PaMUS10 is the functional homolog of PARKIN. Taken together, the results from our analysis provide first evidence for a role of ubiquitination of mitochondrial proteins in *P. anserina* and a role of the UPS in lifespan control.
We would like to thank the LOEWE excellence initiative of the state Hesse (Ub-Net) for support.

**117F Neutral lipid metabolism in the dimorphic fungus *Blastomyces dermatitidis* influences temperature-dependent morphologic development.** Isabelle Cooperstein, Amber Marty, *Gregory Gauthier* Department of Medicine, University of Wisconsin, Madison, WI.

Worldwide, the thermally dimorphic fungi cause several million human infections each year. The temperature-dependent morphologic switch between mycelia (22°C) and yeast (37°C) defines the biology of these fungi and is critical for pathogenesis. Knowledge about the mechanisms underlying temperature adaptation remain poorly understood and represent a significant gap in knowledge. Using *Blastomyces dermatitidis*, the etiologic agent of blastomycosis, as a model system, we identified a GATA transcription factor encoded by *SREB* (sidereophore biosynthesis repressor in *Blastomyces*) that governs the transition from yeast to mycelia following a drop in temperature from 37°C to 22°C. *SREB* null mutants fail to complete the conversion to mycelia, cannot properly regulate iron assimilation, and have reduced biosynthesis of neutral lipids (triacylglycerol, ergosterol) including lipid droplets at 22°C. Altering exogenous iron concentrations did not affect the morphologic defect in *SREB* null mutants. In contrast, treatment with saturated fatty acids (16:0, 18:0) accelerated the morphologic switch and transiently restored lipid droplet abundance in *SREB* null mutants at 22°C. Gene expression microarray and qRT-PCR analyses demonstrated reduced transcription of neutral lipid biosynthetic genes including *ARE*, which is involved with triacylglycerol biosynthesis and ergosterol esterification. Overexpression of *ARE* in *SREB* null mutants accelerated the conversion to hyphae at 22°C and increased lipid droplet formation. To assess the influence of *ARE* on the morphologic switch in wild-type *B. dermatitidis* we targeted it for gene editing using CRISPR-cas9. Edited strains exhibited impaired conversion to mycelia at 22°C but did not have reduced lipid droplet formation. Collectively, these data suggest that neutral lipids including genes involved with neutral lipid biosynthesis influence the temperature-dependent switch to mycelia.

**118W Live-cell imaging of nuclei and filamentous actin in the basidiomycete *Schizophyllum commune*.** Elke-Martina Jung¹, Erika Kothe¹, *Marjatta Raudaskoski* ² 1) Institute of Microbiology, Microbial Communication, Friedrich Schiller University, Neugasse 25, 07734 Jena, Germany; 2) Molecular Plant Biology, Department of Biochemistry, University of Turku, Tykistökatu 6A, 20520 Turku, Finland.

The visualization of cytoskeletal structures in living hyphae of filamentous basidiomycetes can yield additional information that cannot be obtained with ascomycetes. In basidiomycetes, monokaryotic or defined dikaryotic hyphal compartments are found, and the formation of septa is tightly linked to nuclear division. In addition, some model basidiomycetes, including *Schizophyllum commune*, show nuclear division in dikaryotic hyphae linked to clamp formation. Here, we use histone H2B labeling by EGFP to allow for *in vivo* visualization of the elongated nuclei in both, mated dikaryotic and unmated monokaryotic hyphal compartments. In apical cells, the interphase nuclei moved towards the hyphal tip with a rate proportional to tip growth. In some apical cells, the nuclear content showed increased fluorescence due to chromatin condensation. The condensed chromatin gathered at the apical part of the nucleus, while the fluorescence in the rest of the nucleus remained weak and finally became separated from the bright part. The decrease in nuclear size during division has long been known in *S. commune*, but only the observation of the separation of a weakly fluorescent part observed here clearly suggests that nuclear division in filamentous basidiomycetes follows the “(late) open mitosis” principles, as previously suggested also for the yeast like growth phase of basidiomycetes including *Ustilago maydis* or *Cryptococcus neoformans*. The phenomenon was seen more clearly during nuclear division in homokaryotic than in dikaryotic hyphae. Live-cell imaging of filamentous actin with Lifeact::EGFP revealed filamentous actin after nuclear division at the formation of septa both in monokaryotic and dikaryotic hyphae. The dynamics of actin at clamp cell formation suggested a more central role for actin than recorded before. At the initial stage of clamp cell formation, seen as a small peg growing from the mother cell, the proceeding movement of nuclei halts. At the same time, an actin web below the outgrowth becomes visible. This actin web could be responsible for the cessation of nuclear movement. Associated with actin web formation is the movement of one of the nuclei into the clamp initial. Microtubules have been suggested to play a central role for the movement, but here we could show that actin filaments appear to be involved in an essential manner.

**119T Comparative analysis of the Bud Site Selection System and the Septation Initiation Network in the Taxonomically Unrelated β-lactam Producers *Acremonium chrysogenum* and *Penicillium chrysogenum*.** J. Kluge, U. Kueck General and Molecular Botany, Ruhr-University Bochum, Bochum, NRW, DE.

Beta-lactam antibiotics are of major biotechnological and medical relevance and have a huge world market value. Whereas the filamentous fungus *Acremonium chrysogenum* is the primordial producer of cephalosporin C, *Penicillium chrysogenum* is the industrial source of penicillin. A typical morphological feature of *A. chrysogenum* is the fragmentation of vegetative mycelium into arthrospores. It is assumed that the constriction of these uni-or binuclear cells occurs from septa. Due to the known correlation of cephalosporin C production and arthrospore formation, we are interested in the identification of specific regulatory factors affecting both, cephalosporin C biosynthesis and morphological development. To expand the current topic regarding the septation process within arthrospore formation in *A. chrysogenum* and conidiospore development in *P. chrysogenum*, we performed a comparative analysis of both fungi. Here, we present functional characterizations of *Axl2*, a component of the bud site selection system and the germinal center kinase *Mst1*, which act as a connecting protein of the septation initiation network and the morphogenesis network (Heilig et al. 2014). In microscopic analysis and quantitative evaluations, we determined a time-delayed arthrospore development together with an altered conidiophore phenotype. In both fungi, *axl2* and *mst1* deletion mutants show growth defects in response to osmotic and cell wall stress conditions. The observation of the dynamic actin cytoskeleton and associated development of septa was performed by the establishment of the live cell marker Lifeact. The deletion of *mst1* in *A. chrysogenum* leads to an abnormal localization of actin within the hyphae. Our results highlight the similar function of developmental networks in two taxonomically unrelated ascomycetes.

Heilig Y, Dettmann A, Mourino-Perez RR, Schmitt K, Valerius O, Seiler S (2014) Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1. Plos Genet 10 (4):e1004306
**120F Crosstalk between pheromone response pathway and STRIPAK complex regulates developmental processes in *Sordaria macrospora*.** A. Beier, B. Ramšak, I. Teichert, U. Kück Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE e-mail: anna.beier@rub.de.

Developmental processes are often regulated by signal transduction pathways that include phosphorylation as a signal. These pathways are not linear and require additional regulation, which is often provided by feedback loops and crosstalk. In filamentous ascomycetes, the three mitogen-activated kinase (MAPK) cascades that primarily respond to cell wall stress (CWI), pheromone exposure (PR), and osmotic stress (OS). Besides these specific functions, fungal MAPK cascades are involved in e.g. vegetative growth, cell-to-cell communication, as well as sexual and asexual development. MAPK cascades are highly conserved eukaryotic phospho-signaling modules, which are tightly regulated by feedback loops, crosstalk, and phosphatase-catalyzed signal modulations. Here, we show an interaction of the PR pathway and the striatin interacting phosphatase and kinase (STRIPAK) complex, which is a major regulator of hyphal fusion and fruiting body development in the ascomycete *S. macrospora*. Phosphorylation levels of MAK2, the MAPK of the PR pathway, in STRIPAK deletion strains suggest an association of both complexes. In yeast two-hybrid analysis, we tested the composition of the PR module with constructs encoding for MAPKKK MIK2, MAPKK MEK2, MAPK MAK2, and the putative scaffold protein, which is homologous to HAM-5 from *Neurospora crassa*. Further, yeast two-hybrid showed a physical interaction of PR components with the STRIPAK subunit PP2Ac1. This STRIPAK subunit is a catalytic subunit of the S/T protein phosphatase 2A (PP2A) and governs sexual development, hyphal fusion, and cell wall stress response in fungi (1). Deletion strains for PR pathway subunits were generated and functionally characterized. Phenotypic analysis point towards a similar function of PR and STRIPAK subunits in developmental processes. Together with previous results that revealed a connection of STRIPAK to the CWI MAPK cascade, our findings suggest a regulatory function of STRIPAK in phospho-signaling of both MAPK cascades.

1. Beier A, Teichert I, Krisp C, Wolters DA, Kück U. 2016. Catalytic subunit 1 of protein phosphatase 2A is a subunit of the STRIPAK complex and governs fungal sexual development. mBio 7(3):e00870-16. doi:10.1128/mBio.00870-16

**121W Screening for genes important to cold acclimatization in Neurospora crassa.** Victor Manzanilla, Holly Howell, Alexander Mehreteab, Eric Rose, Nichole Walters, Nicholas Seitz, Jacob Nava, *Michael Watters* Dept Biol, Valparaiso Univ, Valparaiso, IN.

The environmental conditions that life must contend with can vary widely. Organisms have evolved a wide range of mechanisms for contending with these changing conditions. For relatively simple, sessile organisms (such as fungi), they must adapt themselves to the extremes of the environment. When subjected to rapid drops of temperature (cold shock), Neurospora responds with a dramatic, but temporary shift in its branching pattern. The fungal response, seems to parallel the physiological response to similar cold shock which has been observed in E. coli in which a set of genes is activated transiently in order to adjust the cell for growth in the cold. While the Neurospora response has been described morphologically, it has yet to be examined to any serious extent genetically. This project aims to begin the genetic characterization of the cold shock response and the associated acclimatization to cold environments. We report here the results of two screens of mutants from the Neurospora knockout library for their response to cold shock. In the first, strains with knockouts in genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes are tested for their response to cold shock. In the second, strains with knockouts of genes homologous to E. coli genes known to alter their expression in response to cold shock are examined for their morphological response. Several were identified with altered responses. The genes impacted in these mutants are listed and discussed.

**122T A downy mildew effector evades recognition by polymorphism of expression and subcellular localization.** S. Asai¹, V. Cevik², O.J. Furzer², D.S. Kim², N Ishaque², K Shirasu¹, J.D.G. Jones² 1) RIKEN CSRS, Yokohama, JP; 2) Sainsbury Laboratory, Norwich, UK.

*Hyaloperonospora arabidopsidis* (Hpa; formerly *Peronospora parasitica*) is an obligate biotrophic oomycete that causes downy mildew in *Arabidopsis thaliana*. The *Arabidopsis-Hpa* pathosystem has been extensively used to study host/pathogen co-evolution, and has enabled identification of cognate host resistance and pathogen avirulence genes. *Arabidopsis* Col-0 is resistant to Hpa isolates Emoy2 and Emwa1 through recognition by a resistance gene *RPP4*, but its cognate avirulence effector(s) were not previously identified. Comparative genomics and transcriptomics among different isolates of Hpa including Emoy2 and Emwa1 uncovered an *Hpa* effector recognized by *RPP4*. Here, we report how its unrecognised alleles evade immune recognition by *RPP4*.

**123F The potential role of an RLXR effector protein in *Phytophthora infestans* sexual reproduction.** George Tzelepis, Anna Åsman, Ramesh Vetukkuri, Johan Fogelqvist, Christina Dixelius Department of Plant Biology, Uppsala Biocenter, Swedish University of Agricultural Sciences, Linnean Center for Plant Biology, P.O. Box 7080, S-75007, Uppsala, Sweden.

*Phytophthora infestans* is a notorious fungal-like plant pathogen, which belongs to the class Oomycota within the Stramenopiles kingdom, responsible for major economic losses in potato and tomato cultivations worldwide. In order to establish a successful infection, this pathogen deploys an arsenal of small-secreted proteins, termed effectors. Among them, effectors harboring the RXLR motif are the best-studied ones. They are highly induced during early biotrophic stage of infection and are able to manipulate plant defense mechanisms operating inside the host cell. *Phytophthora infestans* is a heterothallic species, with two distinguished mating types (A1 and A2), and sexual reproduction plays a crucial role in epidemiology.

In this study we investigated the transcription profiles of different *P. infestans* isolates (originated from different parts in Europe) during the mating process, since mating frequencies are known to differ among European countries. Our results revealed that an RXLR effector-encoding gene is highly induced during mating in most of our crossings. Further analysis showed that oospores production is significantly reduced when this effector has been silenced. Moreover, we observed that this protein is accumulated in hyphal tips during mating, while infection of *N. bethamiana* plants with the GFP-tagged strain and transient expression in potato leaves confirmed the cytoplasmic localization of this effector upon infection. In this study, we could also detect the RXLR protein in the extracellular environment, when *P. infestans* was grown in pea broth medium without forming haustoria, indicating that this cytoplasmic RXLR effector can also be secreted from the hyphal tips. Furthermore, co-immunoprecipitation assays revealed that this RXLR protein putatively binds to a nuclease and to a nucleolus protein during mating. Interestingly, silencing lines displayed reduced virulence compared to wild type.
In conclusion, this study revealed a potential multifunctional role of an RXLR effector. Interestingly, the presence of this effector only in heterothallic closely related to *P. infestans* species, supports its potential role in sexual reproduction. We can speculate that certain effectors might be involved in other aspects of *P. infestans* biology than in oomycete-plant interactions. If this function reflects evolutionary adaptation remains to be clarified.

**124W Origins of novel sexual development phenotypes in the evolution of the transcriptome in five fungi.** Jeffrey P. Townsend¹,
Zheng Wang¹, Kayla Stefanko², Caitlyn Cubba², Frances Trail² 1) Yale University, New Haven, CT; 2) Michigan State University, East Lansing, MI.

Changes in gene expression have been hypothesized to play an important role in the evolution of divergent morphologies. To test this hypothesis, we examined developmental differences in fruiting body morphology of filamentous fungi, culturing five species in a common garden environment and profiling genome-wide gene expression at five stages. We reconstructed ancestral gene expression, identifying genes with the largest evolved increases in gene expression across development. Conducting knockouts and performing phenotypic analysis in two divergent species typically demonstrated altered fruiting body development in the species that had evolved increased expression. Our evolutionary approach to finding relevant genes proved far more efficient than other gene deletion studies targeting whole genomes or gene families. Combining gene expression measurements with knockout phenotypes facilitated the refinement of Bayesian models of the gene network underlying fruiting body development, regulation of which is one of the least understood processes of multicellular development.

**125T Cag1, one of the Tup1 paralogues in *Coprinopsis cinerea*, is required for gill formation in fruiting body development.** H. Muraguchi, R. Masuda, N. Iguchi, K. Tsukuta, T. Nagoshi, K. Kemuriyama Dept Biotechnology, Akita Prefectural Univ, Akita, JP.

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

**126F Environmental and genetic control of the coordinated process of fruiting body development in *Coprinopsis cinerea*.** Shanta Subba, Ursula Kües Molecular Wood Biotechnology, University of Göttingen, Göttingen, DE.

Fruiting body development in *Coprinopsis cinerea* follows a conserved scheme defined by day and night phases, with well predictable distinct stages over the time. Starting with hundreds of structures, every day some will be given up in development with only a few to mature. Fruiting starts with primary hyphal knot (PHK) formation in the dark, followed by light-induced compact aggregates, secondary hyphal knots (SHKs) in which stipe and cap tissues differentiate. Primordium development (P1 to P5) takes five days to culminate on day 6 of development in karyogamy (K) and meiosis (M) within the basidia and subsequent basidiospore production which parallels fruiting body maturation (stipe elongation and cap expansion). Mature fruiting bodies autolyse on day 7 to release the spores in liquid droplets. The developmental pathway is regulated by factors such as light, temperature, humidity and nutrients. Light signals induce formation of SHKs, the differentiation of tissues within the growing primordia and karyogamy within the basidia. Without light so-called 'dark stipes' are formed from SHKs with elongated stipes and underdeveloped caps. Aeration is a new key factor we show here to influence the morphological development of the fungus. Inhibiting passage of air into cultures can alter normal behaviour of the fungus. Hindering air entry before light-induced formation of SHKs fully blocks fruiting and results in the formation of fluffy mycelial growth. When SHKs have been formed blockage of air development arrests. Block in aeration at P1 to P3 leads in light to outgrowth of elongated stipes that are similar to the structures observed in aerated cultures in the dark. When plates are air-sealed at the P4 or the P5 stage fruiting body maturation happens but caps are colourless by reduced spore numbers. Mutants in light reception can form dark stipes in light while the phenotype in others may arise from defects in regulation by aeration. Most mutants in fruiting are however blocked at specific steps in development. In a collection of about 1500 strains, mutations do not evenly distribute over the complete pathway. High numbers of mutants are available from the early developmental stages up to stage P1, comparably few in the subsequent steps up to P3. Larger sets of mutants exist for P4 and P5 when karyogamy and meiosis have to occur in the basidia and fruiting body maturation to be initiated. Mutant numbers may reflect the complexity of specific steps in fruiting.

**127W IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus *Podospora anserina*.** Herve Lalucque¹, Fabienne Malagnac¹,², Kimberly Green³, Valérie Gautier¹, Pierre Grognet¹,², Laetitia Chan Ho Tong¹,⁴, Barry Scott³, Philippe Silar¹ 1) Univ Paris Diderot, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, Case courrier 7040 Lamarck, 75205 Paris, France; 2) Present adress : Institut de Biologie Intégrative de la Cellule, UMR 9198 CEA CNRS Univ Paris Sud, 91405 Orsay, France; 3) Institute of Fundamental Sciences, Massey University, Palmerston North, 4442, New Zealand; 4) Present adress : Institut Français du Pétrole Energies Nouvelles 1-4 Avenue du Bois Préau, 92852 Rueil-Malmaison, France.
Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus *Podospora anserina*. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.

**128T Leveraging the diversity in the hypervariable species *Schizophyllum commune* to understand mushroom development.** Ioana Marian, Jordi Pelkmans, Luis Lugones, Han Wösten, *Robin Ohm* Microbiology, Utrecht University, Utrecht, NL. *Schizophyllum commune* is used as a model system to study mushroom development. A large number of wild isolates of *S. commune* have been isolated from all over the world. These strains show a large diversity in mushroom development phenotypes. For example, some strains develop mushrooms in high CO₂ concentrations, which usually inhibit mushroom development. Moreover, mushroom shape and development varies when grown under lab conditions. Many of these traits show mendelian inheritance, allowing it to be mapped by genome sequencing and bulk segregant analysis.

Novel regulators of mushroom development were identified. The previously identified homeodomain transcription factor Hom2 is involved in the CO₂ sensing pathway as well as subsequent mushroom development. Several subdomains were shown to be indispensable for the correct function of the protein.

**129F Dsciphering the roles of the Phox-homolog domain proteins during the development and pathogenesis of *Fusarium graminearum*.** Y. Lou College of Plant protection, Fujian A & F University, fuzhou, Fujian, CN.

The previous study indicates that retrograde trafficking from endosome to trans-Golgi network mediated by retromer is very important for fungal development and pathogenicity in *Fusarium graminearum*. The Vps5 and Vps17 subunits of the retromer complex which was localized to the endoplasmic reticulum membrane can bind with PtdInsP (phosphoinositides) lipids, indicating that differences in membrane composition may be involved in the morphogenesis and host invasion. The Phox homolog domain protein is localized to different organelles through binding with different types of phosphoinositides, and is involved in cell trafficking, endocytosis, exocytosis and cell morphogenesis. In *Fusarium graminearum*, thirteen Phox-homolog domain proteins have been identified. We currently characterized homologs of *BEM1*, *SPO14*, *GRD19*, *SNX4*, *YPT35*, *MVP1*, *YKR078*, *YPR097*, *SNX14*, *MDM1*, *VPS5* and *VPS17* and determined their functions as well as contribution to the development and pathogenesis in *F. graminearum*. The deletion mutants of Phox homolog domain proteins, besides *YPR097*, showed defect in the pathogenesis. Interestingly, the deletion mutants of *BEM1* may be involved in the regulation of ROS homeostasis. Progress in understanding the roles of Phox homolog domain in cell trafficking and protein interaction will be presented.

