Microascales: Ceratocystidaceae), was described in 1850 and is a serious, mainly root-infecting plant pathogen of many important crop and ornamental plant species. Despite multiple unsuccessful attempts to induce mating, nothing is known regarding the mating strategy of *T. basicola* and it has often been referred to as an asexual fungus. The aim of this study was to determine the mating strategy of the pathogen. DNA, extracted from a culture derived from a single conidium, was sent for genome sequencing using a combination of next generation sequencing platforms. The draft genome was assembled and the mating locus identified by BLAST searches against known mating type genes available for other species in the Ceratocystidaceae. The results showed that the sequenced isolate harbours a *MAT1-2* idiomorph with a *MAT 1-2-1* gene, as well as a gene with strong similarity to *MAT 1-2-7* previously reported from species in the related genus *Huntiella*. The absence of the *MAT 1-1-1* gene suggests that *T. basicola* is most likely heterothallic. Ongoing research is focussed on attempts to locate a *MAT1-1* idiomorph in a large collection of cultures and to further understand whether sexual reproduction might be occurring in this important pathogen.

**606F** Recombination is suppressed on collinear chromosomes in *Neurospora tetrasperma*. Jesper Svedberg¹, Yu Sun², Markus Hiltunen¹, Pádraic Corcoran³, Hanna Johannesson¹ 1) Department of Organismal Biology, Uppsala University, Uppsala, SE; 2) Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, CN; 3) Department of Animal and Plant Sciences, University of Sheffield, UK.

In many eukaryotic genomes meiotic recombination is locally suppressed as a response to various selective pressures. The most well-known examples are non-recombining sex chromosomes, where recombination is hypothesised to be suppressed in response to sexually antagonistic selection, but similar "supergenes" have also been implicated in driving local adaptation and speciation in other systems. The causes of the suppression of recombination are in many of these cases not well understood; structural rearrangements – such as inversions – have often been implicated, but they may also be a consequence of suppression, rather than a cause. In the pseudohomothallic ascomycete *Neurospora tetrasperma* the proper packaging of the two mating types in is ensured by suppressing recombination between the mating-type locus and the centromere. Cytological and crossing data, as well as data on nucleotide divergence and linkage disequilibrium have further shown that recombination is absent in 60-80 % of the chromosome. In an earlier genome study the chromosome carrying one of the mating types in one specific strain was also shown to carry a complex of overlapping inversions, and these inversions were hypothesized to maintain the suppression of recombination in the species. By generating high quality genome assemblies from eight other *N. tetrasperma* strains we can here show that the majority of *N. tetrasperma* strains are collinear, that the previously identified inversions are a derived feature and that the suppression of recombination has to be maintained through non-structural means. We also identify several other derived inversions on the chromosome, supporting the hypothesis that suppression of recombination can relax selection against inversions. These results establish the existence of large-scale, long-term recombination suppression in the absence of structural rearrangements and caution against interpreting the presence of such rearrangements as being causative. The identified mode of suppression of recombination is a new phenomenon that potentially has significant effects on genome evolution and how we interpret signals of divergence.

**607W** Return of the mitochondrial DNA. *B. Brankovics*¹,², P. van Dam³, M. Rep³, S. de Hoog¹,², T. van der Lee⁴, C. Waalwijk⁴, A. van Diepeningen¹,⁴ 1) CBS Fungal Biodiversity Centre, KNAW, Utrecht, NL; 2) Institute for Biodiversity and Ecosystem Dynamics, UvA, Amsterdam, NL; 3) Swammerdam Institute for Life Sciences, UvA, Amsterdam, NL; 4) Wageningen Plant Research, WUR, Wageningen, NL.

The mitochondrial genome is present in high copy number within cells, making it easier to access than single copy nuclear sites. In many
organisms mitochondrial sequences are uniparentally inherited, hence non-recombining, making them ideal for phylogenetic
reconstructions. This is the reason why mitochondrial markers have been widely used in population genetic studies. In the '90s the
mitochondrial encoded COI (cox1) was used as the first barcoding marker. However, amplifying the sequence proved problematic in many
fungal groups, because the frequent insertion of introns into the region made universal primer design difficult. Hence, the mitochondrial
marker was abandoned and the barcoding community choose the ITS region as the official barcode for fungi. Also this barcode proved to
have insufficient resolution in many closely related species. Thus, multi-locus analysis became the new standard, most of which included at
least one mitochondrial marker. There has been no consensus on which mitochondrial loci to include.

With next generation sequencing and new assembly tools it is possible to assemble the complete mitochondrial genome of the isolates, which provide all the benefits that are associated with using mitochondrial markers. In addition, using the complete mitochondrial genome offers better resolution for phylogenetic analyses and with sufficient sampling, it can be placed in the context of previous works done on mitochondrial barcoding markers.

In order to prove the usefulness and applicability of this whole mitochondrial genome analysis approach, we have assembled the complete mitogenomes of 61 *Fusarium oxysporum* strains. Multiple NGS platforms were used and a new assembly pipeline was developed to
reconstruct the mitochondrial genomes. Our findings concurred that complete mitogenomes are a rich source of phylogenetic information that confirm earlier phylogenies and add more resolution. Furthermore, the analysis has revealed the presence of a cryptic parasexual cycle in *F. oxysporum* as revealed by mitochondrial recombination. In conclusion: complete mitochondrial genome sequences offer a stable basis and reference point for phylogenetic and population genetic studies, while still offering compatibility with earlier studies.

**608T Genome-wide identification of horizontal gene transfer in *Fusarium verticillioides*.** S. Gao¹, J Wisecaver², Y Zhang³, L.-J Ma³, A Rokas², S Gold⁴, A Glenn⁴ 1) University of Georgia, Athens, Georgia; 2) Vanderbilt University, Nashville, Tennessee; 3) University of Massachusetts, Amherst, Massachusetts; 4) Richard B. Russell Research Center, USDA-ARS, Athens, Georgia.

Horizontal gene transfer (HGT), the exchange and stable integration of genetic material between different lineages, breaks species boundaries and generates new biological diversity. In eukaryotes, despite potential barriers, like the nuclear envelope and multicellularity, HGT may be facilitated by the intimate contact between organisms and other strategies. Intrigued by the potential role of HGT in the evolution and adaptation of the maize pathogen *Fusarium verticillioides* (*Fv*), we applied a phylogenomic pipeline to identify potential HGT candidates in the genome of *Fv*. From an initial output of 1801 genes, manual curation revealed 117 strong HGT candidates, which tend to be intronless, non-plastid-derived, and bacteria-acquired. Functional category analysis suggested several enriched metabolic pathways (e.g. lysine biosynthesis and nitrogen metabolism) compared to the general frequency of such genes within the *Fv* genome. Interestingly, among the five candidates that were categorized as being involved in lysine biosynthesis, FVEG_09873 has no orthologs in other fungi. This gene was acquired from plant-associated Proteobacteria and putatively encodes a dianinopimelate epimerase in the dianinopimelic acid pathway employed by most bacteria but not commonly found in fungi, further supporting the bacterial origin of this candidate. Another promising HGT candidate acquired from bacteria is FVEG_10494 that encodes a putative class-III aminotransferase, functioning in nitrogen metabolism. Additional analyses will focus on other HGT candidates having limited fungal distribution. Transcriptional profiling and functional characterization are underway to determine the significance of select HGT candidates on the fitness, virulence, and physiological responses of *Fv*.

**609F Repeat Induced Point Mutations: Driving genome divergence of the *Fusarium fujikuroi* species complex.** S. van Wyk¹, E. T. Steenkamp¹, B. D. Wingfield², L. De Vos², N. A. van der Merwe² 1) Department of Microbiology, University of Pretoria, Forestry and Agricultural Biotechnology Institute, Pretoria, Gauteng, ZA; 2) Department of Genetics, University of Pretoria, Forestry and Agricultural Biotechnology Institute, Pretoria, Gauteng, ZA.

The *Fusarium fujikuroi* species complex (FFSC) is a monophyletic assemblage of plant pathogens that threaten sustainable agriculture and forestry. Despite the growing number of genomes available for this complex, the genetic basis and genomic processes underlying species differentiation amongst its lineages remain poorly understood. In this study, we considered genome differentiation by investigating Repeat Induced Point mutation (RIP), which is generally regarded as an irreversible genome defense mechanism acting on duplicated sequences and transposable elements. For this purpose, we investigated the frequency, effect, distribution and genomic context of hyper-mutation associated with RIP in the genomes of four representatives of the FFSC (i.e., *F. circinatum*, *F. temperatum*, *F. fujikuroi* and *F. verticillioides*). The results of our in silico analyses showed that hyper-mutation patterns, indicative of RIP-like genome defense, were present in all the genomes analysed, although the extent and frequencies of mutation varied among genomes. Variable genomic regions such as telomeres and dispensable chromosomes also seemed to be more highly affected by RIP than the so-called core genomic regions. Here, the RIP targeted regions appeared to be distributed throughout core genomic regions. Comparison of the four genomes also revealed that signatures of RIP were more highly pronounced in *F. temperatum* and *F. fujikuroi*, to such an extent that the RIP affected regions mostly did not contain genes. This was in contrast to the situation in *F. circinatum* and *F. verticillioides*, suggesting that rapid sequence divergence and modification of codon usage allowed for the occurrence of genes in their RIP affected regions. Future research will seek to investigate the genetic and biological aspects contributing to this variation within this species complex.

