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FungiDB is supported in part by NIH HHSN272201400030C and the Welcome Trust grant WT108443MA

Authors are individuals present at this meeting and representing the EuPathDB team.

**204F Parallel fitness using barcode sequencing in A. fumigatus: Assessment of the role of Aspergillus Kinases in azole resistance and as Targets for Antifungal Drug Discovery.** N. ALFURAJI, J. Mabey, M. Bromley, P. Bowyer Manchester Fungal Infection Group, Division of Immunity, Infection and Respiratory Medicine, Faculty of Biology, Medicine and Health Sciences, University of Manchester.

Fungi cause a wide range of infections including invasive and life threatening disease, superficial infections of the skin and mucosal membranes, as well as allergic disorders. The mortality rate due to fungal diseases remains unacceptably high, and is thought to exceed one million patients annually. Currently, only four groups of antifungals are available to treat systemic fungal infections: the polyenes, flucytosine, triazoles and echinocandins. The emergence of resistance to the available antifungal drugs and toxicity associated with some classes necessitates the exploration of novel pharmacologically effective antifungal drugs. Protein phosphorylation by protein kinases (PK) impacts all areas of cellular activity. Their critical roles in a wide range of cellular functions, along with the relative ease in which drug inhibitor assays can be developed have highlighted this class of enzymes as potential drug targets.

A bioinformatic analysis of the genomes of 9 Aspergillus species has revealed, in contrast to a previous study, that the number of protein kinases in each species is relatively consistent ranging from a minimum of 140 in *A. nidulans* up to 175 in *A. niger*. The kinases are distributed over 11 sub-groups including genes which apparently encode tyrosine like kinases and tyrosine kinases. We present comparative analysis which suggests that there are a sub-group of kinases that are specific to filamentous fungi.

To assess the role of these kinases in growth, virulence and drug tolerance, we have generated a library of 90 genetically barcoded knockout mutants. We have validated a barcode-sequencing approach to assess in pooled cultures, the fitness defect in each null mutant when in standard culture conditions and in the presence of the antifungal drug itraconazole. We show that 2 genes, the mitogen-activated protein kinase *mpkB* (AFUB_078810) and a serine/threonine protein kinase *ssn3* (AFUB_035220) are critical for azole tolerance. In addition, we have identified 25 genes that encode kinases that are essential for viability. Our assessment of virulence of these strains is currently in progress.

**205W Comparison of a commercial Aspergillus oryzae strain and its degenerated strain by transcriptome analysis.** Y. Zhong, W. Nong, L. Xing, X. Lu, H. Kwan The Chinese University of Hong Kong, N.T., HK.

With a great capacity of enzyme production for macronutrient hydrolysis, *Aspergillus oryzae* is important in Koji-making process in soy sauce fermentation to contribute to subsequent fermentation and the characteristic aroma and flavor. Chinese commercial strain *A. oryzae* RD2 can produce soy sauce with high quality. The strain TS2 was isolated after repetitive use of RD2, which has limited enzyme production and produces poor-quality soy sauce. We aimed to explain the phenotypic differences between two strains at the molecular level by transcriptome analysis. We first cultivated RD2 and TS2 in the mixture of steamed soy beans and wheat flour and sampled in duplicate in three Koji-fermentation stages: mycelium expansion (ME), early sporulation (ES) and mature sporulation (MS). Then we sequenced the extracted RNA in Illumina Hiseq 2000 Platform. Differentially expressed genes (DEGs) were identified: 1589 genes in ME, 686 genes in ES and 225 genes in MS. Among them, we mainly focused on those related to hydrolytic process including amylases and proteinases. The result shows that the active strain RD2 has higher expression level in both types of hydrolases throughout all stages, which explains the better performance of RD2 in Koji fermentation. Interestingly, in the degenerated strain TS2, more genes related to glycolysis and amino acid catabolism are expressed at a higher level compared to RD2. So the off-flavor of soy sauce produced by TS2 is probably due to the poorer hydrolase expression and the overuse of flavor compounds for energy, and RD2 could not compete with TS2. In conclusion, the metabolic saccharolytic and proteolytic activities make RD2 a good fermentative strain in soy sauce production due to its capacity of releasing compounds for flavor formation and subsequent fermentation, while the spontaneous degeneration would cause loss of the industrially favorable phenotypes but gain of growth dominance. Based on the transcriptome comparison, this study provides insights into the identification of molecular markers related to good soy sauce fermentation, which informs the selection and breeding of superior strains. We will explore to find more molecular markers of superior soy sauce production strains, and investigate the metabolism and functions needed for good soy sauce production.

**206T Spatially-resolved gene regulation in wood-degrading fungi highlights carbohydrate-selective machinery in brown rot fungi.** J. Zhang¹, K. Silverstein², M. Figueroa³, J. Schilling¹ 1) Bioproducts and Biosystems Engineering, University of Minnesota, Saint Paul, MN; 2) Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN; 3) Department of Plant Pathology, University of Minnesota, Saint Paul, MN.

Brown and white rot are two typical woody plant cell wall degradation strategies among *Agaricomycotina*. Compared to the simultaneous extraction of all carbon sources of white rot, brown rot selectively utilizes carbohydrates by leaving most of the lignin, potentially a more efficient carbohydrate-converting system. Recent genomic comparisons have noted that losses of ligninolytic genes have occurred in brown rot fungal evolution, corresponding to a surprising increase in decay efficiency. To approach this conundrum, we compared these two wood-decaying strategies, tracking temporal gene regulation at a whole genome-level among fungi from disparate phylogenetic clades. These data show that brown rot fungi up-regulate transcription of reactive oxygen species (ROS) at the advancing hyphal front, with an inducible glycoside hydrolase expression at later stages. In contrast, data showed the white rot fungi expressing laccase and several class II peroxidases at the hyphal front, presumably targeting lignin as a pretreatment. In later stages, white rot fungi coupled the transcriptional up-regulation of lignocellulose-oxidizing genes with glycoside hydrolase genes. In addition, we observed 115 genes up-regulated (2-fold change) only in brown rot fungi in early decay, narrowing perhaps the candidate list of lignocellulose pretreatment genes in these unique, carbohydrate-selective fungi.

**207F Supersize fungi: Rust in the genomics era!** Cecile Lorrain¹, Stephen J. Mondo², Emmanuelle Morin¹, Kurt LaButti², Anna Lipzen², Pascal Frey¹, Joseph W. Spatafora³, Igor V. Grigoriev², *Sebastien Duplessis*¹ 1) INRA, UMR 1136 Interactions Arbres-Microorganismes, INRA/Université de Lorraine, 54270 Champenoux, France; 2) US Department of Energy Joint Genome Institute, Walnut
Creek, CA, USA; 3) Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA.

Rust disease fungi cause consequent losses in plantations of major crops and trees. Despite their importance as plant pathogens, limited progress has been made in understanding molecular bases of host infection. Indeed, rust fungi exhibits the most complex life cycles described among fungi (e.g. heteroecious and macrocyclic) and they are true obligate biotrophs with no culture or manipulation possible in the lab, making their study a real challenge. So far, only a handful of virulence effectors have been described in a limited number of species and no clear characterization of their possible role was established. Rust fungi also exhibit the largest genomes known for fungi so far, full of transposable elements and with many expanded and specific gene families of unknown function. Rust genome sequencing is also most often based on dikaryotic spores, which adds to the difficulty to obtain complete and neat assembly. The 336 Mb genome of the poplar rust fungus *Melampsora allii-populina* recently sequenced in the frame of the US DoE Joint Genome Institute 1KFG project [1] will be introduced to illustrate how comparative genomics within the Melampsoraceae and the Pucciniales allow to explore host specificity in rust gene repertoires, particularly for rust candidate effectors.

[1] Mycocosm: http://jgi.doe.gov/fungi

Pucciniomycotina: *Melampsora allii-populina* 12AY07 v1.0

**208W Genetic analysis of natural variation in Botrytis cinerea field isolates identifies a single gene with a major effect on pathogenicity and sporulation.** Wilson Acosta¹, Francisco Anta¹, José María Díaz Mínguez¹, Michael Thon¹, Jan A.L. van Kan², Ernesto P. Benito¹ 1) CIALE-UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN; 2) WAGENINGEN UNIVERSITY, WAGENINGEN, THE NETHERLANDS.

Natural populations of *Botrytis cinerea* from vineyards of Castilla y León, Spain, are highly diverse both genetically and physiologically. Evaluation of aggressiveness in a representative sample of field isolates collected from different *Vitis* varieties made it possible to identify isolates displaying large differences in their ability to cause infection. In order to perform a genetic analysis of these differences, crosses between a highly aggressive isolate (448) and a non-pathogenic isolate (371) were performed. In the progeny, a 1:1 segregation was observed for the phenotype "ability to infect". Remarkably, the "ability to infect" strictly cosegregated with the "capacity to sporulate". Within the pathogenic progeny large differences in aggressiveness were found, suggesting a key role for a "major pathogenicity gene" essential for the expression of a number of pathogenicity factors which segregate in the progeny. In an attempt to identify the gene responsible, a strategy based on "bulk segregant analysis (BSA)" was designed. First, the sequence of the genomes of both parental strains was determined and a list of SNPs and of small indels was elaborated. Then, two pools of genomic DNA were prepared and sequenced: a first one (P1) from a group of isolates resembling the aggressive parental strain 448, and a second one (P2) from a group of non-aggressive isolates, like parental strain 371. Finally, the distribution in both pools of the polymorphisms found between isolates 448 and 371 was analysed searching for isolate 371 specific variants exclusively found in pool P2 but absent in pool P1 and for isolate 448 specific variants found in pool P1 but absent in pool P2. A candidate region for the major pathogenicity gene to be located in has been identified which is currently being characterized.

This work was supported by grants AGL2012-39876-C02-01 and AGL2015-66131-C2-1-R (MINECO, Spain).

**209T Defining the role of heteroresistance in persistent human fungal infections.** I.V. Ene¹, K. Navarrete¹, E. Fu¹, J.M. Bliss¹,², A.L. Colombo³, C. Cuomo⁴, R.J. Bennett¹ 1) Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA; 2) Women & Infants Hospital, Providence, Rhode Island, USA; 3) Universidade Federal de São Paulo, São Paulo, Brazil; 4) Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

A critical problem in the clinic is the inability of available drugs to clear systemic fungal infections. In some cases, therapeutic failure is due to drug resistance, in which isolates evolve inherent resistance to the drug. However, in many cases persistent infections involve the emergence of tolerant cells (TCs) in the population, a process termed *heteroresistance*. To understand the mechanisms underlying persistence, we performed genome sequencing on a set of *Candida albicans* isolates that were drug susceptible yet persisted during antifungal therapy, and compared this set to isolates that were successfully cleared by drugs. Preliminary analyses indicate that clonal lineages often persisted during the infection, with isolates experiencing large-scale genetic changes including loss of heterozygosity (LOH) across large tracts of the genome. Persistent isolates were less susceptible to drugs and more likely to form TCs compared to isolates that were rapidly cleared from patients upon drug treatment. This implies the existence of a subset of resilient cells with distinct properties from the majority of the cells in the population. TCs formed by persistent isolates grew under much higher drug concentrations than rare TCs formed by cleared isolates. These high resistance levels were gradually lost with growth in the absence of drug, indicating that this phenotype is transient. Similar to bacterial TCs, *C. albicans* TCs have a fitness defect relative to the whole population in the absence of drug, but rapidly outcompete the rest of the population in the presence of drug. To understand how tolerance arises in a population, further genomic and genetic analyses are being performed. This includes screening of a library of all *C. albicans* transcription factors to identify genes that enable TC formation in response to different antifungals. Several gene families modulate tolerance, including telomeric and stress response genes. This study shifts the current paradigm that primary host status dictates persistence, and reveals that intrinsic *C. albicans* properties can also contribute to this important clinical phenomenon.

**210F Systematic genetic analysis in the opportunistic yeast *Candida albicans* reveals evolutionary plasticity of cell size homeostasis mechanisms in eukaryotes.** J. Chaillot, A. Sellam Infectious Diseases Research Centre (CRI), CHU de Québec Research Center (CHUQ), University Laval, Quebec, QC, CANADA.

The basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans, is a critical but still poorly understood aspect of eukaryotic cell proliferation. All eukaryotic cells must grow to a critical cell size before commitment to division occurs. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. To probe the conservation of the extensive size homeostasis network, we performed a quantitative genome-wide analysis of a systematic collection of gene deletion strains in the pathogenic yeast *Candida albicans*. Analysis of the size phenotype uncovered 195 genes that markedly altered cell size. Our results revealed an unexpected high degree of divergence between genes that affect size in *C. albicans* versus the yeast model *Saccharomyces cerevisiae*. In addition to conserved size regulators previously identified in *S. cerevisiae* and metazoans, we uncovered previously undocumented regulatory circuits that govern critical cell size at Start. A comprehensive genetic connectivity, using suppressive-
dosage and epistasis genetic interactions, were used to order the size regulatory network in *C. albicans*. This comprehensive analysis revealed a complex network of novel regulator of Start and cell size the most potent of which were the transcription factors Ahr1, Sfp1, Dot6, the AGC kinase Sch9 and the MAPK Hog1. In particular, we delineate a novel stress-independent function of the p38/HOG MAPK network in coupling cell growth to cell division. Our genetic and biochemical analysis suggests that the HOG module directly and interacts with central components of both the cell growth and cell division machineries. Furthermore, we identify a novel size network where the helix-loop-helix transcription factors Dot6 act as a major effector of the TOR pathway to modulate cell size at Start through control of both cytoplasmic and mitochondrial translation. We also showed that the transcription factor Ahr1 that modulates adhesion genes and virulence in *C. albicans*, control cell size of both yeast and filamentous forms by connecting amino acid metabolism with cell cycle commitment decision. This work establishes the first systematic characterization of the mechanisms underlying regulation of growth and division in a pathogenic fungus and illuminate the evolutionary plasticity of the size control network in eukaryotes.

**211W Functional characterization of chestnut blight fungus, *Cryphonectria parasitica* septin gene, CpSep1.** Myeongjin Jo, Yo-Han Ko, Kum-Kang So, Jyotiranjan Bal, *Jeesun Chun*, Dae-Hyuk Kim Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea.

Chestnut blight fungus, *Cryphonectria parasitica*, and its hypovirus present a useful model system for investigating the mechanisms of hypoviral infection. Pathoproteomics analysis revealed a protein spot, further identified as a septin, showing up-regulation in the presence of *Cryphonectria* hypoviruses (CHV1) and tannic acid supplementation. Blast search revealed its homology towards *AspA* gene of *Aspergillus nidulans* (an ortholog of *Saccharomyces cerevisiae* CDC11). In order to analyze the functional role of this septin gene (*CpSep1*), we attempted to construct its null mutant and a total of eight *CpSep1*-null mutants out of 137 transformants were obtained. All *CpSep1*-null mutants showed retarded growth with less aerial mycelia and intense pigmentation on PDAmb plate. When the marginal hyphae were examined, hyperbranching was observed compared to that of the wild-type. However, the inhibition of colonial growth was partially recovered when the *Cpsep1*-null mutants were cultured in the presence of an osmostabilizer. Conidia production of the *CpSep1*-null mutants was significantly i.e., at least ten-fold more increased and electron microscopy suggested abnormal conidia-bearing structure. Interestingly, conidial morphology of the *CpSep1*-null mutants changed to be circular in comparison to the rod-shaped spores of the wild-type indicating a role of septin in spore morphology of *C. parasitica*. However, no difference in the germination process in terms of germ tube number, polarity, and timing was observed. Mycelial growth of the *Cpsep1*-null mutants on the excised chestnut tree bark was severely affected suggesting that the virulence was affected due to the fungal growth defect in the host tissue. This study indicated that the *CpSep1* gene is required for an appropriate mycelial growth and pigmentation, osmotic stabilizing, spore morphology, and fungal growth in the host plant, which is consistent with the results of up-regulation by the hypoviral infection and tannic acid supplementation.

**212T *Colletotrichum acutatum* species complex genomes reveal signatures of host adaptation in the casual agent of lupin anthracnose.** *R. Baroncelli*¹, F. Pensec¹, S.A. Sukno², B. Henrissat³, S. Haridas⁴, W. Schackwitz⁴, I.V. Grigoriev⁴, S. Sreenivasaprasad⁵, M.R. Thon², G. Le Floch¹ 1) Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (LUBEM), Université de Brest, EA 3882, Brest, France; 2) Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), University of Salamanca, Spain; 3) Aix-Marseille Université, UMR 7257, Marseille, France; 4) US Department of Energy, Joint Genome Institute, Walnut Creek, USA; 5) Institute of Biomedical and Environmental Science and Technology (iBEST), University of Bedfordshire, United Kingdom.

Many species belonging to the genus *Colletotrichum* are associated with plant diseases, commonly referred to as anthracnose. In addition to its economic impact, *Colletotrichum* is one of the most studied genera of plant pathogenic fungi. Closely related species belonging to *Colletotrichum acutatum* species complex (CAsc) can be pathogens of a wide range of hosts or can show a high level of host specialization. *C. lupini* is a species within the CAsc and it is the major threat of its only host *Lupinus* spp. We sequenced and annotated the genomes of two *C. lupini* reference genomes and resequenced three isolates along with twelve closely related CA species characterized by a polyphagous lifestyle. The genome sequences were used to perform a comparative analysis with the aim of exploring genomic signatures associated with host preference in *C. lupini*. Whole genome alignments revealed that only 1.766% of *C. lupini* nuclear genome is species specific (SS) and 0.035% is isolate specific. 117 genes make up the SS portion and only 7 of those have been predicted to be secreted. Manual inspection revealed that among the seven predicted secreted proteins, four do not have similarity with any another sequence in databases, one has similarity with other *Colletotrichum* proteins with unknown functions, one is a metalloproteinase and one is a necrosis and ethylene inducing protein.

Hierarchical clustering of gene family and functional domain assignments revealed expansions of pathogenicity related genes such as necrosis and ethylene-inducing proteins (NEPs) and proteins characterized by CFEM domain (fungal specific cysteine rich domain) and other families with unknown function. We also found contractions in the CAZy repertoire (mainly glycoside hydrolases such as GH43 and GH3), transcription factors and in several clusters conserved within the genus of unknown functions and lacking conserved functional domains. We provide a first look at the adaptations in gene content that are associated with host specialization in *Colletotrichum* spp. This study also illustrates the plasticity of *Colletotrichum* genomes, and shows that relatively recent changes in gene content are associated with major changes in host range. This study also demonstrates the need for higher resolution taxonomic sampling in order to better understand the role of gene gain and loss in the evolution of fungal genomes and the possible association with fungal adaptation.

**213F *Cryptococcus neoformans* genomics influence human survival and immunologic responses.** *Aleeza Gerstein*¹, David Boulware¹, David Meya²,³, Kirsten Nielsen¹ 1) Department of Microbiology & Immunology, University of Minnesota, Minneapolis, MN; 2) Infectious Diseases Institute, Makerere University, Kampala, Uganda; 3) Makerere University College of Health Sciences, School of Medicine, Department of Medicine, Kampala, Uganda.

Patient outcomes are due to a complex interplay between the quality of medical care, the host immunity factors, and the infecting pathogen's characteristics. Pathogen burden is clearly associated with mortality risk, but other pathogen characteristics, such as genotype and phenotype, have not been fully explored. A number of studies have recently suggested that pathogen genotype may directly influence host survival, even with tremendous variability of other host factors. To further probe this connection, we examined *Cryptococcus neoformans* genotypes from Ugandan clinical isolates collected as part of the Cryptococcal Optimal ART Timing (COAT) trial to examine whether there was a genomic signature in the pathogen associated with patient survival and/or immune response. We focused on
ST93, the most frequently observed sequence type (ST) among the isolates, and performed whole-genome sequencing on 41 ST93 isolates, acquired from 22 survivors and 19 from patients who died. Sixteen other isolates from a diversity of ST groups were also sequenced for comparison. We identified 131754 SNPs and 16924 small indels among all isolates, with ~40% in at least one ST93 genome. We used the variants to construct a phylogenetic tree, and surprisingly found that ST93 isolates grouped into two separate clades (clade A and clade B). We focused our analyses on the 219 SNPs and 58 indels that were variable among the ST93 genomes, were unassociated with a centromere, and were predicted to have a fitness effect. Approximately half of these variants deliniated ST93 clade A isolates (123 SNPs and 23 indels). Importantly, a number of these variants were significantly associated with decreased 26-week survival (clade A: 29% vs clade B: 63%, 17 SNPs), decreased CD4 T cell counts nadir (a two base pair deletion), and altered CSF cytokines: a six base pair insertion associated with increased levels of pro-inflammatory interleukin (IL)-6, and a ten base pair deletion associated with increased protective Th1 response of IL-12 and increased protective Th2 response of IL-13. Additional non-clade specific SNPs (n=26) were also each significantly associated with at least two immunologic trait responses. This work demonstrates that fungal genotype may significantly influence human immune responses and survival and provides important candidate genes for future studies on virulence-associated traits in *Cryptococcus*.