**130W Chemical genetics to unravel *Fusarium* development and pathogenesis.** *G. Subramaniam*¹,²,³, C Mogg³, C Bonner², D Desveaux³ 1) Ottawa Research and Development, Agriculture Canada, Ottawa, Ontario, Canada; 2) Carleton University, Ottawa, Ontario, Canada; 3) University of Toronto, Toronto, Onatrio, Canada.

Chemical genetics provides a powerful tool to identify targets and genetic pathways involved in all aspects of cellular development. Unlike classical forward genetics, chemical genetic have an added advantage to identify essential genes involved in cellular pathways. We used chemical genetics to identify genes involved in *Fusarium graminearum* development. One chemical, Antofine, previously characterized to inhibit fungal growth, including *F. graminearum* was used in a haploid insufficiency (HIP) screen in the yeast *S. cerevisiae* to identify targets. We identified genes associated with the target of rapamycin (TOR) pathway as targets of Antofine, implicating this important pathway in *F. graminearum* development. The results of the HIP screen and characterization of the TOR pathway in *F. graminearum* will be presented.

**131T Characterization and functional roles of the flavohemoglobin genes of the plant pathogen *Fusarium oxysporum*.** Eduardo Argotti, Serenella Sukno, *José María Díaz-Minguez*, Ernesto P. Benito CIALE-UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN.

Nitric oxide (NO) is a highly reactive molecule playing important roles in essential developmental processes and stress responses. Among them, it is a crucial component of the plant immune response. This poses an interesting challenge for phytopathogenic fungi: they may use NO as a signalling molecule to modulate development but, concurrently, they have to counteract the plant defensive responses primed by NO. Fungi have evolved several mechanisms against nitrosative stress, among them the detoxification of NO radicals by the enzymes flavohemoglobins is a prominent one.
Genome analysis of several *Fusarium oxysporum* isolates showed that this fungus has four genes encoding flavohemoglobins (*FHG1-4*). *FHG1*, *FHG2* and *FHG3*, encode fungal cytoplasmatic flavohemoglobins, while *FHG4* encodes a mitochondrial flavohemoglobin with a probable bacterial origin. Gene expression analyses performed with weakly virulent (WV) and highly virulent (HV) *F. oxysporum* f. sp. *phaseoli* strains showed that *FHG1* and *FHG2* responded transcriptionally to exogenous NO during spore germination. *FHG3* showed a complex regulation while *FHG4* did not show any transcriptional response in the assayed conditions. Functional analysis of mutants deleted in the *FHG2* gene and silenced in the *FHG1* gene in both strains, confirmed that *FHG1* plays a major role and *FHG2* a minor role in NO detoxification, which confers WV and HV virulent strains resistance to nitrosative stress and prevents the delay in spore germination produced by high levels of NO. Plant inoculation assays performed either with single *FHG2* deleted mutants or double *DFHG2-FHG1RNAi* mutants, showed that these genes are not pathogenicity or virulence factors. However, *FHG1* upregulation during the first week after infection suggests that this flavohemoglobin is required for plant colonization. Finally, it cannot be precluded that flavohemoglobin *FHG4* plays a role in the detoxification of the NO produced during denitrification under low oxygen concentrations.
This work was supported by grants AGL2012-39876-C02-01 and AGL2015-66131-C2-1-R (MINECO, Spain).

**132F Sex pheromone autocrine signaling controls vegetative fungal development.** S. Vitale, A. Di Pietro, *D. Turrà* Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, Cordoba, Spain.
Mating in heterothallic ascomycetes involves paracrine signaling between two opposite mating types producing two different peptide
pheromones, a and α. Inappropriate self-signaling is prevented by protease-mediated pheromone degradation and transcriptional shut-
down of the cognate receptor. Here we show that in the heterothallic fungal pathogen *Fusarium oxysporum*, which lacks a known sexual
cycle, pheromone signaling follows a distinct configuration in which both pheromone/receptor pairs are co-expressed, resulting in autocrine
signaling. Unexpectedly, mutants lacking a given peptide pheromone or its cognate receptor displayed increased sensitivity towards the
opposite pheromone, as well as defects in vegetative hyphal fusion or cell-cell agglutination. Our results support a model in which a and α
pheromone coordinately control key developmental processes in *F. oxysporum*, revealing a new role for autocrine pheromone signaling in
vegetative fungal development.

**133W Structure-activity relationship of α mating pheromone from the fungal pathogen *Fusarium oxysporum* .** S. Vitale¹, A. Partida-Hanon², S. Serrano², A. Martínez-del-Pozo³, A. Di Pietro¹, M. Bruix², D. Turrà¹ 1) Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, Cordoba, Spain; 2) Department of Biological Physical Chemistry, Institute of Physical Chemistry Rocasolano, CSIC, Madrid, Spain; 3) Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutense University, Madrid, Spain.

During sexual development, ascomycetes produce two types of peptide pheromones termed a and α. The α pheromone from the budding
yeast *Saccharomyces cerevisiae*, a thirteen residue peptide which elicits cell cycle arrest and chemotropic growth, has served as paradigm
for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available
for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S.
*cerevisiae* homolog. High-resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete *Fusarium oxysporum*
revealed the presence of a central β-turn resembling that of its yeast counterpart. Disruption of the fold by D-alanine substitution of the
conserved central Gly5-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in
chemoattractant activity. Unexpectedly, the growth inhibitory effect of *F. oxysporum* α-pheromone was independent of the central β-turn but
instead required two conserved Trp1-Cys2 residues at the N-terminus. These results indicate that, in spite of their reduced size, fungal α-
pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as
polarity reorientation and cell division.

**134T Structure-function studies on the components of the Polycomb Repressive Complex 2.** Brian Josephson, Zackary Bango, Allyson Erlendson, Brett Pierce, Lanelle Connolly, Michael Freitag Dept. of Biochemistry and Biophysics, Oregon State University, Corvallis, OR.

Polycomb Repressive Complex 2 (PRC2) is a histone methyltransferase responsible for trimethylating histone 3 on lysine 27 (H3K27me3). Mutations within its three primary subunits, Kmt6, Eed, and Suz12, result in inherited diseases and sporadic cancers. In the fungus *Fusarium graminearum*, deletion of the gene encoding the catalytic subunit, *kmt6*, results in derepression of ~25% of the genome as well as distinct phenotypes. While previous ChIP-seq and RNA-seq has revealed genomic targets for PRC2, little is known about how it localizes to specific areas of the genome and spreads the H3K27me3 mark along the chromosome. We addressed these questions by engineering a set of *kmt6*, *eed*, and *suz12* mutants, for which we used the copper responsive *tcu1* promoter, resulting in tunable control of PRC2 expression. We characterized these mutants by fluorescent microscopy, ChIP-seq, and RNA-seq. Our studies were further guided by a partial crystal structure for PRC2 from the fungus *Chaetomium thermophilum*. We generated several Kmt6 mutants, all tagged with Green Fluorescent Protein (GFP), to evaluate if structure-function relationships proposed *in vitro* are supported by *in vivo* studies. By making minor changes in the primary sequence of Kmt6 important Kmt6-Eed-Suz12 interphases were disturbed and resulted in loss of function and phenotypes similar to that of our kmt6 deletion strain. Cytology showed mislocalization of Kmt6-GFP that is similar to that of Eed-GFP in kmt6 deletion mutants. Further assays will test for PRC2 activity as well as H3K27me3 enrichment by chromatin immunoprecipitation (ChIP).

**135F De-regulation of the tryptophan biosynthesis pathway affects fungal viability and sexual reproduction.** Bastian Dörnte, Ursula Kües Molecular Wood Biotechnology, University of Göttingen, Göttingen, DE.

Aromatic amino acid biosynthesis begins with chorismate as a common precursor. Tryptophan is produced from chorismate in six
consecutive steps which are mediated in *Coprinopsis cinerea* by four different enzymes. Tryptophan synthase encoded by the *trp1*⁺ gene is
a bifunctional enzyme which catalyzes the final two reactions in the biosynthetic pathway to tryptophan. It consists of two distinct functional
domains separated by a central linker region, unlike the situation in bacteria where the two domains are encoded by separate genes. The
N-terminal A-domain (TrpA) of Trp1 is responsible for the conversion of indole-3-glycerol-phosphate (IGP) into indole and the C-terminal B-
domain (TrpB) for subsequent production of tryptophan from serine and indole. The *trp1*⁺ gene is often used in *C. cinerea* as a marker gene
in protoplast transformation of *trp1-1, 1-6* mutants. DNA integrates in transformation at multiple ectopic sites into the *C. cinerea*
chromosomes. This allows cotransformation with two or more vectors. Surprisingly, numbers of transformants are doubled in
cotransformation of a *trp1*⁺-vector with a non-*trp1*⁺-vector as compared to single *trp1*⁺-vector transformations. This paradoxical phenomenon
results from loss of clones in single vector transformations by tryptophan feedback inhibition and de-regulation of cross-pathway control of
aromatic amino acid biosyntheses by insertion of multiple *trp1*⁺ copies into nuclei of *C. cinerea* (Dörnte and Kües 2016, Appl Microbiol
Biotechnol 100:8789-8807). Similar lethal effects were recently reported for *Schizophyllum commune* when the fungus transformed with its
*trp1*⁺ gene (orthologous to *trp2*⁺ of *C. cinerea*). This gene encodes a trifunctional enzyme with glutamine amidotransferase (GATase),
phosphoribosylanthranilate isomerase (PRAI) and indol-3-glycerol-phosphate synthase (IGPS) activities. Transformation with only the *IGPS*
DNA region induced fruiting body development in *S. commune* several days earlier than normal which indicates a link between the
tryptophan biosynthesis pathway and sexual development (Sen et al. 2016, Biosci Biotechnol Biochem 80:2033-2044). Moreover, in *C.
cinerea* sexual development is affected by *trp1*⁺ defects in irregular pigment synthesis of basidiospores. Studies on effects on viability and
sexual development by gene *trp2*⁺ as well as by the *trp3*⁺ and *trp4*⁺ genes of *C. cinerea* are underway.

**136W Uni-directional mating type switching in *Davidsoniella virescens*.** P. Markus Wilken¹, Emma T. Steenkamp², Michael J. Wingfield¹, Brenda D. Wingfield¹ 1) Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa ; 2) Department of Microbiology & Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of
Pretoria, South Africa.

Uni-directional mating type switching is a form of secondary homothallism where self-fertile isolates are able to produce both self-fertile and self-sterile offspring. Although this process has been reported in species in *Ceratocystis*, *Endoconidiophora*, *Davidsoniella*, *Sclerotinia*, *Glomerella* and *Chromocrea*, the *MAT1* locus of only *Ceratocystis* and *Sclerotinia* has been extensively studied. The gene complement of self-fertile strains contains both *MAT1-1* and *MAT1-2* related genes. Two copies of a direct repeat mediate the deletion of part of the *MAT1* locus, resulting in the loss of the *MAT1-2* genes. This renders the isolate self-sterile. In the present study, we examined the *MAT1* locus in *Davisoniella virescens* that is also capable of uni-directional mating type switching. The self-fertile *MAT1* locus in this species was found to include three mating genes in the order *MAT1-2-1*, *MAT1-1-1* and *MAT1-1-2*. Two copies of a 517 bp direct repeat flank the *MAT1-2-1* gene, with one complete copy present as part of the *MAT1-1-1* gene. The switching process results in the deletion of 4.6 kb of the *MAT1* locus and includes the full *MAT1-2-1* gene and flanking regions as well as one copy of the repeat. This is similar to what has been found in *Ceratocystis* and *Sclerotinia*, although the switching process in *D. virescens* produced a truncated *MAT1-1-1* gene that shortens the predicted protein from 322 to 273 amino acids. The effect of this truncation on the sexual cycle of *D. virescens* is unknown, and will form the basis of future studies.

**137T Sensing and signalling intercalary growth in *Epichloë* endophytes: A transcriptomics approach to mechanosensing.** A. Ozturk¹, S. Ariyawansa¹, R. E. Bradshaw², P. Maclean¹, J. Koolaard¹, P. Dupont², C. J. Eaton², N. D. Read³, N. A. Gow⁴, C. R. Voisey¹ 1) AgResearch, Grasslands Research Centre, Private Bag 11-008, Palmerston North 4442, New Zealand; 2) Bio-Protection Research Centre, Institute of Fundamental Sciences, Massey University, Palmerston North 4474, New Zealand; 3) Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester M13 9NT, United Kingdom; 4) The Aberdeen Fungal Group, School of Medicine, Medical Science and Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB252ZD, UK.

Although fungal hyphae normally extend at apical tips, exceptions to polar growth characterise the ecology of many important species. We are investigating the physiology of intercalary growth in *Epichloë festucae*, a seed-transmitted symbiont that colonises the aerial parts of grasses. Developing leaves are initially invaded by hyphal tips, however continued hyphal colonisation occurs through expansion of intercalary compartments. We recently tested the hypothesis that intercalary growth is stimulated by mechanical forces imposed on hyphae through their attachment to expanding host cells. *E. festucae* hyphae were grown *in vitro* on silicon membranes and stretching forces were shown to induce intercalary compartment extension and division, as observed in developing leaves. The aim of this study was to identify the cellular responses that induce intercalary hyphal growth after stretch. *E. festucae* cultures were grown on silicon membranes fitted to a custom-designed stretching frame, stretched by 8%, and then harvested either 5 min or 3 h later. The transcriptomes of stretched hyphae were compared against controls grown on the same devices with no stretch applied. Over 100 genes were differentially expressed in cultures harvested 5 min after stretch, whereas after 3 h over 400 genes were differentially regulated. The results suggest that reprogramming of primary metabolism and plasma membrane organisation occurs almost immediately in response to mechanical stress, and mobilisation of cell wall enzymes and secondary metabolism is observed in the cultures harvested later. These findings will assist in determining the role of mechanical stress in regulating *E. festucae* physiology and growth in plants.

**138F Sterol-rich membrane domains of the basidiomycete *Schizophyllum commune*.** J. Poetschner, K. Krause, E. Kothe Department of Microbiology, Microbial Communication, Friedrich Schiller University, Jena, Germany.

The highly polarized growth of filamentous fungi requires continuous supply of proteins and lipids to the hyphal tip. This transport is managed by a constant flow of secretion vesicles trafficking via the actin and microtubule cytoskeleton. Special microdomains, called lipid rafts are involved in the transport of proteins from the cell body to the growing hyphal tip. The main compounds of these membrane structures are glycosphingolipids and sterol. Apical sterol-rich plasma membrane domains (SRDs) are gaining attention for their important role in polarized growth of filamentous fungi and as receptor platform for signal recognition and forwarding. Although the importance of SRDs is becoming clear due to investigations on ascomycetes, their exact roles and formation mechanisms remain rather unclear. Here we investigated lipid rafts of the basidiomycete *Schizophyllum commune*, which has been in the focus of research for many decades. Due to its fast passage over the entire life cycle under laboratory conditions, it became a model organism for physiological and genetic studies. We used fluorescent dyes like filipin to visualize sterol-rich membrane domains in this saprotrophic white-rot fungus. Furthermore, detection and identification of lipid raft localized signal proteins should give insights into the formation and localization of lipid rafts in basidiomycetes.

**139W SIP-1 and BRO-1 interact with the SO protein and are essential for germling communication in *Neurospora crassa*.** M.R. Schumann¹, A. Oostlander¹, H. Haj Hammadeh¹, J. Wissing², L. Jänsch², A. Fleissner¹ 1) Department of Genetics, TU Braunschweig, Braunschweig, DE; 2) Cellular Proteome Research, Helmholtz Centre for Infection Research, Braunschweig, DE.

Cell fusion is an essential process for the growth and development of eukaryotic organism. In the ascomycete fungus, *Neurospora crassa* germinating conidia sense each other and fuse. As a result, a network of germlings is formed, which further develops into the mycelial colony.

In order to establish physical contact, fusing germlings employ an unusual mode of communication. In a kind of "cell dialog", the two fusion partners coordinately switch between two physiological stages probably related to signal sending and receiving. This involves the alternating recruitment of the MAK-2 MAP kinase module and the SO protein to the plasma membrane of fusion tips. We identified SIP-1 and BRO-1 as new interaction partners of the SO protein. A deletion of the *sip-1* or *bro-1* gene results in a Δso-like phenotype, resulting in the loss of chemotropic interactions and subsequent fusion. Subcellular localization and live-cell imaging revealed that SIP-1 and BRO-1 are recruited to the tips of interacting germlings in the same oscillating manner as SO and co-localizes with it. Surprisingly, in contrast to other known switching proteins, SIP-1 also accumulates in the described oscillating fashion at the membrane of isolated, non-interacting germlings. This surprising finding suggests, that already the individual germlings switch between two physiological stages, and that during cell-cell interaction these switches become coordinated. We hypothesize that SIP-1 contributes to fusion competence and the initiation of cell-cell communication. Future characterization of SIP-1 functions will further our insight into the unique communication mechanism described as the "cell dialog".
**140T Subcellular dynamics of the MAP kinase MAK-2 correlate with its activity during germling fusion, thereby mediating a conserved cell-cell signaling mechanism.** A. Serrano¹, M. Weichert¹, R. Marschall², J. Illgen¹, U. Brandt¹, N. Thieme¹, A. Lichius³, N. Read³, P. Tudzynski², A. Fleissner¹ 1) Department of Genetics, TU Braunschweig, Braunschweig, DE; 2) Institute of Molecular Biology and Biotechnology of Fungi, Schlossplatz 8, Münster, DE; 3) Institute of Inflammation and Repair, University of Manchester, CTF Building 46, UK.

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

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

Together, these data suggest that the "cell dialog" mode of communication is conserved in filamentous ascomycete fungi and that divergent fungal species share a common language.

**141F The gray mold's take on color: Role of the eleven photoreceptors of Botrytis cinerea in photomorphogenesis.** K C Cohrs, L Pape, J Schumacher IBBP, WWU Münster, Münster, DE.

Light is an important environmental signal for fungi to regulate processes such as morphogenetic programs, pigmentation, stress responses and the circadian clock. The plant pathogen *Botrytis cinerea* produces conidia when exposed to light, and sclerotia, strongly melanized enduring structures, in the dark. Induction of conidiation by full-spectrum light is mediated by the light-responsive transcription factor (TF) BcLTF2. The formation of conidia is regulated by at least four different light qualities: exposure to blue light inhibits conidiation and leads to the formation of sterile aerial hyphae, an effect that can be reverted by near-UV (NUV) or far-red light, but not by red light. *B. cinerea* possesses a total of eleven putative photoreceptors (PRs) – more than most other filamentous ascomycetes: two NUV-sensing cryptochromes (CRYs), four blue light-sensing LOV (light, oxygen, voltage domain) proteins, two green light-sensing opsins, and three red/far-red light-sensing phytochromes (PHYs). The role of the PRs in photomorphogenesis was studied by gene deletion and overexpression approaches.

The White Collar Complex (WCC), containing the blue light receptor/ GATA-TF BcWCL1, negatively regulates conidiation through repression of *bcltf2* and acts as a primary sensor of light as it induces the expression of other PR-encoding genes. For example, expression of *bcvvd1*, encoding a putative blue light receptor, depends on the WCC. BcVVD1 in turn acts as a negative regulator of WCC activity, creating a negative feedback loop and allowing for the fine tuning of conidiation. Additionally, the putative NUV PR BcCRY2 acts as a negative regulator of conidiation in the light through a yet unknown mechanism. The role of the red/far-red light-sensing PHYs in the light response system is still unclear as single deletions did not affect conidia formation. Because the three PHYs may have redundant functions, the generation of double and triple mutants has been initiated.

