Thursday, March 16 3:00 PM – 6:00 PM

Kiln

RNA Biology

**RNAi-dependent epimutations evoke transient antifungal drug resistance.** *J. Heitman*¹, *Z. Chang*¹, *S. Lee*¹, *M. Cardenas*¹, *S. Calo*¹, *F. Nicolas*², *A. Vila*², *M. Cervantes*², *S. Torres-Martinez*², *R. Ruiz-Vazquez*² 1) Department of Molecular Genetics and Microbiology, Duke University, Durham; 2) Department of Genetics and Microbiology, University of Murcia, Spain.

Microorganisms evolve via sexual reproduction, mutators, aneuploidy, Hsp90, and prions. We discovered a new mechanism conferring antifungal drug resistance in the human fungal pathogen *Mucor*. In this species, spontaneous resistance to the antifungal drug FK506 evolves via two mechanisms. One involves Mendelian mutations conferring stable irreversible drug resistance; the other occurs via an epigenetic RNA interference (RNAi) pathway resulting in unstable, transient drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks the *Mucor* dimorphic transition to hyphae and enforces growth as yeast. In some FK506-resistant isolates, mutations in the *fkbA* gene encoding FKBP12 or the calcineurin *cnbR* or *cnaA* genes confer FK506 resistance restoring hyphal growth. In other isolates, no mutations occurred in these drug targets. Instead, RNAi spontaneously silenced the *fkbA* gene yielding drug-resistant epimutants that revert to drug-sensitivity in the absence of FK506. Establishment of these epimutants involves generation of abundant *fkbA* small RNAs and requires several known RNAi pathway components whereas other RNAi elements are dispensable. Surprisingly, epimutants occur at a higher frequency and are more stable in mutants lacking RNA-dependent RNA polymerase 1, revealing some RNAi components constrain epimutation. Silencing of the drug target FKBP12 appears to involve generation of a double-stranded RNA intermediate using the *fkbA* mature mRNA as template to produce antisense *fkbA* RNA. Our recent studies reveal additional components required for epimutation, including orthologs of the *Neurospora crassa* quelling inducing protein (QIP) and Sad-3 helicase (RnhA); interestingly *rnhA* is linked to the *Mucor* sex locus, suggesting possible parallels with sex induced silencing in *Cryptococcus*. We also found epimutants occur at a higher frequency and are more stable in two additional RNAi mutants lacking RNA-dependent RNA polymerase 3 or the R3B2 factor. These findings were generalized by isolating epimutations in a second *Mucor* species and identifying epimutations in the *pyrF* or *pyrG* genes conferring 5-FOA resistance. These studies reveal a novel, reversible, transient epigenetic RNAi epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi pathways in fungi and other eukaryotes.

**A non-canonical RNA silencing pathway in *Mucor circinelloides* and its involvement in pathogenesis.** *V. Garre*¹, *F. E. Nicolás*¹, *J. T. Cánovas-Márquez*¹, *C. Pérez-Arques*¹, *M. I. Navarro-Mendoza*¹, *T. A. Trieu*¹,², *J. A. Pérez-Ruiz*¹, *E. Navarro*¹, *S. Torres-Martínez*¹, *R. M. Ruiz-Vázquez*¹ 1) Department of Genetics and Microbiology, University of Murcia, Murcia, ES; 2) Department of Genetics, Faculty of Biology, Hanoi National University of Education, Hanoi, Vietnam.

The mechanism of RNA silencing or RNA interference (RNAi) plays an important role in the maintenance of genome integrity, particularly protection against exogenous nucleic acids such as mobile elements, but also a role in the regulation of gene expression by means of regulatory endogenous small RNAs (esRNAs). *Mucor circinelloides*, an opportunistic human pathogen that is a causal agent for the rare but lethal infection mucormycosis, represents one of the most outstanding examples about the role of RNAi in gene regulation. This fungus exhibits at least three RNAi-related pathways dedicated to regulate several physiological and development processes. Besides *dicer*-dependent RNAi pathways involved in regulation, *Mucor* presents a non-canonical *dicer*-independent RNAi pathway that controls the expression of target genes by promoting the degradation of their mRNAs. This non-canonical pathway in which participate two RNA-dependent RNA polymerases (RdRP) and a putative endonuclease, named R3B2, regulates specific cellular processes, such as sexual interaction and the response to environmental stresses. However, our recent studies implicate this regulatory pathway also in virulence, since mutants for *r3b2* and *rdrp-1* show reduced virulence in a mouse model. Interestingly, R3B2, which is found exclusively in basal fungi, was suggested to play a crucial role in the biogenesis and function of regulatory esRNAs because it is also involved in the *dicer*-dependent RNAi pathways. *In vitro* nuclease activity assays have shown that R3B2 cleaves dsRNA producing sRNAs of a wide range of sizes, whereas recombinant human Dicer produces sRNAs of discrete size in the same analysis. Moreover, mutation of conserved residues in the RNase III catalytic domain-like of R3B2 that blocks its function in RNA silencing *in vivo* produces a loss of dsRNA cleavage activity *in vitro*. These results support an endonuclease role for R3B2 in the non-canonical RNAi pathway that controls expression of genes involved in fungal infection by Mucorales.

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

**Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing.** *Yunkun Dang*¹, *Jiasen Cheng*², *Xianyun Sun*³, *Zhipeng Zhou*¹, *Yi Liu*¹ 1) UT Southwestern Medical Center, Dallas, TX. USA; 2) Huazhong Agricultural University, Wuhan, Hubei, China; 3) Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We previously showed that antisense transcription triggers Dicer-independent small interfering RNA (disiRNA) production and disiRNA loci DNA methylation (DLDM) in *Neurospora crassa*. Here we show that the conserved exonuclease ERI-1 is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and to recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that in the cytoplasm ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long non-coding RNAs-mediated epigenetic modifications and identify ERI-1 as an important regulator of co-transcriptional gene silencing and post-transcriptional RNA metabolism.
**The RNAi system in the Brassicaceae smut fungus *Thecaphora thlaspeos*.** K. Boesch¹,², L. Frantzeskakis¹, K. Courville¹, M. Feldbrügge¹,²,³, V. Göhre¹,²,³ 1) Heinrich-Heine-University, Düsseldorf, NRW, DE; 2) Bioeconomy Science Center (BioSC), Jülich, NRW, DE; 3) Cluster of Excellence on Plant Sciences (CEPLAS), Düsseldorf, NRW, DE.

Smut fungi infect important crop plants, e.g. maize or potato. To develop novel plant protection strategies, the infection process has to be characterized molecularly. Genetic manipulation of the crop host plants is still difficult because of their complex genomes. For this reason, we want to characterize the interaction of the smut fungus *Thecaphora thlaspeos* with the model plant *Arabidopsis thaliana*.

*T. thlaspeos* infects different Brassicaceae throughout Europe, among them the perennial plant *Arabis alpina*. Its lifecycle differs in two important steps from crop smut fungi such as *Ustilago maydis*: First, teliospores only germinate in the presence of a plant-derived signal. Second, there is no dimorphism. Filaments emerge from teliospores that are directly infectious. Importantly, these filaments can be propagated as haploid cultures enabling genome sequencing and genetic manipulation.

Interestingly the genome encodes components of the RNAi machinery, which are absent in *U. maydis* but present in the smut fungus *Sporisorium reilianum*. Cross-kingdom RNAi is used by *Botrytis cinerea* to modulate *Arabidopsis thaliana*'s immune system. Based on reduced virulence of the DICER deletion mutant, it is hypothesized that sRNAs are generated in the fungus, transported into the host plant's cell and there silences expression of specific genes involved in immunity. With our new model system we will study how *T. thlaspeos* uses RNAi and ultimately investigate whether also a smut fungus utilizes cross-kingdom RNAi during infection.

**Endosome-coupled mRNA transport.** *M. Feldbrügge* Institute for Microbiology, Heinrich-Heine University, Düsseldorf, NRW, DE.

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we present new findings obtained in the model organism *Ustilago maydis*. In highly polarized cells of this fungus microtubule-dependent co-transport of mRNAs and endosomes is essential for efficient polar growth. We discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Key players are RNA-binding proteins containing RNA recognition motifs for mRNA binding as well as Mademoiselle domains for protein/protein interaction. Here, new insights on protein RNA as well as protein-protein interactions will be presented. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes suggesting that this phenomenon is a common theme and not an exception restricted to fungi.

**Exp5, a Magnaporthe oryzae nucleo-cytoplasmic receptor involved in non-coding RNA metabolism and plant pathogenesis.** A. Illana, M. Marconi, J. Rodríguez-Romero, M. Wilkinson, A. Sesma Centre for Plant Biotechnology and Genomics, Technical University of Madrid, Pozuelo de Alarcon, Madrid, ES.

Following a genetic screening of a *M. oryzae* T-DNA insertional library of ca. 3,000 transformants looking for pathogenicity-deficient mutants, genes associated with RNA metabolism were selected for further characterization, including the karyopherin *EXP5*. The *M. oryzae* Exp5 protein is the orthologue of the human karyopherin exportin-5 and the *Saccharomyces cerevisiae* Msn5. Karyopherins are involved in the translocation of RNAs and/or proteins between the nucleus and the cytoplasm. To better understand the role of this karyopherin during *M. oryzae* plant infection we set out to identify its cargoes. Several t-RNA synthetases, seven subunits of the 26S proteasome and key components of signal transduction pathways such the MAPK Pmk1 and Mps1 immunoprecipitated with Exp5. An important set of mitochondrial proteins including TIM44 and TOM70 also interacted with Exp5, which suggested that mitochondrial dysfunction is possibly contributing to the strong root infection defects exhibited by Δ*exp5*. The sRNA sequencing analysis pointed out that Exp5 is potentially required for the transport of specific classes of retrotransposons, and allowed us to identify a single-stranded RNA virus present in the *M. oryzae* field isolate Guy11. Northern blots carried out with sRNAs extracted from nuclear and cytoplasmic subcellular fractions suggested deficiencies in the Δ*exp5* mutant such as accumulation of sRNAs derived from transposon elements and tRNA<sup>Met</sup> also supported the involvement of Exp5 in sRNA metabolism and tRNA transport. Our results suggest that Exp5 is required for the translocation of specific classes of proteins, tRNAs and sRNAs, several of which contribute to *M. oryzae* pathogenicity.

**References**

1. Tucker SL, Besi MI, Galhano R, Franceschetti M, Goetz S, Lenhert S, Osbourn, A & Sesma A. (2010) Common Genetic Pathways Regulate Organ-Specific Infection-Related Development in the Rice Blast Fungus. *Plant Cell* **22**: 953-972.

2. Illana A, Marconi M, Rodríguez-Romero J, Ayllón MA, Xu P, Dalmay T, Wilkinson M and Sesma A. Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus *Magnaporthe oryzae* (2016). Archives of Virology. DOI: 10.1007/s00705-016-3144-9
**Introns regulate gene expression in Cryptococcus neoformans.** G. Janbon Institut Pasteur, Paris, FR.

*Cryptococcus neoformans* is a basidiomycetous opportunistic pathogen leaving in the environment responsible for more than 500 000 deaths every year. Our recent work suggests that a fascinating, complex pattern of RNA molecules composes its transcriptome and this fungus is emerging for different aspects as an ideal model to study RNA metabolism in eukaryotes. It is also tempting to hypothesize that this complex RNA metabolism provides a mechanism for this yeast to respond to different environmental cues and to be an efficient pathogen. The most prominent features of this transcriptome are introns. Our recent re-annotation of the *C. neoformans* genome revealed that nearly all the expressed genes contain introns. These introns can be present within the coding sequence but also within UTR regions. We previously demonstrated that these introns are essential for gene expression. Here, we identified a large number of regulated alternative splicing events. However, alternative splicing in this yeast seems to be more means to regulate gene expression than to diversify the proteome.

**Pas3 regulates cryptococcal morphogenesis through Bre1/Npl3-Mediated alternative splicing.** Y. Zhao, X. Lin Biology, Texas A&M University, College Station, TX.

Per-ARNT-Sim (PAS) proteins function as signal receptors, transducers, or regulators. In the human fungal pathogen *Cryptococcus neoformans*, two PAS proteins, Bwc1 and Bwc2 (white collar 1 and 2), are known to regulate mating, virulence, and tolerance of ultraviolet radiation. To comprehensively understand the regulatory role of PAS proteins in regulating morphogenesis and pathogenicity, all 9 PAS domain coding genes were analyzed in *C. neoformans*. One of the PAS domain proteins, Pas3, was found to be critical in cryptococcal filamentation and virulence. Deletion of *PAS3* in either the serotype D reference strain XL280 or the serotype A reference strain H99
severely impaired filamentation during either unisexual (a alone) or bisexual (a-a) reproduction. Interestingly, Pas3 was localized to the
nucleus despite absence of NLS, implicating regulatory roles in addition to transducing signals. Through co-immunoprecipitation (Co-IP)
coupled with mass spectrometry, we identified Bre1 and Npl3 as two potential interacting protein-partners of Pas3. Bre1, as an E3 ligase,
mono-ubiquitinates histone H2B subunit, and may regulate cryptococcal morphogenesis through changing the chromatin structure at
filamentation-associated genetic loci, resulting in altered gene transcription. In addition to chromatin modification effects, Bre1 and Npl3 may
function together in regulating alternative splicing of selective pre-mRNAs. Deletion of *BRE1* and *NPL3* individually partially recapitulated
the filamentation defect of *pas3D*, indicating that Bre1 and Npl3 may function both in parallel and overlapped manners downstream of Pas3
in regulating morphogenesis. Fluorescently labeled Bre1 gave fluorescent signals all over the nucleus and also one bright non-nucleolus
dot, indicating its specific role in regulating alternative splicing in addition to ubiquitination of H2B. Our forward genetic screening for non-
filament mutants identified the splicing factor Cus1 as an important regulator of filamentation. This corroborates the hypothesis that
alternative splicing regulates cryptococcal morphogenesis. Currently we are analyzing the alternative splicing profile in *cus1D* relative to
wild type via RNA-seq. Identification of the genetic connection among Pas3, Bre1 and Npl3, and dissection of mode of action by alternative
splicing in regulating fungal morphogenesis in *C. neoformans* will expand the knowledge about signal transduction and post-transcriptional
regulatory network in fungal development.
Thursday, March 16 3:00 PM – 6:00 PM

Heather

# Sustainable Bio-product Fermentation: From Biomass Conversion to High Value Product Formation

**Substrate engineering as a viable option to complement fungal bio-engineering efforts.** *J. Philipp Benz*, Manfred J. Reppke HFM, Technical University of Munich, Freising, DE.

## Background

Cellulases and hemicellulases have become important industrially used enzymes, such as for feed stock processing, textile applications and the production of biofuels. The applications for these enzymes are mostly limited by the economical aspects of their production. Differently produced celluloses are commonly used as part of the fermentation media for hydrolase production by microorganisms. A wealth of potential celluloses is available, but the relevant physiochemical characteristics determining their effectiveness as inducer or substrate so far remain largely unclear. Moreover, the regulatory networks of the fungal production host strongly affect the response, but have not been fully elucidated. These factors are collectively making it difficult to rationally decide for the correct product to use.

## Results

We have been analyzing different celluloses for their suitability as cellulase-inducing substrates using two filamentous ascomycetes: the genetic model fungus Neurospora crassa and the Trichoderma reesei hypercellulolytic strain Rut-C30. Performance was evaluated by quantifying gene induction, protein secretion and enzymatic activities. Results showed clear differences between the cellulosic substrates. Chemical and physical analyses identified crystallinity as a major factor affecting performance while particle morphology did not seem to have as strong of an impact as expected. Moreover, due to cross-signaling between cellulose and hemicellulose perception pathways, even small amounts of impurities were found to strongly affect the fungal response.

## Conclusions

Our data demonstrate that highly pure celluloses do not necessarily perform well as substrates for the production of cellulases in filamentous fungi and indicate that certain hemicellulose impurities as well as a lower crystallinity are advantageous for their effectiveness. However, what constitutes a beneficial vs. a detrimental impurity strongly depends on the genetic background of the production host. Moreover, the fine structure of the substrate will define how bioavailable each polysaccharide is, and therefore how well it can be perceived. In collaboration with industrial fiber mills, we are now embarking on "substrate engineering" as a viable addition to fungal bio-engineering efforts. The presented results highlight key factors that need to be considered when choosing the optimal cellulosic product for a biotechnological cellulase or hemicellulase production process.

**The molecular response of the white-rot fungus *Dichomitus squalens* to wood and non-woody biomass.** *M.R. Mäkelä*¹, *J. Rytioja*¹, K. Hilden¹, M. Di Falco², M. Zhou³, M.V. Aguilar-Pontes³, O.-M. Sietiö¹, A. Tsang², R.P. de Vries³ 1) Department of Food and Environmental Sciences, University of Helsinki, Helsinki, FI; 2) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands; 3) Centre for Structural and Functional Genomics, Concordia University, Montreal, Canada.

Analyses of ascomycetes have shown that the genomes of the species that occupy highly defined habitats (e.g. the dung fungus *Podospora anserina* and the plant pathogen *Botrytis cinerea*) are well tailored to obtain carbon from the prevailing plant biomass substrate. In contrast, ascomycetes that thrive in a large variety of habitats (e.g. *Aspergillus niger*) possess a diverse enzymatic machinery for the degradation of a wide range of biomass.

Wood-rotting basidiomycetes are exclusively found on wood in nature, where they play a significant role in degradation of all polymeric components of wood cell walls. The white-rot basidiomycete *Dichomitus squalens* is commonly found in the northern regions of Europe, Asia and North America. It is an efficient wood degrader with a genome that possesses an extensive gene set predicted to encode diverse plant cell wall modifying enzymes.

In this study, we examined the transcriptome and exoproteome of *D. squalens* during growth on two wood substrates, aspen and spruce, and two non-woody biomasses, wheat bran and cotton seed hulls. In addition to the higher lignin composition in the wood substrates, these biomasses differ substantially in the structure of their hemicelluloses. The results showed that *D. squalens* upregulates a set of genes, and secretes their encoding enzymes, that are targeted to the degradation of plant cell walls of both wood and non-woody substrates. Moreover, the genes/enzymes that are expressed/produced display functional specificity towards the polymeric components of the substrates. These results indicate that *D. squalens* has preserved its ability to respond adequately to a variety of plant biomass, even to those which it would not be expected to encounter in nature. They also suggest that factors other than the ability to obtain carbon are responsible for its limitation to wood as a habitat.