**610W Population genetic structure and mycotoxin potential of the wheat crown rot and head blight pathogen, *Fusarium culmorum*, in Algeria, Italy, Australia and the United States.** I. Laraba¹, H. Boureghda¹, N. Abdallah¹, F. Obanor², A. Moreti³, D. M. Geiser⁴, A. C. Kelly⁵, S. McCormick⁵, R. H. Proctor⁵, T. J. Ward⁵, K. O'Donnell⁵ 1) Laboratoire de phytopathologie et de biologie moléculaire, Département de botanique, Ecole Nationale Supérieure Agronomique, Algiers, Algeria; 2) Grains Research and Development Corporation, Canberra, Australia; 3) Institute of Sciences of Food Production, CNR, Bari, Italy; 4) Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, Pennsylvania; 5) NCAUR-USDA-ARS, Peoria, Illinois.

Surveys for crown rot (FCR) and head blight (FHB) of Algerian wheat conducted during 2014 and 2015 revealed that Fusarium culmorum producing 3-acetyldeoxynivalenol (3ADON) and nivalenol (NIV) were the primary causal agents of these economically important diseases. Morphological identification of the *F. culmorum* isolates (n FCR = 91, n FHB = 15) was confirmed by sequencing a portion of TEF1. To assess mating type idiomorph composition, trichothecene chemotype potential and global population genetic structure, the Algerian strains were compared with *F. culmorum* collections from Italy (n = 27), Australia (n = 30) and the United States (n = 28). A PCR assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains were segregating in nearly equal proportions, except within Algeria where two-thirds of the strains were MAT1-2. An allele-specific PCR assay indicated that the 3ADON trichothecene genotype was predominant globally (83.8% 3ADON) and in each of the four countries sampled. Moreover, while significant differences in trichothecene genotype frequencies were observed in Algeria, Australia and the United States, none was detected in Italy. Fusarium culmorum strains produced 3ADON (n = 65), NIV (n = 14), or culmorin (n = 79) in vitro; however, 9 strains failed to produce detectable levels of toxins. Global population genetic structure of 191 strains was assessed using 9 microsatellite markers (SSRs). AMOVA of the clone corrected data indicated that 89% of the variation was within populations. Bayesian clustering analysis of the SSR data, using the admixture model with independent allele frequencies, identified two globally distributed, sympatric populations within which both trichothecene chemotypes and mating types were represented.
**611T Experimental evolution of *Fusarium oxysporum* reveals genome rearrangements as the major evolution mechanism governing short-term evolution.** *D.H. Ayhan*¹, *C. López Díaz*², *A. Di Pietro*², *L.-J. Ma*¹ 1) Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA; 2) Department of Genetics, University of Córdoba, Spain.

Natural selection is a fundamental evolutionary process that acts on changes in a genome and results in the adaptation of an organism. The most common forms of genome changes range from single nucleotide polymorphism (SNP), small insertion/deletion, segmental duplication, chromosomal rearrangement, to whole genome duplication. How do these different changes influence the evolutionary processes under different selection pressure? To explore answers to these questions, this study takes an experimental approach to observe evolving processes using a model organism *Fusarium oxysporum*, a highly adaptive species complex containing the mobile and lineage-specific (LS) chromosomes that are rich for transposons and determines host-specific pathogenicity. The same starting population (*F. oxysporum* f. sp. *lycopersici* strain 4287, a tomato pathogenic isolate) was passaged ten times through two distinct serial transfers in its host or on rich media plates. We have sequenced 5 evolved populations from each serial transfer at the end of the experiment. Comparative genomics revealed the presence of segmental duplications and deletions on parts of LS regions, suggesting that genome rearrangements are the major forces that shape short-term evolution. Retaining a relatively constant mutation rate, there is a much lower possibility to accumulate beneficial SNPs in short time frame. As expected, we observed few SNPs among these evolving populations. Interestingly, the populations that evolved in rich media tended to lose parts of or entire LS chromosomes, suggesting the dispensable nature and energy cost associated with these chromosomes when growing on a petri dish. These results highlight that different evolutionary constraints determine the outcome when populations are adapting to different environments.

**612F Chromosome plasticity during experimental evolution of the cross-kingdom pathogen *Fusarium oxysporum*.** *C. Lopez-Díaz*¹, *D. Hazal Ayhan*², *L.J Ma*², *A. Di Pietro*¹ 1) Department of Genetics, University of Córdoba, Cordoba, Spain; 2) University of Massachusetts, Amherst, USA.

Filamentous plant pathogens undergo rapid evolution, leading to expansions or shifts in host range. The *Fusarium oxysporum* species complex collectively causes vascular wilt disease in more than a hundred different crops. Meanwhile, clinical isolates of the fungus cause life-threatening systemic infections in humans. Remarkably, a single strain of *F. oxysporum*, Fol 42-87, is able to kill tomato plants, immunodepressed mice and larvae of the invertebrate animal model *Galleria*. The evolutionary mechanisms underlying host range dynamics remain poorly understood. Here we followed an experimental evolution approach involving serial passages of Fol 42-87 through tomato plants, axenic media plates, or *Galleria* larvae. Independently evolved populations obtained after ten consecutive passages through plants or rich media displayed notable phenotypic differences with respect to the initial clonal isolate, with four of the five plate-passaged populations showing significantly reduced virulence on tomato plants. Resequencing of the evolved populations revealed segmental duplications and deletions on transposon-rich accessory regions of the genome, including loss of entire chromosomes. These findings suggest that chromosome plasticity acts as a major evolutionary driver in *F. oxysporum*, and provide new insights into the genetic mechanisms underlying host adaptation in this cross-kingdom fungal pathogen.

**613W Adaptive Zn tolerance is associated with high expression of a CDF family transporter in the ectomycorrhizal fungus *Suillus luteus*.** *J. Ruytinx*, *L. Coninx*, *H. Nguyen*, *J. Colpaert* Hasselt University, Diepenbeek, BE.

*Suillus luteus* is a cosmopolitan fungal species, symbiotically associated with pine trees. In particular, in primary successions of pines this species is abundant and involved in seedling establishment. On severely metal-contaminated soils, Zn-tolerant *S. luteus* populations evolved by natural selection. Tolerant individuals effectively protect their host tree from Zn toxicity on these soils. However, the molecular-genetic mechanism underlying adaptive Zn tolerance in *S. luteus* is unknown. We hypothesize that the tolerance phenotype is due to an adaptation in the common Zn homeostasis network. By comparative and functional genetics we identified several *S. luteus* genes encoding transporters involved in Zn homeostasis. One of these transporters, SIzNt2, a CDF family transporter exhibits a differential gene expression among Zn-tolerant and Zn-sensitive phenotypes. Zn-tolerant *S. luteus* isolates show a high expression whereas Zn-sensitive isolates show a near to background expression of the gene. The difference in expression level seems to be partly caused by an extensive gene multiplication, partly by differences is cis-regulation. SIzNt2 copy number in Zn-tolerant isolates ranges between two and seven. Zn-sensitive isolates have one copy. Three different promoter genotypes were identified; one of these was exclusively found in Zn-tolerant isolates. SIzNt2 is predicted to be localized on the tonoplast and to move Zn from the cytoplasm into the vacuole. We could not confirm this function by heterologous expression in yeast. In conclusion, Zn-tolerant *S. luteus* isolates are selected for high expression of SIzNt2, a CDF family transporter of unknown function. Further functional analyses are required to define the role of high SIzNt2 expression in Zn tolerance.

**614T Genomic analysis reveals multiple endemic and pandemic lineages of rice blast and gene flow into recombining populations from southeast continental Asia.** *Pierre GLADIEUX*¹, *Maud THIERRY*², *Adrien RIEUX*³, *Sebastien RAVEL*², *Sandrine CROS-ARTEIL*¹, *Henri ADREIT*², *Joëlle MILAZZO*², *Elisabeth FOURNIER*¹, *Ryohei TERAUCHI*⁴, *Didier THARREAU*² 1) BGPI Research Unit, INRA, Montpellier, FR; 2) BGPI Research Unit, CIRAD, Montpellier, FR; 3) PVBMT Research Unit, CIRAD, La Réunion, FR; 4) Iwate Biotechnology Research Center, Kitakami, Iwate, JP.

