**27F Physiological effects of hemicellulosic pentose sugars on glucose metabolism in the maize pathogen *Bipolaris maydis*.** *Hiroshi Yoshida*, Chihiro Tanaka Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

Fungi secrete extracellular hydrolytic enzymes to degrade various environmental substrates, some of which can serve as nutrients or signaling molecules for fungal growth. The filamentous fungus *Bipolaris maydis*, also known as *Cochliobolus heterostrophus*, is the causal agent of southern leaf blight of maize. On *B. maydis*-infected maize leaves, there is observed early development of water-soaked lesions, probably relating to plant cell wall degrading enzymes produced by this fungus. Since arabinoxylan, a hemicellulosic polysaccharide comprising xylose polymer backbone and arabinose side chains, is a major component of maize cell wall, ability of *B. maydis* to degrade this saccharide is likely to be highly involved in fungal nutrient acquisition or physiological regulation during infection. We sought to elucidate the physiological effects of arabinoxylan and its component pentose sugars in *B. maydis* to reveal its post-invasion pathogenic lifestyle. A quantitative RT-PCR (qPCR) analysis and GFP-reporter assay of a β-xylosidase gene of *B. maydis* (*BmXyp1*) showed that it was upregulated immediately after appressorial penetration, suggesting that xylan degradation and enzymatic release of xylose were activated during early infection. Our qPCR analysis also demonstrated that xylose metabolic genes were simultaneously induced with *BmXyp1*. These results possibly indicate the importance of xylan degradation for fungal nutrition in host tissue. However, xylulokinase gene-disrupted strains (Δ*XkiA*), which lacked the ability to grow on xylose, arabinose or xylan, still showed a certain level of virulence on maize, indicating that the fungus did not mainly depend on these sugars for nutrient sources.

Next, we focused on a notable effect of xylose on Δ*XkiA*: "unmetabolizable" xylose strongly inhibited growth, even if in the presence of glucose. A qPCR analysis of glucose metabolic genes demonstrated that a hexokinase gene *BmHxkA* and a glucose-6-phosphate dehydrogenase gene *BmZwf1* were remarkably upregulated in Δ*XkiA* on xylose but not on glucose, compared to in the absence of any sugars. Our results suggest a possible role for hemicellulosic pentose sugars in *B. maydis* physiology, i.e., a part as a signal to induce NADPH production through the hexose monophosphate shunt.

**28W Assessing the role of γ-aminobutyric acid during asexual reproduction and pathogenesis in the wheat pathogen, *Parastagonospora nodorum*.** *O.L. Mead*, S.A. Breen, P.S. Solomon Plant Sciences, Australian National University, Canberra, ACT, AU. *Parastagonospora nodorum* is the causal agent of the Septoria Nodorum Blotch disease on wheat. This disease is prevalent in the U.S.A, Europe, the Middle East and Australia causing significant economic losses to wheat industries globally. Sporulation is critical to disease formation as this necrotrophic fungus relies on multiple cycles of infection, reproduction and re-infection of the host to reduce grain yields. We have previously demonstrated that the non-proteinaceous amino acid, γ-aminobutyric acid (GABA), induces an *in vitro* state of hyper-sporulation in *P. nodorum* under conditions normally producing vegetative growth (Mead *et al.* 2013). GABA is ubiquitous across Kingdoms and is a product of primary metabolism. The anabolic pathway to GABA circumvents a key ATP-producing reaction in the Krebs Cycle and thus imparts a metabolic cost. Pertinently, GABA is produced by plants in response to biotic and abiotic stress. In fungi, the function of this molecule and its associated metabolic pathway are yet to be determined. We show that disruption of key enzymatic steps in GABA metabolism reduces pathogenicity. A transcriptomics approach to identifying mechanisms of GABA-induced sporulation has revealed that the canonical core sporulation pathways in *Neurospora crassa* and *Aspergillus nidulans* are not activated during sporulation in this *Dothideomycete*. We describe a reverse genetics investigation into mechanisms of sporulation in the wheat pathogen, *P. nodorum*.

**29T Antifungal activity of wood extractives.** *M. Morel-Rouhier*¹,², *Nicolas Valette*¹,², *Thomas Perrot*¹,², *Sormani Rodnay*¹,², *Gelhaye Eric*¹,² 1) UMR 1136 Tree-Microbe interactions, Lorraine University, Vandoeuvre les Nancy, FR; 2) INRA, Interactions Arbres–Microorganismes, UMR1136, F-54280 Champenoux, France.

Extractives are non-structural wood molecules that represent a minor fraction in wood. However, they are source of diverse molecules putatively bioactive. Inhibition of fungal growth is one of the most interesting properties of wood extractives in a context of wood preservation, crop protection or medical treatments. The antifungal effect of molecules isolated from wood extractives has been mainly attributed to various mechanisms such as metal and free radical scavenging activity, direct interaction with enzymes, disruption of membrane integrity and perturbation of ionic homeostasis. Lignolytic fungi, which are microorganisms adapted to wood substrates, have developed various strategies to protect themselves against this toxicity. Comparative genomics clearly show that detoxification systems are evolutionary correlated with white rot wood decay. We have shown at the functional level that the neofunctionalization of some enzymes in lignolytic fungi could explain their high resistance capacity to wood extractives.

**30F The cereal pathogen *Fusarium pseudograminearum* produces a mimic of cytokinin plant hormones.** *D.M. Gardiner*¹, J.L. Sørensen², A.H. Benfield¹, R.D. Wollenberg², K Westphal², R Wimmer², K.F. Nielsen³, J. Carere¹, L. Covarelli⁴, G. Beccari⁴, J. Powell¹, T. Yamashino⁵, H. Kogler⁶, T.E. Sondergaard¹ 1) Agriculture and Food, CSIRO, St Lucia, Queensland, AU; 2) Department of Chemistry and Bioscience, Aalborg University, DK-9000 Aalborg, Denmark; 3) Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; 4) Department of Agricultural, Food and Environmental Sciences, University of Perugia, IT-06121 Perugia, Italy; 5) Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Furocho, Chikusa-ku, Nagoya, 464-8601 Japan; 6) Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany.

The necrotrophic pathogen *Fusarium pseudograminearum* infects a broad range of agronomically important crops including barley and wheat. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, which are plant hormones, was identified as being produced by the pathogen during plant infection. Cytokinins are generally thought of as having growth promoting and anti-senescence activity and the production of a cytokinin mimic by a necrotrophic pathogen challenges the view that these pathogens invade by a simple barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the *F. pseudograminearum* genome for the production of these compounds was identified and the biosynthetic pathway established using gene knockouts. The *F. pseudograminearum* cytokinins can activate cytokinin signalling demonstrating their genuine hormone mimicry. *In planta* analysis of the transcriptional response to one of the *F. pseudograminearum* cytokinins suggests extensive reprogramming of the host environment by these molecules, possibly through cross talk with defence signalling pathways.
**31W Clade-specific variations in wood decay strategies among brown rot fungi indicate decay mechanism heterogeneity among brown rot fungi.** G.N. Presley, J. S. Schilling Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN.

Brown rot fungi are a polyphyletic group of wood degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. These mechanisms are incompatible in discrete space, and differential gene expression has been implicated in *Postia placenta* to segregate these reactions. It is unclear, however, how this 'two-step' varies in other brown rot clades with different gene options and distinct niches in nature. Using proteomics, we analyzed the temporal progression of spruce wood decay by brown rot fungi *Serpula lacrymans* (Boletales) and *Gloeophyllum trabeum* (Gloeophyllum clade), using a space-for-time wood wafer design. Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. Each fungus, however, utilized a different wood colonization strategy - *S. lacrymans* invested more in fungal biomass while *G. trabeum* invested more in protein production, with ergosterol/protein ratios 7x higher in *S. lacrymans* than in *G. trabeum*. In line with their native substrate associations, hemicellulase specific activity was dominated by mannanase in *S. lacrymans* and by xylanase in *G. trabeum*. Despite containing a GH 6 cellobiohydrolase (CBH) in its genome, a distinguishing feature of brown rot members of the Boletales, *S. lacrymans* notably did not produce CBH on spruce wafers. This work highlights similarities in a two-step brown rot decay mechanism among distantly related fungi but identifies unique niche-specific variations. It also supports targeting gene discovery efforts on *S. lacrymans*, given the efficacy of its secretome despite its reduced diversity.

**32T Mushroom polyketide synthase produces polyenes for chemical defense.** P. Brandt¹, M. García-Altares², M. Nett³, C. Hertweck², D. Hoffmeister¹ 1) Friedrich-Schiller-Universität, Department Pharmaceutical Microbiology at the Hans-Knöll-Institute, Winzerlaer Straße 2, 07745 Jena (Germany); 2) Leibniz Institute for Natural Product Research and Infection Biology, Department Biomolecular Chemistry, Beutelbergstraße 11a, 07745 Jena (Germany); 3) Technische Universität Dortmund, Department Biochemical and Chemical Engineering, Technical Biology, Emil-Figge-Straße 66, 44227 Dortmund (Germany).

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

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

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

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

**33F Biosynthesis of acurin A and B in *Aspergillus aculeatus*.** M.L. Nielsen, P.P. Wolff, L.M. Petersen, L.N. Andersen, T.I. Petersen, D.K. Holm, U.H. Mortensen, C.S. Nødvig, T.O. Larsen, *J.B. Hoof* Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.

*Aspergillus aculeatus* is known for the commercial utilization in production of several enzymes. We have identified two stereoisomeric compounds of mixed polyketide-nonribosomal peptide origin in the extracts of *A. aculeatus* that we named acurin A and acurin B. The structures of the compounds strongly resemble the structure of the mycotoxin fusarin C produced by several *Fusarium* species. CRISPR-Cas9 was used to construct a non-homologous end-joining deficient strain of *A. aculeatus*, which enabled efficient gene deletions in the acurin gene cluster. Using RT-qPCR in combination with metabolite profiling of gene deletion strains, the acurin producing gene cluster was delineated, which allowed us to propose a biosynthetic pathway for formation of acurin. Our results show that acurin, in contrast to fusarin C, is biosynthesized by an individual polyketide synthase and non-ribosomal synthetase. At least six other enzymatic activities are required for the biosynthesis of acurin. This study shows how we exploit the CRISPR-Cas9 system for the rapid construction of fungal host strains that can be readily engineered to generate valuable knowledge.

**34W Analysis of secondary metabolite gene clusters in *Alternaria alternata*.** B. Voss, R. Fischer Department of Microbiology, Institute for Applied Bioscience, Karlsruhe, Baden-Württemberg, DE.

The filamentous fungus *Alternaria alternata* is an ascomycete that causes huge economic losses every year because of plant infection and food and feed spoilage. *A. alternata* produces more than 60 different secondary metabolites, and more are to be expected because many secondary metabolite gene clusters are not expressed under laboratory conditions. In order to discover new metabolites and/or identify the corresponding genes, different physiological conditions were tested for the induction of certain gene cluster. Using quantitative Real-Time PCR we found that the four non-ribosomal peptide synthases (NRPS) found in the genome, were expressed under glucose-starvation conditions. Nitrate starvation induced two genes (*AAT PG02707*, *AAT PG03557*), whereas no significant difference occurred during phosphate starvation. When *A. alternata* was exposed to osmotic stress, two genes (*AAT PG02707*, *AAT PG04268*) were induced. In order to identify the corresponding metabolites, CRISPR Cas technology was used to inactivate all four NRPS genes (1). The metabolite profile of the mutant strains will be compared to the profile of corresponding wild-type strains by HPLC. In addition, the NRPS genes along with other putative tailoring enzyme encoding genes found in the vicinity of the NRPS genes, will be expressed heterologously in *Aspergillus oryzae* (2). One NRPS (*AAT PG04268*) was already successfully introduced into *A. oryzae* and the metabolite analysis is under way.

(1) Nødvig CS, Nielsen JB, Kogle ME, Mortensen UH (2015) A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS ONE 10(7): e0133085.
(2) Pahirulzaman KAK, Williams K, Lazarus CM (2012) A Toolkit for Heterologous Expression of Metabolic Pathways in *Aspergillus oryzae*. Methods in Enzymology, 517: 241-260

**35T Analysis of the alternariol producing gene cluster in Alternaria alternata.** M. Wenderoth, R. Fischer Department of Microbiology, Institute for Applied Bioscience, Karlsruhe, BW, DE.

The filamentous fungus *Alternaria alternata* is an ascomycete that colonizes many different organic substrates and is pathogenic for many plants. It causes tremendous economic damage every year due to plant infections and post-harvest food and feed spoilage. *A. alternata* produces a variety of different secondary metabolites but only little is known about their biosynthetic pathways and the regulation of the corresponding genes. One of the most prominent toxins is alternariol (AOH). Although the toxin itself is well studied, the way of synthesis and the corresponding gene cluster remained unknown. In the ascomycete *Parastogonospora nodorum* the corresponding PKS was identified recently (1). However, other genes found in the vicinity of the PKS were not studied.

In *A. alternata* ten *pks* gene clusters were identified (2). In order to assign a function to Pks1 and inactivate the gene we adapted the recently developed CRISPR-Cas9 system for *A. alternata* (3). *pks1* is located in a gene cluster consisting of Pks1, three tailoring enzymes and a putative transcription factor. To further understand the mechanisms of AOH and alternariol-monomethyl ether (AME) biosynthesis we expressed *pks1* in combination with three tailoring enzymes in *Aspergillus oryzae* as a heterologous host using a multi expression vector (4). Single expression of *pks1*, lead to AOH production. By co-expression of the O-methyl transferase located in this cluster the production of (AME) was observed. Through multiple co-expression of the remaining tailoring enzymes a new compound was identified. Using a laccase based reporter gene assay, we confirmed that the transcription factor induces *pks1* expression.

(1) Chooi *et al.* (2015). *Appl Environ Microbiol.* 81(16):5309-17

(2) Saha *et al.* (2012). PLoS ONE. 7(7): e40564

(3) Nødvig *et al.* (2015). PLoS ONE. 10(7): e0133085

(4) Pahirulzaman *et al.* (2012). *Methods Enzymol.* 517: 241-260

**36F Gene cluster diversity for indole-diterpene biosynthesis in the grass endophyte Epichloë festucae.** T.A. Miller¹,², W.J Mace¹, B Scott², R.D Johnson¹ 1) Grasslands, AgResearch, Palmerston North, Manawatu, NZ; 2) Institute of fundamental science, Massey University, Palmerston North, Manawatu, NZ.