**214W Genome of the obligately alkaliphilic fungus *Sodiomyces alkalinus* reveals its adaptations to high pH.** A.A. Grum-Grzhimaylo¹, D.L. Falkoski², J. van den Heuvel¹, B. Min³,⁴, I-G. Choi³,⁴, B. Henrissat⁵,⁶, H.J. Franssen⁷, E.N. Bilanenko⁸, R.P. de Vries²,⁹, J.A.L. van Kan¹⁰, I.V. Grigoriev³, A.J.M. Debets¹ 1) Laboratory of Genetics, Wageningen University, Wageningen, NL; 2) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, NL; 3) DOE Joint Genome Institute, Walnut Creek, CA, USA; 4) Computational & Synthetic Biology Laboratory, College of Life Sciences & Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul, South Korea; 5) CNRS, Aix-Marseille Université, AFMB, Marseille, France; Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Marseille, France; 6) Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; 7) Laboratory of Molecular Biology, Wageningen University, Wageningen, NL; 8) Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia; 9) Microbiology and Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, NL; 10) Laboratory of Phytopathology, Wageningen University, Wageningen, NL.

Alkaliphilic fungi, i.e. fungi that grow optimally at high pH, are exceptional with only a handful of species described to date. Here, we sequenced the obligate alkaliphilic ascomycete *S. alkalinus* isolated from alkaline soda soils, and explored the features responsible for its unusual life-style. We found that PacC, the major regulator of alkaline-related genes, is up-regulated at higher pH values than PacC orthologues of neutrophilic species. To assess how this fungus can degrade carbohydrate sources under extremely alkalic conditions, we searched for the CAZymes encoded in the genome of *S. alkalinus* and performed enzyme assays. We revealed many cellulases, but their overall activity was low presumably because of slower induction. Although cellulolytic and hemicellulolytic activity was optimal at pH 6, there still was some activity at pH 10, at which pH it was completely absent in the neutrophilic *A. oryzae*. Excellent *in vitro* growth on xylan indicates that grasses are the preferred nutritional source for *S. alkalinus* in nature. We detected potent proteolytic activity at alkaline pH, compared to the neutrophilic *A. oryzae*, which may reflect the need for obtaining extra nitrogen, as this essential element becomes limiting at alkaline conditions. Proteins of bacterial cells, which are present in bulk at soda lakes, seem the likely source of nitrogen. In support of this hypothesis, we found several instances of horizontal transfer of prokaryotic genes into the genome of *S. alkalinus*, encoding enzymes that degrade bacterial cell walls. The genome of *S. alkalinus* will provide a valuable source to further study the biology and evolution of alkaliphilic trait in fungi with respect to neutrophilic species. In addition, it may provide alkaline-active metabolites of commercial interest.

**215T Investigating outcomes of fungal interactions during wood decay.** G. Powell¹, L. Boddy², E. Dudley³, J. Hiscox², S.C. Moody¹, M. Savoury², D.C. Eastwood¹ 1) Department of Biosciences, Swansea University, Swansea. GB; 2) School of Biosciences, Cardiff University, Cardiff. GB; 3) School of Medicine, Swansea University, Swansea. GB.

Wood decomposition in natural habitats is achieved by a succession of interacting decay species. Species occupy niches which may be loosely defined as primary, secondary, tertiary and quaternary decay depending upon the amount of decomposition of the substrate. Evidence of priority effects have been described, but the process of succession within natural communities is not well understood. Interactions between species within or between decay niches can vary from replacement to deadlock, or mutual replacement, and outcomes can be affected by the nature of the substrate, temperature and water and nutrient availability. Here we report ongoing research on the competitive interaction between wood decay fungi from different decay niches using wood block-based experiments. Transcriptomic and proteomic data is used to analyse the outcomes of different decay scenarios to gain insight into the mechanisms regulating competitive interactions and ultimately start to understand how natural communities develop over time.

**216F Fungal gene clusters adapted to plant secondary metabolites.** E. Gluck-Thaler, V. Vijayakumar, J. Slot Department Plant Pathology, The Ohio State University, Columbus, OH.

In order to successfully colonize plant hosts, fungal saprophytes, symbionts and pathogens often require metabolic capabilities to degrade plant secondary metabolites and/or mitigate their fungitoxic effects. Degradative metabolic pathways are occasionally maintained in gene clusters encoding all of the necessary enzymes, transporters and regulators required for the metabolic pathway to function. We recently identified a type of fungal gene cluster that appears to encode the degradation of stilbenes, a diverse group of plant secondary metabolites that play an important role in the constitutive and induced chemical defenses of plants. This cluster contains a gene encoding stilbene dioxygenase (*sdo*), which is known to degrade stilbene compounds, along with other uncharacterized genes. Using a novel algorithm that detects unexpectedly conserved synteny, we further explored the diversity of *sdo*-containing clusters and identified three new and differently composed cluster types distributed across 78 diverse species. In order to determine whether metabolic pathways encoded by these different clusters are adapted to specific plant metabolites, we simultaneously investigated the substrate specificity of different *sdo* enzymes using *in vitro* assays as well as the evolution and ecological associations of the various cluster types. Given the ecological distribution of *sdo* cluster types, we hypothesized that each cluster type is specialized for the degradation of ecology-specific stilbene molecules. The substrate specificity of four different *sdo* enzymes from the plant pathogens *Magnaporthe oryzae* and *Sphaeropsis sapinea*, the dung decayer *Podospora anserina* and the saprotroph *Penicillum roquefortii* is conserved, providing no evidence of enzyme specialization according to cluster type or ecology. Ancestral state reconstructions of cluster composition, on the other hand, suggest that shifts in fungal
ecology are associated with the overall composition of clusters and the acquisition of clusters by horizontal gene transfer. The association of distinct cluster types but not *sdo* substrate specificity with ecological transitions suggests that processes driving fungal adaptations to plant chemical defenses may act at the level of genome organization, rather than at the level of individual gene function.

**217W Mycorrhizosphere interactions: From genes to proteome to volatilome.** *E. Kothe*, C Henke, D Sammer, K Wagner, K Krause
Dept Microbiol, Microbial Communication, Friedrich Schiller University, Jena, DE.

Ectomycorrhiza establishes a mutual symbiosis that stabilized forest ecosystems. The mycorrhizosphere community can modulate this interaction by interfering with the signaling between both partners. We investigated the association of *Tricholoma vaccinum* with its specific host, *Pinus sylvestris*, addressing the fungal secretome and volatilome specifically. The genome sequence of *Tricholoma* revealed 11,981 genes coding for proteins, 297 of which with prediction to be excreted. Of these, 26 could be verified by 2D gel electrophoresis, with seven specifically up-regulated after addition of spruce root exudates. The fungal signaling components revealed formation of the plant hormone indole acetic acid (IAA) that induced short root formation in the tree and increased branching in the fungus, thereby allowing for the formation of Hartig' net for nutrient exchange. The influence of the mycorrhizosphere was dissected from microbiome analyses, and a zygomycete-derived compound was found to induce the specific transport of IAA from *Tricholoma* cells. All in all, the tripartite interaction between tree, ectomycorrhizal fungus and the mycorrhizosphere community could be shown to regulate ectomycorrhizal formation through specific exchange of signal molecules. Transformants over-expressing a gene involved in fungal IAA synthesis verified the hypothesis of increased Hartig' net formation. A change in the volatilome of ectomycorrhizal, stressed trees was observed compared to non-stressed trees. This supports the role of mycorrhiza in stabilizing the ecosystem.

**218T Rise to the bait: Towards identifying the *Puccinia graminis* effector Avr4/5 “baited in” by the Rpg5 protein kinase integrated decoy domain.** *R. Sharma Poudel*, S. Solanki, S. Shrestha, J. Richards, R. Brueggeman
Department of Plant Pathology, North Dakota State University, Fargo, ND.

Stem rust, caused by *Puccinia graminis* f. sp. tritici (*Pgt*) is a threat to wheat and barley production with virulent races posing a threat to world food security. The barley *rpg4/Rpg5* stem rust resistance locus (*rpg4/5*) locus confers resistance against the majority of the prevalent *Pgt* races, including the recently emerged highly virulent race TTKSK (A.K.A Ug99) and its lineage. The positional cloning of rpg4/5 locus showed that three tightly linked genes, two NBS-LRR (NLR) resistance-like genes, *Rpg5* and *HvRga1*, and the actin depolymerization factor *HvAdf3* are required together to confer resistance. *Rpg5* is the functional resistance gene at this locus encoding a typical NLR with a predicted C-terminal protein kinase (PK) decoy domain. The *rpg4/5* genomic architecture and predicted protein domain function support the integrated decoy domain model. However, the experimental validation of this hypothesis is hampered without identification of the corresponding *Pgt* Avr4/5 effector. This research aims to identify and validate *Pgt* Avr4/5 by utilizing diverse *Pgt* isolates collected in North Dakota since 1970. Thirty-seven *Pgt* isolates showing a differential response on barley genotype with *rpg4/5* were genotyped utilizing Restriction site Associated DNA-Genotyping by Sequencing to identify 24 diverse isolates (9 avrRpg4/5 and 15 AvrRpg4/5). *In planta* RNAseq was conducted by collecting infected tissue from the susceptible barley variety Harrington, 5 DPI (days post inoculation) infected with the 24 *Pgt* isolates. Association mapping conducted using SNPs identified from the RNAseq showed no significant association. However, eleven candidate secreted effector proteins (CSEPs) showing differential presence/absence polymorphism correlating with avrrpg4/5 and AvrRpg4/5 isolates were identified. These eleven Avr4/5 CSEPs are being transformed into *Pichia pastoris* for protein expression and infiltration in barley *rpg4/5+* and *rpg4/5-* genotypes. These CSEPs are also being tested in Yeast two hybrid interaction with the Rpg5 PK domain as bait. A mutation approach involving gamma irradiation (1000Gray) of seven AvrRpg4/5 isolates to induce virulence by mutating the Avr4/5 gene in the avirulent isolates is also being utilized towards Avr4/5 identification.

**219F Mining effectors in *Phyllosticta citricarpa*.** *Paulo Santos*¹,², *Carol Rodrigues*², *Tiago Oliveira*¹,², *Marco Takita*², *Chirlei Glienke*³, *Marcos Machado*²
1) Universidade Estadual de Campinas, Campinas, São Paulo, Brasil; 2) Centro de Citicultura Sylvio Moreira, Cordeirópolis, São Paulo, Brasil; 3) Universidade Federal Do Paraná, Curitiba, Paraná, Brasil.

Citrus black spot (CBS) is one of the most devastating disease of citrus. Caused by the *Phyllosticta citricarpa* (McAlp. Van de Aa) the disease affects all citrus species of economic importance, and its symptoms are marked by dark spots on the rims of fruits, rendering to their depreciation, early fruit drop and reduced crop yield. The molecular mechanisms under the interaction between *P. citricarpa* and citrus remain elusive. We hypothesize citrus plants might recognize effector proteins of *P. citricarpa*, leading to hypersensitivity responses on infected tissues. In order to gain insight into the molecular basis of CBS pathosystem, we obtained the genome sequence using Illumina platform HiSeq2000 and further *de novo* assembly using CLC Genomics Workbench software. For predicting ORFs we used Augustus software. Annotation and categorization of ORFs were performed automatically in Blast2GO. Data from the sequenced genome generated 179,880,616 reads resulting in 19,143 contigs after assembly with 16,267 ORFs and an estimated genome size of 32 Mb. In order to predict candidate effectors in the genome of *P. citricarpa*, we developed a bioinformatics pipeline, taking into account common features found in pathogen effectors. Among the criteria established in our pipeline included: presence of secretion signal, absence of transmembrane anchors, functional domains, presence of nuclear localization signals, and other general structural features. We found 334 candidates secreted effector proteins (CSEP) out of 16.217 total evaluated proteins and selected 15 for further analysis. In order to evaluate the gene expression of CSEP, we established contrasting fungal growth conditions, by supplementing M1 medium with either phenolic compounds or albedo extract of sweet orange (susceptible) or albedo extract of Tahiti lime (resistant), compared to the control; only the medium. We observed that seven genes had their expression induced in the supplemented treatments. Considering these findings, we believe that compounds present in the supplements trigger the upregulation of CSEP genes, which might be recognized by the plant.
**Financial Support:** CNPq; INCT Citrus; Fapesp.

**220W Functional suppression of *B. napus* R genes; a new trick by *L. maculans* to avoid host defense responses.** *K. Ghanbarnia*¹,², *N. Larkan*⁵, *M. Lisong*¹, *P. Haddadi*¹, *W.G.D. Fernanado*², *M.H. Borhan*¹
1) Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada; 2) Dept. of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada; 3) Armatus Genetics Inc., Saskatoon, SK, Canada.

*The ascomycete fungus Leptosphaeria maculans (Lm) is the causal agent of economically important blackleg disease of canola (Brassica*
napus). B. napus immunity to Lm is largely governed by race-specific resistance (R) genes that mediate the recognition of corresponding Lm effector proteins encoded by Lm avirulence (Avr) genes. Two B. napus R genes, LepR3 and Rlm2 and seven Lm Avr genes have been cloned including, AvrLm1 and AvrLm2 that convey avirulence towards LepR3 and Rlm2, respectively. The majority of predicted and all cloned Lm Avr genes are located in repeat-rich regions of the genome, which promotes virulence via mutation or complete deletion of Avr genes. Another mechanism for evolving virulence is by suppression of host R gene response, as was recently reported for AvrLm3, in which its recognition by B. napus Rlm3 is masked in the presence of another Lm effector, AvrLm4-7. Here we provide an additional example of this epistatic suppression of Avr recognition. We took a map based cloning approach to identify the Lm avirulence gene AvrLm9, which encodes a typical small cysteine-rich secreted effector protein. However, when AvrLm4-7 is present Lm is able to avoid Rlm9-mediated recognition of AvrLm9. Hypotheses will be presented for the possible mechanism of suppression of the Rlm3 and Rlm9 defense responses by AvrLm4-7.

**221T Comparative evolutionary histories of fungal proteases reveal gene gains in mycoparasitic and nematode-parasitizing fungi.** M. Iqbal¹, M. Dubey¹, M. Viketoft², D.F. Jensen¹, M. Karlsson¹ 1) Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-75007, Uppsala, Sweden; 2) Department of Ecology, Swedish University of Agricultural Sciences, Box 7044, SE-75007, Uppsala, Sweden.

*Clonostachys rosea* [Bionectriaceae (Hypocreales, Ascomycota)] is a biological control fungus that can control several important plant diseases caused by plant pathogenic fungi and nematodes. In this study, we analysed the evolutionary history of protease gene families in 13 hypocrealean fungi, and *Neurospora crassa*. Proteases were classified into subgroups based on the MEROPS peptidase database. The highest number of protease genes (601) was found in *Fusarium solani*, followed by *C. rosea* with 589 genes. A computational analysis for gene family evolution (using CAFE version 3) identified the five serine protease gene families S08A, S09X, S10, S12 and S33 to be accelerated in gene gain or loss. S09A serine protease gene numbers were specifically increased in *C. rosea*, *Trichoderma virens* and *Hirsutella minnesotensis*, all reported to have the ability to parasitize nematodes. The mycoparasitic species *C. rosea*, *T. atroviride* and *T. virens* contained high gene numbers of S08A and S33 serine proteases. These results suggest that serine proteases contribute to the broad environmental opportunism displayed by *C. rosea* and certain *Trichoderma* spp.

**222F In silico characterisation of Endo-polygalacturonase in members of the Agaricales.** L. Potgieter, B.D. Wingfield, M.A. van der Nest, M.P.A. Coetzee Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Endo-polygalacturonases (endo-PGs) are cell wall degrading enzymes responsible for the degradation of homogalacturonan chains in pectin. These enzymes are important in fungal host infection as they cause tissue maceration that enables the fungi to infect and subsequently colonise plant hosts. Very little is known regarding their characteristics in member species of the Agaricales. The aim of this study was thus, to identify, characterize and study the distribution and evolution of putative endo-PGs from 54 species of the Agaricales for which whole genome sequences are publicly available. Using amino acid similarity and keyword searches, endo-PG genes were identified in most of the genomes. Many of the genes are present as multi copies. In general, saprotrophs, dung decomposing, litter decaying fungi, and mycorrhizal fungi harbored fewer gene copies than pathogens and wood rot fungi. This is expected, as these genes play an important role in host infection and colonizing. Functional prediction showed that the genomes harbor endo-PG isoforms active at different pH, ranging between 3.55 and 9.72 (average: 5.37). All the genomes in which endo-PGs genes were identified, contained endo-PGs predicted to be optimally active at an acidic pH, while genes predicted to encode basic endo-PGs were not present in all the genomes. Endo-PG isoforms active at different pH allow for activity and substrate access at variable ambient pH created by oxalic acid during pathogenesis. The number of introns varied greatly among the endo-PG genes identified in the different species. In comparison to species from other orders, endo-PG genes within the Agaricales contained, on average, four times more introns and the introns are at relatively conserved positions. This suggests that the ancestral endoPG gene in the Agaricales contained several introns prior to gene duplications. Overall, results of this study suggest that gene duplication followed by gene loss and/or gene expansion played an important role in the distribution and evolution of endo-PGs in the Agaricales.

**223W Comparative Genomic Analyses of Two Pyrophilous Fungi.** A. Carver¹,³, K. Stillman¹, H. Liu¹, S. Branco², A. Kuo³, T. Bruns¹, I. Grigoriev¹,³ 1) Plant and Microbial Biology, UC Berkeley, Berkeley, CA; 2) Laboratoire Ecologie, Systématique et Evolution, Un Paris Sud, CNRS-UPS-AgroParisTech, Orsay France; 3) Joint Genome Institute, Lawrence Berkeley National Laboratory, Walnut Creek, CA.

Pyrophilous fungi are filamentous fungi that have historically been found fruiting exclusively on burned soil. Forest soil changes greatly after fire, with an enrichment of dead biomass, partially pyrolyzed carbon compounds and increased hydrophobicity. The fungal species that inhabit such soil comprise a diverse guild, though most are Ascomycota in the order Pezizales and most of these are in the family Pyronemataceae. To better understand the role of pyrophilous fungi in post-fire soil, we compare the genomes of two pyrophilous fungi of the Pyronemataceae family, *Pyronema omphalodes* and *Wilcoxina mikolae*, to 12 non-pyrophilous Pezizales species. Although *P. omphalodes* and *W. mikolae* have otherwise distinct lifestyles as saprotrophs or as ectomycorrhizae respectively, both are dominant in forest soil communities after fires. They share an expansion in a novel chitin-binding gene family that is unique amongst sequenced fungi and may be horizontally transferred, and both genomes contain glycoside hydrolases that are absent in other sequenced Pezizomycetes species. In addition, *P. omphalodes* has an expansion in heat shock proteins over other sequenced Ascomycetes, which may assist in its colonization of burned forest soil. The information from this study will increase our understanding of the lifestyles and metabolic processes of pyrophilous fungi and their role in post-fire carbon cycling.

**224T Patterns of genome evolution across the fungal kingdom.** Michiel Konings, Robin Ohm Microbiology, Utrecht University, Utrecht, NL.

A comparative genomics analysis of over 200 fungal genomes has revealed large differences in genome evolution across the fungal kingdom. Driving forces behind these differences are variable rates of interchromosomal rearrangements and variable inversion lengths in intrachromosomal rearrangements. Each class of fungi follows its own pattern of genome evolution.

For example, it was previously shown in the class Dothideomycetes (phylum Ascomycota) that a low rate of interchromosomal
rearrangements and high rate of intrachromosomal rearrangements leads to mesosyntheny, where gene content but not gene order is conserved. In the class Saccharomycetes (phyum Ascomycota), in contrast, interchromosomal rearrangements occur frequently, quickly degrading conserved syntheny. A similar pattern of genome evolution occurs in the class Agaricomycetes (phylum Basidiomycota) as in Dothideomycetes, with the important difference that the intrachromosomal inversion length is much smaller. This results in a distinct pattern only found in this class. Moreover, in Agaricomycetes several conserved ancestral chromosomes could be identified with varying patterns of genome evolution, possibly due to their difference in sequence composition. On gene level, conserved gene clusters were identified despite the observed intrachromosomal inversions. Notably, certain transcription factors were over-represented in these conserved gene clusters.

**225F Early origins of fungal multicellularity-related genes in eukaryotic evolution.** *E. Kiss*, A. Prasanna N, K. Krizsan, L. G. Nagy Synthetic and Systems Biology Unit, Biological Research Centre, Szeged, HU.