The rice blast fungus *Magnaporthe oryzae* is the most damaging rice pathogen, and a textbook example of widely distributed, rapidly adapting pathogen, despite limited genetic diversity. The aim of our study was to elucidate the factors and evolutionary changes underlying the emergence, diversification and spread of *M. oryzae* in rice agrosystems. Analyses of population structure based on Infinium-genotyping of 5300 SNPs for 970 isolates collected on five continents identified three main pandemic lineages on *indica* and *japonica* rice, coexisting with multiple lineages with more restricted distributions in Asia and sub-saharan Africa. Analyses of recombination based on resequencing data for a subset of 89 isolates revealed that most lineages are highly clonal, although we found evidence for recombination in a widely distributed lineage infecting japonica and hybrid rice in Yunnan, Laos and Thailand. Because the sequenced isolates were collected between 1973 and 2009 and recombination is limited, we could use a phylogenetic approach to date the emergence and global dispersal of *M. oryzae* using dated tips to calibrate tree nodes. Our analysis provided an accurate estimate of the nucleotide substitution rate. Rice- and Setaria-infecting major lineages diverged ~10000 years before present, matching the oldest archaeological evidence for human exploitation of rice, and rice-infecting lineages radiated ~1200 years ago. Probabilistic and non-parametric 'chromosome painting' revealed gene flow
into multiple non-pandemic lineages -including the recombining lineages from southeast continental Asia- from multiple source populations. Gene sets matching putatively migrant mutations were functionally enriched in genes involved in non-self recognition and programmed cell death, suggesting a role of fungal immunity in increasing the genomic diversity of rice blast populations. Despite the general lack of polymorphism and the accumulation of deleterious mutations in rice-infecting lineages, we could identify regions with signatures of balancing selection (increased functional variation) that were functionally enriched in small secreted proteins and proteins involved in oxidation-reduction processes. Our work provides a population-level genomic framework for defining molecular markers to assist in the control of rice blast and for investigating the molecular underpinnings of phenotypic and fitness differences between divergent lineages.

**615F PgtSNP 3.0k chip: A high-throughput SNP genotyping array for *Puccinia graminis* f. sp. *tritici*.** Les J. Szabo¹, Jerry L. Johnson¹, Pablo Olivera², Yue Jin¹ 1) Cereal Disease Laboratory, USDA-ARS, St. Paul, MN; 2) Department of Plant Pathology, University of Minnesota, St. Paul, MN.

Understanding the genetic diversity and population structure of the wheat stem rust pathogen, *Puccinia graminis* f. sp. *tritici* has been hampered by the obligate, dikaryotic nature of this fungus and the lack of a robust high-throughput genotyping tools. A custom Illumina Infinium SNP array (PgtSNP 3.0k chip) was developed based on a *P. graminis* f. sp. *tritici* reference genome (U.S. isolate 75-36-700) and NGS sequence data from selected isolates. A balanced selection scheme was used to identify 3,000 candidate SNP loci evenly distributed (average 27 kb) along the assembled supercontigs, covering greater than 90% of the genome. These loci are partitioned between genic (84.1%) and intergenic (15.9%). This new array represents an expansion of the original PgtSNP 1.5k chip, based on GoldenGate technology. To test the performance of the PgtSNP 3.0k chip a set of 100 *P. graminis* f. sp. *tritici* isolates, derived from wheat stem rust collections from Africa, Asia, Europe, Middle East and North America, were used. In general, the *Puccinia graminis* f. sp. *tritici* isolates were grouped geographically, with the North American isolates forming two genetic clusters and the isolates from Africa, Asia, Europe and Middle East forming three genetic clusters. In addition, DNA extracted from ethanol-killed *P. graminis* f. sp. *tritici* infected wheat tissue (single pustule) was tested and shown to give similar results to DNA extracted from fresh samples (collected spores and infected tissue), thus allowing genotyping of a wider variety of samples. The PgtSNP 3.0k chip provides the wheat rust community a powerful genotyping tool for mapping the genetic landscape of *P. graminis* f. sp. *tritici*.

**616W Molecular analysis of killer DNA from *Neurospora Spore killer-2*.** N. Rhoades¹, A. Harvey¹, D. Samarajeewa¹, P. Manitchotpisit¹, J. Svedberg², D. Brown³, P. Shiu⁴, H. Johannesson², T. Hammond¹ 1) School of Biological Sciences, Illinois State University, Normal, IL; 2) Department of Organismal Biology, Uppsala University, Uppsala, Sweden; 3) National Center for Agricultural Utilization Research USDA-ARS, Peoria, IL; 4) Division of Biological Sciences, University of Missouri, Columbia, MO.

In standard Mendelian inheritance, each allele in a sexual cross has an equal probability of being transmitted to the next generation. However, there are certain "selfish" genes that are able to propagate themselves at a higher frequency than others in a population. Examples include the *Neurospora Spore killers* (Sk): *Spore killer-1* (Sk-1), *Spore killer-2* (Sk-2), and *Spore killer-3* (Sk-3). The work presented here focuses on the *Sk-2* element. Crosses of *Sk-2* x *Sk-3* (*Spore killer*-sensitive) produce asci with four black, viable ascospores and four white, inviable ascospores. The four surviving ascospores inherit the *Sk-2* element, resulting in a nearly 100% transmission of *Sk-2* to the surviving population. Because of this, *Sk-2* is transmitted in a non-Mendelian manner. Previous work has identified one gene and one locus involved in the spore killing mechanism, *rsk* (resistance to *Spore killer*) and *rfk-1* (required for killing). Here, we report the identification of a 1500 bp segment of DNA (designated *AH36*) from the *rfk-1* locus that correlates with a peculiar ascus abortion phenotype. Genetic evidence suggests that *AH36* encodes a transcript that must be expressed for ascus abortion to occur. Preliminary in-silico analyses suggests that *AH36* may encode a 39 amino acid protein that causes the ascus abortion phenotype. Site-directed mutagenesis and sub-cloning assays are in progress as part of an effort to test this hypothesis.

**617T Genomic diversity of *Malassezia* yeasts: implications for epidemiology and the clinic.** Teun Boekhout¹,², Anna Kolecka¹, Bart Theelen¹, Claudia Cafarchia³, Roberta Iatti³, 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.

**618F Population structure of Aspergillus flavus in soil and corn kernels from four different states.** *M.S. Molo*¹, *R. Singh*¹, *J.B. White*¹, *T. Isakeit*², *K.A. Wise*³, *C.P. Woloshuk*³, *B.H. Bluhm*⁴, *B.W. Horn*⁵, *R. Heiniger*¹, *I. Carbonw*¹ 1) North Carolina State University, Raleigh, NC; 2) Texas A&M University, College Station, TX; 3) Purdue University, West Lafayette, IN; 4) University of Arkansas, Fayetteville, AR; 5) United States Department of Agriculture, Agriculture Research Service, Dawson, GA.

*Aspergillus flavus* is a cosmopolitan soil-borne fungus that contaminates oil seed crops by producing carcinogenic aflatoxins (AFs). Ingesting AF-contaminated grains has been linked to hundreds of deaths in Africa. Due to the health risks AFs pose, the FDA has strict regulations on grain contaminated with AFs. Therefore, AFs have a huge economic impact on corn growers through management practices, yield loss, and adverse health effects. Previous reports indicate that certain *A. flavus* genotypes exist in the soil and others are specialized to colonize corn kernels, but this is based on sequence data from a relatively small number of genetic markers. Understanding the genetic differences that separate corn and soil isolates could lead to insights into the development of the next generation of biological control agents. We sampled *A. flavus* from soil early in the growing season and from kernels at harvest in Texas, North Carolina, Arkansas, and Indiana in 2013. This allowed us to examine genetic structure between populations from different states as well as between soil and kernel substrates. We sequenced a total of 246 isolates using double digest restriction-site associated DNA sequencing, which represents the genome in a manageable size by providing markers across each chromosome and the mitochondria. A preliminary analysis revealed that each state had native strains of *A. flavus* as well as shared evolutionary lineages. Further work will examine the distribution of genetic variation between soil and kernel populations.

**619W Influence of farming practices on the genetic diversity of Cercospora zeina in South Africa.** *DL. Nsibo*¹, *N. Kunene*¹, *I. Barnes*², *DK. Berger*¹ 1) Plant and Soil Sciences, FABI, University, Pretoria, Gauteng, ZA; 2) Genetics, FABI, University, Pretoria, Gauteng, ZA.

Grey leaf spot disease (GLS) is an important foliar disease of maize and a threat to global food security by causing up to 65 % yield losses. The increasing prevalence of GLS in Africa, Asia and the Americas is driven in part by increased conservation agriculture.
*Cercospora zeina* is the causal agent of GLS in Africa, and the disease is prevalent on both commercial and smallholder maize farms. Since the latter are more similar to conservation agriculture, with no chemical control and a wider diversity of genotypes within a field, we hypothesized that smallholder farming practices would select for distinct pathogen populations with greater diversity than large-scale commercial farms. Genotyping with 13 microsatellite markers was performed on 347 isolates from seven smallholder farms, each within a 50 km radius of commercial production areas. Only 61% of the 75 alleles obtained for these markers were shared with those identified from commercial farms. Although no population differentiation was observed between the farming systems, a slightly greater gene diversity was observed from smallholder farms (0.44 compared to 0.35) in KwaZulu Natal province, a local hotspot of GLS. Both mating type idiomorphs (*MAT1-1* and *MAT1-2*) were isolated from each field for both farming systems, and complied with the 1:1 mating type frequency at six out of seven sites screened. It is likely that dispersal mechanisms such as wind and the possibility of sexual recombination, influences the admixture of haplotypes into one large mega population across the region. Although a higher genetic diversity was observed under conservation agriculture conditions, the current data did not provide strong support for our hypothesis. Results from this study indicate that farming practices do not have an influence on the genetic diversity of *C. zeina* on maize in South Africa and that the same disease management strategies can be used for both smallholder and commercial farming systems.