**142W Chitin-deacetylase activity has important roles in inducing appressorium formation.** M. Kuroki¹, Y. Shiga¹, K. Okauchi¹, K. Saitoh², T. Teraoka², M. Narukawa¹, T. Kamakura¹ 1) Tokyo University of Science, Noda, Chiba, JP; 2) Tokyo University of Agriculture and Technology, Fuchū, Tokyo, JP.

*Magnaporthe oryzae* (*Pyricularia oryzae*) is a filamentous plant pathogen and causes rice blast. *M. oryzae* differentiates appressorium in order to infect plant cell. *M. oryzae* seems to perceive environmental signals, but induction mechanism of appressorium has remained unrevealed. We focused on *CBP1* (MGG_12939) that have two chitin-binding domains and chitin-deacetylase (CDA) homologous domain. Appressorium formation is delayed in *CBP1* deletion mutant compared with wild type on hydrophobic substrate, but there is no difference between *CBP1* deletion mutants and wild type when cutin monomer was added. We suspect Cbp1 is an important component for induction of appressorium formation.

We previously observed appressorium formation of CDA inactive Cbp1 mutants is low level and chitosan amount is lower in *CBP1* deletion mutant. These data suggest Cbp1 has CDA activity and it has an important role in appressorium formation. In this study, we observed expression patterns of other CDAs in *M. oryzae*. *M. oryzae* has 6 CDA homologous genes except for *CBP1*. These 6 CDAs were not up-regulated in *CBP1* deletion mutant, but some of them were up-regulated more than wild type by adding cutin monomer. We estimated that CDA activity is required to form appressorium in its early stage and 6 CDAs highly express to compensate for deletion of *CBP1* when be added cutin monomer.

In wild type, chitosan was accumulated in tips of germ tubes before it started to differentiate appressorium. On the other hand, in *CBP1* deletion mutant, chitosan amount in tips of germ tubes seemed much less than wild type. In addition, amino acid sequence suggested Cbp1 was GPI-anchored protein and locates cell surface, but other 6 CDAs were free secreted proteins. We presumed location of Cbp1 could explain Cbp1's importance in appressorium formation in contrast to other CDAs. We are going to observe Cbp1's location in detail and verify whether Cbp1 is GPI-anchored protein.

CDAs generally convert chitin into chitosan and release acetic acid together. We focused on acetic acid as inducer of appressorium formation. We are trying to observe appressorium formation under existence of acetic acid.
**143T Chloramphenicol inhibits appressorium formation of Magnaporthe oryzae via a Ser/Thr-protein phosphatase, MoDullard.** A. Nozaka¹, S. Endo¹, N. Tanaka¹, M. Narukawa¹, S. Kamisuki², M. Nakajima¹, H. Taguchi¹, F. Sugawara¹, T. Kamakura¹ 1) App. Bio. Sci., Tokyo Univ. of Sci., Chiba, Japan; 2) Dept. Vet. Med., Azabu Univ., Tokyo, Japan.

The appressorium formation of *Magnaporthe oryzae* is essential for infection and related to a cell differentiation. Some drugs inhibiting appressorium formation will also affect the factor which is involved in eukaryotic cell differentiation in the rice blast fungus. We searched novel targets of preexisting drugs using appressorium formation of *M. oryzae* as an indicator whether the factor relates cell differentiation. As a result, antibiotic Chloramphenicol (Cm) showed remarkable inhibition of appressorium formation. Since Cm interacts to 50S ribosomal subunit in prokaryotes, Cm inhibits protein synthesis of prokaryotes specifically. However, in human Cm often causes a serious side effect aplastic anemia the target of Cm in eukaryote is unknown. If we can discover the unknown eukaryotic target of Cm, it will lead to contribute to clarifying the mechanism of the side effect caused by Cm, to develop a novel pesticide or rice blast specific fungicide, and/or to obtain the knowledge about the regulation of cell differentiation and appressorium formation.

We selected T7 phage display as a tool for searching Cm's target(s). From the candidate, we deduced that a Ser/Thr protein phosphatase Dullard (*MoDullard*: MGG_03646) was a possible binding protein of Cm. Using RTPCR, we detected the expression of *MoDullard* during appressorium formation. Then we acquired the GST-tag fused *MoDullard* expressed by *E. coli* and showed that the tag fused MoDullard protein bound to Cm. Moreover in the appressorium formation assay, *MoDullard* over expressed strains showed the lower sensitivity to Cm than WT. Each results inferred that MoDullard interacts with Cm in *M. oryzae* and affects appressorium formation of the rice blast fungus.

Now we challenge to establish *MoDullard* knockout and knockdown strain, and as soon as obtainment we are going to check the phenotype of them against Cm in appressorium formation assay. We also expect that the analysis of *MoDullard* for the mechanism of appressorium formation will shed new light on the mechanism of side effect of Cm, and contribute to development of antifungal agents.

**144F Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae.** M. Oses-Ruiz, W. Sakulkoo, G. R. Littlejohn, M. Martin-Urdiroz, N. J. Talbot School of Biosciences, College of Live and Environmental Sciences University of Exeter, Exeter, EX4 4QD, United Kingdom.

The rice blast fungus *Magnaporthe oryzae* elaborates a specialised infection cell called an appressorium to infect rice leaves. The appressorium generates enormous turgor, which is focused as physical force to break the rice leaf cuticle allowing the fungus entry to rice cells. We have found that appressorium-mediated plant infection by *M. oryzae* requires two independent S-phase cell cycle checkpoints. During initial formation of appressoria on the rice leaf surface, an S-phase checkpoint operates through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, during appressorium repolarization a novel, DDR-independent S-phase checkpoint is triggered by appressorium turgor generation and melanisation. This checkpoint regulates NADPH oxidase-regulated, septin-dependent, F-actin re-modeling to organise the appressorium pore and bring about entry of the fungus into rice tissue. We will report on the operation of these two checkpoints, the processes they control, and how they regulate rice infection by the *M. oryzae*.

**145W Phenotypic and genotypic analysis of a new set of Aspergillus nidulans mutants defective in hyphal morphogenesis and secretion.** Laure Ries¹, Thaila dos Reis¹, Karthik Boppidi², Anna Lipzen³, Kerrie Barry³, Igor Grigoriev³, Gustavo Goldman¹, Mark Marten², Steven Harris⁴ 1) Faculdade de Ciencias Farmaceuticas de Ribeirao Preto. Universidade de Sao Paulo, Brazil; 2) Department of Chemical, Biochemical and Environmental Engineering, University of Maryland-Baltimore County, Baltimore, MD; 3) JGI-DOE, Walnut Creek, CA; 4) Center Plant Sci Innovation, Univ Nebraska, Lincoln, NE.

Investigation of the mechanisms that coordinate hyphal morphogenesis with vesicle trafficking and secretion is central to understanding how filamentous fungi grow. Reverse genetic approaches that characterize homologues of well-known yeast gene products involved in morphogenesis and vesicle trafficking proteins have yielded significant insight into these mechanisms. Nevertheless, morphogenesis and secretion are clearly more complex in hyphae than in yeast cells. For example, hyphae can simultaneously sustain multiple polarity axes, and there is increasing evidence for the existence of multiple routes of vesicle delivery to the cell surface. To account for the greater complexity of these processes in filamentous fungi, we contend that unbiased forward genetic screens represent an attractive alternative approach that is not constrained by existing knowledge of other systems. This is particularly important given that ~50% of annotated genes have no known function in a typical filamentous fungus, some of which undoubtedly play key roles in morphogenesis and secretion. Here, we present the preliminary characterization of a new set of *Aspergillus nidulans* morphological mutants with associated secretion defects. Mutants were derived from strain FGSC A4 using a filtration-based enrichment followed by secondary plate-based screens for secretion defects. We report here on the phenotypes and genome sequences of 39 mutants. A small number of these mutants exhibit an apparent hyper-secretion phenotype, and possess mutations predicted to impact regulation of the GTPases Cdc42 and Rac1, myosin-based transport, and function of the protein kinase PakB. Other mutants possess mutations in a number of genes not previously implicated in hyphal morphogenesis or vesicle transport. We anticipate that this collection of mutants and genome sequences will prove to be a valuable community resource.

**146T Extensive transcriptional differentiation during acquisition of a plastic developmental competence state in Aspergillus nidulans.** Luke Noble¹,², Linda Holland¹,³, Alex Andrianopoulos¹ 1) School of BioSciences, University of Melbourne, Parkville, Victoria, Australia; 2) Department of Biology, New York University, New York, USA; 3) School of Biotechnology, Dublin City University, Dublin, Ireland.

Development requires precise temporal and spatial timing of gene expression. At the cellular level where differentiation occurs, this is controlled by the presence of inductive and repressive cues, such as hormones and cell-cell contacts but must also be buffered against intrinsic biological noise that can lead to sporadic induction of differentiation. A central mechanism to buffer biological noise is to control when cells become competent to receive developmental cues. We have undertaken a broad analysis of the environmental and genetic basis of 'developmental competence' in the filamentous eukaryote *Aspergillus nidulans*. Density and pH are critical parameters, and we identify 21 genes with effects on competence timing through genetic screens and candidate mutagenesis, from fungal-specific transcription factors to the conserved unfolded protein response regulator HacA, GTPase RasB and ambient pH sensor PalH. Transcriptional profiling and cross-species comparison demonstrates substantial, concordant genome-wide activity, in the absence of morphological differentiation, including a wave of gene expression around centromeres indicative of chromatin remodeling. Species-specific endogenous hormones and
metabolic capacity likely determine competence timing. An understanding of reproductive competence in fungi is of relevance to all biology associated with the 'adult' form – asexual and sexual reproduction, primary and secondary metabolism, and pathogenesis – and provides a new model of nutrient-limited life history phases.

**147F A conserved regulator of meiosis has been rewired to control chlamydosporulation in the fungal pathogen of humans Candida albicans.** A. Hernandez-Cervantes¹, S. Znaidi¹,², V. Basso¹, J. Ropars¹, N. Sertour¹, D. Sullivan³, G. Moran³, F. Vincent⁴, F. Dalle⁴, M.E. Bougnoux¹, S. Bachellier-Bassi¹, C. d'Enfert¹ 1) Fungal Biology and Pathogenicity, Institut Pasteur, INRA, Paris, FR; 2) Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique, Institut Pasteur de Tunis, Tunis, TUN; 3) Trinity College, Dublin, IRL; 4) UMR 1347, Université de Bourgogne, Dijon, FR.

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

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

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

**148W Regulation, localization and stability of the velvet protein VE-1 in Neurospora crassa M. del Mar Gil-Sánchez, Eva M. Luque, Luis M. Corrochano.** M. del Mar Gil Sanchez, Eva M. Luque Fobelo, Luis M. Corrochano Pelaez Genetics, University of Seville, Seville, Seville, ES.

In *Neurospora crassa* light is perceived through the WC complex, a light-dependent transcription factor complex that regulates the transcription of a number of genes. The *Neurospora* genome contains a homolog of the *Aspergillus nidulans* *veA* gene, *ve-1*. In *A. nidulans* mutations in *veA* results in constitutive conidiation that is independent of light, and the VeA protein forms a complex with photoreceptors. VE-1 is a protein with a nuclear localization signal and a velvet factor domain that is highly conserved in fungi. The *N. crassa* *ve-1* mutant has defects in aerial hyphal growth and increased conidiation.

We have characterized the light-dependent accumulation of carotenoids in a strain with a deletion in *ve-1* and in the wild-type. A ten-fold reduction in sensitivity was observed in the *Δve-1* strain, an indication for a role of VE-1 in light sensing in *N. crassa*.

We have created a strain with a tagged version of VE-1 to detect the protein. We observed a minor increase in the accumulation of *ve-1* mRNA after light exposure in vegetative mycelia (30 min), which did not lead to changes in VE-1 accumulation. The mutation in *ve-1* results in decreased light-dependent accumulation of mRNA for several genes, including the carotenogenesis genes, *wc-1*, *vvd*, and *frq*. We have characterized the cellular localization of VE-1 under different light conditions and we have observed that VE-1 is preferentially located in the nucleus under all conditions, but VE-1 was also detected in the cytoplasm. VE-1 was not present during conidial development in the dark, but VE-1 was detected in vegetative mycelia. In the light, however, VE-1 was detected in aerial hyphae and vegetative mycelia and it is localized mostly in the nucleus. Our results suggest that the presence of VE-1 in aerial hyphae could depend on light, given that VE-1 is not detected in light neither in the dark in the ?*wc1* mutant despite the detection of the mRNA for *ve-1*. This could indicate a posttranslational protein regulation. Therefore, we studied the degradation of VE-1 through the signalosome CSN and the FWD-1 pathway. The accumulation of VE-1 is higher in the *fwd-1* mutant than in the wt. In addition, we have observed that light regulates the stability of VE-1 and the protein is more stable in the absence of CSN-5 and FWD-1. The regulation by light of the degradation of VE-1 through the CSN and after interactions with FWD-1 could be key in the regulation of conidial development in *N. crassa*.

**149T Cell-to-cell communication in Neurospora crassa: Transcriptional regulation and the interplay between self and non-self recognition.** M.S. Fischer, N.L. Glass Plant & Microbial Biology, UC Berkeley, Berkeley, CA.

Self/non-self recognition and communication between genetically identical cells is an essential developmental process across the tree of life. We use the model filamentous Ascomycete Neurospora crassa to investigate the genetic and molecular mechanisms of cell-to-cell communication. As *N. crassa* conidia germinate into germlings, they begin to communicate with other nearby germlings. If they recognize that nearby germlings are genetically identical ("self"), then they will fuse and work together to establish a new colony. Several genetic screens and experiments have identified over 60 genes that are required for normal chemotropic growth and fusion between germlings. Among these 60 genes are two transcription factors, ADV1 and PP1. We identified that in germlings PP1 is required for transcription of ADV1, which controls the expression of at least 30% of the known communication and fusion genes (identified via RNAseq). Furthermore, overexpression of ADV1 is sufficient to suppress the pleiotropic phenotype of *Δpp1*, but the inverse is not true. Many of the genes regulated by ADV1 and PP1 are poorly characterized or simply annotated as hypothetical proteins. We hypothesize that some of these uninvestigated genes are involved in germling communication. *ham11*, *doc1*, and *doc2* are all in the PP1/ADV1 transcriptional regulon, and until recently, these genes were also uninvestigated hypothetical proteins. Several germling communication assays with *Δham11*, *Δdoc1*, *Δdoc2* and all possible double and triple mutants led us to the conclusion that HAM11 and DOC1/DOC2 function in parallel to mediate germling communication. HAM11 mediates self-communication by positively reinforcing communication between genetically identical germlings. In contrast, DOC1 and DOC2 strongly repress communication between genetically dissimilar germlings. We are currently working toward a
deeper understand of the interplay between the self and non-self recognition pathways during germling communication and colony establishment.

**150F Neurospora MSH1 protein functions to maintain the mitochondrial DNA.** T. Yokoi, Y. Kojima, S. Tanaka, S.
Hatakeyama Saitama University, Saitama, Saitama, JP.

The *nd* (natural death) strain of *Neurospora* was originally isolated which showed serious growth defect even on complete medium and restrictive temperature. This mutant shows sensitivity to alkylating agent and ceases hyphal growth in approximately two weeks along with large deletion of mitochondrial DNA (mtDNA). Previously, we identified that the *nd* strain harbored the nonsense mutation in *msh1* (*MutS homolog 1*) gene and wild type *msh1* gene could complement phenotypes of the *nd* strain. MSH proteins, MSH1 to MSH6, have identified as eukaryotic homologs of *E. coli* MutS protein, which recognizes the mis-incorporated nucleotide in the newly synthesized DNA strand. Among these, MSH1 is conserved exclusively in fungi, so this protein is implied to have unique role in fungal cell. Knock out of the *msh1* gene in *Neurospora* caused similar phenotypes of the *nd* strain. The predicted amino acid sequence of *Neurospora* MSH1 reveals that mitochondrial localization signal (MLS) and DNA binding motif are well conserved in the N-terminus. Since deletion of the MLS resulted in showing similar phenotypes to those of the *msh1* KO and *nd* strains, MSH1 might be functioned in the mitochondria. Further, in these mutants, not only large size deletion of mtDNA was detected, but also aberrant branched DNA in mitochondria was accumulated. Expression of the fusion protein of wild type MSH1 with GFP showed its localization in mitochondria, but interestingly, MLS deletion version of MSH1 protein also associated to this organelle. From these results, we are forecasting that the MSH1 protein may function in the maintenance of mtDNA, which is indispensable to sustain mitochondrial function one of which correlates to energy supply for the elongation of hyphal tip.

**151W CSE-7, a predicted ER-chaperone, plays an important role in the delivery of CHS-4 to hyphal tips and septa in Neurospora crassa.** A. Rico Ramirez, M. Riquelme Microbiology, CICESE, Ensenada, Baja California, Mexico.

Chitin synthesis is catalyzed by chitin synthases (CHS), a family of plasma membrane (PM) embedded enzymes. In *Saccharomyces cerevisiae*, exit of Chs3p from the endoplasmic reticulum (ER) requires the help of chaperone Chs7p, while at the Golgi, it requires the exomer complex components Chs5p and Chs5p-Arf1p binding proteins or ChAPs (Chs6p, Bch2p, Bch1p and Bud7p). In addition, the localization and activation of Chs3p at the PM depends on its interaction with Chs4p. Corresponding homologues for the yeast proteins involved in Chs3p transport have been identified in *silico* in the *Neurospora crassa* genome: CSE-7 (Chs7p), CBS-5 (Chs5p), BUD-7 (as homologue of the four *Chs5p-Arf1p* binding proteins or ChAPs, Chs6p, Bch1p, Bch2p and Bud7p), and three homologues of Chs4p (CSA-1, CSA-2 and CSR-3).

*N. crassa* CHS-4 (orthologue of the yeast Chs3p) tagged with GFP was located at the core of the Spitzenkörper (Spk) and in septa. CSE-7-GFP was revealed at the Spk core, at developing septa, and additionally, at an extensive subapical endomembranous tubular network, resembling the fluorescence pattern observed for mCherry containing the ER retaining signal HVEL. Total Internal Reflection Fluorescent Microscopy revealed highly dynamic vesicles at hyphal subapical regions for CSE-7-GFP but not for mCherry-HVEL. In addition, fixed hyphae of *N. crassa* expressing CSE-7-mChFP and exposed to ER-Tracker Blue-White-DPX revealed a partial co-localization, confirming that CSE-7 is indeed at the ER. In a *N. crassa* Δcse-7 mutant CHS-4-GFP no longer accumulated at the Spk and septa; instead, fluorescence was observed in the subapical region lighting up tubular structures similar to those observed for CSE-7-GFP. In the complemented strain *Pchs4::chs-4-gfp::natR;Δcse-7::hph*⁺;*his3*⁺;*Δmus51::bar*⁺;*Pccg-1::cse-7::mchfp::his-3*⁺ the localization of CHS-4-GFP at the Spk and septa was restored, providing evidence that CSE-7 is necessary for ER exit of CHS-4. Co-immunoprecipitation (Co-IP) studies are undergoing to confirm interaction between CSE-7 and CHS-4, and also to identify other physiologically relevant interactors.

**152T A HAD family phosphatase PSR-1 regulates circadian output pathway in Neurospora crassa.** X. Zhou¹, B. Wang¹, J. Emerson¹, C. Ringelberg¹, S. Scott¹,³, J Loros², J Dunlap¹ 1) Molecular and System Biology, Geisel School of Medicine at Dartmouth, Hanover, NH; 2) Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH; 3) Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH.