**Rewiring transcriptional control of plant cell wall degrading enzyme networks in *Aspergillus niger*.** Jing Niu¹, Ebru Alazi¹, Ian Reed², Mark Arentshorst¹, Peter Punt³, Jaap Visser¹, Adrian Tsang², *Arthur Ram*¹ 1) Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden, The Netherlands; 2) Centre for Structural and Functional Genomics, Concordia University, Montreal, QC, Canada; 3) Dutch DNA Biotech, Zeist, The Netherlands.

*Aspergillus niger* is an important industrial fungus expressing a broad spectrum of plant cell wall degrading enzymes. The expression of genes encoding these enzymes and the metabolic pathways required for carbon utilization are tightly controlled by pathway specific transcription factors. Well known examples of such transcription factors include AmyR and XlnR, which control the expression of amylylolytic and xylanolytic enzymes networks respectively. We recently identified a galacturonic acid-specific transcription factor named GaaR and showed that it acts as an activator for the expression of genes required for release and utilization of galacturonic acid from pectin (1). We performed a forward genetic screen for the isolation of mutants that constitutively express GaaR-controlled polygalacturonases. Phenotypic, genomic and transcriptomic analyses of the constitutive mutants resulted in the identification of a repressor protein, GaaX, which specifically controls expression of the genes encoding enzymes related to the galacturonate/pectin degradation pathway. The *gaaX* gene is located next to *gaaR* in the genome. Phylogenomic analysis and published reports reveal similar activator-repressor modules among fungi of the subphylum Pezizomycotina, and that they regulate gene expression involved in the utilization of quinate, galacturonate, and possibly
other carbon substrates.

(1) Alazi E, Niu J, Kowalczyk JE, Peng M, Aguilar Pontes MV, van Kan JA, Visser J, de Vries RP, Ram AF. The transcriptional activator GaaR of *Aspergillus niger* is required for release and utilization of D-galacturonic acid from pectin. FEBS Lett. 2016; 590(12):1804-15.

**Sustainable and cost-effective enzyme production by deploying advanced engineering technologies in an integrated approach.** Noel van Peij, Rene Verwaal, Herman Pel, Roel Bovenberg, Hans Roubos DSM Food Specialties, DSM Biotechnology Center, Delft, NL.

Enzymes are widely used in food and bio-based industries for various applications. The industrial production of enzymes by DSM is using fungal microorganisms such as *Aspergillus niger*. The industrial hosts have a long history of safe use and are subject to continuous improvement.

The field of molecular and engineering biology is developing rapidly. More tools become available to design strains in a rationalized and integrated way, build them with high precision in a faster way and increase the throughput of strain testing. The iterative design-build-test-learn cycle concept has significantly accelerated strain and process development and optimization in industry. A few selected examples, how recently developed tools can be used in improved, rationalized and faster strain construction for the development of high-value enzyme products, will be discussed in this presentation.

**Genetic parts screening and artificial N-glycosylation motif engineering for heterologous protein production in *Aspergillus niger*.** Jinxiang Zhang<sup>1,3</sup>, Saori Amaike-Campen<sup>1,2</sup>, Sam Deutsch<sup>4</sup>, Ljiljana Pasa-Tolic<sup>5</sup>, Erika Zink<sup>5</sup>, Jon Jacobs<sup>5</sup>, Blake Simmons<sup>1,6</sup>, John Gladden<sup>1,3</sup>, Jon Magnuson<sup>1,2</sup> 1) Joint BioEnergy Institute, Emeryville, CA; 2) Pacific Northwest National Laboratory, Richland, WA; 3) Sandia National Laboratories, Livermore, CA; 4) Joint Genome Institute, Walnut Creek, CA; 5) Environmental Molecular Sciences Laboratory, PNNL, Richland, WA; 6) Lawrence Berkeley National Laboratory, Berkeley, CA.

*Aspergillus niger* is a genetically tractable model organism for scientific discovery and a platform organism used in industry for the production of enzymes. Expression of secreted native enzymes at tens of grams per liter have been discussed by those in industry, but high level production of heterologous enzymes remains elusive. Strategies to increase production include the use of strong promoters, protease-deficient strains, fusion proteins, multiple gene copies, etc. However, yields of heterologous proteins are still lower than desired.

We generated proteomics data from secretome samples of A. niger grown on a variety of minimal and rich media, with the goal of identifying useful genetic elements for increasing heterologous protein production. Twenty promoters, six signal sequences and four introns from the most highly secreted proteins were identified as candidate genetic elements to enhance heterologous gene expression. These candidate elements were tested for their ability to drive expression of a prokaryotic glycoside hydrolase. A vector was designed to target integration of the modified expression cassette to the native glucoamylase (glaA) gene locus by homologous recombination. Considerable diversity was seen in heterologous protein production driven by these various elements. Interestingly, a signal peptide from GPI-anchored cell wall protein showed promising results.

In addition, based on the 3D structure of heterologous protein, we generated seven individual artificial N-glycosylation motifs on the surface of the heterologous protein. Analysis of these sites for glycosylation via top-down proteomics is just beginning. Correlation of glycosylation with any changes in kinetic and thermodynamic properties of the altered enzymes is the goal of this aspect of the research.

**Protease regulatory factors of *Trichoderma reesei* can be controlled to improve therapeutic protein production.** Christopher Landowski<sup>1</sup>, Ann Westerholm-Parvinen<sup>1</sup>, Bernhard Helk<sup>2</sup>, Juhani Saarinen<sup>3</sup>, Markku Saloheimo<sup>1</sup> 1) VTT Technical Research Centre of Finland, Espoo, FI; 2) Novartis Pharma Ag., Basel, Switzerland; 3) Glykos FInland Oy, Helsinki, Finland.

Protease secretion limits the production of many sensitive therapeutic proteins such as hormones and cytokines that are by nature easy to degrade. There are over 40 potential proteases secreted by *Trichoderma reesei*. We looked for transcriptional regulators of these proteases with the aim to control and reduce the expression of a wide range of proteases. Protease induction studies were set up to trigger protease activity with peptide and protein substrates in liquid cultures of *T. reesei*. Genome-wide expression data was generated and clustered to find out what genes are co-regulated after different treatments. Twelve candidate transcription factors or regulatory proteins were identified. To narrow the selection, the regulator genes were located on the scaffold to see if they were physically near any protease genes. Transiently silencing *ptf1*, *prp1*, and *ptf3* with siRNA downregulated the expression of a selection of protease genes in accordance with the co-regulation observed. Treatment with both *ptf1* and *prp1* siRNAs increased the effectiveness of the knockdown and reduced protease activity. The deletion of single, double, and triple combinations of the regulators successfully reduced protease activity and increased interferon alpha 2b production. For example, the triple deletion $\Delta ptf1 \Delta prp1 \Delta ptf8$ lead to a 3.7-fold improvement in interferon alpha 2b production. This is the first time protease regulatory proteins have been identified in *T. reesei*, and we have demonstrated how silencing or deleting them could broadly reduce protease activity.

**Rhodosporidium toruloides: A new platform organism for production of biofuels and bioproducts derived from depolymerized polysaccharides and lignin.** Junko Yaegashi<sup>1,2</sup>, James Kirby<sup>1,3</sup>, Masakazu Ito<sup>4</sup>, Jian Sun<sup>1,5</sup>, Tanmoy Dutta<sup>1,5</sup>, Mona Mirsiaghi<sup>6</sup>, Eric R. Sundstrom<sup>6</sup>, Edward Baidoo<sup>1,6</sup>, Deepti Tanjore<sup>6</sup>, Todd Pray<sup>6</sup>, Kenneth Sale<sup>1,5</sup>, Seema Singh<sup>1,5</sup>, Jay D. Keasling<sup>1,3,6,7,8</sup>, Blake A. Simmons<sup>1,6</sup>, Steven W. Singer<sup>1,6</sup>, Jon K. Magnuson<sup>1,2</sup>, Adam P. Arkin<sup>4,6,8</sup>, Jeffrey M. Skerker<sup>4,6</sup>, John M. Gladden<sup>1,5</sup> 1) Joint BioEnergy Institute, Emeryville, CA; 2) Pacific Northwest National Laboratory, Richland, WA; 3) California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA; 4) Energy Bioscience Institute, Berkeley, CA; 5) Sandia National Laboratory, Livermore, CA; 6) Lawrence Berkeley National Laboratory, Berkeley, CA; 7) Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA; 8) Department of Bioengineering, University of California, Berkeley, CA.

Maximizing utilization of the different carbon sources present in raw lignocellulosic material into the desired final product is a key aspect to increasing the effectiveness of producing bioproducts from plant biomass. While conventional microbial organisms such as *E. coli* and *S. cerevisiae* are the most commonly used host microbes for bioproduct synthesis, they do not readily uptake and utilize multiple carbon sources simultaneously, especially in the presence of glucose. Other organisms that are innately able to use carbon sources other than glucose tend to lack efficient genetic tools for host engineering.

In this study we explored the utility of *Rhodosporidium toruloides* as a new platform organism for production of terpenes from deconstructed
plant biomass. Remarkably, we found that it is able to simultaneously utilize all three of the major carbon sources that are found in lignocellulosic hydrolysates: glucose, xylose, and *p*-coumaric acid. Furthermore, it is fast-growing, single-celled, and genetically tractable, making it a promising host for pathway engineering. We demonstrate the production of two heterologous terpenes, bisabolene and amorphadiene in *R. toruloides* achieved by random chromosomal integration of their respective terpene synthase expression cassettes. We show its ability to utilize each of the three carbon sources mentioned above individually as well as simultaneously for growth and production of bisabolene. Finally, we demonstrate its ability to be applied to a one-pot pretreatment, saccharification, and fermentation system of lignocellulosic feedstocks. This organism was able to tolerate the ionic liquid that was used for pretreatment and was also able to utilize all of the three major carbon sources in the hydrolysate to support growth and bisabolene production.

**Cycloaspeptides: elucidation of a novel fungal cyclic peptide biosynthetic pathway.** Kate M. J. de Mattos-Shipley¹, Claudio Greco¹, David Heard¹, Nicholas P. Mulholland², Jason L. Vincent², Thomas J. Simpson¹, Christine L. Willis¹, Russell J. Cox¹,³,⁴, Andrew M. Bailey⁵
1) School of Chemistry, University of Bristol, UK; 2) Syngenta, Bracknell, Berkshire, UK; 3) Institute für Organsche Chemie, Leibniz Universität Hannover, Hannover, Germany; 4) BMWZ, Leibniz Universität Hannover, Hannover, Germany; 5) School of Biological Sciences, Life Sciences Building, University of Bristol, Bristol, UK.

The cycloaspeptides are cyclic pentapeptides produced by a range of filamentous fungi that are reported to exhibit various bioactivities, including anti-malarial, anti-tumour and insecticidal. In this work, two psychrotolerant *Penicillium* species, *P. soppii* and *P. jamesonlandense*, were confirmed as producers of cycloaspeptides A and E. Genome sequencing and comparative bioinformatics identified a putative gene cluster, which was confirmed as being responsible for cycloaspeptide biosynthesis by gene knock-outs and heterologous expression. The minimal gene set consists of a 5-module non-ribosomal peptide synthetase (NRPS) and a new type of trans-acting *N*-methyltransferase. Disruption of the *N*-methyltransferase and subsequent feeding studies have uncovered a hitherto unseen trait in fungal secondary metabolism: the ability of the NRPS to adenylate and incorporate *N*-methylated tyrosine and phenylalanine directly into the cyclic peptide. This discovery has been exploited to alter the ratios of cycloaspeptides produced, including a total shift from the non-active major metabolite cycloaspeptide A to the insecticidally active minor metabolite cycloaspeptide E. It also allowed the production of cycloaspeptides B, C and G, compounds not previously detected in a *Penicillium* species. Searches for similar gene clusters in other filamentous fungi identified the known ditryptophenaline gene cluster from *A. flavus*. This pathway was shown to be homologous, in that methylated phenylalanine is also accepted by the ditryptophenaline NRPS. We have also demonstrated that novel NRP natural products can be generated by feeding synthetic amino acid analogues to these systems. This discovery, coupled with the growing field of megasynthase engineering, could potentially be used to introduce methylated amino acids and synthetic amino acids into other NRP natural products of interest.
Thursday, March 16 3:00 PM – 6:00 PM

Nautilus

## Fungal-bacterial Interactions

**NOD-like receptors (NLR) and non self recognition in fungi: genomic and functional insights.** *M. Paoletti* Institut de Biologie et Génétique Cellulaire, CNRS, Bordeaux, FR.

Recognizing other species, be it symbiotic or pathogenic, is essential to initiate an appropriate response. In Plants and animals, if NOD Like Receptors (NLRs) were identified first in the context of innate immunity it now becomes clear (at least for animals) they also play a role in host communication with the microbiota. NLRs display a characteristic tripartite organization with an N terminal effector domain and a C terminal LRR repeat domain surrounding a central Nucleotide Binding domain. Using that tripartite organization as the main criteria we recently identified and described the NLR repertoire of about 200 fungal species. Fungal genomes encode for highly diversified and dynamic sets of NLR encoding genes. Overall diversity goes down to the individual level. Considering the huge diversity of fungal NLRs, one might hypothesize that they function as interspecific communication devices either to activate an innate immune response or to establish symbiosis, the nature of the response presumably depending on the N terminal effector domain.
In an innate immunity context plant and animal NLRs can recognize pathogens either by directly binding a pathogen marker or by recognizing altered self proteins as a consequence of the pathogen's actions. Pathogen markers and host receptors encoding genes may be engaged in an evolutionary arms race, occasionally resulting in host auto-immune diseases. In the model *Podospora anserina* the genes *het-c* encoding a Glycolipid Transfer Protein (GLTP), and *het-e* encoding a NLR were first identified as controlling vegetative incompatibility, a process of non self recognition between individuals of the same species. Their evolutionary signatures led to the idea that incompatible combinations arose as a consequence of their pathogen driven fast evolution. We now investigate this gene pair in the context of innate immunity. The GLTP is required for the proper fungal defense in confrontation with some pathogenic bacteria, and is the target of a bacterial protease known to be a pathogenicity factor. The HET-E cognate NLR may recognize these alterations of the GLTP to initiate a proper response.

**Characterization of the interaction between bacteria and *Magnaporthe oryzae*.** *X. Zhang*, JR Xu Botany and plant pathology, Purdue University, west Lafayette, IN.

Like many other foliar pathogens, the rice blast fungus *Magnaporthe oryzae* form appressoria for plant penetration. Interestingly, bacteria attached to appressoria are often observed in in vitro assays for appressorium formation. In this study, *Pseudomonas fluorescens* strain TR3 was isolated from rice leaves and used for studying its interaction with *M. oryzae*. TR3 could attached to conidia, germ tubes, and appressoria and caused fungal cell death. In co-cultivation assays, TR3 was inhibitory to colony growth in a contact-based manner, excluding the possibility of small antifungal metabolites. Attachment of TR3 to fungal spores was inhibitory to germination and plant infection. Antifungal activities were specifically detected in culture filtrates of TR3 grown in M9 medium together with *M. oryzae*, suggesting the involvement of the type 3 secretion system (T3SS). Mutants detected of the *hrcC* gene encoding the outer membrane pore forming protein of T3SS failed to form flagella and had reduced inhibitory activities against *M. oryzae*. In contrast, deletion of the *pilC* gene that is essential for T4SS had no impact on its antifungal potential although affected attachment to fungal hyphae or conidia. The phosphorylation level of Pmk1 MAP kinase was significantly increased after incubated with TR3 for 2 h, which was not observed in the *sho1* deletion mutant. Taken together, these results indicate that the interaction of *P. fluorescens* with *M. oryzae* is inhibitory to fungal growth, development, and pathogenesis and likely involves T3SS effectors affecting intracellular signaling pathways.

**The fungal holobiont: exploring the mechanisms maintaining the *Rhizopus-Paraburkholderia* symbioses through RNA-Seq and comparative genomics.** J.R. Bermúdez-Barrientos¹,², S.J. Mondo³, T.E. Pawlowska³, C. Abreu-Goodger², L.P. Partida-Martínez¹ ¹) CINVESTAV, Ingeniería Genética, Irapuato, Guanajuato 36821, MX; ²) CINVESTAV, Unidad de Genómica Avanzada (LANGEBIO), Irapuato, Guanajuato 36821, MX; ³) Cornell University, Plant Pathology & Plant-Microbe Biology, Ithaca, NY 14853.

Microorganisms are the most versatile and abundant living organisms on earth. They drive the biogeochemical cycles and constantly interact among themselves and with most, if not all, eukaryotes, including plants and animals. In the last years, the importance of the microbiome to the fitness of eukaryotic hosts has been recognized and new terms such as holobiont, hologenome and symbiogenesis had been introduced. However, these terms have been only applied to animals and plants, but not fungi. Yet, studies on the *Rhizopus-Paraburkholderia* symbioses suggest that fungi may be indeed also considered as holobionts (fungal host + symbionts).

Under the light of the holobiont concept, we will present the influence of symbiosis in the biology of *Rhizopus microsporus*. In these fungi, endofungal bacteria are the actual producers of the "mycotoxins" rhizoxin and rhizonin, and the presence of the bacterial symbionts is required for the asexual reproduction of the host. This dependency assures the vertical transmission of the endosymbionts and opens further questions about the implications of the symbiosis for the involved partners.

Strikingly, our recent RNA-Seq analyses revealed the presence of a *Narnavirus* in *R. microsporus*, suggesting a third partner in the association. Moreover, gene expression in *R. microsporus* suffers dramatic changes correlated with the presence/absence of its bacterial symbionts. We found that nuclear proteins, particularly transcription factors, are over-expressed in the absence of the endosymbiont, while fungal histone methyl-transferases are up-regulated when the endosymbiont is present, suggesting the involvement of epigenetic mechanisms in the regulation of the bacterial-fungal symbiosis.

Comparative genomics across endofungal bacteria *P. rhizoxinica* and *Ca. Glomeribacter* gigasporarum versus free-living *Paraburkholderia* revealed an enrichment of toxin-antitoxin system proteins in endofungal bacteria. We propose that these proteins may help ensure the vertical transmission of the symbionts (symbiont addiction).

Altogether these analyses have opened novel avenues for research and further support the notion that fungi can be true holobionts.