**620T Evidence for sexual reproduction: identification, frequency and spatial distribution of Fusicladium effusum (pecan scab) mating type idiomorphs.** *Carolyn A. Young*¹, *Clive H. Bock*², *Nikki D. Charlton*¹, *Chakradhar Mattupalli*¹, *Kim M. Plummer*³, *Johanna K. Bowen*⁴, *Bruce W. Wood*² 1) Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, U.S.A; 2) USDA-ARS-SEFTNRL, 21 Dunbar Rd, Byron, GA 31008, U.S.A; 3) Department of Animal, Plant and Soil Sciences, La Trobe University, Melbourne, Australia; 4) The New Zealand Institute for Plant & Food Research Ltd, New Zealand.

*Fusicladium effusum* is the causal agent of pecan scab, the most prevalent disease of pecan (*Carya illinoinensis*) in the southeastern USA. Infection by the pathogen can result in serious and even catastrophic yield loss when conditions are favorable for an epidemic.
*Fusicladium effusum* is currently known only by its asexual (conidial) stage. However, the degree and distribution of genetic diversity observed within and among populations of *F. effusum* are typical of a sexually reproducing fungal pathogen, and comparable to other dothideomycetes with a known sexual stage, including the closely related apple scab pathogen, *Venturia inaequalis*. Using the mating type loci from *V. inaequalis* we identified a single mating-type gene, *mtAA* (*mat1-1*, containing alpha-box) idiomorph, in a draft genome of *F. effusum* flanked by two conserved genes encoding a DNA lyase (apnB) and an uncharacterised PH domain-containing protein. The mating type locus, spanning the flanking genes, was amplified and sequenced in 14 isolates, collected from different geographic locations and cultivars. Only seven of these samples contained the *mtAA* idiomorph, whereas the remaining samples contained the *mtBA* (*mat1-2*, containing HMG-box) idiomorph. A multiplex PCR screen was developed to amplify a conserved housekeeping gene (tubB), *mtAA* and *mtBA*, and used to screen 1203 *F. effusum* isolates collected from 13 pecan populations across the southeastern USA. A hierarchical sampling protocol representing region, orchard, tree and leaflet was followed at all sites so the mating type structure at different spatial scales could be assessed. Analysis of this collection revealed the frequency of the mating type idiomorphs is in a 1:1 equilibrium of *MTA:MTB*. In vitro paired matings resulted in the formation of pseudothecia only between isolates of the opposite mating types indicating that *F. effusum* is heterothallic. The apparent equilibrium of the mating type idiomorphs and *in vitro* mating provides impetus to a renewed effort to search for a sexual stage of *F. effusum*. Identification of a sexual stage will provide a fuller understanding of the disease epidemiology and pathogen population genetics, and eventually a basis for better management of existing resistance genes in pecan.
**621F Mating System and Mating Genes Evolution in *Suillus luteus*.** Y.-H. Ke¹, R. Debray¹, K. Hameed¹, A. Senchenkov¹, A. Rojas¹, H.-L. Liao², N. Nguyen³, P. Kennedy³, T. Bruns⁴, R. Vilgalys¹ 1) Duke University, Durham, NC; 2) University of Florida, Quincy, FL; 3) University of Minnesota, Minneapolis, MN; 4) University of California, Berkeley, CA.

The bolete genus *Suillus* is a highly diverse lineage of worldwide distributed ectomycorrhizal fungi (over 50 spp) genus with a worldwide distribution which that has emerge as a model to study ectomycorrhizal symbiosis with different host trees in the Pinaceae system. Mating pattern is an important character in fungi that deeply impacts affecting its population structure, ecology and evolution. Previous study of mating system studies in *Suillus* have been largely restricted to small scale mating experiments that arePrevious works indicate *S. luteus* and *S. granulatus* possessing tetrapolar mating systems. However, the traditional method for mating system is difficult to conduct in *Suillus* because basidiospores hardly germinate poorly on artificial media, and because dikaryotic mycelia don't do produce clamp connections after successful mating. Based on observed changes in mycelial morphology and nuclear cytology, Fries in the 1980's suggest that two species *S. luteus* and *S. granulatus* each have a tetrapolar mating system governed by two mating loci (A and B). However, sThus only mass morphology and cytology are used to conclude mating results before. Strong supports from microscopic and molecular-based information are were not available. In the other hand, former mating system studies in *Suillus* are restricted to small scale mating experiments from a few fruiting bodies. Since *S. luteus* had a rapid habitat expansion within hundreds of years by human activities, by mating gene polymorphism sampling across worldwide populations, we are able to identify gene evolution and allele frequency changes along the process.

InAs an alternative to traditional approaches that rely on pairing of single-spore isolates this research, we introduced developed a new sequencing-based method to easily obtain information on determining mating gene haplotypes and mating system in *S. luteus* based on next-gen sequencing. From By comparing genome data for the A mating locus from 6 species (available at JGI mycocosm website), we designed six primer pairs to that successfully amplify 900-1300 bp fragments spanning the A mating locus. separate mating A loci to 900-1300 bp long fragments. By employing Using NGS techniques, we are able to rebuild reconstruct the two haplotypes of mating gene haplotypessi, and infer patterns of mating allele segregation among sibling progeny and fruit bodies each individual. In the future, we will These data are being used to use them to study the life history and mating biology in of *S. luteus and other Suillus* species. In the first part, we will inoculate in inoculated basidiospores from each spore prints onto one pine seedling roots and allow them to germinate and form mycorrhizae with their pine host through random mating. random mating. Dikaryotic mycelia forming on roots are sequenced to determine genotypes then and to infer inferring mating system. In the second part, we will survey population level mating gene diversity of *S. luteus* to indicate mating gene evolution and allele frequency change during its rapid expansion history across different geographic populations.

**622W wtf causes infertility via a poison-antidote system.** Maria A. Bravo Nunez¹, Nicole L. Nuckolls ¹, Michael T. Eickbush¹, Janet M. Young², Gerald R. Smith², Harmit S. Malik²,³, *Sara H. E. Zanders*¹ 1) Stowers Institute for Medical Research, Kansas City, MO; 2) Fred Hutchinson Cancer Research Center, Seattle, WA; 3) Howard Hughes Medical Insitute, Chevy Chase, MD.

Meiotic drivers are genomic parasites that act selfishly to promote their own transmission into more than half of the gametes (e.g. sperm) generated by heterozygotes. In this work, we identify *Schizosaccharomyces kambucha wtf3* as the first described meiotic drive gene in a yeast species. *wtf3* belongs to the class of 'ultra-selfish' meiotic drivers because it acts by destroying the gametes that fail to inherit the gene. This gamete destruction increases *wtf3* transmission into up to 96% of gametes generated by a heterozygote, while cutting fertility of by nearly 50%. To enact drive, *wtf3* encodes two largely overlapping proteins. The short protein is a trans-acting gamete-killing poison that fills the cytoplasm surrounding and can enter gametes. The long protein is an antidote that becomes enriched only in the gametes that encode *wtf3*, thereby rescuing them from destruction. *wtf3* is a member of the previously uncharacterized large *wtf* gene family. Gamete killing ability is shared by other *wtf* genes, suggesting meiotic drive is the ancestral function of the family. This work provides insight into how meiotic drivers function, shape genome evolution and cause rapid speciation.

**623T Population genomic and transcriptomic analysis of the brown rot fungus Fomitopsis pinicola.** Nils Högberg, Prakash Kancherla, Jan Stenlid, Mikael Brandström Durling Forest mycology & plant pathology, Forest mycology & plant pathology, Uppsala, SE.

The basidomycete Fomitopsis pinicola is an effective wood decomposer and one of the most common brown rot fungi. It is part of a species complex with several closely related species. We have sequenced 28 haploid isolates of this fungus in order to use population genomic methods to reveal footprints of selection especially with respect to wood decay mechanisms. One isolate was sequenced with a combination of Illumina and SOLiD sequencing technology. The resulting genome sequence was 45MB and 13,888 gene models were predicted. A comparison with a previously sequenced *F. pinicola* isolate showed that their CAZyme profiles were relatively similar while the secondary metabolite clusters differed substantially between the two isolates. When *F. pinicola* transcriptomes were compared between a glucose medium and wood decomposition several CAZymes, oxidative enzymes, transporters, lipases and proteases were among the groups of genes which were up-regulated during wood decomposition. The most highly up regulated gene was a copper-dependent lytic polysaccharide monooxygenase. The gene expression pattern was similar to that of other brown rot fungi but differed in important aspects for example with respect to iron reduction and oxalate metabolism.

SNPs from a total of 28 Swedish *F. pinicola* genomes were used to identify selection and for genome wide association of markers linked to the phenotypic trait wood decomposition which differed among the sequenced isolates. Footprints of selected sweeps with elevated levels of linkage disequilibrium were predominantly found around genes involved in secondary metabolite production, transportation and chitin production. When the phenotypic trait wood decay was associated with the genome wide SNP-matrix, markers close to a quinate permease and genes involved in oxidoreductase activity, transportation, alcohol metabolism were linked to this trait.