The emergence of multicellular organisms from single-celled ancestors is considered one of the major transitions in the history of life. It has been suggested that genes underlying several multicellularity-related traits, like cell adhesion, cell-cell communication and programmed cell death already existed in unicellular organisms. These traits might have predisposed them for evolving multicellularity, consistent with the multiple origins of multicellularity in many groups, including fungi. However, so far there have not been detailed studies on the evolution of multicellularity-related gene families in Fungi, where multicellularity has different physiological bases from that of other groups. We obtained information on the genetic background of fungal multicellularity by collecting known and putative multicellularity-related genes from the literature. We identified 995 such genes, involved in signal transduction, gene expression regulation, adhesion and hyphal morphogenesis. The evolutionary origins of these genes were examined using complete genomes of 77 unicellular and multicellular eukaryotes. Through similarity based clustering we found that the 996 genes grouped into 580 clusters of homologues. We next performed phylostratigraphic analysis on these gene clusters using a custom pipeline to uncover the evolutionary origins of their gene families and reconstructed gene duplication/loss histories in 525 clusters using reconciled gene trees, which gave a high-resolution view on the dynamics of these putatively multicellularity-related gene families over time.

The analyses of gene families revealed that part of the genetic toolkit behind fungal multicellularity was already present in ancestral unicellular eukaryotes and we found that several multicellularity related gene families started diversifying before the emergence of multicellular fungi. These observations might explain the convergent evolution of hyphae in early-diverging groups. Further, our results suggest a role of gene regulatory network rewiring in addition to *de novo* gene family birth and gene duplications. Taken together, these results suggest that unicellular fungal ancestors were partially predisposed for evolving multicellularity, which, probably combined with lineage-specific genetic innovations, gave rise to extant filamentous fungi.

**226W Microbotryum-mediated sex conversion of *Silene latifolia*.** A. Harkess¹, P. Baldrich¹, L. Delph², M. Perlin³, B. Meyers¹ 1) Donald Danforth Plant Science Center, St. Louis MO 63132; 2) Indiana University, Bloomington IN 47405; 3) University of Louisville, Louisville KY 40292.

More than a century ago, Strasburger (1900) observed that *Microbotryum lychnidis-dioicae* (formerly *Ustilago violacea*) was able to infect *Silene latifolia* flowers and lead to sexual conversions. This peculiar fungal parasitism can cause *S. latifolia* XX females to produce rudimentary anthers inside which the fungus propagates, but interestingly the infection of XY male plants does not produce female organs. Given that our understanding of the molecular basis of sex determination on the *S. latifolia* X and Y chromosomes is incomplete, this parasitism could be a key to identifying the master sex determination genes on the *Silene* XY sex chromosome pair. To understand the molecular mechanisms of this sexual transition, we are sequencing mRNA, small RNA, and PARE ("degradome") libraries from pre- and post-infection *S. latifolia* flowers, as well as attempting genetic transformation of both species for Cas9-based genome editing. Given that there may exist species co-diversification of both fungus and host, we hypothesize that the transition from hermaphroditism (self-compatibility) to dioecy (outcrossing) in *S. latifolia* may have been spurred by a plant-fungus evolutionary "arms race".

**227T Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.** S.J. Hanson¹,², K.P. Byrne², K.H. Wolfe² 1) Molecular Biology, Colorado College, Colorado Springs, CO; 2) UCD Conway Institute, School of Medicine, University College Dublin, Dublin 4, Ireland.

In *Ogataea* (*Hansenula*) *polymorpha* an environmental signal, nitrogen starvation, induces a reversible change in the physical structure of a chromosome. This process, mating-type switching, involves inverting a 19-kb DNA region to place either *MAT*a or *MAT*alpha genes under centromeric repression of transcription, depending on the orientation of the region. Here, we investigated the genetic pathway that controls switching. We first characterized the transcriptomes of haploid and diploid *O. polymorpha* by RNAseq in rich and nitrogen-deficient media, and found that there are no constitutively a-specific or alpha-specific genes other than the *MAT* genes themselves. We identified a switching defect in a related species (*O. parapolymorpha* strain DL-1) and mapped the defect by interspecies bulk segregant analysis to a frameshift in the transcription factor *EFG1*, which in *Candida albicans* regulates filamentous growth and white/opaque switching. By gene knockout, overexpression and ChIPseq experiments we established that *EFG1* regulates *RME1*, which in turn regulates *STE12*. All three genes are necessary for mating-type switching in response to nitrogen starvation, as well as for mating. Overexpression of *RME1* or *STE12* alone is sufficient to induce switching without a nitrogen signal. Our results show that the pathway controlling switching in *O. polymorpha* is substantially different from that in *S. cerevisiae*, which does not involve an environmental signal, and that it shares some components with mating-type switching in *Kluyveromyces lactis* and with white/opaque phenotypic switching in *C. albicans*. The downstream mechanism by which the chromosomal inversion occurs remains unidentified.

**228F Decoding the pathogenicity of Armillaria species through phylogenomics and transcriptomics.** A. Prasanna N¹, G. Sipos³, B. Kiss¹, B. Bálint², K. Krizsán¹, I. Nagy², L. G. Nagy¹ 1) Synthetic and Systems Biology Unit, BRC, Szeged, Hungary; 2) Seqomics Ltd, Mórahalom, Hungary; 3) Institute of Silviculture and Forest Protection, University of West-Hungary, Sopron, Hungary.

The genus *Armillaria* is a group of pathogenic white rot fungi, imparting significant economic and environmental losses worldwide. They cause root rot disease and propagate as rhizomorphs and infect plants through direct root contact, a unique dispersal mechanism only found in *Armillaria* spp. Here, our aim is to understand genome evolution in Armillaria spp and identify the factors involved in their pathogenicity. For this purpose, we reconstructed genome evolution across 5 *Armillaria* and 22 brown rot, white rot, litter decomposer and
ECM fungi, and analyzed transcriptomes from rhizomorphs, different developmental stages of *Armillaria ostoyae* C18.

We detected a significant genome expansion in the *Armillaria* genus, which comprised 2012 gene duplications affecting several families with pathogen-specific Pfam signatures. Next, we performed a domain based copy number analysis of plant cell wall degrading enzymes (PCWDE) and putative pathogenicity-related genes. Compared to non-pathogens, pathogenic *Armillaria* spp showed higher copy numbers of several putative pathogenicity factors and PCWDE's including expansins, hydrophobins, Trp-halogenases, secondary metabolite genes, genes with chitin bidning ability (LysM), chitosanase (GH75), pectin lyases, pectin methyltransferase, class II peroxidases and cellular transport and detoxification pathway components (p450, MFS_1).

Transcriptome analyses indicated that expression patterns of rhizomorphs are intermediate between fruiting bodies and vegetative mycelia, suggesting a link between fruiting body and rhizomorph development. Several putative pathogenicity genes, mannosyltransferases, hydrophobins, and pore-forming toxin genes were upregulated, indicating exploratory, nutrient uptake, defense and fruiting body-like functionalities for rhizomorphs. Taken together, our results suggest that pathogenic Armillaria species have significantly expanded their putative pathogenicity and PCWDE gene artillery through lineage-specific genetic innovations over the course of evolution, which might explain their saprotrophic and pathogenic potentials worldwide.

**229W Evolutionary strata on young mating-type chromosomes despite lack of sexual antagonism.** S. Branco¹, H. Badouin¹,², J. Gouzy², R. Rodriguez de la Vega¹, G. Aguiletta¹, S. Siguenza², J.-T. Brandenbourg¹, M. Coelho³, M. Hood⁴, T. Giraud¹ 1) Laboratoire Ecologie, Systématique et Evolution, Université de Paris Sud, Orsay, FR; 2) LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, FR; 3) UCIBIO-REQUIMTE, Dept. Ciências Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Caparica, PT; 4) Department of Biology, Amherst College, Amherst, MS.

Sex chromosomes often show exceptional features such as successive steps of suppressed recombination (called "evolutionary strata"), structural dimorphism and degeneration. The current theory for explaining these patterns is a successive linkage of multiple sexually-antagonistic genes to sex-determining genes. However, little evidence supports this explanation and alternative hypotheses have been proposed. Fungi are interesting models to test this theory as there is no male/female role associated with their mating-type chromosomes while they can display extensive suppressed recombination. Here we report the first compelling evidence for multiple evolutionary strata in fungal mating-type chromosomes, by comparing nearly finished genome assemblies of closely related anther-smut fungi. The existence of evolutionary strata in these fungi despite the lack of sexual antagonism calls for a unified theory of sex-related chromosome evolution, incorporating more general hypotheses, such as the sheltering of deleterious alleles or balancing selection maintaining rearrangements across longer evolutionary times than on autosomes.

**230T What drives speciation? Examination into the evolutionary events of more than 100 Aspergillus species.** J.L. Nybo¹, T.C. Vesth¹, S. Theobald¹, I. Kjaerboelling¹, J.C. Frisvad¹, T.O. Larsen¹, R. Riley², A. Salamov², I.V. Grigoriev², S.E. Baker³, M.R. Andersen¹ 1) Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, DK; 2) Joint Genome Institute, Walnut Creek, CA, USA; 3) Joint Bioenergy Institute, Berkeley, CA, USA.

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

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

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

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

**231F Phylogenomic analyses of the genetic bases of ballistospory.** M. Sanchez-Garcia¹, L. Nagy², D. Hibbett¹ 1) Biology Department, Clark University, Worcester, MA; 2) Synthetic and Systems Biology Unit, Biological Research Center, HAS, Szeged, Hungary.

Agaricomycetes (mushrooms) with an exposed hymenium have a complex mechanism of forcible spore discharge, called ballistospory. Structural features associated with this mechanism include short and curved stermigma, spores with a hilar appendage, and production of a droplet of fluid at the base of the spore. Gasteroid forms (such as puffballs) produce spores internally and in most cases have basidia that lack such features and that do not discharge spores forcibly. The mechanism of ballistospory evolved once in the phylum Basidiomycota and has been lost repeatedly during the evolutionary history of this group. It has been suggested that the loss of ballistospory is irreversible. We analyzed 60 genomes of Agaricomycetes including 12 gasteroid forms in order to infer the genetic bases of ballistospory by identifying parallel losses of genes potentially involved in this mechanism. We used a recently developed pipeline called COMPARE (Comparative Phylogenomic Analysis of Trait Evolution) to map gene duplications and losses onto a species tree and to correlate them with the presence of ballistospory.
**232W Predicting dispensable chromosomes(scaffolds) in newly sequenced Fusaarium species.** A. Salamov¹, W. Andreopoulos¹, L-J Ma², J Coleman³, I Grigoriev¹ 1) DOE Joint Genome Insitute, Walnut Creek, CA; 2) Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA, 01003, USA; 3) Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, 36849, USA.

Many Fusarium species have dispensable (or lineage-specific) chromosomes which are not required for normal growth, but may control host-specific pathogenicity of these species. For newly sequenced species from *F.solani* and *F.oxysporum* species complexes we investigate the possibility of predicting dispensable chromosomes (scaffolds) based on genomes sequence features and similarity to two reference genomes of *F.oxysporum f.sp. lycopersici* and *Nectria haematococca* with experimentally determined dispensable chromosomes. We have found that when new strains are close enough to the reference genomes, dispensable chromosomes can be reliably predicted based on combination of genome alignments and mapping of proteins from reference genomes to new sequences. We also built Hidden Markov Models for gene families, specific for dispensable chromosomes of reference genomes, which in combination with other features, such as repeat content, sparsity of genes with known functional domains, codon usage, and GC content can be used for prediction of dispensable chromosomes in more distant species.

**233T Genomic comparison of a dispensable chromosome within the Fusarium fujikuroi complex.** L. De Vos¹, E.T. Steenkamp², M.A. van der Nest¹, W-Y. Chan², S. van Wyk², B.D. Wingfiled¹ 1) Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 2) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

Species within the *Fusarium fujikuroi* complex (FFC) are considered important pathogens of commercially planted plants and trees. Members of this complex typically have 12 chromosomes, of which the 12th chromosome is the smallest and considered to be part of the accessory genome. This hypothesis is based on low sequence similarity among the corresponding chromosome from different species, and the apparently dispensable nature of this chromosome within the FFC. In this study, we investigated this hypothesis by making use of a comparative genomic approach. For this purpose, whole genome sequence data available for representatives of the FFC (i.e., *F. circinatum*, *F. subglutinans*, *F. nygamai*, *F. fujikuroi* and *F. temperatum*) were used. From these data, sequences for chromosome 12 of *F. fujikuroi* and *F. temperatum* were used to assemble the corresponding chromosome in the other three species. Chromosome 12 comparisons within the FFC showed that the density and average size of genes encoded on this molecule were lower when compared to whole-genome data. Assignment of gene functions revealed genes primarily involved in oxidation-reduction and macromolecule metabolic processes, cation and nucleoside phosphate binding, and as intrinsic components of membranes. Phylogenetic analyses also showed that the genes of chromosome 12 had diverse and non-orthologous origins, with only a small proportion apparently sharing an evolutionary trajectory similar to those of the species harbouring them. Taken together, these findings therefore suggest that the ancestor of the FFC likely harboured a version of chromosome 12. Also, consistent with being part of the accessory genome, the evolution of chromosome 12 involved the acquisition of genes from sources outside the FFC. Future research should seek to determine the processes encoded by chromosome 12 and the biological properties this molecule might confer to the species harbouring it.

**234F Genome mining, pathogenicity and secondary metabolism of three strains of Fusarium fujikuroi, the causal agent of bakanae disease on rice.** E. Piombo¹,², H. Banani¹,², I. Siciliano¹, P. Abbruscato³, A. Acquadro², M.L. Gullino¹,², D. Spadaro¹,² 1) Universita degli Studi di Torino, Centro AGROINNOVA; 2) Universita degli Studi di Torino, DISAFA; 3) Bioeconomy Unit, Parco Tecnologico Padano.

Bakanae is an important seedborne disease of rice, caused by *Fusarium fujikuroi*. This pathogen can produce a wide range of secondary metabolites, including fumonisins and gibberellins. In order to gain insight into secondary metabolites (SM) synthesis in *F. fujikuroi*, the genomes of the 3 strains *Augusto2*, *CSV1* and *I1.3* were sequenced to identify the allelic variants in the genes responsible for SM production, and compare the virulence on rice and the SM production *in vitro* and on rice. Sequence analysis was conducted by *de novo* and *reference guided* genome assembly. Three genomes of 42.8 Mb on average were obtained. The gene clusters responsible for fumonisin and gibberellin production, formed respectively by 15 and 7 genes, were analyzed and aminoacidic differences were predicted for *fum1*, *fum13* and *fum21*. The 3 strains also exhibited distinct differences in colony morphology and growth kinetics. The virulence and fumonisin production of the three strains on cultivar "Galileo" of rice were compared. At 3 weeks post germination *I1.3* showed statistically higher virulence compared to *Augusto2* and *CSV1*. *Augusto2* was the major producer of fumonisins both *in vitro* and *in vivo*, followed by *CSV1*, while *I1.3* was unable to produce fumonisins either *in vivo* or *in vitro*. The lack of fumonisin production in *I1.3* is probably caused by a number of identified mutations in the sequence of key genes (*fum1* and *fum21*) in the fumonisin gene cluster, while the difference in the production between *Augusto2* and *CSV1* might be due to a mutation in the gene *VeA*, regulator of SM biosynthesis, including fumonisins. *CSV1* was unable to produce gibberellins *in vivo* and *in vitro* on Petri dish, confirming the different symptomatology of *CSV1* on rice, characterized by dwarfing and chlorosis, but lack of stem elongation. This study showed how *F. fujikuroi* can present a great deal of intra-species variation, both in the induced symptoms and in SM production.

**235W Quantitative mapping of pathogenicity factors in the Fusarium fujikuroi complex with an interspecific genetic cross.** Wei Yue¹, Christopher Toomajian¹, Nik Mohd Izham Mohamed Nor¹,², John F. Leslie¹ 1) Dept Plant Pathology, Kansas State Univ, Manhattan, KS, USA; 2) School of Biological Sciences, Science University of Malaysia, Penang, Malaysia.

The *Fusarium fujikuroi* species complex contains agronomically important plant pathogens that produce secondary metabolites such as mycotoxins and gibberellic acids (GAs) that are involved in pathogen virulence. Two members of this species complex, *F. proliferatum* (*Fp*) and *F. fujikuroi* (*Ff*), are closely related phylogenetically but have different host specificity and produce different mycotoxins. Our long-term aim is to explore the genetic bases behind these differences, and we have begun by conducting QTL analyses of progeny from an interspecific cross. Using the R/qtl software package, we constructed a high density genetic map with data from 6,381 high quality Genotyping-by-Sequencing (GBS) markers and 253 progeny from the cross. The final genetic map consisted of 12 linkage groups, which correspond to the 12 chromosomes present in each species, and had a length of over 1900 cM with only 4 gaps greater than 20 cM. In parallel, we generated next generation sequence data from the *Fp* parent of this cross and performed a draft genome assembly. Our sequence-based markers allowed us to anchor 97.8% (42.2 Mb) of the total assembly onto the 12 linkage groups. Not surprisingly for an
interspecific cross, we observed several from expectations in terms of marker segregation and recombination patterns. Many markers
display significantly skewed segregation towards the *Fp* allele, although we also detected a significant skew towards the *Ff* allele in other
regions. Using our GBS markers mapped to our *Fp* genome assembly and reference *Ff* genomes, we detected heterogeneity in
recombination rates. Though some of this heterogeneity is consistent with recombination patterns observed in other Fusarium species,
certain recombination hotspots appeared to be due to shared recombination events among all progeny collected from the same
peritheium, while the absence of recombination in other regions helped to confirm inferred chromosomal inversions between the species.
We performed QTL mapping of two traits relevant to the pathogenicity of each parent: GA level and pathogenicity to onion. The major peak
for onion pathogenicity co-localizes with a known secondary metabolite cluster. In addition to mapping the biosynthetic gene cluster for GA
production, we also identified an unlinked modifier locus that interacts epistatically with it. This map provides valuable information for
understanding the genetic bases underlying differences between these two closely related *Fusarium* species.

**236T Comparative transcriptomics analysis of two different pathotype strains of Fusarium fujikuroi obtained from rice in Korea.** Hee-Kyoung Kim, *Da-Woon Kim*, Sung-Hwan Yun Dept Med Biotech, Soonchunhyang Univ, Asan, Chungnam, KR.

*Fusarium fujikuroi* is known to be commonly associated with rice, not only causing bakanae disease, but also producing a group of secondary metabolites, such as gibberellins, bikaverin, fumonisins (FB), and fusarin C. Here, we have focused on two *F. fujikuroi* isolates (B20 and B14) obtained from rice grains in Korea, which show different pathogenesis mechanisms on rice seedling. The B20 strain is able to cause a typical stem elongation in rice seedling, while B14 causes no change in the seedling height; rather it causes a discoloration in root of rice seedling. To characterize the difference in pathogenicity between these two pathotype strains, we performed a RNA-seq analysis using total RNA samples obtained from rice seedlings inoculated with these strains. Compared to those expressed during the vegetative growth on PDA, genes encoding cell wall degrading enzymes, fusaric acid biosynthesis proteins, and proteases were up-expressed on rice (at 3 dpi and 6 dpi stages) from both strains. However, the expression patterns of several gene clusters for secondary metabolites (SM) of these strains were different from each other on rice. The expressions of most gibberellin gene cluster members in B14 were highly induced on rice, whereas those in B20 seemed to be constitutively expressed on both PDA and rice. Interestingly, the entire fumonisin gene cluster members in B14 were highly expressed on both PDA and rice, while six genes in the cluster were missing in B20; furthermore the expression level of the rest members on rice were almost undetectable. In addition, the apicidin F cluster, which showed a rice-specific expression pattern in B20, was not physically present in the genome of B14. Further functional analyses including a targeted gene deletion will elucidate if variations in the expression of SM gene clusters would be responsible for the different pathogenesis mechanisms between these two *F. fujikuroi* strains.

**237F Comparative population genomics of *Fusarium graminearum* reveals adaptive divergence among cereal head blight pathogens.** A.C. Kelly, Todd J. Ward ARS, USDA, Peoria.

In this study we sequenced the genomes of 60 *Fusarium graminearum*, the major fungal pathogen responsible for Fusarium head blight (FHB) in cereal crops world-wide. To investigate adaptive evolution of FHB pathogens, we performed population-level analyses to characterize genomic structure, signatures of natural selection and differences in gene content among isolates. Genome-wide patterns of SNP diversity revealed that most isolates with the novel NX-2 (3α-acetoxy. 7α,15-81 dihydroxy-12,13-epoxytrichothec-9-ene) toxin type represent a genetic population (termed NA3) that is distinct from the native (NA1, largely 15-acetyl-deoxynivalenol toxin type) and invasive (NA2, largely 3-acetyl-deoxynivalenol toxin type) populations inhabiting North America, although genetic exchange among populations was documented. The three populations differed in gene content, with 134 genes showing population-specific patterns of conservation. In addition, each population had unique genetic signatures of adaptive selection that were largely focused in hypervariable regions of chromosomes. Sixteen candidate loci, varying in size from 10-40 kb, showed genetic signals of adaptive divergence, in that alleles were highly differentiated among populations but showed reduced diversity within populations. The strongest signals of selective sweeps were observed at the trichothecene biosynthetic gene cluster. However, functional annotation of population-differentiating genomic regions revealed numerous genes involved in host invasion, toxin production and secondary metabolism, and implicated plant hosts, microbial competitors, and temperature and light as major drivers of adaptive divergence. Collectively, our results show that North American populations of *F. graminearum* harbor unique sets of adaptations that contribute to differences in how these pathogens exploit the agricultural landscape.