Indole-diterpenes (IDT) are an important class of bioprotective metabolites synthesized by *Epichloë festucae* in association with *Festuca* and *Lolium spp.* The most abundant product in *L. perenne* infected with strain FI1 is lolitrem B, a potent tremorgenic mycotoxin responsible for the mammalian syndrome 'ryegrass staggers'. Molecular cloning and functional analysis has shown that the genes responsible for lolitrem B biosynthesis are organized in three sub-telomeric gene clusters interspersed with transposon relics. These 11 *idt* genes comprise a set of core genes for the biosynthesis of the first stable intermediate, paspaline, and additional genes that encode products for further elaboration of this core molecule. Genome sequence and PCR analysis has shown that there is remarkable diversity in the *idt* gene profile within *E. festucae*, resulting in a diversity of IDT products synthesized *in planta*. The aim of this work is to analyse the *idt* gene cluster structure in an agriculturally important *E. festucae var loli* (asexual) strain PN3136 and determine whether this explains the observed IDT chemical phenotype. The genome of PN3136 was sequenced using Illumina technology and the *idt* gene cluster analysed. PN3136 was found to have all 11 *idt* genes with conserved synteny to strain FI1. However, *idtP* and *idtQ* (cytochrome P450 monooxygenases) had a lower than expected sequence identity when compared with FI1. In addition *idtF* (encoding a DMAT synthase) and *idtK* (encoding a P450 monooxygenase) have SNPs that introduce frame-shift mutations and premature termination of translation. The *in planta* LC-MS/MS detection of metabolites consistent with IDT products lacking the additional prenylated cyclic ring as found in lolitrem B and E, is consistent with IdtF and IdtK being non-functional. Interestingly, this IDT chemical phenotype is strikingly similar to that previously determined in an *E. festucae* (sexual) strain Fg1. We therefore sequenced the genome of Fg1 and found that the *idt* gene profile was identical to PN3136 except for *idtK*, which had a different SNP that results in earlier termination of translation. PN3136 and Fg1 are therefore two closely related asexual and sexual *E. festucae* strains that have a novel IDT chemical profile.

**37W Transcriptional co-regulation and spatial coordination of mevalonate and sesquiterpene biosynthetic pathway enzymes of Fusarium graminearum based on in vitro RNAseq and organellar proteomics.** Karen Broz¹, Marike Boenisch², H. Corby Kistler¹,² 1) USDA ARS Cereal Disease Laboratory, Saint Paul, MN, USA; 2) University of Minnesota, St. Paul, MN, USA.

Farnesyl pyrophosphate (FPP), a product of the primary metabolic mevalonate pathway, gives rise to both primary and secondary metabolite terpenes. The phytopathogenic fungus *F. graminearum* produces the sesquiterpene mycotoxin deoxynivalenol (DON) during infection of wheat or when induced in culture. Under conditions conducive to DON production, FPP utilization may shift towards secondary metabolites, requiring primary and secondary metabolism pathway coordination. Transcriptome analysis (RNAseq) of *F. graminearum* grown in DON inducing and non-inducing medium over time shows that mevalonate- and DON biosynthetic pathway genes are transcriptionally upregulated during DON induction. At the same time, genes for synthesis of ergosterol, a diterpene primary metabolite, are down regulated, suggesting coordination of terpene pathways and a shift in precursors towards DON production. When reporter strains with fluorescently labeled enzymes are grown in DON induction medium, the mevalonate pathway enzyme HMG CoA reductase (Hmr1) strictly co-localizes with membrane-associated DON biosynthetic cytochrome P450s Tri1 and Tri4 in membrane accumulations of the endoplasmic reticulum (ER). Mevalonate and sesquiterpene biosynthetic pathway enzymes are spatially coincident suggesting that the enzymes may be part of a multi-enzyme complex. To identify further proteins of a potential multi-enzyme complex, fluorescence-activated cell sorting (FACS) was used to enrich for Tri4::RFP labeled ER membranes for proteomic analysis. Proteomics of the RFP-fluorescent ER fraction revealed additional proteins involved in DON synthesis (Tri1, Tri11 and Tri14), as well as Hmr1, and a number of conserved ER proteins. RNAseq data further indicate that genes corresponding to enriched proteins in the ER proteome are transcriptionally upregulated upon DON induction. Transcriptomics and ER proteomics suggest that DON biosynthesis may require not only coordinate expression of genes for specific primary and secondary metabolic pathways, but also co-localization of biosynthetic enzymes to specific organelles.
**38T Thinking outside the box:NADPH oxidases as signaling hubs are more than just simple ROS producer.** R. Marschall, P. Tudzynski Institute of Plant Biology and Biotechnology, Münster, DE.

The filamentous fungus *Botrytis cinerea* is one of the most devastating plant pathogens. Molecules, which are directly involved in the communication between host and pathogen are reactive oxygen species (ROS). They are produced as mere by-products of the respiratory chain or in highly conserved processes by e.g. NADPH oxidase complexes. In *B. cinerea* two different Nox complexes do exist, both regulating a huge variety of developmental processes. While for the NoxA complex the elucidation of the complex composition and interacting proteins (Bclqg1, BcNoxD) is already sophisticated<fnref n="1" />/<fnref n="2" />, there are hardly any information about proteins that directly interact with the second catalytic subunit BcNoxB or the overall regulator BcNoxR.

Most recently, we identified a large set of proteins in a pulldown screening with the catalytic subunit NoxB and the regulator NoxR. While potential interaction partner that were previously postulated as putative Nox complex members could not be verified, other target proteins were fished in the pulldown approach and verified by Y2H experiments.

Surprisingly, in four independent experiments proteins were pulled down with BcNoxB that all belong to a predicted PKS5-NRPS7 cluster. Subsequent Y2H analyses revealed that indeed proteins of the cluster interact directly with BcNoxB, which might indicate a role for BcNoxB in the secondary metabolism. Moreover, different cluster proteins seem to build up a complex since some of them are interacting with each other.

However, not only BcNoxB seems to be more than just a simple ROS producer. The first catalytic subunit BcNoxA was shown to interact with the cytochrome C peroxidase. Thus, for the first time in fungi a direct link came apparent between mitochondria, the respiratory chain and the NoxA complex. Since BcNoxA is additionally associated with the protein disulfide isomerase BcPDI it can be speculated that BcNoxA is contributing to ROS levels, but is also working as a signaling hub, connecting essential intracellular processes such as protein folding, redox homeostasis, respiration and ROS production.

**39F Identification and characterisation of polyketide synthases in the barley pathogen Ramularia collo-cygni.** F. Dussart<fnref n="1" />,<fnref n="2" />, P.N. Hoebe<fnref n="1" />, S.H. Spoel<fnref n="2" />, N.D. Havis<fnref n="1" />, G.R.D. McGrann<fnref n="1" /> 1) Crop and Soil Research Department, SRUC, Edinburgh, GB; 2) School of Biological Sciences, University of Edinburgh, Edinburgh, GB.

The filamentous fungus *Ramularia collo-cygni* (Rcc) causes the late season barley disease Ramularia Leaf Spot (RLS). In the past twenty years it has become an important disease in northern Europe and other temperate regions of the world. RLS can quickly cause substantial yield losses and reduce grain quality. Limited options to control RLS have given this disease a higher profile and attracted scientific attention enhancing the understanding of the Rcc life cycle which indicated that this fungus is an endophyte with a necrotrophic phase.

Rcc produces a series of anthraquinone toxins called rubellins, thought to be involved in RLS development. Anthraquinones are known to be synthesised through the polyketide pathway. In this study, ten putative polyketide synthases (PKS) were identified in the genome of Rcc. Using *in silico* genome walking eight secondary metabolite-related gene clusters were identified near core PKS genes. Clusters with no PKS core genes were also identified. Gene expression of six core PKS genes was assessed during disease development in barley seedlings. Expression of most PKS transcripts declined during disease development. Co-regulation of secondary metabolism-related genes that clustered with core PKS genes was also observed. Together these data imply that, if produced; polyketide-derived secondary metabolites in Rcc might not be associated with disease symptom development. Instead, secondary metabolites may act as antifungal agents, as we found that Rcc inhibited the growth of several major barley fungal pathogens *in vitro*, suggesting these compounds may be important for Rcc niche exploitation.

**40W Elucidating the biosynthetic pathway of the anticancer secondary metabolite calbistrin in Penicillium decumbens.** Sietske Grijseels<fnref n="1" />, Carsten Pohl<fnref n="2" />, Zahida Wasil<fnref n="1" />, Jens Christian Nielsen³, Yvonne Nygård<fnref n="2" />, Jens Nielsen³, Jens C. Frisvad<fnref n="1" />, Kristian Fog Nielsen<fnref n="1" />, Mhairi Workman<fnref n="1" />, Thomas Ostenfeld Larsen<fnref n="1" />, Arnold Driessen<fnref n="2" />, Rasmus John Normand Frandsen<fnref n="1" /> 1) Department of Biotechnology and Biomedicine, Technical University of Denmark, DK 2800 Kgs. Lyngby, Denmark; 2) Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands; 3) Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96 Gothenburg, Sweden.

Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. One interesting secondary metabolite is calbistrin, a compound recently found to have bioactivity against leukemia cells. This compound consists of two polyketides linked by an ester bond; a decalin containing polyketide similar to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin is known to be produced by several uniseriate black Aspergilli, *Aspergillus versicolor*-related species, and several Penicillia. Among the Penicillia, the recently genome sequenced *P. decumbens* is interesting as it produces several putative intermediates of the calbistrin pathway, such as decumbenone A and B and versiol. In this study, the molecular and enzymatic mechanisms underlying the biosynthesis of calbistrin are elucidated using a combinatorial approach of bioinformatics, molecular biology and analytical chemistry. Comparative studies of the polyketide synthase (PKS) sequences from the three genome sequenced species *A. versicolor*, *A. aculeatus* and *P. decumbens* resulted in the identification of a putative gene cluster for production of the decalin part of calbistrin. Implementation of CRISPR/Cas9 technologies in *P. decumbens* facilitated the deletion of the putative PKS in this species. Subsequent UHPLC-MS analysis of extract metabolites revealed that calbistrin and putative intermediate compounds were absent, proving the involvement of the PKS in calbistrin production. Further characterization of the predicted gene cluster is achieved by targeted deletion of the individual biosynthetic genes in the cluster.

**41T Mechanisms of capsaicin tolerance in fungal seed pathogens of wild chili peppers.** C.A. Adams<fnref n="1" />,<fnref n="2" />, K. Zimmerman<fnref n="2" />,⁴, K. Fenstermacher³, D. Geiser³, A. Pringle<fnref n="1" />,⁵ 1) Plant and Microbial Biology, UC Berkeley, Berkeley, CA; 2) Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA; 3) Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA; 4) Gingko Bioworks; 5) Department of Botany and Bacteriology, University of Wisconsin-Madison,

<footnote n="1">¹Marschall et al., 2016: Update on Nox function, site of action and regulation in *Botrytis cinerea*. Fungal Biology and Biotechnology2016:38</footnote>

<footnote n="2">²Marschall and Tudzynski, 2016: Reactive oxygen species in development and infection processes. Semin Cell Dev Biol. 2016 Sep;57:138-46.</footnote>
Madison, WI.

To deter herbivores and pathogens, many plants produce secondary metabolites. However, herbivores and pathogens that commonly associate with plants can evolve resistance to defensive compounds. Such co-evolution is evident in a wild chili pepper, *Capsicum chacoense*. Though plant-produced capsaicinoids slow fungal growth, fungi that infect *C. chacoense* seeds possess much higher capsaicinoid tolerance than other tested microbes. Previous researchers have proposed two separate mechanisms of capsaicin inhibition. A small number of studies propose that capsaicinoids can cause cell membrane disruption, while a separate, larger body of literature has demonstrated that capsaicinoids like capsaicin and dihydrocapsaicin bind to the NADH dehydrogenase (Complex I) of the Electron Transport Chain (ETC), crippling Oxidative Phosphorylation (OXPHOS) and subsequent energy production (ATP). Here, we investigated how fungal seed pathogens of wild *Capsicum chacoense* evolved tolerance to capsaicin. We measured fungal growth rate in the presence of different ETC inhibitors to determine 1) if these fungi possess alternative respiratory enzymes, and 2) if capsaicinoids' effects on the ETC fully explained the effect of these inhibitors. We selected 16 fungal isolates from four *Ascomycete* genera (*Alternaria*, *Colletotrichum*, *Fusarium* and *Phomopsis*). In all isolates, we found evidence for a Complex I alternative NADH dehydrogenase. Unexpectedly, we also found evidence for a Complex III alternative cytochrome oxidase in many isolates. These fungi may achieve additional capsaicin tolerance by degrading capsaicin. Inhibition of OXPHOS appears to weakly explain the mechanism by which dihydrocapsaicin slows fungal growth, but not capsaicin. We hypothesize capsaicin disrupts cell and mitochondrial membranes in addition to inhibiting OXPHOS. Our research also suggests alternative respiratory enzymes are more prevalent in plant pathogens than was previously appreciated.

**42F Identification and characterization of the Aspergillus flavus aspergillic acid gene cluster.** J. Cary¹, M. Lebar¹, C. Carter-Wientjes¹, B. Mack¹, R. Majumdar¹, J. Diana Di Mavungu², S. De Saeger² 1) USDA-ARS-SRRC, New Orleans, LA; 2) Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.

*Aspergillus flavus* produces toxic and carcinogenic aflatoxins. *In silico* analysis of the *A. flavus* genome revealed 56 gene clusters encoding for secondary metabolites (SMs). Although about 20 SMs have been identified from *A. flavus* cultures, studies by our group and others have experimentally assigned metabolites to just ten *A. flavus* gene clusters. We are interested in how these SMs affect fungal development, survival, and virulence. We are particularly interested in *A. flavus* metabolites that are produced during infection of corn seed. RNA-Seq analysis of all predicted *A. flavus* secondary metabolic gene cluster 'backbone' genes during corn kernel infection showed that in addition to the aflatoxin cluster polyketide synthase (PKS) gene, *aflC*, one of the earliest genes expressed was the uncharacterized Cluster 11 nonribosomal peptide synthetase (NRPS) gene, *asaB* (AFLA_023020). SMURF analysis indicated that Cluster 11 may be composed of as many as 12 genes, however we focused on five genes encoding the putative NRPS, desaturase, P450 oxidoreductase, MFS transporter and C6 transcription factor. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized pyrazinone, deoxyaspergillic acid (deoxy-AA), and the antimicrobial hydroxamic acid-containing mycotoxins aspergillic acid (AA) and hydroxy-AA (OH-AA). From the LC-MS data we have proposed a biosynthetic pathway for the production of these metabolites. In addition, we found that AA can form a trimer with one atom of iron to generate ferriaspergillin, perhaps indicating that this cluster is involved in regulation of iron homeostasis in *A. flavus*.

**43W Trichoderma reesei forms surface attachment structures and senses cellulosic biomass using class XIII GPCRs.** Eva Stappler¹, Guofen Li¹, David Turrà², Stefanie Kindel¹, Ursula Sauer¹, Antonio Di Pietro², *Monika Schmoll*¹ 1) Health and Bioresources, AIT Austrian Institute of Technology GmbH, Tulln, Lower Austria, AT; 2) Department of Genetics, University of Cordoba, Campus de Excelencia Internacional Agroalimentario, Córdoba, Spain.