In conclusion, our study found that several groups of genes which were highly up-regulated during wood decomposition also were associated to the wood decay trait in a GWAS analysis, among these were genes which may be linked to the Fenton reaction. Footprints of selected sweeps were mostly found for genes involved in secondary metabolism. Secondary metabolites have been suggested to act as competitive weapons used against other fungi and their rapid evolution may be a factor behind the success of the *F. pinicola* species complex in conifer forests.
**624F** *Mycosarcoma*, a resurrected generic name for corn smut (Ustilago maydis). *D. Begerow*¹, A. McTaggart², R. Shivas³ 1) Geobotany, Ruhr-Universität Bochum, Bochum, DE; 2) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 3) Plant Pathology Herbarium, Biosecurity Queensland, Department of Agriculture and Fisheries, Queensland, Australia.

Ustilago is a polyphyletic genus of smut fungi found mainly on Poaceae. The development of a taxonomy that reflects phylogeny requires subdivision of *Ustilago* into smaller monophyletic genera. Several separate systematic analyses have determined that *Macalpinomyces mackinlayi*, *M. tubiformis*, *Tolyposporella pachycarpa*, *Ustilago bouriquetii* and *U. maydis*, occupy a unique phylogenetic position within the Ustilaginaceae. A previously introduced monotypic generic name typified by *U. maydis*, *Mycosarcoma*, is available to accommodate these species, which resolves one component of polyphyly for *Ustilago* s. lat. in Ustilaginaceae. An emended description of *Mycosarcoma* is provided to reflect the morphological synapomorphies of this monophyletic group. A specimen of *Ustilago* maydis that has had its genome sequenced is designated as a genotype for this species. Taxonomic stability will further be provided by a forthcoming proposal to conserve the name *Uredo maydis* over *Lycoperdon zeae*, which has priority by date, in order to preserve the well-known epithet *maydis*.

**625W** **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*.

**626T** **Comparative genomics of hybrids reveal potential host specificity genes in Microbotryum.** Britta Büker¹, Michael E. Hood², Andreas Brachmann³, Sven Rahmann⁴, *Dominik Begerow*¹ 1) Geobotanik, Ruhr-Universität Bochum, Bochum, DE; 2) Department of Biology, Amherst College, Amherst, MA, USA; 3) Genetics, Faculty of Biology, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany; 4) Genominformatik, Institut für Humangenetik, Medizinische Fakultät, Universität Duisburg-Essen, Essen, Germany.

The evolution of obligate plant pathogens like smut fungi is often characterized by lineage tracking, resulting in host specificity and one-to-one relationships. Therefore, adaptation and specialization to the host plant seems to be crucial and should involve genes or regulatory pathways governing host specificity. To identify genes relevant for host specificity of *Microbotryum* species, we produced artificial hybrids between the two host-specific species *M. lychnidis-dioicae* and *M. silenes-acaulis* and applied strong experimental selection on different host plants to identify genes necessary for successful infections.
Genome comparison of the two species revealed that most gene families are shared and the majority of genes are conserved, indicating very similar biological features of both species, including host adaptation and infection processes. Lower nucleotide identity of genes encoding for secreted proteins might indicate their importance for host specific interaction, as it is known from other plant pathogens.
Moreover, we identified 211 candidate genes that occur in each hybrid and backcross genome that were posed under host-driven selection and might therefore play a crucial role in host specialization. The analysis of hybrid genomes also demonstrates the effect of genetic homogeneity on the fitness of hybrid individuals including the occurrence of species-specific mating type chromosomes.
In conclusion, the combination of comparative genomics with experimental selection and hybridization is a promising way to identify potential host specificity factors. Our data suggest that only a limited set of genes is required for successful infection, but also demonstrate the strong influence of intra-genomic conflicts on the viability of hybrids.

**Synthetic Biology**

**627F** **Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced Gene Editing.** *J. Weber*¹,², V. Valiante³, C.S. Nødvig⁴, D.J. Mattern¹,², R.A. Slotkowski¹,², U.H. Mortensen⁴, A.A. Brakhage¹,² 1) Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI) –, Jena, Germany; 2) Institute of Microbiology, Friedrich Schiller University Jena, Germany; 3) Leibniz Research Group – Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany; 4) Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.

The human pathogenic fungus *Aspergillus fumigatus* is known to produce various spore-borne natural products. One of these is the polyketide trypacidin which has been shown to be involved in the interactions with alveolar macrophages as well as with the amoeba *Dictyostelium discoideum*. Even though recent studies could elucidate the corresponding gene cluster in *A. fumigatus*, it still remained elusive why several isolates do not produce trypacidin. We addressed this question employing a CRISPR/Cas9-based gene editing strategy. Thus, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and also
reconstitute its production in a nonproducing strain. In addition, we developed a split-marker approach for the selection of edited strains, since the selectable marker could not be directly linked to the target site. The here established tool could be useful in next generation fungal genetics e.g. for the investigation of single nucleotide polymorphism, or amino acid substitutions.

**628W Identification of gene targets for improved heterologous enzyme production in Aspergillus niger.** M.C. Reilly¹, J. Kim¹, J. Lynn¹, J.M. Gladden¹,², J.K. Magnuson¹,³, S.E. Baker¹,³ 1) Joint BioEnergy Institute, Emeryville, CA; 2) Sandia National Laboratory, Livermore, CA; 3) Pacific Northwest National Laboratory, Richland, WA.

Efficient and economical deconstruction of biomass is critical for the success of lignocellulosic biorefineries. The use of ionic liquids (ILs) in the pretreatment of biomass leads to increased biomass saccharification efficiency at lower cellulose loadings. However, some ILs can inhibit the activity of commercial cellulases and must be removed from the biomass prior to the application of the lignocellulosic enzyme cocktail, a costly additional step in the conversion process. To overcome this issue, cellulolytic enzymes that can maintain function in the presence of ILs have been identified. Many of these enzymes originate from bacterial species that are difficult to cultivate in the laboratory. One possible solution are filamentous fungi, which have been widely utilized for enzyme production in industry. Here, a forward genetics approach was utilized to increase heterologous enzyme production in *Aspergillus niger*: a strain engineered to secrete an IL-tolerant bacterial beta-glucosidase (BG) was mutagenized and the resulting progeny screened for increased BG activity. Subsequent full-genome sequencing revealed several loci associated with the hyper-production of the heterologous enzyme. Deletion of one of these, a putative sugar transporter, results in a doubling of heterologous BG enzyme activity detected in culture supernatants. Future studies will further characterize the role of this sugar transporter in heterologous enzyme production.

**629T An in-silico reconstructed gene regulation network for Aspergillus niger for the prediction of protein functions.** Sascha Jung, Norman Paege, Paul Schäpe, Vera Meyer Institute of Biotechnology, Department Applied and Molecular Microbiology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.

The genome of *A. niger* belongs to the best annotated genomes among *Aspergillus* species; however, only 2% of its ~14,000 genes are functionally verified and 50% of the predicted open reading frames encode for hypothetical proteins. Hence, the genetic basis for almost all cellular processes in *A. niger* and its physiological peculiarities is unknown. On the other hand, hundreds of post-genomic data including transcriptomic and proteomic data are available for *A. niger* for more than 150 different growth conditions. This holistic dataset can be scrutinized and used to predict gene functions and gene interactions. For a proof-of-concept, we have focused on the *anafp* gene encoding the antifungal protein AnAFP known to selectively inhibit the growth of filamentous fungi. As more than 50 AnAFP orthologs have been identified in many different genera of the *Ascomycota* tree of life, we wished to understand which regulatory systems control expression of the *anafp* gene.

Our analysis uncovered that *anafp* displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we could demonstrate that the *anafp* promoter is indeed under control of CreA and FlbA as predicted by the *in silico* data. A co-expression network analysis further predicted that *anafp* expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators (1). We currently prove these predictions by respective wet-lab experiments. First results match the expected knock out phenotype in *A. niger*.

(1) Paege N, Jung S, et al. A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in *Aspergillus niger*. PloS One 2016; DOI:10.1371/journal.pone.0165755.s009

**630F Genome wide consequences of the deletion of the Aspergilli non-homologous end joining (NHEJ) DNA repair mechanism.** I. Álvarez-Escribano¹, C. Sasse², J. Woo Bok³, A. Lipzen⁴, W. Schackwitz⁴, J. Marin⁴, K. Barry⁴, I. Grigoriev⁴, A.T. Marcos¹, N.P. Keller³, G.H. Braus², D. Canovas¹ 1) Department of Genetics, Faculty of Biology, University of Seville, Spain; 2) Department of Molecular Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University, Göttingen, Germany; 3) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 4) DOE Joint Genome Institute, Walnut Creek, California, USA.

The relevance of *Aspergillus* for human daily life is immeasurable: not only because of its industrial applications but also because of its clinical implications. Most *Aspergillus* laboratories world-wide use mutants in the non-homologous end joining (NHEJ) pathway (*KU70* or *KU80*) to do genetic modifications in the strains under study due to a higher frequency of homologous integrations after transformation of DNA constructs. *KU70* is involved in a DNA repair mechanism highly conserved in eukaryotes, and genetic manipulations are much faster in NHEJ mutant (?KU70) strains in many fungal species.

