# Comparative proteomics and phosphoproteomics to decipher signal transduction in Botrytis cinerea.

Jaafar Kilani, Marlène Davanture, Michel Zivy, Sabine Fillinger

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Jaafar Kilani, Marlène Davanture, Michel Zivy, Sabine Fillinger. Comparative proteomics and phosphoproteomics to decipher signal transduction in Botrytis cinerea.. 29. Fungal genetics conference Asilomar 17, Mar 2017, Pacific Grove CA, United States. p.212. hal-01604794

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March 14-19, 2017

Pacific Grove, CA

**Abstract Book**

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Wednesday, March 15 9:00 AM – 12:00 NOON

Merrill Hall and Chapel

Plenary Session I: Model Fungi: Biological Insights Beyond the Kingdom

**Neurospora** as a model for circadian clocks in higher organisms. J.J. Loros¹, X. Zhou¹, A. Crowell¹, J.M. Hurley², J.M. Emerson¹,
B.R. Crane³, D. Bell-Pedersen⁵, M. Freitag⁴, L.F. Larrondo⁶, J.C. Dunlap¹ 1) Geisel School of Medicine at Dartmouth, Hanover, NH; 2)
Rensselaer Polytechnic Institute, Troy, NY; 3) Cornell University, Ithaca, NY; 4) Oregon State University, Corvallis, OR; 5) Texas A&M
University, TX; 6) Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.

Circadian rhythmicity, the cell-intrinsic ability of organisms to track internal time and thereby coordinate activities with the environment, has arisen at least three times in evolution. Neurospora crassa has been a durable system for understanding the molecular bases of these oscillators within the fungal and animal Kingdoms; Neurospora is perhaps the most intensively studied of all circadian cell types. At the core of the circadian system is the heterodimer of White Collar-1 and -2 forming the White Collar Complex (WCC) that drives the expression of the frequency (frq) gene. FRQ complexes with other proteins, including Frequency Interacting RNA Helicase (FRH) and casein kinase 1 (CK1), and in a time-delayed, and highly regulated manner dictated by progressive phosphorylation the complex reduces the activity of the WCC, thus resulting in a negative auto-regulatory feedback loop.

WC-1 and WC-2 interact via PAS domains as do similar positively acting clock-relevant TFs in animal clocks; conserved within the negative element FRQ-FRH complex is the interaction with CK1 and the extensive phosphorylation of a negative element that generates the time delay. FRQ, an intrinsically disordered protein (IDP), is stabilized by FRH which acts non-enzymatically in the clock; separately FRH has a helicase function required for cell viability. The negative elements in fungal and animal clocks are poorly conserved at the protein level, even just among animals, possibly reflecting the fact that many appear to be IDPs for which there is no inherent structural drive for conservation of sequence.

Circadian clocks entrain to daily environmental cycles of light and temperature. Molecular mechanisms of light responsiveness are well understood in Neurospora and the way in which light resets rhythms in general was first worked out in this system. Of central importance for any organism with a clock is the daily control of cellular activities -growth, metabolism, behavior, development and

beyond. Work on Neurospora set the paradigms for understanding clock output, revealing that most of it is through clock-controlled gene expression. As much as 40% of the Neurospora genome is clock controlled at the level of transcript abundance by greater than 50 TFs. Recently a consortium of laboratories have collaborated in using RNA-Seq, ChIP-Seq and bioinformatics to understand the transcriptional networks through which output from the circadian clock in Neurospora is controlled.

**Molecular control of fungal senescence: macroautophagy as a "double edged" sword in a complex cellular quality control network.**
Heinz D. Osiewacz, Laura Knuppertz, Alexander Jung, Piet Kramer, Andrea Hamann Molecular Biosciences, J W Goethe University, Frankfurt, DE.

*Podospora anserina* is a well established model organism to unravel the complex molecular basis of organismal aging. Research with this system uncovered a strong mitochondrial etiology and provided novel perspectives for the elucidation of molecular mechanisms of aging relevant from yeast to humans.

In the last decade, we used *P. anserina* as an *in vivo* system to study the influence on organismal aging of pathways involved in the control of mitochondrial quality. We observed that pathways active in balancing reactive oxygen species, the degradation of proteins by mitochondrial proteases, mitochondrial fission and fusion, programmed cell death, and recently macroautophagy (hereafter termed autophagy) all influence the aging process. Sometimes we obtained results that were according to our expectations. In other cases, they were counterintuitive and called for further investigations and more complex explanations. Recently, a knockout mutant in which the mitochondrial superoxide dismutase isoform was ablated did not show any effect on lifespan compared to the wild type, although the cellular superoxide anion load was strongly increased and mitochondrial respiration was moderately impaired. We found that in this mutant selective autophagy of mitochondria, termed mitophagy, is increased and acts as a compensating "pro-survival" pathway. We also found that the beneficial effect of mild oxidative stress, which is described in different organisms as mitohormesis, in *P. anserina* depends on a functional autophagy machinery. In contrast, increased oxidative stress or stress caused by excessive overexpression of the gene coding for cyclophilin D turns autophagy to a "pro-death" pathway that leads to "autophagic cell death", also termed "programmed cell death type II". In another mutant, general or "bulk" autophagy was found to be induced while in a third mitochondrial quality control mutant both general autophagy and mitophagy were unaffected. Overall, these observations demonstrate a context-dependent flexible function of autophagy in the control of cellular homeostasis that allows an organism to adapt to changing conditions. The elucidation of the underlying regulatory mechanisms and of crosstalks between different pathways is one of the key challenges in this field of research.

**Phytophthora effectors promote infection by suppressing small RNA silencing in the host.** Y. Zhai¹, D. Choi¹, J. He², Y. Hou¹, W. Ye³, J. Ma², W. Ma¹, Y. Zhai, D. Choi and J. He contribute equally to this work. 1) University of California, Riverside, CA; 2) Fudan University, Shanghai, China; 3) Nanjing Agricultural University, Nanjing, China.

A broad range of parasites rely on the functions of effector proteins to subvert host immunity and facilitate disease development. The
genus *Phytophthora* contains some of the most notorious plant pathogens. Each *Phytophthora* genome is predicted to encode several
hundreds to over one thousand effectors, and the majority of them contained the consensus RxLR motif, which allows effector uptake into
the host cells. This remarkably large effector repertoire reflects the high level of complexity in *Phytophthora*–plant arms race and demands
mechanistic analysis of effector functions in order to gain understanding of *Phytophthora* pathogenesis.

Previously, we discovered RxLR effectors with RNA silencing suppression activity from the soybean pathogen *Phytophthora sojae*.
Interestingly, *Phytophthora* Suppressor of RNA silencing 2 (PSR2) has seven tandem repeats, each containing the so-called L-W-Y motifs.
Crystal structure of PSR2 revealed that each tandem repeat forms a conserved five-alpha-helix bundle. Genome-wide analysis of RxLR
effectors in five *Phytophthora* species identified 20-30 PSR2-like effectors (per species) with various numbers of the repeat units but similar
structural fold, suggesting that PSR2 belongs to a highly conserved and diversified effector family. Other members of the PSR2 effector family also possess RNA silencing suppression activity.

PSR2 promotes infection by specifically affecting the accumulation of secondary small interfering RNAs called phasiRNAs in plants. We characterized PSR2-associating protein(s) in plants in order to understand its virulence mechanism. Our results show that PSR2 interacts with Double-stranded RNA-Binding protein 4 (DRB4), which has a known function in phasiRNA biogenesis by partnering with the endonuclease Dicer-like protein 4 (DCL4). We further determined that genes involved in the phasiRNA pathway positively regulate plant defense against *Phytophthora*. These results suggest that PSR2 promotes infection by targeting phasiRNA biogenesis through its interaction with DRB4.

**The impact of modulating translation initiation, elongation and termination on gene expression.** M.S. Sachs Department of Biology, Texas A&M University, College Station, TX.

The central dogma of molecular biology, generally stated, is that DNA makes RNA (transcription), and RNA makes protein (translation). Studies of model fungi have been instrumental in establishing the strategies and mechanisms that eukaryotes use to control transcription and translation. While the importance of transcriptional control is universally acknowledged, there is now a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. For example, it is now apparent that upstream open reading frames (uORFs), once thought of as rarities, are present in more than 25% of eukaryotic mRNAs, and that the translation of these uORFs can be critical for controlling gene expression levels. It is now apparent that initiation of translation of these uORFs - as well as initiation of translation of the major predicted gene products of mRNAs - can occur at codons other than AUG codons, and these non-AUG initiation events have functional significance. The rate of translation elongation can also be modulated at the level of specific mRNAs by codon-choice or by encoded nascent peptides that stall ribosomes by affecting the function of the ribosome's peptidyltransferase center. Initiation and elongation can both be modulated by controlling the activity of the translation machinery. Events associated with translation termination can have regulatory functions critical for controlling gene expression and also can have a major impact on mRNA stability through the mRNA quality control pathway known as nonsense-mediated mRNA decay (NMD). Here we discuss these processes of translational control and studies in the model fungi that have contributed to our general understanding of how they impact gene expression.

**Autocatalytic backbone N-methylation hallmarks a distinct family of ribosomally encoded peptides.** N. van der Velden, N. Kalin, M. Helf, J. Piel, M. Freeman, *M. Kunzler* Institute of Microbiology, ETH Zurich, Zurich, CH.

N-Methylated backbone amides alter the physicochemical properties of peptides and are critical for the activity and stability of blockbuster drugs like the immunosuppressant cyclosporin A. In nature, backbone N-methylations have not been observed in proteins, but only in peptides not synthesized by the ribosome. Here, we show that omphalotins, cyclic backbone N-methylated peptides from the mushroom *Omphalotus olearius*, are ribosomally synthesized and post-translationally modified peptides (RiPPs). Expression of a single gene, *ophA*, in *Escherichia coli* revealed auto-α-N-methylation activity of the encoded protein. Remarkably, the sequence destined to be the nematotoxic natural products is encoded in the C-terminus of OphA, and we demonstrate that OphA catalyzes iterative α-N-methylation of this sequence. Thus, OphA is the first observed case of backbone N-methylations as post-translational modifications (PTMs) and the first RiPP precursor found to be capable of catalysis or self-modification. The omphalotins represent, therefore, a new RiPP family, one of the few so far identified in fungi. We also report that OphA can methylate engineered peptide sequences exchanged for the core omphalotin sequence, providing a straightforward and convenient biotechnological strategy to produce custom peptides with backbone N-methylation.
Wednesday, March 15 3:00 PM – 6:00 PM

Merrill Hall

Plant-Fungus Interactions

**The two-speed genome of Verticillium dahliae mediates emergence of potent virulence factors.** B.P.H.J. Thomma, L. Faino, J. Li, X. Shi-Kunne, J. Depotter, M. Kramer, G.C. van den Berg, D.E. Cook, H. Rovenich, M.F. Seidl Laboratory of Phytopathology, Wageningen University, Wageningen.

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, virulence genes reside in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of such compartments, and the role of transposons therein, remains limited. We show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen *Verticillium dahliae*, and that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. Remarkably, LS regions are enriched in active transposons, which may contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of *V. dahliae* virulence. Based on this knowledge, we are now able to identify crucial virulence factors of *V. dahliae*, which also allows investigating causal relationships between particular effectors and pathotypes.

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

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

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

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

**Field pathogenomics of wheat yellow rust.** Diane Saunders¹,² 1) Crop Genetics, John Innes Centre, Norwich, Norfolk, GB; 2) Earlham Institute, Norwich, Norfolk, GB.

Emerging and re-emerging pathogens pose a continuous threat to food security and human health. Recent advances in next-generation sequencing technologies have provided new opportunities to integrate genomics into the tracking of emerging filamentous plant pathogens. Accordingly, we are leading the genome sequencing of hundreds of isolates of the wheat yellow rust pathogen *Puccinia striiformis* f. sp. *tritici* (PST), aimed at improving our understanding of the molecular mechanisms that drive PST evolution. Furthermore, we have developed a robust and rapid "field pathogenomics" strategy to improve filamentous pathogen surveillance. Using gene sequencing of PST-infected wheat leaves taken directly from the field, this technique enabled us to gain insight into the population structure of PST over successive seasons. Our analysis uncovered a dramatic shift in the PST population in the UK and supports the hypothesis that a recent introduction of a diverse set of exotic PST lineages may have displaced the previous populations. Furthermore, we uncovered potential seasonal and varietal specificity for specific genotypes of PST. This knowledge regarding which wheat varieties are susceptible to specific PST isolates, and when those isolates are prevalent throughout the year, represents a powerful tool for disease management.

**Historic transposon-driven evolution of a novel non-ribosomal peptide.** D. Berry¹, H.B. Bode², K. Grage¹, W. Mace³, C.A. Young⁴, C.L. Schardl⁵, P.P. Dijkwel¹, B. Scott¹ 1) Massey University, Palmerston North, New Zealand; 2) Goethe University, Frankfurt, Germany; 3) AgResearch, Palmerston North, New Zealand; 4) The Samuel Roberts Noble Foundation, Ardmore, OK, USA; 5) University of Kentucky, Lexington, KY, USA.

*Epichloë* fungi are endophytic symbionts of grasses that produce a variety of bio-protective secondary metabolites. One such metabolite, peramine, is an insect-deterring cyclic dipeptide synthesised by the two-module non-ribosomal peptide synthetase (NRPS) PerA. A historic small transposable element insertion into the 3' end of the *perA* gene resulted in deletion of sequence encoding the C-terminal reductase domain of PerA, which is required for product release during peramine synthesis. This truncated allele, named *perA-2*, is the only known widespread peramine-negative allelic variant of *perA*; balancing selection and recombination has maintained both *perA* and *perA-2* alleles across multiple speciation events in an example of trans-species polymorphism. We have conducted extensive experiments utilising *Penicillium paxilli* as a heterologous expression system for *perA*, *perA-2*, and a number of synthetic *perA/perA-2* chimeric alleles. We report results demonstrating that the transposon-mediated deletion in *perA-2* is associated with substrate specificity alterations and methylation domain inactivation in PerA-2 proteins. In contrast to previous reports characterising *perA-2* alleles as non-functional, we demonstrate that these evolutionary changes culminate in production of a novel cyclic dipeptide. The structure of this new secondary metabolite will be
described, as will the spontaneous release mechanism that is proposed to take the place of a dedicated product release domain in PerA-2
proteins.

**Dissecting the role of the Tox3 effector in the *Parastagonospora nodorum*-wheat interaction.** Susan Breen¹, Megan Outram²,
Bostjan Kobe², Simon Williams¹, *Peter Solomon*¹ 1) Research School of Biology, The Australian National University, Canberra, ACT, AU;
2) School of Chemistry and Molecular Biosciences, The University of Queensland, QLD, AU.

It had long been thought that necrotrophic pathogenic fungi use a barrage of lytic enzymes to break down plant cells releasing nutrients for growth. However, in recent years it has emerged that some necrotrophic fungi facilitate disease through a strict gene-for-gene mechanism as observed in biotrophic pathogens. For the wheat pathogen *Parastagonospora nodorum*, the basis of this host specific interaction is small cysteine-rich effector proteins secreted during infection (ToxA, Tox1 and Tox3). These effectors interact with specific dominant susceptibility genes in the host leading to a programmed cell death response and disease. However, whilst we now understand the requirement of these effector proteins for disease, their modes of action remain poorly understood.

To characterise these necrotrophic effectors, a search for potential host protein binding partners for the Tox3 effector was conducted. From this work, the wheat TaPR1-1 protein was validated through two independent approaches to interact with Tox3. Subsequent analysis confirmed that Tox3 was able to interact with most acidic and basic TaPR1 proteins in wheat, but not those harbouring a C-terminal extension. Mutagenesis and protein modelling using interacting and non-interacting TaPR1 proteins has predicted several amino acids involved in this interaction and we have now made significant progress in characterising these proteins at the structural level using a crystallography approach.

These findings have raised the question as to the role of the TaPR-1 proteins during infection and the consequence of its interaction with the Tox3 effector. A recent study by Chen *et al*, 2014 showed that the CAPE1 peptide derived from the C-terminus of the tomato PR-1b regulates immune signalling. This peptide is conserved in the TaPR1 proteins but is not required for the interaction with Tox3. The exogenous application of the peptide on wheat leaves significantly enhanced *P. nodorum* disease in a Tox3-dependent manner tentative linking the interaction of Tox3 and TaPR1 with CAPE1 defence signalling. We have now also shown that the CAPE1 peptide directly impacts on the outcome of other diseases, both necrotrophic and biotrophic. These data raise important questions as to the function of the enigmatic PR1 proteins in plant-microbe interactions and highlight the intriguing prospect that they potentially function as carrier proteins to localise the CAPE1 defence signalling peptides to the host apoplast.