*Trichoderma reesei* (syn. *Hypocrea jecorina*) is one of the most prolific producers of plant cell wall degrading enzymes. Production of these enzymes is induced in response to the presence of carbon sources related to degradation production of plant biomass. Here we tested chemotropic growth of *T. reesei* germ tubes towards different compounds, and observed a robust response to glucose, the organic nitrogen source glutamate and the presence of a mating partner. However, no chemotropic response was observed for the cellulase-inducing disaccharide sophorose or to the non-fermentable sugar lactose. Recently, comparison of transcript data from different inducing carbon sources indicated, that the class XIII G protein-coupled receptors (GPCRs) CSG1 and CSG2 are involved in transcriptional activation of cellulases by the substrate cellulose. Mutants lacking CSG1 or CSG2 were impaired in the chemotropic response to glucose. Moreover, whereas the wild-type strain formed broadened appressoria-like structures at the hyphal tips during growth on a natural cellulosic substrate (maple leaves), the two mutants failed to differentiate these structures. Contact angle analysis suggested that regulation of hydrophobin expression did not play a role in this difference. Our results suggest that *T. reesei* can chemotropically sense cellulosic plant biomass through its degradation product glucose, and that this sensing requires the GPCRs CSG1 and CSG2. Transcriptome data of ?csg1 reveal the molecular pathways involved in the response to cellulose.

**44T Sensing and responding to hypersaline conditions: same signal but different mechanisms of activation of HOG pathway in halotolerant and halophilic fungi, isolated from hypersaline environments.** A. Plemenitas¹, N. Gunde-Cimerman², M. Lenassi¹, T. Konte¹ 1) Institute of Biochemistry, University of Ljubljana, Faculty of Medicine, Ljubljana, SI; 2) Biology Department, University of Ljubljana, Biotechnical Faculty, Ljubljana, SI.

The main signaling pathway in fungi responsible for cellular responses to increased NaCl concentrations is high osmolarity glycerol (HOG) pathway. Here we present the architecture of HOG signal transduction pathways for extremely halotolerant *H.wernecki*, moderately halotolerant *A pullulans* and for halophilic *W.ichthyophaga*, which were all isolated from the environments, characterized by extremely high NaCl content, like solar salters. Our studies were mostly focused on extremely halotolerant *H.werneckii* and halophilic *W.ichthyophaga* which both tolerate NaCl up to 5M concentration. Moderately halotolerant *A pullulans* was included for comparative studies. With the availability of the genomes of these fungi, the presence of known components of HOG pathway, as well as novel HOG components were confirmed through homology searches. We show that although there are many similarities between halotolerant and halophilic fungi, important differences were found in components of HOG pathway as well as in phosphorylation of MAPK Hog1 that might also explain the different halotolerant/ halophilic characters of the studied fungi. At the protein level, we observed in *H. werneckii* a phosphorylation pattern similar to that of *S. cerevisiae*, with the exception, that the HwHog1 kinase was fully phosphorylated only when *H. werneckii* cells were exposed to high salt concentrations, 3 M NaCl or higher. In *W. ichthyophaga*, a completely opposite phosphorylation pattern was observed:
WiHog1 kinase is dephosphorylated after hypo-osmotic or hyperosmotic shock, and is constitutively phosphorylated only under optimal salinity concentration (3.4 M NaCl). We also demonstrated that HwHog1 is activated via both, SHO and SLN branches of HOG pathway, in *W. ichthyophaga*, on the other hand, it appears that only SLN branch is involved in the activation of WiHog1. Importance of HOG pathway in adaptation to high NaCl concentrations in halotolerant/halophilic fungi was confirmed by applying specific inhibitor of MAPK Hog1.

**45F Hemolytic activity of Mannosylerythritollipids is based on mono-acetylated variants.** T. Deinzer¹, U. Linne², M. Bölker¹, *B. Sandrock*¹ 1) Department of Biology - Genetics, Philipps-University Marburg, Marburg, DE; 2) Department of Chemistry, Philipps-University Marburg, Marburg, DE.

Mannosylerythritollipids (MELs) are secondary metabolites produced by fungi. MELs are used as biosurfactants in industrial processes for the production of e.g. detergents or pharmaceuticals. In the phytopathogenic fungus *Ustilago maydis* biosynthesis of these glycolipids can be studied under nitrogen starvation conditions. A gene cluster composed of five genes has been identified and is necessary for MEL biosynthesis. The present work was initiated by characterisation of the major facilitator transporter protein Mmf1 and its function during MEL production. Cells carrying a deletion of *mmf1* produced only di-acetylated MEL variants, which are less hemolytically active. The transporter could be functionally replaced by Mmf1 of *Ustilago hordei* but not by Mfs1, a closely related major facilitator transporter encoded in the genome of *U. maydis*. Strains capable of producing predominantly either non-, mono- or di-acetylated MEL isoforms were constructed demonstrating that mono-actylated MELs contain the highest degree of hemolytical activity. Therefore we suggest that active transport of these MEL variants by Mmf1 is necessary to prevent self destruction by cell lysis.

**46W Trichothecene mycotoxin transport in the phytopathogen *Fusarium graminearum*.** Sean P. O'Mara¹, Karen Broz², Yanhong Dong³, H. Corby Kistler²,³ 1) Department of Plant Biology, University of Minnesota, St. Paul, MN, USA; 2) USDA ARS Cereal Disease Laboratory, St. Paul, MN, USA; 3) Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA.

*Fusarium graminearum* is a necrotrophic pathogen and the causal agent of Fusarium Head Blight disease of small grain cereals. *F. graminearum* infection results in the contamination of grain with potent trichothecene mycotoxins. These mycotoxins are protein synthesis inhibitors and when ingested, trichothecene toxicosis can lead to ribotoxic stress, cellular apoptosis, and immuno-modulation. Accumulation of the trichothecene deoxynivalenol (DON) by *F. graminearum* during infection is essential for full virulence to wheat (*Triticum aestivum*). While the biosynthetic pathway of DON production has been well studied, potential modes of DON transport and cellular sequestration have not. Previous evidence indicated that the major facilitator superfamily (MFS) transporter, Tri12, whose gene is located within the core trichothecene biosynthetic cluster of *F. graminearum*, is not the sole transporter of DON. Thus, we aim to elucidate other modes of DON transport and cellular sequestration in *F. graminearum* utilizing both *in vitro* and *in vivo* methods. Genes for six proteins predicted to be involved in DON transport or sequestration (three ATP binding cassette (ABC) transporters, one MFS transporter, one t-SNARE protein, and one Rab GTPase) were deleted using the split-marker homologous recombination technique. These mutants were then tested for DON accumulation *in vitro* using toxin-inducing medium, and *in planta* using a 14-day wheat point-inoculation bioassay. Our results indicate that the ABC transporter *Abc1* as well as *Rab7*, a Rab GTPase essential for vacuolar biogenesis, are crucial proteins allowing for DON accumulation. Individually, these two mutants showed significantly lowered virulence *in planta* (p<0.05). The virulence on wheat of *Abc1* mutants was only ~28% of wild-type *F. graminearum* PH1, and *rab7* mutants were completely incapable of spreading beyond the inoculated floret. Future work aims to generate double mutants of DON transporter proteins to analyze their relative contribution to DON export as well as the potential redundant or synergistic function of these multiple modes of transport.

## Biotechnology

**47T Development of CRISPR-Cas9 for targeted gene disruption in the multinucleate filamentous pathogen *Sclerotinia sclerotiorum*.** Jingtao Li¹,², Yanhua Zhang¹,², Hongyu Pan¹, Jeffrey A. Rollins² 1) College of Plant Science, Jilin University, Changchun, Jilin, China; 2) Department of Plant Pathology, University of Florida, Gainesville, Florida, USA.

*Sclerotinia sclerotiorum* (Lib.) de Bary is a necrotrophic fungal plant pathogen of Arabidopsis and many agriculture crops. With the development of complete genome sequencing and annotation many potentially important genes for pathogenicity and development have been discovered. Conventionally, knockout or disruption mutants of target genes have been created by homologous recombination, knockdowns by RNAi, and random insertion mutations introduced by T-DNA. These approaches, while utilized effectively, have been of low efficiency. The emerging technologies of CRISPR-Cas9 represent efficient, convenient, timely, and cost-saving approaches for gene manipulation. In this research, we developed a CRISPR-Cas9 tool based on modification of existing plasmids. This vector was used to explore the efficiency of target gene mutation using the oxalate biosynthesis *oah1* gene as a test target. We found that transformation with a circular plasmid resulted in a high frequency (40%) of targeted gene mutation among transformants. Unexpectedly, 100% of these mutants integrated large segments of the transforming plasmid at the target site. This result was confirmed among multiple target sites within the *oah1* gene and among three independent isolates of *S. sclerotiorum*. The efficiency of creating CRISPR-Cas9 insertion mutants has allowed us to confirm the requirement of *oah1* for oxalic acid biosynthesis in multiple isolates of *S. sclerotiorum* and to begin characterization of shared phenotypes among isolates. One such phenotype not previously reported is the overproduction of compound suppressor. These mutants are capable of infecting healthy host tissue allowing us to explore the early compatibility events and factors associated with host infection independent of oxalic acid. Comparative transcriptomic analysis between *oah1* mutants and wild type is being pursued to identify these factors.

**48F 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.

**49W Fast and highly efficient gene replacement in Aspergillus niger using CRISPR/Cas9.** Jean Paul Ouedraogo, Yun Zheng, Tricia John, Letian Song, Adrian Tsang Concordia University, CSFG, 7141 Sherbrooke St.W. Montreal, QC, H4B1R6.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is transforming biology by providing a simple and efficient method to precisely edit the genome of any organism. As an important bio industrial workhorse, *Aspergillus niger* has attracted widespread interest for exploring broaden application in the sectors of industrial enzymes and organic acids. The availability of its genome sequence enables the redesign and engineering novel stains of *A. niger* based on CRISPR/Cas9 tools. In this study, we have extended CRISPR/Cas9 system for gene editing in *A. niger*. The endogenous tRNA gene as RNA polymerase III promoter was employed to lead the expression of guide-RNA (gRNA) cassette to delete and replace gene by homologous recombination in *Aspergillus niger*. The combination of CRISPR/Cas9 system and non-homologous end joining deficient strain allows fast and highly efficiency gene replacement in an organic acids producer *A. niger* strain with 100 % of transformants showing correct gene replacement.

**50T 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.

**51F Genomic and transcriptomic analysis of Aspergillus niger producing thermophilic bacterial cellulases.** J. Kim<sup>1,2</sup>, S. A. Campen<sup>1,2,5</sup>, B. A. Simmons<sup>1,3</sup>, S. E. Baker<sup>1,2</sup>, J. M. Gladden<sup>1,4</sup>, J. K. Magnuson<sup>1,2</sup> 1) Joint BioEnergy Institute, Emeryville, CA; 2) Pacific Northwest National Laboratory, Richland, WA; 3) Lawrence Berkeley National Laboratory, Berkeley, CA; 4) Sandia National Laboratories, Livermore, CA; 5) J. Craig Venter Institute, La Jolla, CA (current affiliation).

Enzymatic hydrolysis of pretreated lignocellulosic biomass is important in efficient conversion of lignocellulosic biomass to biofuel. Thermophilic bacterial cellulase enzymes have advantages in industrially relevant conditions due to their stability in high temperature and tolerance to inhibitory compounds generated during biomass pretreatment. *Aspergillus niger* is a filamentous fungus known for its ability to produce high levels of enzymes, and it has been used for the industrial production of many useful enzymes. In this work, we employ genomics and transcriptomics approaches to understand and improve the production of thermophilic bacterial cellulases in *A. niger*.

Comparative transcriptomic analysis of the *A. niger* ATCC 11414 parent strain and an engineered strain expressing a thermophilic bacterial cellulase using RNA-Seq shows that more than two-thirds of the genes are differentially expressed. We also investigated differential RNA polymerase binding and histone modification using ChIP-Seq, in order to examine these potential causes of differential expression. Using whole-genome resequencing, we identified the genetic variation in the engineered *A. niger* strain as well as the copy number and integration locus of the introduced bacterial cellulase gene. These genetic changes could be responsible for the epigenetic changes and subsequent differential gene expression in heterologous enzyme producing *A. niger* strain.

**52W Milk-clotting enzymes from Aspergillus oryzae and Aspergillus luchuensis.** Yoko Takyu<sup>1</sup>, Taro Asamura<sup>1</sup>, Ayako Okamoto<sup>1</sup>, Hiroshi Maeda<sup>1</sup>, Michio Takeuchi<sup>1</sup>, Ken-Ichi Kusumoto<sup>2</sup>, Hitoshi Amano<sup>3</sup>, Hiroki Ishida<sup>4</sup>, Youhei Yamagata<sup>1</sup> 1) Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchū, Tokyo, JP; 2) NFRI, Ibaraki, JP; 3) Amano Enzyme Inc., Gifu, JP; 4) Research Institute, Gekkeikan Sake Company Ltd., Kyoto, JP.

Milk-clotting enzymes were used for the cheese production. Chymosin is an aspartic endopeptidase functioned as a key enzyme for curdling process. The enzyme is found in rennet made from the abomasum of calves. Chymosin specifically cleaves κ-casein between Phe<sup>105</sup>-Met<sup>106</sup>. The scission causes collapse of the casein micelles in the milk and curd formation. Although the clotting enzyme from *A. oryzae* was reported, the enzyme was not identified. We searched aspartic endopeptidases with milk-clotting activity in *A. oryzae*. The genome project of *A. oryzae* revealed that the fungus has 134 proteolytic enzyme genes, and the aspartic endopeptidase genes are 11. We overexpressed the enzymes in *A. nidulans* as a host and found two enzymes showing the milk-clotting activity. One enzyme showed a
high curding activity and another showed a weak activity. The high-activity enzyme (AOchymosin) was purified and characterized. On the other hand, we also found the homologous enzyme genes in *Aspergillus luchuensis* (formerly known as *A. awamori*) used for production of *awamori*, a kind of *shochu*. The similarity of amino acid sequence between the AOchymosin and the *A. luchuensis* counterpart (ALchymosin) was 95%. ALchymosin was also overexpressed in *A. nidulans* and purified as a single band in SDS-PAGE. The molecular masses of the both purified fungal chymosin were about 40 kDa. The enzymes showed maximum milk-clotting and proteolytic activities at pH 5.5 and 3.0, respectively. The peptide analysis of the digested k-casein showed that the cleavage sites were coincide, Phe¹⁰⁵-Met¹⁰⁶ and Lys²⁴-Tyr²⁵. We concluded the both enzymes would be able to use for cheese production.
This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

**53T Expression of an AT-biased *Rhizopus* glucoamylase gene in *Aspergillus* oryzae using codon optimization.** *D. Zawil, M. Tanaka, T. Shintani, K. Gomi* Grad. Sch. Agric. Sci, Tohoku Univeristy, Sendai shi, Miyagi, JP.

Glucoamylase from *Rhizopus oryzae* is of great importance in biotechnological applications because of its strong hydrolyzing activity toward raw starch compared to other glucoamylases. However, despite such an advantage of *Rhizopus* glucoamylase, *R. oryzae* produces low amounts of glucoamylase in submerged cultures. To overcome some of the obstacles of mass production of *Rhizopus* glucoamylase, we try to produce it at high levels using *Aspergillus oryzae* as a host.