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Although the mechanism of the core oscillator is becoming understood, little is known regarding pathways connecting the core clock to its output pathways. Here, we demonstrate the HAD family phosphatase PSR-1 is involved in circadian clock output but not in the core oscillator. The *Δpsr-1* deletion mutant has a circadian output defect on race tubes under free running conditions such that constant conidiation is observed instead of typical clock-controlled rhythmic banding. However, further analysis indicates that the FRQ-WCC (FREQUENCY-WHITE COLLAR COMPLEX) Oscillator functions normally in *Δpsr-1* strains albeit with a minor 3.5-hour phase delay. PSR-1 physically interacts with WHI-2, the *Δwhi-2* mutant has phenotypes similar to those observed in *Δpsr-1*, and analysis of the *Δpsr-1*, *Δwhi-2* double mutant reveals PSR-1 plays the major catalytic role in the PSR-1/WHI-2 complex. PSR-1 primarily localizes to the nucleus and directly interacts with WC-1, it is essential for maintaining WC-1 protein and phosphorylation levels. PSR-1 is also important for the interaction between VVD and WC-1. Double mutant *Δpsr-1*, *Δvvd* strains showed further reduced WC-1 protein amounts and phase delays compared to either single mutant suggesting that the reduced amount of WC-1 may factor in determining the phase delay. Consistent with this, when WC-1 protein level is increased by use of a *qa-2*-induced promoter in the *Δpsr-1* mutant, the phase delay phenotype is partially rescued although not the defect in rhythmic banding. Loss of overt banding but not of core clock function in *?psr-1* suggested a discrete loss of circadian control of transcriptional output from the clock, and screening of candidate clock-controlled transcription factors identified loss of control of ADV-1, a direct target of the WCC, as responsible for the loss of overt rhythmicity. The PSR-1/WHI-2 complex participates in *Neurospora* clock output, regulating the level and phosphorylation status of WC-1 and thereby promoting proper transcriptional/translational activation of *adv-1/ADV-1*. These data provide a precedent for an essential role of post-translational regulation in the early steps of circadian output.
**153F** Is auxin biosynthesis in *N. crassa* connected to the eisosomal localized BEM46 protein? *F. Kempken*, K. Kollath-Leiß, P. Sardar, Q. Yang Institute of Botany, Christian-Albrechts University, Kiel, DE.

We previously showed tryptophan-dependent auxin (IAA) production in *Neurospora crassa* by HPLC and TLC analyzes (1). Further we identified the complete IAA-biosynthesis network by bioinformatical tools. These pathways were confirmed by quantitative and qualitative determination of the produced indolic compounds in newly generated double and triple mutants. BEM46 (2), a protein that influences the expression of genes involved in the IAA-biosynthesis network and also of the *mtr* gene encoding a tryptophane transporter (1) is a conserved α-β-hydrolase in *N. crassa* (3). Over-expression of *bem46* led to loss of ascospore germination and a clear reduction of hyphal elongation. RNAseq data have recently been obtained are being analysed. *bem-46*-knock-out strains show a reduced ascospore germination and a slightly but significant higher elongation of vegetative hyphae. Y2H and split-YFP experiments demonstrated the interaction of BEM46 with anthranylate synthase, which is involved in the tryptophan biosynthesis of the fungus (1).

BEM46::eGFP was found to be localized to the ER (2) and to eisosomes (1). The latter are static, plasma membrane associated protein complexes with a hypothetical function of protecting particular proteins from endocytosis (4). For a better understanding of the eisosomal composition in a filamentous fungus, GFP and RFP reporter constructs of various transport proteins have been constructed and protein localization in different developmental stages is being analyzed. Most interestingly, MTR, a tryptophane transporter is also localized in the eisosome.

1. Kollath-Leiß K, Bönniger C, Sardar P, Kempken F. 2014. BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in *Neurospora crassa*. *Eukaryot Cell* 13:1051–1063.

2. Mercker M, Kollath-Leiß K, Allgaier S, Weiland N, Kempken F. 2009. The BEM46-like protein appears to be essential for hyphal development upon ascospore germination in *Neurospora crassa* and is targeted to the endoplasmic reticulum. *Curr Genet* 55:151–161.

3. Kumar A, Kollath-Leiß K, Kempken F. 2013. Characterization of bud emergence 46 (BEM46) protein: sequence, structural, phylogenetic and subcellular localization analyses. *Biochem Biophys Res Commun* 438:526–532.

4. Lee J-H, Heuser JE, Roth R, Goodenough U. 2015. Eisosome ultrastructure and evolution in fungi, microalgae, and lichens. *Euk Cell* 14:1017–1042.

**154W** Characterization of the mutant *dnf-4*, a phospholipid flippase in the filamentous fungus *Neurospora crassa*. *O.A Callejas-Negrete*¹, I Murillo-Corona¹, B.D Shaw², R.R Mouriño-Pérez¹ 1) Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada. Ensenada B. C. México. ; 2) Department Plant Pathology and Microbiology. Texas A&M University College Station, TX, US.

Phospholipid flippases are transmembrane proteins that are conserved among eukaryotic organisms; they translocate specific population of phospholipids from the luminal leaflet to the cytosolic leaflet and have been associated with apoptosis signaling, protein sorting, vesicular traffic and secretion. The *dnf-4* gene of *Neurospora crassa* has a high identity with the *neo-1* gene of *Saccharomyces cerevisiae* that is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. We studied the dynamics of DNF-4 and the effect of the mutation of the *dnf-4* gene in *Neurospora crassa*. The Δ*dnf-4* mutant had a decrease of 41% in cell growth, 30% in biomass production and 76% in conidia production (p<0.05). Branching rate was three-fold higher in the Δ*dnf-4* mutant (p<0.05). Mature hyphae showed a Spk smaller and less stable and also presented a distorted morphology, presenting bulging sites. DNF-4-GFP was present as bright spot surrounding the endoplasmic reticulum and the Golgi, it was not present in the apex or the plasma membrane. The mutation *dnf-4* gene in *N. crassa* is not essential but affects growth rate and morphology and severely reduced the production of conidia. It is a phospholipids translocator of the ER and Golgi membrane.

**155T** Nod-like receptor mediated allorecognition induces germling regulated death in *Neurospora crassa*. *J. Heller*, N. L. Glass Plant and Microbial Biology, University of California at Berkeley, Berkeley, CA.

Allorecognition-induced death in filamentous fungi has been proposed to function as a type of fungal innate immunity system, whereby recognition of non-self reduces the risk of transmission of pathogenic elements between colonies and exploitation by aggressive genotypes. Cellular fusion of vegetative cells is an essential process for fungal development and can occur between hyphal cells of an individual colony or between hyphal cells of genetically distinct colonies. Heterokaryon incompatibility (HI), a form of allorecognition, is one mechanism to restrict the propagation of fusion products between genetically dissimilar individuals by inducing a programmed cell death (PCD) response. HI and PCD have been considered to only occur after the fusion of mature hyphae while being suppressed during germling fusion. Here, we present the identification of allorecognition mechanisms that act at the germling stage. If germlings of various wild isolates are of different specificity at so-called *germling regulated death* (GRD) loci, allorecognition mediates rapid cell death following membrane merger between germlings (~20 min). Flow cytometry experiments showed that death is 100% penetrant (all fused germlings of different GRD background die). By bulk segregant analysis (BSA) of progeny from a cross between two wild isolates followed by whole genome re-sequencing we identified two linked loci (*plp-1* and *sec-9*) that segregated with the GRD phenotype. The genes *plp-1* and *sec-9* have typical characteristics of allorecognition genes: they show discrete haplotypes that have a high level of polymorphism with long-term persistence through multiple speciation events (trans-species polymorphism), supporting the hypothesis that balancing selection is acting on these loci. SEC-9 is an essential SNARE protein predicted to be involved in membrane fusion and exocytosis. PLP-1 is a nod-like receptor similar to intracellular pattern recognition receptors (NLRs) that mediate recognition of microbe-associated molecular patterns, the initial step of innate immunity in animals and plants. Analyses of death frequencies in various mutants of *plp-1* and *sec-9* confirmed that non-allelic interaction between *plp-1* and *sec-9* is required for allorecognition and GRD. By creating chimeric proteins we identified domains in SEC-9 and PLP-1 that are essential for the PCD induction and specificity. We propose a model in which PLP-1 acts as a NLR in fungal innate immunity mediating recognition of SEC-9.

**156F** Role of class II myosin in apical growth and morphogenesis of Neurospora crassa. Fernando Nuñez-Moreno, Salomon Bartnicki-Garcia, Olga Callejas-Negrete, Rosa. Mouriño-Pérez Dept Microbiology, CICESE, Ensenada, MX.
Evidence continuous to mount for the central role of the actin cytoskeleton and its binding proteins (ABP) in the growth of fungal
hyphae. Here we present evidence for the participation of MYO-2, a type II myosin, in the actin cytoskeleton of *Neurospora crassa*, and its role in growth and morphogenesis. We generated a strain expressing MYO-2 fused to green fluorescent protein (GFP) and a *myo-2* deletion mutant (Δ*myo-2*). MYO-2-GFP was present in hyphal tips, in septa and in microconidia. By confocal microscopy, we discovered that MYO-2-GFP colocalizes sharply with the FM4-64-stained Spitzenkörper (Spk). FRAP analysis of the MYO-2-GFP fluorescent signal in the Spk yielded a 50% recovery after 50 sec and a total recovery after 130 sec, about twice as long compared to the known recovery times of CHS-GFP signals in the Spk of *N. crassa*. By blocking the binding of ATP to MYO-2-GFP with blebistatin, the fluorescence in the Spk became dispersed showing that Spk localization was dependent on ATPase activity. *Myo-2* deletion caused severe losses: biomass was decreased by 57%, while colony radial growth was reduced by 75%; this was accompanied by a 45% decrease in hyphal diameter and only about half as many branches were produced per hypha. Hyphal septa disappeared in the Δ*myo-2* mutant together with an almost complete inhibition of aerial mycelium formation (97%), and no conidiation was observed. In the absence of MYO-2, the Spk appeared normal though smaller in size in concordance with the smaller hyphal diameter. Mutant hyphae showed an increased tendency for meandering. Although similar findings have been reported for *myo-2* deletion causing severe reduction in growth and conidiation of other fungi (*Aspergillus nidulans*, *Penicillium marneffei* and *Fusarium graminearum*) this is the first report on the localization of MYO-2 in the Spk of a fungus. The exact function of this myosin in the Spk is uncertain but MYO-2 seems not to be essential for hyphal growth though its presence may contribute to Spk stability thus assuring full hyphal growth potential. In contrast, MYO-2 does play an essential role in septation and conidiation as a component of the septal actomyosin constricting ring as was previously demonstrated.

**157W The penta-EF-hand Protein PEF-1 is part of the membrane damage response during cell fusion- induced lysis in Neurospora crassa.** M. R. Schumann, L. Hartung, A. Fleissner TU Braunschweig, Braunschweig, DE.

During colony establishment, germinating vegetative spores of *Neurospora crassa* fuse with each other to form a supracellular network, which further develops into the mycelial colony. Fusion pore formation is mediated by a controlled cell wall breakdown and plasma membrane merger. These steps bear the risk of cell lysis and death by membrane rupture. Earlier studies indicated that in the plasma membrane fusion mutant ΔPrm1, lysis of fusion pairs occurs more frequently. In the absence of calcium, lysis rates increased even further, suggesting the presence of Ca²⁺-mediated membrane repair mechanisms.

We identified the Ca²⁺-binding penta-EF-hand protein PEF-1 as a potential part of this proposed repair mechanism. Subcellular localization and live-cell imaging revealed that PEF-1 is recruited to the fusion point of lysing germling pairs and accumulates at the plasma membrane after treatment with the anti-fungal and membrane destabilizing drug nystatin. Deletion of the *pef-1* gene in the *DPrm-1* mutant had a comparable effect to the depletion of calcium and resulted in an increased lysis rate. In contrast to recent publications on Pef-1 functions in the budding yeast *Saccharomyces cerevisiae*, a deletion of *pef-1* in *N. crassa* does not result in reduced stress tolerance. Interestingly, a deletion of *Pef-1* in the important human fungal pathogen *Candida albicans* results in reduced growth in the presence of cation-chelating agents EGTA and membrane- destabilizing SDS. The mammalian PEF-1 homolog ALG-2 is thought to link calcium-dependent signaling with vesicle trafficking. In addition, vesicles commonly contribute to membrane repair, for example by the formation of wound patches in response to a calcium influx.

Based on these observations, we hypothesize that in *N. crassa*, membrane damage results in the influx of calcium, which activates PEF-1, which in turn mediates vesicle-based plasma membrane repair. Further studies aim to fully characterize this repair mechanism, which is also activated in response to the important anti-fungal drug nystatin.

**158T Chitin synthase regulator CSR-3 and the protein SO function together in cell-cell fusion and stress-induced cell wall remodeling in Neurospora crassa.** S. Herzog, T. Sedlacek, A. Hirsch, U. Brandt, A. Fleissner Department Of Genetics, TU Braunschweig, Braunschweig, Niedersachsen, DE.

The filamentous ascomycete fungus Neurospora crassa is a widely used model system to study cell-cell fusion. Genetically identical germinating spores fuse with each other and form a subcellular network resulting in an interconnected mycelium. The interacting cells establish an unusual mode of communication by switching between signal sending and receiving. During this 'cell dialog' the MAK-2 MAP kinase module and the protein SO are recruited to the interacting cell tips in an alternating manner. After cell-cell contact, the MAK-1 MAP kinase module which is part of the cell wall integrity (CWI) pathway localizes together with MAK-2 and SO to the forming fusion pore. Despite the described function of SO as a scaffold for the CWI protein kinases in S. macrospora, its molecular function during these processes is still unknown.

In an Y2H screen we identified in addition to MEK-1 and MIK-1, the up-stream MAP kinases of MAK-1, the chitin synthase regulator CSR-3 as potential interaction partner of SO. CSR-3 transiently co-localizes with these proteins to the prospective fusion site after cell-cell contact and remains there until pore formation is completed. The lack of this regulator increase lyses of fusing cell pairs suggesting that CSR-3 stabilizes fusion pore formation. The protein exhibits three potential phosphorylation sites whose potential role in this process will be analyzed via targeted mutagenesis. In addition, CSR-3 co-aggregates with SO and MEK-1 in complexes which form at the cell periphery in response to cell wall stress. In hyphae, CSR-3 and its potential target the chitin synthase CHS-2 are also involved in septa formation, a well described process that requires new synthesis of cell wall compounds.

Together, these findings suggest that cell wall reconstruction during cell fusion is based on a fine-tuned equilibrium between cell wall synthesis and degradation. Both fusion pore formation related cell wall remodeling and the response to cell wall stress share common molecular signaling networks, in which SO might mediate the interaction of the MAK-1 MAP kinase module with its targets.

**159F Membrane asymmetry markers and polarized growth in Neurospora crassa.** R.R. Mouriño.Pérez¹, I. Murillo-Corona¹, Z. Schultzhaus², B.D. Shaw², O.A. Callejas-Negrete¹ 1) Microbiology, CICESE, Ensenada, BC, MX; 2) Department of Plant Pathology and Microbiology, Program for the Biology of Filamentous Fungi, TAMU.

In fungal cells, specialized proteins gather in specific places to break cell symmetry and produce hyphae. This organization includes the orchestration of two distinct vesicle processes, endocytosis and exocytosis that take place in tandem in different areas of the apical compartment in growing hyphae. Part of the signals for endocytosis and endocytosis include the asymmetry of the plasma membrane phospholipid bilayer. We studied the flippases, DNF-1 and DRS-2 that seems to be responsible for this membrane asymmetry. The mutation of *dnf-1* and *drs-2* genes produced alteration in the maintenance and stability of the Spitzenkörper and affected the actin
cytoskeleton organization in the apical compartment. Surprisingly, neither of the flippases DNF-1 and DRS-2 was present in the plasma membrane, both were localized in different layers of the Spitzenkörper, associated to different secretory vesicles. DRS-2 was associated to vesicles transporting chitin synthases. These results indicate that phospholipid flippases (P4 ATPases) may be important for polarity on secretory vesicles, Spitzenkörper integrity and thus for the localization of many tip growing proteins.

**160W The protein disulfide isomerase of Botrytis cinerea: An ER protein involved in protein folding and redox homeostasis mediates NADPH oxidase signaling processes.** R. Marschall, P. Tudzynski Institute of Plant Biology and Biotechnology, Münster, DE.

One of the most devastating pathogens, which is responsible for tremendous crop losses of edible fruits and vegetables, is *Botrytis cinerea*, causative agent of the grey mold disease. The fungus has a necrotrophic lifestyle and benefits from humid air moisture and moderate temperatures. Central molecules within pathogenic processes are reactive oxygen species (ROS). ROS are ubiquitous present in all cells that depend on molecular oxygen and are known to work as signaling molecules in appropriate concentrations while they are harmful to macromolecules in elevated amounts. While ROS result from the activity of the respiratory chain as mere byproducts, there are enzymes such as the NADPH oxidase (Nox) complexes, which actively forming superoxide ($O_2^-$) by transporting electrons across the lipid bilayer onto molecular oxygen. In *B. cinerea*, Nox complexes are contributing to a wide range of vegetative and pathogenic processes such as the formation of infection structures and the sporulation. However, despite of major advances regarding the composition and function of fungal Nox complexes¹, there are still open questions concerning their integration in the complex signaling network.

Recently, we identified the protein disulfide isomerase (PDI) BcPdi as protein, which directly interacts with the catalytic subunit BcNoxA in Y2H and co-IP assays. PDIs are highly conserved among different species. They are involved in essential processes such as the protein folding and the UPR and are responsible for the maintenance of the redox homeostasis. By characterizing deletion mutants we found out that *Δbcpdi* and *ΔbcnoxA* exhibit a quite similar phenotype. Moreover, we elucidate that upon calcium stimulation BcNoxA, BcPdi and the second catalytic subunit BcNoxB work closely together, resulting in ROS production and an oxidative burst inside the cytosol. The activity of the three proteins might be regulated by the phosphatase calcineurin, which came up as an additional interaction partner of BcPdi. Taken together, we postulate an essential role for BcPdi in the Nox signaling machinery and speculate that Nox driven developmental processes are regulated by PDI. Further on, we highlight an additional linkage between ROS and calcium dependent signaling pathways, which supports the hypothesis that both pathways are interconnected.

¹) Marschall and Tudzynski, 2016: Reactive oxygen species in development and infection processes. Semin Cell Dev Biol. 2016 Sep;57:138-46.

**161T LaeA/1-velvet interplay in Aspergillus and Trichoderma: Regulation of secondary metabolites and cellulases.** R. Karimi

Aghcheh¹, C. P. Kubicek², J. Strauss³, G. Braus⁴ 1) Department of Molecular Microbiology and Genetics, Georg August University Goettingen, Goettingen, DE; 2) Institute of Chemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a A-1060 Vienn, Austria; 3) Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, BOKU University Vienna, Austria; 4) Department of Molecular Microbiology and Genetics, Georg August University Goettingen, Goettingen, DE.

Filamentous fungi are of considerable economic and social significance for human health, nutrition and in white biotechnology. These organisms are dominant producers of a range of primary metabolites such as citric acid, microbial lipids (biodiesel) and higher unsaturated fatty acids (HUFAs). In particular, they produce also important but structurally complex secondary metabolites with enormous therapeutic applications in pharmaceutical industry, for example: cephalosporin, penicillin, taxol, zeranol and ergot alkaloids. Several fungal secondary metabolites, which are significantly relevant to human health do not only include antibiotics, but also e.g. lovastatin, a well-known antihypercholesterolemic agent produced by *Aspergillus terreus*, or aflatoxin, a carcinogen produced by A. flavus. In addition to their roles for human health and agriculture, some fungi are industrially and commercially important: Species of the ascomycete genus *Hypocrea spp.* (teleomorph of *Trichoderma*) have been demonstrated as efficient producer of highly active cellulolytic enzymes. This trait makes them effective in disrupting and depolymerization of lignocellulosic materials and thus applicable tools in number of biotechnological areas as diverse as clothes-washing detergent, animal feed, and pulp and fuel productions.