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

Polyamines are a group of ubiquitous and essential metabolites found in all living organisms. In fungi, the most abundant molecules - putrescine, spermidine, and spermine - have roles in conidiation and spore germination in *Aspergillus*, *Mucor*, and *Phycomyces* species. Little is known about how polyamine metabolism affects the development of fungal infection structures. Here, we report that a spermine synthase - encoded by *SMT1* - is required by the rice blast fungus *Magnaporthe oryzae* to generate spermine from spermidine and is essential for developing functional infection cells (appressoria) on rice leaf surfaces. *M. oryzae*, the most important pathogen of cultivated rice, elaborates appressoria at the tips of germ tubes emerging from spores on the leaf surface. The melanin-rich wall of the appressorium allows solutes such as glycerol to accumulate and generate enormous internal turgor pressure. This pressure is translated into mechanical force acting on the penetration peg emerging from a pore at the base of the appressorium, forcing it through the plant cuticle. In contrast, incipient cytorrhysis experiments determined that the appressoria of Δ*smt1* mutant strains generated turgor pressures that were 1.8 higher than wild type (WT), but no penetration pegs were observed breaching rice leaf cuticles by confocal microscopy. These developmental defects were not due to misregulated lipid mobilization or cell cycle progression, which occurred like WT in the *Dsmt1* mutant strains. Rather, by calculating the ratio of appressoria plasmolysis to cytorrhysis following PEG treatment, we determined that appressorial cell wall porosity was increased 3.5 in *Dsmt1* mutant strains compared to WT. We conclude that polyamine metabolism in *M. oryzae* appressoria, like in *Arabidopsis*, might control cell wall porosity. Perturbing porosity in *Dsmt1* appressoria could affect turgor and/or the rigidity of the cell wall. In turn, these structural defects might affect the polarized growth of the penetration peg from the appressorial pore. Ongoing studies of *Dsmt1* will, compared to WT, examine the structure of the appressorial cell wall, examine the pore at the base of the appressorium, and determine the status of penetration peg formation. Taken together, our results reveal novel roles for polyamine metabolism in appressorium function that enrich our understanding of the metabolic pathways intrinsic to rice cell colonization by *M. oryzae*.

**Characterization of effector candidates from the soybean rust fungus that suppress plant immunity.** M. Qi¹, T Link², R Voegele², T Baum¹, S Whitham¹ 1) Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA; 2) Institut für Phytomedizin, Universität Hohenheim, Otto-Sander-Straße 5 70599 Stuttgart, Germany.

Soybean rust (SBR) is an important disease caused by *Phakopsora pachyrhizi* (*Pp*). Like other rust fungi, *Pp* forms specialized cells called haustoria, which are key hubs of molecular exchange with host cells. Haustoria express effector proteins that are transferred to plant cells where they promote fungal growth. We have recently sequenced the *Pp* haustorial transcriptome and predicted candidate effector proteins. To gain insights into functions of *Pp* effector candidates (*PpECs*), we have cloned 82 *PpEC* coding sequences and delivered the proteins into host and non-host plants using a bacterial type III secretion system-based delivery vector. Several *PpECs* suppressed immune responses associated with effector- and pattern-triggered immunity, and we are in the process of characterizing these further. One interesting example is *PpEC23*, a small, cysteine-rich effector candidate that attenuated the hypersensitive response caused by *Pseudomonas syringae* pv *tomato* DC3000 on soybean and tobacco, and it also suppressed basal defense on tobacco and *Arabidopsis*. *PpEC23* interacts with a transcription factor that our data suggests negatively regulates soybean defenses. We are now using host-induced gene silencing and transcriptomics to determine the effects of *PpEC23* and the interacting soybean transcription factor in immunity or virulence. The results are expected to provide mechanistic insights into the molecular basis of SBR and may be relevant to other rust diseases.
**A small secreted protein in *Zymoseptoria tritici* is responsible for avirulence on wheat cultivars carrying the *Stb6* resistance gene.**
Ziming Zhong¹, Thierry Marcel², Clémence Plissonneau¹, Bruce McDonald¹, Daniel Croll¹, *Javier Palma-Guerrero*¹ 1) Plant Pathology Group, ETH Zürich, Switzerland; 2) UMR BIOGER, INRA, , France.

*Zymoseptoria tritici* is the causal agent of Septoria tritici blotch (STB), a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. The *Z. tritici* infection cycle includes an extended asymptomatic phase characterized by slow apoplastic growth followed by a switch to necrotrophy that coincides with the onset of plant cell death. Gene-for-gene (GFG) interactions between this apoplastic pathogen and resistant wheat cultivars have been postulated for many years, but until now none of the genes involved in this interaction were identified. We identified *AvrStb6* by combining QTL mapping based on a cross between two Swiss strains with a genome-wide association study (GWAS) using a natural population of ~100 strains coming from France. We functionally validated *AvrStb6* using ectopic transformations. *AvrStb6* encodes a small, cysteine-rich, secreted protein, with features of fungal effector proteins, which produces an avirulence phenotype on wheat cultivars carrying the *Stb6* resistance gene. *AvrStb6* shows peak of expression during the transition to the necrotrophic phase suggesting that the protein may act as a necrotrophic effector in susceptible cultivars. We found 16 non-synonymous SNPs among the tested strains, indicating that *AvrStb6* is evolving very rapidly. By analyzing the finished genomes of both parental strains used to establish the cross, we found that *AvrStb6* is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements. This genomic environment may facilitate the rapid evolution of *AvrStb6* and enable *Z. tritici* to rapidly adapt to overcome *Stb6* resistance. *AvrStb6* is the first avirulence gene to be functionally validated in *Z. tritici*, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms of GFG interactions between *Z. tritici* and wheat.
Wednesday, March 15 3:00 PM – 6:00 PM

Chapel

Mating and Clonality in Fungi

**Functional link between NADPH oxidase complex and pheromone perception machinery in chemotropic sensing of host plant signals by Fusarium oxysporum.** Daniela Nordzieke, Stefania Vitale, Mennat El Ghalid, Maria Crespo Ruiz-Cabello, David Turrà, Antonio Di Pietro
*Departamento de Genetica, Universidad de Cordoba, 14071 Cordoba, Spain.*

Chemotropism, the ability to re-orient hyphal growth in response to chemical cues, is critical for many aspects of the fungal lifestyle including sexual development and host infection. We previously showed that the root-infecting pathogen *Fusarium oxysporum* senses heme peroxidases secreted by plant roots as chemoattractant signals to direct hyphal growth towards the host. This chemotropic response requires the two pheromone receptors *Ste2* and *Ste3*, as well as elements of the conserved fungal cell wall integrity mitogen-activated protein kinase (CWI MAPK) cascade. Because peroxidases catalyze the reductive cleavage of reactive oxygen species (ROS), we examined the role in chemotropism of the NADPH oxidase complex (NOX), which generates superoxide by transferring electrons across the membrane. Using deletion mutants in different components of the *F. oxysporum* NOX complex, we found that *NoxB*, *NoxR* and the tetraspanin *Pls1*, but not *NoxA*, are specifically required for peroxidase-induced chemotropism. NOX mutants were impaired in the peroxidase-triggered phosphorylation of the CWI MAPK *Mpk1* and the high osmolarity MAPK *Hog1*. Importantly, directional growth towards peroxidase was restored by exogenous addition of hydrogen peroxide in the *noxBΔ* and *noxRΔ* mutants, but not in *pls1Δ* and *ste2Δ*. Addition of DETC, a specific inhibitor of superoxide dismutase (SOD), an enzyme that converts NOX-derived superoxide to hydrogen peroxide, also abolished peroxidase-induced chemotropism. Our findings reveal a new role for NOX- and SOD in peroxidase-triggered chemotropism and suggest a possible functional link between ROS and pheromone sensing machinery in hyphal growth of a plant pathogen towards the host plant.

**Resurrecting a sexual cycle in the human fungal pathogen Candida glabrata.** Jane Usher¹, Alison Day³, Judith Berman², Janet Quinn³, Ken Haynes¹
1) Biosciences, University of Exeter, Exeter, Devon, UK; 2) Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel; 3) Institute for Cell and Molecular Biosciences, Faculty of Medicine, Newcastle University, UK.

To date no sexual cycle has been described in the pathogenic yeast *Candida glabrata*. This is curious as the population structure contains evidence of recombination, the genome contains mating-type loci that can switch at sites of infection and orthologues of the majority of genes involved in fungal sexual reproduction and meiosis, including many of those missing in other *Candida* species. The lack of a complete sexual cycle in some hemiascomycete yeasts is surprising as they contain many of the required genes. We naively hypothesized that the functions of the encoded proteins may have changed, as has been previously described for regulators of ribosome biosynthesis in *C. albicans*, resulting in an inability to complete a sexual cycle. To test this hypothesis, we cloned each of the *C. glabrata* genes, functionally annotated to the GO terms mating or meiosis, and attempted to systematically complement the mating/meiosis defects of the orthologous *S. cerevisiae* mutants, as has previously been done for *ste11*, *ste12* and *ste20* mutants in *C. glabrata*.

Here we show that a complete sexual cycle can be engineered in *C. glabrata*. IME1, encoding the principle regulator of meiosis and IME2, encoding a meiosis specific kinase from *C. glabrata* are unable to complement the mating defects of the orthologous *S. cerevisiae* null mutants. The replacement of the native *C. glabrata* IME1 and IME2 with their orthologs from *S. cerevisiae* results in *C. glabrata* cells that can mate, form stable diploids and undergo sporulation, to give tetrad-like structures containing viable haploid progeny. These progeny, when sequenced show evidence of having undergone recombination. Our results demonstrate that in addition to the loss of orthologous mating and/or meiosis proteins seen in some fungi, those retained may have had their function reassigned, thereby resulting in a reliance on asexual reproduction.

This is the first report of an apparently asexual ascomycetous yeast that has been engineered to successfully complete a sexual cycle. This will not only allow for forward gentic analysis in *C. glabrata*, but offers an approach that may be successful in revealing hidden mating pathways in other species such as *C. albicans* in which stable haploids have recently been described. This data also highlights key evolutionary changes between pathogenic and non-pathogenic fungi.

**Genome evolution in asexual laboratory populations of Saccharomyces cerevisiae.** Gregory Lang
Biological Sciences, Lehigh University, Bethlehem, PA.

Natural selection optimizes an organism's genotype within the context of its environment. Adaptations to one environment can decrease fitness in another, revealing evolutionary trade-offs. We show that the cost of gene expression underlies a trade-off between growth rate and mating efficiency in the yeast, *Saccharomyces cerevisiae*. During asexual growth, mutations that eliminate the ability to mate provide a fitness advantage. We have developed a method to track the fraction of sterile cells in a population by assaying for the presence of a mating-specific fluorescent reporter. Using this system, we tracked the emergence and fate of spontaneous sterile mutations in ~600 populations through 1,000 generations. We sequenced forty of these populations at 80-generations resolution and identified hundreds of successful mutations (including sterile mutations). We are currently reconstructing all possible combinations of observed mutations to quantify the fitness effects of each mutation individually and in combination.

**MAT genes in a lineage of the Basidiomycota unusually enriched in homothallic species.** M. David Palma, M. A. Coelho, J. P. Sampaio, P. Gonçalves
UCIBIO-REQUIMTE, Departamento de Ciências de Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal.

Most fungal species are heterothallic, requiring a combination of two mating types for sexual reproduction. However, a minority of species is homothallic, which means that a single individual is capable of completing the sexual cycle without the need for a compatible partner. The mechanistic basis of homothallism may vary, including mating type switching as observed in *Saccharomyces cerevisiae* or unisexual mating as recently described in *Cryptococcus neoformans*, along with primary or "true" homothallism in which an haploid genome expresses all the
molecular components required for sexual reproduction. Basidiomycetes evolved a complex mating system, usually comprising two genetically independent *MAT* loci encoding two distinct mating compatibility check points, whose molecular underpinnings are known in detail for numerous heterothallic species. However, studies showing how basidiomycete *MAT* genes participate in primary homothallic sexual cycles were lacking. This prompted us to unravel the molecular basis of sexual reproduction in *Phaffia rhodozyma*, a genetically accessible, homothallic yeast species in the Cystofilobasidiales (Agaricomycotina, Basidiomycota). Six *MAT* genes encoding pheromone receptors, pheromone precursors and HD transcription factors were uncovered in the draft genome of *P. rhodozyma*. All were shown by genetic dissection to be functional and involved in sexual reproduction. We subsequently used comparative genomics to uncover *MAT* genes in other species belonging to the Cystofilobasidiales, a lineage comprising an unusually high proportion of homothallic species. Our results indicate that transitions to homothallism probably occurred several times independently in this lineage and reveal a particularly dynamic pattern of *MAT* gene evolution, with high turnover of pheromone receptor alleles and exceptionally large numbers of mature pheromones encoded in the genomes of some of the species.

**Whole genome DNA-methylation (methylome) profiling during heterokaryosis in the Agaricomycetes.** R.A. Powers, T.Y.

James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

DNA methylation of the 5' position of cytosines, a well-known form of 'epigenetic' modification, has been shown to be important in such diverse processes as the formation of human cancers, development in multicellular eukaryotes, and the silencing of transposons and repetitive elements in plants, animals, and fungi. Despite the importance and apparent conservation of DNA methylation across diverse clades of eukaryotes, we still lack a basic understanding of its roles in the mushroom-forming fungi of the Agaricomycetes. While it has been shown that DNA methylation in CpG (5' Cà G 3') contexts is important for the silencing of repetitive DNA elements in fungi, we lack a comprehensive understanding of DNA methylation at the whole genome level in the Basidiomycota. In particular, we know little of how whole genome DNA methylation ("methylome") patterning differs both during different life-stages within a species, as well as how methylomes differ between taxa. Using whole-genome bisulfite sequencing (WGBS), which involves the treatment of genomic DNA with sodium-bisulfite followed by whole genome next generation sequencing, we have generated genome-wide methylation profiles of two haploid homokaryotic isolates from five taxa across diverse clades of the Agaricomycetes: *Coprinopsis cinerea*, *Heterobasidion irregulare*, *Wolfiporia cocos*, *Coprinellus disseminatus* and *Cyathus stercoreus*. Here, we report that the amount of genomic methylation of cytosines in CpG contexts is species specific and ranges from of a low of ~2% in *Heterobasidion irregulare* to ~12.5% in *Cyathus stercoreus*. Further, we show that there is a high degree of variation in CpG methylation percentages between haploid strains of the same species for most of the species tested. Lastly, we examined the role of changes in methylome patterning during mating and heterokaryosis and show that DNA methylation patterns change during the transition for haploid to heterokaryon, and that these changes are also species specific.

**Unpacking the molecular mechanism behind unisexual reproduction in *Huntiella moniliformis*.** A. Wilson¹, M. van der Nest¹, P.M.

Wilken¹, M.J. Wingfield², B.D. Wingfield¹ 1) Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa ; 2) Department of Microbiology & Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

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

**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.

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

*Cryptococcus neoformans* and *Cryptococcus deneoformans* are two sibling species belong to the opportunistic human fungal pathogen *Cryptococcus* species complex. *C. neoformans* mainly undergoes bisexual reproduction, whereas *C. deneoformans* undergoes both unisexual and bisexual reproduction. During both sexual cycles, a dramatic yeast-to-hyphal morphological transition takes place and generates either monokaryotic or dikaryotic hyphae, and this process is regulated by a common set of genetic circuits. The unisexual cycle can also generate genotypic and phenotypic diversity de novo. Despite the similarities between unisexual and bisexual cycles, there are still genetic and morphological differences, such as an absence of an opposite mating partner and monokaryotic instead of dikaryotic hyphae. The natural populations for both species are mainly α mating type; however, the unisexual cycle is well established in *C. deneoformans* but not in *C. neoformans*, suggesting differences in mating mechanisms may contribute to this. To understand what distinguishes the different modes of sexual cycles, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and nuclear fusion. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both *Cryptococcus* species, and showed that they play conserved roles in cell fusion and karyogamy during sexual reproduction. The involvement of Prm1 and Kar5 indicates that there are fundamental differences between unisexual and bisexual reproduction, and between bisexual reproduction of the two sibling species. Cell fusion and karyogamy are largely dispensable for unisexual reproduction, and the unisexual cycle achieves diploidization early during hyphal development, likely through endoreplication. During the bisexual cycles, in *C. deneoformans*, karyogamy occurs early during the hyphal development whereas it occurred inside the basidium in *C. neoformans*.
Wednesday, March 15 3:00 PM – 6:00 PM

Fred Farr Forum

Circadian Rhythms and Photobiology

**Phytochrome-dependent light signaling in Aspergillus nidulans.** R. Fischer, A. Ali, Ch. Streng, Z. Yu Dept of Microbiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, BW, DE.