Working with two organisms of different GC content, however, codon optimization is a must to be able to achieve highly efficient production of the glucoamylase, according to our previous studies (1, 2) suggesting that AT-biased heterologous gene transcripts tend to be polyadenylated prematurely and be degraded rapidly. Hence, we firstly compare the production level of *Rhizopus* glucoamylase overexpressed in *A. oryzae* and in the yeast *Saccharomyces cerevisiae*. So far we have succeeded in transforming the *Rhizopus* glucoamylase in both yeast and *A. oryzae*. Proceedingly we are checking for the glucoamylase activity and its transcript level by northern blot analysis in the transformants.

1) Tokuoka et al, *Appl. Environ. Microbiol.*, **74**, 6538?6546 (2008)

2) Tanaka et al, *Appl. Microbiol. Biotechnol.*, **96**, 1275?1282 (2012)

**54F Adsorption kinetics and self assembled structures of *Aspergillus* oryzae hydrophobin RolA on chemically modified solid surfaces.** Y. Terauchi¹, M. Nagayama¹, T. Tanaka¹, H. Tanabe¹, T. Arita², H. Higuchi², F. Hasegawa³, *K. Abe*¹,³ 1) Dept. Microbial biotechnology, Grad. Sch. Agricult. Sci., Tohoku Univ., Sendai, Miyagi, JP; 2) IMRAM, Tohoku University; 3) NICHe, Tohoku University.

Hydrophobins are amphipathic proteins that are ubiquitous among filamentous fungi, and required for the formation of fungal aerial structures (1). Hydrophobins also play an important role in the infection of pathogenic fungi because hydrophobins are not recognized by pattern recognition receptors of animals and plants (2, 3). The industrial fungus *Aspergillus oryzae* produces a hydrophobin RolA (4). RolA attaches and self-assembles on solid surfaces such as polyesters, and recruits an esterase CutL1, which results in stimulation of the hydrolysis of polyester by CutL1 (4). RolA also evades from recognition by mouse immune system (unpublished results). Because of these properties, RolA is expected to be applicable to industrial and medical fields. Therefore, kinetic properties and self-assembled properties of RolA-adsorption on solid surfaces are important. To investigate kinetic properties and self-assembled structures of RolA on solid surfaces, we constructed self-assembled monolayers (SAM) of 1-undecanethiol (hydrophobic), 11-amino-1-undecanethiol (cationic), and 10-carboxy-1-undecanethiol (anionic) on the electrodes of a Quartz Crystal Microbalance (QCM). We analyzed kinetics of RolA-adsorption on SAM-electrodes at various pH, and then observed self assembled structures of RolA by an AFM. Affinities between RolA and all SAM-electrodes were highest at pH 4 and lowest at pH 10. The forms of self-assembled structure of RolA depended on the pH values, amounts of adsorbed RolA, and functional groups of SAMs. Rod-like structures of self-assembled RolA were observed on all SAM-electrodes at pH 4. In addition, surface tensions of SAM-electrodes were modified by RolA adsorption. Overall, these results suggest that hydrophobic and electrostatic interactions between RolA molecules adsorbed on SAM-electrodes and soluble RolA molecules in water-phase depend on pH values and mainly contributed to the kinetics of RolA adsorption and self-assembly of RolA molecules on solid surfaces.

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

2) Aimananda V. et al., Nature. **460**:1117 (2009)

3) Martellini F. et al., Mol Biotechnol. 55: 27 (2013)

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

**55W A novel constitutive promoter for engineering *Trichoderma* to control biological contaminants in industrial processes and as a biostimulant in the Phosphite metabolizing Crops Platform.** *N. Carreras-Villaseñor*¹, G. Rico-Ruiz¹, M. Leyva-González¹, A. Herrera-Estrella², D. López-Arredondo¹, L. Herrera-Estrella² 1) StelaGenomics Mexico, Irapuato, Guanajuato, MX; 2) LANGEBIO-UGA Irapuato, Guanajuato, MX.

Members of the genus *Trichoderma* are used in the industry as producer of homologous and heterologous enzymes due to its high secretory capacity. Achieving high expression of heterologous genes requires a strong promoter to drive robust transcription, in this regard, the use of constitutive promoters for expression of recombinant proteins has advantages, such as, the avoidance of the use of inducers and the complex gene regulation. Based in transcriptional data of *T. atroviride* subjected to many growth conditions, we selected the promoter of one of the most expressed gene in any of the tested conditions. This promoter is called Pta1. To evaluate the strength of Pta1 promoter, we used it to express the coding sequence of the *ptxD* gene in *T. atroviride*. PtxD is a bacterial Phosphite dehydrogenase that oxidizes Phosphite (Phi) into Phosphate (Pi). *Trichoderma* transgenic strains using the Pta1 promoter have expression levels of *ptxD* comparable with those observed when *ptxD* was placed under control of Ppki, one of the strongest promoters available for expression of native or foreign proteins in this fungus. *T. atroviride* is unable to metabolize Phi and use it as a sole phosphorus source. By contrast transgenic strains expressing PtxD under control of Pta1 and Ppki promoters could grow in Phi as a sole source of P. Expression of *ptxD* did not alter the native characteristics of *T. atroviride*, as the transgenic strains preserve their abilities as biocontrol agent and plant growth promoting organisms. Therefore, Pta1 is a good candidate as constitutive promoter for the expression of homologous and heterologous proteins with industrial importance. In this poster, to produce proteins or spores in a system that does not require sterilization to prevent biological contaminations and thus significantly reducing the cost of production. We will also report on the progress of using *Trichoderma*-Phi metabolizing strains as a biostimulant and biocontrol agent for crop production on a Phi-based fertilizer system.
**56T Selective cleavage of β-O-4 lignin bond by novel β-etherase of the white-rot fungus *Dichomitus squalens*.** *M. Marinovic*¹, M.R. Mäkelä¹, P. Nousiainen⁴, A. Dilokpimol², R. Moore⁴, J. Sipilä⁴, R.P. de Vries²,³, K. Hildén¹ 1) Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Finland; 2) Fungal Physiology, CBS-KNAW, Utrecht, The Netherlands; 3) Fungal Molecular Physiology, Utrecht University, The Netherlands; 4) Department of Chemistry, Laboratory of Organic Chemistry, University of Helsinki, Finland.

Lignin is a recalcitrant aromatic polymer present in woody plant cell walls. It is depolymerized by classical lignin-modifying oxidoreductases (laccases and class II heme peroxidases) produced by wood decaying fungi, which catalyze non-selective cleavage of different types of bonds present in lignin. Novel enzymes catalyzing aromatic conversions are needed to unlock the potential of lignin as a major renewable source of bulk and fine chemicals.

Glutathione S-transferases (GSTs; E.C. 2.5.1.18) constitute a complex and diverse superfamily of multifunctional enzymes with essential roles in cellular detoxification. These proteins are widespread in animals, plants, bacteria and fungi. Some of the bacterial GSTs possess β-etherase activity that can catalyze selective cleavage of β-O-4 aryl ether linkages present in lignin. However, only few fungal GSTs have been characterized so far, and little is known about these enzymes. In this work, putative GST-encoding genes from the lignocellulose degrading basidiomycete *Dichomitus squalens* were cloned and heterologously expressed in *Escherichia coli*. One of the enzymes that showed a weak amino acid level homology to bacterial β-etherase ligE was shown to cleave β-O-4 bond in α-oxo erol lignin model compound. To our knowledge, this is the first report of eukaryotic β-etherase.

**57F Expanding feruloyl esterase gene family of Aspergillus niger: characterization of a new feruloyl esterase, FaeC.** A. Dilokpimol¹,², M. R. Mäkelä¹,²,³, S. Mansouri³, O. Belova¹,², M. Waterstraat⁴, M Bunzel⁴, R. P. de Vries¹,²,³, K. S. Hildén³ 1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; 2) Fungal Molecular Physiology, Utrecht University, Utrecht, the Netherlands; 3) Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, Finland; 4) Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Germany.

Ferulic acid (FA) and to a lesser extent other hydroxycinnamic acids (e.g. *p*-coumaric acid) are ester-linked to plant cell wall polymers, mainly to (hetero)xylans and pectins, forming polysaccharide-polysaccharide and polysaccharide-lignin cross-links. Phenolic cross-links increase the physical strength and integrity of plant cell walls and reduce their biodegradability by microorganisms. Feruloyl esterases (FAEs) [E.C. 3.1.1.73] are able to release FA and other phenolic acids from natural plant sources and agro-industrial by-products and are therefore widely used in food, feed, pulp-paper, bioethanol and pharmaceutical industries.

Two FAEs from *Aspergillus niger* (FaeA and FaeB) have been previously characterized and are among the best-studied FAEs. Phylogenetic analysis of fungal FAEs revealed numerous FAE candidates, including one from *A. niger* (FaeC).¹,² Recombinantly produced FaeC was most active at pH 7.0 and 50°C and showed broad substrate specificity. The enzyme released both ferulic acid and *p*-coumaric acid from wheat arabinoxylans and sugar beet pectins, and acted synergistically with a commercial xylanase. The expression profile of *faeC* on a set of phenolic compounds differed from those of *faeA* and *faeB*, indicating that these FAE isoenzymes may target different substrates in a complementary manner. FaeC showed promising potential for applications, particularly because of its broad substrate profile and its maximum activity at neutral pH.³

**Acknowledgement**

This work was supported by the European Union, Grant agreement no: 613868 (OPTIBIOCAT).

**Reference**

¹ Benoit, I., Danchin, E.G.J., Bleichrodt, R.J., de Vries R.P. 2008. Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity *Biotechnol Lett.* 30:387-396.

² Dilokpimol, A., Mäkelä, M.R., Aguilar-Pontes, M.V., Benoit-Gelber, I., Hildén, K.S., de Vries R.P. 2016. Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties and industrial applications. *Biotechnol Biofuels.* 9:1-18.

³ Dilokpimol, A., Mäkelä, M.R., Mansouri, S., Belova, O., Waterstraat, M., Bunzel, M., de Vries, R.P., Hildén, K.S. Expanding the feruloyl esterase gene family of *Aspergillus niger* by characterization of a feruloyl esterase, FaeC. *In revision*.

**58W Disruption of the glucose repressor gene cre1 in Trichoderma harzianum CFAM-422 and its effect on plant cell wall degrading enzymes production.** M.S. Tamietti¹, G.E.O. Midorikawa¹, T.D. Mendes¹, M.C.T. Damaso¹, E.P.S. Bon², A.S. Silva³, L.M.F. Gottschalk⁴, E.F. Noronha⁵, L.C.L. Fávaro¹ 1) Embrapa Agroenergia, Brazilian Agricultural Research Corporation (Embrapa), Brasília, DF, BR; 2) Biochemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, BR; 3) Catalysis and Biochemical Processes, National Institute of Technology (INT), Rio de Janeiro, RJ, BR; 4) Food Agroindustry, Brazilian Agricultural Research Corporation (Embrapa), Rio de Janeiro, RJ, BR; 5) Molecular Biology, University of Brasília (UnB), Brasília, DF, BR.

The Carbon Catabolite Repression (CCR) is a mechanism by which saprophytic fungi and bacteria can utilize preferrably highly energetic compounds over compounds of difficult degradation. For *Trichoderma reesei*, the protein that acts as the repressor in the presence of glucose is CRE1. In this project, we aim to disrupt the *cre1* gene in the strain CFAM-T422 of *Trichoderma harzianum*, obtaining mutants with enhanced plant cell wall degrading enzymes production. The disruption of *cre1* in *T. harzianum* CFAM-422 was performed by transforming protoplast cells with a cassette containing a hygromycin B phosphotransferase gene (*hph*) flanked by *cre1* gene sequence, favoring its integration in the fungal genome by homologous recombination. A stability test was performed by successive transferences to nonselective medium (without hygromicin B) for ten generations, the last one being transferred to medium containing hygromicin B (200 µg/mL). Mutants genetically stable and the parental strain were tested for enzyme production in both inducing and repressing conditions with four different carbon sources (carboxymethylcellulose, pectin, starch and xylan). Their enzymatic indexes (e.i.), the ratio between the degradation halo diameter over the colony diameter, were determined. The glucose content for the test under repressing condition was determined as the minimal concentration that triggers CCR in CFAM-T422. The totality of the stable transformants showed increased enzymatic index in all carbon sources tested. For the xylan containing media, which showed the most expressive differences between wild and mutant strains, the i.e. for the parental was 1 and transformants varied from 2,0 to 2,9 regardless of the presence of glucose. These
findings indicate that the *cre1* gene in *T. harzianum* regulates the expression of carbohidrate active enzymes and its deletion can result into mutant strains with enhanced cellulase, hemicellulase and pectinase production with lower glucose inhibition.

**59T Comparing Fungal Capacity for Bioremediation of Human Solid Waste.** Alexander Mehreteab, Victor Manzanilla, *Michael Watters* Dept Biol, Valparaiso Univ, Valparaiso, IN.

*Neurospora crassa* can be used to reduce human solid waste while converting it into fungal biomass which has the potential to be used as a dietary supplement. Typically portrayed as an environmental hazard and source of undesirable odor, solid waste represents an untapped resource. Filamentous fungi are natural decomposers with the ability to use this resource and reduce its environmental impact.

We report here on experiments to determine the conditions which maximize yield of fungal biomass. We compared the effect of the length of incubation and alternative methods of aeration as well as choice of fungal species on fungal growth. Rates of conversion were variable, with Neurospora producing the highest average rate of conversion (grams of dry fungal biomass produced from grams of dry waste in the media) of fungi tested. Additionally fungal growth reduced the characteristic odor of the media. Although this project was initially proposed to address problems inherent to long-term space flight: food storage & waste management, it holds potential benefit in diverse situations including livestock confinement operations.

**60F Genome analysis and gene silencing of the citrus pathogen *Colletotrichum abscissum*.** Eduardo Goulin¹,², Marco Takita¹, Marcos Machado¹ 1) Biotechnology, Centro de Citicultura "Sylvio Moreira", Cordeirópolis, Cordeirópolis, BR; 2) State University of Campinas.

The citrus crop is a worldwide important crop. But, it is constantly affected by several pathogens, and fungi is an economically relevant class of citrus pathogens. New technologies are being applied every year to better understand the pathogens biology, and it can contribute to plant diseases control. The whole genome sequencing become a popular technology making possible and fast many knowledge advances. Therewith, we used NGS to investigate the RNA interference canonical machinery genes in the Post-Bloom Fruit Drop causal agent, *Colletotrichum abscissum*, as well as targets to RNAi silencing. The genome sequencing was performed in Illumina HiSeq platform, followed by de novo assembly using CLC genomics workbench software, and ORFs prediction by Augustus. The Local Blast were used for gene sequences investigation. The genome of *C. abscissum* is well coverage (169 times), which allowed the gene prediction, checked by RNAi machinery proteins search and confirmed by sanger sequencing of Argonaute, Dicer and RdRp. The RNAi functionality was proved after RT-qPCR of several genes and also transforming the fungus with a hairpin construction to induce the gene silencing. The fungus had a previously inserted report gene silenced after transformation attesting the machinery functionality. The genome provided a range of genes that can become a target to RNAi. Therefore, the genome of *Colletotrichum abscissum* was efficiently sequenced, assembly and used to a relevant investigation, providing information about the fungus biology, and also a tool to gene function studies and pathogen control.