**Bacteria induce pigment formation in the basidiomycete *Serpula lacrymans*.** J. Tauber¹, V. Schroeckh², D. Hoffmeister¹, E. Shelest², A. Brakhage² ¹) Friedrich-Schiller-University, Jena, Germany; ²) Leibniz Institute for Natural Product Research and Infection Biology– Hans Knöll Institute, Jena, Germany.
The basidiomycete *Serpula lacrymans* produces chromophoric natural products derived from the quinone precursor atromentin such as the pulvinic acid-type variegatic acid. These compounds have been shown to function in redox cycling and were induced from nutritional cues [1,2,3]. Atromentin is a widespread pigment. Hence, we addressed the question if there are other environmental cues, such as microbes, that could also cause induction of these pigments and how such induction may be regulated for Basidiomycota. *Serpula lacrymans* was co-incubated with one of three diverse terrestrial bacteria, *Bacillus subtilis*, *Pseudomonas putida*, or *Streptomyces iranensis* [4]. Analysis of pigment induction was accomplished two-fold. First, gene expression was quantified by qRT-PCR from the clustered genes encoding a quinone synthetase, an aminotransferase, and an alcohol dehydrogenase. Secondly, pigment secretion was measured chromatographically by HPLC. We showed that the atromentin gene cluster of *S. lacrymans* was up-regulated and that secreted pigments accumulated (variegatic, xerocomic, and atromentic acid) during co-incubation with each bacterium indicating induction by a diverse set of microbes. Analysis of the promoter region of each clustered gene across 12 different basidiomycetes containing putatively orthologous atromentin gene clusters indicated a common motif, and thus conserved regulation. We concluded that because a diverse set of bacteria caused pigment induction (in addition to nutritional cues), and given the widespread, conserved presence of functional atromentin-derived pigments and its regulation, these pigments are an essential aspect for basidiomycetes in nutrition uptake, protection, or both.

[1] Eastwood DC., et al. The plant cell wall-decomposing machinery underlies the functional diversity of forest fungi. Science. 2011 333:762-765.

[2] Shah F., et al. Involutin is a Fe³⁺ reductant secreted by the ectomycorrhizal fungus *Paxillus involutus* during Fenton-based decomposition of organic matter. Appl. Environ. Microbiol. 2015 81:8427-8433.

[3] Braesel J., et al. Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete *Paxillus involutus*. Chem Biol. 2015 22:1325-34.

[4] Tauber, JP., et al. Bacteria induce pigment formation in the basidiomycete *Serpula lacrymans*. Environ Microbiol. 2016.

**Bacterial biofilm formation on soil fungi: a widespread ability under controls.** Aurelie Deveau¹, Cora Miquel Guennoc¹,², Jessy Labbé² 1) INRA Université de Lorraine, Interactions Arbres-Microorganismes, UMR 1136, Champenoux, France; 2) Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

Although bacterial biofilms are ubiquitous, little is known about their occurrence and their involvement in bacterial/fungal interaction in soil. In order to contribute to fill this gap, we combined confocal and electronic microscopy to describe the structure, dynamic and specificity of *in vitro* biofilm formation by soil bacteria on fungal hyphae. We show that the ability to form biofilms on fungal hyphae is widely shared among soil bacteria. In contrast, some fungi, mainly belonging to the Ascomycete class, did not allow the formation of bacterial biofilms on their surfaces. The formation of biofilms was also strongly modulated by the presence of tree roots and by the development of the ectomycorrhizal symbiosis, suggesting that biofilm formation does not occur randomly in soil but that it is regulated by several biotic factors. All the biofilms formed on fungal hyphae were structured by dense networks of DNA containing filaments. Those filaments were essential for the formation and the structural maintenance of the biofilms and were produced by both gram positive and negative bacteria. Our data coupled to those produced by other groups strongly suggest that DNA is widely used by prokaryotes and eukaryotes as a structural molecule.

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

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

**Antifungal activity of the human gut metabolome.** C.E. Garcia-Rangel¹, F Tebbji¹, MC Daigneault², NN Liu³, JR Koähler³, E Allen-Vercoe², A Sellam¹ 1) Infectious Diseases Research Centre-CRI, CHU de Québec Research Center, Quebec City, Quebec, Canada; 2) Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada; 3) Boston Children's Hospital/Harvard Medical School, Boston, MA, USA.

The human gut contains a variety of commensal microbes which are composed of diverse organisms that belong to all three domains of life with Eukaria primarily represented by fungi. The commensal/opportunistic yeast *Candida albicans* has been reported as the most common fungus in the gut of healthy humans. Recent study has shown that commensal microbiota play a critical role in the protection of the gut against colonization by other bacterial pathogens and pathobionts. However, so far, whether *C. albicans* overgrowth or pathogenicity are controlled by other fecal microbiota is not known. In this study, we showed that the secreted human gut metabolome (HGM) exerts an
antifungal activity against different intestinal-resident yeasts including *C. albicans*, *C. tropicalis* and *C. parapsilosis*. The HGM inhibited the growth of both sensitive and drug-resistant strains of *C. albicans*. To uncover the mechanism of action of the HGM associated with its antifungal property, a genome-wide genetic screen was undertaken and identified key components of the conserved TOR (Target Of Rapamycin) pathway as required for HGM tolerance. The HGM reduced the phosphorylation state of the known TOR effector, RPS6 confirming thus that the HGM inhibit *C. albicans* growth through TOR pathway. Given the central role of TOR pathway in modulating cell growth in response to nutrients cues, these data support a model where gut microbial cohabitants control *C. albicans* nutritional competitiveness through the modulation of TOR pathway activity. Furthermore, hyphal growth, a critical virulence trait of *C. albicans*, as well as the inducibility of hyphae-specific genes were significantly reduced. In accordance with the inhibitory effect on hyphae formation, we also showed that the HGM reduced significantly the damage inflicted to the human colon epithelial cells by *C. albicans*. To identify microbial specie(s) that produce the antifungal molecule(s), individual isolates form continuous-culture chemostat of fecal extracts of two healthy human donors were screened. Our data revealed that common butyrate-producing bacteria from the two donors exhibited an apparent antifungal activity. Together, these emphasize that the novel cross-kingdom interaction mediated by butyrate-producing bacteria contribute to the control of both the growth and virulence traits of *C. albicans* and provide a new paradigm where the commensal growth of this major human pathogen in the gut is dictated by the surrounding microbiota.

**'Life, Love, and Murder' - logistics of fungal bacterial interplays for soil ecosystem functioning.** L.Y. Wick Environmental Microbiology, Helmholtz Centre for Environmental Research UFZ, Leipzig, DE.

Fungal bacterial interactions are highly diverse and contribute to many ecosystem processes. Their emergence in structurally heterogeneous soil structures or under common environmental stress scenarios, however, still remains mainly elusive. In this contribution we exemplify emerging fungal bacterial interplays by the ecosystem function of contaminant biodegradation in soil. The structural heterogeneity of soil results in the formation of a high variety of soil micro-environments and hotspots of microbial diversity and activity. In heterogeneous environments microbial activity can be considered as a 'logistic' problem, as effective biotransformation will evolve only if water, chemicals and nutrients are sufficiently available to actively degrading microbial communities. In order to cope with heterogeneous environments mycelial soil fungi have developed a unique network-based growth form. Unlike bacteria, hyphae spread efficiently in the soil, penetrate air-water interfaces and cross over air-filled pores. Here we demonstrate the role of mycelial networks for preferential bacterial colonization of subsurface interfaces and discuss its effects on contaminant degradation. In particular, we show that mycelia (i) act as hotspots for horizontal gene transfer, (ii) shape predator-prey interactions and concomitant compound turnover, and (iii) passively and actively enable the functionality of bacterial ecosystems when stressed by low nutrient availability or at low matric potentials. Given the ubiquity and length of up to 500-1000 m g⁻¹ dry soil of hyphae, we conclude that transport and dispersal processes by mycelia not only play a significant role for the ecosystem service of biodegradation of chemicals but also for the nutrient and carbon turnover in soil.
Thursday, March 16 3:00 PM – 6:00 PM

Scripps

Intracellular Mobility, Traffic, and Secretion

**Modelling of whole-cell endosome trafficking reveals molecular adaptation of dynein.** C. Lin², P. Ashwin², M. Schuster¹, G.

Steinberg¹ 1) School of Biosciences, University of Exeter, Exeter, GB; 2) Mathematics, University of Exeter, Exeter, GB.

Fungal early endosomes (EEs) move rapidly along microtubules, thereby organising other organelles, such as peroxisomes and lipid droplets. In *Ustilago maydis*, kinesin-3 and dynein cooperate to mediate EE motility along complex bi-polar microtubule array. Here, we develop a mathematical model of the whole-cell EE motility along various MT arrays, which is based on numerous experimental parameters, including microtubule organisation, EE numbers and motility parameters. In simulations, the model predicts EE run-length, spatial EE distribution, peroxisome distribution and dynamics in wildtype cells and kinesin-3 mutants with high accuracy. We next asked if there are alternative ways of organising organelles. In 23 simulations, we altered motor characteristics (e.g. numbers on organelle, binding rates or run-length) and rearranged the microtubule arrays (e.g. swapping polarity). While only few of these altered scenarios support even EE and PO distribution and motility, all *in silico* solutions require an extended run-length of dynein, not known from other cells. Indeed, experiments confirm that single dynein motors transport over up to 30 µm, which is due to a modified dynactin, characterised by an extended basic domain. Thus, motors have adapted to optimise long-range membrane trafficking in fungal hyphae.

**Dissemination of *Cryptococcus neoformans* to the Central Nervous System is dependent on activation of phosphate uptake at alkaline pH by the transcription factor Pho4.** *Julianne Djordjevic*¹,²,⁶, *Sophie Lev*¹, *Keren Kaufman-Francis*¹, *Desmarini Desmarini*¹, *Pierre Juillard*¹, *Cecilia Li*¹, *Sebastian Stifter*³, *Carl Feng*³, *Tania Sorrell*¹,²,⁶, *Georges Grau*⁴, *Yong-Sun Bahn*⁵ 1) Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Sydney, NSW, AU; 2) Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, NSW, Australia; 3) Immunology and Host Defense Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, NSW, Australia; 4) Vascular Immunology Unit, Department of Pathology, School of Medical Sciences, The University of Sydney, NSW, Australia; 5) Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03 722, Republic of Korea. ; 6) The Westmead Clinical School, Sydney Medical School, The University of Sydney NSW, Australia.

Phosphate acquisition by fungi is regulated by the PHO signaling pathway. *Cryptococcus neoformans* disseminates from lung to brain and is the most common cause of fungal meningitis world-wide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we identified a mutant strain (*hlh?/phoΔ*) from a transcription factor knockout library, which is defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, *Hlh3/Pho4* functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. *pho4Δ* was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH and, under these conditions, phosphate uptake and growth were compromised in the *pho4Δ* mutant. *pho4Δ* was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. *Pho4Δ* was not detected in blood nor did it proliferate when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on the activation of phosphate uptake and stress tolerance at alkaline pH, both of which are dependent on Pho4.

Vacuolar protein sorting VPS-27 identifies putative multivesicular bodies in *Neurospora crassa* hyphae. *M. Riquelme*¹, A. Aguilar-Romero¹, B. Bowman², L. Martínez-Núñez¹ 1) Department of Microbiology, Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE, Baja California, MX; 2) Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California, USA.

Multivesicular bodies (MVBs) are formed when the membrane of the late endosomal compartment invaginates within its own lumen forming intraluminal vesicles (ILVs). This process requires both the ubiquitination of the cargoes destined to the ILVs and the participation of multiprotein Endosomal Sorting Required for Transport (ESCRT) complexes: ESCRT-0, I, II and III. The MVB sorting pathway participates presumably in many important cellular processes, most of which remain unknown in filamentous fungi. To study the morphology and distribution of MVBs and their role in the recycling of plasma membrane (PM) enzymes in *Neurospora crassa* we have analyzed the homologue of the yeast class E Vps27 protein. The FYVE domain of Vps27 mediates its interaction with endosomal PI(3)P. VPS-27 (NCU04015), a putative component of ESCRT-0, tagged with either GFP or mCherry, was found at fluorescent puncta all along the subapical cytoplasm excluding the apex. In further distal regions, VPS-27 was found at the membrane of pleomorphic bodies, presumably MVBs, which while advancing remained at a constant distance from the hyphal apex. VMA-1 and RAB-7, both previously identified at prevacuolar compartments (PVCs), showed partial co-localization with VPS-27 at the membrane of the putative MVBs. Different high-resolution live-imaging approaches are currently being used to investigate the fate of the MVBs.

**The direct pathway connecting trans-Golgi cisternae and the plasma membrane with RabE<sup>RAB11</sup> secretory vesicles.** *M.A. Penalva*¹, *M. Pinar*¹, *M. Hernández*¹, *H.N. Arst*², *J. Zhang*³, *X. Xiang*³, *A. Pantazopoulou*¹ 1) Department of Cellular and Molecular Biology, CSIC Centro de Investigaciones Biológicas, Madrid, Madrid, ES; 2) Section of Microbiology, Imperial College London, London, UK; 3) Department of Biochemistry and Molecular Biology, The Uniformed Services University of the Health Sciences, Bethesda, MD.

The trans-Golgi network of *Aspergillus nidulans* gives rise to secretory vesicles (SVs) bound to the apical plasma membrane. These SVs bud off the Golgi after a maturation process by which cisternae gradually acquire post-Golgi identity by recruiting RabE<sup>RAB11</sup>. Shortly after this recruitment SVs undergo transport towards the apex using myosin-5 and kinesin motors. TRAPPII, an oligomeric GEF containing Trs120 mediates the exchange of GDP for GTP on RabE. Trs120 is notable in that the gene encoding this TRAPPII component, denoted *hypA* in *Aspergillus*, was initially identified in a screen for conditional mutations affecting hyphal morphology. HypA<sup>TRS120</sup> is recruited to the TGN preceding the departure of SVs. Mutations in *rabE* that enable growth in the absence of *hypA/TRAPPII* promote spontaneous guanine nucleotide exchange on the GTPase. Transport of SVs to the Spitzenkörper involves the myosin-5 MyoE, one of the likely effectors of
RabE<sup>RAB11</sup>. It also involves kinesin-1 (KinA), which is sufficient to sustain apical extension in the absence of myosin-5. Lastly SVs can also engage dynein, which mediates their retrograde transport away from the apex. Dynein loading on SVs involves the p25 subunit of dynactin.

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

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

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

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

**Internuclear diffusion of histone H1 within cellular compartments in *Aspergillus nidulans*.** Alexander Mela, Michelle Momany Plant Biology, University of Georgia, Athens, GA.

Histone H1 is an evolutionarily conserved 'linker' histone protein which serves the important dynamic function of arranging and stabilizing chromatin structure. The mechanism by which histone H1 carries out its function remains unclear. One thing that is certain is the common use of fluorophore-tagged histone H1 protein in microscopy to track nuclei within cells. In time-lapse analyses, we observed stochastic exchange of photo-activated Dendra2-Histone-H1 protein between nuclei. We also observed heterogeneous mixing of histone proteins between nuclei in hyphae and conidiophores derived from heterokaryon fusions of histone H1-RFP and H1-GFP strains. Subsequent analysis of the resulting conidia that contained both RFP- and GFP-labeled histone H1 proteins, showed only parental genotypes. These data together suggest the stochastic exchange of histone H1 protein between nuclei is likely a result of diffusion rather than genetic recombination during karyogamy.

**Microtubules are reversibly depolymerized in response to changing gaseous microenvironments within *Aspergillus* biofilms.** N. Shukla¹,², A. Osmani², S. Osmani¹,² 1) The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210; 2) Department of Molecular Genetics, The Ohio State University, Columbus Ohio 43210.

Fungal persistence and drug resistance during infections occurs mainly due to their ability to form biofilms. Concentration gradients of oxygen and nutrients caused by cell crowding and potentially secreted signaling factors cause cells in different areas of the biofilm to experience different microenvironments. The goal of our study was to understand how microenvironments within different areas of a fungal biofilm impact cell physiology.

Using confocal microscopy and genetic analyses we have uncovered a new physiological response that *Aspergillus nidulans* cells in a biofilm undergo in response to their microenvironment. We found that after adhesion to a surface, cells at the base of a forming biofilm first halt their growth and later on depolymerize their microtubules (MTs) in a cell-autonomous manner. During such MT-disassembly, MT plus-end binding proteins like EB1, ClipA and Dynein transiently locate to unique bar-like structures. To our surprise, we also found that biofilm mediated MT-disassembly involves soluble gases since cells instantly repolymerize their MTs upon simple air exchange above the static biofilm media. Further experiments revealed that biofilm-mediated MT disassembly occurs as a regulated response to hypoxia. Upon further maturation, and possibly due to the formation of the extracellular matrix (ECM), cells no longer respond to simple gaseous exchange above the biofilm media. At this time, physical removal of a part of the biofilm is required to promote MT reassembly that then occurs in cells at the new biofilm edge. In experiments aimed at further understanding the volatile agent involved in the regulation of MT dynamics within biofilms
we tested the effects of the gaseous signaling agent, hydrogen sulfide (H₂S). We find that all aspects of MT depolymerization and repolymerization, including EB1, ClipA and Dynein bar formation, can be mimicked in growing cells under normoxia by H₂S addition and removal. Collectively, our study has implications in the areas of MT regulation, responses to hypoxia, gaseous signaling, and potentially fungal pathogenesis.
Friday, March 17 9:00 AM – 12:00 NOON

Merrill Hall and Chapel

**Plenary Session III: Systems Biology: Genes, Genomics and Genome Structure**

**Sequence all the fungi: what can you do with 1000 fungal genomes?** Jason Stajich¹, Joseph Spatafora², Zygolife and 1000 Fungal Genomes Consortia 1) Plant Pathology & Microbiology, Univ California-Riverside, Riverside, CA; 2) Botany & Plant Pathology, Oregon State University, Corvallis, OR.

Fungi lead the way in number and phylogenetic breadth of sequenced eukaryotic genomes. Worldwide efforts have generated many hundreds of fungal genome sequences for research ranging from biotechnology, plant and animal disease, to basic fungal and evolutionary biology. The 1000 Fungal Genomes project (1KFG; http://1000.fungalgenomes.org) is one effort to broadly sample the fungal tree of life aiming for at least 2 genomes sampled from every taxonomic family. Additionally the Zygolife project (http://zygolife.org) is an effort to increase sampling and understanding of zygomycete fungi phylogeny and evolution. These genome and organismal data are useful in a variety of questions. **Evolutionary Relationships** Using a dataset of 350+ genomes we have revised the fungal phylogeny and reclassified early branching zygomycete lineages into two new phyla. These genome data have also supported development of a broad set of conserved gene markers useful for phylogenetics and genome completeness assessment. **Comparative Biology.** Genomic comparisons in protein coding gene content has revealed changes in ecological niche lifestyles and primary metabolisms. Contrasts of mycoparasite, plant and animal associated, or saprotrophic lifestyles is also revealing how evolutionary shifts in trophic associations are reflected in genomic composition and changes. **Metagenomics and classification methods.** A rich set of reference genome data is essential when analyzing gene content in metagenomic or transcriptomic datasets. We have tested these improved resolutions with rapid sequencing technologies to more quickly identify likely species classifications. Efforts to use light coverage genome sequencing coupled with reference genome resources is also proving useful for resolving phylogenetic relationships and simple gene inventories at low costs.