Bacteria often use two component systems (TCS) as phosphorylation relays to transmit environmental signals from the cell surface to the inner cell. In comparison, microbial eukaryotes use MAP kinase phosphorylation cascades, although TCS are commonly found in the fungal kingdom¹. Interestingly, in the case of stress-sensing, fungi use a composite signaling cascade comprised of a TCS plus a downstream MAP kinase cascade to trigger gene expression. Besides osmolarity or oxidative stress, fungi sense many other environmental factors, one of which is light²,³. Light controls morphogenetic pathways but also the production of secondary metabolites such as penicillin. Here we show that phytochrome-dependent light signaling in *Aspergillus nidulans* uses the stress-sensing signaling cascade to transmit the signal from the cytoplasm into nuclei⁴. In a screening for recessively blind mutants, the MAP kinase HogA/SakA was identified by whole-genome sequencing. The phytochrome FphA physically interacted with the histidine-containing phosphotransfer protein YpdA and caused light-dependent phosphorylation of the MAP kinase HogA/SakA and its shuttling into nuclei. In the absence of FphA, HogA/SakA still responded to osmotic stress but not to light. The HogA pathway thus integrates several stress factors and can be considered as a hub for environmental signals. We present evidence that conformational changes of FphA lead to the initiation of the signaling process starting with the phosphotransfer from YpdA to the FphA response regulator. In a screening for dominant mutants, in which light-dependent genes are expressed constitutively, a PHD-finger protein was identified, probably acting as a repressor.

1. Bahn, Y., Xue, C., Idnurm, A., Rutherford, J. & Cardenas, M. E. Sensing the environment: lessons from fungi. *Nat. Rev. Microbiol.* **36**, 57-69 (2007).

2. Rodriguez-Romero, J., Hedtke, M., Kastner, C., Müller, S. & Fischer, R. Fungi, hidden in soil or up in the air: light makes a difference. *Annu. Rev. Microbiol.* **64**, 585-510 (2010).

3. Dasgupta, A., Fuller, K. K., Dunlap, J. C. & Loros, J. J. Seeing the world differently: variability in the photosensory mechanisms of two model fungi. *Environ. Microbiol.* **18**:5-20 (2015).

4. Yu, Z., Armant, O. & Fischer, R. Fungi use the SakA/HogA pathway for phytochrome-dependent light signaling. *Nature Microbiol.* **1**:16019 (2016).

**Light conservatively regulates the ergosterol biosynthetic pathway in the mold pathogens Aspergillus and Fusarium.** Kevin Fuller¹, Mike Zegans², Robert Cramer³, Jennifer Loros⁴, Jay Dunlap¹ 1) Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH; 2) Department of Ophthalmology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; 3) Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH; 4) Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH.

We have shown that light serves as an important environmental cue that regulates growth, development, and stress resistance in the mold pathogen *Aspergillus fumigatus* (Fuller et al., 2013 & 2016). Whole-genome microarray analysis revealed that several genes in the ergosterol biosynthetic pathway are down-regulated by light in this fungus, including those encoding the sterol 14-demethylase (Erg11/Cyp51b), the hydroxymethylglutaryl CoA synthase (Erg13), and the C4-methyl sterol oxidase (Erg25). qRT-PCR analysis confirmed the light-repression of these genes in the two commonly studied *A. fumigatus* strains, Af293 and CEA10. Furthermore, the influence of light on their expression was lost upon deletion of the *lreA* (blue light receptor) and *fphA* (red light receptor), indicating that these discrete photosystems cooperatively regulate sterol metabolism. Because Erg11 is the target of the azole class of antifungals, we next hypothesized that the light environment could augment the sensitivity of *A. fumigatus* to these drugs. Indeed, E-test and microbroth dilution assays demonstrated that the fungus was more sensitive to voriconazole when cultured in the light. We reasoned that the influence of light on fungal sensitivity to sterol-targeting drugs may be particularly relevant in the context of fungal eye infections (keratitis). Accordingly, we sought to determine whether the light-repression of ergosterol genes observed in *A. fumigatus* was conserved in species commonly associated with fungal keratitis (namely *Fusarium* species). We identified orthologs of *erg11*, *erg13*, and *erg25* for both *F. oxysporum* and *F. solani* and found that, in both species, all three genes were down-regulated upon light exposure when assessed by qRT-PCR. Taken together, these data suggest that light may play a conserved role in regulating sterol metabolism in fungi and that this could be exploited to a therapeutic end in certain clinical contexts.

**Circadian clock control of translation in Neurospora crassa.** K.D. Castillo, S.Z. Caster, D Bell-Pedersen Biology, Texas A&M University, College Station, TX.

The circadian clock controls daily rhythms in a wide variety of physiological and behavioral processes in diverse organisms, allowing anticipation of daily environmental cycles and maintenance of internal temporal order. Rhythms in mRNA accumulation are observed for up to 40% of the eukaryotic genome. Interestingly, about 50% of the oscillating proteins in the mouse liver are produced from mRNAs that are not clock-controlled, suggesting a prominent role for clock regulation of post-transcriptional processes. While the basic mechanisms of circadian transcriptional control are known, the extent of clock control of mRNA translation is understudied. The filamentous fungus *Neurospora crassa* is a simple model organism for determining how the clock controls gene expression. In *N. crassa*, we discovered that the phosphorylation of the highly conserved translation elongation factor 2 (eEF-2), by the serine/threonine kinase RCK-2, is rhythmic in wild-type cells, but not in *Δrck-2* or clock mutant *Δfrq* cells. These data suggested that clock control of eEF-2 activity plays a key role in rhythmic translation of all, or specific, mRNAs. RNA-seq and ribosome profiling experiments from cultures harvested at different times of the day revealed that clock regulation of eEF-2 activity influences temporal translation of specific mRNAs, including glutathione-S-transferase 3 (*gst-3*). Experiments are currently underway to determine the mechanism of this specificity.
**Light-induced gene expression in *Botrytis cinerea* involves GATA-transcription factors and the stress-activated MAP kinase module.** *Julia Schumacher*¹, *Kim Cohrs*¹, *Adeline Simon*², *Jaafar Kilani*², *Muriel Viaud*², *Sabine Fillinger*² 1) IBBP, WWU, Muenster, Germany; 2) BIOGER, INRA, Grignon, France.

*Botrytis cinerea* is a plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/ pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modifiers is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs *BcWCL1* (as part of the White Collar complex (WCC)) and *BcLTF1* are important regulators as their deletions result in light-independent conidiation (“always conidia”) due to the deregulation of *BcLTF2* (Schumacher et al. 2014; Canessa et al. 2013; Cohrs et al. 2016). Study of light-induced gene (LIG) expression in both deletion mutants highlighted the role of the TFs in activating EARLY and in repressing LATE LIG expression, including *bcltf2* encoding the master regulator of conidiation and further LTFs that may function downstream or in parallel with *BcLTF2* in regulating the conidiation process. As the group of LIGs also contained genes that are induced by various stresses in a *BcSAK1*-dependent manner, the phosphorylation status of the stress-activated MAPK *BcSAK1* was studied and shown to increase after exposure to light. Deletion of *bcsak1* impairs LIG expression suggesting that *BcSAK1* functions as a co-activator of the WCC in inducing EARLY genes, and as a component releasing the LATE promoters (e.g. *bcltf2*) from repression by *BcLTF1* and the WCC resulting in the expression of the conidiation genes. This preliminary model is in agreement with the observations that the deletion of *BcSAK1* results in a “never conidia” phenotype (Segmüller et al. 2007), and that the exposure of the wild type to heat and osmotic stress bypasses the requirement of light for conidiation.

**Circadian proteomic analysis demonstrates a role for post-translations mechanisms in circadian regulation.** *Jennifer M. Hurley*¹,
*Alexander Crowell*², *Sam Fordyce*¹, *Sam Purvine*³, *Errol Robinson*³, *Anil Shukla*³, *Erika Zink*³, *Scott Baker*³, *Jennifer Loros*⁴, *Jay Dunlap*² 1) Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY; 2) Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; 3) Environmental Molecular Sciences Laboratory, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA; 4) Department of Biochemistry and Cellular Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.

The fundamental model of the eukaryotic circadian clock is comprised of a transcriptional/translational negative-feedback loop that controls rhythmic transcription of a substantial fraction of the transcriptome. Based on this model, the prevailing hypothesis in circadian biology has been that by tracking transcriptional rhythms over circadian time, a fundamental understanding of how circadian output mediates behavior, physiology, and metabolism can be obtained. However, data shows that concordance between mRNA and protein levels, including the relationship between circadianly-oscillating mRNAs and proteins, is far from a one-to-one correlation. Therefore, in order to better understand the relationship between circadian output at the level of expression and the level of the proteome, we used Tandem-Mass-Tag Mass Spectrometry to identify global protein levels over a period of 48 hours with a resolution of 2 hours. When we compared this data to our previously-published transcriptomic data, we found significant discordance between rhythmic mRNAs and proteins, demonstrating there are additional factors that determine rhythmicity at the protein level outside of transcription. However, despite this discordance, many trends that are present at the transcriptional level are mirrored at the proteomic level, including that daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. When we analyzed these rhythmic proteins using Gene Ontology (GO), we demonstrated that circadianly-regulated proteins were enriched in many ontological classifications with a great deal of that enrichment in metabolic categories. Due to the strong link between the clock and metabolism, we analyzed proteomic oscillations in a strain that had the gene *csp-1* knocked out, as *csp-1* has been shown to be a potential link between the circadian clock and metabolism. We found that when *csp-1* is knocked out, many metabolic genes are no longer rhythmic, demonstrating that *csp-1* has significant control over the cellular output that is regulated by the circadian clock.

**Light controls dimorphic transitions in the human fungal pathogen *Histoplasma capsulatum*.** *S. Beyhan*¹,², *M. Voorhies*², *A. Sil*² 1) J. Craig Venter Institute, La Jolla, CA; 2) University of California, San Francisco, San Francisco, CA.

*Histoplasma capsulatum* (*Hc*), a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent host. In the soil, *Hc* grows in a filamentous form. Once inhaled, the filaments and the associated spores convert to a yeast form that expresses virulence genes and causes disease. Host temperature is the key signal that triggers a developmental switch in *Hc*. In the laboratory, cells grow in the filamentous form at room temperature (RT), whereas growth at 37°C is sufficient to trigger growth in the yeast form. Through previous studies, we identified four key regulators, Ryp1,2,3,4, that are required for yeast-phase growth at 37°C. Ryp1 and Ryp4 are transcriptional regulators that are conserved in the fungal kingdom. Ryp2 and Ryp3 are both members of the Velvet protein family, which regulates development and secondary metabolism in many fungi. Interestingly, in *A. nidulans*, Velvet family proteins can form a complex with various photosensors to regulate sexual and asexual development in response to light. In *Hc*, the role of light in regulating cell morphology and virulence has not been fully explored. Furthermore, it is unknown whether Velvet family proteins are involved in light response in *Hc*. In this study, our goal is to investigate the role of light in the regulation of phenotypic traits in *Hc*. In our preliminary studies, we found that conidiation was dramatically affected by light. Additionally, we found that the yeast-to-filament transition of *Hc* is light-regulated. Wild-type yeast cells (grown at 37°C) were shifted to RT under light and dark conditions. Morphological changes were monitored by microscopy and transcriptional changes were monitored by RNAseq. As a result, we found that cells that were grown in the dark persisted in the yeast form for a longer period of time before they ultimately switched to filaments. These morphological changes were accompanied by changes in transcript levels of *RYP* genes. We found that *RYP2* and *RYP4* transcript levels were lower in light compared to dark conditions at RT, suggesting that accumulation of these transcripts is light-responsive. Overexpression of *RYP2* or *RYP4* in light conditions was sufficient to prevent the yeast-to-filament transition at RT. Taken together, our results suggest that dark promotes (or light inhibits) yeast-phase growth via regulation of Ryp transcript accumulation, and that light is an important environmental signal for the physiology of *Hc*.
**Facultative heterochromatin at the clock gene frequency.** William Belden Dept of Animal Science, Rutgers, The State University of New Jersey, New Brunswick, NJ.

The circadian clock allows organisms to anticipate daily changes in environmental conditions and controls developmental and physiological processes. In *Neurospora*, the core mechanism of the circadian clock is a transcriptional negative feedback loop where White Collar-1 (WC-1) and WC-2 drive rhythmic expression of the *frequency* (*frq*) gene. Part of circadian clock-regulated gene expression involves facultative heterochromatin at the central clock gene; A process that is conserved in species ranging from *Neurospora* to mammals. Facultative heterochromatin formation at *frq* is distinct from heterochromatin at repeated regions throughout the genome and requires the *frq* natural antisense transcript (NAT) *qrf*, components of the COMPASS complex, including the histone H3 lysine 4 methyltransferase SET1, SWD1, and SWD3. Despite these differences, there still a requirement for components of the DCDC complex including the H3 lysine 9 methyltransferase DIM-5. The requirement for both H3K4me and H3K9me in proper regulation of *frq* has led us to examine the genome-wide importance of these modifications in transcriptional regulation. Efforts are currently underway to further define the molecular mechanisms of facultative heterochromatin at *frq* and attempts are being made to understand the enigmatic role of COMPASS.

**Investigating the complex regulatory network that controls photoinduction of carotenogenesis in *Fusarium*.** Javier Avalos¹,
Obdulia Parra-Rivero¹, Javier Pardo-Medina¹, Macarena Ruger-Herreros¹, Steffen Nordzieke¹, Francisco J. Romero-Campero², M. Carmen Limón¹ 1) Genetics, University of Seville, Seville, ES; 2) Computing Sciences and Artificial Intelligence, University of Seville, Seville, ES.

Carotenoids are terpenoid pigments produced by many fungi. As *Neurospora crassa*, species of the genus *Fusarium* accumulate neurosporaxanthin and other carotenoids under light. This production is due to the induced expression of the structural genes *carRA*, *carB*, and *carT*, needed to synthesize neurosporaxanthin. The response is accompanied by the induction of the genes *carX* and *carO*, needed to produce retinal and CarO rhodopsin and organized in a cluster with *carRA* and *carB*. This communication deals with our current knowledge of the molecular mechanisms of this photo-regulation in *F. fujikuroi* and *F. oxysporum*. In contrast to *N. crassa*, carotenogenesis is still able to respond to light in the mutants of the White collar 1 ortholog WcoA, although this protein is mostly responsible for the transcriptional photoinduction. Kinetics experiments on the response to light revealed a biphasic response in *F. fujikuroi*, with an early stage mostly dependent of WcoA and a later stage in which participates the DASH cryptochrome CryD, both stages modulated by the Vvd ortholog VvdA. In addition, the pathway is down-regulated by the RING-finger protein CarS. RNAseq analyses showed that either light or the loss of *carS* function exert mainly an activating effect on the transcriptome of *Fusarium*, with a greater functional diversity in the case of the genes induced by light. The number of the latter decreases drastically in the *carS* mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the *carS* mutation affects the light response of many genes. Approximately 27% of the genes activated at least 2-fold by light or by the *carS* mutation are coincident, raising to 40% for an activation threshold of 8-fold, confirming regulatory connections between the regulations by light and by the CarS protein. Upstream to *carS*, we found a previously unidentified transcript, presumably a long non-coding RNA, whose deletion abolishes the photoinduction of the structural genes. Moreover, the deletion of another linked gene coding for a protein of unknown function results in a lower carotenoid accumulation, which was not due to a decreased mRNA photoinduction of the structural genes. Therefore, the available data point to a complex regulation exerted by different interacting elements, either at transcriptional and post-transcriptional levels.
Wednesday, March 15 3:00 PM – 6:00 PM

Kiln

**Primary Metabolism and Metabolic Engineering**

**Expansions and reductions in fungal primary metabolism studied across 100 fungal species.** Julian Brandl, Jane Nybo, Tammi C.

Vesth, Mikael Rørdam Andersen Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs Lyngby, DK.

The primary metabolism of fungi is the power house that drives nearly all cellular functions. Primary metabolism is involved in converting the surroundings of the fungus to a food source as well as delivering metabolite precursors for everything from cellular growth and maintenance over biological responses to external stimuli to producing secreted secondary metabolites and protein effectors. Furthermore, it is known that fungal metabolism is highly versatile. Saphrophytic fungi can grow on a very wide range of carbon and nitrogen sources and utilize this for production of even more diverse range of secondary metabolites and secreted proteins.

Within this project, we have been interested in two main things: 1) What is the "roadmap" of fungal metabolism within a single species? What is the total sum of genes involved in primary metabolism? For studying this, we have been focusing on the industrial workhorse and model fungus Aspergillus niger. 2) What is the diversity of metabolism across fungi? Which gene functions are expanded, reduced, added and lost across species? For this we have used the metabolic "roadmap" of *Aspergillus niger*, and used it to query the genomes of approximately 100 different species, primarily of the genus Aspergillus.