Fungal LaeA/LAE1 (Loss of *aflR* Expression A) homologs their gene products act at the interphase between secondary metabolisms, cellulase production and development. Lack of the corresponding genes results in significant physiological changes including loss of secondary metabolite and lignocellulose degrading enzymes production. At the molecular level the encoded proteins are presumably methyltransferases or demethylases which act directly or indirectly at heterochromatin and interact with velvet domain proteins. Velvet proteins bind to DNA and affect expression of secondary metabolites (SMs) genes and cellulases. The dynamic interplay between LaeA/LAE1, velvet proteins and additional interaction partners is the key for an understanding of the coordination of metabolic and morphological functions of fungi and is required for a biotechnological control of the formation of desired bioactive products. *Aspergilli* and *Trichoderma* represent different biotechnologically significant species with significant differences in the LaeA/LAE1-Velvet protein machinery and their target proteins. We therefore performed a comparative study of the interaction partners of this machinery and the dynamics of the various protein-protein interactions using our robust proteomic and mass spectrometry techniques. This enhances our knowledge about the fungal coordination of secondary metabolism, cellulase production and development and thereby will certainly improve recombinant fungal strain construction for the production of industrial secondary metabolite or lignocellulose hydrolytic enzymes.

**162F Internuclear diffusion of histone H1 within cellular compartments in Aspergillus nidulans.** Alexander Mela, Michelle

Momany Plant Biology, University of Georgia, Athens, GA.

Histone H1 is an evolutionarily conserved 'linker' histone protein which serves the important dynamic function of arranging and stabilizing chromatin structure. The mechanism by which histone H1 carries out its function remains unclear. One thing that is certain is the common use of fluorophore-tagged histone H1 protein in microscopy to track nuclei within cells. In time-lapse analyses, we observed stochastic exchange of photo-activated Dendra2-Histone-H1 protein between nuclei. We also observed heterogeneous mixing of histone proteins between nuclei in hyphae and conidiophores derived from heterokaryon fusions of histone H1-RFP and H1-GFP strains. Subsequent analysis of the resulting conidia that contained both RFP- and GFP-labeled histone H1 proteins, showed only parental genotypes. These data together suggest the stochastic exchange of histone H1 protein between nuclei is likely a result of diffusion rather than genetic recombination during karyogamy.
**163W Genes that modulate unconventional secretion of GAPDH.** *M.J. Cohen*¹,², *A Chichester*¹, *P Lipke*¹,² 1) Biology, Brooklyn College, Brooklyn, NY; 2) Molecular, Cell, and Development, CUNY Graduate Center, New York, NY.

Extracellular proteins with a signal sequence exit yeast through a well characterized classical secretory system. Many cell wall proteins in *Saccharomyces cerevisiae* lack a signal sequence, and reach the extracellular environment by uncharacterized mechanisms. We screened *S. cerevisiae* with deletions in vesicular transport genes for changes in activity of the unconventionally secreted cell wall protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase, encoded by the genes *TDH1*, *TDH2* and *TDH3*). Deletions of *VPS21* and *YPT7* Rab GTPases involved in endosomal transport to the vacuole, the GTP Exchange Factor *VAM6* (which encodes a subunit of the HOPS endocytic tethering complex), and *BRO1* (a homolog of mammalian ALIX which may be involved in sorting proteins into the multivesicular body) decreased extracellular GAPDH activity. *S. cerevisiae* lacking the GEFS Vps9, Muk1 or the ESCRT-II and III components Snf7 and Snf8 had increased extracellular GAPDH activity.

Additionally, *S. cerevisiae* lacking autophagy genes *ATG5*, *ATG7*, and *ATG12* have normal levels of GAPDH activity in the cell wall. These genes were previously reported to be required for unconventional secretion of Acb1. This result suggests that GAPDH and Acb1 reach the cell surface of *S. cerevisiae* by different mechanisms. Thus, GAPDH reaches the cell wall by a process that is positively and negatively impacted by different elements of the vacuolar protein sorting pathway.

**164T Nanoscale clustering of the cytosolic enzyme trichodiene synthase to a trichothecene biosynthetic enzyme complex at the endoplasmic reticulum resolved by superresolution microscopy and FRET.** *Marike Boenisch¹, Karen Broz², Ailisa Blum³,⁴, Donald Gardiner³, H. Corby Kistler¹,² 1) University of Minnesota, St. Paul, MN, USA; 2) USDA ARS Cereal Disease Laboratory, St. Paul, MN, USA; 3) CSIRO Agriculture & Food, St. Lucia, Brisbane, Queensland, Australia; 4) SAFS, University of Queensland, St. Lucia, Brisbane, Queensland, Australia.*

The phytopathogenic fungus *Fusarium graminearum* produces the trichothecene mycotoxin deoxynivalenol (DON) during infection of wheat and when induced *in vitro*. Enzymes in the primary mevalonate pathway and in the secondary DON pathway catalyze key steps for fungal DON production and are transcriptionally co-regulated. Mevalonate pathway enzyme HMG CoA reductase (Hmr1) and the DON biosynthetic pathway enzymes trichodiene oxygenase (Tri4) and calonectrin oxygenase (Tri1) are membrane associated proteins. When labeled with fluorescent proteins and grown in DON induction medium, Hmr1, Tri4, and Tri1 strictly co-localize at the endoplasmic reticulum (ER), suggesting they may be part of a multi-enzyme complex within the ER membrane. This hypothesis is supported by positive fluorescence resonance energy transfer (FRET) between either Hmr1 or Tri1 and Tri4, suggesting they are less than 10 nm apart. However, GFP tagged mevalonate pathway enzyme HMG CoA synthase (Hms1) and DON pathway enzyme trichodiene synthase (Tri5) are localized in the cytosol under DON inducing conditions. To determine if these enzymes were located proximal to the ER, 3D SIM superresolution microscopy was applied to a Tri5-GFP/Tri4-RFP or a Hms1-GFP/Tri4-RFP dual tagged strain grown in DON inducing medium. Measuring mean GFP intensity (n=91) in 250 nm cytosolic subregions, Tri5-GFP intensity was significantly higher (P<0.001) surrounding ER membranes (visualized by Tri4-RFP) than elsewhere in the cytosol, while the opposite effect was detected with Hms1-GFP. Tri5 thus may preferentially accumulate at and surrounding the ER membranes that harbor the postulated DON biosynthetic enzyme complex, allowing pathway intermediates to be spatially concentrated at particular regions of the cell. Compartmentalization of toxic pathway intermediates to particular organelles and cytosolic subregions might facilitate DON biosynthesis and self-protection of the fungus.

**165F Distinctive nuclear localization signals in the oomycete *Phytophthora sojae*.** *Y. Fang*¹,²,⁵, *H. Sang*³, *G. Watson*⁴, *D. Wellapilli*⁵, *B.M. Tyler*¹,² 1) Interdisciplinary Ph.D. Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA; 2) Center for Genome Research & Biocomputing and Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR; 3) Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, OR; 4) Biological & Population Health Sciences, Oregon State University, Corvallis, OR; 5) Current address: Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC.

Nuclear localization signals (NLSs) are stretches of residues that direct proteins from the cytoplasm into the nucleus in eukaryotic cells. Despite their critical roles in protein regulation, NLSs in oomycetes have not been defined. Here, we use the soybean pathogen *Phytophthora sojae* as a model to investigate these sequences in oomycetes. By establishing a reliable *in vivo* NLS assay based on confocal microscopy, we found that *P. sojae* uses NLS for translocation of proteins into the nucleus that differs from conventional well-characterized NLSs. Most tested classical NLSs and proline-tyrosine NLSs (PY-NLSs) from model organisms showed poor nuclear import activities in *P. sojae*. In comparison, functional nuclear localization sequences from *P. sojae* nuclear proteins resembled conventional NLSs but required additional basic residues, either within the NLS motif or nearby. In addition, multiple weak NLSs could also work collaboratively for the efficient nuclear import of proteins. To learn more about the features of NLS-mediated transport of nuclear proteins in *P. sojae*, we further characterized in depth the nuclear import mechanism of a *P. sojae* basic leucine zipper transcription factor, PsbZIP1. We found that the nuclear translocation of PsbZIP1 was determined by a central conserved region. Mutational analysis of this region indicated its nuclear translocation was mediated by four distinct motifs but was independent of conserved DNA-binding residues. Three motifs showed autonomous NLS activity and the fourth motif served as a nuclear localization enhancer. Sequence comparison and mutational analysis of these nuclear localization motifs revealed a new form of bipartite NLS consisting of a triplet of basic residues followed by a tail of scattered basic amino acids.

Manuscripts under revision:

Distinctive Nuclear Localization Signals in the Oomycete *Phytophthora sojae*. *Frontiers in Microbiology*.

Nuclear localization of a putative *Phytophthora sojae* bZIP1 transcription factor is mediated by multiple targeting motifs. *Molecular Microbiology*.
**166W Phosphoproteomic analysis of Penicillium chrysogenum reveals 383 phosphorylated proteins.** U. Carrasco, *J. E. Zuniga*, F. J. Fernandez, F. Fierro Universidad Autonoma Metropolitana, UAM-I, Iztapalapa, DF.

**Introduction:** Penicillium chrysogenum is used for the industrial production of the b-lactam antibiotic penicillin. We have recently performed a proteomic analysis of the Pga1-mediated heterotrimeric G protein signaling pathway of this fungus (1), finding two phosphorylated proteins as effectors of the pathway. To advance in the characterization of this signaling pathway we have performed a phosphoproteomic analysis. Here we describe the identification of a total of 383 phosphorylated proteins and their functional classification.

**Methods:** After trypsin digestion, phosphopeptide enrichment was carried out by metal oxide affinity chromatography with TiO₂, then the phosphopeptides were submitted to LC-MS/MS for identification, Validation of protein identification from MS/MS was carried out with the program Scaffold v4.0.5 (Proteome Software Inc.), Confirmed identification of a protein was considered when the probability was above 95 % and at least one peptides from the protein were positively identified. **Results:** 383 phosphorylated proteins were identified from 8343 total spectra. The classification of the proteins according to their biological function showed that 28% of the proteins are involved in the primary metabolism, and 1.96% are proteins related to signal transduction Approximately 20 % of the proteins are described as located in the nucleus. Fifteen probable transcription factors and chromatin remodeling proteins were identified. It is interesting to note that several phosphorylated proteins are described as involved in polarized growth and morphogenesis. **Conclusions.** We have performed the first analysis of the *P. chrysogenum* phosphoproteome, identifying 383 phosphorylated proteins. **References:** 1) Microb Cell Fact. 2016;15(1):173.

**167T Unpacking the molecular mechanism behind unisexual reproduction in Huntiella moniliformis.** A. Wilson¹, M. van der Nest¹, P.M. Wilken¹, M.J. Wingfield², B.D. Wingfield¹ 1) Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa ; 2) Department of Microbiology & Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Unisexual reproduction is a reproductive strategy where an individual fungus is able to produce sexual offspring despite possessing genes that represent a single MAT idiomorph. This form of reproduction has been described in a few species of fungi, but was most recently observed in MAT2 individuals of the filamentous ascomycete; *Huntiella moniliformis* (Wilson *et al.* 2015. Fun. Genet. Biol. 80:1-9). This fungus is a member of the family Ceratocystidaceae, a group including well-known pathogenic fungi that infect a wide variety of economically important plants. The underlying molecular mechanisms responsible for unisexuality in filamentous fungi are poorly understood and this study aimed to elucidate these mechanisms using a comparative transcriptomics approach. By sequencing the mRNA from vegetative and sexually-competent cultures of *H. omanensis*, a strictly heterothallic relative of *H. moniliformis*, we were able to identify some of the genes that are important for sexual reproduction in these fungi. By comparing these data to similar data obtained from cultures of *H. moniliformis*, we were able to detect significant differences in gene expression between the heterosexual and unisexual pathways. Most notable was the mating type-independent expression of both the α- and a-factor pheromone genes in the unisexual *H. moniliformis*. This was in contrast to *H. omanensis*, were MAT1 individuals produce the α-factor pheromone and MAT2 individuals the a-factor pheromone in a manner similar to other heterothallic species such as *Neurospora crassa*. *H. moniliformis* cultures also expressed both pheromone receptors at a constitutive level during vegetative growth and sexual reproduction compared to the potentially mating type-specific expression observed in *H. omanensis*. The results suggest that mating type-independent expression of these pheromones plays an important role in the unisexual capabilities of *H. moniliformis*. This can be compared to a unisexual pathway in *C. albicans* where endogenous pheromone production allows for self-activation.

**168F Yeast colony life style is affected by the environment.** L. Vachova¹, Z. Palkova² 1) Institute of Microbiology CAS, v.v.i. - Biovec, Prague, Czech Republic; 2) Faculty of Science, Biovec, Charles University, Prague, Czech Republic.

Wild strains of *Saccharomyces cerevisiae*, which are adapted to hostile natural environment, are able to form structured biofilm colonies, composed of aerial part and of pseudohyphae that invade the agar and attach the colony to the substratum. These colonies evolve a number of protective strategies, such as production of Flo11p adhesin, production of extracellular matrix by cells localized to the colony interior and activation of multidrug resistance pumps in cells localized to the colony surface (*J Cell Biol* 194:679, 2011). Rich nutrient supplies induce the process of domestication in wild strains leading to formation of cells that start to form smooth colonies similar to colonies of laboratory strains. This domestication is accompanied by switching off of most protective mechanisms (*Fungal Genet Biol* 47:1012, 2010). Domestication can be reversed under adverse conditions when feral subclones start to appear that reacquire the ability to form biofilm colonies with wild type-like biofilm colony architecture (*BMC Genomics*. 15:136, 2014). We compared various features of colony cell populations formed by wild, domesticated and feral strains by transcriptomic and confocal microscopy approaches, combined with other analytical methods. Colonies formed by the feral strain resemble biofilm colonies of the wild strain in several ways, including restoration of the major features that are switched off during domestication, such as formation of extracellular matrix, production of Flo11p adhesin and ability to absorb high amount of water. The main functional groups of genes, typical of colonies with a "structured" morphotype, include genes linked to cell wall remodeling and plasma membrane functions as well as genes involved in signaling cascades. This research was funded from the Norwegian Financial Mechanism 2009-2014 under Project Contract no. 7F14083 and by GACR 13-08605S.

**169W Structural reorganization of the endoplasmic reticulum upon induction of mycotoxin biosynthesis in Fusarium graminearum.** Marike Boenisch¹, Karen Broz², H. Corby Kistler¹,² 1) University of Minnesota, St. Paul, MN, USA; 2) USDA ARS Cereal Disease Laboratory, St. Paul, MN, USA.

The ascomycete fungus *Fusarium graminearum* causes disease on wheat and barley and contaminates grains with trichothecene (TRI) mycotoxins. To understand how toxin biosynthesis is spatially regulated in the fungal cell and to explore potential self-protection mechanisms of the fungus, the subcellular localization of TRI biosynthetic enzymes was determined and cellular changes during toxigenesis were studied. Using fluorescence tagged proteins and the blue fluorescent dye ER-Tracker, enzymes, catalyzing early and late steps in trichothecene biosynthesis, hydroxymethylglutaryl CoA reductase (Hmr1), trichodiene oxygenase (Tri4), and calonectrin oxygenase (Tri1), were observed to co-localize with the endoplasmic reticulum (ER) when TRI is induced artificially *in vitro* and naturally *in planta*. In addition to these enzymes, a protein of unknown function, Tri14, encoded by a gene within the trichothecene gene cluster, also co-localizes with Tri4 at the ER during TRI production. Interestingly the trichodiene synthase Tri5, while localized in the cytosol, appears to cluster at the
ER. Applying 3D SIM superresolution microscopy to the wild type strain, we demonstrate that, upon TRI induction, the ER stained with ER-Tracker shifts from being reticulate to being thickened with pronounced perinuclear and peripheral ER. This reorganization of the ER was confirmed with reporter strains containing the ER resident protein Sec22-GFP, as well as with strains having GFP linked to the ER retrieval sequence (HDEL) and expressed in the fungal ER lumen. Transmission electron microscopy revealed that organized smooth ER membranes (OSER) are formed in TRI producing cells, but not in non-TRI producing cells. OSER are lamellar or concentric stacks of smooth perinuclear or peripheral ER membranes, with a ~10 nm cytoplasmic space between each ER sheet. Since enzyme active sites for Tri1, Tri4, and Hmr1 are predicted to be on the cytoplasmic side of the ER membrane, we conclude that TRI is likely synthesized in the cytoplasmic spaces within OSER. Thus, TRI metabolites may be sequestered from targets of TRI inhibition e.g. ribosomes and mitochondria and might facilitate TRI biosynthesis and self-protection of the fungus.

**170T Naming mating genes: A review of Pezizomycotina MAT gene nomenclature.** Markus Wilken¹, Emma Steenkamp², Michael Wingfield¹, Brenda Wingfield¹ 1) Dept Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 2) Department of Microbiology & Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

The sub-phylum Pezizomycotina (Ascomycetes) includes fungi with a diverse range of sexual strategies, all regulated by the mating (MAT) genes. Naming these genes is dictated by a nomenclature system that has been applied for more than 15 years. Contemporary techniques including whole genome sequencing have resulted in the description of numerous novel MAT genes, which in many cases have been erroneously named. Reasons for this include the low levels of sequence similarity between putative homologs, the misidentification of genes in public sequence repositories and misinterpretations of the nomenclature rules. This situation emphasises the need to re-evaluate and revise the manner in which the nomenclatural system for MAT genes is applied. In this study, we examined all genes that have been described from the *MAT1* locus in the Pezizomycotina. Our results showed that the 15 known mating genes described in the Pezizomycotina *MAT1* locus represent at least 24 distinct genes, some of which have known homologs outside of the *MAT1* locus. To address this discrepancy, we propose a revision of the nomenclature system that would involve renaming some of these genes. This revision would result in the addition of two *MAT1-1* and four *MAT1-2* gene names, as well as the removal of four currently used MAT gene names. The overall outcome would bring the total number of unique mating genes to 18. This study is being prepared as a review with firm recommendations on changes to the current names of these genes. Our hope is that such a revision will clarify existing problems and provide clear guidelines as well as reference sequences to be used when these important genes are named in the future.

**171F Characterization of two ferroxidases and one ascorbate oxidase belonging to the multicopper oxidase family in the filamentous fungus *Podospora anserina*: a new role in fungal development.** N. Xie¹,², G. Ruprich-Robert³, P. Silar², E. Herbert², R. Ferrari², F. Chapeland-Leclerc³ 1) Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China; 2) Univ Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, 75205 Paris, France; 3) Univ Paris Descartes, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, 75205 Paris, France.

Our previous phylogenetic analyses showed that the *Podospora anserina* genome contains a large family of 15 MCOs, including two genes encoding FET3-like proteins and one gene encoding an AO-like protein. FET3-1, FET3-2 and AO1 are involved in global laccase activity, since deletion of the relevant genes led to a decrease of activity. However, contrary to the *P. anserina* MCO proteins previously characterized, none of these three MCOs seemed to be involved in lignocellulose degradation and in resistance to phenolic compounds and H₂O₂. Deletion of the *ao1* gene led to an almost total loss of AO activity. No direct involvement of AO1 in fungal developmental process in *P. anserina* was evidenced, except in a *fet3-1*⁴ background. We showed that the bulk of FET3 activity was clearly due to FET3-1, and only in minor part to FET3-2, although FET3-1 does not contain all the residues typical of FET3 proteins. Moreover, we showed that FET3-1, closely related to the *Aspergillus fumigatus* ABR1 protein, was clearly and specifically involved in pigmentation of ascospores. Surprisingly, phenotypes were more severe in mutants lacking both *fet3-1* and *ao1*. Overall, unlike other previously characterized MCOs, we thus evidence a clear involvement FET3-1 protein in fungal development.