A guide to some of these large datasets of genomic resources for fungi will be described, how they are generated and how others can contribute species to be sampled and sequenced, how the data are disseminated and analyzed, and what ways it can be most useful to the broad community.

**Epigenetic memory over geological timescales.** S. Catania¹, P. Dumesic¹, C. Stoddard¹, G. Narlikar¹, C. Cuomo², H. Madhani¹ 1) Dept. of Biochemistry and Biophysics, UCSF, San Francisco, CA; 2) Broad Institute, Cambridge, MA.

It is well established that evolution operates on natural variation that is produced by variation in DNA sequence (genetic variation). An alternative form of heritable variation is epigenetic variation, defined here as heritable differences between individuals that are not due to differences in the DNA sequence. Whether such variation can be maintained accurately over a sufficient number of generations to enable evolution is not clear. Here we provide an example in which modification of DNA, cytosine methylation, can be epigenetically inherited over a time scale of millions of years. We have characterized mechanisms of establishment and maintenance of DNA methylation in the basidiomycetous yeast *Cryptococcus neoformans*, whose single predicted cytosine methyltransferase, Dnmt5, is responsible for all symmetric CG methylation. Our detailed genetic and biochemical experiments indicate that, like the mammalian Dnmt1 maintenance methylase, Dnmt5 maintains DNA methylation through two partially redundant mechanisms, which respectively involve the recognition of heterochromatin marks and of prior DNA methylation. Despite the ability of Dnmt5 to maintain cytosine methylation, it cannot perform *de novo* methylation *in vivo* or *in vitro*. Genetic experiments in which the DNMT5 gene is removed or repressed and then restored demonstrate that once lost, DNA methylation cannot be regained *in vivo*. Consistent with these results, purified full-length Dnmt5 exhibits robust cytosine methyltransferase activity on hemimethylated DNA substrates, but has no detectable activity on unmethylated substrates. By examining the genomes of species in the order of Tremellales, of which *C. neoformans* is a part, we find that ancestors of *Cryptococcus* harbored two enzymes: a Dnmt5 ortholog and second predicted cytosine DNA methyltransferase, a presumptive *de novo* methylase. Our phylogenetic analysis reveals that the gene coding for this second enzyme was lost prior to the divergence of *C. neoformans* and the genus *Kwoniella*, which is estimated to have occurred between 50 and 150 million years ago. We propose that DNA methylation has been propagated epigenetically by Dnmt5 via a "maintenance-only" mechanism since the loss of the presumptive *de novo* methylase gene.

**Systems biology illuminates the mechanistic basis of host damage during fungal diseases of the human lung.** E.M.

Bignell Microbiology Section, Imperial College London, London, GB.

The human lung is continually exposed to spores of the airborne mould *Aspergillus fumigatus*. Inhaled spores are small enough to bypass mucociliary clearance mechanisms and reach the alveoli of the lung where interaction between host and pathogen cells can lead to fungal clearance, or to development of inflammatory or invasive fungal diseases. *A. fumigatus* is an accidental pathogen whose encounters with the host, although frequent, are circumstantial in nature. The capacity of this organism to cause human disease is unique amongst a cohort of several hundred related Aspergillus species and relative to closest sequenced relative there are no large scale genetic events which signify recent evolution of pathogenicity. However, multiple, recently evolved telomere-associated genes are preferentially upregulated in the host environment, including those which drive production of secondary metabolites. Clinical relevance of aspergillus species does not correlate with fungal growth rate, but is positively correlated with thermotolerance, and epithelial toxicity.
Fuelled by the results of extensive fungal transcriptome research we have developed, in recent years, a programme of research which seeks the mechanistic basis of lung damage during *A. fumigatus* infection. In order to find out why *A. fumigatus* is cytotoxic to human epithelia we have observed the interaction between host and pathogen in laboratory culture and in mouse disease, and measured epithelial decay, lytic death of host cells, host cell signalling and cytokine degradation in response to fungal challenge. This research has revealed multiple mechanisms involved in eliciting epithelial damage, occurring at different stages of the host-pathogen interaction and involving different fungal morphotypes. In this talk I will explain how systems approaches have led us to ask the following questions: Why is *A. fumigatus* cytotoxic to human cells? Does the host or the pathogen drive epithelial damage and how? How do pathogen-derived proteins shape the immune environment? How is damage driven in the whole animal host? Which elicitors of host damage are produced by *A. fumigatus*, and have they recently evolved? I will finish by introducing a new high throughout study addressing the *A. fumigatus* regulatory
network driving epithelial damage in cultured human lung tissue and in the whole animal, and examine how this knowledge might lead to
novel therapeutic interventions.

**How to make a fruiting body: comparative genomics and transcriptomics in ascomycetes.** David Schumacher, Stefanie Traeger, Stefan Gesing, Ramona Lütkenhaus, Florian Altegoer, Daniel Schindler, *Minou Nowrousian* Department of General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany.

The development of complex multicellular structures has evolved many times in eukaryotes, and at least twice in fungi. The fruiting bodies of filamentous ascomycetes are one example, and even though many genes essential for their differentiation have been identified, a model describing the spatio-temporal regulation of this developmental process at the molecular level has yet to emerge. Our aim is to identify evolutionary trends and core processes that regulate fruiting body morphogenesis through comparative genome and transcriptome mining. We performed RNA-seq on young fruiting bodies isolated by laser microdissection from the Sordariomycete *Sordaria macrospora* and the Pezizomycetes *Pyronema confluens* and *Ascodesmis nigricans*. Comparative expression analysis of genes conserved in all three species shows structure-specific expression when looking at relative expression changes, but species-specific expression with respect to overall expression levels. Genes involved in chromatin organization and regulation of gene expression, and genes involved in the organization of endomembrane systems make up more than 40 % of genes specifically upregulated in young fruiting bodies in all three species. For further functional analysis, our focus is on genes involved in chromatin-related processes. Among the genes with conserved expression patterns identified in previous comparative studies are the histone chaperone gene *asf1* and the transcription factor gene *pro44*, both of which are essential for fruiting body development in *S. macrospora*. RNA-seq analysis of *pro44* and *asf1* mutants showed that many genes with overall weak expression are upregulated in the *asf1*, but not the *pro44* mutant, suggesting that ASF1 acts as a repressor of weakly transcribed genes. To analyze whether this transcriptional derepression might be caused by problems with nucleosome deposition in the histone chaperone mutant, we performed micrococcal nuclease sequencing (MNase-seq). Genome-wide nucleosome patterns are wild type-like in the *asf1* mutant, indicating that the transcriptional changes and developmental defects of the mutant are not caused by an overall change in nucleosome distribution. Future studies will focus on a potential role of *asf1* in nucleosome turnover and histone modifications.

**Bidirectional cross-kingdom RNAi and spray-induced gene silencing for plant disease control.** Ming Wang¹, Arne Weiberg¹,⁴, Feng-Mao Lin², Bart Thomma³, Hsien-Da Huang², *Hailing Jin*¹ 1) University of California, Riverside, CA; 2) Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsin-Chu 300, Taiwan; 3) Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; 4) Current Address: Institute of Genetics, University of Munich Martinsried, Germany.

Small RNAs (sRNAs) are a class of short non-coding RNAs that mediate gene silencing in a sequence-specific manner. We have demonstrated that some sRNAs from eukaryotic pathogens, such as *Botrytis cinerea*, the fungal pathogen that causes grey mold disease on more than 1000 plant species, could be translocated into host plant cells and suppress host immunity genes for successful infection. This finding represented the first example of naturally occurring Cross-Kingdom RNAi in host-pathogen interactions. These sRNAs act as a new class of pathogen effectors that translocate into host cells to suppress host immunity.

Recently we have found that transgenic plants expressing hairpin RNAs that targeting *Botrytis Dicer* 1 and Dicer 2 genes could effectively block the generation of fungal sRNA effectors and suppress grey mold disease. This strategy can be adapted to simultaneously control multiple fungal diseases. We show that Arabidopsis plants expressing hairpin RNAs that simultaneously target DCL genes of B. cinerea and V. dahliae show enhanced disease resistance to both pathogens. These results also suggest that sRNA trafficking is bi-directional and sRNAs could move from the host cells to the interacting pathogens. Most strikingly, we discovered that *Botrytis* could take up double-stranded RNAs and sRNAs from the environment. Applying sRNAs or dsRNAs that target *Botrytis Dicer* genes on the surface of fruits, vegetables and flowers significantly inhibits grey mold disease. Such pathogen gene-targeting RNAs represent a new generation of environmentally friendly fungicides.
Friday, March 17 3:00 PM – 6:00 PM

Merrill Hall

Evolution and Mechanisms of Antifungal Drug Resistance

**Adaptive landscapes in fungicide resistance.** Nichola Hawkins, Bart Fraaije Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, GB.

The use of fungicides generally exerts a strong selective pressure for resistance, and in many cases this results in the rapid spread of a single mutation conferring resistance across a mode of action. However, in other cases the evolution of resistance has been less straightforward, resulting in more durable effectiveness of some fungicides. The application of evolutionary concepts such as adaptive landscapes and fitness penalties can help to explain such differences, and to inform future resistance risk assessment and management. In the case of the QoI and MBC fungicides, the repeated parallel evolution of few, simple resistance mechanisms across many plant pathogens indicates a relatively smooth adaptive landscape with consistent functional constraints. The more complex evolution of azole resistance in *Zymoseptoria tritici* results from a more rugged adaptive landscape, with the fitness effect of each mutation varying due to epistatic interactions with other mutations and the genetic background.

The new ascomycete-active SDHI fungicides are considered to be at high risk for the emergence of resistance. Target-site mutations conferring high levels of resistance have already been reported in some key pathogens, but the rate of spread of these mutations and the effectiveness of resistance management approaches will depend upon the underlying adaptive landscape. A combination of evolutionary concepts and functional genetic tools will help to predict this.

**Unveiling a novel regulatory role of the pH regulator PacC and the CCAAT-Binding-Complex in *Aspergillus fumigatus* 5-Flucytosine resistance.** F. Gsaller, T. Furukawa, P.D. Carr, M.J. Bromley Manchester Fungal Infection Group, Division of Infection, Immunity & Respiratory Medicine, University of Manchester, Manchester, GB.

Annually >1.5 million people die from fungal disease, a big proportion results from invasive and chronic mould infections, predominantly caused by *Aspergillus fumigatus*. Only four classes of agents, the azoles, polyenes, candins and 5-Flucytosine (5FC) are available to treat clinical infection. 5FC, a derivative of the nucleobase cytosine is rarely used as a monotherapeutic agent to treat *Aspergillus* infection as resistance develops rapidly. In addition at neutral pH (pH 7.0) the *in vitro* activity of 5FC against *A. fumigatus* is insignificant, however, 5FC is highly active at low pH (pH 5.0).

In this work we provide an explanation for the low efficacy of 5FC at pH 7.0 and uncover two transcription factors that mediate resistance to 5FC - The CCAAT binding complex (CBC) and the pH regulator PacC. Genetic inactivation of the CBC and PacC results in derepression of the purine-cytosine permease encoding gene *fcyB*, which results in 5-FC hypersusceptibility phenotypes. We show that *fcyB* expression is repressed at pH 7.0 and transcriptionally activated at pH 5.0. Disruption of *fcyB* results in hyperresistance to 5FC in wt and, moreover, significantly increases 5FC resistance in both the CBC and PacC mutant backgrounds independent of the pH. In contrast, CBC/PacC independent overexpression of *fcyB* using a conditional expression system results in 5FC hypersusceptibility. ChIP-Seq based genome-wide binding analysis using a strain expressing *gfp*-tagged *hapC* (*hapC***<sup>GFP</sup>* uncovers the CBC as direct regulator of *fcyB*.

Collectively, within this study we characterised the cytosine permease encoding gene *fcyB* the expression of which is under combinatorial transcriptional control of the CBC and PacC. Furthermore, we show that both regulators act as repressors of *fcyB* during pH 7.0, which results in 5FC resistance.

**Chloramphenicol inhibits appressorium formation of *Magnaporthe oryzae* via a Ser/Thr-protein phosphatase, *MoDullard*.** A. Nozaka¹, S. Endo¹, N. Tanaka¹, M. Narukawa¹, S. Kamisuki², M. Nakajima¹, H. Taguchi¹, F. Sugawara¹, T. Kamakura¹ 1) App. Bio. Sci., Tokyo Univ. of Sci., Chiba, Japan; 2) Dept. Vet. Med., Azabu Univ., Tokyo, Japan.

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

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

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

**Plasticity of the MFS1 promoter leads to multi drug resistance in the wheat pathogen *Zymoseptoria tritici*.** Selim Omrane¹, Colette Audéon¹, Amandine Ignace¹, Clémentine Duplaix¹, Lamia Aouini², Gert Kema², Anne-Sophie Walker¹, Sabine Fillinger¹ 1) BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, FR; 2) Wageningen University, Plant Research International, Wageningen, NL.

The ascomycete *Zymoseptoria tritici* is the causal agent of septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide application. The fungus however displays a high potential to circumvent both methods. Resistance against all major fungicides has been observed and studied over decades. Especially the adaptation to DMIs, inhibiting sterol biosynthesis, implying modifications of the *CYP51* target gene has revealed a large spectrum of resistance mutations. Recently introduced fungicides are facing
resistance through target gene mutations as well. A different type of resistance has evolved among *Z. tritici* populations affecting multiple chemical families (Leroux & Walker, 2011). Strains considered as multi-drug resistant have been isolated since 2009.

Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. MDR is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. We showed that active drug efflux was at play in *Z. tritici* MDR field strains and we identified the major-facilitator gene, *MFS1*, as principal player of this emerging resistance mechanism (Omrane et al., 2015). The *MFS1* gene was found overexpressed in all tested field strains. At the origin of the overexpression and the MDR phenotype is a 519 bp insert in the *MFS1* promoter, an LTR reminiscence of a recent retro-transposition event. The insert harbors four copies of a highly conserved transcription-factor binding-site that may explain *MFS1* overexpression. Besides this type of insert, we found a different type of promoter insert in more recent MDR strains. Interestingly, this last insert was found upstream of other genes in different *Z. tritici* strains. It also contains potential transcription factor binding sites. Finally, a 3rd type of insert was identified in two other *MFS1* overexpressing MDR strains.

Altogether, these results underline the extremely high adaptive potential of *Z. tritici* to fungicides through the plasticity of the *MFS1* promoter leading to its over-expression, to fungicide efflux and, ultimately, to MDR (Omrane et al., in prep).

Leroux & Walker, (2011) *Pest Manag Sci* **67**: 44-59.

Omrane, et al. (2015) *Env. Microbiol.* **17**: 2805-2823.

**Defining the role of heteroresistance in persistent human fungal infections.** I.V. Ene¹, K. Navarrete¹, E. Fu¹, J.M. Bliss¹,², A.L.

Colombo³, C. Cuomo⁴, R.J. Bennett¹ 1) Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA; 2)

Women & Infants Hospital, Providence, Rhode Island, USA; 3) Universidade Federal de São Paulo, São Paulo, Brazil; 4) Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

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

**Functional and subcellular localization analyses of transcription factors, AtrR and SrbA, involved in azole drug resistance in Aspergillus oryzae.** K. Sugiyama¹, A. Ohba-Tanaka¹, D. Hagiwara², S. Kawamoto², M. Tanaka¹, T. Shintani³, K. Gomi¹ 1) Graduate School of Agricultural Science, Tohoku University, Sendai, JP; 2) Medical Mycology Research Center, Chiba University, Chiba, JP.

We previously demonstrated that a novel Zn(II)₂Cys₆ transcriptional factor, AoAtrR, regulates gene expression of the ABC transporters that would function as drug efflux pumps and contributes to the azole drug resistance in *Aspergillus oryzae*, and a disruption mutant of the *AoatrR* ortholog (*AaftrR*) in *Aspergillus fumigatus* was similarly hypersensitive to azole drugs. In addition, RNA-seq analysis in *A. fumigatus* showed that AtrR regulated several ergosterol biosynthetic genes including *erg11*. Surprisingly, most of these ergosterol biosynthetic genes regulated by AtrR were nearly identical to those regulated by the basic helix-loop-helix transcription factor, SrbA (1). Therefore, we are interested in and investigate the difference in function between AtrR and SrbA in *Aspergillus oryzae*.

The expression of ergosterol biosynthetic genes such as *erg11*, *erg24*, and *erg25*, as well as ABC transporter genes, was significantly down-regulated in the *AoatrR* disruptant. Similarly, *AosrbA* disruption resulted in remarkable down-regulation of ergosterol biosynthetic genes same as in *A. fumigatus*, but not ABC transporter genes. In contrast, *AoatrR* overexpression did not resulted in up-regulation of ergosterol biosynthetic genes. These results suggested that AtrR and SrbA coordinately regulate ergosterol biosynthetic genes in aspergilli.

However, *AoatrR* or *AosrbA* disruption had apparently no effect on another gene expression level, suggesting their expression would be regulated independently of each other. On the other hand, the *AoatrR* disruptant was more hypersensitive to azole drugs compared to the *AosrbA* disruptant and sensitive comparable to the *AoatrR/AosrbA* double disruptant, suggesting that hypersensitivity of the *atrR* disruptant to azole drugs is attributed not only to lowered ergosterol levels owing to down-regulation of ergosterol biosynthetic genes, but also to reduced efflux transport of the drugs owing to down-regulation of ABC transporter genes. Subcellular localization analysis of AoAtrR and AoSrbA showed that AoAtrR was constitutively localized in the nucleus and AoSrbA was likely localized in the nuclear envelope and/or endoplasmic reticulum.

(1) Hagiwara et al., *PLoS Pathogens*, in press.