Three *Aspergillus* species were selected for this study to allow for comparative genetics and genomics: *A. flavus*, *A. fumigatus* and *A. nidulans*. Wild-type and NHEJ mutants were grown on solid media for 60 growth passages using asexual spores (ca. 3,000 mitosis). In the case of *A. nidulans* ten passages with sexual cleistothecia were also independently performed. Genome sequences were obtained and analyzed. In both *A. flavus* and *A. fumigatus*, the *KU70* mutants accumulated on average more mutations than the wild type strains (8.37 +/- 1.80 vs 6.12 +/- 1.76 in *A. flavus*, and 5.75 +/- 2.33 vs 4.12 +/- 1.76 in *A. fumigatus*). The higher number of non-synonymous mutations in *A. fumigatus* and of mutations in the intergenic regions in *A. flavus* in the *KU70* mutants was statistically significant. None of the sexual passages of the *A. nidulans KU70* strain could be completed due to the lack of cleistothecia formation, while the majority of the wild type passages reached ten passages. Collectively here we provide an assessment of the effects of the NHEJ pathway in the genomic stability in Aspergilli.

**631W CRISPR/Cas9 systems can be used to manipulate gene function in the thermally dimorphic fungus Histoplasma capsulatum.** Saori Amaike Campen, Sinem Beyhan J Craig Venter Institute, La Jolla, CA.

The thermally dimorphic fungus, *Histoplasma capsulatum* is a causal agent of the severe pulmonary and systemic human disease histoplasmosis. *H. capsulatum* can cause disease in both immunocompromised and immunocompetent individuals and is endemic in the
U.S. It estimated that over twenty-five thousand individuals acquire life-threatening infections each year with up to 50% mortality rates. The pathogen grows in the filamentous form in soil. Upon inhalation by a mammalian host, the fungus switches its morphology from filamentous form to yeast form, and continues to grow intracellularly within host macrophages. Despite its high impact on human health, there is limited research done in *H. capsulatum* due to the limited reverse genetic tools available for this organism. Targeted allelic replacements have been performed in *H. capsulatum* but their success varies greatly among *H. capsulatum* strains. Importantly, the highly virulent North American G217B strain remains largely refractory to gene knockouts by targeted allelic replacements. To study gene function, we frequently utilize RNA interference and generate knockdown mutants in the highly virulent G217B strain; however, the available tools often result in unstable phenotypes over time due to insufficient knockdown levels, or integration or eventual loss of the knockdown plasmid. In this study, our goal is to develop robust gene editing technologies to manipulate gene function in *H. capsulatum*. Recently, cutting-edge gene editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system have been used successfully to manipulate gene function in filamentous fungi. Here, we employ this technology to target two well-characterized genes in *H. capsulatum*: *URA5*, encoding an orotidine-5'-monophosphate pyrophosphorylase that is required to grow in the absence of uracil and *RYP1*, encoding a transcription factor that is required for yeast-phase growth in *H. capsulatum*. Our preliminary results show that the transformants carrying a *Cas9-URA5* construct exhibit uracil auxotrophy and the transformants carrying a *Cas9-RYP1* construct exhibit filamentous phenotype at 37°C. We are currently quantifying the frequency of these phenotypes and optimizing the efficiency of the CRISPR/Cas9 systems in *H. capsulatum*. Overall our results suggest that CRISPR/Cas9 systems can be used to manipulate gene function in *H. capsulatum*.

**632T A Universal Expression System for Protein Production.** Mari Valkonen, Anssi Rantasalo, Salla Koskela, Marika Vitikainen, Jussi Jäntti, Christopher Landowski, Dominik Mojzita Industrial Biotechnology, VTT Technical Research Centre of Finland Ltd., Espoo, Finland.

*Trichoderma reesei* is a filamentous fungus that is used world-wide as a host for industrial enzyme production. The enzymes produced are used, for example, in pulp and paper production, in the food and feed industries, and in the textile industry. *T. reesei* enzymes are also increasingly important because of their ability to turn lignocellulosic biomass into sugars that can be used to produce biofuels and chemicals. *Pichia pastoris* is another well-known protein production organism important for industrial enzymes and biopharmaceuticals. The strongest promoters used in both of these systems typically require the use of inducing molecules, which create limitations and generate extra cost in the process. To simplify and improve the production process we have employed a novel orthogonal expression system based upon a synthetic transcription factor (sTF) that regulates expression of the target gene via a sTF-dependent promoter. The great benefit is that new expression allows for high level, constitutive expression in diverse growth conditions and growth stages. Multiple sTF-binding sites can be designed to enable selection of a wide range of target gene expression levels, from very low to extremely high. The same system functions in a wide spectrum of eukaryotic microorganisms providing a simpler genetic toolbox for host strain construction. We have taken advantage of this unique expression system to improve native and heterologous protein production in the industrial microbes *T. reesei* and *P. pastoris*. We have been able to produce high levels of secreted proteins without the use of inducing molecules, which are typically used for these production organisms. For instance, with *T. reesei* we could produce the native CBH1 cellulase at high levels when grown in glucose medium. The new universal expression system allows for simpler media and bioprocesses to be used for industrial protein production.

**633F Adapting filamentous fungi to an automated environment for the application of a Design-Build-Test-Analyze-Learn approach to strain optimization.** K.S. Bruno, E. Szewczyk, K Rothschild-Mancinelli, P.J. Westfall Zymergen, Inc, Emeryville, CA.

Identifying, understanding and ultimately engineering the complex set of interacting genes responsible for any cellular phenotype is extremely difficult and traditionally requires years of study. For example, optimizing metabolic flux to a native or non-native small molecule, or increasing the diversity of carbon sources that a microbe can use are well studied topics and yet very few novel compounds have made it through the high risk path to commercialization. To address this problem, Zymergen has built a platform for automated and high-throughput microbial strain improvement that is focused on improving the economics of large-scale fermentation processes and accelerating the industrialization of novel bio-products. The platform has been designed to be flexible, highly reliable, and host-agnostic.

Filamentous fungi are sought after as production hosts for their ability to tolerate a variety of feedstocks as well as their capacity for high production of enzymes and other small compounds. However, adapting fungi to modern automation platforms for high throughput manipulation poses some unique challenges. Large scale, routine preparation of protoplasts, automated liquid handling during transformation, reduction of heterokaryon formation and minimizing cross-contamination with volatile spores are just some of the engineering challenges that need to be overcome to ensure successful strain improvement outcomes. Here we describe the adaptation of Aspergillus niger to an automated liquid handling and data capture environment that allows large scale implementation of a Design-Build-Test-Analyze-Learn cycle for strain improvement and pathway engineering.

**634W A CRISPR-Cas9 platform for Genetic Engineering of Diverse Filamentous Fungi.** Matthew Entler¹,², Peter Meidl¹,², Jessy Labbé¹,² 1) Biosciences Division, ORNL, Oak Ridge, TN; 2) University of Tennessee, Knoxville, TN 37996.

Fungi represent one of the most essential groups of organisms on the planet, playing vital roles in the biosphere. The development of effective methods for genetic engineering of fungi over the past two decades has demonstrated the importance of such tools to alter both the detrimental and beneficial activities and metabolic processes of fungi. Nonetheless, detailed knowledge into the molecular biology and biochemistry is only available for a few fungal models. Full genome sequencing efforts (e.g., 1KFG project) are rapidly increasing the ability to explore the fungal kingdom beyond the handful that are currently available. However, the sparsity of Basidiomycete and Zygomycete biochemical knowledge can be partly explained by a lack of well-developed genetic tools which are hampered by low gene targeting frequencies. Therefore, we established an easy and versatile fungal genetic engineering platform that works for all major phyla (i.e., Ascomycetes, Basidiomycetes and Zygomycetes) and based on the CRISPR-Cas9 system that has been successfully adapted to a wide range of organisms. We created modular CRISPR-Cas9 vectors for directed mutagenesis across multiple fungal phyla and optimized versatile fungal CRISPR-Cas9 gene-editing methods. Development of these tools is critically necessary to position ORNL at the cutting edge of systems and synthetic biology. The versatility of our system of assembled vectors, which contain a cas9 gene codon optimized for
fungi, three promotors, different selections markers and a GFP-reporter, is being tested into four fungal species representatives of Ascomycetes (Trichoderma reesei), Basidiomycetes (L. bicolor and Lactarius psammicola) and Zygomycetes (Mortierella elongata) phyla. Here, we will show that it is possible to introduce defined point mutations in specific genes using this versatile system in several genes and discuss efficiencies.

**635T 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.

Other

**636F Molecular mechanisms of demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. teres.** Wesley Mair¹, Weiwei Deng¹, Jonathan Mullins², Samuel West², Penghao Wang³, Naghmeh Besharat¹, Simon Ellwood¹, Richard Oliver¹, Francisco Lopez-Ruiz¹ 1) Department of Environment and Agriculture, Centre for Crop and Disease Management, Curtin University, Bentley, WA, Australia; 2) Institute of Life Science, School of Medicine, Swansea University, Swansea, UK; 3) School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA, Australia.