**238W Functional genomics of lipid accumulation in *Rhodosporidium toruloides*.** Samuel Coradetti¹,², Dominic Pinel², Gina Geislman², Adam Arkin², Rachel Brem¹,³, Jeffrey Skerker² 1) Buck Institute for Research on Aging, Novato, CA; 2) Department of Bioengineering, University of California, Berkeley, CA; 3) Plant and Microbial Biology, University of California, Berkeley, CA.

Wild isolates of the basidiomycete yeast *Rhodosporidium toruloides* can accumulate more than 70% of their biomass as nuetral lipid in the form of triacylglycerol and produce relatively high concentrations of carotenoids. *R. toruloides* can also utilize a wide spectrum of plant cell wall sugars and lignin derivatives, and has a natural tolerance for common inhibitors in biomass hydrolysates. These properties make *R. toruloides* an attractive host for sustainable production of hydrocarbon chemicals and fuels from low-value feedstocks. While the *R. toruloides* research community is growing rapidly, relatively sparse biochemical data limits current engineering efforts. To rapidly interrogate gene function on a genomic scale, we applied high efficiency *Agrobacterium tumefaciens* mediated transformation to build a 300,000+ member strain library of barcoded random insertions. By tracking the relative abundance of barcoded strains in competition experiments and physical enrichments, we can simultaneously assign phenotypes for deletions of thousands of genes in a given condition. We have used this technique to identify a large cohort of genes (both novel and homologous to well-characterized genes in other systems) with roles in various aspects lipid metabolism including central carbon metabolism, nucleation and maturation of lipid bodies, protein trafficking, and nutrient sensing.

**239T Genomic analysis of phenotype reversion in *cuf1*- strains of *Cryptococcus neoformans* var *neoformans*.** Shannon Ellis², Morgan Coslett³, Jocelyn Teagarden¹, Tanner Bross¹, *Jeramia Ory*¹ 1) Department of Basic Sciences, Saint Louis College of Pharmacy, Saint Louis, MO ; 2) Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 3) Department of Biology, King's College, Wilkes-Barre, PA.
*Cryptococcus neoformans* is a ubiquitous soil fungus that is a major cause of meningitis in immunocompromised patients worldwide. One virulence factor responsible for the pathogenesis of *C. neoformans* is the fungus' ability to produce melanin using a wide variety of environmental substrates. The gene responsible for melanin synthesis, *CNLAC1*, has been identified and shown by others to encode a copper dependent laccase. In addition, proper copper import and processing has been shown by multiple studies to be necessary for pathogenesis. *CUF1* codes for the major copper sensing transcription factor, and controls expression of numerous genes related to copper metabolism. To further explore the role of copper metabolism in cell fitness and pathogenesis, we constructed a *CUF1* deletion strain of *C. neoformans var neoformans* (*cuf1*). Studies by our lab and others show that the *cuf1* strain grows quite poorly on Yeast Peptone Dextrose (YPD) media without supplemented copper. To characterize the growth phenotype and identify possible gene regulatory networks, we measured microarray whole genome expression of both wild type (JEC21) and *cuf1* strains in various copper supplemented conditions. Expression patterns are similar to those found for *C. neoformans var grubii*, but analysis is hindered by metabolic stress found in the *var neoformans* *cuf1* strain that is not present in the *var grubii* *cuf1* strain. In low copper environments the *var neoformans* *cuf1* strain is unable to express copper import genes as expected, but also shows lower levels (compared to wild type) of citric acid cycle and glycogen storage enzymes and higher levels of saccharide processing and breakdown enzymes. The metabolic stress caused by insufficient copper import also appears to impart significant selective pressure. We find the *var neoformans* *cuf1* strain repeatedly and stably reverts to fitness levels close to wild type when serially passaged on YPD without supplemented copper. We are currently collecting whole genome short read data on independent revertants, and are analyzing them for mutations or duplication events that would explain the mechanism of reversion.

**240F Adaptations of *Exophiala dermatitidis* in stressful environments.** Jyothi Kumar, Anthony Mosher, Steven D Harris
Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE.

Fungi are known to inhabit extreme environments that are constantly exposed to high salt, high temperature, cold and desiccation stress. A multitude of factors enable colonization of these environments, including melanization and UV resistance. *Exophiala dermatitidis* is a model black yeast extremophile that causes cutaneous and subcutaneous infections in humans. Studies indicate that it can produce carotenoids, melanins, and oxidative enzymes in response to stress and as a means to survive extreme environments. Our objectives are to investigate the mechanisms that underlie stress resistance in *E. dermatitidis*. In this study, we performed a transcriptomic analysis of *E. dermatitidis* using RNA sequencing (RNA-seq) to investigate the role of the conserved GTPases Cdc42 and Rac1 in stress tolerance. Differential expression analyses revealed important genes / processes involved in light responses, carotenoid synthesis, oxidative stress, heat shock proteins and MAPK signaling pathways. Transcription factors like RLM1, SMP1, PHO2 involved in the regulation of melanin synthesis, the HOG1 pathway and phosphate synthesis were also modulated. *E. dermatitidis* requires photoinduction for carotenogenesis and has an intriguing set of light sensing genes. We studied the role of light in regulating pigmentation by phenotypic and expression profiling of genes involved. Our results show that light and maltose repress the morphological transition from yeast to filamentous form and CDC42/Rac1 module is a positive regulator for the switch from yeast to hyphal form.

**241W Improved genome sequence of *Diplodia seriata*, a tool for comparative genomics of *Diplodia* species.** G. Robert¹, J. Vallet², E. Abou-Mansour³, J. Xu⁴, P. Rey⁵, C. Bertsch⁶, C. Rego⁷, P. Larignon⁸, F. Fontaine², MH. Lebrun¹
1) BIOGER, INRA, Thiverval-Grignon, FRUMR 1290 BIOGER, INRA, AgroParisTech, Campus AgroParistech, Ave Bretignières, 78850, Thiverval-Grignon, France; 2) Université de Reims Champagne-Ardenne, URVVC EA 4707, Laboratoire Stress, Défenses et Reproduction des Plantes, BP 1039, 51687 Reims Cedex 2, France; 3) Plant Biology Department, University of Fribourg, Chemin du Musée 10, 1700 Fribourg, Switzerland; 4) BGH-Shenzhen, 518083 Shenzhen, China; 5) UMR1065 SAVE, Santé et Agroécologie du Vignoble, INRA, Bordeaux Sciences Agro, F-33140, Villenave d'Ornon, France; 6) Université Haute Alsace, Laboratoire Vigne Biotechnologie et Environnement EA 3991, 33, rue de Herrlisheim, 68008 Colmar Cedex, France; 7) Institut Supérieur d'Agronomie, Tapada da Ajuda, 1349-017 Lisbonne, Portugal; 8) Institut Français de la Vigne et du Vin Pôle Rhône-Méditerranée, 7 avenue Cazeaux, 30230 Rodilhan, France.

The Ascomycotina *Diplodia seriata* is one of the causal agents of grapevine trunk diseases. Here, we present an improved genome sequence of *D. seriata* isolate F98.1, isolated in 1998 at Perpignan (France) from the trunk of a diseased grapevine. *D. seriata* is one of the most common *Botryosphaeriaceae* species associated with grapevine trunk diseases. This species is also frequently isolated from woody tissues of trees such as *Acer* sp., *Prunus* sp. or *Quercus* sp.. Sequencing was performed using Illumina HiSeq 2500 at a coverage of 270 X. Assembly with SOAPdenovo 1.05 led to 512 contigs and 112 scaffolds (37.27 Mb, GC%: 56.8). This high quality genome (Scaffold N50, 2.9 Mb; Min scaffold length, 1.007 bp; gaps, 250 kb) has 13 scaffolds with a size higher than 1 Mb (90% of total sequence), likely corresponding to chromosomes. Using GLEAN, 8.087 CDS were identified, 93 % being supported by RNAseq (mycelium on PDB or MM for 4 days). Recently, the genome sequence of *D. seriata* isolate DS831 from an infected grapevine (USA, 2011) was released. The genome size of DS831 (37.13 Mb) is almost similar to F98.1. However, its assembly is 5 times more fragmented (1391 contigs, 695 scaffolds) and it carries 9398 CDS. Bidirectional best blast hit (BDBH) analysis revealed that 82% of F98.1 and DS831 CDS are similar, 1507 genes are specific to F98.1 strain, and 2763 are specific to DS831 strain. Using Blast, we showed that 2686 (97.2%) of DS831 apparently specific genes were present in F98.1 genome sequence, and reversely 1440 (95.6%) of F98.1 apparently specific genes are present in DS831 genome sequence. Therefore, the difference in CDS numbers between DS831 and F98.1 is likely a consequence of using different annotation software (Glean vs Augustus). Fusion of two annotation leads to a total of 10,500 genes. The genomes of the two species *Diplodia sapinea* and *Diplodia scrobiculata* closely related to *D. seriata* have been sequenced. These two genome have a size of, respectively, 36,97 and 35,85 Mb, and 13020 and 13624 genes respectively. These differences could reflect recent massive gene losses in *D. seriata*.

**242T Unravelling potato wart disease; genome assembly and annotation of an obligate parasitic chytrid fungus.** B.T.L.H. van de Vossenberg¹,², H. Nguyen³, M. van Gent Pelzer¹, S. Warris¹, L.V. Bakker¹, H.C. van de Geest¹, D.S. Smith⁴, D. Joly⁵, C.A. Levesque³, J.H Vossen¹, R.G.F. Visser¹, T.A.J. van der Lee¹
1) Wageningen University, Wageningen, Gelderland, NL; 2) National Reference Centre, Dutch National Plant Protection Organization, Wageningen, Gelderland, NL; 3) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ca; 4) Pathogen Identification Research, Canadian Food Inspection Agency, Ottawa, Canada; 5) Université de Moncton, Moncton, New Brunswick, CA.

The obligate fungal parasite *Synchytrium endobioticum* (Chytridiomycota) is the causal agent of potato wart disease, a severe disease of
cultivated potato that leads up to 100% yield loss. Over the last decades a multitude of pathotypes emerged that break resistance. Once introduced into the soil, the soil itself cannot be used for further crop production due to robust resting spores that can remain viable and infectious for over four decades. Worldwide quarantine measures are in place to restrict the spread of *S. endobioticum*. In addition, the pathogen is included on the HHS and USDA Select Agent list.

To better understand the obligate parasitic lifestyle of the fungus and its interaction with potato, we sequenced and assembled the genomes of two different strains: MB42 from the Netherlands and LEV6574 from Canada. As this pathogen cannot be cultured, generating DNA for this fungus free from its host and other contaminants proved to be impossible. A comparative read-mapping approach (referred to as ZOO selection) with 21 HiSeq or Miseq datasets generated from 17 *S. endobioticum* strains, RNAseq of both reference strains, and HiSeq data from healthy potato allowed the identification of contaminant scaffolds. The effect of ZOO selection was visualised using the Blobology tool. The resulting genome size ranged from 21.6 to 23.5 Mb and show only low levels of DNA polymorphisms between the two strains. RNAseq-based structural annotation using the BRAKER pipeline resulted in 8400-9000 predicted gene models for the respective strains. The annotation completeness of the gene dense and intron rich genomes was assessed using the BUSCO v2 benchmark, resulting in 91.4% and 92.1% annotation completeness for the respective strains. InterProScan was used to functionally annotate the predicted genes and to assign them to KEGG pathways.

Orthologous gene families shared between *S. endobioticum* and culturable saprobic or pathogenic chytrid species were predicted using Orthofinder and allowed the identification of genes present in all culturable chytrids, but absent in both obligate parasitic *S. endobioticum* genomes. In addition, orthologues gene families with transposons were found to be highly expanded in both *S. endobioticum* genomes compared to the other chytrid genomes. We hypothesise that these transposons may have had a significant impact on the genome evolution and perhaps its reduced genome size.

**243F Genetic diversity of 100+ Aspergillus species - the aspMine analysis resource.** *T. C. Vesth*¹, J. L. Nybo¹, S. THEOBALD¹, R. P. DE VRIES⁴, I. V. GRIGORIEV³, S. E. BAKER⁵, M. R. ANDERSEN¹ 1) Department of Bioengineering, Technical University of Denmark, Lyngby, Denmark; 2) Joint Bioenergy Institute, Berkeley, CA, USA, Berkeley, CA, USA; 3) Joint Genome Institute, Walnut Creek, CA, USA, Walnut Creek, CA, USA; 4) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands, Utrecht, The Netherlands.

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

**244W Insight into Cryptococcus neoformans lineages with different clinical patterns using 800 whole genome sequences.** *P.M. Ashton*¹, P.H. Trieu¹, L.T. Thanh¹, D.V. Anh¹, T.T. Chau¹, S. Baker¹,², G.E. Thwaites¹, F.M. Kibengo⁴, N.T.K. Cuc⁵, W. Chierakul⁶, A.K. Chan⁷, M. Mayxay⁸, J. Beardsley¹,², S. Harris³, J.N. Day¹,² 1) Oxford University Clinical Research Unit, Ho Chi Minh city, VN; 2) Centre for Tropical Medicine, University of Oxford, UK; 3) Wellcome Trust Sanger Centre, Hinxton, UK; 4) MRC/UVRI Uganda Research Unit on AIDS, Entebbe, Uganda; 5) Hospital for Tropical Disease, Ho Chi Minh City, Vietnam; 6) Mahidol Oxford Tropical Medicine Research Unit, Bankgkok, Thailand; 7) Dignitas International, Zomba, Malawi; 8) Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Vientiane, Laos.

The vast majority of cryptococcal meningitis in immunodepressed patients is caused by *C. neoformans* var. *grubii*. In Viet Nam, we see disease in apparently immunocompetent patients due to a specific cluster of *C. neoformans* var. *grubii* (VNly, lineage 5). This causes 84 % of infections in HIV-negative patients, but only 35% in HIV-positive patients. These data suggest that VNly is not the dominant genotype in the environment, but is associated with HIV-negative patients. We hypothesize that the VNly cluster has increased ability to infect immuno-competent people compared with other VNI strains. Here, we report initial results from the use of whole genome sequencing of 800 isolates of *C. neoformans* var *grubii* collected as part of clinical trials to investigate this hypothesis.

Gene multiplication has been shown to be a source of genetic diversity in *C. neoformans* which can result in significant phenotypic shifts (Chow et al., 2012). Here, we examined our dataset for other gene multiplication events. There were a total of 1267 putative multiplication events affecting 501 genes identified in this dataset. Most genes are only multiplied in one strain (73%). Furthermore, 93% of the singletons are accounted for by just 5 strains, with one large 465 kb duplication accounting for 46% of duplications present in only one strain. However, some genes are duplicated in a larger number of strains, suggesting that these genetic events have been selected for. There was an average of 17 duplication events per genome, with a maximum of 370 and a minimum of 0.

Our phylogenetic analysis shows that *C. neoformans* var *grubii* is divided into at least 5 lineages, separated by considerable genetic distance. Each lineage caused infections in both South East Asia and East Africa, but their prevalence differs markedly. The fact that all
lineages were present in both SE Asia and East Africa, but the prevalences varied markedly suggests that the different lineages have characteristic environmental or host adaptations that result in this difference.

In conclusion, whole genome sequencing of 800 *C. neoformans* isolates has generated a rich resource for the investigation of this organism and will allow us to further investigate the clinical differences between lineages of this species.

**245T Whole genome sequence of the causal agent of Asian pear scab disease, *Venturia nashicola* for comparative genomics analyses toward identifying host-specificity determinants in pear scab fungi.** *Shakira Johnson*¹,², *Joanna Bowen*³, *Helge Dzierzon*³, *Dan Jones*³, *Carl Mesarich*⁵, *Vincent Bus*⁴, *Hideo Ishii*⁶, *Kim Plummer*¹,² ¹) Animal, Plant and Soil Sciences, La Trobe University, Bundoora, VICTORIA, AU; ²) Plant Biosecurity Cooperative Research Centre, Canberra, Australia; ³) Plant and Food Research, Auckland, New Zealand; ⁴) Plant and Food Research, Hawke's Bay, New Zealand; ⁵) Massey University, Palmerston North, New Zealand; ⁶) Kibi Internation University, Takahashi, Okayama, Japan.

Pear scab is a major disease of pears worldwide; it is caused by two distinct fungal species, *Venturia pirina* (causes European pear scab) and *V. nashicola* (causes Asian pear scab, a quarantine threat outside Asia). The two species display host specificity: *V. pirina* exclusively infects European pears (*Pyrus communis*); while *V. nashicola* exclusively infects Asian pears (*P. bretschneideri*, *P. ussuriensis* and *P. pyrifolia*). The distinction between the host specificity of *V. pirina* and *V. nashicola* is likely to be determined by differences in their effector gene arsenal. It is hypothesized that these effectors are important factors in determining the non-host resistance relationships (i.e. in the incompatible interactions). The *Pyrus-Venturia* patho-system provides a unique opportunity to dissect the underlying genetics of non-host interactions and to understand host pathogen coevolution of this potentially more durable form of resistance. Genome sequencing technologies have greatly increased the power to perform comparative analyses that assist in the understanding of the molecular mechanisms involved in pathogenesis. Here we report the first draft genome sequence of *V. nashicola*, which will be used to perform comparative genomics with a WGS of *V. pirina* to identify unique and essential effectors that govern host-specificity of these pear-infecting fungi. In addition to these sequences, we have re-sequenced a number of *V. pirina* and *V. nashicola* isolates to reveal effector candidates under either diversifying or purifying selection pressure, with the aim to use these effector candidates to screen experimental *Pyrus* germplasm for durable resistance.

**246F Understanding genome evolution and adaptation in the Dothideomycetes.** *S. Haridas*¹, *Ryan Albert*², *Asaf Salamov*¹, *Pedro Crous*³, *Joseph Spatafora*⁴, *Igor Grigoriev*¹,² ¹) DOE Joint Genome Institute, Walnut Creek, CA. USA; ²) Plant and Microbial Dept., University of California Berkeley, Berkeley, CA. USA; ³) CBS Fungal Biodiversity Centre, Utrecht, The Netherlands; ⁴) Oregon State University, Corvallis, OR. USA.

Dothideomycetes is the largest and most diverse class of ascomycete fungi with 23 orders 110 families, 1300 genera and over 19,000 known species. We have sequenced over 50 Dothideomycetes genomes across the phylogenetic tree. This, along with the previously published 40 Dothideomycetes genomes provides an opportunity and challenge for large scale genomic studies across 300 million years of evolutionary history. We were able to clarify the phylogenetic positions of several species whose origins were unclear in previous morphological and sequence comparison studies. We also show some of the challenges in analyzing such a diverse dataset and present some recent breakthroughs in understanding genome evolution and adaptation.

**247W Wild-isolates of nematode-trapping fungi in Taiwan.** *S. Lin*, *Y. Hsueh* Institute of Molecular Biology, Academia Sinica, Taipei, TW.

Nematodes- trapping fungi use different mechanisms to trap and digest nematodes. However, little is known about how they evolve to identify their prey in different environments. To understand the distribution and ecological niche of nematode-trapping fungi, we collected ~60 soil samples from 12 locations in Taiwan and isolated 6 different *Arthrobotrys* species including *A. oligospora*. Among these species, *A. oligospora* appeared to be the most ubiquitously distributed one. Characterization of the phenotypes of *A. oligospora* wild-isolates revealed that these wild isolates exhibited significant difference in certain phenotypic traits such as growth at high temperature and nematode-trapping ability. We plan to use GWAS to understand the potential links between the genotypes and phenotypes in these nematode-trapping fungi.

**248T A new genetic code in yeasts.** *Robert Riley*¹, *Sajeet Haridas*¹, *Kenneth H. Wolfe*², *Mariana R. Lopes*³, *Chris Todd Hittinger*³, *Asaf Salamov*¹, *Christopher H. Calvey*⁴, *Aisling Y. Coughlan*², *Cletus P. Kurtzman*⁵, *Meredith Blackwell*⁶, *Igor V. Grigoriev*¹, *Thomas W. Jeffries*³,⁴ ¹) US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Walnut Creek, CA; ²) University College Dublin, Dublin, Ireland; ³) University of Wisconsin-Madison, Madison, WI; ⁴) Xylome Corporation, Madison, WI; ⁵) US Department of Agriculture ARS NCAUR, Peoria, IL; ⁶) Louisiana State University, Baton Rouge, LA.