**Molecular mechanisms of multidrug resistance conferred by xenobiotic detoxification in Sclerotinia homoeocarpa.** Geunhwa Jung¹, Hyunkyu Sang¹, Jon Hulvey², Robert Green¹, Taehyun Chang³ 1) University of Massachusetts, Stockbridge School of Agriculture, 161 Holdsworth Ways, Amherst, MA; 2) University of Massachusetts, Biology Department, 611 North Pleasant Street, Amherst, MA; 3) Kyungpook National University, School of Ecology & Environmental System, Sangju, South Korea.

Dollar spot, caused by the “sterile” ascomycete fungus *Sclerotinia homoeocarpa* is the most economically important disease on high amenity turfgrass. Fungicide resistance in *S. homoeocarpa* has developed in multiple fungicide classes through target gene mutations and
multidrug resistance (MDR) is conferred by a xenobiotic detoxification system. Multidrug resistance has been a major impediment in treating cancer patients and is an emerging problem in plant and human pathogenic fungi. In cancer cells, a single nuclear receptor, PXR (pregnane X receptor) has been known to play a pivotal role in concomitantly regulating drug-induced elevated changes in Phase I metabolizing enzymes, Phase II conjugating enzymes, and Phase III efflux transporters. We also uncovered a mechanistic analogue of PXR, single fungal-specific transcription factor (ShXDR1) in *S. homoeocarpa* that coordinately regulates Phase I cytochrome P450 (CYP450) enzymes in drug metabolism, and Phase III ATP-binding cassette (ABC) transporters in drug secretion, with a differential substrate specificity for detoxification of multiple xenobiotics. This appears to be the first report establishing a molecular mechanism for the regulation of antifungal/xenobiotic detoxification conferring multidrug resistance in filamentous plant pathogenic fungi.

**Drug resistance via RNAi-dependent epimutations in *Mucor circinelloides*.** Z. Chang¹, B. Billmyre¹, S.C. Lee², J. Heitman¹ 1) Duke University, Durham, NC; 2) University of Texas at San Antonio, San Antonio, TX.

The opportunistic fungal infection mucormycosis is notable for high morbidity and mortality as well as increasing incidence. Treatment is complicated by the fact that *Mucor circinelloides*, a major cause of mucormycosis, demonstrates high intrinsic resistance to the majority of antifungal agents. However, the mechanisms driving this extensive resistance remain poorly understood. Previous work demonstrated that *Mucor* is capable of developing transient resistance to the antifungal FK506 through a novel, RNA interference-dependent mechanism known as epimutation. Epimutants silence the drug target gene and can be selected by exposure to FK506; the target gene is re-expressed in these strains following passage in the absence of FK506 selective pressure. This silencing process involves the generation of small RNAs (sRNA) against the target gene via core RNAi pathway proteins including RNA-dependent RNA polymerases, Dicer, and Argonaute. To further investigate the role of epimutation in *Mucor's* intrinsic antifungal resistance, we studied the development of resistance to a second drug, 5-fluoroorotic acid (5-FOA). We have identified epimutants that exhibit resistance to 5-FOA without mutations in either of the target genes, *pyrF* or *pyrG*. We conducted sRNA hybridization analysis to document the presence of sRNA against *pyrF* or *pyrG* in these epimutants, and demonstrated this sRNA is lost after reversion to drug sensitivity. Analysis of sRNA libraries generated from *pyrF* and *pyrG* epimutants demonstrated expression of sRNA against the *pyrF* and *pyrG* loci, respectively. From this work, we conclude that epimutation is a general mechanism through which *Mucor* can develop resistance to multiple antifungal agents. Elucidation of the role of RNAi in epimutation will afford us a fuller understanding of mucormycosis and also fungal pathogenesis and drug resistance more generally.
Friday, March 17 3:00 PM – 6:00 PM

Chapel

Cell Communication

**Cell differentiation and interaction within developing yeast colonies.** Zdena Palkova¹, Libuse Vachova²
1) Faculty of Science, Biocev, Charles University, Prague, Czech Republic; 2) Institute of Microbiology CAS, v.v.i., Biocev, Czech Republic.

Yeast colonies have become an excellent model for investigating various aspects of microbial multicellularity, including processes involved in cell differentiation, signaling and interaction. Two major types of *Saccharomyces cerevisiae* colonies can be classified according to their architecture: smooth colonies formed by laboratory strains and structured biofilm colonies, formed by wild strains. We have analyzed these colonies and identified several specifically localized cell subpopulations that have distinct properties and functions and even mutually interact (reviewed in *FEMS Yeast Res* 14, 96–108, 2014). Smooth *S. cerevisiae* colonies are composed of the two major cell subpopulations: U cells in upper regions that activate adaptive metabolism and gain a longevity phenotype and L cells in lower regions that behave like starved and stressed cells, activating different hydrolytic mechanisms and providing nutrients to U cells (*Mol Cell* 46:436, 2012). Using transcriptomic and confocal microscopy approaches, combined with other analytical methods and modified yeast strains, we have identified metabolic and regulatory pathways that are advantageous to the long-term vitality of these subpopulations. We have shown that differently modified mitochondria of U and L cell subpopulations are involved in the control of cellular reprogramming and the activation of specific metabolic pathways via retrograde (RTG) signaling (*Oncotarget* 7: 15299, 2016). We have identified three distinct pathways of mitochondrial RTG signaling that are all dependent on functional Rtg1p, Rtg2p and Rtg3p regulators. However, each of these pathways activates expression of specific gene targets and thus leads to divergent metabolic reprogramming and subsequent effects on the survival of specific cell subpopulations in developing colonies. These data showed that the RTG response in yeast is more complex than previously assumed, involves a number of yet to be identified regulatory elements and affects different cellular processes and the subsequent fate of differentiated yeast cells. This research was funded from the Norwegian Financial Mechanism 2009-2014 under Project Contract no. 7F14083 and by GACR 15-08225S.

**Crosstalk between pheromone response pathway and STRIPAK complex regulates developmental processes in *Sordaria macrospora*.** A. Beier, B. Ramšak, I. Teichert, U. Kück Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE e-mail: anna.beier@rub.de.

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

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

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

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

**Sex pheromone autocrine signaling controls vegetative fungal development.** S. Vitale, A. Di Pietro, *D. Turrà* Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, Cordoba, Spain.

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

**Fungal-plant communication in the arbuscular mycorrhizal symbiosis.** *N. Requena*, R Betz, S Heidt, M Hartmann, S Walter Karlsruhe Institute of Technology, Karlsruhe, DE.

The arbuscular mycorrhiza (AM) symbiosis represents one of the most ancient and widespread symbiosis on the earth, characterized by an intimate life-long association between Glomeromycotan fungi and most plant roots. The establishment and maintenance of the symbiosis requires a complex communication exchange between symbionts and secreted fungal effector proteins might be key to facilitate colonization and nutrient exchange. Compared to other biotrophic fungi, the effectome of the AM fungus *Rhizophagus irregularis* seems to be rather small, considering the small number of proteins identified containing a secretion signal. Interestingly, *R. irregularis* is able to colonize the majority of land plants in contrast to most plant colonizing fungi that have a quite narrow host range. We thus hypothesize that the few effector candidates identified are conserved in other AM fungi and might play crucial roles in rewiring the plant physiology. We previously identified the effector SP7 (secreted protein 7) the first characterized AM effector, and a group of related proteins (SP7-like2, SP31 and SPrubi). All members of this family show the same basic protein structure with a central domain of tandem, hydrophilic and imperfect repeats and a nuclear localization signal. Although there is evidence of a positive effect on symbiosis for SP7, the molecular mechanisms underlying the symbiotic function of the SP family remain elusive. Detailed in planta co-localization experiments with defined cell markers together with interactomic studies revealed a direct association of the SP effectors with spliceosomal components, the nonsense mediated decay and plasmodesmata. Thereby Processing bodies (specific cytoplasmic RNA-Protein granules) could be identified as a novel plant target of fungal effectors. Our results point out towards a significant involvement of AM fungal SP effectors in the conserved plant mRNA processing machinery leading to translational control. Recent results will be presented.

**More than just a funny smell: filamentous fungi use molecular vapors to communicate.** *J.W. Bennett* Dept Plant Biol & Pathology, Rutgers Univ, New Brunswick, NJ.

Fungal volatile organic compounds (VOCs) are found as odiferous mixtures of alcohols, aldehydes, acids, ethers, esters, ketones, hydrocarbons, terpenes and sulfur compounds, and produced at varying concentrations depending on the species, substrate, incubation period and other environmental parameters. They are known as aroma compounds in foods and beverages and also have been used as indicators of fungal growth. There is increasing evidence that many of them serve as chemical signals influencing growth, germination, and pathogenicity. Our laboratory has used genetic models for testing the bioactivity of fungal VOCs. Certain mixtures of VOCs emitted from living cultures of molds isolated after Hurricanes Katrina and Sandy, as well as low concentrations of chemical standards of some individual VOCs, are toxic in *Drosophila melanogaster*. For example, low concentrations (0.5-2.8ppm) of "mushroom alcohol" (1-octen-3-ol) exhibit potent neurotoxicity in flies, specifically against dopaminergic neurons. When a yeast knock out library is screened for resistance to 1-octen-3-ol, ninety-one resistance genes are identified, the majority of which are involved in protein trafficking. Specifically, when either the retrograde (retromer and GARP complexes) or anterograde (ESCRT) sorting pathways are disrupted, yeast cells are resistant to 1-octen-3-ol.

Screening of twenty *Trichoderma* isolates for VOC-induced growth of *Arabidopsis* identified 9 growth promoting isolates increasing plant biomass (up to 41.6%) and chlorophyll content (>89.3%). GC-MS analysis of *Trichoderma* isolates identified over 147 compounds and several unknown terpenes. Twenty six chemical standards, representing common and uncommon fungal VOCs, were evaluated for their effects on *A. thaliana* seeds and vegetative plants growth. Several individual compounds were able to induce similar growth promoting effects at 0.5 µg and 10 ng concentrations. Due to plant growth promoting effects at relatively high and low concentrations, 1-decene was selected for plant gene expression studies, RNA sequencing data identified several candidate genes inducible by the plant hormone auxin, as well as down regulation of a large number of genes related to plant defense and stress responses.

We propose the term "mycosemiotics" to describe the study of vapor-phase signaling molecules, as well as better known hormone and secondary metabolites, that fungi use in intra- and inter-specific communication.

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

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

**Membrane traffic in *Candida albicans* hyphal growth and virulence.** Hayet Labbaoui¹, Stephanie Bogliolo¹, Rohan Wakade¹, Daniele Stalder¹, Norma Solis², Scott Filler², Robert Arkowitz¹, *Martine Bassilana*¹ 1) University of Côte d'Azur/ CNRS/ INSERM, Institute of Biology Valrose, Nice, France. ; 2) Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.

Virulence of the human fungal pathogen *Candida albicans* depends, in particular, on the switch from budding to filamentous growth, which requires both polarized growth and sustained membrane traffic. Endo- and exocytosis are critical for hyphal growth (1) and the balance between these two processes is likely to be regulated by Rab (Ras-related proteins in brain) (2) and Arf (ADP-ribosylation factor) GTPases, which are key players in membrane traffic. Here, we focused on the role of Arf proteins in *C. albicans*. Previous studies have determined that the putative Arf GAP (GTPase activating protein) Age3 is required for filamentous growth, cell wall integrity and virulence, as well as hypersensitivity to antifungal drugs (3, 4). Our results indicate that of the 3 Arf proteins, Arf1-Arf3, and the 2 Arf-like proteins, Arl1 and Arl3, Arf2 is required for viability and hypersensitivity to antifungal drugs. We also show that Arf2 and Arl1 are required for invasive hyphal growth, with an *arf2* repressible mutant unable to form hyphae and the hyphae of an *arl1* deletion mutant 2-fold shorter than that of the wild-type. Both mutants are drastically reduced in virulence, with *arl1* particularly defective in oropharyngeal candidiasis. Further characterization indicated that only the *arf2* mutant is altered in hyphal-specific gene induction and Golgi integrity. In contrast, the *arl1* mutant is defective in restricting growth to a single site and exhibits increased secretion. Together, these results indicate that Arf2 and Arl1 function in different processes to regulate filamentous growth. To investigate the function of Arl1, we examined the impact on filamentous growth of a number of potential Arl1 interactors, such as that of the sole Golgin protein Imh1, the lipid flippase Drs2 and the Rab GTPase Ypt6, and are currently using non-biased approaches to probe genetic/biochemical interactions.

Schultzhau & Shaw, 2015, Fungal Biology Reviews, **29**, 43-53.

Johansen *et al.*, 2016, PLoS Biology, **14**(8): e1002534.

Epp *et al.*, 2010, PLoS Pathogens, **6**(2):e1000753.

Lettner *et al.*, 2010, PLoS One, **5**(8):e11993.
Friday, March 17 3:00 PM – 6:00 PM

Fred Farr Forum

Epigenetics and Post-Transcriptional Control Mechanisms

**The histone methyltransferases FfSet2 and FfAsh1 deposit H3K36 methylation at specific loci, having a major impact on vegetative growth, sporulation, secondary metabolism and virulence in *Fusarium fujikuroi*.** S. Janevska<fnref n="1" />, L. Baumann<fnref n="1" />, J. Ulrich<fnref n="2" />, J. Kaemper<fnref n="2" />, B. Tudzynski<fnref n="1" /> 1) Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, DE; 2) Institute for Applied Biosciences, Karlsruhe Institute of Technology, Karlsruhe, DE.

*Fusarium fujikuroi* is well-studied due to its ability to produce highly bioactive plant hormones, gibberellic acids, that cause the *bakanae* disease of rice plants. In addition, *F. fujikuroi* possesses a great potential to synthesize a vast range of other secondary metabolites (SMs). The manipulation of chromatin-mediated regulation represents a powerful tool for the activation of "cryptic" SM gene clusters without yet assigned product. In the present work, we focused on the histone 3 lysine 36 methylation (H3K36me) mark which is generally associated with transcriptional activation. In budding yeast, one single methyltransferase ScSet2 confers H3K36me, while there are several enzymes dedicated to this methylation in higher eukaryotes. In filamentous fungi, only the Set2-homolog has been described as H3K36-specific methyltransferase so far.

We show that the *F. fujikuroi* Set2-homolog FfSet2 is responsible for conferring the bulk amount of H3K36me2/me3, however a significant activity remains in Δ*ffset2* mutants. Bioinformatic analysis identified a second homolog of the Set2-family in *F. fujikuroi*, FfAsh1, which is conserved among filamentous fungi. Deletion of *FfASH1* resulted in only a slight decrease of global H3K36me3. But ChIP-Seq analysis revealed that both methyltransferases deposit H3K36me3 at different and very specific loci. Δ*ffset2* and Δ*ffash1* mutants are viable, however strongly attenuated in their vegetative growth and conidiation. Microarray analysis underlined a large impact on the expression of both known as well as unknown SM gene clusters. Production of gibberellic acids is nearly fully abolished *in vitro*, going in line with a decreased pathogenicity of the deletion mutants in rice infection assays.

Furthermore, to shed more light onto the role of H3K36me in the regulatory network of gene expression, we also analyze the H3K36-specific demethylase, which counteracts the activity of FfSet2 and FfAsh1.

**Regulators of histone H3K4 trimethylation influence transcription of secondary metabolite gene clusters in *Aspergillus* and *Fusarium* species.** L. Studt<fnref n="1" />, S. Bachleitner<fnref n="1" />, A. Gacek-Matthews<fnref n="1" />, Z.A. Lewis<fnref n="2" />, J. Strauss<fnref n="1" /> 1) BOKU University of Natural Resources and Life Science Vienna, Tulln, AT; 2) University of Georgia, Athens, GA.

Fungi produce a vast array of secondary metabolites (SMs) that contribute to the fitness of fungi in their natural environment. To prevent an unnecessary use of resources in times their beneficial properties are not required, SM gene expression is adapted to environmental and developmental signals by pathway-specific as well as wide-domain regulators operating also at the level of chromatin. One of the crucial histone posttranslational modifications (PTMs) in active chromatin is trimethylation of lysine 4 in histone H3 (H3K4me3), although this mark was also shown to be necessary for subtelomeric gene silencing. While this histone PTM was shown to be associated with active transcription also in *Aspergillus* and *Fusarium* species, it was surprising to observe that in both fungi SM gene clusters are largely devoid of this histone mark even under activated conditions<fnref n="1" />⁻<fnref n="3" />. Intriguingly, lack of CclA, a regulatory component of COMPASS (complex associated with Set1) and required for genome-wide H3K4me3, resulted in induction of subtelomeric SM gene clusters in *A. nidulans*<fnref n="4" />. We observed a similar phenotype also for the two plant-pathogenic fungi *Fusarium fujikuroi* and *Fusarium graminearum*. At the opposite side of the pathway, enzymes of the KDM5-family of histone demethylases remove one or two methyl groups from trimethylated H3K4 thereby functioning mainly as repressors. We studied the homologous protein designated KdmB in *A. nidulans* and showed that it indeed negatively regulates hundreds of genes but appears to be a specific activator of many SM gene clusters<fnref n="1" />. We started to dissect the function of KdmB domains and inactivated the highly conserved catalytic JmjC demethylase domain by point mutations. Subsequent functional characterization suggests that KdmB-mediated repression is independent of the catalytic function at selected SM gene clusters, whereas the activating role requires the integrity of the JmjC domain. Whether the KdmB-homolog Kdm5 has a similar role in SM gene regulation in *F. graminearum* is currently under investigation. Taken together, this comparative view indicates that proteins regulating the H3K4me2/me3 status play roles in the regulation of SM gene expression despite the fact that the clusters themselves are largely devoid of this histone mark.

**Influence of histone modifications on effector gene expression in the plant pathogenic fungus *Leptosphaeria maculans*.** Jessica L. Soyer<fnref n="1" />, Colin Clairet<fnref n="1" />, Julie Gervais<fnref n="1" />, Marie-Hélène Balesdent<fnref n="1" />, Lanelle R. Connolly<fnref n="2" />, Michael Freitag<fnref n="2" />, Thierry Rouxel<fnref n="1" />, Isabelle Fudal<fnref n="1" /> 1) INRA / AgroParisTech, UMR 1290 BIOGER, Thiverval-Grignon, France; 2) Department of Biochemistry and Biophysics, Oregon State University, Corvallis, USA.