**61W Parasitism of *Trichoderma* on closely related fungi (adelphoparasitism) is linked to intensive interfungal DNA exchange.** Irina S. Druzhinina Institute of Chemical Engineering, TU Wien, Vienna, Austria.

The remarkable cellulolytic activity of the initially fungicolous fungus *Trichoderma* (Hypocreales, Pezizomycotina, Ascomycota) arose as a result of unprecedented intensive lateral transfer of genes (LGT) that are required for the degradation of plant biomass. The evolutionary analysis of all lignocellulose degrading carbohydrate active enzymes (IclCAZymes) of *Trichoderma* showed that one half of more than 120 genes was obtained through LGT from herbivore Pezizomycotina fungi (such as Eurotiomycetes, Sordariomycetes incl. Hypocreales, Leotiomycetes and others) but none from other fungi or prokaryotes. Here we review the ecology of *Trichoderma* and present details on ultrastructure of its parasitism on different fungi. We provide evidence that the high frequency of LGT of IclCAZymes is linked to the unique ability of *Trichoderma* to parasitize on genetically closely related fungi that is extended up to adelphoparasitism in its strict sense. The data suggests that the taxonomically and functionally biased genome enrichment of IclCAZome by LGT allowed *Trichoderma* to expand its diet from plesiomorphic fungivory/carnivory up to apomorphic phytosaprotrophy, thus resulting in the emergence of its outstanding ecological versatility referred as environmental opportunism.

The case study on *Trichoderma* allows us to assume that intracellular adelphoparasitism may be a prerequisite for the lateral transfer of genes also in other organisms. Although such parasitic interactions are rare in nature, potential systems in other low eukaryotes will be discussed.

**62T Marker recycling through CRIME.** A.P. Mitchell, M. Huang Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.

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

To use CRIME, we first created marker cassettes flanked by direct repeats. We used one repeat-flanked marker to create a homozygous mutation in our favorite gene, *OFG1*, by CRISPR-Cas9 transformation. Then, in the *ofg1/ofg1* strain, we made a homozygous mutation in a second gene, *OFG2*, by use of a different repeat-flanked marker. In that second transformation, we also included a guide RNA gene to direct cleavage of the marker that lay in the *ofg1* mutant alleles. The idea was that either single-strand annealing or homology-directed repair would then excise the marker we cleaved and leave behind only a copy of the flanking repeats. The idea proved correct: we recovered *ofg1/ofg1 ofg2/ofg2* double mutants from the transformation that had only the marker in the *ofg2* alleles; they had lost both copies of the marker that had been in the *ofg1* alleles. In fact, we have been able to create successive homozygous mutations in three genes by use of two markers, and still ended up with one marker available for further selection in the triple homozygote. That sequence of steps and analyses required 3 weeks total. Our findings illustrate that CRIME pays, at least in this one context.
We are now modifying our approach to enable wild-type allele reconstruction through a process that we call TIME. We feel that this capability will be critical for geneticists of the future, because CRISPR-Cas9 systems can have off-target effects. Our hope is that geneticists of the future will always remember that, if they do the CRIME, they must do the TIME.

**63F A flexible expression platform for Aspergillus based on CRISPR mediated homologous recombination.** F.H. Kirchner, Z.D. Jarczynska, C.D. Nødvig, U.M. Mortensen Danish Technical University, Copenhagen, DK.

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

**64W Protease regulatory factors of Trichoderma reesei can be controlled to improve therapeutic protein production.** Christopher Landowski¹, Ann Westerholm-Parvinen¹, Bernhard Hellg², Juhani Saarinen³, Markku Saloheimo¹ 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.

**65T Identification and demonstration of a single nucleotide substitution with significant impact on specific productivity in Trichoderma reesei.** R. Rabinovich, A. Virag, E. Bodie, Z. Chen, M. Ward DuPont Industrial Biosciences, Palo Alto, CA.

Within our perpetual quest to increase amounts of secreted protein produced by our strains, we chemically mutagenized *Trichoderma reesei* strain RL-P37 and obtained a daughter *T. reesei* strain with improved specific productivity. To find the gene(s) responsible for the improved specific productivity, the genomes of the daughter strain and its parent strain RL-P37 were sequenced, and a large number of intragenic and intergenic mutations were identified. A subset of non-synonymous substitutions were individually introduced into the parent RL-P37 strain and assessed for specific productivity. Introduction of a single nucleotide substitution in a histidine kinase gene (*hkmutant*) into strain RL-P37 brought specific productivity up to levels typical for the daughter strain. Besides increasing specific productivity, introduction of the *hkmutant* mutation caused sensitivity to high osmolarity, which is in line with the function of histidine kinase homologs as osmosensors in other filamentous fungi. Deletion of the native *hkwt* allele in strain RL-P37 significantly reduced specific productivity, indicating that the *hkmutant* allele contains a gain of function mutation. Replacing the mutant *hkmutant* allele with the wild type *hkwt* allele in the daughter strain significantly reduced specific productivity, demonstrating that the mutated *hk* gene is the main gene responsible for the specific productivity improvement in the daughter strain. To demonstrate that we can get the same benefit in other host backgrounds and strains overexpressing proteins of interest, we replaced the native *hkwt* allele with the *hkmutant* allele in a *T. reesei* strain over-expressing Cbh1. The increase in specific productivity that we observed confirmed that we can combine the *hkmutant* allele with other useful host modifications. The use of the discovered *hkmutant* allele, alone or combined with other beneficial mutations, will significantly improve secreted protein production.

**66F Rhodosporidium toruloides: A new platform organism for production of biofuels and bioproducts derived from depolymerized polysaccharides and lignin.** Junko Yaegashi¹,², James Kirby¹,³, Masakazu Ito⁴, Jian Sun¹,⁵, Tanmoy Dutta¹,⁵, Mona Mirsiaghi⁶, Eric R. Sundstrom⁶, Edward Baidoo¹,⁶, Deepti Tanjore⁶, Todd Pray⁶, Kenneth Sale¹,⁵, Seema Singh¹,⁵, Jay D. Keasling¹,³,⁶,⁷,⁸, Blake A. Simmons¹,⁶, Steven W. Singer¹,⁶, Jon K. Magnuson¹,², Adam P. Arkin⁴,⁶,⁸, Jeffrey M. Skerker⁴,⁶, John M. Gladden¹,⁵ 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.

**67W Universal expression system for eukaryotic microorganisms.** *A. Rantasalo, C. Landowski, J. Kuivanen, J. Jäntti, D.*
Mojzita VTT Technical Research Centre of Finland Ltd.

We have developed a novel orthogonal expression system that functions in a wide spectrum of eukaryotic microorganisms. The expression system is based upon a synthetic transcription factor (sTF) that regulates expression of the target gene via a sTF-dependent promoter. The sTF expression is driven by an engineered, universal core promoter that provides a low, but sufficient expression level of the sTF. The sTF expression is constitutive and thus present in the cells in all growth conditions and in all developmental and growth stages. The sTF-dependent promoter regulating the expression of the target gene also contains a similar type of universal core promoter functional in diverse species. This core promoter can be further modified by inserting synthetic repressor (sR) binding sites, through which an additional expression control is gained upon expression of the sR. The combination of multiple sTF-binding sites and the core promoters (including the repressible versions) enables specific adjustment over a wide range of target gene expression levels, from very low to very high. This expression system provides robust, stable, and tuneable expression levels of the target genes in a broad spectrum of host organisms. Further it simplifies the genetic tools needed for the construction of new protein expression hosts, including those with undeveloped know-how. The method for selecting the universal core promoters, constructions of the expression systems, and demonstrations of their performance in diverse hosts, including yeasts and filamentous fungi, will be presented.

**68T Transcriptome analysis of two unrelated fungal β-lactam producers *Acremonium chrysogenum* and *Penicillium chrysogenum*: Velvet-regulated genes are major targets during conventional strain improvement programs.** *D. Terfehr, T.A.*
Dahlmann, U. Kück Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE.

*Acremonium chrysogenum* and *Penicillium chrysogenum* are the industrial producers of the β-lactam antibiotics cephalosporin C and penicillin G, which are used worldwide for the treatment of bacterial infections in human and veterinary medicine. Beside the ability to generate anti-infectives, both species do not have much in common and are members of distinct taxonomic groups within the Ascomycota. Previously, wild-type isolates from *A. chrysogenum* ATCC11550 and *P. chrysogenum* NRRL1951 were randomly mutated during several rounds of conventional mutagenesis to reach economically relevant titers of the corresponding β-lactam antibiotics. Although, previous research has shed light on the biosynthesis pathway as well as parts of the regulatory processes involved in β-lactam production, there is still a lack of knowledge how the increased β-lactam production was achieved during conventional strain improvement programs. Here, we performed RNA-seq analysis of the two wild-type strains and industrial strains of both species (*A. chrysogenum* A3/2 and *P. chrysogenum* P2niaD18) to address the question whether both fungi have undergone similar expressional changes during strain improvement. Furthermore, our analysis includes the investigation of two mutants (ΔAckeA and ΔPcvelA) that lack Velvet, a global regulator of fungal secondary metabolism. Within the set of differential expressed genes during strain improvement, both fungi show an intersection of genes associated directly or indirectly to industrial β-lactam production. Furthermore, we will demonstrate that genes assigned to specific functional categories are also regulated by Velvet. Interestingly, Velvet regulated genes seem to be one of the primary targets of classical strain improvements programs in both fungi. This assigns Velvet an important role in strain improvement of industrial β-lactam producers, although components of the Velvet complex might be different in both fungi. Our results will contribute to develop alternative strategies for strain improvements of these β-lactam antibiotic producers.

**69F Cycloaspeptides: elucidation of a novel fungal cyclic peptide biosynthetic pathway.** *Kate M. J. de Mattos-Shipley*¹, *Claudio Greco*¹, *David Heard*¹, *Nicholas P. Mulholland*², *Jason L. Vincent*², *Thomas J. Simpson*¹, *Christine L. Willis*¹, *Russell J. Cox*¹,³,⁴, *Andrew M. Bailey*⁵
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.

**70W Resistance Gene-Guided Genome Mining: Serial Promoter Exchanges in Aspergillus nidulans Reveal the Biosynthetic Pathway for Fellutamide B, a Proteasome Inhibitor.** *Hsu-Hua Yeh*¹, *Manmeet Ahuja*², *Yi-Ming Chiang*²,³, *C. Elizabeth Oakley*², *Shauna Moore*², *Olivia Yoon*², *Heather Hajovsky*², *Jin-Woo Bok*⁴, *Nancy P. Keller*⁴, *Clay C. C. Wang*¹,⁵, *Berl R. Oakley*²
1) Department of
Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, United States; 2) Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States; 3) Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan City 71710, Taiwan; 4) Department of Bacteriology and Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706, United States; 5) Department of Chemistry, University of Southern California, Dornsiehe College of Letters, Arts, and Sciences, Los Angeles, California 90089, United States.

Fungal genome projects are revealing thousands
of cryptic secondary metabolism (SM) biosynthetic gene
clusters that encode pathways that potentially produce valuable compounds. Heterologous expression systems should allow these clusters
to be expressed and their products obtained, but approaches are needed to identify the most valuable target clusters. The inp cluster of
Aspergillus nidulans contains a gene, inpE, that encodes a proteasome subunit, leading us to hypothesize that the inp cluster produces a
proteasome inhibitor and inpE confers resistance to this compound. Previous efforts to express this cluster have failed, but by sequentially
replacing the promoters of the genes of the cluster with a regulatable promotor, we have expressed them successfully. Expression reveals
that the product of the inp cluster is the proteasome inhibitor fellutamide B, and our data allow us to propose a biosynthetic pathway for the
compound. By deleting inpE and activating expression of the inp cluster, we demonstrate that inpE is required for resistance to internally
produced fellutamide B. These data provide experimental validation for the hypothesis that some fungal SM clusters contain genes that
encode resistant forms of the enzymes targeted by the compound produced by the cluster.

**71T The chromatin remodeler suberoyl bis-hydroxamic acid (SBHA) induces changes in the pattern of metabolites produced by Acremonium chrysogenum.** A. Barron Gutierrez¹, W. Perez Perez¹, J. Mendoza-Espinoza²,³, F. Fierro Fierro¹, F. Fernandez Perrino¹ 1) Departamento de Biotecnologia, Universidad Autonoma Metropolitana - Iztapalapa, Mexico city, Mexico city, MX; 2) Departamento de Ciencias de la Salud, Universidad Autonoma Metropolitana - Iztapalapa, Mexico city, Mexico city, MX; 3) Universidad Autónoma de la Ciudad de México, Mexico city, Mexico city, MX.

**Introduction.** Many new fungal metabolites are being discovered by genome mining approaches. One strategy to find new secondary metabolites is the use of chemical chromatin remodelers, which would result in the activation of silenced gene clusters and the production of the "cryptic" metabolites. One of these chromatin remodelers is the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA). Inhibition of histone deacetylase activity would maintain an acetylated state of histones, which is associated with a less condensed chromatin state and higher transcriptional activity.

*Acremonium chrysogenum* is used in the industry to produce the antibiotic cephalosporin, the sequencing of its genome revealed the presence of 42 biosynthetic clusters for secondary metabolites. The objective of this work was to study the changes in the chemical pattern, as observed by HPLC, of the metabolites produced by *Acremonium chrysogenum* in cultures added with SBHA.

**Methodology.** The wild type strain of *A. chrysogenum*, ATCC 11550, was used in this study. To obtain the Ultraviolet-Visible (UV) spectra, 10 mL of the extract were taken and read in a GenWay spectrometer. HPLC was performed with a CLAR equipment (Agilent 1260), using an elution gradient of water-acetonitrile on a C-18 column.

**Results.** The UV absorption spectra showed that the absorption values were between 200 and 480 nm. These spectra are considered as a chemical fingerprint of the extract and allow to fix the sweep of wavelengths for the HPLC analysis. CLAR analysis at 215 nm revealed changes in the chemical profile related to the time of culture and concentration of SBHA.

**Conclusions.** The use of suberoyl bis-hydroxamic acid (SBHA) revealed the feasibility to elicit the production of "cryptic" secondary metabolites in *A. chrysogenum*, and can be considered as a genome mining strategy applicable to this fungus.

**72F Aspergillus niger versus Aspergillus oryzae: Expression platforms for heterologous secondary metabolite production.** E. Geib, M. Brock Fungal Genetics and Biology, University of Nottingham, Nottingham, GB.

Filamentous fungi are treasure chests for novel secondary metabolites and genome mining has uncovered a multitude of yet unexplored secondary metabolite biosynthesis gene clusters. Their identification and characterisation is crucial for the development of drugs that combat various kinds of diseases. Unfortunately, most of these gene clusters appear silent under laboratory cultivation conditions, which requires heterologous gene expression in well-characterised expression systems. Previously, we developed such an expression system in *Aspergillus niger*, which bases on regulatory elements from the *Aspergillus terreus* terrein biosynthetic gene cluster. We used this platform to produce polyketides (e.g. lecanoric acid), non-ribosomal peptide synthetase-like products (e.g. aspulvinone E) and reconstituted the Asp-melanin biosynthesis pathway from *A. terreus*.