The ascomycete yeasts, which include the model organism *Saccharomyces cerevisae*, are a diverse group of fungi spanning a billion years of evolution, and with enormous biotechnological potential. Unique among eukaryotes, some yeasts are known to have a genetic code change, in which CUG codons are translated to serine rather than the usual leucine. Such genetic code changes are interesting due to their extreme rarity, but may also complicate efforts to exploit the yeasts' biotechnological potential through exogenous expression of genes (which might have genetic codes incompatible with hosts). We sequenced the genomes of 16 ascomycete yeasts with the goal of discovering biotechnologically useful genes. From the genome sequences, we predicted that a xylose-fermenting yeast, *Pachysolen tannophilus*, translates CUG codons neither to canonical leucine nor previously known serine, but to alanine. Mass-spectrometry proteome analysis and genetic transformation experiments confirmed this prediction. The existence of multiple genetic codes in the ascomycete yeasts has implications for their exploitation in biotechnology and synthetic biology.

**249F Resolving the Mortierellaceae phylogeny through Multi-Locus Sequence Typing (MLST) and phylogenomics.** *Natalie Vande Pol*¹, *Jason Stajich*², *Kerry O'Donnell*³, *Alessandro Desiro*¹, *Gregory Bonito*¹ ¹) Michigan State University, East Lansing, MI; ²) University of California at Riverside, Riverside, CA; ³) Mycotoxin Prevention & Applied Microbiology Research Unit, NCAUR-ARS-USDA, Peoria, IL.
The Mortierellaceae (Mortierellomycotina) are a diverse family of fungi that are of evolutionary and ecological relevance. They are the closest lineage to the arbuscular mycorrhizae (Glomeromycotina) and include some of the first species to evolve fruiting body production. The Mortierellaceae are estimated to contain at least 100 species classified within six polyphyletic genera that cannot be resolved with ribosomal markers. With advances in DNA sequencing technology, it is now feasible to generate sequence data from many loci in parallel (MLST), or to perform low-coverage genome (LCG) sequencing to identify phylogenetically informative loci. In collaboration with the ZyGo Life consortium and the Joint Genome Institute, we sequenced 68 LCGs representing 50 unique species of Mortierellaceae. From these, we identified 400 informative loci and used RaxML to build a concatenated tree. The resulting phylogeny has very strong bootstrap support and a very different structure from existing ribosomal trees. In parallel, we analyzed three *de novo* sequenced *Mortierella* genomes and extracted 13 informative loci. For each locus, we designed PCR primers for multiplexed PCR amplification across 333 Mortierellaceae isolates, which included the 68 LCG isolates. We will discuss the strengths and limitations of these two approaches.

**250W Comparative genomics and transcriptomics of Russulaceae.** Brian Looney¹,², Dan Jacobson¹, Kerrie Barry³, Alan Kuo³, Igor Grigoriev³, Francis Martin⁴, Jessy Labbé¹,² 1) Bioscience Division Oak Ridge National Laboratory, Oak Ridge TN 37830; 2) University of Tennessee, Knoxville, TN 37996; 3) US DOE Joint Genome Institute, Walnut Creek, CA 94598; 4) INRA UMR1136, 54280 Champenoux, France.

*Russulaceae* is a diverse fungal family including the genera *Russula*, *Lactarius*, *Lactifluus*, and *Multifurca*, and is one of the most widespread and species rich ECM lineages. In a recent collaborative effort, the Joint Genome Institute has sequenced genomes and transcriptomes of representative groups across *Russulaceae*, including a saprotrophic outgroup. We present here an overview of the first insight into the dense genome sampling within the family to capture specific genomic features and investigate i) to what extent genes involved in plant biopolymer degradation have been maintained within a single, diverse, ECM lineage and ii) to examine functional diversity within this ecologically important clade, specifically focused on their ability to scavenge nitrogen sources from recalcitrant organic matter. Indeed, preliminary evidence suggests that members of this family, though being mutualists, have retained a restricted set of genes coding for lignin peroxidases and copper oxidoreductases which may be responsible for the degradation of lignin derivatives accumulating in soil organic matter.

**251T Genome sequencing reveals a heterothallic mating system in the Eucalyptus pathogen Chrysoporthe austroafricana.** A.M. Kanzi¹, B.D. Wingfield¹, E.T. Steenkamp², N.A. van der Merwe¹ 1) Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, Gauteng, ZA; 2) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, Gauteng, ZA.

*Chrysoporthe austroafricana* is a fungal pathogen that affects commercial forestry establishments of *Eucalyptus* species and native species of *Myrtales* in Southern Africa by causing deadly tankers. The mating system of *C. austroafricana* has not been characterized, but sexual structures have been observed on native *Syzygium* trees. Homothallism was proposed as the mating system in this species, but this hypothesis could not be fully tested due to the lack of mating-type information. Thus, genome sequence data of two *C. austroafricana* isolates and two close relatives, *C. cubensis* and *C. deuterocubensis*, which are also known to cause disease on *Myrtales*, were analyzed. A putative *MAT1-1* idiomorph was found in one of the *C. austroafricana* isolates and a putative *MAT1-2* idiomorph, containing a truncated *MAT1-1-1* gene, was observed in the other *C. austroafricana* isolate. The presence of a single idiomorph in each isolate suggests that this fungus is heterothallic. The ratio of *MAT1-2* to *MAT1-1* isolates in a South African population of *C. austroafricana* was 2:1. In contrast, the ratios of *MAT1-2* to *MAT1-1* in populations from neighbouring Southern African countries ranged from 1:1 to 1:6. The non-proportional ratios in some populations suggest that these populations may be the result of recent introductions. The sibling species *C. cubensis* and *C. deuterocubensis* are apparently homothallic, since the *MAT1* locus of these isolates included genes associated with both *MAT1-1* and *MAT1-2* idiomorphs.

**252F Comparative genomics to assess myco-parasitism and pathogenicity in understudied Oomycetes.** Ramesh R. Vetukuri¹, Kurt H. Lamour², Laura J. Grenville-Briggs¹ 1) Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden; 2) Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, USA.

The oomycetes are a lineage of filamentous Eukaryotes that resemble fungi, but are most closely related to the heterokont (brown) algae. Many oomycetes are plant pathogens including the new threat, *Phytophthora colocasiae* causing taro blight, however, some Pythium species are parasitic to fungi and other oomycetes (*Pythium oligandrum* and *Pythium periplocum*) whilst others such as *Lagenidium giganteum* are parasites of insects including mosquitos that vector Zika virus and Dengu fever. We have carried out *de novo* genome sequencing and genome-wide transcriptomics (RNA-seq) of four understudied oomycetes, *P. oligandrum*, *P. periplocum*, *L. giganteum* and *P. colocasiae* using the SciLifeLab Illumina Hiseq platform using both paired and mate pair sequencing. We are currently investigating the genetic and molecular determinants of host specificity in all the four sequenced oomycetes and mycoparasitism in the Pythium species. The sizes of the these four genomes range from 36 Mb to 100 Mb. RxLR class effectors were found in *P. colocasiae* but not in the three Pythium species. Crinkler (CRN), and elicitin proteins were detected in all the four oomycetes sequenced. Novel effector families are present in the insect pathogen *L. giganteum*. RNA-seq data from *P. oligandrum* myco-parasitising the potato late blight oomycete *Phytophthora infestans* shows specific up-regulation of 1940 *P. oligandrum* genes 12 hrs after infection and 300 genes at 24hrs. Some of these genes are involved in secondary metabolism and terpenoid biosynthesis, transport and cell wall degradation. Novel transcripts from *P. infestans* reveal how this oomycete defends itself against mycoparasitic attack and may provide vital clues to control potato late blight in a more durable way in the future. Comparative analyses of the hyperaggressive mycoparasite *P. oligandrum* versus the weaker mycoparasite *P. periplocum* are providing vital clues to the success of *P. oligandrum*. Identification of common and specific pathogenicity determinants and defence pathways opens the way for more sustainable oomycete control measures and better use of oomycetes as biological control agents.

**253W Pervasive Glyphosate Resistance in S. cerevisiae Lays Outside the Chorismate Pathway.** J. Gallagher, X. Rong-Mullins, C. Blackwood, K. McNeal, Z. Lonergan, J. Creamer, A. Biega, A. Ravishankar Dept Biology, West Virginia Univ, Morgantown, WV.

*S. cerevisiae* are associated with different crops and environments and are consequently subject to a wide range of selective pressures whether intentional or by happenstance. First marketed as RoundUp™, glyphosate is the most widely used herbicide. Glyphosate inhibits
the chorismate pathway which is present in many organisms but not in mammals. Agriculture-associated isolates of *S. cerevisiae* isolated during the last forty yeast had high glyphosate resistance. Despite sequence similarity between the target protein, the plant EPSPS and the yeast orthologous protein Aro1, and the prediction that yeast would be resistant to glyphosate, there was a wide dose range of yeast growth inhibition to a commercial preparation of glyphosate. Growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in a multiple-drug transporter and amino acid permease. Recently collected yeast from conventional and organic farms, urban and rural areas had glyphosate resistant yeast. However, in remote areas or areas only recently exposed, the number of glyphosate resistant yeast was significantly lower. Therefore, close association with challenging environments is changing the wild *S. cerevisiae* populations either through selection by directed application of glyphosate or other unforeseen selective pressures.

**254T Comparison of orthologous gene clusters and carbohydrate-active enzymes of *Ophiosphaerella* spp. the causal agents of spring dead spot of bermudagrass.** *N. Graf Grachet*¹, B. Couger², C. D. Garzon¹, S. Marek¹, N. Walker¹ 1) Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK; 2) High Performance Computing Center, Oklahoma State University, Stillwater, OK.

In the United States, turfgrasses and their maintenance are estimated to be a $40 billion industry. Bermudagrass (*Cynodon dactylon* and *C. dactylon* x *C. transvaalensis*) is a warm-season grass commonly cultivated as turfgrass in the southern US. Spring dead spot is considered the most destructive disease of bermudagrass in this region. Control of this disease requires high rates of fungicides. Spring dead spot is caused by three fungi: *Ophiosphaerella herpotricha*, *O. korrae*, and *O. narmari*. The fungi infect and colonize below ground organs of bermudagrass. Although in the same genus, the species vary in asci and melanin formation when cultured. In addition, the species differ in virulence, origin, and host range. The objective of this study was to examine genomic information of each species. Whole-genome of 11 isolates and total RNA of 6 isolates of *Ophiosphaerella* spp. were sequenced using Illumina HiSeq and PacBio SMRT technology. Average genome size was 47 Mb in *O. narmari*, 65 Mb in *O. herpotricha*, and 70 Mb in *O. korrae*. Number of protein coding genes varied from 12 to 14 thousand across all species. The difference in genome size was due to expansion of intergenic bases. Ortholog gene clustering in OrthoMCL revealed that approximately 85% of the protein coding genes were shared between the three *Ophiosphaerella* spp. The same analysis showed that *O. korrae* had more unique genes (638) than *O. narmari* (147) and *O. herpotricha* (77). Preliminary results of SignalP predicted an average of 1,000 signal peptides among all three species. *Ophiosphaerella* spp. shared similar abundances of the CAZymes. There was an expansion of auxiliary activities families 3, 4, and 11, and of carbohydrate esterases family 1. The majority of *Ophiosphaerella* spp. enzymes were homologs of plant-acting enzymes. It was hypothesized that these groups act on plant substrates such as lignin to facilitate root colonization.

**255F Genome sequence and its use in developmental, genetic and population studies of Shiitake mushroom *Lentinula edodes*.** *H.S. Kwan*¹,², *Yang Xiao*¹,³, *Lei Li*¹,², *Chun Hang Au*¹,², *Man Chun Wong*¹,², *Qianli Huang*¹,², *Xuanjin Cheng*¹,², *Man Kit Cheung*¹,², *Jinhui Chang*¹,², *Wenyan Nong*¹,² 1) School of Life Sciences, Chinese Univ of Hong Kong, Hong Kong, SAR; 2) Food Research Centre, Chinese Univ of Hong Kong, Hong Kong SAR; 3) Institute of Applied Mycology, Huazhong Agricultural University, 430070, Hubei Province, P. R. China.

The Shiitake mushroom *Lentinula edodes* is one of the most popular edible mushrooms in the world. We sequenced the genome of the *L. edodes* monokaryon L54A. Comparative analyses on genome sequences of basidiomycetes and ascomycetes revealed genes expanded in genomes of mushroom-forming fungi. Four functional categories, Signal transduction mechanisms [T], Posttranslational modification, protein turnover, chaperones [O], Transcription [K] and Carbohydrate transport and metabolism [G], dominate in the expanded families. We examined kinome, ubiquitome, transcription factories and CAZymes. AGC kinase subfamily, F-box and paracaspase domain-containing E3 like proteins are significantly expanded in mushroom-forming genomes. We also performed RNA-Seq of multiple stages and identified genes differentially expressed. Transcriptome age index (TAI) profile and transcriptome divergence index (TDI) profile showed a molecular hourglass pattern over the developmental stages. Young fruiting body stage was the "waist" of the hourglass that expressed the evolutionarily oldest transcriptome. The genome sequence is used to generate a high-density genetic map for Expression Qualitative Trait (eQTL) studies. In addition, the genome sequence also served as the reference sequence for analysis of wild and cultivated populations in China. Our works have generated rich resources for genomics, transcriptomics, and population studies of mushrooms.

**256W Genome sequence of the pathogenic white-rot fungus *Phellinus noxius* OVT-YTM/97 reveals insights on pathogenesis.** *K.S.W. Tsang*¹, *Y.C. Lam*², *W.Y. Nong*¹, *C.K. Wong*¹, *H.S. Kwan*¹ 1) The Chinese University of Hong Kong, Hong Kong SAR; 2) Muni Arborist Limited, Hong Kong SAR.

*Phellinus noxius* is a pathogenic white-rot fungus of the phylum Basidiomycota that causes brown root rot disease, common to pan-tropical and subtropical regions with a host range of over 200 plant species. The fungal pathogen causes irrevocable damage in plant hosts and if left untreated, can lead to swift deterioration of the host health within a year. In Hong Kong, occurrence of *P. noxius* infected trees has increased dramatically in the past several years and many of which are old and valuable with cultural or historical significance. Currently there is no cure for this disease, despite the development of several biocontrol methods and fungicides.

We have assembled the first genome sequence of *P. noxius* OVT-YTM/97, isolated from a root sample obtained from an infected *Ficus microcarpa* tree in Hong Kong. This strain was found to have a particularly aggressive pathogenicity and exhibited resistance towards biocontrol agents. The genome was sequenced on Illumina Mi-Seq and PacBio RSII platforms, followed by *de novo* assembly using the Celera assembler v8.3 and annotated.

A total of 9,957 protein-coding genes were predicted using GeneMark-ES and carbohydrate-active enzyme (CAZyme). Analyses using dbCAN v5 revealed 478 CAZymes and a strong resemblance to that of necrotrophic plant pathogens, with large proportions of glycoside hydrolases and carbohydrate esterase families 1 and 10, which are required for the complete breakdown of plant cell walls for infection. In addition, analyses using the Database of Fungal Virulence factors (DFVF) indicated an abundance of β (1-3)-glucan synthases and class I and II chitin synthases, which control cell wall synthesis and septa production, providing structural stability for the hyphae. The presence of these carbohydrate-active enzymes and virulence factors could explain the high virulence of the pathogen and its ability to cause catastrophic fatalities in infected trees. The results generated from this study allow us to have a deeper understanding of the mechanisms of
action and the hypervirulence of this pathogen. It also provides a basis for the development of prevention and treatment to protect trees from this pathogen.

**257T Comparative genomics integrated with association analysis identifies candidate effector genes corresponding to *Lr20* in phenotype-paired *Puccinia triticina* isolates from Australia.** *J.Q. Wu*¹, *S. Sakthikumar*², *C.A. Cuomo*², *R.F. Park*¹ 1) Plant Breeding Institute, Faculty of Agriculture and Environment, The University of Sydney, Narellan, NSW, Australia; 2) Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA 0214, USA.

Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, *P. triticina* (*Pt*). In the present study, 20 *Pt* isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for *Lr20*, were analysed using whole genome sequencing. Compared to the reference genome of the American *Pt* isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) per isolate was found and the proportion of heterozygous SNPs was around 87% in the majority of the samples, demonstrating a high level of polymorphism and a high rate of heterozygosity. From the genome-wide SNPs, a phylogenetic tree was inferred, which consisted of a large clade of 15 isolates representing diverse presumed clonal lineages including 14 closely related isolates and the more diverged isolate 670028 and a small clade of five isolates characterised by lower heterozygosity level. Principle component analysis detected 3 distinct clusters, corresponding exactly to the two major subsets of the small clade and the large clade comprising all 15 isolates without further separation of isolate 670028. While genome-wide association analysis identified 302 genes harbouring at least one SNP associated with *Lr20* virulence (p<0.05), a Wilcoxon rank sum test revealed that 36 and 68 genes had significant (p<0.05) and marginally significant (p<0.1) differences in the counts of non-synonymous mutations between *Lr20* avirulent and virulent groups, respectively. Twenty of these genes were predicted to have a signal peptide without a transmembrane segment, which were identified as candidate effector genes corresponding to *Lr20*. Future studies are thus warranted to investigate the biological functions of the candidate effectors as well as the gene regulation mechanisms at epigenetic and post-transcription levels. Our study is the first to integrate phenotype-genotype association with effector prediction in *Pt* genomes, an approach that may circumvent some of the technical difficulties in working with obligate rust fungi and accelerate avirulence gene identification.

**258F Blue light perception in the maize foliar pathogen, *Exserohilum turcicum*.** *J. Liversage*¹,², *S.H. Stoychev*³, *B.G. Crampton*¹,² 1) Department of Plant and Soil Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; 2) Genomics Research Institute, University of Pretoria, South Africa; 3) Council for Scientific and Industrial Research, Biosciences, Pretoria, South Africa.

Northern corn leaf blight (NCLB) is a devastating maize foliar disease in South Africa, with the causative agent being *Exserohilum turcicum*. To understand how the fungus infects maize and how this links to its photobiology is unexplored. Here we firstly set out to identify all the *E. turcicum* circadian clock proteins, which include the White collar-1 (blue light photoreceptor), White collar-2 (forms part of light responsive transcription factor) and Vivid (photoadaptation). Lastly, we aimed to identify and characterize an alternative blue light photoreceptor, CRY-DASH, the function of which remains unexplained in the circadian clock and pathogenicity. Local databases were set up of each of the genes from other fungi, after which a BLAST search against the sequenced genome of *Setoshearia turcica* ET28A v1.0. was performed. Retrieved gene sequences were analyzed for correct domain architecture and there after qPCR primer sets were designed. *E. turcicum* cultures were grown in constant darkness and flash frozen following light pulses of 15, 30 and 60 min as well as a 24 hr pulse. Following, RNA extraction and cDNA synthesis the light response profile of the clock genes were generated. The function of CRY-DASH was determined through producing a knock-out via Agrobacterium-mediated transformation and the generation of a Green Fluorescent CRY-DASH fusion protein. All the classical clock genes are present in the genome sequence of *E. turcicum*. The traditional light expression pattern of these genes differ from that in literature. Cry-DASH is a promising alternative blue light photoreceptor in fungi.

**259W Developing durable disease control strategies for rice blast disease in Sub-Saharan Africa by genetic and genomic analysis of *Magnaporthe oryzae* populations.** *V.M. Were*¹, *D. Mwongera*¹, *S. Mutiga*²,⁴, *F. Rotich*⁴, *M.J. Kershaw*¹, *D.M. Soanes*¹, *G.R. Littlejohn*¹, *X. Yan*¹, *B. Zhou*³, *J.W. Harvey*², *I. Ouédraogo*⁶, *G-L. Wang*⁵, *T. Mitchell*⁵, *J. Correll*⁴, *N.J. Talbot*¹ 1) Biosciences, University of Exeter, UK; 2) BecA-ILRI, Nairobi, Kenya; 3) International Rice Research Institute, Los Baños, The Philippines; 4) Department of Plant Pathology, University of Arkansas, USA; 5) Department of Plant Pathology, Ohio State University, Columbus, Ohio; 6) Institute of Environment and Agricultural Research, Ouagadougou, Burkina Faso.