For the initial roadmap of fungal metabolism, we have reconstructed the metabolism of *A. niger* ATCC 1015 at genome-scale. The metabolic reconstruction covers 1801 metabolic conversions, 997 genes, and 1411 metabolites across six compartments. Phenotype arrays have been applied to evaluate the ability to germinate on 180 carbon sources and 92 nitrogen sources. Examining the metabolism shows a high degree of isoenzymes across both central and outer metabolism.

Employing this metabolic network to our database of fungal genomes allows us to examine the diversity of metabolic strategies through the identification of orthologs across species and map this to the species. Our analysis shows that some specialized fungi have a more reduced genome and survives with 1-2 isoenzymes for most of central metabolism, while others apply a diversification strategy and often have 2-5 isoenzymes, even for the highly conserved functions in central metabolism.

**Engineering the smut fungus *Ustilago maydis* for the degradation of pectin.** P. Stoffels¹,³, E. Geiser²,³, L.M. Blank²,³, M.

Feldbrügge¹,³, N. Wiercx²,³, K. Schipper¹,³ 1) Institute for Microbiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany ; 2) iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany ; 3) Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich, 52425 Jülich, Germany.

The microbial conversion of plant biomass components to valuable products in a consolidated bioprocess would greatly increase the ecologic and economic impact of a biorefinery. Plant-pathogenic fungi are promising candidates for biomass valorization, because they contain a vast repertoire of hydrolytic enzymes to sustain their lifestyle. However, expression of the corresponding genes is usually tightly regulated and mostly restricted to the pathogenic phase. We use the biotrophic smut fungus *Ustilago maydis* for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is equipped with a potent set of hydrolytic enzymes, and moreover, it naturally produces value-added substances such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of biomass components like xylan, cellobiose, and polygalacturonic acid to fermentable sugars. Currently, the intrinsic fungal repertoire is activated and in parallel supplemented with potent heterologous enzymes for the degradation of pectin. Importantly, this can be achieved using both conventional secretion for enzymes of eukaryotic and unconventional secretion for enzymes of bacterial origin. In summary, our results demonstrate the potential applicability of activating the expression of native hydrolytic enzymes from phytopathogens in a biocatalytic process.

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

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

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

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

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

**Identifying carbohydrate sensing pathways in Neurospora crassa.** L.B. Huberman, S.T. Coradetti, N.L. Glass Plant and Microbial Biology, UC Berkeley, Berkeley, CA.

Identifying and utilizing the nutrients available in the most efficient manner is a challenge common to all organisms. The model filamentous fungus, Neurospora crassa, is capable of utilizing a variety of carbohydrates: from simple sugars to the complex sugar chains found in plant cell walls. Expression of the genes necessary for degrading these complex carbohydrates is energetically expensive, so tight regulation of these genes is important. Several transcription factors necessary for cellulose utilization in N. crassa have been identified, but the method by which these transcription factors are activated is not clear. We identified a novel repressor of the cellulolytic response, which in combination with other, more general nutrient sensing pathways appears to play an important role in regulating cellulase expression in N. crassa.

**Global transcriptional regulation and chromatin dynamics of primary metabolism in Aspergillus nidulans.** Koon Ho Wong¹, Ang Li¹, Yingying Chen¹, Liguo Dong¹, Md Ashiqul Alam³, Fang Wang¹, Kaeling Tan¹, Michael Hynes⁴, Joan Kelly³, Richard Todd² 1) Faculty of Health Sciences, University of Macau, Macau; 2) Department of Plant Pathology, Kansas State University, KS; 3) Department of Genetics and Evolution, School of Biological Science, The University of Adelaide, Australia; 4) School of BioSciences, University of Melbourne, Australia.

Fungi adapt their metabolism to nutrient availability primarily via transcriptional control of nutrient uptake and metabolic genes. In Aspergillus nidulans, genes involved in carbon and nitrogen metabolism are tightly controlled by carbon catabolite repression (CCR) and nitrogen metabolite repression (NMR), respectively. Two key global regulators are involved in CCR and NMR; the repressor CreA controls expression of carbon metabolic genes, while the activator AreA modulates nitrogen metabolic gene expression. An extensive set of genes is expected to be under the control of the two regulators, which often act in concert with different pathway-specific transcription factors to turn on different subsets of metabolic genes according to nutritional conditions. In an attempt to identify the genome-wide targets of the global and pathway-specific regulators and to learn how they collaborate with each other to bring about the regulation, we have mapped whole-genome binding sites of the two global regulators and some pathway specific factors under different nutrient conditions and integrated the information with genome-wide transcription profiles of wildtype, *creA* and *areA* strains. In addition, we have also mapped global chromatin dynamics and chromatin modifications under different nutrient conditions in order to understand the mechanism of regulation at the chromatin level. A summary of CreA and AreA global regulation and chromatin dynamics will be presented.

**Aspergillus fumigatus carbon catabolite repression is essential for virulence in established infection microenvironments.** S. Beattie¹, K. Mark², L. Reis³, S. Dhingra¹, C. Black⁶, C. Cheng²,⁴,⁵, G. Goldman³, R. Cramer¹ 1) Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH USA; 2) Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH USA; 3) Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil; 4) Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH USA; 5) Institute for Quantitative Biomedical Sciences, Geisel School of Medicine at Dartmouth, Lebanon, NH USA; 6) Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH USA.

**Purpose:** The filamentous fungus Aspergillus fumigatus is responsible for a disproportionate number of cases of invasive mycoses, relative to other filamentous fungi found in the environment in similar or even higher quantities. Therefore, unique aspects of A. fumigatus biology must account for the ability to grow within the host and cause disease. Here, we hypothesize that carbon availability during an infection is dynamic, and the ability to optimize carbon utilization within the host is critical for optimal virulence throughout the fungus-host interaction.

**Methods:** To test this hypothesis, we characterized the carbon catabolite repression (CCR) system of A. fumigatus through generation of a creA genetic null mutant. Murine models of invasive aspergillosis were used to assess the contribution of CCR to pathogenesis. Transcriptomics, metabolomics, and in vitro growth and stress susceptibility assays were further utilized to determine the role of CCR in A. fumigatus fitness.

**Results:** In contrast to what has been observed in yeasts, we identified a novel role for CreA and CCR in the virulence of A. fumigatus. While CCR was not required for the establishment of infection, loss of CreA dramatically reduced virulence after infection establishment. Therefore, we hypothesized the virulence defect to be a result of altered *in vivo* host carbon metabolism and observed a critical role for CreA in controlling redox homeostasis in the face of a dynamic nutrient environment.

**Conclusions:** Collectively, our results support the conclusion that transcriptional regulation of fungal bioenergetics to optimize *in vivo* utilization of carbon and nitrogen sources is critical for human fungal virulence.

**Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi.** F. Bi², S. Barad¹,³, A Dubay¹, D. Kumar¹, J.D. Mínguez⁴, E.A. Espeso⁵, R. Fluhr⁶, D. Prusky¹ 1) Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Rishon LeZion, IL; 2) Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, and Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture, Guangzhou 510640, China; 3) Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel; 4) Department of Microbiology and Genetics, CIALE, Universidad de Salamanca, Salamanca, Spain; 5) Department of Molecular and Cellular Biology, Centro de Investigaciones Biológicas (C.I.B.), Madrid, Spain; 6) Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot.

Fruit pathogens can either acidify or alkalinize the pH of the colonized host environment. This capability has been used to divide fungal pathogens into acidifying and alkalinizing classes. In the present study, we show that diverse classes of fungal pathogens, including; *Colletotrichum gloesporioides*, *Penicillium expansum*, *Aspergillus nidulans*, *Sclerotinia sclerotiorum*, and *Fusarium oxysporum*—secrete small pH-effector molecules. These molecules modify the environmental pH that dictates acidic or alkaline colonization patterns and select for the activity of PACC-dependent genes. We show that the secretion of pH-effector molecules that contribute to fruit pathogenicity is carbon-dependent. In multiple cross-class organisms, acidification is induced under excess, e.g. 175 mM sucrose. This carbon source is
oxidized by glucose oxidase (*gox2*) to gluconic acid, contributing to medium acidification. In contrast, alkalization occurs under conditions of carbon deprivation, e.g., less than 15 mM sucrose, where catalyzed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (*gdh2*) results in the secretion of ammonia. Functional *?gdh2* mutants show reduced alkalization and pathogenicity during growth under carbon deprivation, but not in high-carbon media or on fruit rich in sugar, whereas analysis of *?gox2* mutants show reduced acidification in carbon-rich media. The induction pattern of *gdh2* was negatively correlated with expression of the zinc finger global carbon regulator repressor *creA*. The present results indicate that differential pH modulation by fungal pathogens is a universal host-dependent mechanism that modulates environmental pH as a tool to enhance host plant colonization.

**Role of the urea cycle in the synthesis of nitric oxide.** *D. Canovas*¹,², A.T. Marcos¹, J.F. Marcos³, T. Schinko², J. Strauss² 1)

Department of Genetics, University of Sevilla, Sevilla, Spain; 2) Division of Microbial Genetics and Pathogen Interactions, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Sciences Vienna, Austria; 3) Department of Food Science, Institute of Agrochemistry and Food Technology (IATA), Valencia, Spain.

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

**Reference:**

Marcos AT, Ramos MS, Marcos JF, Carmona L, Strauss J, Cánovas D. Nitric oxide synthesis by nitrate reductase is regulated during development in *Aspergillus*. Mol Microbiol (2016) 99:15-33.
Wednesday, March 15 3:00 PM – 6:00 PM

Heather

## Magnitude of Fungi Biodiversity

**Continental-level population differentiation and environmental adaptation in the mushroom *Suillus brevipes*.** S. Branco¹, K. Bi², H.-L. Liao³, P. Gladieux⁴, H. Badouin¹, C. Ellison⁵, N. Nguyen⁶, R. Vilgalys⁷, K. Peay⁸, J. Taylor⁹, T. Bruns⁹ 1) Departement Genetique et Ecologie Evolutive, Universite de Paris Sud, Orsay, FR; 2) Computational Genomics Resource Laboratory, University of California, Berkeley, CA; 3) North Florida Research and Education Center, University of Florida, Quincy FL; 4) INRA, UMR BGPI, Montpellier, FR; 5) Department of Genetics, Rutgers University, Piscataway, NJ; 6) Department of Tropical Plant and Soil Sciences, University of Hawai'i at Manoa, Honolulu, HI; 7) Department of Biology, Duke University, Durham, NC; 8) Department of Biology, Stanford University, Stanford, CA; 9) Department of Plant and Microbial Biology, University of California, Berkeley, CA.

Recent advancements in sequencing technology allowed researchers to better address the patterns and mechanisms involved in microbial environmental adaptation at large spatial scales. Here we investigated the genomic basis of adaptation to climate at the continental scale in *Suillus brevipes*, an ectomycorrhizal fungus symbiotically associated with the roots of pine trees. We used genomic data from 55 individuals in seven locations across North America to perform genome scans to detect signatures of positive selection and assess whether temperature and precipitation were associated with genetic differentiation. We found that S. brevipes exhibited overall strong population differentiation, with potential admixture in Canadian populations. This species also displayed genomic signatures of positive selection as well as genomic sites significantly associated with distinct climatic regimes and abiotic environmental parameters. These genomic regions included genes involved in transmembrane transport of substances and helicase activity potentially involved in cold stress response. Our study sheds light on large-scale environmental adaptation in fungi by identifying putative adaptive genes and providing a framework to further investigate the genetic basis of fungal adaptation.

**Genetic diversity in fungal plant pathogens: interspecific gene exchange, genome plasticity and mobile elements.** Eva

Stukenbrock Environmental Genomics, Max Planck Inst Evolutionary Biology, Plön, Germany.

Population genomic data provide information about past and ongoing evolutionary dynamics of plant pathogens. In the recent years a large number of publications have shed light on evolutionary processes in fungal pathogens. A general conclusion from these studies is that these pathogens have the capacity to evolve very rapidly. Interspecific gene exchange, genome plasticity and mobile elements appear to be important drivers of rapid evolution and adaptation to an extent so far not observed in other Eukaryotes. We study genome evolution in a set of closely related grass pathogens of the genus *Zymoseptoria* to infer mechanisms leading to genetic diversity within and between species. The genus comprises species with distinct population structure and notably different effective population sizes. We have used population genomics analyses and coalescence models to reconstruct the population history of four *Zymoseptoria* species. An extreme case is *Z. pseudotritici*, a young hybrid species. We previously reported a striking pattern of genomic variation in 5 isolates with 1-100 kb regions with no variation at all alternating with similar size regions with 1%-5% variable sites, always arranged into two divergent haplotypes. This is consistent with *Z. pseudotritici* having arisen by hybridization of two divergent haploid spores less than 500 sexual generations ago. Our recent analyses include full genome sequences from additionally 22 isolates. We show that the loss of variation in the hybrid is highly non-random with respect to chromosomes and genes and that selection seems to maintain certain gene combinations. We show that this data set allows us to use the variable regions to study mutation accumulation since speciation, and the size distribution of regions with and without variation illustrates both the process of recombination and mutation in this species. We conclude that *Z. pseudotritici* will be an important model species for studying the consequences of hybrid speciation and that such processes may be common in fungi.

**Comparative genomics and transcriptomics of *Russulaceae*.** Brian Looney¹,², Dan Jacobson¹, Kerrie Barry³, Alan Kuo³, Igor Grigoriev³, Francis Martin⁴, Jessy Labbe¹,² 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.

**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.

**Elucidating fungal endosymbiont interaction mechanisms between *Mortierella elongata* and *Mycoavidus cysteinexigens***. Jessie Uehling¹, Fred Dietrich¹, Pawel Misztal², Gregory Bonito³, Tim Tschaplinski⁴, Allen Goldstein², Chris Schadt⁴, Jessy Labbe⁴, Rytas Vilgalys¹ 1) Genetics & Genomics, Duke University, Durham NC 27708, USA; 2) University of California at Berkeley, Berkeley, CA 94270 USA; 3) Michigan State University, East Lansing, MI 48823 USA; 4) Biosciences Division, Oak Ridge National Laboratory, PO Box 2008 MS 6445, Oak Ridge, TN 37831-6445, USA.

Recently efforts to catalog beneficial microbes associated with *Populus* have yielded cultures of diverse fungi including several *Mortierella elongata* (*Mortierellomycotina*) isolates. Some of these fungi including *M. elongata* strain AG77 harbor bacterial endosymbionts belonging to the Burkholderiales. We have sequenced the genome of *M. elongata* AG77 and its bacterial endosymbiont *Mycoavidus cysteinexigens* (AG77), and used comparative phylogenomics to infer this symbiosis is ancient and grounded in shared metabolites. We have used and developed methods to assemble single genomes from meta-genomic data and inferred several gene losses that make culturing endosymbiont *M. cysteinexigens* (AG77) challenging. To circumvent this research obstacle, we used an antibiotic treatment to create fungal strains with and without their long-term endosymbionts for comparative multi-omic studies. The ability to contrast cleared *M. elongata* strains and their wild type, endosymbiont-housing relatives in the same genetic background allows us to ask the following questions: 1. *How does the presence or absence of long-term, co-evolved endosymbionts impact health and physiology of host fungi?* 2. *Which currently available methods can be used and modified to study interaction dynamics for systems involving multiple intractable microbes?* Our research goals through this work were to develop omics and imaging pipelines that allow the study of bacterial fungal interaction mechanisms. To this end we have used genome sequencing, transcriptomics, metabolomics, and volomics for studying fungal endosymbiont interaction dynamics. The results of these experiments will be further discussed.

**Horizontal gene transfer: resolving functional diversity in fungal communities.** J.C. Slot Plant Pathology, The Ohio State University, Columbus, OH.

Horizontal gene transfer (HGT) may have multiple impacts on the functional diversity within fungal communities. HGT may increase the functional diversity among related species through recombination with foreign genetic material. However, HGT may decrease diversity at the molecular and community levels by allowing natural selection to favor the most adaptive genetic mechanisms among species in a community. Gene clusters (co-localized genes contributing to a common function) introduce additional complexity to HGT-enabled functional diversification; they increase the HGT dispersal of individual genes by providing a more complete selectable function than isolated genes, and they may enable complex functional diversity in populations when they exist as 'supergene' polymorphisms. Recent analyses suggest HGT is widespread among fungi with overlapping ecology, implying that many fungal genes and gene clusters have evolutionary ecologies independent of their host lineages. Here, we present novel genetic mechanisms discovered through targeted and systematic searches for likely HGT events involving gene clusters. Specifically, we describe a large diversity of plant secondary metabolite degradation clusters, and a biosynthetic gene cluster responsible for the production of psilocybin, a bioactive component of hallucinogenic mushrooms. We provide evidence that the ecological distributions of gene clusters and their histories of HGT events can indicate the ecological functions of plant and fungal secondary metabolites.