*Leptosphaeria maculans*, the fungus responsible for stem canker of oilseed rape (*Brassica napus*), presents a particular genome structure divided into gene-rich and transposable element (TE)-rich regions. The latter are proposed to be heterochromatic domains, as they are gene-poor, TE-rich and present lower recombination rates than gene-rich regions. While overall gene-poor, TE-rich regions are enriched for putative effector genes specifically expressed during the early stages of plant infection ('early' effector genes). In contrast, effector genes expressed during the late stages of infection (endophytic growth and stem necrosis, 'late' effector genes) are located in gene-rich regions. We investigated the concerted expression of 'early' effector genes by assessing whether TE-rich regions could be targets of reversible chromatin modifications that affect the regulation of effector gene expression. We first examined the involvement of one histone modification, histone H3 lysine 9 tri-methylation (H3K9me3) in chromatin-based regulation of effector gene expression. For this purpose, we silenced expression of two key players in heterochromatin assembly and maintenance, *HP1* and *KMT1*, by RNAi. Silencing of *HP1* and

<footnote n="1">¹Gacek-Matthews et al. 2016, PLoS Genet. 12:e1006222</footnote>

<footnote n="2">²Connolly et al., 2013, PLoS Genet. 9:e1003916</footnote>

<footnote n="3">³Wiemann et al., 2013 PLoS Pathog. 9:e1003475</footnote>

<footnote n="4">⁴Bok et al. 2009, Nat Chem Biol. 5:462-464</footnote>
KMT1 led to an over-expression of 'early' effector genes in TE-rich regions during *in vitro* growth (but no induction of 'late' effector gene expression). The increase of expression was associated with a reduction of H3K9 tri-methylation at two effector-encoding gene loci. Our data strongly suggest that a chromatin-based control, mediated by HP1 and KMT1, represses the expression of at least part of the 'early' effector genes located in TE-rich regions during growth in axenic culture. We are currently investigating the role of another key player in heterochromatin assembly, KMT6, involved in the heterochromatic-associated histone modification H3K27me3, on the control of effector gene expression.

**Mapping of defective in silencing (dis) mutants in *Fusarium graminearum* by bulk segregant analysis and high-throughput sequencing.** Kristina Smith, Lanelle Connolly, Xiao Lan Chang, Brett Pierce, Corinne Fargo, Brian Josephson, Zackary Bango, Madison Esposito, Michael Freitag
Dept. of Biochem & Biophysics, Oregon State University, Corvallis, OR.

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

**Rad51 plays a role in copy number-dependent de novo DNA methylation of a retrotransposon.** B. Vu, Q. Nguyen, Y. Kondo, A. Iritani, T. Murata, H. Nakayashiki
Faculty Agriculture, Kobe Univ, Kobe, JP.

Repetitive sequences are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here we show that Rad51, which is a key enzyme in search for homologous sequences during DNA repair, is involved in copy number-dependent *de novo* DNA methylation of the retrotransposon MAGGY in the fungus *Magnaporthe oryzae*. Double knock-out (KO) of the *M. oryzae* Rad51 (Rhm51) and Ddnm1 (Deficient in De Novo Methylation 1) genes, resulted in a drastic reduction in the level of copy-number dependent-*de novo*, but not maintenance, DNA methylation of MAGGY. A single rhm51 or ddnm1 KO mutant had a defect in DNA repair but showed only a slight reduction in the level of MAGGY DNA methylation. Co-immunoprecipitation and yeast-two-hybrid assays indicated that Ddnm1 and Rhm51 physically interacted with each other. The decreased level of MAGGY DNA methylation was associated with a reduction in epigenetic marks for heterochromatin such as tri-methylation of histone H3 on lysine 9. Overall our results present a possible link between repeat-sensing machineries for DNA repair and heterochromatin formation at repetitive sequences.

**Regulation of fungal effector gene expression through chromatin de-condensation.** M. Kramer¹, D.E. Cook¹,², M.F. Seidl¹, B.P.H.J. Thomma¹
1) Laboratory of Phytopathology, Wageningen University & Research, Wageningen, NL; 2) Department of Plant Pathology, Kansas State University, Manhattan, KS.

Fungal plant pathogens require tight control over the expression of their effector genes which encode secreted proteins that facilitate host invasion. Failure to express such genes at the appropriate time or location during host invasion may lead to interception by the plant host, and thus failure of the infection. In many fungal plant pathogens, effector genes are not randomly distributed over the genome, but localized in distinct genomic regions that are enriched in transposable elements (TEs). TEs can transpose over the genome and thereby affect gene expression or functionality, which may negatively affect the organism. To control TE activity, TE-containing genomic regions are usually structured as heterochromatin, a highly condensed genomic structure that is not accessible to the transcription machinery. Consequently, TEs are generally silenced. Due to the close proximity of many effectors to TEs, they may be held in a co-silenced state. Consequently, upon encountering a host-plant, pathogens will require de-condensation of heterochromatin to appropriately express effector genes. To investigate whether the genomic regions containing effector genes are actively de-condensed during host colonization, we are using chromatin immunoprecipitation (ChIP) to selectively isolate heterochromatic DNA of the broad host-range fungus *Verticillium dahliae* grown *in vitro* and *in planta*. Additionally, we map the genome-wide positioning of nucleosomes to investigate differences between effector genes in TE-rich regions and genes that reside within core regions of the genome. This research will lead to a better understanding of the regulation of effector genes and reveal the importance of chromatin dynamics in this process.

**The constitutive heterochromatin-defined genome organization in *Neurospora crassa* depends on subtelomeric facultative heterochromatin.** A. D. Klocko¹, J. M. Galazka², T. Ormsby¹, S. Honda³, M. Freitag², E. U. Selker¹
1) Institute of Molecular Biology, University of Oregon, Eugene, OR; 2) Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR; 3) Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

Eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that likely result from interactions between the chromatin constituents -proteins, DNA, and RNA- within the physical constraints of the nucleus. We used chromosome conformation capture (3C) followed by high-throughput sequencing (Hi-C) with wild-type and mutant strains of *Neurospora crassa* to understand the role of heterochromatin in genome organization and function. The average genome configuration of wild type revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, yet elimination of H3K9me3 or its binding partner HP1 – both prominent features of constitutive heterochromatin – minimally alter the Hi-C pattern. It remained possible that the facultative heterochromatin mark H3K27me2/3, which is redistributed to constitutive heterochromatin upon H3K9me3 or HP1 loss, is necessary for genome organization. We found that H3K27me2/3, catalyzed by the PRC2 member SET-7, is
important for the Hi-C pattern of wild type but its redistribution upon H3K9me3 loss does not maintain genome architecture. The Hi-C pattern of a mutant defective in the PRC2 member Neurospora p55 (NPF), which is required for subtelomeric H3K27me2/3, was equivalent to that of the Δset-7 strain, suggesting that subtelomeric facultative heterochromatin is paramount for normal chromosome conformation. Cytological observations suggested loss of H3K27me2/3 partially displaces telomere clusters from the nuclear periphery. Thus, associations between heterochromatic regions are a major component of the Neurospora chromosome conformation but while two constitutive heterochromatin proteins are not necessary, H3K27me2/3 is required for the normal 3D genome organization in Neurospora. Also, the overall architecture of the genome remains largely intact even when key features of both constitutive and facultative heterochromatin are eliminated. Additional studies are needed to explore the functional significance of eukaryotic genome organization.

**DNA sequence homology triggers cytosine-to-thymine mutation by a heterochromatin-related pathway.** Eugene Gladyshev,
Nancy Kleckner Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA.

In many eukaryotes, chromosomal regions with highly repetitive DNA are normally associated with cytosine methylation (5mC) and histone H3 lysine-9 di-/trimethylation (H3K9me2/3) in the context of heterochromatin. In the fungus Neurospora crassa, where the pathway of heterochromatin assembly has been characterized in great detail, H3K9me3 and 5mC are catalyzed, respectively, by a conserved SUV39 lysine methylase DIM-5 and a DNMT1-like cytosine methylase DIM-2. Here we show that DIM-2 can mediate cytosine-to-thymine (C-to-T) mutation of repetitive DNA during a process known as "RIP" (Repeat-Induced Point mutation). We further show that DIM-2-mediated mutation requires DIM-5 as well as a number of additional heterochromatin factors including HP1. The pathway can act effectively on tandem repeat arrays as well as on individual pairs of closely positioned or widely separated sequence copies. These results, when combined with our model for repeat recognition during RIP, define a new process by which heterochromatin can be nucleated specifically on repetitive DNA. Existing molecular mechanisms rely on cis-acting proteins or non-coding RNAs. We propose, instead, that formation of constitutive heterochromatin can be initiated by direct interactions between homologous double-stranded DNA segments. Given that the number of potential homologous interactions will scale with the number of repeat copies, this process could be particularly effective and important in heterochromatic silencing of large repetitive regions such as those associated with (peri)centromeric and (sub)telomeric regions. Also, irrespective of the repeat-recognition mechanism, our results implicate the DIM-5/DIM-2 pathway in the accelerated evolution of pathogenic genes that are typically found near repetitive elements in the genomes of many filamentous fungi.
Friday, March 17 3:00 PM – 6:00 PM

Kiln

Cytoskeletal Dynamics and Septa

**The TOG protein Stu2 interacts with SUMO via multiple mechanisms.** Rita Miller¹, Matt Greenlee¹, Annabel Alonso¹, Maliha Rahman¹, Savannah Morris¹, Nida Meednu² 1) Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK; 2) Department of Biology, University of Rochester, Rochester, New York.

Microtubule-associated proteins (MAPs) regulate a variety of functions for microtubule polymers. Many MAPs are regulated by phosphorylation, but little is known about how MAPs are regulated by SUMO, the Small Ubiquitin-like Modifier. SUMO can interact with proteins two ways, covalently or non-covalently. Covalent conjugation of SUMO occurs on the target protein through an isopeptide bond formed between the carboxyl group of the terminal glycine of SUMO and the epsilon amino group of the conjugated lysine residue. A non-covalent interaction occurs thorough the hydrophobic and ionic interactions of a "SUMO-Interacting Motif" within the interacting protein. The Miller lab has identified five new classes of MAPs that interact with SUMO. Here, we show that the TOG protein Stu2p interacts with SUMO by multiple mechanisms. Stu2 is the yeast homologue of the CKAP5/XMAP215 class of proteins that promotes microtubule dynamics by facilitating the loading of tubulin dimers onto microtubule ends. Although Stu2p plays a vital role in microtubule function, little is known about how it is regulated. Using two-hybrid analysis, we show that Stu2 interacts with Smt3/SUMO and several key members of the sumoylation system. Domain mapping of amino- and carboxy- terminal truncations show that the dimerization region of Stu2 is important for its interaction with SUMO. Using an *in vitro* sumoylation assay, four shifted bands of Stu2 can be observed. Higher molecular weight forms of Stu2 are seen in vivo. Pull-down assays in denaturing buffer containing urea demonstrate that an anti-SUMO reactive band co-migrates with Stu2. Inactivation of the Ulp1 SUMO protease with a temperature sensitive mutant reduces the levels of the higher molecular weight form of Stu2. Additional copies of SUMO further reduce the higher molecular weight form. Using mass spectrometry, we analyzed Stu2 for the expected signature "stub" of the wild-type SUMO motif. Stu2 also interacts with a SUMO-Targeted Ubiquitin Ligase that adds ubiquitin to SUMOylated proteins. Together, these results indicate that Stu2 is conjugated by SUMO. We hypothesize that SUMO conjugation destabilizes Stu2. To test whether Stu2 could interact non-covalently with SUMO, we analyzed Stu2 interaction with a non-conjugatable form of SUMO. Stu2 bound to the non-conjugatable SUMO but not to GST alone. Together our work suggests that Stu2 can interact with SUMO by multiple mechanisms, covalent and non-covalent.

**Understanding the role of septin AspD in the early vegetative growth of Aspergillus nidulans.** I. Dorter, M. Momany Plant Biology, UGA, Athens, GA.

Basic research on septin dynamics in the model organism *Aspergillus nidulans* helps us better understand the cellular and molecular mechanisms of these cytoskeletal elements and can help develop new therapeutic approaches against diseases. Under the right conditions *Aspergillus nidulans* spores emerge a germ tube to explore new growth medium. Thereby fungal hyphal tips grow into the food substrate forming a hyphal network with evenly spaced lateral branches. This basic process requires a fine and precise coordination of biochemical and cellular processes. We performed single deletions of the genes encoding for the core septins in *A. nidulans* (*AspA-D*). In the absence of *AspA*, *AspB* and *AspC* we could observe an abnormal increase of lateral branches, which is also known as hyperbranching. The knock-out of the fourth core septin gene *AspD* did not result in obvious phenotypic changes whereas fluorescence microscopy revealed abnormal nuclear structures that worsened with each mitotic division. In contrast to WT the average distance between two neighboring nuclei was greatly reduced in the *?aspD* mutant background, whereas the nuclei themselves were slightly elongated. Time-lapse analyses with a strain expressing *AspD*-GFP fusion protein showed septin bars contacting both nucleus and cell cortex. Shortly after nuclear division the septin bar located between the two newly divided nuclei suggesting that the septin AspD plays a role after mitosis. In addition, the disruption of the septins revealed an increased number of cells harboring an odd number of nuclei. This may point to an asynchronous nuclear division cycle of the multinucleated fungus in the absence of core septins. We asked the question whether septins could be part of a checkpoint that regulates nuclear division by monitoring cell cycle progression. We treated WT and septin deletion mutants with benomyl, which depolymerizes microtubules and inhibits spindle formation thereby activating the spindle assembly checkpoint. In contrast to the other cells all *ΔaspD* mutants showed changes in conidiospore density and color, a sectoring phenotype, which is the result of chromosome missegregation. This supports the idea that the SAC checkpoint did not function properly in the absence of AspD. We were able to construct a strain, which expresses both *AspB*-CFP and *AspD*-GFP and could confirm that at least two distinct septin heteropolymer populations co-exist. Future time-lapse analyses will show if these two populations play different roles during nuclear division in *A. nidulans*.

**Function of the dynactin complex in dynein-mediated intracellular transport.** Jun Zhang¹, Rongde Qiu¹, Xuanli Yao¹, Herb Arst², Miguel Penalva³, Xin Xiang¹ 1) 1Department of Biochemistry and Molecular Biology, The Uniformed Services University of the Health Sciences- F. Edward Hébert School of Medicine, Bethesda, Maryland 20814, USA; 2) Microbiology Section, Department of Medicine, Imperial College London, London, SW7 2AZ, UK; 3) 3Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain.

Filamentous fungi are excellent model organisms for studying the mechanism of microtubule-based distribution of organelles/vesicles. In recent years, we used *Aspergillus nidulans* as a genetic system to dissect how the minus-end-directed motor dynein physically interacts with early endosomes. Our studies revealed that the dynein-binding complex, dynactin, is critical for the interaction between dynein and early endosomes. Within the vertebrate dynactin complex, multiple Arp1 (actin-related protein 1) subunits form an Arp1 mini-filament whose barbed end is occupied by capping protein and whose pointed end occupied by a pointed-end complex containing Arp11, p62, p25 and p27. In contrast to budding yeast dynactin that does not contain capping protein, p62, p25 or p27, our recent proteomic analysis combined with previous studies indicate that *A. nidulans* dynactin complex contains all these components. Our genetic screen identified the Hook protein complex that links early endosome to dynein-dynactin, and the Hook-dynein-dynactin interaction requires both dynein and the p25 subunit of dynactin. p25 is critical for dynein-mediated transport of early endosomes, and our recent data suggest that p25 is not only involved in HookA binding but also in regulating dynactin-microtubule interaction. In contrast, capping protein and p27 are not important for early endosome transport in *A. nidulans*.
**Live cell imaging of the cytoskeleton in *Phytophthora* pathogens reveals unique actin and microtubule configurations. K. Kots*¹,²,**
T. Ketelaar², D. J. Van den Hoogen¹, H. J. G. Meijer¹, F. Govers¹ 1) Laboratory of Phytopathology, Wageningen University, Wageningen, Gelderland, NL; 2) Laboratory of Cell Biology, Wageningen University, Wageningen, Gelderland, NL.

The cytoskeleton is a dynamic but well organized intracellular network that is essential for proper functioning of eukaryotic cells. We study the cytoskeleton in *Phytophthora* species, oomycete plant pathogens that cause devastating diseases worldwide. We use Lifeact-eGFP expressing *Phytophthora infestans* for live cell imaging of the actin cytoskeleton in various developmental stages. Previously we identified actin plaques as highly immobile, long-lived structures that are unique for oomycetes. Here we present two other unique actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there is a typical local actin accumulation and during plug deposition actin remains associated with the leading edge. In appressoria we observed an aster-like actin configuration that is localized at the contact point with the underlying surface. These actin configurations strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration. For live cell imaging of the microtubule cytoskeleton we have generated a *Phytophthora palmivora* transformant expressing GFP-α-tubulin which allows us to visualize the dynamics of microtubules in oomycetes for the first time. Diseases caused by oomycetes are difficult to control and this emphasizes the need to find novel potential drug targets specific for oomycetes. Although actin and tubulin are conserved proteins, many structural and regulatory proteins interacting with the cytoskeleton are unique for specific phylogenetic groups. The data presented here are our first attempts to better understand the structure and functioning of the *Phytophthora* cytoskeleton. The long term goal is to uncover oomycete or *Phytophthora* specific features that might be instrumental for drug design.

**Cell shape is sensed using the septin cytoskeleton.** Kevin Cannon, *Amy Gladfelter* Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We have found that fungal septins are able to distinguish between different degrees of micron-scale curvature. Additionally, using supported lipid bilayers on beads of different curvature, we can reconstitute and measure the intrinsic septin curvature preference. We have found that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape. Our current focus is on identifying the molecular mechanisms that govern the preference for septins to polymerize on curved surfaces.

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

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

**Analysis of septins during hyphal growth of *Ustilago maydis*. Sabrina Zander¹, Sebastian Baumann², Michael Feldbrügge¹ 1)**
Heinrich Heine University, Düsseldorf, Institute for Microbiology, 40225 Düsseldorf, Germany; 2) CRG – Center for Genomic Regulation, 08003 Barcelona, Spain.