The rice blast fungus, *Magnaporthe oryzae*, causes the most serious disease of cultivated rice. We are characterising effector proteins from *M. oryzae* and using this knowledge to develop new strategies for durable rice blast disease control in Sub-Saharan Africa (SSA). We have collected more than 500 rice blast isolates from 9 countries and carried out extensive pathotype analysis on monogenic rice lines carrying 24 rice blast resistance genes *Pia*, *Pib*, *Pik*, *Pik-h*, *Pi-km*, *Pik-p*, *Pik-s*, *Pish*, *Pit*, *Pita*, *Pita-2*, *Piz*, *Piz-5*, *Piz-t*, *Pi1*, *Pii*, *Pi3*, *Pi5* (t), *Pi7* (t), *Pi9*, *Pi11* (t), *Pi12* (t), *Pi19* (t), and *Pi20*. We have used this analysis to identify gene combinations that might lead to durable resistance. We have found that *Pi-9*, *Pita-2*, *Pi-12*, *Piz* and *Pi-z5* offer the greatest possibility for exclusion of the prevailing rice blast population. We have begun to introgress these genes and other potential sources of resistance into high yielding locally adapted rice cultivars. To understand the relationship between the effector repertoire of *M. oryzae* and their potential recognition as avirulence determinants in blast populations in SSA, we have sequenced the genomes of 21 *M. oryzae* isolates. We have combined this comparative genome analysis with RNA-seq analysis of rice blast infections to identify a set of putative avirulence gene candidates. We have characterised allelic variation at these loci and then carried out targeted gene deletion and complementation analysis to determine their functions. Progress towards the characterisation of these effectors and their use to guide future rice breeding programmes in Africa will be reported.

**260T MagNet: the integrated gene network of the rice blast fungus *Magnaporthe oryzae*.** *H. Kim*¹, *K. Cheong*¹, *K.T. Kim*², *J. Jeon*¹, *G. Choi*¹, *Y.H. Lee*¹,²,³ 1) Interdisciplinary Program in Agricultural Genomics, Seoul National University, Seoul 08826, Korea; 2) Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea; 3) Center for Fungal Genetic Resources, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

*Magnaporthe oryzae*, the rice blast fungus, plays a role as a model organism in the area of molecular plant-microbe interaction research.
Studies on pathogenic mechanism of this fungus revealed many genes in signaling pathways are essential for appressorium formation and penetration. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological process during the pathogenesis of *M. oryzae*. Identifying genome-wide protein-protein interaction (PPI) network is one of omics-level approaches which can help to understand signaling and regulatory pathways. However, existing biological network resources of *M. oryzae* are not sufficient to decipher molecular mechanisms of pathogenesis. In this study, PPI network analysis platform of *M. oryzae*, MagNet, was constructed with three methods: homology-based 'Interolog' search, co-expression network construction, and domain-domain interaction based prediction. Interologs within *M. oryzae* proteins which have orthologues in model species were predicted with six PPI repositories. Co-expression networks were built with RNA sequencing data from the infection stages and those from the vegetative stage. In addition, we gathered the information of domain-domain interactions (DDIs) from two integrated DDI databases, and predicted interactions between domain-annotated proteins. With three approaches all together, the pan-network with 6,600,976 interactions was generated including highly confident 215,731 interactions found in more than three sub-networks. Experimental data on *M. oryzae* PPIs support that our highly confident PPI network can predict PPIs with higher sensitivity (89.65%) and specificity (78.57%) compared to the previously constructed databases. MagNet would provide integrated biological network data which can help to understand the molecular mechanisms of the rice blast fungus and beyond.

**261F Global proteome analysis of *Magnaporthe oryzae* during nitrogen starvation.** Yeonyee Oh¹,²,⁵, Suzanne Robertson³,⁴,⁵, Jennifer Parker³,⁴,⁵, David Muddiman³,⁴,⁵, Ralph Dean¹,²,⁵ 1) Center for Integrated Fungal Research; 2) Department of Entomology and Plant Pathology; 3) W. M. Keck FT-ICR Mass Spectrometry Laboratory; 4) Department of Chemistry; 5) North Carolina State University, Raleigh, NC.

Fungi are constantly exposed to nitrogen limiting environments and the efficient regulation of nitrogen metabolism is essential for fungal survival, growth, development and pathogenicity. To understand how the rice blast pathogen, *Magnaporthe oryzae* deals with nitrogen shortage, we performed global proteome analyses. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified a total of 5,498 *M. oryzae* proteins during nitrogen starved or non-starved conditions. Comparative analysis showed 363 proteins were significantly induced or uniquely expressed under the nitrogen starved condition and 266 proteins under non-starved condition. A functional analysis of differentially expressed proteins suggested major biological pathway changes during nitrogen starvation including nitrogen repression, melanin biosynthesis, protein degradation and protein translation. Nitrogen starvation induced accumulation of various extracellular proteins including a number of small extracellular proteins suggesting a link between nitrogen starvation and pathogenicity in *M. oryzae*. The results from this study provide a better understanding of fungal response to nitrogen availability.

**262W Genome and Transcriptome Analysis of *Phellinus linteus* for a Biosynthetic Pathway for Hispidin Compounds.** D. Pathiraja, H. Park, B. Min, K. Jang, J. Byun, I. Choi Korea University, Seoul, KR.

*Phellinus linteus* is a medicinal mushroom used as traditional medicine in East Asia. *Phellinus linteus* has been studied for various favorable pharmacological effects such as anti-inflammatory, anti-allergic, anticancer, immunomodulatory and anti-oxidant effects. These effects were found to be associated with bioactive secondary metabolites of *Phellinus linteus*. We carried out genome and transcriptome sequencing to identify and characterize those metabolic pathways. Here, we report a comparative genomics of *Phellinus linteus* strains known as 'Changsu' and 'Goryo' in South Korea. Genome sequencing of two medicinal mushrooms was performed in the Illumina MiSeq platform. The size of *P. linteus* Changsu was estimated as 40.6 Mb, whereas *P. linteus* Goryo was estimated as 31.9 Mb. Unique genetic patterns of these two genomes were surveyed by comparing to well-studied mushroom genomes: *Ganoderma lucidum*, *Agaricus bisporus*, and *Laccaria bicolor*. We have identified several important secondary metabolic pathways in these genomes. Among them, the gene cluster responsible for the biosynthesis of Hispidin, a well-known anti-oxidant, was predicted in both Changsu and Goryo strains.

**263T Genomics of mycorrhizal fungi.** S. Unruh¹, F. Martin², L. Zettler³, S. Perotto⁴, C. Hudson⁵, G. Conant¹, J.C. Pires¹ 1) Biological Sciences, University of Missouri, Columbia, MO; 2) Institut National de la Recherche Agronomique (INRA), Nancy, France; 3) Department of Biology, Illinois College, Jacksonville, IL; 4) University of Turin, Italy; 5) Systems Biology Research Group, Sandia National Laboratories, Livermore, CA.

Orchids have a unique relationship with a certain subset of fungi in that the seeds of these plants rely on nutrients from fungi to germinate and develop. The fungi involved are not all in other more typical mycorrhizal interactions, so the criteria for what makes a fungus an orchid mycorrhizal fungus is a mystery. With a Community Sequencing Project, my team is attempting to better understand the genomes of various orchid mycorrhizal fungi, their relationships to each other, and what genes are expressed solely in the symbiosis. To do this, we are sequencing whole genomes of 15 fungi, shallowly sequencing up to 400 fungal isolates to perform phylogenetic analysis, and performing RNAseq on orchid protocorms infected with mycorrhizal fungi.

The whole genome sequencing is underway, the shallow sequencing is also in progress and new barcodes are being developed that are more useful in this group than ITS. The RNAseq experiment will start shortly after the fungal genetics conference.

**264F Understanding the evolution and functioning of symbiosis : *Pisolithus sp.* as model.** A. Kohler¹, M. de Freitas Pereira¹, E. Morin¹, V. Singan², A. Kuo², J.M. Plett³, M.D. Costa⁴, I. Grigoriev², F. Martin¹ 1) UMR IAM, LABEX ARBRE, INRA Centre de Nancy, Champenoux, Lorraine, FR; 2) US Department of Energy, Joint Genome Institute (JGI), Walnut Creek, California, USA; 3) Hawkesbury Institute for the Environment, University of Western Sydney, Richmond NSW, Australia; 4) Departamento de Microbiologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil.

All ectomycorrhizal fungi sequenced so far exhibit a reduced set of genes encoding plant cell wall-degrading enzymes (PCWDE) compared to their ancestral wood decayers and the analysis of mycorrhizal transcriptomes revealed the involvement of both conserved and clade-specific genes. Induced are genes coding for the same functions but without orthology, like (often clade-specific) small-secreted proteins, transporters, redox metabolism or carbohydrate active enzymes, suggesting a convergent evolution. These findings are also true for two *Pisolithus* genomes, *Pisolithus tinctorius* and *P. microcarpus*, sequenced in the frame of the JGI Mycorrhizal Genomics Initiative (MGI). Both species are frequent fungi in forest ecosystems but colonize different host trees, pine and eucalypt, respectively. Comparison of the *Pisolithus* mycorrhizal transcriptomes revealed a set of small-secreted proteins induced in interaction with their hosts that are conserved
between the two genomes but expanded in gene copy number compared to other genomes.

Further, a *Pisolithus microcarpus* monocaryotic strain collection isolated from the same fruiting body showed striking differences in the ability to form mycorrhiza. Genome resequencing and gene expression analyses during mycorrhiza formation allowed further insights into genetic differences within this *Pisolithus* progeny.

**265W Extensive genome diversity among isolates of a model arbuscular mycorrhizal fungus.** *E.Chun-Hung. Chen¹, S.*

Ndikumana¹, E. Morin², F. Martin², J. Noel¹, D. Beaudet¹, J. Giorgi¹, N. Corradi¹ 1) Biology, University of Ottawa, Ottawa, Ontario, CA; 2) UMR 1136, INRA-Nancy Université, Interactions Arbres/Microorganismes, Champenoux, FR.

Arbuscular mycorrhizal fungi (AMF) are best known for their beneficial effects on the health of many land plants and their surrounding ecosystem through establishment of the mycorrhizal symbiosis. It has been proposed that that intra-specific diversity is an essential component of a successful symbiosis with the plant host. Recent evidence of a mating-driven processes in a model AMF has revealed some of the mechanisms involved in creating intra-specific diversity in this group, but how the genome content and structure varies within one species is still poorly understood. To address this question, we compared the genome sequences of five isolates of previously proposed to be part of the *Rhizophagus irregularis* clade. Our analyses show that inter-isolate genome diversity is present at all levels, and includes large amounts of isolate-specific genes, sequence and gene copy number polymorphisms and inter-isolate rearrangements indicative of inter-isolate recombination. Plasticity affects all biological functions, leads to re-interpretations of population-based, and underscores potential problems with the species concept in these widespread plant symbionts.

**266T PhytoPath: harnessing community expertise to fight plant disease.** *Helder Pedro¹, Uma Maheswari¹, Nishadi De Silva¹, Paul Kersey¹, Kim Hammond-Kosack², Alayne Cuzick², Martin Urban² 1) EMBL-EBI, Hinxton, Cambridge, GB; 2) Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK; 3) Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ.*

Plant pathogens continue to threaten food security and have a significant economic impact. For instance, it is estimated that late blight of potato and tomato (caused by *Phytophthora infestans*) incur annual global losses in excess of US$6.7 billion. With burgeoning populations, maintaining (and increasing) the yields of crop plants through pesticide research and plant breeding has never before being more important. For this, an understanding of gene function and host-pathogen interactions is critical, and accurate annotation of genomic sequence is important to both enable and disseminate this knowledge.

PhytoPath (www.phytopathdb.org), established in 2010, is a bioinformatics resource for genome scale data for important fungal and protist plant pathogens. It integrates resources from the Ensembl Genomes and PHI-base databases to organise genome sequence data, genetic variation, comparative analyses and phenotypic data to facilitate research on plant pathogenesis. Currently PhytoPath houses over 250 genomes from 115 plant pathogen species, from which over 1800 genes have been associated with disease phenotypes. We run automated pipelines at regular intervals to acquire and analyse new fungal and protist genomes deposited in the public data archives, and provide visualisations using the Ensembl software suite and numerous routes for programmatic data access.

Furthermore, we facilitate the refinement of genome annotations by capturing manual community curation through WebApollo, an online gene editing tool. In 2015, we trained close to 50 species experts from the *Botrytis cinerea* community who systematically reviewed and revised the entire gene set for this organism. Following a thorough QC process, these updates have been integrated into subsequent releases of PhytoPath and Ensembl Genomes and made available to the wider research community. Following this successful initiative, we are currently in the process of engaging with the communities for *Zymoseptoria tritici*, *Blumeria graminis* and *Fusarium culmorum* and expect to commence curation of these species in the near future.

**267F HAP3 of *Phomopsis longicolla* influences growth, development, and pathogenesis.** *M. Zaccaron, W Hawkins, B Dhillon, Y Ramegowda, J Ridenour, B Bluhm* Plant Pathology, University of Arkansas, Fayetteville, AR.

*Phomopsis longicolla* (Hobbs), the most important soybean seed pathogen, is the causal agent of *Phomopsis* seed decay. Currently, little is known about the molecular basis of pathogenesis in *P. longicolla*, in part because crucial tools for molecular genetics, such as targeted gene deletion, have not been demonstrated in this organism. In other filamentous fungi, the heterotrimeric CCAAT-binding complex is involved in diverse aspects of growth and development, including secondary metabolism, morphogenesis, and pathogenesis. In this study, a putative component of the CCAAT-binding complex (HAP3) was identified in *P. longicolla* and characterized through functional genomics. With agrobacterium-mediated transformation, *HAP3* gene was successfully deleted via homologous recombination, and the mutant was genetically complemented via reintroduction of the wild-type gene. Deletion of *HAP3* substantially impaired growth and development as well as the ability of *P. longicolla* to colonize soybean seeds and stems. A RNAseq experiment reviewed massive reprogramming in a ?*hap3* strain (H101) transcriptome while colonizing soybean seeds. A total of 2080 upregulated and 1732 downregulated genes were found in H101, accounting, respectively, for 12 and 14% of *P. longicolla* predicted protein coding genes (adjusted *P* <0.05). This study reviews the important role of the *HAP3* component of the CCAAT-binding complex in growth, development and pathogenesis of *P. longicolla*. More importantly, our work illustrates the feasibility of targeted gene deletion in *P. longicolla*. Phenotypic similarities between *HAP3* deletion mutants of *P. longicolla* and other plant pathogenic fungi potentially indicate broad involvement of the CCAAT-binding complex in plant pathogenesis.

**268W DNA methylation and chromatin architecture contribute to pathogenic fungal genome organization and adaptation.** *David E. Cook¹,², Michael F. Seidl¹, Martin Kramer¹, Bart PHJ Thomma¹ 1) Laboratory of Plant Pathology, Wageningen University, Wageningen, Netherlands; 2) Department of Plant Pathology, Kansas State University, Manhattan, KS USA.*

Fungal pathogens have evolved diverse strategies to overcome host immunity. During fungal-plant interactions, invading fungi utilize secreted proteins, termed effectors, to avoid or subvert the plant immune response through varied biochemical mechanisms. Effector genes are not randomly distributed across a genome, but often reside in polymorphic regions of the genome, clustering with repetitive DNA. Despite the ubiquity and importance of fungal effectors, our mechanistic understanding of their transcriptional regulation and genome organization remains inadequate. As such, we are addressing two key questions 1) How are *in planta* effectors transcriptionally regulated? 2) How does repetitive DNA contribute to the evolution of highly variable genomic regions that contribute to fungal virulence? Using a variety of genetic and computational approaches, we are characterizing how DNA modifications and chromatin structure (the organization of
DNA in a cell) contribute to the evolution of virulence using the soil-borne fungal pathogen *Verticillium dahliae*. The genome of *V. dahliae* is predicted to express numerous homologs of known DNA and chromatin modifying proteins, including three putative DNA methyltransferases. We have identified that a single DNA methyltransferase controls a significant portion of the observed DNA methylation at repetitive DNA. Interestingly, repetitive DNA arising from recent segmental genome duplications are devoid of DNA methylation and are more transcriptionally active relative to repetitive DNA at other loci. Additionally, we are assaying the genome for open chromatin to develop a comprehensive view of how gene regulation and chromatin architecture impacts the evolution of fungal virulence.

**269T A virulence interactome of the wheat pathogen Fusarium graminearum.** *A. Mirmiran*¹,², *C. Tsai*¹, *C. Mogg*¹,², *G. Subramaniam*¹,², *D. Desveaux*¹,³ ¹) University of Toronto, Toronto, ON, Canada; ²) Agriculture and Agri-Food Canada, Ottawa, ON, Canada; ³) Centre for the Analysis of Genome Evolution and Function, Toronto, ON, Canada.

Fusarium Head Blight (FHB) is a disease affecting wheat (*Triticum aestivum*) and other cereal crops worldwide resulting in the loss of over $3 billion in the US alone since the 1990s. This disease caused by the fungal pathogen *Fusarium graminearum* results in the loss of yield, quality, and food safety in wheat due to its production of trichothecene mycotoxin. The trichothecene, deoxynivalenol (DON), is responsible for both the spread of the disease within the host as well as the inhibition of protein synthesis when consumed resulting in symptoms such as anorexia, vomiting, and immune response alteration. As the primary agent responsible for FHB in wheat, *F. graminearum* forms its toxin while under specific environmental conditions such as low pH, light, and nutrient-limited environments. It has been established that the trichothecene biosynthetic pathway is expressed through the TRI gene cluster Expression of this pathway is regulated by two particular members of the TRI family: TRI6 and TRI10. Studies have shown that disruption of either regulator results in complete suppression of DON production rendering the fungus non-pathogenic. Our interests lie in further exploring the regulation of DON synthesis to uncover novel regulatory pathways that contribute to mycotoxin production and *F. graminearum* virulence. To do so, we have created a protein-protein interaction map of genes that are co-regulated during DON production. We will discuss the hypotheses we have generated from our preliminary analysis of this network and our efforts to functionally test them.

**270F Characterization of a 26S protease ortholog, CpPts1, from Cryphonectria parasitica in response to several stress.** *Yo-Han Ko*¹, *Jung-Mi Kim*², *Jyotiranjan Bal*¹, *Kum-Kang So*¹, *JeeSun Chun*¹, *Seung-Moon Park*³, *Moon-Sik Yang*¹, *Dae-Hyuk Kim*¹ ¹) Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea; ²) Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, Korea; ³) Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570-752, Republic of Korea.

The chestnut blight fungus, *Cryphonectria parasitica*, and its hypovirus comprise an useful model system to study the mechanisms of hypoviral infection. Pathoproteomic analysis revealed a protein spot, showing upregulation in the presence of Cryphonectria hypovirus1 (CHV1) and tannic acid supplementation. The corresponding protein spot was identified as the 26S protease homologue of *Cryphonectria parasitica* by tandem mass analysis. Sequence analysis of cDNA revealed that, it comprised of four exons, and three introns. It also contained a canonical AAA (ATPase associated with various activities) domain, consisting of Walker A motif and Walker B motif, which are involved in binding and hydrolysis of ATP. The encoded protein had a predicted mass of 51.8 kDa and a pI of 5.05. Sequence similarity and phylogenetic analysis indicated that the cloned gene encodes a homolog of the 26S protease of *C. parasitica* and refers as *CpPts1*. Northern blot analysis confirmed that the transcript of the cloned gene was upregulated by CHV1. Functional analysis of the cloned gene was conducted using gene deletion analysis. We obtained a *CpPts1*-null mutant among 192 candidates. The *CpPts1*-null mutant showed similar phenotype as that of wild type strain on PDAmb media. However, when the *CpPts1*-null mutant was challenged by the CHV1 infection, dramatic changes in colonial growth and morphology were observed.

**271W Comparative Phosphoproteomics of the Pga1-mediated heterotrimeric G protein signaling pathway in Penicillium chrysogenum.** *U. Carrasco-Navarro*¹, *R. O. García-Rico*², *R. Vera-Estrella*³, *B. J. Barkla*⁴, *F. Fierro*¹ ¹) Departamento de Biotecnología, Universidad Autónoma Metropolitana, Unidad Iztapalapa, México; ²) Departamento de Microbiología, Universidad de Pamplona, Colombia; ³) Instituto de Biotecnología, Universidad Nacional Autónoma de México, México; ⁴) Southern Cross Plant Science, Southern Cross University, Australia.