**The exciting content in your cup of tea.** I. Skaar¹, Y Zhang², M Sulyok⁴, X Liu⁵, M Rao⁶, J Taylor³ 1) Mycology, Norwegian Veterinary Institute, Oslo, NO; 2) School of Life Sciences, Shanxi University, Taiyuan, Shanxi, China; 3) Department of Plant and Microbial Biology, University of California, Berkeley, California, USA; 4) Center for Analytical Chemistry, Department of Agrobiotechnology, University of Natural Resources and Life Sciences, Tulln, Austria; 5) State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China and; 6) Bureau of Culture and Sports, Puer, Yunnan, China.

Tea is one of the most popular and widely consumed beverages in the world. Tea has important physiological effects on consumers due to the presence of compounds such as polyphenols, amino acids, vitamins, carbohydrates, caffeine, and purine alkaloids, all of which can have health benefits On the other hand, tea may also offer a complex microbiological diversity which can both contribute to the terroir of the tea, but also lead to presence of adverse secondary metabolites. Some teas are produced by microbiological fermentation. An example of such teas is the Pu-erh tea, produced in Yunnan, China, by fermentation of fresh *Camellia sinensis* leaves. The production and quality of Pu-erh tea is consequently closely related to microbial activity, making it important to understand the Pu-erh microbiome. The fungal diversity in teas and the corresponding fungal metabolites as revealed both by culture dependent and culture-independent approaches will be discussed.

We will present a study of fungal and bacterial communities leaves and two types of Pu-erh teas by high-throughput, rDNA-amplicon sequencing and by quantitative liquid chromatography-tandem mass spectrometry of their metabolites.

We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs. Major findings were: 1) fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2) fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3) aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4) ripened and well-aged raw, but not young, Pu-erh have similar microbial communities. Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating. We show that principles of community ecology can be applied to microbially fermented foods, that the acceleration of Pu-erh fermentation by the ripened process approximates the slower, traditional process, and that discarding the first preparation of Pu-erh tea and saving the wetted tea to brew a second cup for consumption has a sound mycotoxicological basis.
**Environment and host specificity shape a global diversity of marine fungi.** *Anthony Amend*, Kaho Tisthammer, Benjamin

Wainwright, Geoffrey Zahn University of Hawaii at Manoa, Honolulu, HI.

Fungal diversity in the marine realm has received considerably less attention than terrestrial environments, and has figured rarely into global estimates of fungal diversity. A critical mass of high-throughput sequencing datasets, however, has helped fill in some of the gaps in our knowledge of this often overlooked realm. Over the past five years, our group has examined fungal communities present in the water column, sediments, on corals, sponges and marine algae, while others have conducted systematic global scale surveys of the world's oceans. Using these datasets we address some of the most fundamental questions about marine fungal diversity and the factors that shape it. We show that environmental factors, not geographic distance, describe the majority of variance in community dissimilarly, and that habitats such as sediments, water columns and invertebrate hosts harbor dissimilar communities and unique taxa. We show evidence for host specificity of fungi associated with coral and macro-algal systems, suggesting that species-rich substrates such as coral reefs likely harbor extensive and novel fungal biodiversity. Last, we showcase the prevalence of putatively amphibious fungi, abundant in water and on land. The view that emerges from a synthetic analysis of marine fungal diversity and distributions is that while there is substantial taxonomic overlap between marine and terrestrial systems, biogeographic patterns differ between these realms.
Wednesday, March 15 3:00 PM – 6:00 PM

Nautilus

## Fungi in the Microbiome

**Crosstalk between gut microbiota and host immunity during fungal dysbiosis.** Iliyan Iliev, Jill Roberts Institute for Reserach in IBD, Department of Medicine, Weill Cornell Medicine, New York, NY.

Commensal microbiota has an enormous effect on wide range of physiological processes. Recent studies demonstrate that together with bacteria and viruses, fungi are important, although smaller, component of the intestinal microbial community. It is currently unknown whether disruption of fungal communities occupying the gastrointestinal tract would affect host immunity. We have previously shown that a polymorphism in the human gene encoding the anti-fungal receptor Dectin-1 is strongly associated with severity of Ulcerative Colitis (UC) and that in a mouse model of colitis fungi can contribute to intestinal inflammation, suggesting that anti-fungal immunity may be an important factor in intestinal inflammation.

Here we show that fungal community dysbiosis takes place during intestinal inflammation. Using in vivo modeling, targeting of gut fungi with drugs and sequencing of gut fungal communities, we identified a define signature associated with targeted or with inflammation-induced gut mycobiota dysbiosis. We further show that targeted disturbance of healthy gut fungal populations resulted in increased intestinal inflammation. We identified a pathway responsible for the regulation in these upstream immunological events. Finally, fungal dysbiosis affected gut commensal bacterial populations suggesting that these communities are interdependent. Taken together our results indicate that disruption of commensal fungal populations in the gut can influence host immunity, affect commensal bacterial communities and contribute to intestinal disease.

**The association of fungal communities with chronic non-healing wounds.** L. Kalan¹, M. Loesche¹, B. Hodkinson¹, G. Ruthel³, S. Gardner², E. Grice¹ 1) Department of Dermatology, University of Pennsylvania, Philadelphia, PA; 2) University of Iowa, College of Nursing, Iowa City, IA; 3) School of Veterinary Medicine, PennVet Imaging Core, University of Pennsylvania, Philadelphia, PA.

Wounds are an under-appreciated but serious complication for a diverse spectrum of diseases. Wound healing requires the execution of highly coordinated and sequential events. Increased microbial bioburden can stall this process in the inflammatory stage resulting in chronic delayed healing, even in the absence of overt infection. As a result, chronic wounds such as diabetic foot ulcers (DFUs) have been heardalied a silent epidemic causing significant morbidity and mortality. Advances have been made characterizing the bacterial component of DFUs but few studies have evaluated the fungal component. We longitudinally profiled 100, non-healing DFUs with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus. DFUs were sampled every 2 weeks until wound closure, amputation, or the end of the study (26 weeks). We estimate that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The mycobiome exhibited high levels of temporal instability and heterogeneity between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. Proportions of the phlyum Ascomycota were significantly greater (p=0.015) at the study onset in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal communities (p=0.0001), while a second major group of taxa identified as allergenic filamentous fungi, were associated with low levels of systemic inflammation. Microbe-microbe interactions are hypothesized to promote pathogenesis that impairs wound healing thus we employed directed culturing of wound specimens stably colonized by pathogens. This approach revealed novel inter-kingdom interactions faciliating biofilm formation of *Candida albicans* with *Citrobacter freundii* and *Trichosporon asahii* with *Staphylococcus simulans*. This study reports that a large proportion of DFU's are colonized by fungal species comprising ubiquitous environmental moulds, skin associated species, and opportunistic pathogens. Little is known about their contribution to stalled healing or interactions with the bacterial microbiome, and may be recognized as important targets for intervention.

**Fungi in the built environment.** Rachel Adams, Despoina Lymeropoulou, Iman Sylvain, John Taylor, Tom Bruns Plant & Microbial Biology, University of California Berkeley, Berkeley, CA.

Built environments harbor an array of fungi to which occupants are exposed. While most fungi we encounter indoors likely do not affect our health, we would like to understand better the situations in which fungi and the built environment interact to affect occupant wellbeing. I will present recent work we are doing to characterize fungi (and bacteria) in residences. In water-damaged buildings, we are working to identify fungi that have originated indoors, for this is key to understanding what is harmful about water-damaging housing. Moreover, the biological state of the fungi (and other microorganisms) detected in these indoor environments through amplicon-based microbiome surveys is often unknown. We have undertaken sampling of different environments within typical residences (non-water damaged) and applied various molecular approaches to try to better understand if the microbes encountered are alive, dormant, or dead. The fungi in wet settings, such as kitchens and bathrooms, as well as dry areas, such as bedrooms and living rooms, were collected and processed using techniques that detect cells with intact membranes (PMA) or that are metabolically active (RNA). These results will help inform to what extent built environments are true ecological habitats or more often an ecological sink of outdoor-sourced fungi. Our work seeks to inform the causes and consequences of fungi in the built environment.

**The structure and function of fungal decomposer communities.** J.M. Talbot¹, C.A. Zeiner¹, R Bandy¹, S. Rainsford¹, D. Segre¹, D. Cullen² 1) Biology, Boston University, Boston, MA; 2) USDA Forest Products Laboratory, Madison, WI.

Niche differentiation among species is a key mechanism by which microbial biodiversity may be linked to ecosystem function. We tested a set of widely invoked hypotheses about the extent of niche differentiation in one of the most diverse communities on Earth – decomposer microorganisms – by measuring their response to changes in three abundant litter resources; lignin, cellulose, and nitrogen (N). To do this, we used the model system *Arabidopsis thaliana* to manipulate lignin, cellulose, and N availability and then used high-throughput sequencing to measure the response of microbial communities during decay in the field. In addition, we grew select species in the lab and measured their resource use profiles, as well as competitive ability on *Arabidopsis* litter.

Low concentrations of lignin, cellulose, and N in the litter each caused unique shifts in decomposer community composition after one year
of decay. Fungal communities were dominated by taxa with unique and complementary substrate use profiles. Competitiveness of species in the field mirrored competitive outcome in the laboratory, with species showing unique enzymatic profiles on litter in microcosms. While growth in culture was related to genes coding for metallopeptidases, competitive dominance was achieved by species with high numbers of genes coding for glycosyl hydrolases. Competitive dominance also correlated with increased secretion of fungal hydrocarbons onto litter, suggesting that fungal metabolites and resource exploitation influenced decomposer community composition during decay. Our results contrast the assumption in ecosystem ecology that major C and N degradation mechanisms are uniform across whole decomposer communities and instead suggest that ecosystem functions arise from complementary among groups of dominant and metabolically diverse taxa.

**Intestinal mycobiome variation across geography and phylogeny in the snail genus *Conus*.** A. Quandt, A. Glasco, T.

James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

This project seeks to use the extensive collections of a hyper-diverse, predatory genus of marine snails, *Conus*, at the University of Michigan Museum of Zoology to fill a gap in our understanding of the fungi that associate with mollusks and how the mycobiome (fungal community) varies across geographic space, phylogenetic diversity, and ecological niches of the hosts. Several species of *Conus* (including *C. ebraeus*), have near global distributions and are particularly well represented in the collections, yet we have no knowledge of whether the mycobiome of such animals will be driven by their geographic location or their phylogenetic relationships. To determine mycobiome compositions, we dissected the intestines and digestive gland tissues from more than 30 *Conus* spp. from over 20 different countries, extracted DNA, and used fungal-specific amplicon-based high throughput sequencing of the ribosomal DNA region. First, we identify which fungi are commonly and uniquely associated with the digestive tracts of *Conus* spp., all of which are dominated by *Malassezia* spp. We also explore whether *Conus* phylogeny or geography causes greater variation in fungal community structure, and discuss techniques for targeted sequencing of preserved museum specimens for studies.

**Microbial communities associated with external fur surfaces of bats and the discovery of novel isolates with antifungal activity against *Pseudogymnoascus destructans*.** Paris Hamm¹, Shiloh Lueschow², Ara Winter³, Nicole Caimi³, Diana Northup³, Ernest Valdez⁴, Debbie Buecher⁵, Christopher Dunlap², David Labeda⁶, Andrea Porras-Alfaro¹ 1) Department of Biological Sciences, Western Illinois University, Macomb, IL; 2) Crop Bioprotection Research Unit, US Department of Agriculture, Peoria, IL; 3) Department of Biology, University of New Mexico, Albuquerque, NM; 4) United States Geological Survey, Fort Collins Science Center, Albuquerque, NM; 5) Buecher Biological Consulting, Tucson, AZ; 6) Mycotoxin Prevention and Applied Microbiology Research Unit, US Department of Agriculture, Peoria, IL.

White-nose Syndrome (WNS), a bat fungal disease caused by the psychrophilic fungus *Pseudogymnoascus destructans*, has been estimated to have caused the death of more than six million bats in the eastern U.S. and Canada. Fungal and bacterial surveys were conducted to explore bats' natural microbial communities as a possible defense against this pathogen. Our WNS research efforts have mainly focused on the study of microbial communities associated with bats using next generation sequencing, metagenomic analysis, and the evaluation of cultured Actinobacteria to determine antifungal activity against *P. destructans*. Metagenomic analysis showed that fungi on bat fur surfaces belong to Chytridiomycota, Ascomycota, Basidiomycota, Glomeromycota, and Neocallimastigomycota. Distinct fungal and bacteria communities were detected on WNS (+) bats from Illinois and Indiana and WNS (-) bats from New Mexico and Arizona using 454 pyrosequencing. Further, we evaluated the antifungal potential of naturally occurring Actinobacteria isolated from WNS (-) bats. Bacteria colonizing bat fur and membranes were isolated from 12 healthy bat species providing approximately 2,700 isolates. We have screened over 1,300 bacterial isolates using a bi-layer method, of which 36 isolates show antifungal activity against *P. destructans*. Of the antifungal producing Actinobacteria, 32 (89%) were from the genus *Streptomyces*, known for their antibiotic production. Fifteen of the isolates with antifungal activity against *P. destructans* were identified as novel *Streptomyces* species after morphological and multi-gene phylogenetic analysis. This study includes one of the largest surveys of microbial communities associated with healthy bats. Our results show that bats in western North America possess novel bacterial microbiota with the potential to inhibit *P. destructans*.

**Functional ecology of mycobiome shifts associated with plant senescence - linking environmental and experimental re-synthesis metatranscriptomics.** Ko-Hsuan Chen¹, Hui-Ling Liao², Hailee B. Korotkin³, P. Brandon Matheny³, Francois Lutzoni¹ 1) Department of Biology, Duke University, Durham, NC, USA; 2) Soil and Water Sciences Department, University of Florida, Gainesville, FL, USA; 3) Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN, USA.

The same fungi are often found in healthy and dead tissues of the same plant species. Because plant tissues at different stages of senescence are usually separated by time and space, tracking functional transitions of their mycobiomes is often confounded with extraneous factors. The perennial moss *Dicranum scoparium* has a growth form with a continuous senescence gradient, making it an ideal system to study fungal trophic transitions. Our goal is to understand the function of fungi associated with healthy photosynthetic tissues to advanced states of decomposition. A metatranscriptomic approach was first applied to obtain the metabolic (rRNA) and functional (mRNA) activities of fungi associated with top (photosynthetic), middle (senescent), and bottom (decomposing) layers for the naturally occurring *D. scoparium*. Our study of nutrient-related gene expression in each layer, revealed that the highly expressed genes in the top layer are mostly related to carbon exchange, whereas in the middle and bottom layers we also found highly expressed genes that are involved in nitrogen and phosphate transport. In parallel to our metatranscriptomic study of environmental samples of *D. scoparium*, we isolated fungal strains from the three layers of this moss and grew *D. scoparium* in vitro from spores. By comparing and integrating the fungal rRNA sequences generated by metatranscriptome and culture-based approaches, we assigned fungi into three types of association categories: 1) High activity in photosynthetic tissues, 2) High activity in decomposing tissues and 3) Low activity throughout the gametophyte but abundant when using culture-based detection. Based on these categories, seven fungal strains representing distinct fungal lineages (four Ascomycota, two Basidiomycota, one Mortierellomycota) were selected to establish fungus-plant re-synthesis pairs in the laboratory with both living and dead gametophytes. For every fungus-plant pairs, growth rates were monitored for two months after fungal colonies reached the moss. Metatranscriptomes of these fungus-plant pairs were obtained at the end of this period.
**Fungal-bacterial interactions drive community assembly in cheese rind microbiomes.**

Cosetta, Erik Kastman Biology, Tufts University, Medford, MA.

Benjamin Wolfe, Yuanchen Zhang, Casey

Despite a growing appreciation of the widespread co-occurrence of bacteria and fungi in many microbiomes, mechanisms that drive the ecology of bacterial species are largely studied while excluding neighboring microbial eukaryotes. My lab is using tractable microbiomes based on fermented foods to identify the processes and molecular mechanisms by which diverse fungi can shape the ecology of bacterial species and communities. We first use in situ metagenomics and culture-based approaches to identify putative bacterial-fungal interactions in naturally forming communities. We then combine in vitro community reconstructions, comparative genomics, transcriptomics, and metabolomics to identify the molecular mechanisms driving fungal-mediated bacterial competition and cooperation. Results from a variety of cheese rind biofilm models illustrate the diverse ways that fungi drive bacterial community development. In biofilms dominated by closely-related *Staphylococcus* species, the fungus *Scopulariopsis* allows *Staphylococcus* species that are weak competitors and colonizers to outcompete neighbors by altering iron and amino acid availability. In biofilms composed of both non-motile Firmicutes and Actinobacteria as well as motile Proteobacteria, the biophysical networks formed by *Mucor* can facilitate the dispersal of motile community members, leading to communities dominated by Proteobacteria. Most recently, we have found that volatile organic compounds produced by fungi can impact the growth of individual bacterial species and alter the development of multispecies bacterial communities. Collectively, our results demonstrate how fungi can mediate bacterial competition and community development in multispecies microbiomes through a variety of contact-dependent and contact-independent mechanisms. Ongoing work is exploring how manipulation of the bacterial-fungal interactions we have identified can be used to better manage the quality and safety of food microbiomes.
Wednesday, March 15 3:00 PM – 6:00 PM

Scripps

**Population Genomics**

**A framework for comparing genomic rearrangement rates.** C.T. Hann-Soden¹, L.A. Montoya¹, I Liachko², S.T. Sullivan³, J.W. Taylor¹ 1) Plant and Microbial Biology, UC Berkeley, Berkeley, CA; 2) University of Washington, Seattle, WA; 3) Phase Genomics, Inc., Seattle, WA.