The latter studies led to an interest in understanding the chemistry of NRPS-like enzymes that produce metabolites with antifungal, cytotoxic, antitumorigenic and antiviral activity. Enzymes of this class may accept the same substrate, but form different products depending on their thioesterase domain. To study these domains, we compared the aspulvinone E synthetase MeIA from *A. terreus* with the atromentin synthetase InvA5 from *Paxillus involutus*. While recombinant expression of *melA* in *A. niger* resulted in aspulvinone E production, expression of *invA5* led to a range of yet unknown products, but failed to produce atromentin. In contrast, recombinant and purified InvA5 produced atromentin *in vitro*. We therefore speculated that the physiology of *A. niger* might lead to a modification of the InvA5-derived metabolite. Consequently, the expression system was transferred to the alternative host *Aspergillus oryzae*. Indeed, *A. oryzae* produced aspulvinone E from MeIA and, even more, atromentin from InvA5.

In conclusion, our recombinant expression system is perfectly suited for heterologous production of secondary metabolites. However, the metabolic physiology of *A. niger* and *A. oryzae* differs and at least two different expression platforms should be selected when aiming in the characterisation of novel secondary metabolite biosynthesis genes.

**73W Development of a T. reesei "Sexual reproduction toolkit" enabling BSA-seq technology and industrial strain improvement.** L. Chan Ho Tong¹, D. Naquin², P. Silar³, A. Margeot¹, F. Bidard¹ 1) IFP Energies nouvelles, Biotechnology Department, France; 2) Institute for Integrative Biology of the Cell, High throughput sequencing platform, France; 3) Paris-Diderot University, Paris Interdisciplinary Energy Research Institute, France.

The *Sordariomycetes Trichoderma reesei* is a mesophilic filamentous fungus currently used to produce enzymes hydrolysing lignocellulosic biomass to glucose to be subsequently fermented to ethanol.
*T. reesei*, originally described from a single wild type isolate called QM6a, was believed to be devoid of a sexual cycle whereas its teleomorph, *Hypocrea jecorina*, undergoes an heterothallic sexual cycle involving MAT1-1 and MAT1-2 loci. However, after identification of a MAT1-2 loci in QM6a, a sexual reproduction has been obtained successfully with a MAT1-1 natural isolate. Further experiments have shown that QM6a and its derivatives are female-sterile but male-fertile.

Sexual reproduction in industrial strains is of a great economical and fundamental importance, as it makes a valuable genetic tool. In order to apply sexual reproduction as a tool in *T. reesei*, we developed a toolkit : ascospores isolation and conservation, optimization of nutritional and environmental factors to get a precise control of stromata production, characterization and sequencing of a wild type MAT1-1 strain sexually compatible with QM6a and its derivatives. This toolkit enables exploration of new genetic strategies.

Two examples of applications have been implemented and are described here. First, we successfully carried out a Bulk Segregant Analysis associated with high-throughput sequencing (BSA-seq) to identify the known causative mutation of the cellulase negative QM9136 strain. Secondly, we generated genetic diversity by crossing the RutC30 industrial strain with A2 wild type isolate. Improved progenies in cellulase production were obtained.

Materials and methods developed in our toolkit are available for the *T. reesei* community.

**74T Functional dynamics of wood-decay through genome-wide analysis of the white rot Polyporales species Phlebia radiata.**

*Mari Mäkinen*¹, *Jaana Kuuskeri*¹, *Netta Risulainen*¹, *Pia Laine*², *Olli-Pekka Smolander*², *Lars Paulin*², *Markku Varjosalo*³, *Petri Auvinen*², *Taina Lundell*¹

1) Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki; 2) DNA Sequencing and Genomics Laboratory, Institute of Biotechnology, University of Helsinki; 3) Proteomics Unit, Institute of Biotechnology, University of Helsinki.

White rot Agaricomycetes of Basidiomycota secrete carbohydrate-active enzymes and auxiliary oxidoreductases to decompose wood lignocellulose. With genome sequencing projects and accumulating omics data, fungal metabolism and degradation processes are under intensive research. *P. radiata* is an ecologically important fungus able to degrade efficiently the main components of plant cell wall, presenting high applicability in biotechnological processes, and has a systematic importance as type species of phlebioid clade of Polyporales (1). The aim of the study was to identify main activities and proteins for wood-decay in *P. radiata* cultivated on Norway spruce and to study gene expression and enzyme activities in the presence of lignocellulosic waste material. Next-generation sequencing of the isolate 79 resulted first in construction of the mitogenome (2), then assembling and gene annotating the nuclear genome (3).

Time-scale analysis of transcriptome by RNA-seq and proteomics analysis upon a six weeks' cultivation period on spruce wood presented a set of genes that were up-regulated during the fungal colonization of wood and expressed as proteins (3). According to the proteome analyses, especially the lignin-modifying class-II peroxidases together with glyoxal and alcohol oxidases were abundantly produced on wood. Dynamic changes e.g. enhanced production of LiPs and MnPs on week 2 of growth on wood were detected. Comparative analysis of transcriptome and proteome data showed that majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant-cell wall and included the main activities of white rot decay. Our study indicates an initial strong oxidative attack mainly against lignin then followed by longer-term expression of hydrolytic CAZymes. Among others, the wood transcriptome revealed expression of AA9 LPMOs thus indicating their importance in oxidative cleavage of carbohydrate polymers.

(1) Kuuskeri J (2015) BMC Microbiol, 15:217

(2) Salavirta H (2014) PLoS ONE, 9(5): e97141

(3) Kuuskeri J (2016) Biotech Biofuels, 9:192

**75F Overexpression of a C₄-dicarboxylate transporter is the key for converting citric acid production to C₄-dicarboxylic acid production in Aspergillus carbonarius.**

Lei Yang, Eleni Christakou, Jesper Vang, Mette Luebeck, *Peter Luebeck* Chemistry and Bioscience, Aalborg University, Aalborg, DK.

C₄-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. *Aspergillus carbonarius* is capable of producing high amounts of citric acid, however, all attempts to change the citric acid production into C₄-dicarboxylic production by pathway engineering have been with very limited success. In this study, a glucose oxidase deficient strain of *A. carbonarius* was used as the parental strain to overexpress a native C₄-dicarboxylate transporter and the *frd* gene encoding fumarate reductase from *Trypanosoma brucei* individually and in combination to investigate their impacts on organic acid production. Overexpression of the C₄-dicarboxylate transporter alone and in combination with the *frd* gene significantly increased the production of C₄-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the *frd* gene alone did not result in any significant change of the organic acid production profile.

This study demonstrates that the key to change the citric acid production into production of C₄-dicarboxylic acids in *A. carbonarius* is the C₄-dicarboxylate transporter. Furthermore it shows that the C₄-dicarboxylic acid production in *A. carbonarius* can be further increased via metabolic engineering.

**76W Engineering the smut fungus *Ustilago maydis* for the degradation of pectin.**

P. Stoffels¹,³, E. Geiser²,³, L.M. Blank²,³, M. Feldbrügge¹,³, N. Wierckx²,³, K. Schipper¹,³

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

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

**77T Upscaling of cellulase enzyme production using Thermoascus aurantiacus.** Timo Schuerg¹,², Raphael Gabriel¹,², Jan-Philip Prahl¹,², Simon Harth¹,², Ziyu Dai³, Beth Hofstad³, Jon Magnuson³, Steven Singer¹,² 1) Lawrence Berkeley National Laboratory, Berkeley, CA; 2) Joint BioEnergy Institute (JBEI), Emeryville, CA; 3) Pacific Northwest National Laboratory, Richland, WA.

Thermophilic filamentous fungi are a valuable source of thermostable cellulases. *Thermoascus aurantiacus*, often isolated from self-heating compost piles, is a true thermophile with a growth optimum at 50°C. Here we report successful scale-up of enzyme production by *T. aurantiacus* to 20 L scale. We demonstrated that MXP (Methyl beta-D-xylopyranoside), a non-metabolizable xylose analog, strongly induces cellulases in batch cultivations, whereas metabolizable xylose is much less potent. Based on our observations we hypothesized that carbon catabolite repression (CCR) might be responsible for the inhibited induction of xylose. To test our hypothesis, we established an easily deployable fed-batch system for the 50 mL shake flask scale, which allowed us to circumvent CCR by feeding only small amounts of xylose over time. When continuously feeding only 78 mg xylose h⁻¹ L⁻¹, a 5x increase in cellulase induction was achieved as compared to feeding the same amount of xylose in one pulse. The system proved to be successful in optimizing fed-batch cultivation conditions at 50 mL scale and was successfully transferred to 2 L and 20 L pilot scales with crude enzyme titers ranging from 1.1 - 3.2 g/L. In parallel, we established a transformation system and successfully engineered a *Dku70* strain, which will facilitate strain engineering strategies based on homology directed repair. Current efforts focus on genetically disrupting CCR, overexpressing the two major activators of cellulase genes, *clr1* and *clr2*, and disrupting unfavorable protease activity. Furthermore, we established procedures for classical mutagenesis and screening and developed a system for obtaining recombinant progeny from sexual crosses. Establishing CRISPR/Cas9 genome editing technology is ongoing. In summary, *T. aurantiacus* has great potential to become an attractive production platform for thermostable cellulases.

**78F Expression platforms impact the surface modulating properties of Trichoderma class II hydrophobins.** Agnes Przylucka¹,², *Gunseli Bayram Akcapınar*¹,⁷, Klaus Bonazza³,⁸, Thiago M. Mello-de-Sousa⁴, Astrid R. Mach-Aigner⁴, Erik Reimhult⁵, Hinrich Grothe⁶, Christian P. Kubicek¹,²,⁹, Irina S. Druzhinina¹,² 1) Microbiology Group, Research Area Biochemical Technology, Institute of Chemical Engineering, TU Wien, Vienna, AT; 2) ACIB GmbH, c/o Institute of Chemical Engineering, TU Wien, Vienna, AT; 3) *klaus.bonazza@tuwien.ac.at*, TU Wien, Vienna, AT; 4) Synthetic Biology and Gene Technology Group, Research Area Biochemical Technology, Institute of Chemical Engineering, TU Wien, Vienna, AT; 5) Institute for Biologically Inspired Materials, Department of Nanobiotechnology, University of Natural Resources and Life Science, Vienna, AT; 6) Institute of Materials Chemistry, TU Wien, Vienna, AT; 7) present address: Department of Statistics and Medical Informatics, School of Medicine, Acibadem University, Istanbul, TR; 8) present address: Program in Cellular and Molecular Medicine and Division of Hematology, Department of Medicine, Boston Children's Hospital, and Department of Biological Chemistry and Pharmacology, Harvard Medical School, 3 Blackfan Circle, 02215 Boston, M; 9) present address: Steinschötelgasse 7, 1100 Vienna, AT.

Hydrophobins are small proteins secreted by filamentous fungi that are characterized by eight conserved cysteine residues forming four intramolecular disulfide bonds; they show an extremely high surface activity and spontaneous formation of amphipathic layers that convert the surface from hydrophilic to hydrophobic and vice versa. These remarkable properties have consequently been investigated for many industrial and medical applications. Despite this interest, few studies have been conducted towards production of hydrophobins. In this paper, we have compared the heterologous production of two class II hydrophobins from *Trichoderma virens* (HFB4 and HFB7) in two canonical hosts, *Escherichia coli* and *Pichia pastoris*. While the production in both systems was rather similar (0.25 – 0.4 g/L), the ability to bind to hydrophilic and hydrophobic surfaces differed depending on the host used. Structural analysis of the produced proteins in solution by circular dichroism and dynamic light scattering, as well as analysis of surface binding by atomic force spectroscopy and quartz crystal microbalance with dissipation analysis revealed significant differences between the proteins produced in *E. coli* and *P. pastoris*. Our data show that the expression platform significantly influences the structure of hydrophobins, their surface modulating properties and behaviour in solutions; however, the impact is also protein-specific. This effect should be considered when hydrophobins are produced for particular applications.

**79W Lichen symbiosis between Caloplaca flavorubescens and Trebouxia gelatinosa.** S.Y. Park¹, J.A. Kim¹, M.H. Jeong¹, C. H. Park¹, J. J², Y.H. Lee², *J.S. Hur*¹ 1) Korean Lichen Research Institute, Sunchon National University, Suncheon, KR; 2) Dept. of Agricultural Biotechnology, Fungal Bioinformatics Laboratory, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Korea.

Lichens are symbiotic organisms, composed of a fungal partner (the mycobiont) and at least one eukaryotic algal or cyanobacterial species (the photobiont). As demonstrated by the world-wide distribution of lichens in various kinds of habitats from the tropics to the Polar region, lichen symbiosis seems to be a highly successful adaptation to a diverse range of environmental conditions. For establishment of successful lichen symbiosis, it requires a mycobiont to associate with an appropriate free-living photobiont or arrest lichenized photobiont cells. In order to understand the symbiosis, a total of five lichen-forming fungal isolates and their partner algal isolates were selected to analyze in ultrastructural and molecular levels using our developed resynthesis method. A typical secondary metabolites of *Caloplaca flavorubescens* were produced and their morphological developments showed soredia-like clusters after 4 to 6 weeks co-culturing combination in *C. flavorubescens*. Thus, we monitored the lichenization processes by light microscopes, scanning electron microscope, and transmission electron microscope. RNA sequencing analysis was used to investigate gene expression profiles during resynthesis of *C. flavorubescens*. We then analyzed expression patterns of effector-type small secreted proteins (SSPs). We reveal that the six most highly
expressed SSPs with unknown function are consistently expressed during resynthesis. Using antibody of the six SSPs, we provide evidence for the six genes involved in resynthesis of *C. flavorubescens* with their partner alga. This finding will enhance our understanding of the adaptive evolution of the lichen-forming fungi with the algae to their ecological niches.

**80T Growing *Trichoderma reesei* on a mix of carbon sources reveals links between development and cellulase production. A. Pirayre, D. Ivanoff, E. Jourdier, A. Margeot, L. Duval, Frederique Bidard** Biotechnology, IFP Energies nouvelles, Rueil-Malmaison, FR.

The degradation of cellulose and hemi-cellulose molecules into simpler sugars such as glucose takes part of the biofuel production process. Hydrolysis of lignocellulosic substrates is naturally performed by enzymes produced and secreted e.g. by fungus *Trichoderma reesei*. For decades, numerous strains of *T. reesei* have been developed in order to improve its enzyme production level. Here, we focus on the hyper-producer strain RutC30, which notably differs from the wild type QM6a by three rounds of mutagenesis leading to many genetics alterations (SNV, chromosomal translocations, deletions). It is important to note that one of the key mutations is the truncation of Cre1, which is responsible of the carbon catabolite repression.