**Introduction.** The *Penicillium chrysogenum* heterotrimeric Gα protein Pga1 regulates several cellular processes and the biosynthesis of penicillin (1, 2). We have recently performed a proteomic analysis of the Pga1-mediated signaling pathway, finding two phosphorylated proteins as effectors of the pathway (3). Protein phosphorylation plays an important role in signal transduction pathways. To advance in the characterization of the Pga1 signaling pathway we have performed a comparative phosphoproteomic analysis using mutant strains with different levels of Pga1 activity and varying cAMP intracellular concentrations, aiming to find changes in the phosphorylation patterns of proteins between strains in order to identify new effectors and gain insight in the functioning of this signaling pathway. **Methods:** Two different methodologies were used: 2D-DIGE with phosphoprotein-enriched extracts followed by identification of individual spots by LC-MS/MS, and a *Label-Free* phosphoproteomics approach with phosphopeptide-enriched extracts (using TiO₂ affinity chromatography) submitted to LC-MS/MS. Three strains were used: Wis54-1255, *Δpga1* and G42Rpyr-T, plus a fourth condition: *Δpga1+cAMP* (increased cAMP intracellular concentration). **Results:** The 2D-DIGE analysis showed changes in abundance of phosphorylated proteins when comparing the strains, but little information on differential phosphorylation. The *Label-Free* phosphoproteomic analysis showed changes in the phosphorylation patterns of proteins between strains, which allowed to identify new effectors of the Pga1 signaling pathway. One of these proteins was Pc16g10400 (protein with a Bromo Adjacent Homology domain and a PHD zinc finger domain), with two phosphorylation sites at S-738 and S-742, of which the S-742 site is present only in the *Δpga1* strain. **Conclusions:** New effectors of the Pga1 signaling pathway were identified by comparative phosphoproteomic analysis. **References:** 1) *Res Microbiol.* 2007;158(5):437-46. 2) *Microbiology.* 2008;154:3567-78. 3) *Microb Cell Fact.* 2016 Oct 6;15(1):173.
**272T Proteome adaptations during growth on beech wood of the early sapwood decay fungi *Trametes versicolor* and *Schizophyllum commune*.** Amjad Zia, Andrzej Majcherczyk, Ursula Kües Molecular Wood Biotechnology, University of Göttingen, Göttingen, DE.

*Trametes versicolor* with 14,296 predicted genes in its genome is an aggressive white rot wood degrader. It reduced beech wood weight within 90 days by 51% in dark, by 43% in light and by 47% in a 12 h dark/12 h light scheme. Light conditions had effects on the fungal morphology. The surface mycelium in light and in light/dark was massive and primordial fruiting body structures were regularly formed. In dark, surface mycelium was sparse without any aggregated structures. Therefore, distinct extra- and intracellular proteomes are expected to be produced under different illumination schemes. In first attempts to unravel the secretome of *T. versicolor* by LC-MS, extracellular proteins were isolated by different protocols from wood samples incubated in dark. Results distinguished in the number of isolated secreted proteins (between 181 and 224) and contaminations by intracellular proteins (between 77 and 414). In total, 142 secreted proteins were common in all extractions. Among the extracellular proteins identified so far from *T. versicolor* are ligninolytic enzymes (laccase, peroxidase; 6%), enzymes acting in carbohydrate metabolism (e.g. carbohydrate esterase, glycoside hydrolase, glucose dehydrogenase; 44%), cell wall/membrane biogenesis proteins (lysophospholipase, acetylcholinesterase; 4%), protein degrading enzymes (e.g. serine protease, peptide hydrolase, aspartyl peptidase, carboxypeptidase; 14%), phosphatases (acid phosphatase, phosphoglycerate mutase; 3%), and a few enzymes of other functions (6%). 23% of the identified secreted proteins have no known function. *T. versicolor* is in nature often found on wood together with *S. commune*. This fungus is positioned in between white and brown rot and is depicted as weak wood degrader (weight losses of beech wood within 90 days: 4% in dark, 3% in light and in a 12 h dark/12 h light scheme). Surface mycelium of *S. commune* on wood under all conditions was thin and there were no fruiting bodies. The fungus (13,210 predicted genes) is equipped with restricted groups of lignocellulolytic genes. Further experiments will target on production of proteins from early to late wood decay of both species alone and in dual culture in order to study the possible types of interactions that occur between the fungi.
Our work is supported by the Ministry of Lower Saxony by the MaFoHolz PhD program and by DFG funding for a TripleTOF 5600 mass-spectrometer (DFG-GZ: INST 186/1085-1 FUGG).

**273F Using meta-transcriptomics to dissect the *Ocimum basilicum* and *Peronospora belbahrii* interaction.** G. Delulio¹, R Pyne², J Simon², L Ma¹ 1) UMass Amherst, Amherst, MA; 2) Rutgers University, New Brunswick, NJ.

The production of sweet basil (*Ocimum basilicum*) is threatened by the Oomycete downy mildew pathogen *Peronospora belbahrii*. However, economically viable cultivars lack usable resistance traits while some economically inviable cultivars display resistance. Through a comprehensive genetic breeding program, a resistant cultivar MRI showing resistance to *P. belbahrii* was selected. A previous genetic study suggested the presence of two potential resistance genes in MRI. This research dissects the *Ocimum basilicum* and *P. belbahrii* interaction to identify these potential resistant genes. We will compare the meta-transcriptomics of *P. belbahrii* infecting the resistant cultivar MRI with *P. belbahrii* infecting a closely related but susceptible sweet basil cultivar SB22. We have sequenced total mRNA of *P. belbahrii* infected tissue from cultivars MRI and SB22 at four times over the course of 72 hours post inoculation. Using meta-transcriptomics we will identify candidate basil resistance genes *P. belbahrii* resistant cultivar MRI for further downstream characterization.

**274W Evolution of Plant Penetration by Fungal Pathogens.** Cristina Miguel Rojas¹, Zheng Wang², Jeffrey P. Townsend², Frances Trail¹,³ 1) Department of Plant Biology, Michigan State University, 612 Wilson Rd, East Lansing, MI, 44824; 2) Department of Biostatistics, Yale School of Public Health, Yale University, 135 College Street, New Haven, CT 06520-8106; 3) Department of Plant, Soil and Microbial Sciences, Michigan State University, 612 Wilson Rd, East Lansing, MI, 44824.

Fungal spores are responsible for initiation and propagation of the majority of biotic plant diseases. Although spore germination is the first step in most fungal diseases, the genetics of spore germination has never been comparatively explored across multiple fungal lineages. Here we use comparative transcriptomics of spore germination among five fungal plant pathogens and one saprotroph representing two fungal classes, to determine how expression of orthologous genes has changed during evolution, and to predict genes whose knockouts will exhibit phenotypic differences in the spore germination and host penetration processes. We have chosen fungi which represent different approaches to plant penetration. *C. graminicola* and *M. oryzae* form melanized appressoria, while *F. graminearum* penetrates through natural openings associated with flowers, lacking melanization. Finally, *V. inaequalis* produces an intermediate form, with a melanized ring at the base of the appressorium. To provide a basis for comparison among species and to identify infection-specific expression patterns, we compare transcriptional profiles during germination on a single defined medium (saprotrophic conditions) as well as differences during germination on hosts (plant pathogens) and natural substrates (saprotroph). We will estimate ancestral gene expression for orthologous genes common among all species to identify genes that undergo transcriptional shifts during the spore germination process, as well as those that are unique to infective germination, and those that are unique to specific fungi. Lastly, functional assays of a subset of genes exhibiting species-specific and infection-type specific upregulation will be performed to assess the roles of these genes in conidial germination in these fungi. These experiments contribute to our understanding of how shifts in gene expression drive the evolution of conidial germination in a wide range of fungi.

**275T Comparative genomics of *Ashbya gossypii* and *Saccharomyces cerevisiae*.** Fred S. Dietrich, Jonathan Grego, Andrew Wilcox MGM, Duke Univ, Durham, NC.

The hyphal fungus *Ashbya gossypii* is a member of the Saccharomycetaceae. While it is closely related to *S. cerevisiae* it has an unduplicated genome that is overall about 3Mb smaller than *S. cerevisiae*. While *S. cerevisiae* lives in a wide range of environments, *A. gossypii* lives only in the gut of specific insects, the Milkweed bug and members of the genus *Dysdercus*. In comparison of the gene sets between these species *A. gossypii* share a core set of genes, but there are many additional genes found in *S. cerevisiae*. We are using this comparison to identify genes of novel function including a putative epoxide hydrolase of bacterial origin in some strains of *S. cerevisiae*, and a 5'-oxoprolinase gene of bacterial origin in other *S. cerevisiae* strains. We will be discussing what is the core set of genes and what are the variable genes in these organisms, and the mechanisms by which additional genes are acquired in these fungi. These comparisons take advantage of the genome sequences species related to *A. gossypii*, including *A. aceri*, *Holleya sinecauda*, *Eremothecium coryli*, and *Eremothecium cymbalariae*, as well as the tremendous diversity seen among the more than 100 *S. cerevisiae* genomes that have been sequenced.
**276F Evolution of secondary metabolism and host association in insect pathogens.** *Kathryn E. Bushley*¹, *Stephen A. Rehner*², *Joseph W. Spatafora*³ 1) Plant and Microbial Biology, University of Minnesota, Saint Paul, MN; 2) Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, MD; 3) Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR.

Fungal secondary metabolite genes including nonribosomal peptide synthetases, polyketide synthases, terpenes, and alkaloids are among the most rapidly evolving of fungal genes. They respond to selective pressures in the environment, often enabling fungi to adapt to specific environments or parasitize specific hosts. We investigate population genomic variation of secondary metabolite genes and clusters in two insect pathogenic fungi, the beetle pathogen *Tolypocladium inflatum* and the wide host-range insect pathogen and biocontrol agent *Beauveria bassiana*. Using Pac Bio single molecule real time sequencing, we improved the resolution of the sequenced reference strain *T. inflatum* NRRL 8044 and have assembled de novo genomes of additional strains isolated from distinct environments. For the reference strain and one additional strain, we have also used a Hi-C chromosome mapping approach to provide evidence for chromosomal level assemblies. The nearly complete chromosomal assemblies produced using these methods have allowed investigation of fine-scale evolutionary genetic mechanisms contributing to the rapid evolution of secondary metabolite genes and clusters and to evaluate the relative contributions of transposition, duplication/deletion, cluster rearrangement, and horizontal transfer in cluster evolution. Similarly, using Illumina technology, we have sequenced ten strains of *B. bassiana* and several outgroup *Beauveria* species (*B. brongniartii*, *B. asiatica*, *B. australis*) to analyze the evolution of secondary metabolite clusters and other genes involved in host-specific interactions (e.g. G-protein coupled receptors, SSPs). In addition to being an insect pathogen, *B. bassiana* has recently been shown to colonize a wide-range of plant species as an endophyte. Differential expression in plant and insect infection assays examines the role(s) of specific metabolites and other genes in recognition, colonization, and pathogenesis of plant versus insect hosts. The results of our analysis shed light on the role of metabolites in shaping the interaction of these fungi with distinct hosts and in turn on the effects of host-specialization versus generalism in driving the evolution of metabolite clusters.

**277W Identifying more than 300 biosynthetic gene clusters with potential resistance genes in over 75 Aspergillus species using resistance gene-guided genome mining.** *I. Kjaerboelling*¹, *T.C. Vesth*¹, *J.C. Frisvad*¹, *J.L. Nybo*¹, *S. Theobald*¹, *I.V. Grigoriev*², *A. Salamov*², *T.O. Larsen*¹, *U.H. Mortensen*¹, *M.R. Andersen*¹ 1) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, DK; 2) Joint Genome Institute, Walnut Creek, CA, USA.

With the increasing number of genome sequences, an increasing number of orphan /cryptic secondary metabolite biosynthetic gene clusters (BCGs) has followed. These clusters potentially encode pathways for bioactives, however linking genes to compound and elucidating bioactivity requires extensive work. Thus, the question remains how to select the most interesting clusters in a rational manner and find their bioactivity. By using Resistance Gene-Guided Genome mining, it is possible to identify novel bioactive compounds and their clusters.

The hypothesis for this approach is that some BGCs include a gene which is a resistant form of the enzyme targeted by the compound produced by the cluster. In filamentous fungi, this mechanism has been seen for mycophenolic acid as well as Fellutamide B. By using this knowledge and a large number of fungal genomes, we have set up a pipeline, which extracts predicted clusters with potential resistance genes.

A total of over 300 clusters were found to exhibit this specific pattern by running our algorithm on more than 75 *Aspergillus* species and 5000 (SMURF) predicted gene clusters. With further filtering and analysis, we ended up with a manageable number of potential resistance genes and clusters. The filters used in this algorithm will return clusters where one gene in the cluster has a homolog conserved in 98% of the examined species. Although we use a conservative approach it ensures that the identified clusters are most likely of interest. Selected clusters and resistance genes are now being validated experimentally both to confirm the resistance mechanism and to identify the compounds produced.

**278T Genus level analysis of secondary metabolism reveals the origin of hybrid NRPS-PKS gene clusters.** *S. Theobald*¹, *T.C. Vesth*¹, *J.L. Nybo*¹, *I. Kjærboelling*¹, *J.C. Frisvad*¹, *K.F. Nielsen*¹, *T.O. Larsen*¹, *I.V. Grigoriev*², *A. Salamov*², *U.H. Mortensen*¹, *S.E. Baker*², *M.R. Andersen*¹ 1) Department of Biotechnology and Biomedicine, Technical University of Denmark, Lingby, DK; 2) Joint Bioenergy Institute, Berkeley, CA, USA; 3) Joint Genome Institute, Walnut Creek, CA, USA.

The World Health Organization is reporting a rising number of multiple drug resistant pathogens every year, increasing the need for new drug development. However, current methods for natural product discovery rely on time consuming experimental work, making them unable to keep up with this demand.

In the Aspergillus genus sequencing project, we are sequencing and analyzing over 300 species of Aspergilli, a group of filamentous fungi rich in natural compounds. Natural products are encoded by genes located in close proximity, called secondary metabolic gene clusters (SMGC), which makes them interesting targets for genomic analysis. Important classes of SMGC include non ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and hybrids thereof.

We use a modified version of the Secondary Metabolite Unique Regions Finder (SMURF) algorithm, combined with InterPro annotations, network analysis and approximate maximum likelihood (ML) trees of conserved domains, giving insights into the secondary metabolism gene diversity and evolution. In this study we describe the diversity of hybrid SMGC among Aspergilli, horizontal gene transfers within and outside Aspergilli and identify possible ancestors of hybrids.

ML analysis indicated PKS-NRPS hybrids being of bacterial origin and having an ancestor cluster in the Streptomyces and Achromobacter genus. NRPS-PKS hybrids showed a wide diversity with orthologs being found in different Ascomycete classes like Dothideomycetes, Eurotiomycetes, Sordariomycetes and others. Here, our genus level study enables us to determine which hybrids might have been horizontally transferred to the precursor of a whole section, driving speciation of this section by a selective advantage. Examining these events helps us to identify the origin of secondary metabolites in Aspergilli and also provides us with new variants of compounds for industrial applications.
**279F** Comparative genomic analysis of secondary metabolite biosynthetic gene clusters in 207 isolates of *Fusarium*. *H.-S. Kim*, R.H. Proctor, D.W. Brown Mycotoxin Prevention and Applied Microbiology, USDA, ARS, NCAUR, Peoria, IL.

*Fusarium* species are known for their ability to produce secondary metabolites (SMs), including plant hormones, pigments, mycotoxins, and other compounds with potential agricultural, pharmaceutical, and biotechnological impact. Understanding the distribution of SM biosynthetic gene clusters across the phylogenetic diversity of *Fusarium* should provide insight into how changes in selection for production of SMs affect genome dynamics in the fungus. Using genome mining tools, such as antiSMASH and OrthoFinder, we identified known, novel, and putative SM gene clusters in 207 genomes from 159 species representing 25 species complexes of *Fusarium*. We found multiple homologs of 45 gene clusters responsible for biosynthesis of known mycotoxins and other SMs (i.e., trichothecenes, fumonisins, zearalenone, and fusaric acid). A total of 9403 SM gene clusters were detected in the 207 genomes, including 2276 clusters with non-ribosomal peptide synthetase genes, 2267 with polyketide synthase genes, and 1743 with terpene synthase genes. Closely related species within a species complex have similar numbers of SM clusters. For example, members of the *F. fujikuroi* species complex had 43–61 SM clusters, whereas distantly related species from different species complexes had 26–68 SM clusters. The comparative genomic analysis indicated that the gibberellin (plant hormone), fumonisin and zearalenone (mycotoxins) gene clusters have limited distribution, whereas the carotenoid and fusarubin (pigments) clusters are present in all members of most species complexes; they are absent only in some basal lineages of *Fusarium*. These data indicate that SM gene clusters differ markedly in when they were acquired and how long they have persisted during the evolutionary diversification of *Fusarium*.

**280W** Genomic and bioinformatics analyses of biosynthesis and production enhancement of a novel antifungal antibiotics, FR901469, from a filamentous fungus. *Masayuki Machida*¹,⁴, *Makoto Matsui*²,⁴, *Tatsuya Yokoyama*²,⁴, *Hiroya Itoh*²,⁴, *Akira Ohyama*³,⁴, *Takashi Shibata*²,⁴ 1) Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan; 2) Biotechnology labs, Astellas Pharma Inc., Japan; 3) in silico biology, inc., Yokohama, Japan; 4) Technology Research Association of Highly Efficient Gene Design (TRAHED), Tokyo, Japan.

A novel antifungal antibiotics, FR901469, is a non-ribosomal peptide (NRP) compound produced by a filamentous fungus, No. 11243. We have sequenced an entire genome of this strain, revealing genome size of 21.7 Mb with 9,920 predicted genes. Of the 4 putative non-ribosomal peptide synthetase (NRPS) genes existing on the genome, we have identified a 45 kbp NRPS gene for the core biosynthesis of FR901469 and associated nine genes as a gene cluster of 79 kbp in length. To date, fourteen mutants producing higher amount of FR901469 than the parental strain (up to 10 times) have been obtained by UV irradiation. Genome-wide mutation analysis revealed 14-183 mutations for each strain, among which those for genes categorized as "Replication, recombination and repair", "Signal transduction mechanisms" and "Transcription" are highly enriched. Interestingly, the region consisting of approximately 1.6 Mbp in size that showed 50% higher depth of mapping of the short reads from NGS possessed the FR901469 biosynthesis gene cluster above. Transcription expression analysis by RNA-sequencing revealed strong relationship between the expression of NRPS and the FR901469 productivity. In accordance of this result, artificial overexpression of a C6 zinc cluster transcription factor found in the gene cluster of the parental strain showed 3- to 4-times higher production of FR901469. Besides the enhancement of transcription expression of the cluster member genes, we have found genes relating to amino acid biosynthesis were overexpressed commonly in the mutant strains as well. During the progress above, we have developed a software tool to further analyze secondary metabolism (SM) gene clusters, in silico Molecular Cloning Genome Design Suite (IMCDS). Based on the unique feature of this tool, by which multiple clusters are aligned according to the sequence similarity of each cluster member gene, and in combination with MIDDAS-M and MIPS-CG, which we previously developed to predict SM gene cluster in a sequence motif-independent manner, further characterization of the FR901469 biosynthesis gene cluster will be discussed

**281T** Genetic drivers of diversity in secondary metabolic gene clusters in Aspergillus fumigatus populations. *Abigail Lind*¹, Jennifer Wisecaver², Fernando Rodrigues³, Gustavo Goldman⁴, Antonis Rokas² 1) Biomedical Informatics, Vanderbilt University, Nashville, TN; 2) Biological Sciences, Vanderbilt University, Nashville, TN; 3) Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 4) Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil.

Filamentous fungi produce a diverse array of secondary metabolites that play ecological roles in defense, virulence, and inter- and intra-species communication. The biosynthetic genes required for the production of individual secondary metabolites are arranged in contiguous genomic clusters. These pathways are narrowly taxonomically distributed and highly diverse between species, and are among the most fast-evolving protein-coding elements in filamentous fungal genomes. To gain insight into the diversity of secondary metabolic gene clusters in fungal populations and its drivers, we examined the conservation and divergence of secondary metabolic gene clusters across the genomes of 40 representative isolates of the cosmopolitan opportunistic pathogen *Aspergillus fumigatus*. We found that a core set of secondary metabolic gene clusters were present in all isolates, but that other gene clusters were only present in subsets of isolates. We additionally identified multiple different types of gene cluster polymorphisms, including fusion of different gene clusters, alternative genomic locations for gene clusters, and clusters with multiple alternative idiomorphs (i.e., non-homologous alleles). In particular, two gene clusters flanked by transposable elements were consistently found in different genomic locations; one of these clusters showed a phylogenetic pattern consistent with horizontal gene transfer between fungi. Our results suggest that multiple genetic factors, including recombination, gene loss, and horizontal gene transfer, drive the diversification of secondary metabolism pathways.

**282F** A forward genetic screen coupled with a novel target enrichment sequencing approach reveals new insight into the regulation of cercosporin biosynthesis in *Cercospora zeae-maydis*. *B.H. Bluhm*, S. Sharma, J. Ridenour, B. Dhillon Plant Pathology, University of Arkansas, Fayetteville, AR.