While sophisticated models exist for inferring the pace and pattern of substitution mutations, methods for inferring genomic rearrangements are being actively developed. We present a distance based framework for estimating the rate and mean size of genomic rearrangements along a phylogeny from draft whole genome sequences. Our framework is analogous to the distance based frameworks developed for estimating substitution rates along a phylogeny. We apply this framework to studying rearrangement rates within the genus *Neurospora*, where numerous transitions from a highly recombinating lifestyle to a largely clonal lifestyle have occurred. We find evidence that there are more and larger rearrangements in the clonal lineages relative to the recombinating lineages, reflective of weaker selection against rearrangements in clonal lineages. We compare the application of this distance-based framework to the application of more sophisticated models that infer individual rearrangement events within a phylogeny.

**Adaptation by mitotic recombination in evolving yeast populations.** T.Y. James Dept Ecology Evolutionary Bio, Univ Michigan, Ann Arbor, MI.

The role of both mutation and sex in adaptation during microevolution is well known and supported by numerous experimental studies of evolution. However, how often mitotic recombination events are adaptive are poorly studied. Mitotic recombination is here defined to include many forms of loss of heterozygosity (LOH), such as gene conversion, whole chromosomal loss, and crossing over. Why LOH should be adaptive is somewhat counterintuitive, because it leads to a loss of allelic diversity. However, LOH may expose beneficial recessive mutations as well as change epistatic interactions between loci. Moreover, it is observed in essentially all of the species of fungi which have diploid vegetative states, many of which are important pathogens, such as *Candida albicans* and *Batrachochytrium dendrobatidis*. Here, we discuss the development of a model system for studying the role of mitotic recombination during the evolution of diploid fungal pathogens using a yeast-insect system. We show that the system is able to identify genes essential for pathogenicity, and show evidence for LOH during adaptation to the host. These results are compared to adaptation during a 500 generation experiment to diverse in vitro conditions. The data are used to address what form of LOH predominates during the adaptive process, an important question for understanding how precisely genotypes can be fine-tuned by LOH.

**Diversity among species of *Coccidioides*: population genomics analysis and local population structure.** B.M. Barker¹, M.deM. Teixeira¹, D.M. Engelthaler², P. Keim¹,² 1) PMI, Northern Arizona University, Flagstaff, AZ; 2) TGen North, Flagstaff, AZ.

*Coccidioides posadasii* and *C. immitis* are both filamentous fungi and the causative agents of coccidioidomycosis, commonly known as valley fever. These fungi are primary pathogens that can be responsible for morbidity and mortality in otherwise healthy patients. Over the past several years, valley fever cases have increased dramatically, and the endemic range has expanded. Disease can vary from asymptomatic to lethal. The underlying reasons for disease variation are due in part to intrinsic genetic factors in both the pathogen and host, and represent a major gap in our knowledge. Our overall knowledge of variance in clinically relevant phenotypes, especially in relation to the biology of *Coccidioides*, is limited. We have begun to assess genetic variation within and among species and populations of *Coccidioides*. Our approach is focused on identifying SNPs associated with clinically relevant phenotypes by screening variation found within and among species. The strategy should elucidate novel candidate genes in *Coccidioides* stress tolerance using an unbiased approach. Additionally, we have observed local population structure at a smaller scale than previously appreciated. Within Arizona, we detected restricted gene flow among Tucson human clinical, Phoenix human clinical and environmental/veterinary clinical isolates, suggesting that human clinical isolates may not represent total diversity.

**Genomic analysis reveals multiple endemic and pandemic lineages of rice blast and gene flow into recombinating populations from southeast continental Asia.** Pierre GLADIEUX¹, Maud THIERRY², Adrien RIEUX³, Sebastien RAVEL², Sandrine CROS-ARTEIL¹, Henri ADREIT², Joëlle MILAZZO², Elisabeth FOURNIER¹, Ryohei TERAUCHI⁴, Didier THARREAU² 1) BGPI Research Unit, INRA, Montpellier, FR; 2) BGPI Research Unit, CIRAD, Montpellier, FR; 3) PVBMT Research Unit, CIRAD, La Réunion, FR; 4) Iwate Biotechnology Research Center, Kitakami, Iwate, JP.

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

**Evolutionary pathways leading to panzootics of amphibian fungal disease.** Matthew Fisher¹, Simon O'Hanlon¹, Rhys Farrer¹, Adrien Rieux²

1) Dept Infectious Disease Epidemiology, Imperial Col London, London, GB; 2) CIRAD, La Reunion.

The Kingdom Fungi is a biodiverse and essential component of our habitable Planet. However, the last 100 years have witnessed an increasing number of virulent emerging pathogenic fungi across ecosystems, with these infections causing the greatest disease-driven losses of biodiversity ever documented. Fungal life-history characteristics, namely high virulence, environmental persistence, broad host-ranges and flexible genomic architecture, predispose this kingdom to emergence as terminal pathogens across susceptible populations of hosts. Anthropogenic activity is a key factor that perturbs natural cycles of infection by increasing long-distance dispersal of inocula and through environmental forcing of infection dynamics. I demonstrate these concepts by analysing patterns and processes across the backdrop of globally-emerging amphibian-parasitising chytrid fungi. We use comparative genomics to characterise the major evolutionary innovations that have led to the acquisition of pathogenicity across the genus *Batrachochytrium*. Population genomics defines ancient hotspots of amphibian/chytrid biodiversity and pinpoints the origins of panzootic lineages. Tip-dating approaches to calibrating molecular clocks then, for the first time, accurately date the timing of these expansions. These studies answer the questions 'why', 'where from' and 'when' that have long plagued ecologists investigating global amphibian declines. Our findings also argue that ongoing 'fungal pollution' will increasingly cause the attrition of biodiversity unless further steps are taken to tighten global biosecurity for this rapidly emerging class of pathogens.

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

Selfish elements can bias their own transmission from one generation to the next in a process referred to as meiotic drive. The driving element will be passed to the majority of offspring, thereby increasing in frequency over generations. The drive can occur regardless of its effect on fitness and may be a detriment to the organism as a whole. This intragenomic conflict could lead to strong selection against individuals carrying the element and promote reproductive isolation between so called killer and sensitive strains.

In ascomycetous fungi, meiotic drive manifests itself as a spore killing phenomenon. Briefly, ascospores carrying a killer element will induce the death of ascospores in the same ascus that do not carry the element. The killing is assumed to have a large cost to fitness, as a cross between killer and sensitive strains would result in the death of roughly half of all sexual progeny. We have examined 4 different spore killers in both *Neurospora* and *Podospora*. All of these examples of drive show patterns that suggest they may promote reproductive isolation between killer and sensitive strains.

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

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

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

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

**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.
**Cryptococcus neoformans** genomics influence human survival and immunologic responses. Aleeza Gerstein¹, 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*.
Thursday, March 16 9:00 AM – 12:00 NOON

Merrill Hall and Chapel

**Plenary Session II: Applied Mycology: Superpowers of Fungal Heroes and Villains**

**Plant biomass conversion by fungi: a highly diverse and complex process.** *Ronald de Vries*¹,²,³ 1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; 2) Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands; 3) Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.

The conversion of plant biomass by fungi has been a topic of interest for many decades, mainly due to the application of fungal plant biomass degrading enzymes in many biotechnology processes, such as the production of food & feed, paper, textiles, beverages, wine and more recently biofuels and biochemicals. The availability of an increasing number of fungal genome sequences and post-genomic datasets has provided an unprecedented understanding of this process. It revealed that the range of enzymes employed by fungi is significantly higher than was assumed before the availability of genome sequences, but also that the strategies employed by different fungi to degrade similar biomass substrates is highly diverse.

In our group we are particularly interested in understanding the molecular mechanisms underlying this diversity and how the different approaches match the prevailing biomass components. A special emphasis in our studies is in the regulation of the production of the enzymes, as this relates directly to the 'choices' a given species has made with respect to conversion approach. In recent years, several regulators have been identified that are involved in plant biomass conversion and while some are conserved among filamentous ascomycetes, their specific role and set of target genes are far less conserved. The conservation of enzyme systems in basidiomycetes and ascomycetes, while most ascomycete regulators are not present in basidiomycetes, raises questions about the regulatory mechanisms in the latter group and the evolution of the enzyme families versus the regulators that control their production.
In this talk I will present some of our recent data aimed at understanding the diversity of plant biomass conversion by fungi and the highly complex regulatory system that controls this process. This novel insight in how fungi have evolved their plant biomass conversion approaches will provide direct leads on how to improve applications of fungal plant biomass degrading enzymes.

**Resistance to antifungal drugs in fungal pathogens.** *Dominique Sanglard* Univ of Lausanne and Hosp Ctr, CH-1011 Lausanne.

Diseases caused by fungal pathogens necessitate the use of antifungal agents. The treatment of these diseases by antifungal agents remains a major challenge in hospitals given the restricted arsenal of therapeutic options and the continuing occurrence of drug resistance. We identified in the past several resistance mechanisms in fungal species relevant for human diseases using isolates from treated patients. Our current research has focused on 1) the impact of drug resistance on fungal-host interactions and 2) the understanding of fungal multidrug resistance (MDR) development in the host.

With regards to 1), we showed that the human pathogen *Candida glabrata* can develop rapidly resistance to azoles, which are compounds still widely used in hospitals. Resistance is mediated by several mutations in the transcription factor *PDR1*, which results in the overexpression of targets genes (ABC transporters). The same mutations alter fungal-host interactions by increasing the virulence of *C. glabrata* in several infection models. *PDR1* mutations can augment the expression of at least an important adhesin (*EPA1*) which contributes to establish a more efficient disease. Whole genome sequence analysis of *C. glabrata* strains suggest that this yeast species has accumulated more adhesins than previously anticipated, which may also impact on host-fungal interactions.

2) MDR is a current concern among fungal pathogens. A recent example of MDR by *Candida lusitaniae* came across our investigations. *C. lusitaniae* isolates were obtained during a 3-months therapy with amphotericin B (targeting ergosterol), azoles (targeting sterols) and candins (targeting cell wall). Four resistance profiles from 5 distinct isolates (P1-P5) were identified against 2 to 3 drug classes. To investigate MDR, analysis of specific drug resistance genes or comparisons of genomes by PacBio sequencing were used. The PacBio assembled genomes were compared with each other and with P1, a susceptible isolate. Ten non-synonymous SNPs were detected in these comparisons and revealed i) changes in *CIMRR1* that encodes a putative transcriptional activator in azole-resistant isolates P3 and P4 which correlated with upregulation of a MDR transporter (*MFS7*); ii) Three different mutations in *FKS1* (glucan synthase) in P2, P3 and P5 known to participate to resistance; iii) stop codons in *ERG4* and *ERG3* each of which were related to amphotericin B resistance in P2 and P5. Thus, isolates P2 to P5 accumulated six different drug resistance mutations as compared to P1 that emerged during the short time lapse therapy. This case study illustrates the capacity of a specific species to adapt to drug pressure within a host. MDR emergence is now also becoming a concern in the therapy of *C. glabrata* infections worldwide.

**Malassezia – what’s the turning point from commensal to pathogen?** *A. Scheynius*, the Malassezia Research Consortium Karolinska Institutet, Stockholm, SE.

*Malassezia* is a commensal yeast that colonizes the human skin right after birth and predominates the human fungal skin microflora. Fourteen species have so far been identified. One of the species most frequently isolated from human skin is *Malassezia sympodialis*, which is associated with several common skin disorders such as atopic dermatitis (AD) (Saunders CW *et al* 2012). We have found that *M. sympodialis* secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses (Gehrmann U *et al* 2011). To elucidate *M. sympodialis* host-microbe interactions we assessed whether small RNAs are present in MalaEx and, if so, whether the levels of these RNAs differ in MalaEx isolated from *M. sympodialis* cultured at normal skin pH compared to the higher pH on the skin of AD patients. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides (Rayner S *et al* in press). Bioinformatics analysis indicated that these RNA features appear to have an RNAi-independent route for biogenesis. No significant differences were observed between the MalaEx and their cargo of small RNAs isolated from *M. sympodialis* cultured at the two different pH levels indicating that they are not influenced by the elevated pH level observed on the AD skin.

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Through long-read DNA sequencing of *M. sympodialis* (ATCC 42132), we have obtained a gap-free genome assembly comprising eight nuclear and one mitochondrial chromosome (Zhu Y *et al* submitted). Proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using
transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9%. All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The *M. sympodialis* genome assembly and annotation is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.

**Fusarium fujikuroi** - a treasure box for new secondary metabolites and non-canonical regulatory mechanisms. *Bettina*

Tudzynski¹, Eva-Maria Niehaus¹, Slavica Janevska¹, Andreas Pfannmüller¹, Lena Studt¹,², Sarah Rösler¹ 1) Institute of Biology and Biotechnology of Plants, University Münster, Münster, DE; 2) Institut für Angewandte Genetik und Zellbiologie, BOKU Tulln, Austria.

The fungus *F. fujikuroi* causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a class of phytohormones. However, recent genome sequencing revealed the genetic capacity of this pathogen for the biosynthesis of a whole arsenal of secondary metabolites (SMs). Many of the gene clusters are silent under laboratory conditions, and their products and their roles in the fungus-rice interaction were unknown. To activate these SM gene clusters, we studied the multiple regulatory layers which coordinately control the biosynthesis of each single gene cluster. Key players in their regulation are cluster-specific transcription factors (TFs), mostly belonging to the Zn2-Cys6 family, global regulators such AreA, PacC and Sge1, components of the Velvet complex, such as Vel1 and Lae1, and histone modifying enzymes affecting the chromatin status in response to environmental conditions. We found that all these factors play differing roles in regulating each individual cluster, and that all clusters are regulated by a cluster-specific network of regulators. For instance, both deletion of the histone deacetylase *HDA1* and replacement of the repressive histone mark H3K27me3 led to the specific activation of the silent beauvericin cluster due to increased acetylation at H3K27. In addition, the expression of beauvericin cluster genes is regulated by the cluster-specific Zn2-Cys6-type TF and, surprisingly, by the cluster-specific ABC transporter, both acting as repressors. Furthermore, while the majority of SM biosynthetic genes respond to nitrogen availability, only some of these nitrogen-regulated SM genes depend on the major nitrogen regulators AreA and/or AreB. In contrast to our expectations based on the yeast model, both GATA TFs served as activators for most of these clusters. In addition, genetic manipulation of global regulators (e.g. Vel1, Lae1, AreA and Sge1) affected not only the expression levels of SM genes, but led to altered regulatory mechanisms. For example, nitrogen repression of GA biosynthesis was overcome by over-expressing Lae1 and Sge1. In addition, AreA and AreB affected the accessibility of target gene promoters for pathway-specific TFs, e.g. for the fumonisin-specific TF Fum21. These results suggest that the expression of each single SM gene cluster in *F. fujikuroi* is under control of manifold regulatory mechanisms, all different, and sometimes unexpected.

**Symbiosis between a bacterial endophyte and the ancient African crop finger millet results in dramatic trapping and killing of the fungal pathogen Fusarium graminearum.** Walaa Mousa¹, Charles Shearer¹, Victor Limay-Rios², Cassie Ettinger³, Jonathan Eisen³, Manish Raizada¹ 1) Plant Agriculture, University of Guelph, Guelph, ON, CA; 2) Plant Agriculture, University of Guelph, Ridgetown Campus, Guelph, ON, CA; 3) University of California, Davis Genome Center, Davis, CA.