In this work, we studied the impact of the carbon source on the cellulase production. For this purpose, different mixtures of glucose and lactose were used as a substrate for the cellulase production phase in fed-batch mode (after an initial growth phase on glucose in batch mode), while keeping the global amount of carbon source constant. RNA extraction was performed 24h and 48h after production start, and a transcriptomic analysis was carried out. We found 650 genes differentially expressed in at least one of the studied condition, where the 100% glucose was taken as reference. A clustering (K-means algorithm) allows us to separate the 650 genes into 5 classes. In addition, the gene regulatory network (GRN) built using the BRANE Cut software reveals three kinds of sub-networks linked to i) cellulase mechanism, ii) beta-glucosidase mechanism and iii) development. These results improve our understanding regarding the regulation of cellulase production in *Trichoderma reesei*.

**Cell Biology and Development**

**81F Shrink, sputter and fade away: How azoles kill *Aspergillus fumigatus*.** Bernadette Geißel¹, Laura Sturm¹, Zhaojun Zhu¹, Cees A. M. J. J. van den Hondel², Johannes Wagener¹ 1) Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Munich, Germany; 2) Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, Netherlands.

*Aspergillus fumigatus* is an airborne opportunistic pathogen. In immunocompromised patients this mold causes severe systemic infections, a disease that is called invasive aspergillosis. Azole antifungals such as voriconazole are currently recommended as first-line treatment of invasive aspergillosis. This drug class exerts a fungicidal activity on *Aspergillus* species. In *Candida* species, however, azoles are primarily fungistatic. Azoles disrupt the ergosterol biosynthesis pathway by inhibition of the lanosterol 14-α-demethylase. While this mechanism is well characterized, the exact physiological consequences remain largely unknown. In order to understand the increased azole resistance of mitochondrial dynamics mutants (Neubauer *et al.*, Mol Microbiol. 2015 Dec;98(5):930-45) we studied the effect of the azole voriconazole on the fungal physiology of *A. fumigatus*. By exploiting new reporter systems we identified different variants of fungal death caused by the exposure to voriconazole. In addition, we observed an irregular deposition of cell wall polymers which indicates a defect in cell wall biogenesis and could contribute to the fungicidal activity. All the death variants are ultimately linked to the specific inhibition of the lanosterol 14-α-demethylase because conditional downregulation of this enzyme invokes very similar phenotypes. Furthermore, our results indicate that the increased azole resistance of the mitochondrial dynamics mutants is probably not directly linked to dysfunctional mitochondrial fission or fusion processes. In parallel, we performed an RNA-Seq analysis of the mitochondrial dynamics mutant transcriptome. Our results indicated the upregulation of various efflux pumps (approx. 8 ABC and 18 MFS transporters) as well as differential regulation of multiple transcription factors and thereby suggest the activation of a multidrug resistance network. To further dissect this possible network we constructed a number of conditional ABC transporter and transcription factor mutants to characterize their roles in drug resistance. We could identify several genes whose expression influences the azole resistance of *A. fumigatus*.

**82W An oxylipin signal mediates hyphal branching in pathogenic Aspergilli. M. Niu¹, G. Fischer¹, N. Keller¹,²** 1) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA; 2) Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.

Oxylipins are a group of diverse oxygenated polyunsaturated fatty acids found in all eukaryotes that modulate growth, development, and cellular communication. Three oxylipin generating oxygenases, PpoA (Psi producing oxygenase A), PpoB, and PpoC, mediate development and stress responses in pathogenic *Aspergillus* species. However, the cellular targets of these oxylipin metabolites, their cellular functions, and signal transduction pathway(s) transmitting the signal are yet to be investigated. Our laboratory has recently identified that exogenous treatment of 5(S),8(R)-dihydroxide octadecadienoic acid (5,8-diHODE), the final oxylipin product of PpoA, resulted in stunted apical growth, increased lateral growth or hyper-branching, and decreased septal distance in the human fungal pathogen *A. fumigatus* and plant pathogen *A. flavus*. Our results suggested that the observed hyperbranching phenotype is specific to C18 diol-oxylipin acids with specific structural features yet to be identified. In addition, the branching phenotype by 5,8-diHODE treatment was remediated by high amount of Ca²⁺ to the wildtype level, suggesting that Ca²⁺ is involved in oxylipin signal transduction in the pathogenic Aspergilli species.

**83T Understanding cell tropisms in *Aspergillus fumigatus* hyphae. P. Geranios¹, N. Al-Furaji¹, K. Lord², M. Bromley¹, N. Read¹** 1) Manchester Fungal Infection Group, University of Manchester, Manchester, GB; 2) University of Edinburgh, Edinburgh, GB.

Fungal cell tropisms involve directional changes of growing fungal cells or hyphae in response to an external stimulus. Negative cell tropisms are ubiquitous in filamentous fungi. Two clear examples are: (1) avoidance of adjacent germ tubes during colony initiation; and (2) avoidance of adjacent vegetative hyphae and branches at the colony periphery. Both tropisms have been proposed to be important in reducing the competition of neighbouring germ tubes/hyphae for nutrients. Despite the widespread occurrence of negative tropisms in fungi, little is known about the signalling processes governing the phenomenon. The aim of our work is to understand the mechanistic basis of negative cell tropism (self-avoidance) during spore germination and hyphal growth in the human pathogen Aspergillus fumigatus. To this
end, confocal live-cell imaging and quantitative image analysis was employed to image and measure the angles formed between germlings when visualized in both 2D and 3D as the conidial germlings invade the agar substrata of different hardnesses. The angles formed between germlings were found to be dependent on the number of germlings in a given group. Our time-lapse imaging data indicated that re- arrangement of the growth axis occurs almost immediately when hyphae approach each other. Furthermore, the avoiding hyphae also exhibited a propensity to invade their agar substratum and this was influenced by its hardness. Mutants blocked in secondary metabolite synthesis or in the presence of a NO scavenger or CO2 indicated that the avoidance signal was probably not a secondary metabolite or NO. A kinase knockout library of 96 mutant strains of A. fumigatus has been screened with the aim of identifying strains defective in hyphal self- avoidance. The initial screening has involved classifying the mutants into three categories based on their growth phenotype: i) no obvious effect on polarized growth, ii) possibly defective in self-avoidance, and iii) major growth defects. During the kinase mutant screen we have found out that the best way to visualise negative tropisms in a screen like this is to image leading hyphae at the periphery of approaching colonies.

The chemical signal responsible for these negative tropisms has not yet been identified but is suspected to be likely a volatile because it occurs on cellophane overlying growth medium. Currently, the volatiles released by the developing colony are being identified and analysed.

**84F Components of the Aspergillus fumigatus Ras Post-translational modification pathway regulate conidial viability, hyphal growth, and virulence.** Q. Al Abdallah, TA Norton, JR Fortwendel Department of Clinical Pharmacy, UTHSC, Memphis, TN.

The Ras superfamily is comprised of highly conserved, membrane-associated GTPase proteins and activate several downstream signaling cascades. Localization of Ras proteins to the plasma membrane (PM) is an essential step for fungal growth and pathogenesis. In order to be properly localized, Ras proteins must undergo multiple steps of post-translational modifications (PTMs) at the conserved C-terminal CAAX box. In yeast and mammalian cells, these steps include farnesylation, followed by protease-mediated cleavage of the -AAX amino acids, carboxymethylation, and palmitoylation. Our *in silico* analysis suggests that this pathway is conserved in *Aspergillus fumigatus*. Additionally, our previous work shows that palmitoylation of *A. fumigatus* RasA is required for PM localization and for full virulence. The purpose of this study is to investigate the roles of the sole farnesyltransferase complex, as well as the CAAX protease and the methyltransferase in the growth of *A. fumigatus*. To achieve this, we deleted the genes that encode for RasA PTM homologues in *A. fumigatus* and characterized the deletion mutants for their growth rates, RasA localization and activation, and fungal virulence. Conidial germination, overall growth rate, and virulence were all reduced in the farnesylation-deficient mutant, although hyphal morphology appeared normal. Conidial viability was also decreased in the farnesylation mutant and was associated with formation of anuclear conidia. As expected, loss of farnesylation led to decreased RasA PM association. Surprisingly, fungal growth and development was not affected by either single or double deletion of the enzymes contributing to CAAX proteolysis. In addition, RasA localization to the PM and steady-state activation of RasA in the double mutant was unperturbed. However, loss of one of the CAAX proteolysis homologs, *rce1*, is associated with increased sensitivity to cell wall disruption. Together, our results suggest that CAAX proteolysis is not an essential step for fungal growth or regulation of RasA in *A. fumigatus*, whereas farnesylation contributes heavily to these processes.

**85W A palmitoylated protein ArkA and its putative targeted proteins in Aspergillus.** Yuanwei Zhang¹,², Congcong Sun¹, Ling Lu¹ 1) Nanjing Normal University, Nanjing, China; 2) University of Dundee, Dundee, GB.

Finely tuned changes in cytosolic free calcium ([Ca²⁺]ᵣ) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation involves the reversible posttranslational modification that addition of palmitate to cysteines and promotes protein binding and subcellular localization, yet the relationship between palmitoylation and calcium signaling is poorly understood. Here, we present proof that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in *Aspergillus nidulans*, regulates [Ca²⁺]ᵣ homeostasis and plays an important role in hyphal growth and conidiation under low calcium conditions. We utilized calcium reporter aequorin expression system and showed that the [Ca²⁺]ᵣ responses in *akrA* mutants to high extracellular calcium or ER/plasma membrane stress were defective. Furthermore, we demonstrated that all of these effects on the [Ca²⁺]ᵣ responses mediated by AkrA were tightly related to the cysteine of the DHHC motif which is required for AkrA auto-palmitoylation. When auto-palmitoylation of AkrA was inhibited, by either mutation of cysteine or truncated the DHHC motif, the susceptibility of the mutants to azole drugs was increased. By acyl-biotin exchange assay, we identified potential substrates palmitoylated by AkrA including calcium-related proteins and ergosterol biosynthetic proteins which involved in azole drug resistance in *A. nidulans*. In addition, deletion of AkrA homolog in *A. fumigatus* displays a similar phenotype of the colony growth defect to that in *A. nidulans*, indicating function of AkrA in *Aspergillus* or in fungi might be conserved. Thus, this study provides new insights into the relationship among palmitoylation, calcium homeostasis and azole resistance, which has implications for the development of novel antifungal drugs and therapeutic methods. Further details about the cellular function for putative proteins targeted by AkrA in *A. fumigatus* are ongoing.

**86T Comparative analysis of the function of α-1,3-glucan synthases, AgsA and AgsB, in Aspergillus nidulans.** K. Miyazawa¹, A. Yoshimi², S. Yano³, S. Kasahara⁴, F. Hasegawa², K. Abe¹,² 1) Grad. Sch. Agric. Sci., Tohoku Univ., Sendai, JP; 2) NICHe, Tohoku Univ., Sendai, JP; 3) Yamagata Univ., Yonezawa, JP; 4) Miyagi Univ., Sendai, JP.

Although α-1,3-glucan (AG) is one of major polysaccharides in the cell wall of *Aspergillus* species, the biological function of AG remains unclear, except for the role as a virulence factor in some pathogenic fungi. Previously, we carried out functional analysis of two α-1,3-glucan synthase (AGS) genes (i.e. *agsA* and *agsB*) in the model filamentous fungus *Aspergillus nidulans*. The *agsB*? strain lost most cell wall AG, suggesting that a main AGS in this fungus is AgsB. Interestingly, the hyphae of the *agsB*? strain was dispersed under liquid culture conditions, whereas the wild-type strain formed hyphal pellets under same culture conditions. These results suggest that AG has the role as an adhesive factor for hyphal cells. On the other hand, because the expression of *agsA* gene was scarcely detected under normal growth conditions and the *agsA*? strain did not show phenotypic defects, the role of *agsA* remains unclear. In this study, in order to investigate the roles of AgsA and AgsB in cell wall AG synthesis, we comparatively analyzed cell wall polysaccharides synthesized by AgsA and AgsB. First, we constructed *agsA* or *agsB* gene overexpression (O/E) strain by replacing the promoter region of *agsA* or *agsB* with *tet1* promoter under the genetic background of the other AGS gene disruption, and confirmed the high expression of either of the AGS genes in the O/E strains. The O/E of *agsA* restored the growth characteristics of the *agsB*? strain under liquid culture conditions: the O/E *agsA* strain formed the hyphal pellets. This suggests that the *agsA* gene encodes a functional AGS. To elucidate the differences of cell wall structure between
these two strains, we performed the alkaline-fractionation of cell wall and analyzed the sugar composition of the fractions. The carbohydrate analyses revealed that the sugar compositions of the AS2 fraction were similar in these two strains, but the texture of the AS2 fraction derived from these two strains was markedly different from each other, suggesting that the detailed chemical structure of AG obtained from the O/E *agsA* differs from that of AG derived from the O/E *agsB* strains.

**87F 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 Δ*aspD* 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*.

**88W ZtfA is a novel regulator of asexual development, secondary metabolism and oxidative stress defense in Aspergillus nidulans.** K.G. Thieme, J. Gerke, C. Sasse, S. Thieme, O. Valerius, G.H. Braus Institute of Microbiology and Genetics, Georg-August University, Göttingen, DE.

In the filamentous fungus *Aspergillus nidulans*, asexual and sexual development are tightly interconnected with secondary metabolism, orchestrated by the velvet protein transcriptional network. Here, we analyze the new Zn(II)₂-Cys₆ transcription factor ZtfA, which regulates asexual development, secondary metabolite production and oxidative stress response downstream of the velvet factor VosA. A *ztfA* deletion strain produces drastically diminished numbers of conidiospores, whereas a *ztfA* overexpression strain forms conidiophores even in vegetative growth, where normally only vegetative mycelia is formed. Moreover, conidiospores show a rapid loss in viability in ?*ztfA*. Genetic analyses indicate that ZtfA activates the conidiation pathway via the major regulator encoding *brlA*. In the absence of *ztfA* the fungus is not able to produce the secondary metabolites austinol and dehydroaustinol, whereas the biosynthesis of sterigmatocystin in ?*ztfA* as well as in the overexpression of *ztfA* is increased. In addition, ZtfA is involved in the oxidative stress response system. In the absence of *ztfA*, the fungus shows an increased tolerance towards H₂O₂ compared to the wild type.

**89T Control of growth and development by lipid asymmetry in Aspergillus nidulans.** Z.S. Schultzhaus¹, W. Zheng², G. Cunningham¹, Z. Wang², R. Mouriño-Pérez³, B. D. Shaw¹ 1) Plant Pathology and Microbiology, Texas A&M University, College Station, TX; 2) State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China; 3) Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, México.