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

The genome of the filamentous fungus *U.maydis* encodes four septin proteins: Cdc3, Cdc10, Cdc11 and Cdc12. In this study the influence of septin deletions were analyzed, during the hyphal state of *U. maydis*. All four septins are needed for efficient unipolar growth indicating a common function for septins during hyphal growth. Furthermore, the subcellular localization was analyzed with Gfp fusion proteins. All septins showed the same localization in cytoplasmic rings, at septa, in filaments and on moving early endosomes. Colocalization studies revealed that all septins localize interdependently in the same structures. Furthermore, FRET analysis showed that Cdc3 and Cdc12 interact directly *in vivo*. Endosomal transport was not only dependent on each individual septin, but also dependent on the RNA-binding protein Rrm4. RNA live imaging of all four mRNAs showed also an Rrm4-dependent transport. FRAP experiments demonstrated that recovery of Cdc3-mCherryN and Cdc12-GfpN occurs simultaneously at distinct subcellular sites and that the rate of recovery was facilitated by the endosomal RNA transport. Based on these results, a model was proposed where local translation promotes the assembly of newly synthesized septins in heteromeric structures on the surface of endosomes. This is important for the long-distance transport of septins and the efficient formation of the septin cytoskeleton.
**Complex nuclear motility in a fungal hypha explained by an elegant mechanism.** R. Gibeaux¹,³, A. Z. Politi¹, P. Philippsen², F. Nedelec¹
1) Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany; 2) Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; 3) Department of Molecular and Cell Biology, University of California, CA-94720, Berkeley.

Today, biologists want to understand the mechanisms of subsystems in cells. Biochemistry, for a long time extremely successful in developing amazing in vitro systems, can no longer handle mechanistic question above a certain complexity. The only way to move forward is to build computer models to systematically assess if our current concepts are compatible – or not – with the experimental data, and to reconstruct the system in a mathematical model. This is essential to curate and assemble the information that has been gathered in the past.

Our work demonstrates this approach by studying the coordination of movements of multiple nuclei in a common cytoplasm which up to now has not been solved for any multinucleated cell. Our aim was to understand the underlying mechanism(s) of the bi-directional short- and long-range movements of nuclei including nuclear by-passing in multinucleated hyphae of *Ashbya gossypii*. Experimental data, accumulated over the past years, had pinpointed one motor (dynein), an evolved microtubule dynamic instability, yeast-like spindle pole bodies as sole microtubule organization centers, and a potential cortical dynein anchor as key elements for the complex nuclear movements. This knowledge was implemented to run for the first time realistic simulations of movements of multiple nuclei in a common cytoplasm which generated in vivo-like nuclear migration patterns. The simulations could also verify mutant phenotypes. We then exploited the simulation set up to study the influence of the number of microtubules per nucleus, an increase in cytoplasmic flow, and changes in organelle concentration on nuclear motility.
Friday, March 17 3:00 PM – 6:00 PM

Heather

## Symbiosis and Commensalism

**Symbiotic options for the conquest of land.** *Katie Field*¹, *Martin Bidartondo*²,³, *Jeff Duckett*⁴, *William Rimington*²,³, *Silvia Pressel*⁴ 1) School of Biology, University of Leeds, Leeds, UK; 2) Department of Life Sciences, Imperial College London, London, UK; 3) Jodrell Laboratory, Royal Botanic Gardens, Kew, UK; 4) Department of Life Sciences, Natural History Museum, London, UK.

The colonisation of Earth's continental land masses by plants >475 Ma marked a turning point in the development of the terrestrial biosphere, with widespread consequences for the future of terrestrial life. It has long been hypothesised that the terrestrialisation of plants was facilitated by nutritional symbioses with Glomeromycota fungi to form arbuscular mycorrhiza-like associations. The establishment of this symbiosis would likely have enhanced access to mineral nutrients for early, rootless plants in exchange for carbon fixed through photosynthesis. Our recent discoveries that the earliest diverging clade of land plants (Haplomitriopsida liverworts) forms mutualistic symbioses with partially saprotrophic Mucoromycotina fungi challenge this long-standing paradigm. Using a combination of microscopic, molecular and isotope tracer techniques, we have shown that other lineages of early-diverging liverworts form simultaneous mutualistic associations with both Glomeromycota and Mucoromycotina fungi. Together with recent fossil evidence, these findings bring into question the identity, biology and functioning of the fungal symbionts of the earliest land plants. Here, we suggest that there were symbiotic options available to the earliest land plants and that such relationships may have been far more varied and transient than hitherto assumed.

**Extensive genome diversity among isolates of a model arbuscular mycorrhizal fungus.** *E.Chun-Hung. Chen*¹, *S. Ndikumana*¹, *E. Morin*², *F. Martin*², *J. Noel*¹, *D. Beaudet*¹, *J. Giorgi*¹, *N. Corradi*¹ 1) Biology, University of Ottawa, Ottawa, Ontario, CA; 2) UMR 1136, INRA-Nancy Université, Interactions Arbres/Microorganismes, Champenoux, FR.

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

**Functional characterization of CRN-like effector proteins in the arbuscular mycorrhiza symbiosis.** *S. Walter*¹, *R. Betz*¹, *N. Corradi*², *N. Requena*¹ 1) Karlsruhe Institute of Technology, Karlsruhe, DE; 2) University of Ottawa, Canada.

The arbuscular mycorrhiza symbiosis (AMS) between fungi of the phylum Glomeromycota and more than 80% of all living land plants is characterized by the formation of tree-like fungal structures, arbuscules, within cortical root cells, that function as the interface for the bidirectional nutrient exchange. Prior to this, a highly complex communication is necessary in order to establish the interaction. In plant-pathosystems, effector proteins are secreted during colonization that lead to modifications in the plant cell program, essentially to facilitate the infection process. Evidence is now accumulating that mutualistic fungi also use effector proteins when colonizing their host plants. Recent success in sequencing the genome of *Rhizophagus irregularis* revealed a new effector protein family, which resembles the modular structure of a protein family called Crinkler (CRN). CRNs are present in all pathogenic oomycetes and in some chytridiomycetes. Many contain a signal peptide for secretion and share a highly conserved N-terminal region, with a characteristic LFLAK domain for translocation, and a divergent C-terminal region to execute effector activity. Expression analyses of *R. irregularis* CRN-like genes showed two of them being highly expressed in mycorrhizal roots, suggesting their involvement in the symbiosis. To functionally characterize them, expression of *RiCRN1* and *RiCRN2* in *planta* was carried out. Unlike many oomycete CRNs neither *RiCRN1* nor *RiCRN2* induced cell death in *Nicotiana benthamiana* but both led to a strong phenotype in transgenic *Arabidopsis thaliana* that were smaller and displayed anthocyanin accumulation. Fusions with GFP and transient expression in *N. benthamiana* leaves showed nucleo-cytoplasmic localization for *RiCRN1* and *RiCRN2*. Most interestingly preliminary experiments to downregulate these CRN-like proteins during symbiosis in *M. truncatula* using host induced gene silencing showed a severe impairment of the symbiosis. We therefore postulate that these CRN-like proteins work to promote symbiosis. Experiments are in progress to identify the *in planta* targets responsible for such a positive role.

**Host specificity and effector warfare: a dissection of the signaling events during the mutualistic interaction between the ectomycorrhizal genus *Pisolithus* and their hosts.** *J.M. Plett*¹, *M. Freitas Pereira*²,³, *K.L. Plett*¹, *E. Morin*², *M Dutra Costa*³, *I. Grigoriev*⁴, *A. Kohler*², *F. Martin*², *I.C. Anderson*¹, *Pisolithus Pan-genome Consortium* 1) Hawkesbury Institute for the Environment, Western Sydney University, Richmond, AU; 2) INRA, Nancy, FR; 3) Universidade Federal de Viçosa, Viçosa, BR; 4) Joint Genomes Institute, Walnut Creek, CA.

In forest ecosystems the roots of most trees and shrubs are colonized by mutualistic ectomycorrhizal (ECM) fungi. Little is understood concerning the host or fungal cellular reprogramming that favors the formation of ECM colonized root tips in compatible plant:fungal interactions and what prevents the formation of ECM root tips in incompatible interactions. Using the newly available genomic and transcriptomic resources for two species within the *Pisolithus* genus, we have begun dissecting the symbiotic toolbox used by this globally dominant fungus. We have been able to define the core transcriptional responses of the plant and fungus during symbiosis and to characterize some of the proteomic modification necessary for mycorrhizal formation. As in other ECM systems, we have identified a number of effector-like small secreted proteins that are induced during symbiosis and have begun to characterize their roles in negotiating (or forcing?) symbiosis with a host tree. I will discuss some of our most recent advances in understanding ECM host specificity against the backdrop of previous findings with other mycorrhizal fungi.

**Purification and characterization of a symbiosis-induced endocellulase from the ectomycorrhizal symbiont *Laccaria bicolor*.** *F. Zhang*¹, *C Champion*², *M Haon*², *G Anasontzis*², *M Kemppainen*³, *A Pardo*³, *Y Daguerre*¹, *A Deveau*¹, *C Veneault-Fourrey*¹, *A Kohler*¹, *MN*
Rosso², B Henrissat⁴, JG Berrin², F Martin¹ 1) UMR 1136 INRA-Université de Lorraine 'Interactions Arbres/Microorganismes', Laboratoire d'Excellence ARBRE, Centre INRA-Lorraine, 54280, Champenoux, France; 2) UMR 1163 INRA-Biodiversité et Biotechnologie Fongiques, Polytech Marseille, Faculté des Sciences de Luminy, , Marseille, France; 3) Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Provincia de Buenos Aires, Argentina; 4) CNRS, UMR 7257 & Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Marseille, France.

In forest soils, ectomycorrhizal fungi establish a mutualistic symbiosis with tree roots. The mutualistic fungi trade host photoassimilates against soil nitrogen and phosphorus. Differentiation of symbiotic roots induces extensive cell wall architectural modifications in the host apoplastic space. The origin of enzymes involved in these cell wall modifications has been the subject of debate for several decades. The ectomycorrhizal basidiomycete *Laccaria bicolor* has a restricted set of carbohydrate-active enzymes (CAZymes) degrading plant cell wall polysaccharides. However, several of those genes are upregulated upon symbiosis. We speculate that several of the symbiosis-induced CAZymes are involved in the remodeling of the host apoplastic space. Here, we characterize the sole GH5 endoglucanase with a cellulose-binding motif (CBM1) domain (LbGH5) identified in the genome of *L. bicolor*. We showed that the *LbGH5* gene is induced five-fold in ectomycorrhizal roots using qPCR and RNA-Seq. RNAi mutants with a decreased expression of *LbGH5* have a lower ability to form ectomycorrhizal roots. Yeast secretion trap (YST) functional screen confirmed that LbGH5 is a secreted protein. We then produced and purified the recombinant protein LbGH5 with and without its CBM1 domain in *Pichia pastoris*. The recombinant LbGH5 displayed highest activities towards carboxymethyl cellulose (CMC) and cellulose extracted from aspen roots. In contrast, LbGH5 displayed no activities toward *L.bicolor* cell walls or aspen hemicellulose. *In situ* localization of LbGH5 in ectomycorrhizal roots by indirect immunofluorescence confocal microscopy demonstrated that the enzyme accumulates in hyphal cell walls forming the mantle and Hartig net. These data suggest that cell wall modifications within ectomycorrhizal roots arise from cell wall-modifying enzymes of fungal origin.

*Acknowledgements.* This project is funded by the French Agence Nationale de la Recherche and the Laboratory of Excellence ARBRE.

**The fungal specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants.** S. Wawra¹, P. Fesel¹, H. Widmer¹, M. Timm², G. Langen¹, A. Zuccaro¹,³ 1) Univ of Cologne, Botanical Institute, Cluster of Excellence on Plant Sciences (CEPLAS), Cologne, Germany; 2) Univ of Würzburg, Inst of Organic Chemistry, Würzburg, Germany; 3) Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany.

β-glucans are well-known modulators of the immune system in mammals, but little is known about β-glucan triggered immunity *in planta*. We report that the secreted plant-induced protein FGB1 from the root endophyte *Serendipita indica* is a novel type of conserved β-glucan binding lectin in fungi that increases resistance to cell wall stress and is able to efficiently suppress β-glucan triggered immunity in different plant hosts. Our results highlight the importance of β-glucan as a fungal MAMP and the necessity of mutualistic and pathogenic fungi to protect β-glucan polymers from recognition during colonization of different plants. Here we will present our latest data.

**Genetics-based interactions structure foliar fungal communities of Populus trichocarpa.** P.E. Busby¹, Shawn Brown¹, Naupaka Zimmerman², Gregory Crutsinger³, Matthew Barbour³, Kabir Peay⁴, George Newcombe⁵ 1) Botany & Plant Pathology, Oregon State University, Corvallis, OR; 2) Department of Biology, University of San Francisco; 3) Department of Zoology, University of British Columbia; 4) Department of Biology, Stanford University; 5) College of Natural Resources, University of Idaho.

Fungal endophytes form diverse communities within plants, with individual endophytes playing functionally important roles in plant growth, development, and resistance to abiotic and biotic stressors. Despite the ecological importance of endophytes, little is known about the mechanisms that control endophyte community composition. In a manipulative common garden field experiment, we evaluated the impact of host genotype, endophyte inoculation (Cladosporium), and presence of herbivory on foliar fungal community composition. The twelve genotypes included in the experiment varied in tannin levels, phenological shifts in timing of bud flush, and rust disease severity. We found that community structure was most strongly influenced by host-genotype, tannin levels, and phenology (perMANOVA). The endophyte inoculation and herbivory treatments did not produce a detectable shift in endophyte community structure in our system, but these interactions were occurring naturally across all plants and could occlude observable treatment effects. Host-genotype was by far the most influential factor in structuring fungal endophytes (explaining 21.81% of community variation) based on our variance partitioning analyses (PVCA). The relatively weak effect of tannins and phenology on endophyte communities was confirmed using mixed model ANOVAs of NMDS axes loading scores. Additionally, using LEfSe analyses, we identified OTUs that are strongly associated with particular genotypes, providing targets for mapping genetic controls of community members. Taken together, our results indicate that unmeasured host genetic effects are far more important in determining endophyte communities than the traits measured in this study. To further investigate and disentangle host genetics/gene expression from environmental constraints on endophytic communities, we are using both a Genome Wide Association Study (GWAS) and RNA-Seq analysis of endophyte-inoculated leaves.

**Comparative genomics of lichen mycobionts.** A. Kuo¹, D. Culley², O. Mueller³, P. Dyer⁴, J. Magnuson², F. Lutzoni³, I. Grigoriev¹ 1) DOE JGI, Walnut Creek, CA; 2) Pacific Northwest National Lab, Richland, WA; 3) Duke Univ, Durham, NC; 4) Univ of Nottingham, Nottingham, UK.

Lichens are mutualistic symbioses usually between an alga (the photobiont) and a fungus (the mycobiont). The photobiont contributes fixed carbon to the partnership, while the mycobiont provides a physical structure that encloses the photobiont, roots the partners to a substrate, and acquires nutrients often of air-borne origin. Lichens are an ancient (possibly 600 Mya) and highly successful adaptation, covering 8% of terrestrial ecosystems, and comprising as much as 20% of fungal species diversity. The lichen lifestyle has evolved multiple times and occurs in at least 5 fungal classes. As part of an ongoing effort to represent the full phylogenetic and ecological diversity of fungi in the Joint Genome Institute MycoCosm and the 1000 Fungal Genomes Project, we have partnered with members of the lichen research community to sequence, assemble, and annotate several lichen mycobionts and photobionts. Initial comparison of 5 publicly available mycobiont genomes from 3 distinct lichen-forming clades with their nearest non-lichenizing relatives has revealed varied combinations of secondary metabolite synthase, transporter, and protease gains and losses. The diversity of gains and losses suggest that the independent evolutionary transitions to the lichen lifestyle may have involved different gene-level adaptations. We expect to expand on these findings by examining additional functional characters, and by sequencing more genomes, as well as importing more genomes from our partners and from the public domain.
Friday, March 17 3:00 PM – 6:00 PM

Nautilus

## Effectors and Small Secreted Cysteine-Rich Proteins

**SnTox1, a cysteine rich small secreted protein provides in planta protection from wheat chitinases.** Timothy L. Friesen¹,²,
Yuanyuan Gao², Zhaohui Liu², Jonathan Richards², Justin D. Faris¹ 1) USDA-ARS, Northern Crop Science Lab, Cereal Crops Research Unit, Fargo, ND, 58102, U.S.A; 2) Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108, U.S.A.

*Parastagonospora nodorum* is a necrotrophic fungal pathogen and the causal agent of Septoria nodorum blotch (SNB) on wheat. *P. nodorum* produces necrotrophic effectors (NEs) that are critical to the virulence of the pathogen. The NEs produced by *P. nodorum* are recognized directly or indirectly by dominant sensitivity genes contributing to susceptibility in wheat. Nine NE-host dominant sensitivity gene interactions have been identified, and three NE genes have been cloned, including *SnToxA*, *SnTox1* and *SnTox3*. SnTox1 is a cysteine rich small secreted protein that interacts directly with the wheat protein Snn1. In the presence of Snn1, SnTox1 triggers programmed cell death (PCD) involving hallmarks of the defense response including an oxidative burst and up regulation of wheat pathogenesis related (PR) proteins including chitinases. The triggering of PCD involves membrane disruption and electrolyte leakage, and unlike a biotrophic interaction, provides a nutrient source for the pathogen. However, a pathogen with this lifestyle must also be able to protect itself from the negative effects of this defense response including the accumulation of chitinases in its growth environment. In addition to triggering PCD, we have shown that SnTox1 binds chitin, providing protection to the growing fungus in the apoplast during infection. Several additional genes encoding small, secreted, cysteine-rich proteins are present in the *P. nodorum* genome and are predicted to be localized to the apoplast during infection. Some of these proteins could play a role in protecting this pathogen from the host defense response.

**Ustilago maydis effectors targeting the auxin signaling pathway.** A. Djamei, F. Navarrete, M. Gallei, J. Bindics, S. Uhse Gregor Mendel Institute of Molecular Plant Biology, Vienna, AT.

Biotrophic plant pathogenic fungi employ a battery of small secreted molecules, so called effectors, to suppress host defense responses and to redirect the host metabolism in favor of the invader. Although effector proteins are shaping the interaction between the pathogen and the host, it is challenging to elucidate their function as they largely show no sequence homology to proteins with known functional domains. The maize infecting tumor inducing fungus *Ustilago maydis* became in the past decades a model to study biotrophic interactions. Hormone signaling pathways in plants are prime targets for biotrophic pathogens in order to suppress defense responses, reshape host metabolism and host development systemically. In a systematic approach, we study the involvement of *U. maydis* effectors in hormone signaling pathways. Here we report about the identification of several auxin signaling inducing effectors of *U. maydis* and their host sided plant interaction partner. The plant target identified is highly conserved across plant species which opens new applications as a tool for plant biologists.

## Towards understanding the mechanism of cytoplasmic effector translocation during biotrophic development of *Magnaporthe oryzae*.