Unlike its genetic relatives such as corn and wheat, the ancient African crop, finger millet, shows resistance to the fungal pathogen, *Fusarium graminearum*. We recently reported (*Nature Microbiology* 2016; 1:16167) the discovery of a novel plant defence mechanism in which a root-inhabiting bacterial endophyte, strain M6 (*Enterobacter* sp.), detects invading *Fusarium*, and in response stimulates the dramatic growth of nearby root hairs. The root hairs then bend parallel to the root axis, forming a multi-layer, lasagna-like formation, in which the "noodles" are the root hairs and the "filling" consists of microcolonies of the endophyte, a structure we have termed RHESt (root-hair endophyte stacking). The RHESt traps and kills *Fusarium*. Tn5-mutagenesis shows that M6-endophyte killing requires diverse fungicides including phenazine, and resistance to a *Fusarium*-derived antibiotic (fusaric acid), suggestive of long-term, host-endophyte-pathogen co-evolution. This fascinating symbiosis results in reduced deoxynivalenol mycotoxin, and may be protecting millions of subsistence farmers and livestock.
Thursday, March 16 3:00 PM – 6:00 PM

Merrill Hall

Human Pathogenic Fungi

**Marker recycling through CRIME.** A.P. Mitchell, M. Huang Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.

We describe here a new approach to marker recycling, a controlled sequence of manipulations in which a genetic marker is selected, then lost, then selected again. Our work was carried out with the fungal pathogen *Candida albicans*, which is typically a diploid and has no complete sexual cycle. The approach makes use of the RNA-guided DNA cleavage activity of CRISPR-Cas9, which was recently engineered for use in *C. albicans* by Valmik Vyas and colleagues (PMID: 25977940). We used the high-speed adaptation for gene deletion described by Kayden Min in our lab (PMID: 27340698) for the specific studies that will be presented. Because the CRISPR-Cas9 system has been implemented in diverse fungi, we believe that CRIME may be applicable to diverse fungi.

To use CRIME, we first created marker cassettes flanked by direct repeats. We used one repeat-flanked marker to create a homozygous mutation in our favorite gene, *OFG1*, by CRISPR-Cas9 transformation. Then, in the *ofg1/ofg1* strain, we made a homozygous mutation in a second gene, *OFG2*, by use of a different repeat-flanked marker. In that second transformation, we also included a guide RNA gene to direct cleavage of the marker that lay in the *ofg1* mutant alleles. The idea was that either single-strand annealing or homology-directed repair would then excise the marker we cleaved and leave behind only a copy of the flanking repeats. The idea proved correct: we recovered *ofg1/ofg1 ofg2/ofg2* double mutants from the transformation that had only the marker in the *ofg2* alleles; they had lost both copies of the marker that had been in the *ofg1* alleles. In fact, we have been able to create successive homozygous mutations in three genes by use of two markers, and still ended up with one marker available for further selection in the triple homozygote. That sequence of steps and analyses required 3 weeks total. Our findings illustrate that CRIME pays, at least in this one context.

We are now modifying our approach to enable wild-type allele reconstruction through a process that we call TIME. We feel that this capability will be critical for geneticists of the future, because CRISPR-Cas9 systems can have off-target effects. Our hope is that geneticists of the future will always remember that, if they do the CRIME, they must do the TIME.

**Candida albicans Hyr1 binding to c-Met induces oral epithelial cell invasion while inhibiting neutrophil killing.** Scott Filler¹,², Quynh Phan¹, Norma Solis¹, Marc Swidergall¹, Ashraf Ibrahim¹, Aaron Mitchell³ 1) Dept of Medicine, Los Angeles Biomedical Res Inst, Torrance, CA; 2) David Geffen School of Medicine at UCLA, Los Angeles, CA; 3) Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.

**Background.** During oropharyngeal candidiasis (OPC) *C. albicans* invades oral epithelial cells. Previously, we found that *C. albicans* Als3 and Ssa1 mediate invasion of oral epithelial cells by binding to E-cadherin and EGFR/HER2. Recently, we discovered that the c-Met receptor tyrosine kinase also mediates epithelial cell endocytosis of *C. albicans*, but functions in a separate pathway from EGFR/HER2. We set out to find the *C. albicans* ligand for c-Met.

**Methods.** Tyrosine phosphorylation of c-Met and EGFR/HER2 was assessed by immunoblotting with phosphospecific antibodies. To identify the *C. albicans* ligand for c-Met, we used far Western blotting with recombinant c-Met followed by protein sequencing. Epithelial cell endocytosis of *C. albicans* and latex beads coated with recombinant Hyr1 was quantified by a differential fluorescence assay. The extent of *C. albicans* killing by human neutrophils was determined by quantitative culture.

**Results.** Both the c-Met inhibitor SGX523 and the EGFR inhibitor gefitinib reduced epithelial cell endocytosis of *C. albicans* by ~50%, whereas the combination of SGX523 and gefitinib inhibited endocytosis by ~70%, indicating that c-Met and EGFR function in separate pathways. *C. albicans* hyphae induced tyrosine phosphorylation of c-Met by a process that was independent of EGFR/HER2, Als3 and Ssa1, suggesting that c-Met has a novel ligand. By far Western blotting, we found that the *C. albicans* ligand for c-Met is Hyr1. To verify this result, we determined that an *hyr1Δ/Δ* mutant had reduced capacity to induce phosphorylation of epithelial cell c-Met and invade epithelial cells. Also, latex beads coated with recombinant c-Met were endocytosed by epithelial cells more avidly than control beads coated with BSA. As reported by others, the *hyr1Δ/Δ* mutant had increased susceptibility to killing by human neutrophils. This increased susceptibility was due to failure to activate neutrophil c-Met because inhibition of c-Met with SGX52 enhanced neutrophil killing of WT *C. albicans*, phenocopying the effects of the *hyr1* mutation. Furthermore, activation of c-Met with its natural ligand, hepatocyte growth factor, reduced neutrophil killing of the *hyr1Δ/Δ* mutant to WT levels.

**Conclusions.** *C. albicans* Hyr1 is the fungal ligand for c-Met and contributes to virulence by two mechanisms. In epithelial cells, activation of c-Met by Hyr1 enhances the endocytosis of *C. albicans*. In neutrophils, activation of c-Met by Hyr1 impairs killing of *C. albicans*.

**Taking a host genetics approach to understand how the intracellular fungal pathogen *Histoplasma capsulatum* survives within and lyses host macrophages.** A.L. Cohen¹, N.V. Prooyen¹, M.C. Bassik², A. Sil¹ 1) Microbiology and Immunology, UCSF, San Francisco, CA; 2) Department of Genetics, Stanford University School of Medicine, Stanford, CA.

*Histoplasma capsulatum* (*Hc*) is a pathogenic soil fungus that is inhaled by mammalian hosts, including humans. Subsequently, *Hc* is phagocytosed by macrophages, where it utilizes largely unknown strategies to survive and replicate within the macrophage phagosome. Robust proliferation of *Hc* within the phagosome is followed by host-cell death and the release of live fungal cells. Our goal is to understand how *Hc* manipulates host cell processes, including phagosome maturation, to facilitate intracellular replication within macrophages and successful infection. Our lab and others have shown that, unlike most microbes ingested by macrophages, *Hc* resides within a neutral phagosome, and *Hc*-containing phagosomes do not acquire late lysosomal markers such as cathepsins. However, little is known about the molecular players underlying host manipulation by this fungus. We are taking a two-pronged approach to elucidate host pathways that are targeted by *Hc* during intracellular growth and lysis. First, we are conducting a pooled CRISPR-Cas9 based gene disruption screen in human macrophage-like cells to identify host genes required for death following *Hc* infection. We will then study how the corresponding gene products and pathways are directly or indirectly manipulated by *Hc* to promote intracellular infection. Second, we have discovered that a host lysosomal trafficking regulator, LYST, is required for *Hc* to lyse host macrophages during infection. We are investigating the role
of LYST in allowing *Hc* to promote host cell lysis. These studies will hopefully uncover molecular mechanisms of intracellular fungal pathogenesis as well as novel insights into host cell biology.

**Cryptococcus neoformans** suppresses immune cell infiltration into the brain via a secreted immunomodulatory polysaccharide. S.T. Denham, S. Verma, J.M. Daugherty, R. Reynolds, T.E. Lane, J.C.S. Brown Pathology, University of Utah, Salt Lake City, UT.

The basidiomycetes yeast *Cryptococcus neoformans* is the most prevalent cause of fungal meningitis, responsible for ~1 million diagnosed cases and ~600,000 deaths annually. *C. neoformans* infection begins in the lungs after the inhalation of infectious particles. In immunocompromised individuals, where symptomatic disease is most prevalent, *C. neoformans* systemically disseminates from the lungs. Disseminating *C. neoformans* exhibits a particular tropism for the brain, leading to cryptococcal meningitis (CM). CM patients often suffer high titers of infecting fungal cells within the brain, but exhibit limited neuroinflammation and relatively mild initial symptoms. Consequently, many patients do not seek care until fungal burden is damagingly high and therefore difficult to treat. *C. neoformans* conditionally produces a protective polysaccharide capsule, consisting primarily of the polysaccharide glucuronoxylomannan (GXM). GXM is both attached to the cell surface and secreted into the extracellular space as exo-GXM. GXM has numerous immunosuppressive properties, and its levels in the cerebrospinal fluid (CSF) of human patients negatively correlates with CSF white blood cell count. We thus hypothesized that exo-GXM suppresses inflammation and immune infiltration into the brain during infection. Moreover, this pauci-inflammation of CM is likely not only due to the immunocompromised state of patients, as we and others have found that *C. neoformans* suppresses the immune response of immunocompetent mice.

After infecting C57BL/6J mice intranasally with wild-type *C. neoformans*, fungal cells eventually disseminate to the brain. Upon analysis of infected brains, we discovered very little immune infiltration (neutrophils, macrophages, CD4+, and CD8+ cells), despite high fungal burden and histopathology. We also microscopically observed significant levels of exo-GXM in the brain. When mice were infected intracranially with an acapsular strain (*cap60Δ*), numbers of brain infiltrating immune cells significantly increased over wild-type. Likewise, administration of purified GXM to *cap60Δ* infected mice suppressed immune infiltration and increased fungal burden. We have also identified mutants deficient in exo-GXM production and the regulation of the switch between cell surface retention and exo-GXM secretion. Our results support a model in which *C. neoformans* actively secretes exo-GXM to modulate the host's immune response and promote fungal survival in the brain.

**Construction and efficacy of Δcps1, a live attenuated vaccine for coccidioidomycosis.** M. A. Mandel¹,⁴, L. F. Shubitz¹, L. Lewis¹, H. T. Trinh¹, A. S. Buntzman², J. A. Frelinger³, J. N. Galgiani², M. J. Orbach¹,⁴ 1) The Valley Fever Center for Excellence, Tucson, AZ; 2) Department of Medicine, University of Arizona, Tucson, AZ; 3) Department of Immunobiology, University of Arizona, Tucson, AZ; 4) The School of Plant Sciences, University of Arizona, Tucson, AZ.

Coccidioidomycosis (Valley Fever, VF) is a fungal disease of mammals endemic to the SW US and parts of Mexico, Central and South America, caused by *Coccidioides immitis* and *C. posadasii*. While most people experience mild flu-like symptoms, this pathogen can cause meningitis and is responsible for 160 deaths and $170 million in hospital costs in CA and AZ annually. Infection results in lifelong immunity, suggesting a vaccine against VF is attainable. We have developed a live attenuated vaccine by deletion of *CPS1*, the ortholog of a *Cochliobolus heterostrophus* virulence factor. Our *Δcps1* strain is non-pathogenic in susceptible mice, and provides dramatic protection against WT infection. Current analyses indicate that *Cps1* is a member of the AMP-dependent synthetase/ligase family of proteins related to Dip2 in animals. The role of *Cps1* in the parasitic phase is unknown, but evidence shows that the *Δcps1* strain initiates spherule formation but fails to propagate beyond a first generation in the host. We present data showing that vaccination results in long-term survival in mice given lethal doses of *Coccidioides spp.*, and that protection lasts at least 6 months following a single dose of live spores. The *Δcps1* strain forms defective thin-walled spherules that fail to endosporulate and that lyse prematurely. *In silico* analysis suggests *Cps1* is a transmembrane protein and transcriptional analysis supports a role as a transcriptional regulator. The original *Δcps1* strain has the complete ORF replaced by the hygromycin phosphotransferase marker. The presence of this marker is a safety and regulatory concern for a live vaccine. To create a new *Δcps1* strain lacking an antibiotic resistance marker, we used Biolog nutritional and genomic data to determine that *Coccidioides spp.* are unable to use sucrose as a carbon source. We demonstrate that the *A. niger sucA* gene can be introduced into *Coccidioides*, allowing selection on sucrose and are using this gain-of-function strategy for the new vaccine strain design.

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

We investigated two compost-heaps, one containing azoles and one without azoles, for the presence of azole-resistant and azole-sensitive *A. fumigatus* isolates. The azole-free compost yielded 98% (49/50) sensitive and 2% (1/50) azole resistant isolates, whereas the azole-containing compost yielded 8% (4/45) sensitive and 92% (41/45) resistant isolates. From the latter, 84% (37/45) were of the highly resistant TR₄₆ Y121F/T289A genotype and 8% (4/45) had a novel pan-triazole-resistance harbouring a triple tandem repeat: TR₄₆³Y121F/M172I/T289A/G448S. The stark contrast between the two composts indicates that azole-containing compost is a hot spot for the development and maintenance of azole resistance. Subsequent screening of the clinical *A. fumigatus* collection from the Dutch national surveillance programme indicated that this resistance mechanism was already present in 2012, and is now found in all participating medical centres. We were able to recover this novel TR₄₆³ mutation among the sexual progeny in the lab, from a cross between two TR₄₆ isolates of opposite mating type that were from the same compost. This strongly indicates a role of sex in the emergence of this novel azole-resistance mechanism in *A. fumigatus*. Furthermore, we provide further evidence indicating the potential occurrence of sexual reproduction in compost, but this still needs further confirmation. Our findings alarm the fast development of the azole resistance issue in *A. fumigatus*
and further indicate the role of sexual reproduction of *A. fumigatus* in the development of azole resistance.

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

**Comparative genomics of CUG-Ser clade species.** *Geraldine Butler* Conway Institute, Univ Col Dublin, Dublin, IE.

It has been known for some time that *Candida albicans* and close relatives translate the codon CUG as serine rather than leucine, associated with changes in the tRNA<sub>CAG</sub>. Recent proteomic and bionformatic analysis has shown that the switch to CUG serine occurred at the base of the Metschnikowiaceae/ Debaryomycetaceae. The discovery of a second codon change (CUG Alanine) in two species outside the clade means that the Metschnikowiaceae should now be referred to as the “CUG-Ser” clade, rather than the “CUG clade” or “CTG clade” as used previously. Metschnikowiaceae include many fully sexual species such as the *Hyphopichia* group and the *Metschnikowia* group. *C. albicans* and closely related asexual species belong to the Candida/Lodderomyces clade, which also includes other major human fungal pathogens, such as *Candida tropicalis* and *Candida parapsilosis*. We used comparative genomic analysis to expand our study of the evolution of mating type in sexual and asexual species within the CUG-Ser clade. We are also exploring within-species diversity using genome sequencing.

**Regulation of epigenetic switching and mating in *Candida* species.** Matthew Anderson¹, Christine Scaduto¹, Allison Porman¹, Eugenio Mancera², Christina Cuomo³, *Richard Bennett*¹ 1) Brown University, Providence, RI; 2) UCSF, San Francisco, CA; 3) Broad Institute, Cambridge, MA.

We are interested in defining the mechanisms that contribute to both genetic and epigenetic variation in pathogenic *Candida* species. Several *Candida* pathogens, including *C. albicans* and *C. tropicalis*, exhibit “phenotypic switching”, in which cells undergo epigenetic and reversible changes in cell state. Such changes impact key phenotypic properties such as sexual competency and interactions with host immune cells. Here, we addressed the molecular mechanisms underlying tristable switching in *C. tropicalis*, in which cells undergo reversible transitions between three different cell states. Our experiments suggest that tristable switching is achieved via a symmetrical self-activating toggle switch (SATS) involving two antagonistic transcription factors, Wor1 and Efg1. In support of this model, ectopic expression of Wor1 or Efg1 is shown to drive transitions between each of the three cell states. Interestingly, stable inheritance of induced cell states is dependent on synergistic cues between expression of transcription factors and chromatin remodeling. This result thereby establishes parallels with the regulation of cell differentiation events in multicellular organisms. We also address how phenotypic switching regulates entry into the program of sexual reproduction in *Candida*. We reveal that differences in pheromone MAPK signaling are responsible for the distinct mating efficiencies of *Candida* white and opaque cell types. Together, these studies establish close links between cell differentiation events in fungi and higher eukaryotes, and define the molecular mechanism by which different cell types display distinct sexual behavior.
Thursday, March 16 3:00 PM – 6:00 PM

Chapel

**Big Data (omics)**

**Big data in MycoCosm.** Igor Grigoriev, Frank Korzeniewski, Igor Shabalov US Department of Energy Joint Genome Institute, Walnut Creek, CA.