*Pyrenophora teres* f. sp. *teres* is a necrotrophic fungal pathogen and the causative agent of net form of net blotch (NFNB) that, together with spot form of net blotch, is one of the most important diseases of barley (*Hordeum vulgare*). Net blotch diseases are mainly controlled using site-specific systemic fungicides. Unfortunately, disease management has been compromised by the emergence of fungicide resistance. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor (DMI) resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of DMI fungicides. A novel mutation in the gene encoding the DMI target enzyme CYP51A was present only in resistant strains. This amino acid substitution F489L was orthologous to F495I in *Aspergillus fumigatus* and F506I in *Penicillium digitatum*. Structural in silico modelling of CYP51A proteins docked with different DMI fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two *Cyp51A* paralogs and of the *Cyp51B* gene. Under fungicide induction, expression of *Cyp51A1*, *Cyp51A2* and *Cyp51B* was higher in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. Analysis of simple-sequence repeat markers indicated that the allele derives from a single mutational event, with the mutant allele subsequently recombining into different genetic backgrounds. A digital PCR assay has been developed for the high-throughput detection of mutant populations in the field.

**637W NSG2 (ORF19.273) encoding protein controls sensitivity of Candida albicans to azoles through regulating the toxic C14-methylated sterols synthesis.** Q.Z. Lv, L. Yan, Y.Y. Jiang School of Pharmacy, Second Military Medical University, Shanghai, China.

*Candida albicans* is the major opportunistic pathogen that causes serious candidemia, candiduria or organic abscesses in the immunocompromised individuals. Currently, azoles are the most common used antifungal drugs in clinic which is targeted the rate-limiting enzyme of the ergosterol biosynthesis, Erg11p (lanosterol demethylase). But as a consequence of prophylactic use and prolonged treatment regimens, the azoles resistance in *C. albicans* has become a thorny problem. Regardless of mammals or yeast, cells regulate sterol homeostasis by multiple feedback mechanisms through transcriptional and posttranscriptional mechanisms. The transcriptional regulation of the enzymes involved in the biosynthesis of ergosterol in *C. albicans* is mainly dependent on the zinc cluster transcriptional factor Upc2p, which is induced by the ergosterol depletion. On the other hand, the level of sterol could be controlled by insulin-induced genes (INSIGs) interacting directly with proteins containing sterol-sensing domains (SSDs) in a posttranscriptional manner in *Saccharomyces cerevisiae*. But the post-transcriptional regulation of the enzymes in *C. albicans* has been demonstrated rarely. Through homologous comparison, we identified that C3_02820C_A (ORF19.273), containing the INSIG domain, is homologous to *NSG2* in *S. cerevisiae* and therefore we designated it as *CaNSG2*. Using the strategy of fusion PCR and auxotrophic markers, we obtained the null mutant of *NSG2*. In contrast with the functions of NSGs in *S. cerevisiae*, *NSG2*-deficient *C. albicans* strain only improved the sensitivity to azoles and showed no difference to other stresses compared with its parental strain SN152. Hypersensitivity of *NSG2Δ/Δ* to azoles was caused by the imbalance of sterols, especially the increase of toxic eburicol and obtusifoliol. Finally, we concluded that *NSG2* is responsible for reducing the aberrant sterols with a C-14-methyl group and maintaining its resistance to azloes in *C. albicans*. Our study gives a new insight to understand the toxic sterols synthesis in *C. albicans* and the roles of sterols homeostasis in resistance to azoles stress.

**Methods:** *NSG2* in *C. albicans* is disrupted by the strategy of HIS1-ARG4-LEU2 auxotrophic markers and two alleles were disrupted with the *Candida maltosa* LEU2 and *Candida dubliniensis* ARG4 marker. To determine the drug sensitivity, we used the spot assay method that plated serial concentrations of different strains on the YPD agars containing the indicated agents. The transmission electron microscopy was used to observe the membrane changes of yeast cells in response to fluconazole. The obtained cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4°C for 24 h. Quantitation of the percentage of ergosterol was determined by the gas
chromatography mass spectrometer (GC-MS) analysis. The total intracellular ergosterol amount was quantified in the presence and absence of fluconazole and the percentage of different sterols was calculated. The yeast cells were treated with solutions that contains 15% NaOH dissolved in 90% ethanol and reacted in 80% for 1h. Petroleum ether (boiling range 30 ~ 60 °C) was added to the extract for 3 times, and the combined extractions was washed with ethreum ether, ether layer was then evaporated in the 60 °C water bath. Systemic murine candidiasis model was used to evaluate the virulence of different strains. The BALB/C mice were injected with 5×10⁵ C. albicans cells through tail vein.

**Results:**

**Verification of ORF19.273 disruption in C. albicans**

*C. albicans* ORF19.273 is the only ortholog of the mammalian INSIG genes, which is homologous to *NSG2* in *S. cerevisiae*. Although ORF19.273 is significantly diverged from *S. cerevisiae* *NSG2* with only 27% identity throughout, we designated it as *NSG2* and predicted it to be a sterol-biosynthetic-process-related gene. So we used the nutritional markers of HIS1-ARG4-LEU2 and fusion PCR method to acquire homologous recombination fragments. Replacements of the *NSG2* alleles with fusion PCR fragments were verified by PCR. The *nsg2* null mutant strain (*NSG2Δ/Δ*) and the re-introduction strain (*NSG2Δ/Δ+* *NSG2*) were then obtained.

We then investigated the growth rate, hyphae and biofilm formation of *nsg2* null mutant. Our results indicated that *NSG2* has no influence on the proliferation and morphological transformation of *C. albicans*.

**The deficiency of NSG2 specifically increased sensitivity of C. albicans to azoles**

In *S. cerevisiae*, the disruption of *NSG1* and *NSG2* leads to an increased sensitivity to lovastatin. Statins are widely used drugs in the treatment of human hyperlipemia, which reduce the cholesterol levels through a competitive inhibition of HMG-CoA reductase. Lovastatin (8µg/ml) strongly inhibited the growth of SN152 and *NSG2Δ/Δ* equally. This is a significant difference between the functions of INSIGs in *S. cerevisiae* and *C. albicans*. There are the other three classes of antifungal drugs targeted to the sterol synthetic pathway: allylamines (terbinafine, butenafine) targeted to the squalene epoxidase (Erg1p), triazoles (fluconazole, ketoconazole, etc) targeted to the lanosterol 14-alpha-demethylase (Erg11p) and morpholines (amorolfine, fenpropimorph) targeted to C-8 sterol isomerase (Erg2p) and C-14 sterol reductase (Erg24p). So we examined the sensitivity of *NSG2Δ/Δ* to these drugs. In contrast with the stains of *NSG2 Δ/Δ* in *S. cerevisiae*, there are no differences in the sensitivity to lovastatin between the NSG2-dificient stain and SN152. However, the sensitivities of azole drugs were significantly different between the NSG2-disrupted and SN152. [a1] Using two different clones *NSG2Δ/Δ*-1 and *NSG2Δ/Δ*-2, we concluded that *NSG2* is essential in *C. albicans* in maintaining the resistance to azoles specifically. The sensitivities to the upstream or downstream inhibitors of Erg11p (terbinafine, fenpropimorph) showed no difference between the wild type and *NSG2Δ/Δ* strains. From this point, it is possible that *NSG2* interacts with Erg11p in response to azoles.

**The depletion of NSG2 destructed the cell membrane integrity of C. albicans**

Ergosterol plays an important role in cell membrane integrity, permeability and cell polarization. To verify the integrity of plasma membrane, transmission electron microscopy was performed to observe the ultra-structure changes of SN152 and *NSG2Δ/Δ* strains in the presence or absence of fluconazole. In the absence of fluconazole, the integrant *NSG2Δ/Δ* displayed slightly damage in plasma membrane. This effect was amplified in the presence of 8 µg/ml of fluconazole. The cell membranes of the SN152 strain modestly damaged and a small amount of cell contents leaked into the gap between cell membrane and the cell wall when treated with 8 µg/ml of fluconazole. In contrast, the membranes of *NSG2Δ/Δ* treated with 8 µg/ml of fluconazole seriously collapsed and bulks of the cell contents leaked into the gap. These results indicated that the major function of *NSG2* is to maintain the integrity of the plasma membrane, especially in response to azoles.

The biosynthesis of ergosterol effects the polarization of *C. albicans* directly. We stained the initial hyphae of *NSG2Δ/Δ* with filipin, which combines with ergosterol specifically. It is indicaed that *NSG2* polarizes normally with an ergosterol-rich domain between the yeast cell and hyphae.