**172W Functions of PRM1 and KAR5 in cell-cell fusion and karyogamy drive distinct bisexual and unisexual cycles in the Cryptococcus pathogenic species complex.** Ci Fu¹, Joseph Heitman ¹) Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC.

*Cryptococcus neoformans* and *Cryptococcus deneoformans* are two sibling species belong to the opportunistic human fungal pathogen *Cryptococcus* species complex. *C. neoformans* mainly undergoes bisexual reproduction, whereas *C. deneoformans* undergoes both unisexual and bisexual reproduction. During both sexual cycles, a dramatic yeast-to-hyphal morphological transition takes place and generates either monokaryotic or dikaryotic hyphae, and this process is regulated by a common set of genetic circuits. The unisexual cycle can also generate genotypic and phenotypic diversity de novo. Despite the similarities between unisexual and bisexual cycles, there are still genetic and morphological differences, such as an absence of an opposite mating partner and monokaryotic instead of dikaryotic hyphae. The natural populations for both species are mainly α mating type; however, the unisexual cycle is well established in *C. deneoformans* but not in *C. neoformans*, suggesting differences in mating mechanisms may contribute to this. To understand what distinguishes the different modes of sexual cycles, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and nuclear fusion. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both *Cryptococcus* species, and showed that they play conserved roles in cell fusion and karyogamy during sexual reproduction. The involvement of Prm1 and Kar5 indicates that there are fundamental differences between unisexual and bisexual reproduction, and between bisexual reproduction of the two sibling species. Cell fusion and karyogamy are largely dispensable for unisexual reproduction, and the unisexual cycle achieves diploidization early during hyphal development, likely through endoreplication. During the bisexual cycles, in *C. deneoformans*, karyogamy occurs early during the hyphal development whereas it occurred inside the basidium in *C. neoformans*.
**173T** A genetic screen uncovers inositol-phosphate signaling as essential for sexual reproduction in *Podospora anserina*. N. Xie¹,², G. Ruprich-Robert³, F. Chapeland-Leclerc³, H. Lalucque², E. Coppin⁴,⁵, S. Brun², R. Debuchy⁵, P. Silar² 1) Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China; 2) Univ Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, 75205 Paris, France; 3) Univ Paris Descartes, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain (LIED), UMR 8236, 75205 Paris, France; 4) CNRS, Institut de Génétique et Microbiologie UMR8621, Orsay, France; 5) Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, UMR 9198, 91405 Orsay, France.

The molecular pathways involved in the development of multicellular fruiting bodies in fungi are not yet well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe *PM154*, a new strain of the model ascomycetes *Podospora anserina* that mimics a homothallic and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of *spod1*, one of the new mutants, we identified an inositol phosphate polykinase gene that plays a critical role for fruiting body development and growth. A factor present in the wild type and diffusible into mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in *spod1*, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in *spod1*. Overall, the data indicated that inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. Key role of inositol signaling during fruiting body maturation is conserved during the evolution of *Sordariales* fungi.

**174F** Are RIP and heterochromatin involved in *Podospora anserina* sexual development? F. Carlier, V. Contamine, F. Malagnac Institute for Integrative Biology of the Cell (I2BC, UMR 9198), CEA, CNRS, Université Paris Sud, 91400 Orsay CEDEX, France.

In Pezizomycotina, DNA repeats are subjected to a silencing mechanism called RIP (Repeat Induced Point mutation). RIP introduces mutations, preferentially C/G to A/T transitions and sometimes, in species such as *Neurospora crassa*, RIPed sequences are *de novo* methylated. The putative DNA-methyltransferase Rid protein of *N. crassa* is essential for RIP. Besides, when methylated, the RIPed sequences are marked as constitutive heterochromatin (H3K9me3) by histone methyltransferase DIM5 and recognized by the chromodomain containing protein 1 (HP1). In *Podospora anserina*, although no significant levels of DNA methylation have been detected yet, repeats are subjected to RIP during sexual reproduction. Unlike the *N. crassa rid* mutants, the *P. anserina rid* mutants are unable to complete sexual development and thus to produce ascospores. Moreover, fertility of *P. anserina rid* mutants is restored only if PaRID harbors a functional DNA methyltransferase catalytic domain.

To better understand the relations between RIP, chromatin remodeling and *P. anserina* sexual development, we constructed the Δ*PaDim5* and Δ*PaHp1* null mutants. Although H3K9me3 can be detected by Western-blot analysis in wild-type *P. anserina* strains, the single mutant strains, as well as the corresponding double mutant strain, are not affected in sexual reproduction and present mild growth defects. As expected, in a wild-type genetic background, PaDim5-mCherry and PaHp1-GFP proteins are nuclear. The PaHp1-GFP proteins form foci, a pattern that is disrupted in a Δ*PaDim5* genetic background but not in the Δ*PaRid* background. These results suggest that the epigenetic component of the *P. anserina* developmental pathway is PaDim5 independent. Then, we explored the role of the facultative heterochromatin mark H3K27me3 set up by the PaKmt6 histone methyltransferase. The Δ*PaKmt6* null mutant causes a significant growth defect and is impaired in sexual reproduction which suggests that H3K27me3 plays a central role into *P. anserina* sexual development. Identification of the PaRid target during sexual reproduction might contribute to complete our understanding of this crucial developmental pathway.

**175W** Forward genetics identifies developmental genes in the filamentous fungus *Sordaria macrospora*. I. Teichert¹, M. Lutomski¹, A. Hamann², R. Märker¹, M. Nowrousian¹, H.D. Osiewacz², U. Kück¹ 1) Ruhr-University Bochum, Allgemeine und Molekulare Botanik, Bochum, DE; 2) Molecular Developmental Biology, Faculty of Biosciences and Cluster of Excellence 'Macromolecular Complexes', Johann Wolfgang Goethe University, Frankfurt am Main.

The filamentous ascomycete *Sordaria macrospora* is a model system for studying fungal sexual development. *S. macrospora* forms perithecia within one week in the laboratory. A large collection of sterile mutants is available for this fungus, with mutants blocked at different stages of fungal development. Focusing on mutants of the “pro” type, which generate only immature spherical pre fruiting bodies (protoperithecia) already identified quite a number of developmental factors. However, many sterile mutants have not been characterized yet, and functional characterization of many developmental factors is still not complete [1, 2].

Here, we analyzed two further mutants, spadix (spd) and pro34, to identify the underlying genotype. Pooled DNA from progeny of crosses of the mutants to wild type was sequenced by Illumina sequencing. Bioinformatics analysis identified the mutations underlying the mutant phenotypes. The sterile phenotype of mutant spd is caused by deletion of the *spd4* gene. SPD4 is required for fruiting body formation, but not vegetative cell fusion in *S. macrospora*, although both processes have long been thought to be linked. SPD4 displays a nuclear localization and is restricted to ascogonial tissues during sexual development [3]. The sterile phenotype of pro34 is caused by a deletion in the *cia84* (complex I assembly) gene. PRO34 displays a mitochondrial localization, and the mutant shows a respiratory defect. Like SPD4, PRO34 is not required for cell fusion in *S. macrospora*. Thus, sequencing the genomes of two sterile mutants identified additional genes involved in sexual development in a model fungus. Both, SPD4 and PRO34, are required for fruiting body formation, but not cell fusion. These two proteins therefore provide specific insights into the regulation of sexual development in fungi.

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

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

[3] Teichert et al. 2016 Mol Genet Genomics, in press

**176T** Courtship ritual of male and female nuclei during fertilization in *Neurospora crassa*. S. Brun¹, Hsiao-Che Kuo³, Chris Jeffree⁴, Nick Read² 1) LIED-UMR 8236, Univ Paris-Diderot, Paris, FR; 2) MFIG, manchester University, UK; 3) Fungal Cell Biology Group, University of Edinburgh, UK; 4) Institute of Plant Sciences, University of Edinburgh, UK.

Sexual reproduction is a key event in the life cycle of fungi. It relies on very elaborated genetic regulation systems as well as complex cytological events. Fertilization starts by the recognition of “male” and “female” haploid cells, namely the conidium and the trichogyne
emitted by the protoperithecum, respectively. Then fusion of these cells (plasmogamy) follows. However, the fusion of the two parental
nuclei (karyogamy), giving rise to the diploid nucleus undergoing meiosis, will take place within the protoperithecum.
In laboratory conditions, fertilization in the heterothallic model fungus *Neurospora crassa* can be observed few hours after inoculation of
petri dishes bearing protoperithecia by conidial suspensions of opposite mating type (Bistis, 1981). Using parental strains in which nuclei
were tagged with either the Green Fluorescent Protein or the Red Fluorescent Protein, we achieved for the first time live imaging of male
and female nuclei during fertilization. Our study reveals that the parental nuclei behave totally differently and that the movement of male
nuclei down through the trichogyne is a complex process. Indeed, while male nuclei migrate down through the trichogyne changing of
shape from rounded to stretched in an inchworm-like movement, female nuclei "freeze" just prior the entry of male nuclei into the trichogyne
and remain totally immobilized throughout the process. Furthermore, protoperithecia usually emit several trichogynes that are often
branched. Hence, multi-branched trichogynes can fuse to several macroconidia allowing the discharge of multiple male nuclei into a same
network connected to a single protoperithecum. In this labyrinth, we could trace male nuclei and follow their "orienteering race", changing
direction at junctions and probably competing with each other to fertilize the protoperithecum.

Our pioneering study based on live imaging of nuclei during sexual reproduction in *N. crassa* addresses fundamental questions regarding the identity of male and female nuclei as well as which components of the cytoskeleton are involved in nuclei movements during fertilization. It also raises brand new questions about i) the signal guiding male nuclei to the protoperithecum and ii) a possible signal emitted by the fertilized perithecum that inhibits polyspermy.

Bistis, G.N. (1981). Chemotropic Interactions between Trichogynes and Conidia of Opposite Mating-Type in Neurospora crassa. Mycologia 73, 959-975.

177F ChIP-Seq analysis identifies components of the cell wall integrity, NADPH oxidase and pheromone signaling integrity pathways as target genes of the fungal developmental regulator PRO1. U. Kück, S. Steffens, K. Becker, S. Krevet, I.

Teichert Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE.

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

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

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

178W Sex prepares the plant pathogenic fungus *Botrytis cinerea* for host invasion. Alexander Y. Rodenburg¹, Razak Terhem¹,²,
*Jan A.L. van Kan*¹ 1) Phytopathology, Wageningen University, Wageningen, the Netherlands; 2) Department of Forest Management,
Faculty of Forestry, Universiti Putra Malaysia, Serdang, Malaysia.

*Botrytis cinerea* is a plant pathogenic ascomycete producing apothecia as sexual fruiting bodies. We used RNAseq to analyse expression
profiles in sclerotia, three stages of apothecium development (primordia, full grown stipe, apothecial disk), and mature ascospores. Very
pure tissue samples of the five developmental stages could be isolated and transcriptional changes between successive developmental
stages were analysed. Changes were observed in the expression levels of as many as 15 secondary metabolite gene clusters, in different
stages. The most profound overall changes in transcript profiles were at the initiation of sexual development (transition from sclerotia to
primordia, >2500 differentially expressed genes) and even more so, at the completion of sexual reproduction in the mature ascospores:
1424 genes were upregulated and 2485 genes were downregulated in ascospores as compared to mature apothecial disks. Among the
genes upregulated in ascospores were many genes encoding virulence factors (plant cell wall hydrolases, proteinases, multidrug efflux
ABC transporters, superoxide dismutase, phytoalexin-oxidising laccase, an oxalic acid biosynthetic enzyme), several hexose transporters
and a number of signal transduction components known to be involved in virulence. These observations clearly suggest that ascospores
are transcriptionally primed for infection already prior to their arrival on host plants.

Strikingly, the large transcriptional changes at the initiation and the completion of the sexual cycle often affected numerous clusters of
genes (not only genes in secondary metabolite clusters), rather than genes randomly dispersed through the genome. Sexual development
also coincided with changes in the expression of several genes potentially involved in chromatin organization (C-5 cytosine methylase,
several histone acetyltransferases, dicer-like protein), suggesting an epigenetic regulation of gene expression during the sexual cycle.

179T Deciphering the *Teratosphaeria destructans* mating system: a genomics approach. C.T. Tatham, M.A. van der Nest, P.M
Wilken, M.J. Wingfield, B.D. Wingfield Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Fungal pathogens are increasingly threatening global plantations of intensively propagated non-native *Eucalyptus* spp. In this regard, one
of the most destructive shoot and leaf pathogens is *Teratosphaeria destructans*, a fungus of unknown origin that has devastated plantations
particularly in southeast Asia. Despite this, virtually nothing is known regarding its biology or genetics. In this study, we sought to explore
the details of the mating strategy of *T. destructans*. The genome of a *T. destructans* isolate was sequenced and assembled. The draft
genome sequence was used to identify and characterize the mating locus. This isolate contained only the *MAT1-2-1* gene, and was used to
describe this isolate as a MAT 2 isolate containing a *MAT1-2* idiomorph. Other genes such as DNA lyase, APC5 and cytochrome oxidase C
that are often linked to the MAT1 locus in fungi were present in the vicinity of the *T. destructans MAT1* locus. PCR analysis of 21 *T.
destructans* isolates showed that the MAT1-2-1 gene was present in only about half of the isolates. This suggests that *T. destructans*
reproduces in a heterothallic manner, and that the remaining isolates are likely to harbour the *MAT1-1* idiomorph. Collectively, the results
suggest that *T. destructans* would be capable of reproducing sexually in regions where isolates of opposite mating types are present. This could explain why the pathogen has commonly adapted to infect clones selected for resistance.

**180F The phosducin-like protein PhnA of Botrytis cinerea is involved in fungal development and pathogenicity.** *J. Kilani*¹,², M. Miazzi³, C. Audeon¹, S. Fillinger¹ 1) UMR BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France; 2) Univ. Paris-Sud, Université Paris-Saclay, France; 3) Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy.

*Botrytis cinerea* is a necrotrophic, polyphageous plant pathogen, that causes gray mold disease and can infect over 1000 plant species including several agronomically important crops (grapevine, strawberry, tomatoes ...). Fungicides remain the most effective means to combat this disease. However *B. cinerea* rapidly adapts to fungicides. Presently, the phenylpyrrole fludioxonil is one of the most efficient fungicides against *B. cinerea*. Therefore deciphering the response to fludioxonil in *B. cinerea* is crucial.

Fungi rapidly adapt to their environment involving to signalling pathways like those of mitogen activated protein kinases (MAPKs). In *Botrytis cinerea*, the fungicide fludioxonil activates the Sak1 and Bmp3 MAPKs, which are respectively involved in osmoregulation, cell wall integrity, development and pathogenicity. In order to trace the transduction of fludioxonil to the MAPK pathways, we have started a phosphoproteomic approach and the functional analysis of key genes.

A phosphoproteomic pilot project of the wild-type strain subjected to fludioxonil, lead to the identification of "fludioxonil" candidate signalling proteins such as the phosducin-like protein PhnA. In eukaryotes, phosducin allows the dimerization between the subunits Gβ and Gy in the G-protein signalling pathway. In the phytopathogenic fungi *Cryphonectria parasitica* and *Fusarium graminearum*, phosducin is involved in pigmentation, sporulation, and pathogenicity while in *Aspergillus nidulans* this protein is involved in growth, development and mycotoxin production.

We have initiated the functional analysis of *phnA* in *B. cinerea*. Its deletion revealed that *phnA* is involved in vegetative growth, pathogenicity, and in development. This functional analysis of *phnA* highlighted it as a new pathogenicity factor in *B. cinerea*. The role of PhnA-phosphorylation for its activity and the link to fludioxonil, G-protein, and cAMP signalling is under investigation by site-directed mutagenesis of the identified phosphorylation site.

**181W Plant sensing enhances sexual development and overrides nutrient sensing in Trichoderma reesei.** *G. Li*¹, D. Turra², A. Di Pietro², M. Schmoll¹ 1) Austrian Institute of Technology GmbH, Tulln, AT; 2) University of Cordoba, Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Córdoba, Spain.

The natural environment of the potent cellulase producer *Trichoderma reesei* (syn. *Hypocrea jecorina*) is a tropical forest, where it degrades cellulosic plant biomass. However, related *Trichoderma* spp. are also known as efficient plant protection agents, antagonizing fungal pathogens.

In the plant pathogen *Fusarium oxysporum*, plant sensing is accomplished by pheromone receptors responding to secreted plant peroxidases. Here we investigated the relationship between plant sensing, sexual development and nutrient detection in *T. reesei*. We found that *T. reesei* is able to sense plant root exudates and peroxidases via pheromone receptors, suggesting that this mechanism is conserved between *Trichoderma* and *Fusarium*. Moreover, *T. reesei* also showed a chemotropic response to glucose. Interestingly, a nutrient-rich medium such as malt extract (ME) resulted in bipolar and multipolar germination without discernible chemotropism, whereas exudates from soybean roots or from a compatible mating partner in the presence of ME caused unipolar germination with a robust chemotropic response. Hence, signals related to sexual development and plant sensing override nutrient regulated germination.

Accordingly, we found that the presence of soybean germlings promotes sexual development of *T. reesei* as do compounds releasing nitric oxide, a signaling molecule, which is produced by plants to counteract oxidative stress and involved in plant pathogen interaction. Also reacting oxygen species were found to play a role in sexual development, which may explain the relevance of sensing of a peroxidase by pheromone receptors.

We propose that *T. reesei* is attracted to plants by a mechanism that integrates directed hyphal growth and sexual development for efficient adaptation to the host and potentially also for optimized antagonism of competitors and plant pathogens. Thereby, association with a plant has higher priority than availability of nutrients.

**182T Investigation and visualisation of signal transduction elements in Schizophyllum commune.** *Elke-Martina Jung*, Sophia Wirth, Lisa-Marija Ahrens, Erika Kothe Department of Microbiology, Friedrich Schiller University, Jena, DE.

The white rot basidiomycete *Schizophyllum commune* is an early colonizer of tree wounds and after forest fires with competitive abilities based on the production of specific extracellular metabolites and a strategy of fast growth. In addition, recognition of extracellular signals is essential for mating, directed growth, and fruiting body production. G-protein coupled receptors like Bar2 were visualized by GFP-tagging and indirect immunofluorescence in the cell membranes of clamp cells. Downstream signalling is mediated by heterotrimeric G-proteins. An RGS (regulator of G-protein signaling) protein, Thn1, acts as GTPase activating protein (GAP) inhibiting Gα signalling. The interaction of this RGS protein and the pheromone receptor is shown using bimolecular fluorescent complementation (BIFC), establishing this technique for the first time in fruiting body forming, filamentous basidiomycete. Additional steps in signal transduction are mediated by Small GTPases from the Ras superfamily. The *S. commune* genome encodes more than 30 putative Ras GTPases, containing the Ras, Rho, Rab, Ran and Arf subfamilies. The function of the *S. commune* small G-proteins Ras1 and Ras2 have been postulated in MAPK signalling during pheromone response, in morphology, asexual development and mating. Constitutively active Ras1 mutant strains show phenotypes like reduced growth rates, hyperbranching and abnormal growth pattern. Furthermore, the mutants produce abnormal fruiting bodies with weakly developed subhymenium and aberrant spore formation. Thus, both Ras GTPases are major regulators of cellular development.

**183F Deciphering the HET domain-mediated programmed cell death in Neurospora crassa.** *A.I. DASKALOV*, N. L. GLASS Plnat and Microbial Biology, UC Berkeley, Berkeley, CA.

Programmed cell death (PCD) plays a critical role in homeostasis and immunity in Eukaryotes. In Fungi, PCD has been described in the phenomenon of Heterokaryon Incompatibility (HI) – a cell death reaction occurring between somatic fusions of genetically distinct conspecific strains. Currently, there is a relatively good description of the genetic control of HI and a number of incompatibility factors have
been characterized at molecular level. However, the molecular mechanisms and the signal-transducing pathways remain, overall, poorly
understood.