There are 700+ fungal genomes currently available from the US Department of Energy Joint Genome Institute's MycoCosm portal, from projects like the 1000 Fungal Genomes, 300 Aspergillus genomes, and others, which target different scales of fungal diversity. For the majority of these genomes, their annotation employs transcriptomes, often sampled over a range of conditions with most deep sampling for Aspergillus niger and Neurospora crassa (JGI Fungal ENCODE project). For these sequenced genomes, a partnership between two DOE User Facilities, JGI and EMSL, produces proteomics and metabolomics data. In addition, the radidly evolving next generation sequencing platforms enable efficient production of epigenomic data such as DNA methylation. These new types of OMICS data add dimensions to analysis and enable solutions for biological questions of different scales. At the same time, this scale of OMICS data production offers new challenges in data integration, visualization, and analysis.

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

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

**Integrating ecological and -omics approaches to understand fungal responses to global change.** Serita Frey Natural Resources and the Environment, University of New Hampshire, Durham, NH.

Soil fungi play key roles in ecosystem C and N cycles through decomposition of organic matter and nutrient exchange with plants. Soil fungi are the primary decomposers of cellulose and lignin, two of the most abundant compounds in plant material. Together with hemicellulose, these compounds form the complex structure referred to as lignocellulose, the decay of which involves a suite of extracellular enzymes. Fungi are the primary producers of these enzymes, and the presence of enzyme-coding genes determines whether a particular fungal species has the genetic machinery to decompose plant tissues. Any environmental disturbance that selectively impacts one group of fungi with a specific genetic capacity will likely influence organic matter decay, and ultimately, rates of soil nutrient cycling. For example, fungal taxa harbor different numbers and types of genes coding for lignocellulolytic enzymes. If taxa harboring these genes decline in abundance or are lost completely from the community, the capacity of the community to decompose plant material will be compromised. This talk will (1) synthesize several fungal -omics datasets generated from metabarcoding, metatranscriptomics, metabolomics, and whole genome sequencing analyses, and (2) discuss how coupling -omics approaches with biogeochemical analyses has accelerated our research to understand how soil fungal communities are responding to environmental change.

**MagNet: the integrated gene network of the rice blast fungus Magnaporthe oryzae.** H. Kim¹, K. Cheong¹, K.T. Kim², J. Jeon¹, G. Choi¹, Y.H. Lee¹,²,³ 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.

**Chromosome plasticity during experimental evolution of the cross-kingdom pathogen *Fusarium oxysporum*.** C Lopez-Diaz¹, D Hazal Ayhan², L.J Ma², A Di Pietro¹ 1) Department of Genetics, University of Córdoba, Cordoba, Spain; 2) University of Massachusetts, Amherst, USA.

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

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

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

**From genes to molecules: Assigning natural products to gene clusters by removing gene silencing.** Michael Freitag¹, Donovon Adpressa², Lanelle Connolly¹, Kristina Smith¹, Sandra Loesgen² 1) Dept. of Biochemistry and Biophysics; 2) Dept. of Chemistry, Oregon State University, Corvallis, OR, USA.

Fungi are widely known as producers of a large variety of natural products, many of which have been developed into pharmaceuticals. Pathogenic fungi on plants, animals and humans use their arsenal of secondary metabolites to aid in infection, establishment and maintenance of disease. Aided by high-quality draft genome sequences, much progress has been made over the past decade to sort specific metabolites to corresponding gene clusters, but in many cases annotated clusters remain transcriptionally silent even when strains are grown on a wide variety of media. In previous work, we found that several *Fusarium* species maintain a repressive chromatin mark, histone H3 lysine 27 methylation (H3K27me3) on ~33% of their genome. Deleting genes responsible for H3K27 methylation resulted in overexpression of more than 25% of all genes in *F. graminearum*. This discovery gives us new opportunities to express the "cryptic genome". We have generated additional mutants that are defective in H3K27me3-dependent gene silencing and have analyzed them for secondary metabolite profiles. While some previously undetected metabolites emerge from these mutants, we found that it is not always the end product that is overproduced. In several cases intermediates of well-studied pathways accumulate or new shunts result in accumulation of compounds previously not known from *F. graminearum*. Here, we outline strategies to streamline secondary metabolite and "genes-to-molecules" approaches.

**Using big data to compare evolution of fruiting body development across the Sordariomycetes.** Frances Trail¹, Wonyong Kim¹, Zheng Wang², Jeffrey Townsend² 1) Plant Biology, Michigan State University, East Lansing; 2) Department of Biostatistics, School of Public Health, Yale University, New Haven.

Diversity in fungi is most plainly manifested in fruiting body morphology. The Sordariomycetes produce predominantly perithecia, which are designed in a niche specific way for different mechanisms of spore dispersal. Previously, we examined shifts in gene expression that underlie the evolutionary diversity of fruiting body development in two genera: *Neurospora* and *Fusarium*, and identified genes that were critical to morphological differences between the genera. Three additional genera (*Magnaporthe*, *Chaetomium*, and *Neocosmospora*) are being used for a systems level analysis to reveal the genetic basis of a greater scope of morphological features for fungal fruiting bodies. In addition, we are constructing transcription factor networks and comparing the roles of transcription factors in the evolution of the fruiting body development. Through these approaches, we are dissecting the process of fruiting body development and function, identifying the common and distinct features of this process across a broad range of fungi, ultimately defining the underlying genetic networks that result in the successful adaptation of fruiting body structures and functions in nature.
Thursday, March 16 3:00 PM – 6:00 PM

Fred Farr Forum

Secondary Metabolism

**Genomics and evolution of secondary metabolism in *Fusarium*.** R.H. Proctor, H.-S. Kim, D.W. Brown Mycotoxin Prevention and Applied Microbiology, USDA ARS NCAUR, Peoria, IL.

*Fusarium* is a species-rich genus that causes disease on virtually all plant crops and produces diverse secondary metabolites (SMs), including pigments, plant hormones, and some of the mycotoxins of greatest concern to food and feed safety. To better understand the potential SM diversity in *Fusarium* as well as the distribution and evolution of SM biosynthetic genes, we have assessed the presence and absence of known and novel SM biosynthetic genes and gene clusters in genome sequences of >200 isolates representative of 25 lineages (species complexes) of *Fusarium*. antiSMASH analysis indicates that 1.1 – 6.4% of the genes in *Fusarium* genomes are involved in secondary metabolism, and that collectively *Fusarium* species have the genetic potential to produce hundreds of structurally distinct families of SMs, including analogs of multiple SMs described in other fungi. *Fusarium* SM clusters exhibit marked variation in distribution within and among species complexes: some are present in almost all members of all species complexes; others exhibit largely continuous distributions within one or several complexes; and others exhibit discontinuous distributions within one or more complexes. Phylogenetic analyses indicate that vertical inheritance, horizontal transfer, and gene loss have been major contributors to the current distribution of SM clusters among fusaria, but that recent duplication events have contributed little to the current distribution of clusters. Further, if the ancestral *Fusarium* had a similar number (26 – 68) of SM gene clusters as extant species, hundreds of horizontal transfer and loss events would be necessary to account for the current diversity and distribution of SM gene clusters in *Fusarium*.

**Evolution of secondary metabolism and host association in insect pathogens.** Kathryn E. Bushley¹, Stephen A. Rehner², Joseph W. Spatafora³

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. brongnarii*, *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.

**Tracing evolution of the *Cochliobolus heterostrophus* race T genes for T-toxin biosynthesis using phylogenetic analyses.** J.B. Gonzalez, B.J. Condon, B.G. Turgeon Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY.

In 1970, a new race (Race T) of the Dothideomycete *Cochliobolus heterostrophus* caused the Southern Corn Leaf Blight epidemic, the most devastating epidemic in US history. Decades of research has revealed that Race T harbors ten novel genes on four scaffolds and two different chromosomes for biosynthesis of T-toxin, a host selective toxin (HST) with exquisite specificity for Texas male sterile corn (Tcms), widely planted at that time. The evolutionary origin of the *Tox1* genes enabling extreme virulence to Tcms remain a mystery, as they are not found in other Cochliobolus species, including race O, a non-aggressive version of the pathogen. Previously, a subset of the genes was discovered in *Didymella zeae-maydis*, which produces PM-toxin, an HST with the same biological specificity. More recently, the genes were found in the Dothideomycetes, *Ampelomyces quisqualis*, *Ascochyta rabiei*, *Clohesyomyces aquaticus*, *Corynespora cassiicola*, *Leptosphaeria maculans*, *Lindgomyces ingoldianus*, and two Eurotiomycetes, *Penicillium raistrickii*, and *Penicillium brevicompactum*, none of which are known to produce T-toxin. Notably, recently discovered gene cohorts are co-linear, an arrangement that likely reflects the ancestral organization. The identification of the cohort in Eurotiomycetes allowed us to ask if the evolutionary path to a functional T-toxin metabolite was vertical transmission of the genes from an ancestor of the two lineages, or horizontal transmission (HGT) between the two. We evaluated this question by examining phylogenetic congruence between a species tree constructed using 19 conserved proteins, individual *Tox1* protein trees, and a tree constructed using a concatenated alignment of all *Tox1* proteins. Comparison of topologies revealed incongruence between the species tree and the individual protein and full-cohort trees, with the Eurotiomycetes separate from the Dothideomycetes in the former, but clustered within the Dothideomycetes in both of the latter. These findings, plus low conservation of the *Tox1* proteins among species, and that the gene cohort is thus far found predominantly in Dothideomycetes, are suggestive of an HGT event, likely ancient, from a Dothideomycete to the Eurotiomycetes. Our analyses also indicate other likely HGT events between taxa possessing *Tox1*-like genes. Acquisition of the *Tox1* genes in *C. heterostrophus* by vertical inheritance, recombination, and gene loss in intervening species versus HGT remain equally plausible.
**The cereal pathogen *Fusarium pseudograminearum* produces a mimic of cytokinin plant hormones.** *D.M. Gardiner*¹, *J.L. Sørensen*², *A.H. Benfield*¹, *R.D. Wollenberg*², *K Westphal*², *R Wimmer*², *K.F. Nielsen*³, *J. Carere*¹, *L. Covarelli*⁴, *G. Beccari*⁴, *J. Powell*¹, *T. Yamashino*⁵, *H. Kogler*⁶, *T.E. Sondergaard*¹ 1) Agriculture and Food, CSIRO, St Lucia, Queensland, AU; 2) Department of Chemistry and Bioscience, Aalborg University, DK-9000 Aalborg, Denmark; 3) Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; 4) Department of Agricultural, Food and Environmental Sciences, University of Perugia, IT-06121 Perugia, Italy; 5) Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Furocho, Chikusa-ku, Nagoya, 464-8601 Japan; 6) Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany.

The necrotrophic pathogen *Fusarium pseudograminearum* infects a broad range of agronomically important crops including barley and wheat. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, which are plant hormones, was identified as being produced by the pathogen during plant infection. Cytokinins are generally thought of as having growth promoting and anti-senescence activity and the production of a cytokinin mimic by a necrotrophic pathogen challenges the view that these pathogens invade by a simple barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the *F. pseudograminearum* genome for the production of these compounds was identified and the biosynthetic pathway established using gene knockouts. The *F. pseudograminearum* cytokinins can activate cytokinin signalling demonstrating their genuine hormone mimicry. *In planta* analysis of the transcriptional response to one of the *F. pseudograminearum* cytokinins suggests extensive reprogramming of the host environment by these molecules, possibly through cross talk with defence signalling pathways.

**Conserved veA-dependent genetic elements, rtfA and mtfA, regulate secondary metabolism, morphogenesis and virulence in Aspergillus spp.** *A.M. Calvo* Dept Biological Sci, Northern Illinois Univ, Dekalb, IL.

In the model fungus *Aspergillus nidulans* the global regulatory gene veA is necessary for the biosynthesis of several secondary metabolites, including the mycotoxin sterigmatocystin (ST). In order to identify additional veA-dependent genetic elements involved in regulating ST production, we performed a mutagenesis on a deletion veA strain to obtain revertant mutants (RM) that regained the capability to produce toxin. Genetic analysis and molecular characterization of two of the revertant mutants, RM3 and RM7, revealed that point mutations occurred at the coding region of the *rtfA* and *mtfA* genes respectively. *rtfA* encodes a RNA-pol II transcription elongation factor-like protein, similar to *Saccharomyces cerevisiae* Rtf1, while *mtfA* encodes a novel putative C2H2 zinc finger domain transcription factor. Both genes are conserved in *Aspergillus* spp and in other fungal genera. Further research revealed that in a veA wild-type background, *rtfA* controls the production of several secondary metabolites, or natural products, in *A. nidulans*, as well as in the agriculturally and medically important *A. flavus* and *A. fumigatus*. *mtfA* also governs the biosynthesis of natural products in *Aspergillus* spp by regulating the expression of secondary metabolite gene clusters, as demonstrated in *A. nidulans* and *A. fumigatus* functional genomics studies. Additionally, both regulators strongly influence other biological processes, including morphological development and virulence.

**Mushroom polyketide synthase produces polyenes for chemical defense.** *P. Brandt*¹, *M. García-Altares*², *M. Nett*³, *C. Hertweck*², *D. Hoffmeister*¹ 1) Friedrich-Schiller-Universität, Department Pharmaceutical Microbiology at the Hans-Knöll-Institute, Winzerlaer Straße 2, 07745 Jena (Germany); 2) Leibniz Institute for Natural Product Research and Infection Biology, Department Biomolecular Chemistry, Beutenbergstraße 11a, 07745 Jena (Germany); 3) Technische Universität Dortmund, Department Biochemical and Chemical Engineering, Technical Biology, Emil-Figge-Straße 66, 44227 Dortmund (Germany).

Basidiomycetes have evolved a diverse repertoire of bioactive chemical defense compounds. After wounding of its mycelium, the taxonomically undetermined white-rotting basidiomycete BY1 produces yellow pigments *de novo*, which massively inhibit the pupation of insect larvae. These natural products were identified as the polyunsaturated fatty-acid like polyenes, 18-methyl-19-oxoicosaoctaenoic acid and 20-methyl-21-oxodocosanaenoic acid. [1]

The objective of this study was to understand the genetic and biochemical basis of this basidiomycete defense compounds. We succeeded in identifying both alleles of a candidate gene, *PPS1*, in the genome of BY1, coding for a six-domain reducing polyketide synthase (HR-PKS). Quantitative real-time PCR showed a 9.5-fold upregulation of *PPS1* expression 48 hours past injury of the BY1 mycelium. To verify that *PPS1* has polyene synthase activity, the polyene biosynthesis was heterologously reconstituted in *Aspergillus niger*. To that end, *PPS1* was placed under the control of the *ter*A promoter and *trpC* terminator using plasmid SM-Xpress. [2] Combining liquid chromatography, mass spectrometry, and NMR, the structures of the *PPS1* products were elucidated, and proved identical to the polyenes initially isolated from BY1. MALDI-MS imaging indicated polyene accumulation in the wounded mycelial area. Our work represents the first characterized basidiomycete HR-PKS and sets the stage for a more profound understanding of basidiomycete chemical ecology.

[1] D. Schwenk, M. Nett, H.-M. Dahse, U. Horn, R. A. Blanchette, D. Hoffmeister, *J Nat Prod* **2014**, *77*, 2658-2663.

[2] M. Gressler, P. Hortschansky, E. Geib, M. Brock, *Front Microbiol* **2015**, *6*, 184.

**Copper-responsive isocyanide biosynthetic cluster in Aspergillus fumigatus.** *F. Lim*¹, *J. Baccile*², *T. Won*², *P. Wiemann*¹, *A. Lind*³, *A. Rokas*³, *F. Schroeder*², *N. Keller*¹ 1) University of Wisconsin-Madison, Madison, WI, U.S.A; 2) Cornell University, Ithaca, NY, U.S.A; 3) Vanderbilt University, Nashville, TN, U.S.A.

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

Jarczynska, C.D. Nødvig, U.M. Mortensen Danish Technical University, Copenhagen, DK.

Fungi produce a wide variety of secondary metabolites and enzymes. For many of these secondary metabolites, the clusters that are responsible for their production are unknown. One way to elucidate the pathway is to make gene disruptions in the producer stain, but this requires that the species can be grown in the laboratory, that it is transformable and that a genetic tool box is available. Alternatively the cluster can be transferred to a well characterized host where the genes can be heterologously expressed. To facilitate the latter method, we created a flexible expression platform with integration sites in different well characterized Aspergillus species. The integration sites contain a common integration target site that contains the colorimetric marker *lacZ* flanked by long overhangs for homologous recombination. Hence, hosts strains are blue, but if new genes are inserted correctly into the expression sites, the strains will turn white as the *lacZ* gene is eliminated as a result of the integration process. Integrations can be performed in wild-type strains using marker- free gene-targeting by using CRISPR stimulated homologous recombination. Importantly, by using our system, the same gene-targeting construct can be inserted into several different hosts; and the strain with the best product performance can then be selected for further analysis.