**The deficiency of NSG2 increased the accumulation of the toxic C14-methylated sterols**

The fungistatic function of azoles is caused by the reduction of ergosterol in the membranes and the accumulation of the toxic sterols, which disturb the fluidity and permeability of the plasma membrane. We measured the compositions of sterols by gas chromatography-mass spectrometer (GC-MS) analysis. In the absence of fluconazole, the major sterol is still ergosterol in both SN152 and *NSG2Δ/Δ* strains. But the percentage of sterols located in the upstream of Erg11p was a little higher in *NSG2Δ/Δ*, which included obtusifoliol and 14α-methylfecosterol. What's more, these sterols accumulated in *NSG2Δ/Δ* always contained a 14α-methyl group and this structure is fungistatic for yeast. These data suggest that *NSG2* plays a role in regulating the balance of different sterols and this function is more obvious in *C. albicans* in response to fluconazole. When treated with 8µg/ml of fluconazole, the percentage of ergosterol in SN152 and *NSG2Δ/Δ* has decreased to about 4% and some sterols with a C14-methyl group increased obviously. There is a significant difference in the distribution of sterols with 14α-methylation. Fluconazole inhibited the activity of lanosterol 14-alpha-demethylase (Erg11p) and induced the accumulation of lanosterol obviously in SN152. When *NSG2* is disrupted, the proportion of eburicol, obtusifoliol and 14α-methylfecosterol significantly enhanced. In consistent with this, the percentage of lanosterol in *NSG2Δ/Δ* is lower compared with SN152. When using the squalene epoxidase inhibitor terbinafine, the distinctions of distribution and compositions of sterols in either SN152 or *NSG2Δ/Δ* was not obvious. Finally, we concluded that the specific hypersensitivity of *NSG2Δ/Δ* to azoles is attributed to the slight reduction of ergosterol and the accumulations of toxic sterols with 14α-methyl group.

The regulation of *NSG2* on the content of sterols is not dependent on the ERG genes transcription, as the transcriptional levels of *ERG1*, *ERG7*, *ERG11*, *ERG2*, and *ERG3* had no change in the *NSG2Δ/Δ* strain.

**The disruption of NSG2 in C. albicans increased efficacy of fluconazole treatment in a murine systemic candidiasis.**

To investigate the effects of *NSG2* in maintaining the virulence of *C. albicans* in murine, we compared the survival rate and the renal fungal burden in BALB/C mice. Without treatment with fluconazole, the survival curve of *NSG2Δ/Δ* is similar with that of SN152 and the reintroduction strain. In order to evaluate the in vivo sensitivities of *NSG2Δ/Δ*, the BALB/C mice was delivered with 2mg/kg of fluconazole for 7 days following intraperitoneal injection after 2 hours infected with 3×10⁵ cells of SN152, *NSG2Δ/Δ* or *NSG2Δ/Δ+NSG2*. We measured the fungal burden in kidney after stopping the fluconazole delivery in 2 days. The results showed that *NSG2Δ/Δ* was almost all cleared in kidney and the survival outcome of fluconazole treatment was about 90% during the 50 days observation. Corresponding to this, only 30%-40% of the mice infected with SN152 or *NSG2* reintroduction strain was alive after 50 days. Overall, *NSG2* maintained the *C. albicans* resistance to azoles no matter in vitro or in vivo.
**638T** **Purification and characterization of killer toxin from *Debaryomyces hansenii* isolated from cheese.** *N. Banjara*, H.E. Hallen-Adams Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE.

Some yeasts produce toxic proteins or glycoproteins called killer toxins, which can kill sensitive yeast isolates. The yeast *Debaryomyces hansenii* Dh-237 (CBS 14264) isolated from cheese secretes killer toxin which has inhibitory activity in the lab against pathogenic *Candida* spp. including *C. albicans*, *C. pelliculosa*, and *C. tropicalis*. In order to gain more information on the nature of *D. hansenii* killer toxin, we purified and characterized the killer toxin. To purify and characterize the toxin, *D. hansenii* was grown in liquid YPD media, pH 4.5, at 25 C for 72 hours and culture supernatant was concentrated by ultrafiltration with 3 kDa membrane and purified by chromatographic separations, which includes one anion exchange and two size exclusion chromatography steps. Every purified and concentrated product was tested to ensure it retained killer activity against *C. albicans* and *C. tropicalis*. SDS-PAGE of the purified killer toxin indicated a protein with apparent molecular mass of 11 kDa. The purified killer toxin was active at acidic pH, up to 5.0, and killing activity was highest at lower temperatures (25 C and below). However, pure toxin retained stability at 37 C for 3 hours. The ability to act at 37 C, even transiently, coupled with human gut mycobiome data suggest both a negative correlation between foodborne *D. hansenii* and gut commensal *Candida* levels, and a mode of action for such correlation.

**639F** **Determinants of pheromone-independent dimorphism in the human pathogen *Cryptococcus neoformans*.** *J. Lin*, Y. Zhao, X. Lin Department of Biology, Texas A&M University, College Station, TX.

The human pathogen *Cryptococcus neoformans* infects more than a million people worldwide, resulting in annual deaths of over 600,000. This highlights the pressing need to better understand cryptococcal pathogenesis. Like many fungal pathogens, morphological transition from yeast to hypha is associated with virulence shift in *Cryptococcus*. Although mating is historically associated with dimorphism in *Cryptococcus*, the pheromone pathway has minimum impact on virulence. We previously identified transcription factor Znf2 that directs switch to the hyphal form independent of the pheromone cascade. As a potent anti-virulence factor, $ZNF2^{2e}$ can also serve as a vaccine agent against cryptococcosis in animal models. To understand how Znf2 directs morphogenesis and virulence, we decided to identify its downstream targets critical for morphogenesis.

We designed a T-DNA insertional mutagenesis screen in a parental strain where mCherry-Znf2 is under the control of an inducible promotor. We generated and screened 88,000 mutants and isolated 84 filamentation-defective mutants under Znf2 inducing condition. Secondary screens for the examining yeast cell morphology and the presence of fluorescence under Znf2 inducing condition narrowed the candidates to 25. We further confirmed 8 mutants with single T-DNA insertion that is linked to the nonfilamentous. Sequencing of pooled genomic DNA identified the affected genes in these 8 mutants. Four genes were found disrupted by insertions in the 8 mutants, with two of the genes being independently inserted twice. One gene is *SNF5*, which was previously shown to be critical for filamentation. The other gene *ZAF1* is predicted to encode a transcriptional corepressor. Targeted deletion of *ZAF1* recapitulated the non-hyphal phenotype without impairing the pheromone response, like the *znf2Δ* mutant. Thus, our screen has identified key genes required for Znf2 to drive filamentation. Currently, we are characterizing the genetic relationship between our candidates and Znf2. Understanding the Znf2-regulatory network will advance our knowledge of cryptococcal pathogenesis and guide new drug/vaccine development.

**640W** **Investigation of Meiosis in *Cryptococcus*.** *Y. Zhao*¹, *S. Upadhyay*¹, *C. Xue*², *X. Lin*¹ 1) Biology, Texas A&M University, College Station, TX; 2) Public Health Research Institute, Rutgers Biomedical and Health Sciences, Newark, NJ.

*Cryptococcus neoformans* claims ~600,000 people's lives each year, mostly in regions where AIDS are prevalent. This ubiquitous environmental fungus is acquired by humans through inhalation and it can stay dormant in the lungs for decades. Reactivation of latent infections occurs primarily in immunocompromised individuals (e.g. due to HIV infection), resulting in fatal systemic diseases. This fungus typically grows in a haploid state, but polyploid *Cryptococcus* cells are observed in infected lungs and they are proposed to promote cryptococcal latency and persistence. These polyploid cells give rise to haploid cryptococcal populations. What triggers polyploidization and how ploidy reduction is achieved during infection are unclear.

We recently found that *Cryptococcus* increased cell size when cultured in serum, and blocking meiosis by deleting conserved meiosis-specific genes *SPO11* or *DMC1* resulted in larger proportion of cells with elevated size. Remarkably, severe genotoxic stress efficiently induced polyploidization in *Cryptococcus*. Interestingly, we observed meiosis-like features during ploidy reduction *in vitro*. Consistently, meiosis mutants showed delayed kinetics in ploidy reduction *in vitro* and these mutants showed reduced fungal burden in the lungs compared to WT when polyploid cells were used as inoculum. Most excitingly, using the recombination-based *in vivo* expression technology that couples the transient expression of the meiosis-specific gene *DMC1* with a permanent genetic change that yields a drug-resistance phenotype, we detected the activation of *DMC1* in *Cryptococcus* during infection in the mouse model of cryptococcosis. We believe *Cryptococcus* could complete sexual cycle after increased ploidy by meiosis to return to the haploid state.

**641T** **Evolution of the ascomycete specific sex pheromone receptor Ste2.** *B.P. Johnathan*¹, *B.D. Wingfield*², *G. Fourie*², *E.T. Steenkamp*¹ 1) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 2) Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

Seven Transmembrane proteins (7TMPs) are an important group of proteins that act as transporters or response mediators to environmental factors. The low sequence similarity between the various classes of 7TMPs has made it difficult to obtain support for hypotheses regarding their origin(s). Although the availability of genome sequence information and sensitive alignment tools have allowed for the detection of homologues even above the superfamily level, the overall evolution of fungal 7TMPs remains unclear and understudied. One such group of fungal 7TMPs is the ascomycete specific α-sex pheromone receptor commonly annotated as Ste2 or Pre-2. Although previous research has successfully established a link between the α-sex pheromone receptor (Ste3 or Pre-1) and the cyclic AMP receptor, no close relative has been determined for Ste2. In this study, we examine a possible link between Ste2 and the fungal pH sensor PalH. This link was initially discovered using hidden Markov model profiles to search protein databases for possible Ste2 homologues. Further analysis was conducted to investigate the possible relationship between these two proteins which included sequence alignments, motif searches as