Several of the allorecognition determinants share a common domain, termed HET (~150 aa), instrumental for the triggering of PCD during
HI. Intriguingly, the HET domain is ubiquitous in fungal genomes and although its cellular function remains unknown, it has been shown in
silico to bear structural similarity to the Toll/interleukin-1 receptor (TIR) domain – an important effector domain in plant innate immunity. For
those reasons, we decided to better characterize the PCD reaction mediated by the HET domain in *Neurospora crassa*.

First, we analyzed ~65 HET domain-containing proteins, encoded in the genome of *N. crassa*, using the online MEME suite tool and identified 25 significantly conserved (e-value <2.3e-002) amino acid motifs outside of the HET domain. Then, we used the patterns of these motifs to defined three major protein families (A, B and C) and to identify additional putative domains, systematically associated with the HET domain. Here, of particular interest is a putative domain in family A (~40 members) consisting of Zn-finger-like motifs (CxxC—CxxxHxxC) situated at the N-terminus of the proteins, that we termed ZHAD (Zn-finger-like HET-associated domain). We investigated the involvement in HI of the newly identified and widespread ZHAD domain using the mating-type HI determinant TOL. We showed – using site-directed mutagenesis – that the ZHAD domain of TOL is important for the PCD reaction, which was also dependent, unsurprisingly, on the HET domain of TOL. Further analyses will define the precise molecular function of the ZHAD domain and will help address some long-standing questions regarding the molecular mechanism of HET domain-mediated PCD in fungi.

**184W The plant phenolic ferulic acid modulates gene expression and the phosphoproteome of Cochliobolus heterostrophus.** Samer Shalaby, Hiba Simaan, Orit Goldshmidt-Tran, *Benjamin Horwitz* Faculty of Biology, Technion - IIT, Haifa, IL.

Ferulic acid (FA) belongs to a group of phenolics widespread in plants, which are perceived by the necrotrophic maize pathogen *C. heterostrophus* as a stress signal inducing programmed cell death (PCD) (Shalaby et al. 2016, Environ. Microbiol). The mechanisms of signaling and toxicity are unknown. We used RNAseq to study the transcriptomic signature of the early (30 min) response to exposure to a 0.5-2 mM range of concentration of FA. Genes related to metabolism of phenolics, major facilitator family transporters and acetyl CoA metabolism were prominent among the large number of differentially expressed genes. Following the intriguing earlier observation that FA promotes nuclear retention of the redox-sensitive transcription factor ChAP1 without up-regulating genes for oxidant tolerance, we are using the RNASeq dataset to identify specific ChAP1-dependent targets of FA. The time-dependent phosphoproteome was determined for the 5 to 30 min time points at 2 mM FA, the highest concentration tested in the transcriptome study. Of a little over 5000 phosphopeptides detected, about 400 showed differential regulation by FA in two experiments. Integration of these large datasets should generate an initial picture of the events downstream from exposure to FA.

Supported by the Israel Science Foundation, ISF & INCPM 332/13; proteomics by Rina Zuchman & Tamar Ziv, Smoler Protein Center; RNASeq by Olga Karinski, Maor Hatoel, Anastasia Diviatis, Avital Polsky & Tal Katz-Ezov, Technion Genome Center.

**185T Lipid biosensors: a novel tool for investigating the composition of fungal membranes.** A.V. *Candy*¹,², A. Ram¹, PY. Dupont¹,², B. Scott¹,², C.J. Eaton¹,² 1) Institute of Fundamental Sciences, Massey University, Palmerston North, Manawatu-Wanganui, NZ; 2) Bio-Protection Research Centre, Massey University, NZ.

In plant-associated fungi, the production of reactive oxygen species (ROS) by the NADPH oxidase (Nox) complex plays an integral role in controlling the growth of both beneficial and pathogenic fungi within their plant host. Therefore, specific regulation of the Nox complex is essential to maintaining a successful symbiotic relationship. Whilst much is known about the importance of ROS in plant-fungal interactions, comparatively little is known about how its production is regulated. In plant and mammalian systems, production of ROS by the Nox complex is regulated via the recruitment of its cytosolic components. This recruitment is controlled by lipid signalling, with cytosolic components containing sequences which target them to specific phospholipids enriched at certain locations within the plasma membrane. It is hypothesised that similar regulation also occurs in fungi.

Very little is known about the lipid composition of fungal membranes, so the aim of this project is to comprehensively analyse membrane lipid composition in the beneficial fungal symbiont of ryegrass, *Epichloe festucae*, using a suite of biosensors. These biosensors consist of highly specific mammalian lipid binding domains fused to a fluorophore, enabling live cell imaging of phospholipid localisation via fluorescence microscopy. This approach also allows for analysis of membrane lipid composition of hyphae growing *in planta*. Using a phospholipase C-eGFP biosensor, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) was detected in the plasma membrane and at septa of hyphae grown in culture and *in planta*. Similar to yeast and mammalian systems, phosphatidylinositol 4-phosphate (PI[4]P), detected with mCherry-Plekha3 and Pleckha3-eGFP, was found to be localised to golgi vesicles in culture grown hyphae. Biosensors to other key modified phospholipids, namely phosphatidylinositol 3-phosphate (PI[3]P), phosphatidylinositol 3,4-bisphosphate (PI[3,4]P₂), and phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P₃), localised to the cytoplasm of hyphae in culture and *in planta*, suggesting that these phospholipids are absent under these growth conditions in *E. festucae*.

**186F Are dormant spores really dormant? A perspective from the transcription level.** *Fang Wang*, *Liguo Dong*, *Chris Koon Ho* Wong Faculty of Health Sciences, University of Macau, Macau SAR, China.

Fungal spores are specialized reproductive cells of filamentous fungi with properties for dispersal as well as for survival under harsh conditions and over a long period of time. It has been shown in *Aspergillus niger* that the resting spores have little metabolic activities and consume minimal energy. Previous studies have detected mRNA transcripts in dormant spores of some filamentous fungi, but it is not clear whether the transcripts were carried over from the process of conidiation or were actively synthesized in the resting spores. Moreover, a previous study in *A. niger* has been shown that genes involved in protein biosynthesis, RNA degradation, respiration and nitrogen metabolism are induced within an hour upon shifting to favorable growth conditions, suggesting that spores are constantly sensing and readily responding to the environment for growth. In this work, we aim to find out 1) whether resting spores of *Aspergillus nidulans* have any active transcription, 2) whether dormant spores can respond to stresses during dormancy and 3) whether resting spores of different ages and spores kept under different stresses behave differently during the break of dormancy. To address these, we have used Chromatin Immuno-precipitation followed by Next Generation Sequencing (ChIPseq) against RNA polymerase II (Pol II) to map active transcription events in spores kept under different conditions (e.g. fresh spores at 37?, spores aged at RT or 4? with or without oxygen) and during the
germination process. Our preliminary results show that Pol II is engaged on many genes genome-wide in resting spores. Gene Ontology analysis shows that these genes are enriched with stress response, ATP generation and the conidiation process. Unexpectedly, we found a significant level of Pol II accumulation near the 5' end of many genes in resting spores but absent in germinated spores and mycelia. The accumulation pattern is similar to the promoter-proximal pausing phenomenon in higher eukaryotes. We are in the process of confirming and understanding the functional significance of this phenomenon. We speculate that Pol II may be preloaded to specific promoters during the spore maturation process such to reduce the steps and energy needed by activation of genes for growth and survival in energetically dormant spores. Therefore, this Pol II "pausing" phenomenon might have important implications in the physiology and durability of spores.

**187W** **White collar proteins are involved in basidiocarp development, and other responses in *Ustilago maydis*.** *J. Sanchez-Arreguin*¹,², *J. Cabrera-Ponce*¹, *C. León-Ramírez*¹, *E. Arechiga-Carvajal*², *J. Ruiz-Herrera*¹ 1) Genetics engineering, CINVESTAV - CIE6010281U2, Irapuato, Guanajuato, México; 2) Universidad Autónoma de Nuevo León. San Nicolas de Los Garza, Nuevo León, México.

Description of fruiting body development in *Ustilago maydis*, was a report out of the ordinary, since the Class Ustilaginomycetes (Subphylum Ustilaginomycotina) has been described as a non-forming basidiocarps group. Based on the observation that illumination was required for the formation of fruiting bodies (basidiocarps) of *U. maydis* under laboratory conditions, in this study we explored whether the homologues of the White Collar (WC) photoreceptors played a role in this process. Accordingly, we obtained mutants in each one of its three WC homologue genes, *WC1a*, *WC1b*, and *WC2*, and analyzed the phenotype of the corresponding mutants. It was observed that the *wc1b* and *wc2* mutants were severely affected in basidiocarp formation when illuminated with white, blue or red light, whereas *wc1a* mutants formed basidiocarps in the three conditions; a result that suggests that *Wc1b* acts as a photoreceptor or/and that *Wc1a* and *Wc2* are involved in a photoreaction dependent on a phytochrome. *Wc1b* was also involved in virulence to maize. In addition, *Wc1a* and *Wc2*, in a light independent mode, were involved in H₂O₂ protection, and in the control of maximal growth of the fungus.

**188T** **The transcription factor Ztf1 (UMAG_02301) is involved in the pathogenesis and stress response of Ustilago maydis.** *D. Martinez-Soto*¹, *J. Velez-Haro*², *J. Ruiz-Herrera*¹, *J. Velez-Haro* 1) Genetics engineering, CINVESTAV - CIE6010281U2, Irapuato, Guanajuato, MX; 2) Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya.

Gene UMAG_02301 from *Ustilago maydis*, encoding a transcription factor (named Ztf1) was found to be differentially regulated during the infection of *U. maydis* in the experimental host *Arabidopsis thaliana*. Further on, we found that the gene was also differentially regulated during *U. maydis* infection of the natural host *U. maydis*.

With these data we proceeded to delete the gene in FB1 and FB2 strains, and analyze the phenotypic characteristics of the mutants obtained. We observed that mutants devoid of the transcription factor were more sensitive than the wild type strain to different kinds of stress, among them: ionic stress, osmotic stress, oxidative stress, acid stress, stress by compounds that affect the membrane or the cell wall. It was also observed that the mutant strain was affected in the yeast-to mycelium dimorphic process induced by growth in the presence of an fatty acid as carbon source. It was also observed that the haploid mutant strains were not affected in mating, but they showed a significant reduction in their virulence during the infection of *Zea mays* and *A. thaliana*.

These results and the previous transcriptomic analyses, demonstrate that this transcription factor is important in different aspects of the physiology of *U. maydis* being involved at least in dimorphism, stress resistance, and in the advanced stages of the pathogenic process specifically after the fungus has penetrated and has become established within of the host tissues.

**189F** **The peroxisomal import machinery enables contact sites between peroxisomes and different organelles.** *T. Stehlik*¹, *M. Boelker*¹, *J. Freitag*² 1) Biology - Genetics, University of Marburg, Marburg, Hessia, DE; 2) Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, USA.

Peroxisomes are nearly ubiquitous and dynamic organelles that harbor diverse metabolic pathways such as β-oxidation of fatty acids and partial reactions of the glyoxylate cycle. They are surrounded by a single membrane and the lack of a genome necessitates the import of all proteins that are destined for the peroxisomal matrix. The import is facilitated by the coordinated action of a membrane localized protein complex and a cytosolic receptor that binds and translocates newly synthesized cargos. In most cases cargo recognition depends on the presence of a C-terminal peroxisomal targeting signal type 1 (PTS1) with the prototypical sequence Ser-Lys-Leu which is recognized by the import receptor Pex5p. Recently, we found that several organisms possess membrane proteins with a potential PTS1 signal and asked whether such proteins might facilitate the formation of contact sites between peroxisomes and other organelles. Here we show that in both *Ustilago maydis* and *Saccharomyces cerevisiae* the protein phosphatase Ptc5p with the afore-metioned architecture tethers peroxisomes to mitochondria *in vivo*. Further, we demonstrate that tethering depends on (i) a functional PTS1 signal and (ii) the presence of the import receptor Pex5p and (iii) that this mechanism does not require specific protein domains by generating a synthetic ER-peroxisome tether.

**190W** **Analysis of septins during hyphal growth of *Ustilago maydis*.** *Sabrina Zander*¹, *Sebastian Baumann*², *Michael Feldbrügge*¹ 1) Heinrich Heine University, Düsseldorf, Institute for Microbiology, 40225 Düsseldorf, Germany; 2) CRG - Center for Genomic Regulation, 08003 Barcelona, Spain.

Septins are GTP-binding cytoskeletal proteins with functions in cell polarity, membrane remodeling, cytokinesis and cell morphology. Conserved across eukaryotes, except higher-order plants, septins assemble in nonpolar, heteromeric complexes. These further assemble end-to-end to form filaments and a variety of higher-order structures. The heterooligomeric structure of septins and their subcellular localization have already been extensively studied. However, a precise mechanism of their subcellular assembly and their intracellular transport are unknown.

The genome of the filamentous fungus *U.maydis* encodes four septin proteins: Cdc3, Cdc10, Cdc11 and Cdc12. In this study the influence of septin deletions were analyzed, during the hyphal state of *U.maydis*. All four septins are needed for efficient unipolar growth indicating a common function for septins during hyphal growth. Furthermore, the subcellular localization was analyzed with Gfp fusion proteins. All septins showed the same localization in cytoplasmic rings, at septa, in filaments and on moving early endosomes. Colocalization studies revealed that all septins localize interdependently in the same structures. Furthermore, FRET analysis showed that Cdc3 and Cdc12 interact directly *in vivo*. Endosomal transport was not only dependent on each individual septin, but also dependent on the RNA-binding protein Rrm4. RNA live imaging of all four mRNAs showed also an Rrm4-dependent transport. FRAP experiments demonstrated that
recovery of Cdc3-mCherryN and Cdc12-GfpN occurs simultaneously at distinct subcellular sites and that the rate of recovery was facilitated by the endosomal RNA transport. Based on these results, a model was proposed where local translation promotes the assembly of newly synthesized septins in heteromeric structures on the surface of endosomes. This is important for the long-distance transport of septins and the efficient formation of the septin cytoskeleton.

**191T Role of polyamines in Ustilago maydis mating.** F. Perez-Rodriguez, J. Ruiz-Herrera Genetics engeniering, CINVESTAV - CIE6010281U2, Irapuato, Guanajuato, MX.

Polyamines are low molecular weight molecules possessing several amino groups, and which are indispensable for the normal growth of living beings. At physiological pH polyamines interact with negatively charged macromolecules such as nucleic acids, proteins, etc.; being involved in a variety of cellular functions such as regulation of macromolecule synthesis, proliferative phenomena, and cell differentiation. Despite these roles, the precise mechanism by which polyamines are involved in these phenomena remains rather unknown. In order to investigate this problem, we proceeded to utilize *Ustilago maydis* mating as a simple model. The mating process in *U. maydis* involves two unlinked mating loci, denominated *a* and *b*; the first one with 2 alleles, and the second one with about 27. Successful mating between two partners occurs when they have different *a* and *b* alleles. To test the role of polyamines in the process we utilized two odc- (ornithine decarboxylase) polyamine auxotrophic strains, one *a1b1* (LG1), and the other *a2b2* (LG4). Mating was determined by the Fuz reaction on solid MM with activated charcoal, and by microscopic observation in liquid MM, both containing low (0.1 mM) or high (5mM) putrescine (the auxotrophs are unable to grow in the absence of polyamines). It was observed that with low putrescine, Fuz reaction was almost negative, and in liquid medium the number and length of the conjugation tubes were highly reduced. When mating involved the LG4 strain and a w.t. strain labeled with mCherry fluorescent protein, the number and length of germination tubes were lower in the mutant, compared with the w.t. fluorescent strain. Expression of *mfa*, *pra*, *bW* and *bE* genes of LG1 and LG2 mating partners at low or high putrescine levels was measured by semi-quantitative PCR. The results showed that expression of all genes was reduced at low levels of polyamines; but levels of *maf1* and *maf2* genes expression were the most affected ones. These results show that *U. maydis* mating requires polyamines, and that their role occurs at the level of selective gene expression.

**192F The Nma1 protein of *Ustilago maydis*: a novel player in vesicular transport processes.** T. Obhof¹, K. Schneider¹, N. Pinter¹,², N. Kellner¹, O. Valerius², G. Braus², J. Kâmer¹ 1) Institute for Applied Biosciences, Karlsruhe Institute of Technology, Karlsruhe, Germany; 2) Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Germany.

We have identified the Nma1 protein in *Ustilago maydis* as an interactor for Num1 (Kellner et al., 2014), a protein that functions both as a component of the NineteenComplex splicing apparatus and in cytoplasmic transport of early endosomes (EEs). Co-IP and MS analysis revealed that Nma1 interacts with Vps3, Vps11 and Vps18, core components of the CORVET complex involved in tethering and fusion of EEs. Concomitantly, Nma1 co-localizes to Rab5 and Yup1(tSNARE) positive EEs. Deletion of Rab5, the EE-localized Rab-GTPase tethering the CORVET complex to EEs, or of Vps3, the Rab5 interacting subunit of CORVET, abolishes the localization of Nma1 to EEs. Deletion of Nma1 affects the movement of EEs and recycling endosomes (REs), but not on secretory vesicles or late endosomes, implicating a functional role in transport of a defined set of vesicles. Interestingly, during mitosis, Nma1 localization is restricted to spindle pole bodies (SPBs); together with Nma1, we observed also the localization of Rab5 marked EEs at SPBs, implicating a role for EEs (or associated proteins) during mitosis, as recently reported for mammalian cells (Das et al., 2015). As Nma1 interacts with the vesicle population localizing to the SPB, we envision a functional connection with the so far poorly understood role of membrane trafficking during mitosis.

Das et al., 2015, Small GTPases, 6:1, 11-15

Kellner et al., 2014, PLoS Genet 10(1): e1004046

**193W The role of Pep4 in autophagy and programed cell death in *Ustilago maydis*.** C. Soberanes-Gutierrez, J Ruiz-Herrera Genetics engeniering, CINVESTAV - CIE6010281U2, Irapuato, Guanajuato, MX.

*Saccharomyces cerevisiae* is currently a well-established eukaryotic model organism used in the elucidation of the molecular mechanisms underlying necrosis, autophagy, and apoptotic pathways. Autophagy is a complex degradative process in which cytosolic material is randomly sequestered within autophagosomes, which are targeted to the vacuole. After degradation by resident hydrolases, the contents are recycled.

In order to determine whether the *U. maydis* *pep4* gene, that encodes the acid vacuolar proteinase A, ortholog to human Cathepsin D, was important for autophagy in *U. maydis*, we compared the capacity of the wild-type, and *Dpep4* strains to accumulate autophagic bodies within the vacuoles when the cells were exposed to carbon stress conditions. Wild type and *Dpep4* cells were incubated for 5 h in minimal medium lacking a carbon source (MM-C), and processed for TEM. Our observations showed the accumulation of autophagic bodies within the vacuole of *Dpep4* cells, in contrast to the absence of these structures into vacuoles of wild-type cells, demonstrating that the proteinase A deficient mutant is affected in its capacity to lyse the autophagic bodies, and all the cytosolic material is accumulated in the autophagosomes present into the vacuole.

In addition we must cite that in several systems it has been concluded that the acidic protease vacuolar is involved in the programmed cell death. Accordingly, it was decided to determine cell death in *U. maydis* FB2 strain and the *Δpep4* mutant. To analyze cell viability the cells were taken to the stationary stage, and subsequently they were washed and resuspended in minimal synthetic medium pH 7, and followed their viability at different incubation times at 28°C. We observed that the *Δpep4* mutant conserve its viability for a longer time than the wild type strain. This was consistent with the observation of cell staining with berberine sulphate, a non-permeating fluorescent compound. It was also observed that the *Dpep4* mutant strain was more resistant to H₂O₂ and acetic acid treatment than the wild type strain. All these data are evidence that proteinase A is involved in programed cell death of *U. maydis*.