*Cercospora zeae-maydis* causes gray leaf spot of maize (*Zea mays*), a disease that affects maize production throughout the world. During pathogenesis, *C. zeae-maydis* produces cercosporin, a non-host-specific phytotoxin that is an important virulence factor for many species of *Cercospora*. Although the gene cluster underlying cercosporin biosynthesis has been identified, the genetic regulation of cercosporin biosynthesis has not been elucidated. In this study, a forward genetic screen for aberrant cercosporin production was coupled with a novel, target enrichment sequencing approach to identify genes involved in the regulation of cercosporin biosynthesis. Over 3000
genetically tagged mutants of *C. zeae-maydis* were created and screened for altered levels of cercosporin production. A total of 75 mutants produced significantly higher or lower levels of cercosporin compared to the wild-type strain. To characterize the site(s) of insertion in each mutant, biotinylated oligonucleotides were designed to probe the borders of the mutagenesis cassette and selectively enrich cassette-genome break junctions. After target enrichment, bar-coded libraries were sequenced with the Ion Torrent PGM platform. This approach identified 49 mutants with single insertions of the mutagenesis cassette, and 26 mutants with multiple insertions. Novel genes potentially regulating cercosporin biosynthesis were identified, including genes involved in signal transduction, primary and secondary metabolism, growth and development, and stress responses. Additionally, one mutant contained an insertion in *CTB1*, a polyketide synthase gene previously demonstrated to be involved in cercosporin biosynthesis. RNAi lines created for seven of the single-copy mutants confirmed associations between genes disrupted by insertional mutagenesis and cercosporin-related phenotypes. This approach has shed new light on the genetic regulation of cercosporin biosynthesis, and could easily be adapted to dissect the genetic basis of secondary metabolism in a wide range of filamentous fungi.

**283W Genome-wide survey of sexual stage-dependent non-coding RNAs in *Fusarium graminearum*. W. Kim, F. Trail**

Department of Plant Biology, Michigan State University, East Lansing, MI.

Although the global functional significance of non-coding RNAs (ncRNAs) remains controversial, there has been growing evidence that ncRNAs play critical roles in morphological transition and meiosis in yeasts. In filamentous fungi, however, little is known about the role of ncRNAs during the sexual development. Here we identified sexual stage-dependent ncRNAs in *F. graminearum*, a model for studying sexual development. To discover novel ncRNAs expressed during the sexual development of *F. graminearum*, transcripts were assembled from time-series transcriptome data across the sexual development, and compared to the reference annotation. Among the novel transcripts not annotated in the reference genome, transcripts harboring little coding potential and differentially expressed (DE) in at least one-time point during the developmental stages were classified as sexual stage-dependent ncRNAs (DE-ncRNAs). A total of 578 DE-ncRNAs were identified and the expression patterns of the DE-ncRNAs were largely developmental stage-dependent, while only 20% of the DE-ncRNAs (113 out of 578) were expressed during asexual spore germination. Interestingly, many of the DE-ncRNA expressions (243 out of 578) peaked at the stage of ascus formation during which meiosis occurs. Many of the DE-ncRNAs were antisense to protein-coding genes, forming 357 sense-antisense pairs. Functional enrichment analyses showed that the sense genes were significantly enriched for the Gene Ontology term 'DNA metabolism', raising a hypothesis that DE-ncRNAs play regulatory roles in DNA synthesis and degradation. In addition, DE-ncRNAs were searched against the RNAcentral database v5 (http://rnacentral.org) to find ncRNAs conserved in other organisms. Functional roles of selected DE-ncRNAs are currently under investigation. This study will provide a foundation for functional characterization of ncRNAs during the sexual development.

**284T Comparative genomics and transcriptomics of sexual development in a nematode-associated strain of *Fusarium neocosmosporiellum*. W. Kim¹, B. Cavinder¹, R.H. Proctor², K O'Donnell², Z. Wang³, J.P. Townsend³, F. Trail¹**

1) Department of Plant Biology, Michigan State University, East Lansing, MI; 2) Mycotoxin Prevention and Applied Microbiology Research Unit, USDA, Peoria, IL; 3) Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT.

*Fusarium neocosmosporiellum* (formerly *Neocosmospora vasinfecta*) is a ubiquitous saprobic fungus that has been isolated from plants, fungi, nematodes, dung and soil. It is phylogenetically placed in a clade near a lineage of fusaria farmed by ambrosia beetles within the *F. solani* species complex. Here we obtained the genome sequence of *F. neocosmosporiellum* NRRL 22166 strain isolated from soybean cyst nematode to conduct a comparative study with *F. graminearum*, a model for studying sexual stage (perithecium) development. Completeness of the sequenced genome was quantified by the presence of core eukaryotic genes and shown to be comparable to that of the finished *F. graminearum* genome. The *F. neocosmosporiellum* mating type locus included both *MAT1-1* and *MAT1-2* idiomorphs, but the gene order differed from that in *F. graminearum*. We identified several secondary metabolite gene clusters that exhibited increased expression during perithecal development, including *pksN* gene responsible for red perithecal pigmentation. To dissect transcriptional changes during perithecal development, we performed a time-series transcriptomic analysis and identified genes that are differentially expressed (DE) between two successive developmental stages. Overall, many DE genes were upregulated in later stages. Functional enrichment analyses showed that upregulated DE genes were enriched for Gene Ontology (GO) terms 'protein phosphorylation' and 'lipid metabolism' at the beginning of perithecia formation, and enriched for GO terms related to meiosis during ascus formation. Unlike *F. graminearum*, perithecia of *F. neocosmosporiellum* exhibited a marked increase in size at the stage immediately preceding ascus formation. Concordantly, *F. neocosmosporiellum* genes upregulated during this increase were enriched for GO terms 'transcription' and 'carbohydrate transport'. This study provides a comprehensive understanding of transcriptional changes during sexual development in *F. neocosmosporiellum*, a potential biological control agent for soybean cyst nematode.

**285F The Bck1 homolog gene, *CpBck1*, from the chestnut blight fungus *Cryphonectria parasitica* is related to sectorization and changes in DNA methylation. Kum-Kang So¹, Yo-Han Ko¹, Jyotiranjan Bal¹, Jeesun Chun¹, Junhyun Jeon², Jung-Mi Kim³, Jaeyoung Choi⁴, Yong-Hwan Lee⁴, Jin Hoe Huh⁵, Dae-Hyuk Kim¹**
1) Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Chonbuk 561-756, South Korea; 2) Department of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, 38541, South Korea; 3) Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, South Korea; 4) Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea; 5) Department of Plant Science, Seoul National University, Seoul 151-921, Korea.

Mutation in *CpBck1*, an ortholog of the cell wall integrity MAPKKK of *Saccharomyces cerevisiae*, of the chestnut blight fungus *Cryphonectria parasitica* resulted in a sporadic sectorization as the culture proceeded. The progenies from the sectored area maintained the characteristics of sector, a massive morphogenetic change including robust mycelial growth without differentiation. Epigenetic changes as the genetic reason for this sectorization were investigated. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed the genome-wide DNA methylation of the wild-type at the each nucleotide level and changes in the DNA methylation of the sectored progeny. Compared to the wild type, the sectored progeny exhibited marked genome-wide DNA hypomethylation. In trans complementation of the sectored progeny of the *CpBck1*-null mutant using the wild-type *CpBck1* gene recovered the phenotype as well as methylation indicating that the phenotypic and epigenetic changes was due to the *CpBck1* gene. In addition, functional analysis using
mutant phenotypes of corresponding DNMTases demonstrated that a mutant of *CpDmt1*, an ortholog of RID of *Neurospora crassa*, resulted in the sectored phenotype but the *CpDmt2* mutant suggesting the genetic basis of fungal sector appeared to be more complex. The current study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

**286W Functional characterization of CpSlt2, yeast SLT2 homologue, from the chestnut blight fungus Cryphonectria parasitica.** Kum-Kang So¹, Jung-Mi Kim², Kwang Yeop Jahng³, Dae-Hyuk Kim¹ 1) Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea; 2) Department of Bio-Environmental Chemistry, Institute of Life Science and Natural Resources, Wonkwang University, Iksan, Chonbuk 570-749, Korea; 3) Department of Life Sciences, Chonbuk National University, Jeonju-si, 561-756, Republic of Korea.

In fungi, mitogen-activated protein kinases (MAPKs) are involved in pathways that are required for numerous processes related to growth and differentiation. The sequential activation of the MAPK cascade eventually results in the activation of transcription factors and the expression of specific set of genes in response to environmental stimuli. Interestingly, the MAPKs play important roles in the pathogenicity of various pathogenic fungi. The *SLT2* mitogen-activated protein kinase has been well studied in *Saccharomyces cerevisiae* and is required for cell wall integrity. Functional characterization of the *SLT2* homologue in several pathogenic fungi has indicated that this MAPK is well conserved among fungal pathogens and plays important roles during plant infection. In this study, we cloned a yeast *SLT2* homologue, *CpSlt2*, from the chestnut blight fungus *Cryphonectria parasitica* and disrupted *CpSlt2* gene for examining gene functions. Analysis of *CpSlt2* mutant revealed defects in sporulation, vegetative growth, aerial mycelia, and pigmentation. The *CpSlt2*-null mutant showed increased sensitivity to the cell wall-disturbing agents and electron microscopy revealed the presence of intrahyphal hyphae. In addition, the sectorization was observed in *CpSlt2*-null mutant and sectored phenotypes were maintained in the progenies. The phenotypic changes of the *CpSlt2*-null mutant were recovered through *in trans* complementation of the *CpSlt2* gene, indicating the *CpSlt2* gene was responsible for these changes. These results indicate that putative biological functions of *CpSlt2* are related to cell wall integrity and sectorization.

**287T Identification and characterization of necessary sites on Ran-binding protein 1, essential for survivability of Cryphonectria parasitica, through heterokaryon and gene disruption mutant analysis.** So Yeun Choi¹, Yo-Han Ko¹, Kum-Kang So¹, Myeongjin Jo¹, Jeesun Chun¹, Jung-Mi Kim², Dae-Hyuk Kim¹ 1) Institute for Molecular Biology and Genetics, Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Chonbuk 54896, Korea; 2) Department of Bio-Environmental Chemistry, Institute of Life Science and Natural Resources, Wonkwang University, Iksan, Chonbuk 54538, Korea.

The chestnut blight fungus, *Cryphonectria parasitica*, and its hypovirus is known to be a useful model system to study the mechanisms of hypoviral infection. So we attempted a proteomic analysis of the virus-free EP155/2 strain and its isogenic virus-infected UEP1 strain in response to tannic acid(TA), which is abundant in the bark of the chestnut trees. The patho-proteomic analysis presented a differential-regulated protein spot, which showed a high similarity to the known fungal Ran-binding protein 1. The corresponding gene was determined by inspection of the draft genome sequence of *C. parasitica* (http://genome.jgi-psf.org/Crypa1/Crypa1.home.html). The cloned gene was predicted to encode a protein product containing a unique Ran-binding domain. Sequence similarity and phylogenetic analysis indicated that the cloned gene encodes a homolog of the Ran-binding protein 1 of *C. parasitica* and refers as *CpRbp1*. For function analysis, we constructed the *CpRbp1*-null mutant through homologous recombination and obtained two mutant candidates. PCR analysis and southern analysis revealed that both transformants were heterokaryons consisting of mixture of the wild-type and null-mutant nuclei. We designed several complementing vectors of *CpRBP1* based on the conserved Ran-binding protein 1 domains. The complementing experiments allowed function test of specific domain and amino acid residue of the *CpRbp1* gene, which helps us to understand the essential domains or residues within a lethal gene in a filamentous fungus

**288F Characterization of a GSP1/Ran ortholog gene, CpRan1, from a Chestnut blight fungus, Cryphonectria parasitica through point-mutation based heterokaryon analysis.** Yo-Han Ko¹, Jung-Mi Kim², Jyotiranjan Bal¹, Kum-Kang So¹, So Yeun Choi¹, Dae-Hyuk Kim¹ 1) Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Department of Bioactive Material Sciences, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea; 2) Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, Korea.

Pathoproteomic analysis revealed a protein spot down regulated by the presence of Cryphonectria hypovirus1 (CHV1) and tannic acid supplementation. The corresponding protein spot was identified as the GSP1/Ran homologue of *Saccharomyces cerevisiae* by tandem mass analysis. The Ran (Ras-related nuclear protein) also known as GTP-binding nuclear protein is a protein that is involved in transport into and out of the cell nucleus during interphase and also in mitosis. It is a member of the Ras superfamily. Through genomic sequence analysis, a near full-length cDNA clone was obtained. As a result, sequence comparison with the corresponding genomic sequences revealed that the cloned gene consisted of five exons, with four intervening sequences. The deduced protein product consisted of 216 amino acids, with an estimated molecular mass of 24.5 kDa and a pI of 6.11. Homology searches using the deduced amino acid sequence indicated that the protein product of the cloned *CpRan* gene is related to other fungal GSP1/Ran homologs from *Verruconis gallopava* (93%), *Fusarium avenaceum* (94%), *Colletotrichum gloeosporioides* (94%), *Trichoderma virens* (95%), *Aspergillus nidulans* (91%) and *Saccharomyces cerevisiae* (87%). Functional analysis of the cloned *CpRan1* was conducted using gene deletion analysis. We obtained the ?CpRan1 mutant from heterokaryotic transformants consisting of mixed nuclei of wild-type and ?CpRan1 genome. Single-spore analysis followed by genetic identification indicated that the cloned *CpRan1* gene is essential for growth. We design several point-mutation structures of the *CpRan1* gene to confirm that which is an essential part of the CpRan1 for succinylation, ubiquitination, and phosphorylation.

**289W Regulation of plant biomass degradation in Ascomycete fungi.** T. Benocci¹, M V Aguilar Pontes¹, B Seiboth², R De Vries¹ 1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, Utrecht, Netherlands; 2) Vienna University of Technology - Institute of Chemical Engineering. Vienna, Austria.

Fungi play a major role in the global carbon cycle because of their ability to utilize plant biomass (polysaccharides, proteins, and lignin) as
a carbon source and convert it to small molecular compounds. Due the complexity and heterogenic composition of plant biomass, fungi need to produce a broad range of degrading enzymes, matching the composition of (part of) the prevalent substrate. This process is dependent on a network of regulators that not only control the extracellular enzymes that degrade the biomass, but also the metabolic pathways needed to metabolize the resulting monomers.

Genomic and phylogenetic analysis of the currently known transcriptional regulators involved in this process have revealed a high level of diversity among Ascomycete fungi, with respect to the presence and absence of orthologs of these regulators. Together with results from functional studies this revealed that the organization of plant biomass conversion varies from species to species and is likely linked to their natural environment. Highlights will be presented.

**290T Complete genome sequences and comparative analyses of red and white *Antrodia cinnamomea* polyporus mushrooms reveal new insights for fungal life cycle and evolution.** *Chia-Ling Chen*¹, *Chien-Hao Huang*¹,², *Chung-Yu Chen*⁵, *Ko-Yun Lo*¹, *Wan-Chen Li*¹,³, *Yi Yang*¹, *Guo-An Chang*⁶, *Wen-Der Yang*⁷, *Zong-Ming Yeh*⁵, *Ching-Hua Su*⁴, *Ting-Fang Wang*¹ 1) Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan; 2) Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan; 3) Institute of Life Sciences, National Defense Medical Center, Taipei 115, Taiwan; 4) Department of Microbiology and Immunology, Taipei Medical University, Taipei 110, Taiwan; 5) Shen Nong Fungal Biotechnology Co. Ltd., Taoyuan 330, Taiwan; 6) KFK Biotech Co. Ltd., Kaohsiung 802, Taiwan; 7) HIMA Foundation, Taipei 111, Taiwan.

*Antrodia cinnamomea* (syn. *Taiwanofungus camphoratus*), an endemic mushroom-forming fungus, has long been used in traditional medicine in Taiwan. Most wild dikaryotic strains produce lustrous and orange-red fruiting bodies in 1-2 years or even longer. Up to now, only the genome draft sequence (32.15Mb, 360 scaffolds and 9254 genes) of an orange-red monokaryon has been reported. Deletion of *pks63787*, one of the polyketide synthesis genes, displayed a reduced red phenotype and deficient in the biosynthesis of several aromatic metabolites. There are also few wild dikaryotic strains can develop mushrooms faster (within 3-6 months), but the fruiting bodies are in milky white color. To reveal the phylogenetic relationship between orange-red and white *A. cinnamomea* strains, we isolated four sexually compatible monokaryons (W1, W2, V5, V7). W1 and W2 are derived from the arthorspores of a white dikaryotic strain (SN1), whereas V5 and V7 are from the arthospores of an orange-red dikaryotic strain (HC1). Next, we applied PacBio RSII and Illumina sequencing platforms to these four monokaryons for high-quality genome assembly. Our results reveal that these four monokaryons all have 14 chromosomes. Comparative analyses of gene organization and expression in the four monokaryons and the corresponding dikaryons will be presented.

**291F The dynamic and varied genome of the oomycete pathogen *Phytophthora infestans*.** *M.E.H. Matson*¹,², *J. Shrivastava*¹,², *H.S. Judelson*¹,² 1) Genetics, Genomics, & Bioinformatics Graduate Program, University of California-Riverside, Riverside, CA; 2) Department of Plant Pathology & Microbiology, University of California-Riverside, Riverside, CA.

As the causal agent behind the late blight disease of tomato and potato, the oomycete *Phytophthora infestans* causes considerable crop losses around the world. Controlling epidemics via host-mediated resistance is hampered by the remarkable adaptability of the *P. infestans* genome, since new R gene introductions into commercial varieties are quickly overcome. To investigate mechanisms driving genome plasticity, we used whole genome Illumina sequences of progeny from two sexual crosses and >50 diverse global individuals to identify copy number variation (CNV), structural variation (SV), and loss of heterozygosity (LOH) events. We determined through examining SNP allele ratios that the four parental isolates behind our two sexual crosses were mostly 2N, but that about 10% of progeny were mostly 3N. Local CNVs larger than 5kb comprised around 4.5% of genomic regions which could be uniquely mapped, and affected as many as 400 individual genes depending on the strain. Structural variants in the form of inversions and deletions were also identified among nine examined diverse global isolates using Pindel and Breakdancer. Finally, we observed considerable tracks of LOH among our progeny using the YMAP toolset. To investigate the functional consequence of LOH, we sequenced four progeny which spontaneously produced fast-growing sectors in the presence of the fungicide metalaxyl. These exhibited numerous sequence differences which may reveal the basis of resistance to the fungicide.

**292W Comparative Genomics and Transcriptomics of *Panellus stipticus* Strains for Elucidating Fungal Bioluminescence.** *B. Min*¹, *H. Park*¹, *J. Byun*¹, *D. Pathiraja*¹, *R. Ohm*²,³, *R. Riley*², *I. Grigoriev*², *I. Choi*¹ 1) Korea University, Seoul, KR; 2) Joint Genome Institute, Walnut Creek, CA, USA; 3) Utrecht University, 3508 TB Utrecht, Netherlands.

Bioluminescence occurs in various clades of organisms, such as marine vertebrates and invertebrates, microorganisms, and in some fungi. Fungal bioluminescence has been reported in mostly mushroom-forming fungi and a few ascomycete. Genome sequences of several bioluminescent fungi are available, including *Omphalotus*, *Armillaria*, and *Panellus*, but the molecular mechanism and genetic basis of bioluminescence has been hardly understood yet. We announced genome sequences of two *Panellus* strains representing luminescent *Panellus stipticus* LUM and non-luminescent *Panellus stipticus* KUC 8834 isolated in Korea. Two strains have similar genome sizes of 57 Mbp and 53 Mbp, respectively. It is assumed that at least two enzymes are related to fungal bioluminescence such as reductase and luciferase enzymes. We predicted candidate gene families involved in bioluminescence from comparative genome analysis of two strains. We focused on differentially expressed transcripts and orphan genes in the LUM genome. Transcriptome analysis was performed to retrieve expression patterns for targeting metabolic pathway for bioluminescence. By comparing two genetically similar but phenotypically different genomes, we suggest potential genetic basis of fungal bioluminescence.

**293T Assembling the *Parastagonospora nodorum* genome to the bitter end: long reads and optical mapping for reference genome completion and genome structure insights.** *S. Bertazzoni*¹, *K.C. Tan*¹, *R.A. Syme*¹, *T.L. Friesen*², *B.A. McDonald*³, *R.P. Oliver*¹, *J.K. Hane*¹ 1) Centre of Crop Disease Management, Dept. of Environment and Agriculture, Curtin University, Bentley 6102, Western Australia; 2) United States Department of Agriculture-Agricultural Research Service, Northern Crop Science Laboratory, Cereal Crops Research Unit, Fargo, ND, 58102; 3) Institute of Integrative Biology, Plant Pathology Group, Swiss Federal Institute of Technology, ETH Zentrum, LFW, CH-8092 Zürich, Switzerland.

*Parastagonospora nodorum* causes septoria nodorum blotch (SNB), a prevalent disease inflicting damage to wheat crops worldwide. Necrotrophic fungal pathogens such as *P. nodorum* exploits an inverse gene-for-gene interaction where fungal effectors must be recognised by a specific host receptor for infection to occur. A quality annotated reference genome is critical to discover the pool of