Rice blast caused by *Magnaporthe oryzae* (synonym of *Pyricularia oryzae*), a hemibiotroph and facultative pathogen, is the most destructive disease of rice worldwide. During the infection process, *M. oryzae* secretes various effectors, which are hypothesized to be involved in effective host infection. Effectors are classified by their destinations in the interaction court, with apoplastic effectors residing in the extracellular plant compartment and cytoplasmic effectors translocating into the cytoplasm of living plant cells. Notably, cytoplasmic effectors of *M. oryzae* are associated with a specialized interfacial structure, the biotrophic interfacial complex (BIC). To date, little is known about the mechanisms of effector uptake into plant cells during fungal infection. Here we show evidence for translocation of the cytoplasmic effectors Bas1, Pwl1 and Pwl2 in vesicles from BICs to rice cytoplasm during biotrophic development. Using fluorescent protein tagging, we found that cytoplasmic effectors Bas1, Pwl1 and Pwl2 are sorted into different vesicles in BICs formed on primary hyphae (PH), revealing new levels of functional complexity for this biotrophic structure. In contrast, most of the vesicles from BICs on mature bulbous hyphae showed colocalization of the cytoplasmic effectors. Whereas BICs on primary hyphae deliver effectors in micro-vesicles, BICs on mature bulbous hypha deliver effectors in macro-vesicles, at times reaching diameter sizes over 3 µm. Furthermore, we demonstrate that endocytosis inhibitors Cantharidin, Triclosan and Wortmannin induce abnormally-shaped and swollen BICs as well as the accumulation of cytoplasmic effectors in the BICs. Moreover, Cantharidin, Triclosan and Wortmannin treatment induced the accumulation of the cytoplasmic effectors under penetration pores, suggesting that effector uptake begins even before host penetration. Based on these results, it appears that cytoplasmic effector translocation is mediated by vesicle formation and may be characteristic of appressoria as well as biotrophic invasive hyphae. Our results also suggest a potential role of *M. oryzae* effectors for manipulation of the host cell endocytosis process.

**Functional characterization of a protein complex formed by four Ustilago maydis effectors essential for virulence.** N. Ludwig¹, L. Liang¹, K. Schipper¹,², S. Reißmann¹, D. Aßmann¹, T. Glatter¹, J. Altmüller³, R. Kahmann¹ 1) Max Planck Institute for Terrestrial Microbiology, Marburg, Germany; 2) Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 3) Cologne Center for Genomics (CCG), Cologne University, Cologne, Germany.

The fungus *U. maydis* causes smut disease in maize. *U.maydis* is a biotrophic pathogen requiring living plant tissue for colonization. For a successful infection *U.maydis* needs to suppress plant defense responses and manipulate host physiology for its own benefit. To accomplish this, *U.maydis* secretes a cocktail of about 300 effector proteins. The majority of these proteins lack a functional annotation and their function remains to be uncovered. Our current work focuses on effectors expressed early during infection. Systematic deletion of the most highly expressed effector genes in this class resulted in the discovery of three mutants unable to cause disease. Their mutant phenotype resembled previously identified *stp1* (stop after penetration) mutants, and the newly identified genes were designated *stp2*, *stp3* and *stp4*. A similar phenotype was also observed for mutants lacking the essential effector *pep1* (Döhlemann *et al.*,2009). *stp1-4* deletion strains were able to form appressoria that penetrate maize epidermal cells, but their growth was arrested in epidermal tissue. Their growth arrest was accompanied with the elicitation of plant defense responses and plant cell death. Co-IP with individually tagged effectors
followed by mass-spectroscopic analysis revealed that Stp1, Stp3, Stp4 and Pep1 form a complex, while Stp2 is not part of the complex. We will discuss our current efforts to localize the complex and to functionally characterize its components as well as Stp2. Furthermore, we are testing possible theories for the existence of the complex, such as a function in translocation or the shielding of Avr proteins.

**An antimicrobial and phytotoxic ribonuclease secreted by the wheat pathogen Zymoseptoria tritici.** G.J. Kettles, C. Bayon, C.A. Sparks, K. Kanyuka, J.J. Rudd Plant Biology & Crop Science, Rothamsted Research, Harpenden, Hertfordshire, UK.

The dothidiomycete fungus *Zymoseptoria tritici* (*Z. tritici*) is amongst the most prevalent and damaging pathogens of wheat throughout the world. During early (symptomless) colonisation of wheat leaves, there is minimal activation of the host immune system. Later, there is a switch to necrotrophy, accompanied by host defence gene induction and extensive cell death in infected tissues. We recently identified a secreted ribonuclease (Zt6) as a candidate effector protein based on transcriptomic analysis of *Z. tritici* infection of susceptible wheat. Zt6 displays an unusual double-peak expression pattern during wheat infection, with maximal expression both shortly after spore germination on the leaf surface (1 dpi) and after the transition to necrotrophy (14 dpi). Expression at transition (9 dpi) was comparatively low, in contrast to the majority of putative secreted protein effectors identified to-date.

Agrobacterium-mediated transient expression of Zt6 in the non-host model plant *Nicotiana benthamiana* revealed this effector protein to be a highly potent inducer of cell death. Both full-length protein and its mature form (lacking signal peptide) were equally potent in cell death induction. Particle co-bombardment of GFP- and Zt6-expressing constructs into wheat leaves resulted in no GFP expression, indicating Zt6 phytotoxicity against the natural host. Further co-bombardment experiments utilising a 22-amino acid N-terminal loop deletion (?1-22) and catalytic mutation (H52A) revealed both regions of Zt6 to be essential for full functionality and suggested a role for the N-terminal loop in Zt6 translocation into the host cytoplasm. Interestingly, recombinant Zt6 protein showed a ribotoxin-like activity by cleaving native rRNA semi-specifically, with production of distinct cleavage fragments. Finally, microbial toxicity assays revealed Zt6 to be highly toxic to both bacteria and yeast but not to more complex filamentous fungi, including *Z. tritici* itself. Together, our data reveal a new multi-functional cytoplasmic ribotoxin-like effector that is toxic to both host and non-host plants, and also to microbial competitors.

**Learning from nature: establishment of a library of natural and rationally designed hydrophobins from Trichoderma spp. with diverse surface activity.** Gunseli Bayram Akcapinar¹,², Agnieszka Przylucka¹,³, Cai Feng⁴, Thiago Machado Mello De Sousa⁵, Hinrich Grothe⁶, Erik Reimhult⁷, Astrid R. Mach-Aigner⁵, Qirong Shen⁴, Irina S. Druzhinina¹ ¹) Microbiology Group, Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria; ²) Department of Medical Informatics and Biostatistics, Acibadem University, Kayisdagi Cad. No:32, 34752 Istanbul, Turkey; ³) Austrian Centre of Industrial Biotechnology (ACIB) GmbH c/o Institute of Chemical Engineering, University of Technology of Vienna, Vienna, Austria; ⁴) Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing, China; ⁵) Gene Technology Group, Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria; ⁶) Research Division Physical Chemistry, Institute of Materials Chemistry, Vienna University of Technology, 1060 Vienna, Austria; ⁷) Department of Nanobiotechnology, Institute for Biologically Inspired Materials, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria.

Hydrophobins (HFBs) are the small, secreted cysteine rich, amphiphilic proteins which exhibit high surface activities. They are mainly produced by fungi. Their ability to self-assemble at hydrophobic/hydrophilic interfaces makes them ideal candidates for a diversity of industrial applications. In this study, two complementary approaches were used for the construction of a library of HFB4 proteins from a diverse array of *Trichoderma* species. In the first approach, positive evolution, structure guided protein engineering was employed to generate five mutants of HFB4 from *T. virens* (HFB4_vir) (HFB4_vir-V19I, HFB4_vir-V19Q, HFB4_vir-V19R, HFB4_vir-L64Q, HFB4_vir-L64R) using a newly identified repertoire of 160 orthologous genes encoding a novel HFB4 in different species of *Trichoderma*. Molecular Dynamics simulations were performed to decipher the functionally important residues and to study the effects of proposed mutations. In the second approach, eight different natural HFB4 orthologues exhibiting diverse properties were selected from the newly identified repertoire of *Trichoderma* species based on pl and hydrophobicity using bioinformatics methods. Natural and tailor-made HFB4s were heterologously expressed in *Pichia pastoris*. A collection of biochemical and biophysical techniques were employed to verify the expression and surface activity of these proteins and to analyse the differences in the surface activity. Three mutants of HFB4_vir (HFB4_vir-V19I, HFB4_vir-V19R, HFB4_vir-L64R) were found to exhibit improved surface activity on glass. All HFB4 orthologues were shown to increase the water contact angle (WCA) of the glass to different extents. Two HFB4 orthologues, HFB4_kon and HFB4_est, exhibited the highest surface activity on glass by increasing the WCA by almost 70° and 40°, respectively.

**Functionalization of surfaces using hydrophobin-fused laccase from Aspergillus nidulans.** O. Fokina, L. Winandy, R. Fischer Inst. for Appl. Biosciences / Dep. of Microbiology, Karlsruhe Institute of Technology - KIT, Karlsruhe, Baden-Wuerttemberg, DE.

Hydrophobins are small amphiphilic proteins that can self-assemble into monolayers on hydrophilic and hydrophobic surfaces and change their properties. They are secreted by fungi to reduce surface tension at the medium-air interface during hyphal growth and are also responsible for the hydrophobicity of the conidiospore surface. Hydrophobins are divided into two classes, depending on their structural characteristics. Especially class I hydrophobins are interesting for biotechnology, because their layers are stable at high temperatures and can only be removed with strong acids.

Here we developed a system to functionalize surfaces with enzymes using hydrophobin as anchor protein or "glue". To this end *Aspergillus nidulans* laccase C (LccC) was fused to the class I hydrophobins DewA or DewB and used for specific functionalization of hydrophobic/hydrophilic polystyrene and glass surfaces. Fusion proteins were secreted into the culture medium, which was directly used for coatings without additional purification steps. Protein immobilization using hydrophobins provides a stable binding of the fused enzyme to the surface without additional chemical treatments that could potentially be harmful to the enzyme or the surface. Also the spatial orientation of the enzyme is predetermined by the design of the fusion construct. This study presents an easy-to-use alternative to classical enzyme immobilization techniques and can probably be applied not only for laccases, but also for other biotechnologically relevant enzymes.
**Recruitment of esterase by fungal hydrophobins bound on polyesters and subsequent promotion of polyester degradation.** K. Abe<sup>1,2</sup>, T. Tanaka<sup>1</sup>, Y. Terauchi<sup>1</sup>, T. Takahashi<sup>1</sup>, F Hasegawa<sup>2</sup> 1) Dept Microbial Biotechnology, Grad. Sch. Agricult. Sci., Tohoku Univ., Sendai, Miyagi, JP; 2) New Industry Creation Hatchery Center, Tohoku University.

Hydrophobins are amphipathic proteins and are ubiquitous among filamentous fungi [1]. When the industrial fungus *Aspergillus oryzae* is grown in liquid media containing the polyester polybutylene succinate co-adipate (PBSA) as a sole carbon source, *A. oryzae* produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase (polyesterase) [2]. We previously discovered that RolA promotes the CutL1-dependent hydrolysis of PBSA by attaching to the surface of PBSA and interacting with CutL1 to concentrate CutL1 on the PBSA surface [2]. To elucidate the underlying mechanism of RolA-CutL1 interaction, we analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a Quartz Crystal Microbalance (QCM). The QCM analyses revealed that D142, D171 and E31, located on the hydrophilic molecular surface of CutL1, and H32 and K34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions [3]. Phylogenetic and alignment analyses showed that the N-terminal regions of several RolA/RodA orthologs contained positively charged residues and that the corresponding negatively charged residues on the molecular surface of CutL1 that were essential for the RolA-CutL1 interaction were conserved in several CutL1 orthologs [4]. A PBSA degradation assay, a pull-down assay using Teflon particles, and a kinetic analysis using a QCM revealed that recombinant *Aspergillus nidulans* RodA interacted via ionic interactions with two recombinant *A. nidulans* cutinases. Recently, hydrophobin-dependent promotion of polymer degradation by cutinases has been reported in other fungi [5, 6]. Together, these imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

1) Wösten H. A. *Annu Rev Microbiol*. 55: 625 (2001)

2) Takahashi T. et al., *Mol. Microbiol*. 57: 1780 (2005)

3) Takahashi T. et al., *Mol Microbiol*. 96: 14 (2015)

4) Tanaka T. et al., *Appl Microbiol Biotechnol*. In press

5) Espino-Rammer L. et al., *Appl Environ Microbiol*. 79:4230 (2013)

6) Pham C.L. et al., *Sci Rep*. 6:25288 (2016)
Friday, March 17 3:00 PM – 6:00 PM

Scripps

**Fungi in Society**

**Old collection, new discoveries.** *Kyria Boundy-Mills*¹, Luis A. Garay¹, Irnayuli R. Sitepu¹,², Tomas Cajka³, Oliver Fiehn³ 1) Food Science, University of California Davis, One Shields Ave, Davis, CA USA 95616; 2) Department of Biotechnology, Indonesia International Institute for Life Sciences, Jalan Pulo Mas Barat Kav. 88, East Jakarta, DKI 10 Jakarta, Indonesia; 3) UC Davis Genome Center-Metabolomics, University of California, Davis, 451 Health Sciences Drive, Davis, California 95616 USA.

The Phaff Yeast Culture Collection at the University of California Davis is the fourth largest public collection of its kind in the world, containing over 7,500 strains belonging to over 800 different species. The yeasts were gathered, characterized and preserved over the last century by University of California personnel, primarily by Herman Phaff (1913-2001), from environmental sources such as plants, insects, and foods and beverages. The collection is expanding through rescue of over 3,000 yeasts from retiring professors W. T. Starmer (Syracuse University) and P. Ganter (Tennessee State University). Ribosomal sequences (partial 26S and ITS) have been generated for thousands of yeast strains. The yeasts are utilized by academic, government agency and industrial researchers around the world in a wide range of areas such as functional genomics, metabolomics, biogeography, ecology, food fermentations, and metabolomics. The yeast strains and associated data are available to the scientific community through the collection website, http://phaffcollection.ucdavis.edu. The broad diversity enables in-house research including studies of stress tolerance, yeast lipid accumulation, synthesis and secretion of glycolipids, and development of starter cultures for olive fermentations. Several yeast species were identified that are able to synthesize and secrete significant quantities of a class of glycolipids called polyol esters of fatty acids (PEFA), consisting of a 3-hydroxy fatty acid and either mannitol or arabitol, with varying degrees of acetylation. Because the molecule is amphipathic, it has biosurfactant activity. These discoveries are possible thanks to the accumulation, characterization and preservation of yeasts by forward-thinking scientists over many decades.

**Feeding fellow biologists their fungal vegetables.** *C. Adams* Plant and Microbial Biology, UC Berkeley, Oakland, CA.

Among eukaryotes, Kingdom Fungi is extremely diverse in terms of estimated species numbers, as well as ecologies, genetic systems, and more. Yet despite their comparable importance with, for example, animals and plants, the U. S. public knows very little about fungi. Though much remains to be learned about fungi, many aspects are well-understood, such as their importance in nutrient cycling and decomposition, heterothallic mating systems, and production of socially important compounds such as antibiotics. Then why are fungi relatively absent from primary education, and often even college-level biology? This illiteracy may be partially due to the fact biologists have historically understudied fungi, which were long mistaken for "lower plants" of little value or importance. This knowledge gap among biologists themselves hinders not only teaching mycology to the public, but conducting interdisciplinary research. In this talk, I will create a dialog to discuss the aspects of fungal biology that mycologists consider critical for other biologists to know. I will also present strategies that mycologists can use to better communicate with other biologists across a range of social media platforms.

**Diversity of spalting fungi in the Peruvian amazon.** Sarath M. Vega Gutierrez, *Sara C. Robinson* Wood Science & Engineering, Oregon State University, Corvallis, OR.

Most of the research related to fungi of the Peruvian Amazon is focused on edible mushrooms and pathogens. Other important fungi, such as spalting fungi, are not broadly studied because most of them do not produce fruiting bodies, are difficult to locate, and do not present an immediate value to the forager. Finding spalting fungi involves a complete sampling of downed trees and branches in a given area through the opening of the xylem to identify potential pigmenting and zone line producing fungi. This methodoly was employed in the district of Las Piedras, Madre de Dios, Peru. Fungi suspected of causing internal pigment and zone lines were collected, cultured, isolated, and sequenced. The species found belonged to the orders of the Helotiales, Xylariales, Hypocreales, Russulales, Polyporales, Botyosphaeriales and two specimens of the class Leotiomycetes. Most of the fungi produced pigments or zone lines in wild conditions and all of them were part of the wood decomposing fungi. Findings from this research will benefit Amazonian communities as well as Peru as whole, through identification and location of value-added wood products that can widen the commercial wood market currently focused on dark, old growth trees. This also gives more inherent value to the forest, potentially saving the land from being converted to plantation or pasture.

**Improving STEM student retention via early research engagement: a pilot.** *Michael Watters*, Patrice Bouyer Dept Biol, Valparaiso Univ, Valparaiso, IN.

We describe here the results of a pilot study, the goal of which was to expose freshman to an ongoing research project during the academic year to promote student growth and improve retention in the STEM disciplines. Freshmen worked with a faculty mentor and were also chaperoned by a more senior student researcher in order that they learn lab techniques and the capacity to work independently. Participants were fully engaged in a research project (performing experiments, analyzing and discussing results), not a classic classroom projects, but discovery based projects. By bringing students into the research lab at this early stage, our aim was to improve retention by allowing science students to actually *act* as scientists, providing an enhanced experience over the usual freshman survey course content. Of the 6 students who joined the program as freshmen, 5 are still in their major, 4 are still actively engaged in research with a faculty member and have co-authored 11 different papers and conference presentations as of their junior year.

**Genetic diversity of 100+ *Aspergillus* species - the aspMine analysis resource.** *T. C. Vesth*¹, J. L. Nybo¹, S. THEOBALD¹, R. P. DE VRIES⁴, I. V. GRIGORIEV³, S. E. BAKER², M. R. ANDERSEN¹ 1) Department of Bioengineering, Technical University of Denmark, Lyngby, Denmark; 2) Joint Bioenergy Institute, Berkeley, CA, USA, Berkeley, CA, USA; 3) Joint Genome Institute, Walnut Creek, CA, USA, Walnut Creek, CA, USA; 4) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands, Utrecht, The Netherlands.

The filamentous fungal species of the Aspergillus genus are of broad interest to the scientific community including applied, medical and basic research. These fungi are prolific producers of native and heterologous proteins, organic acids, and secondary metabolites (including