**194T Morphological characterization, transcription profiling and metabolic expression during ascospore formation within fertilized sclerotia of *Aspergillus flavus*.** J.M. Luis¹, I. Carbone¹, G.A. Payne¹, D. Bhatnagar², J. Cary², M. Lebar², G.G. Moore², P.S. Ojiambo¹ 1) Center for Integrated Fungal Research, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, 27695; 2) USDA/ARS/SRRC, New Orleans, LA 70124.
Sclerotia of *Aspergillus flavus* can give rise to new hyphae, asexual conidia, or stroma containing sexual spores after fertilization by conidia of the opposite mating type. The transition from asexual to sexual reproduction can have broad implications on the survival and genetic diversity of the fungus, but this process is not well understood. Shifts between sexual and asexual reproduction affect the quantity and diversity of secondary metabolites produced by the fungus, including the carcinogen aflatoxin, which accumulates in field and tree crops infected with *A. flavus*. As asexual structures, the thick-walled and darkly pigmented sclerotia of *A. flavus* are important for survival in the soil. Upon fertilization with conidia of the opposite mating type, the sclerotia can become ascospore-bearing ascocarps. While there is information on the genetic aspects of sexual development in *A. flavus*, there is little to no information on biochemical, transcriptional, or morphological changes that occur between fertilization of sclerotia and the formation of ascopores. This study takes advantage of differences in female fertility between reciprocal crosses to examine morphological, biochemical, and transcriptional differences during sexual reproduction. Specific objectives were to: (i) identify differentially expressed genes between mating pairs with high and low levels of sexual fertility, and (ii) characterize metabolite production when sclerotia are fertilized. These changes were observed every two weeks from the time of fertilization of sclerotia for a total of eight weeks. We hypothesize that genes involved in mating, sexual reproduction, ascocarp development and secondary metabolism will show increased expression in highly fertile sclerotia at the time of ascocarp development, and that there are marked differences in the metabolic profiles of fertilized sclerotia over time. Results from the study will facilitate our understanding of the transitional phase of asexual to sexual reproduction in *A. flavus* and this information could be useful in screening of atoxigenic strains of *A. flavus* with high sexual fertility.

**195F Clampless mutants of the self-fertile *Coprinopsis cinerea* homokaryon AmutBmut.** Weeradej Khonsuntia, Ulrich van Campe, *Ursula Kües* Molecular Wood Biotechnology, University of Göttingen, Göttingen, DE.

A clampless and fruiting-defective mutant E3 was originally obtained by REMI mutagenesis of homokaryon AmutBmut which is self-compatible by mutations in the mating type loci. E3 grew initially with a normal fluffy aerial mycelium and produced oidia in light. In aged cultures, mycelia glued together into rubber-like films that could be lifted off from the agar. The original E3 strain was however lost while two morphological different lines, JE3 and J2E3, remained from sub-culturing E3. Both lines were clampless, did not initiate fruiting but had lost the ability to form rubber-like films. JE3 grew faster with aerial mycelium (4.3 mm/day) than J2E3 (2.7 mm/day) that had barely any aerial mycelium and showed invasive growth into agar. JE3 produced large amounts of oidia in dark (2.6x10⁹ per plate) and even more in light (9.6x10¹⁰). JE3 and J2E3 were also not stable. Spontaneous sectoring was observed in cultures of both strains. Sectorial isolate JE3-1 had a denser mycelium compared to the parental JE3. It lacked clamp cells, produced massive amounts of oidia in dark and formed large swollen cells on thick-walled swollen hyphae. J2E3-1 from a J2E3 sector produced fluffy mycelia, had fused clamp connections at hyphal septa and fruited. Comparable to typical dikaryons, J2E3-1 produced nearly no oidia in dark (1.3x10⁵ per plate) but some in light (3.3x10⁶ per plate). Recurrence of clamp connections and an ability to initiate fruiting body development indicates that the mutations in the *A* and *B* mating type loci from strain AmutBmut are still extant. To test the situation in JE3-1, crosses were performed with monokaryon PG78. All progeny of the cross was clampless, suggesting that possibly the *A* mutation to self-fertility had been reversed to self-sterility. Progeny was sorted into categories thick and thin mycelium and into evenly and irregular growing. Both types of categories split 1:1. Thick mycelium and evenly growing were linked (25 cases of 69) and thin mycelium and irregular shape (26 cases of 69). Furthermore, 13 colonies with thick mycelium formed rubber-like films in aging cultures. The results suggest that the mutation from the original E3 mutant was still present. The repeated changes in morphology and other phenotypes in sub-culturing of E3 and mycelial descendants possibly indicate stress situations caused by genetic defect(s) in complex regulatory networks. Sectoring into new growth types might help to relieve pressure from such stress.

**196W Developmental regulators in the mushroom *Coprinopsis cinerea*.** Weeradej Khonsuntia, Bastian Dörnte, *Ursula Kües* Molecular Wood Biotechnology, University of Göttingen, Göttingen, DE.

Four putative regulatory genes involved in developmental processes in *Coprinopsis cinerea* are being investigated, the duplicated genes *FLU1-1I.1* and *FLU1-1I.2* as homologs of *Aspergillus nidulans* *fluG* for a regulatory protein with a C-terminal glutamine synthase I (GSI)-like domain, *crg1* as homolog of *A. nidulans* *flbA* for a regulator of G-protein signaling, and *NWD2* which encodes a NACHT-NTPase and suppresses a defect in primary hyphal knot formation (*pkn1*) in *C. cinerea* mutant Proto159. All four genes were subcloned for overexpression behind the *gpdII* promoter of *Agaricus bisporus* into a plasmid with a *pab1*⁺ selection marker and transformed into different strains of *C. cinerea*, i.e. into the self-compatible homokaryon AmutBmut, its fruiting-defective mutant Proto159 and the normal monokaryon FA2222. A *pab1*⁺ plasmid pYSK7 habouring the laccase gene *lcc1* was used as a control for effects of random DNA integration in transformation. Transformation of all four genes changed mycelial morphologies of homokaryon AmutBmut on a minimal medium used for selection as compared to the control transformants. Transformation of *Flu1-1I.1*, *Flu1-1I.2* and *crg1* lead to extended aerial hyphal growth, transformants of *NWD2* produced less dense mycelium and shorter aerial hyphae. On YMG/T complete medium, further phenotypes were observed such as in mycelial color, in increased sclerotia production and in massive secretion of yellow liquid droplets. Effects of the genes on fruiting are in test. Transformants of Proto159 of all four genes and of the laccase gene *lcc1* had an enhanced aerial mycelium on YMG/T complete medium as compared to Proto159. Mutant Proto159 produces a strong pigmentation of the YMGT agar medium during aging. The majority of transformants of genes *crg1* (87%), *Flu1-1I.2* (85%), *Flu1-1I.1* (82%), and *NWD2* (73%) but also of the control vector (75%) delayed the pigmentation as compared to Proto159. We conclude that the *pab1*⁺ selection marker possibly strengthen the strain by complementing its *pab1* auxotrophy. Transformants of monokaryon FA2222 were also obtained for all four genes and await their morphological characterization.

**197T Identification of recessive lethal alleles linked to unidirectional loss-of-heterozygosity events in *Candida albicans*.** *Timea Marton*¹,², Adeline Feri¹,², Pierre-Henri Commere³, Corinne Maufrais⁴, Natacha Sertour¹, Gavin Sherlock⁵, Marie-Elisabeth Bougnoux¹, Christophe d'Enfert¹, Melanie Legrand¹ 1) Institut Pasteur, INRA, Labex IBEID, Unité Biologie et Pathogénicité Fongiques, Paris, France; 2) Univ. Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, rue du Docteur Roux, Paris, France; 3) Institut Pasteur, Imagopole, Plate-Forme de Cytométrie, Paris, France; 4) Institut Pasteur, Centre d'Informatique pour la Biologie, Paris, France; 5) Department of Genetics, Stanford University, Stanford, California, USA.

*Candida albicans* is a frequent human commensal yeast responsible for both mucosal and the majority of life-threatening nosocomial fungal infections. Its heterozygous diploid genome is highly plastic, with frequent aneuploidies and loss-of-heterozygosity (LOH) events
commonly associated with acquired antifungal resistance. Although LOH events can be seen on all 8 chromosome pairs, a chromosome homozygosis bias is observed for certain chromosomes regarding the retained homolog in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) that would prevent large-scale LOH events from being stably maintained.
To further explore the impact of LOH on *C. albicans* biology, we took advantage of the *Saccharomyces cerevisiae* I-Scel endonuclease and fluorescent markers (GFP/BFP) to establish a system that allows 1) the induction of a DNA double-strand break (DSB) at a specific location in the genome and 2) the detection of the occurrence of long-tract homozygosis resulting from repair of the induced DNA-DSB.
This approach was applied to identify RLAs on Chr7, where haplotype B (HapB) is never found in the homozygous state, unlike haplotype A, suggesting the presence of RLA on Chr7B. I-Scel successfully targeted and induced a DNA-DSB on both Chr7 homologs as illustrated by an increase in LOH. However, our inability to recover cells homozygous for the right arm of Chr7B confirmed the existence of RLA(s).
Mining of genome sequencing data for 154 *C. albicans* isolates allowed identifying a nonsense-generating SNP within the HapB allele of C7_03400c, whose ortholog in *S. cerevisiae* ScMTR4 encodes for an essential RNA helicase involved in nuclear RNA processing. As well, an RLA and a deleterious recessive allele on another chromosome (Chr4B) have been found explaining why Chr4A cannot be lost. This methodology also allowed highlighting that repeated regions, such as major repeat sequences, are hotspots for homologous recombination between homologs and play a role in LOH dynamics by generating new allele combinations upon haplotype swapping. Overall, our work uncovers regions whose homozygosis might impact *C. albicans*'s ability to persist as a commensal or to cause infections.

**198F** *Mauriceville plasmid-induced senescence impacts nuclear genome stability.* Amani Alharthi, Natasha Nazir, Kala Chinnaswarmy, Charles D. Baudo, John C. Kennell Biology, Saint Louis University, Saint Louis, MO.

Strains of *Neurospora crassa* containing the Mauriceville plasmid are prone to senescence due to plasmid integration into the mitochondrial genome or as a result of plasmid overreplication. Next-generation sequencing (NGS) of pre-senescent cultures revealed an unexpected high number of single nucleotide polymorphisms (SNPs) in the nuclear genome. The number of SNPs in senescent cultures was found to be significantly greater than those associated with plasmid-free strains, suggesting that Mauriceville plasmid leads to increased rates of nuclear mutation. To assess this hypothesis, the viability of multi-nucleate macroconidia versus uni-nucleate microconidia was compared. The results showed that as cultures were transferred, the viability of microconidia decreased much sooner than macroconidia, which may reflect the lack of additional nuclei to complement mutations in essential genes. In addition, rates of reversion of a nuclear auxotrophic mutation in pre-senescent cultures were also determined. The plasmid-containing strains were found to have up to a 40-fold increase in reversion rates in transfers close to senescence suggesting that mitochondrial dysfunction influences the frequency of nuclear mutation. Together, these findings suggest that mitochondrial dysfunction associate with plasmid-induced senescence impacts nuclear genome stability.

**199W** *Mitochondria-mediated nuclear genome instability associated with plasmid-induced senescence in Neurospora crassa.* C. D. Baudo¹, S. Ryu¹, N. M. Dutken¹, K. McCluskey², S. E. Baker³, J. C. Kennell¹ 1) Department of Biology, Saint Louis University, Saint Louis, MO; 2) Fungal Genetics Stock Center, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri; 3) Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington.

Senescence in *Neurospora crassa* is associated with dysfunctional mitochondria and most commonly observed in strains harboring specific mitochondrial plasmids. Variant forms of the Mauriceville retroplasmid (pM) trigger senescence by integrating into the mitochondrial genome disrupting essential mitochondrial genes or by over-replicating and interfering with protein synthesis. The transcriptomes of Mauriceville cultures undergoing senescence and wild-type plasmid-free cultures exposed to two mitochondrial inhibitors were analyzed by RNA-sequencing. Approximately 10% of nuclear encoded genes were regulated similarly under plasmid-induced senescence, inhibition of the electron transport chain, and inhibition of mitochondrial protein synthesis. Critical pathways involved in antioxidant defense, DNA damage response, and mitochondrial homeostasis were upregulated and validated using quantitative PCR. In addition, anomalous SNP accumulation was observed in pre-senescent cultures. Transcriptomic profiling and examination for signatures of oxidative DNA damage indicate that these SNPs do not result from ROS-mediated mutagenesis until stages of growth immediately prior to senescence. The rate of SNP formation in senescent cultures was compared with plasmid-free strains and to a long-lived (LL) derivative of Mauriceville capable of escaping senescence. Surprisingly, the rate of SNP formation in pre-senescent cultures was up to 50 fold greater than plasmid-free and LL strains indicating that plasmid-related mitochondrial stress mediates mutations in the nuclear genome. The mechanisms that allow the LL culture to tolerate and withstand SNPs, despite having abundant levels of plasmid variants, remain to be identified. Further characterization of longevity mutants should help to elucidate mitochondrial-nuclear communication pathways that respond to mitochondrial dysfunction and potentially lead to new therapies for human mt diseases.

## Comparative and Functional Genomics

**200T** Exploring fungal dark matter using single-cell genomics. S. Ahrendt¹,², C. A. Quandt³, D. Ciobanu², A. Clum², A. Salamov², J-F. Cheng², T. Woyke², T. James³, I. Grigoriev¹,² 1) Department of Plant and Microbial Biology, University of California, Berkeley, CA; 2) DOE Joint Genome Insititute, Walnut Creek, CA; 3) Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

Current estimates suggest that only approximately 100,000 of the estimated 5 million fungal species worldwide have been described, and the overwhelming majority of those fall within the Dikarya. Thus, the diversity of the non-Dikarya lineages has been poorly explored. Our lacking insight into the metabolic potential of these fungi is a reflection of their absence from sequence databases, limiting our ability for meaningful comparative analyses and lifestyle predictions. Exacerbating this issue is the fact that a substantial fraction of these organisms is largely uncultivated, challenging genomic exploration, despite being detected in environmental PCR surveys as a significant component of a community. The difficulties inherent in exploring the genetic make-up of this "fungal dark matter" can be overcome using single-cell sequencing to reconstruct genomes of uncultivated organisms directly isolated from the environment. Environmental fungi, particularly among the zoosporic and other early-diverging fungal lineages, make exceptional targets for single-cell genomic techniques; however, as much of the current single-cell genomic work focuses on mammalian, bacterial, and archaeal systems, there is a pressing need to adopt these protocols for fungi. Here we developed and tested these single-cell methods to sequence the genomes of seven species which cannot be grown in pure culture, including the first representatives of the previously unsequenced Zoopagomycotina subphylum. We show
that although there is a large variation in gene space recovery from each single cell (ranging from 6 - 88%), combining multiple cell libraries can increase this recovery to around 90%. Phylogenomic analyses allowed us to place previously unsampled lineages within the fungal tree of life, even when considering partially complete genomes derived from single individual cells. Additionally we explored gene family expansions to identify patterns consistent with lifestyle aspects of these biotrophic organisms.

**201F PHI-base - the Pathogen-Host Interactions database.** Kim Hammond-Kosack¹, Alayne Cuzick¹, Kim Rutherford², Helder Pedro³, Martin Urban¹
1) Dept of Plant Biology and Crop Sciences, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK; 2) Cambridge Systems Biology and Department of Biochemistry, University of Cambridge, Sanger Building, 80 Tennis Court Road, Cambridge, Cambridgeshire CB2 1GA, UK; 3) The European Molecular Biology Laboratory, The European Bioinformatics Institute, Hinxton, Cambridgeshire, CB10 1SD, UK.

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

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

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

**202W Developing a new genome annotation for improved gene prediction of *Cryphonectria parasitica*.** Di Ren, Angus
Dawe Biological Sciences, Mississippi State University, Mississippi State, MS.

*Cryphonectria parasitica*, causal agent of chestnut blight, devastated the American Chestnut population in the early 20th century. Hypoviruses that reduce the severity of infection offer potential for biological control. However, their spread is hampered by a diverse nonself-recognition system, vegetative incompatibility (VI), among strains of *C. parasitica*. CpVIB-1 was identified as a likely transcription regulator with an important role in mediating programmed cell death in response to allelic variation at the *vic4* locus. However, challenges for obtaining further insights into the VI mechanism by identifying the targets of CpVIB-1 through transcriptome comparison arose when performing the analysis using the currently available genome annotation provided by the Joint Genome Institute (JGI). This JGI version (v2.0, released in 2010; http://genome.jgi.doe.gov/Crypa2/Crypa2.home.html) has been found to include some incorrect predictions and is also limited in gene structural annotation details when compared to the information that can be provided by more recent annotation pipelines. A configurable genome annotation and curation pipeline, Two-pass MAKER2, was applied to annotate and mask repetitive elements in the genome, align protein and RNA evidence in a splice-aware manner, and accurately identify genes. This pipeline improves gene predictions by modifying gene models from GeneMakr-ES, SNAP and Augustus gene predictors with evidence from RNA-seq transcriptome data, and protein information from the Uniprot/SwissProt, OrthoMCL, and *Magnaporthe oryzae* protein databases. The reference genome used was the 2010 v2.0 *C. parasitica* genome assembly from JGI. Gene numbers predicted varied between 11704 and 11792 (~0.75 %) using the three gene predictors. 91% had Annotation Edit Distance (AED) values < 1, indicating strong support, with the OrthoMCL data. Uniprot/Swissprot and *M. oryzae* data yielded 86.7% and 88.9% of predicted genes with AED values < 1, respectively. The new output included gene structural features such as mRNA, exons, and untranslated regions (UTRs), along with a quality metric system for each prediction and the detailed evidence used to support the prediction. New unique IDs for each prediction have also been linked to InterPro ID by blastp. Using this new annotation has more accurately predicted certain known genes and will provide the basis for improved transcriptional profiling to address questions related to VI and resulting cell death.

**203T FungiDB: An integrated functional genomics database for fungi and oomycetes.** Evelina Basenko¹, Omar Harb²,
Achchuthan Shanmugasundram¹, David Roos²
1) Centre for Genomic Research, Functional and Comparative Genomics, University of Liverpool, Liverpool, UK; 2) Penn Center for Bioinformatics and Department of Biology, University of Pennsylvania, Philadelphia, PA.

FungiDB (http://FungiDB.org) is a free online resource for data-mining and functional genomics analysis. As part of the Eukaryotic Pathogen Bioinformatics Resource Center (http://EuPathDB.org), FungiDB provides an easy to use and interactive interface to explore genomes, annotation, functional data (transcriptomics or proteomics), metabolic pathways and results from numerous genome wide analyses (ie. InterPro scan, signal peptide and transmembrane domain predictions, orthology, etc.). FungiDB contains an expanding number of genomes from species spanning the Ascomycota, Basidiomycota, zygomycete, and chytrid fungi; including pathogenic species from the *Cryptococcus*, *Histoplasma*, and *Coccidioides* genera.

The graphical user interface in FungiDB allows users to conduct *in silico* experiments that leverage the available data and analyses. For example, a search in FungiDB can identify all genes in *Candida albicans* that do not have orthologs in mammals, have a predicted signal peptide, are annotated as a kinase and are expressed under conditions of high oxygen stress. Results from any search can be further analyzed visually using a companion genome browser or through analysis tools such as genome ontology and metabolic pathway enrichment. All results and searches can be saved in a user's profile or downloaded in multiple formats.

Users of FungiDB can also privately analyze their own data (ie. RNAseq or genomic sequence) via the EuPathDB galaxy implementation. All genomes in EuPathDB are preloaded into this galaxy instance allowing more streamlined data analysis. Results from galaxy workflows can be downloaded or analyzed within FungiDB, such as through visualization of RNAseq read coverage plots in the genome browser.

Input from the community (images, files, PubMed records, etc) can be easily added to FungiDB records (ie. gene pages) via user