The constituents of the plasma membrane, primarily lipids, are continuously in flux through vesicle fusion, budding, diffusion, chemical alterations, and transport between leaflets of the bilayer. The mechanisms that maintain order in fungal plasma membranes, particularly at the actively growing hyphal tip, are not well understood. One group of proteins that regulates eukaryotic plasma membranes is the phospholipid flippases, which are a distinct class of P-type ATPases (a superfamily of transporters which includes Na⁺/K⁺ pumps and calcium pumps) that move phospholipids toward the cytosolic plane of membranes (e.g. Golgi, endosomes, and the plasma membrane). Two of the four *Aspergillus nidulans* flippases, DnfA and DnfB, were previously found to in different regions of the hyphal tip and to share a function essential for hyphal growth. Here, we report on the function and localization of DnfA and DnfB, and another *A. nidulans* flippase, DnfD, in more detail. DnfA and DnfB exhibited different recovery rates within the Spitzenkörper, in agreement with them being transported on different populations of vesicles. Deletion of *dnfB* alone does not affect Spitzenkörper dynamics, but deleting *dnfA* results in a Spitzenkörper that is unstable and diminished. Only a simultaneous downregulation of both (through repression of *cdc50*), however, results in a dramatic mislocalization of secretory and endocytic proteins, suggesting that their roles in the secretory and endocytic pathways overlap substantially. Next, we looked at the localization and function of another flippase, DnfD. DnfD localizes earlier in the secretory pathway (almost overlapping exclusively to the TGN/late Golgi), but appears to play no role in hyphal growth. Rather, a *dnfD* deletion exhibits severe defects in both production of conidia (asexual reproduction) and conidiophore morphology. Combinatorial analysis of flippase mutants, additionally, suggests unique relationships are shared between these proteins in fungal growth and development.

**90F Proteins TaoA and EcoA localize to the apex and sub-apical collar of growing hyphae in Aspergillus nidulans.** B. Commer, Z. Schultzhaus, B. Shaw Plant Pathology & Microbiology, Texas A&M University, College Station, TX.

Filamentous fungi produce polarized cells called hyphae. Polarization of hyphae is dependent on a balance between endocytosis and exocytosis that may be maintained through apical recycling. This balance requires the Spitzenkörper (SPK), a secretory body found only in growing hyphae, and a sub-apical collar 1-5µm immediately distal to the SPK that is enriched for endocytosis. Evidence of this relationship is observed through the investigation of landmark proteins along the plasma membrane that mark areas of growth. These proteins are
displaced along the membrane as growth occurs and new membrane is added. Here, an informatics approach was used to identify proteins in *Aspergillus nidulans* that contain an endocytic NPFxD motif. The long-term goal of this project is to observe the kinetic localization and examine the predicted endocytic association of these NPFxD motif-containing proteins during hyphal growth using fluorescent markers and live-cell imaging. Two of the 42 proteins, TaoA and EcoA, were selected based on previous studies of orthologs in yeast. The TaoA ortholog, Tao3, is a component of the RAM signaling network, which is involved in cell morphology and localizes to polarization sites. The EcoA ortholog, Pal1, is of unknown function and is found to localize to endocytic sites in yeast. Here, strains expressing either TaoA::GFP or EcoA::GFP were constructed and visualized to document dynamic localization during hyphal growth. TaoA::GFP localization was observed at the apical crescent, a cortical area found at the extreme apex in growing hyphae, proximal to the SPK. TaoA::GFP localization terminated in an area that corresponds to the sub-apical collar, suggesting that it may be cargo for endocytic uptake. EcoA::GFP localized to the endocytic collar, and was not observed in the apical crescent. Continuing work for both proteins will include verification of the endocytic association and co-localization with known tip-localized endocytic and exocytic machinery.

**91W Developmental regulation by constitutive activation and inactivation of MpkB MAPK pathway in Aspergillus nidulans.** Sang-Cheol Jun¹, Kwang-Yeop Jahng², Jong-Hwa Kim³, Kap-Hoon Han³ 1) Institute for Genetic Engineering, Chonbuk National University, Jeonju, South Korea; 2) Department of Life Sciences, Chonbuk National University, Jeonju, South Korea; 3) Department of Pharmaceutical Engineering, Woosuk Univ, Wanju, Jeonbuk, South Korea.

The *mpkB* gene is a member of the multiple MAPK pathways in *Aspergillus nidulans*. The MpkB has been known to play a key role in the asexual and sexual development and secondary metabolite production. The MAPK kinase MkkB can be phosphorylated at Ser218 and Thr222 residues within the activation loop site by SteC MAPKK kinase and consequently, activates MpkB by phosphorylating at threonine and tyrosine residues of MpkB in *A. nidulans*. By introducing negatively charged (aspartic acid) or non-polar (proline and lysine) residues that may mimic the effect of phosphorylation at positions 218 and 222 in MkkB, we constructed mutants which constitutively phosphorylate MpkB MAPK and constitutively non-phosphorylate MpkB MAPK. Expression of the constitutively activated MkkB in *A. nidulans* led to the hyper-activation of MpkB MAPK and resulted in a great elevation of sexual development, while asexual sporulation was remarkably reduced. In contrast, constitutively inactivated MkkB mutant could not produce sexual organs, while asexual sporulation and sterigmatocystin production were normally processed. Differential expression pattern of proteins among these mutants were analyzed by using the 2D proteomic analysis, which revealed that expression level of 375 spots are significantly different.

**92T An intrinsically disordered domain of the VelB velvet domain provides specificity for heterodimer formation in Aspergillus nidulans.** S. Thieme, J. Gerke, C. Sasse, O. Valerius, K. G. Thieme, G. H. Braus Molekulare Mikrobiologie und Genetik, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany.

Genetic networks are often controlled by DNA binding proteins, which act as homo- or heterodimers. The velvet domain represents a conserved fungal DNA binding and dimerization domain of approximately 150 amino acids, which is reminiscent to the *rel* domain of NF-kB in the mammalian immune and infection response [1]. Aspergilli possess four members of velvet family proteins (VeA, VelB, VelC and VosA), which can form homo- or heterodimers and are required to control development coordinated with the appropriate interconnected secondary metabolism and spore viability [2]. VeA interacts with several epigenetic methyltransferases and shuttles VelB as a VeA-VelB heterodimer into the nucleus [3, 4]. VelB is also part of the VelB-VosA complex supporting reduced and delayed asexual spore formation and promoting spore viability. Only the velvet domain of VelB is interrupted by an insertion of additional amino acids in ascomycetes. The *A. nidulans* insert consists of 99 amino acids and is conserved among Aspergilli. We compared a VelB with or without insert and found that the insert is required for an accurate light control which normally promotes asexual development and reduces sexual development as well as secondary metabolite production [5]. This is reflected on the molecular level by the finding that the VelB interaction partners with insert differ to the VelB interaction partners without insert. This suggests that the insertion changes the potential of velvet domains and provides a surface for altered protein-protein interactions.

[1] Ahmed et al. (2013) Fungal *velvet* regulators contain a DNA binding domain reminiscent of NF-kB. **PLoS Biol.** 11, e1001750.

[2] Bayram and Braus (2012) Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. **FEMS Microbiol. Rev.** 36, 1-24.

[3] Sarikaya-Bayram et al. (2015) One Juliet and four Romeos: VeA and its methyltransferases. **Front. Microbiol.** 6, 1. 1-7.

[4] Bayram et al. (2008) VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. **Science.** 320, 1504-1506.

[5] Bayram et al. (2016) Changes of global expression and secondary metabolite accumulation during light-dependent *Aspergillus nidulans* development. **Fung. Genet. Biol.** 87, 30-53.

**93F Molecular characterization of microtubule organizing centers(MTOCs) in the filamentous fungus Aspergillus nidulans.** Xiaolei Gao, Ying Zhang, Marjorie Schmid, Reinhard Fischer Department of Microbiology, Institute for Applied Bioscience, Karlsruhe, DE.

Microtubule polymerization starts in fungal and animal cells from large multi-subunit protein complexes, the microtubule-organizing centers (MTOCs). In the model fungus *Aspergillus nidulans* septum-associated MTOCs (sMTOCs) together with the spindle pole bodies nucleate microtubules. We focused on the structure and composition of sMTOCs and how they are formed during septation. We show that in *A. nidulans* several SPB outer plaque proteins are conserved at sMTOCs, such as the gamma-tubulin ring complex (γ-TuRC), the centrosomin-domain (CM) containing protein ApS and the disordered protein Spa18. The latter two proteins were required for recruiting the γ-TuRC component GcpC to sMTOCs and are necessary for microtubule formation from septa. Another intrinsically disordered protein, Spa10, colocalized with tropomyosin (TpmA) at the beginning of septation, while ApS and Spa18 were only found at mature septa. Spa10 was necessary for ApS and Spa18 recruitment and thus essential for septal microtubule nucleation. Whereas Spa10 formed a central disk in the septal pore, Spa18 and ApS formed a discontinuous ring structure with several punctate foci attached to the rim of the Spa10 disk. Different from eMTOCs in *Schizosaccharomyces pombe*, which are only transiently formed during septation, sMTOCs in *A. nidulans* are assembled after septum formation and represent stable structures in hyphal compartments.
**94W** Identification and functional characterization of a Bcl-2 associated athanogene in *Aspergillus nidulans*. *Sachin Jain*¹,
Nancy Keller², Mehdi Kabbage¹ 1) Plant Pathology, University of Wisconsin-Madison, Madison, WI; 2) Medical Microbiology and
Immunology, University of Wisconsin-Madison, Madison, WI.

The Bag (Bcl-2 associated athanogene) family is a multifunctional group of proteins that is involved in multiple cellular processes ranging from apoptosis to tumorigenesis. As evolutionarily conserved proteins, they are distinguished by a common conserved region known as the Bag domain. In animals and plants, Bag genes are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In this study, we focus on the model filamentous fungus *Aspergillus nidulans* to study the function of Bags in fungi. The *A. nidulans bagA* gene expression was strongly induced during sexual development and apoptosis inducing insults. In order to characterize this gene, we generated over-expression and knock-out strains of *bagA* in *A. nidulans*. Our results indicate that *ΔbagA* strains are negatively impacted in their sexual development and had an altered secondary metabolite (SM) profile in both sexual and vegetative growth phases when compared to wild type strain. Interestingly, over-expression of BagA leads to increase in sexual/asexual spore ratio. LC-MS analysis suggests that *bagA* may be a negative regulator of secondary metabolism since several SMs were enriched in the *ΔbagA* strain. SMs from microorganisms play a role in niche securement and those produced by fungi have been particularly rich source of useful natural products. Thus, our results suggest that the disruption of cell death pathways could alter the SM profile of fungi and might help uncover novel bioactive compounds, including antimicrobials and pharmaceutical drugs.

**95T** Citrate overflow by *Aspergillus niger* is associated with extended pre-autolytic growth. *Baljinder Kaur*, Narayan S Punekar Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India-400076.

The filamentous fungus *Aspergillus niger* is commercially important for its production of enzymes, metabolites and organic acids. It has been exploited at industrial scale for the production of citric acid. The citric acid is produced by *A. niger* under a set of stringent fermentation conditions optimized over a century but the mechanisms responsible for this metabolic overflow remain tentative. The two key features of the media for fermentation are limiting nitrogen and trace metal deficiency. Both of them affect the gross morphology of the fungus. In order to better understand the physiological relevance of *A. niger* mycelial morphology on acidogenic growth a detailed microscopic analysis was carried out. This was assisted by using a strain expressing EGFP in the cytoplasm. The major cellular compartments namely, cytoplasm, mitochondria, vacuole and nucleus were monitored under normal and acidogenic growth conditions. While the intracellular morphology remained unaltered throughout the idiophase of fermentation, it corresponded to the pre-autolysis growth phase of mycelia frown on normal media. The hyphae were thin and heavily loaded with vacuoles but were maintained away from immediate autolysis. The continuance of mycelia in the pre-autolysis stage could be a feature responsible for citrate overflow. Possible involvement of autophagy growth during acidogenesis by *A. niger* is explored using fluorescence microscopy and suitable gene deletion strains.

**96F** A metalloprotease, AdmB, involves cell wall construction and carbon starvation stress response in *Aspergillus oryzae*. *T. Kobayashi*, T. Oiwa, H. Maeda, M. Takeuchi, Y. Yamagata The United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchū, JP.

*Aspergillus oryzae* is a mold necessary for Japanese food and brewing. The fungi sense external environment during growing. Genome sequencing and analysis of *A. oryzae* was reported in 2005. As a result, 126 genes predicted to encoding proteases in a total of about 12,000 genes. We found two "a disintegrin and metalloproteinases (ADAMs)" in *A. oryzae* and named them AdmA and AdmB. The ADAMs are membrane proteins and contain two characteristic domains, metalloprotease and disintegrin domain. In mammals, ADAMs play important roles in cell adhesion, protein processing and signaling. Especially, they are able to cleavage and release a ligand for signaling on the cell surface and so are called "sheddase". However, the function of fungal ADAMs has been unknown. We aimed to elucidate biological functions of AdmA and AdmB in *A. oryzae*.

Firstly, to analyze AdmB localization, we constructed an *admB*-enhanced green fluorescence protein (EGFP) strain that highly expressed the AdmB-EGFP fusion protein. Fluorescence microscopy analysis showed that AdmB-EGFP localized to the plasma membrane and some vesicles. Next, we examined *admA* and *admB* expression kinetics by quantitative RT-PCR. As a result, we revealed that the transcription of *admB* was increased in a late growth stage and induced by a carbon starvation stress. Finally, we produced single- and double-gene deletion strains, ?*admA*, ?*admB* and ?*admA*?*admB* using *pyrG* marker recycling. These strains showed normal growth like a control strain, while two strains, ?*admB* and ?*admA*?*admB* were sensitive to cell wall perturbing agents, calcofluor-white and congo red. Moreover, ?*admB* showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. Taken together, these results suggest that AdmB involves in not only the carbon starvation stress response but also cell wall construction and its integrity. Incidentally, AdmB may play roles in these biological processes by acting like mammalian ADAMs.

**97W** Identification and characterization of a vegetative incompatibility locus in *Botrytis cinerea*. *Saadiah Arshed*¹,², Ross Beever³, Murray Cox⁴, James Lee¹,², Michael Pearson², Joanna Bowen¹, Matthew Templeton¹,² 1) The New Zealand Institute for Plant & Food Research, NZ; 2) The University of Auckland, New Zealand; 3) Landcare Research, New Zealand; 4) Massey University, New Zealand.

Vegetative incompatibility (VI) is a fungal non-self recognition system characterized by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon, and is controlled by multiple polymorphic loci termed *vic* (vegetative incompatibility) or *het* (*heterokaryon incompatibility*). We report here the first *vic* locus that has been genetically identified and characterized in the economically important plant pathogenic, necrotrophic fungus *Botrytis cinerea*. We generated near isogenic lines of interacting compatible and incompatible strains that differed at a single *vic* locus and employed a bulk segregant analysis approach coupled with whole genome Illumina sequencing to map a 55-kb genomic region containing the *vic* locus. Within that locus, we identified two adjacent highly polymorphic open reading frames encoding predicted proteins that contain domain architectures implicated in VI in other filamentous fungi, *Bcvic1* and *Bcvic2*. Deletion of *Bcvic1* and *Bcvic2* individually did not have any effect on vegetative incompatibility. However, deletion of the region containing both *Bcvic1* and *Bcvic2* resulted in gene deletion lines that were severely restricted in growth and also showed loss of VI. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype indicating that *Bcvic1* and *Bcvic2* are interacting and controlling VI at this *vic* locus.
**98T Pharmacological and transcriptomic analyses of a *B. cinerea* flavohemoglobin deficient mutant demonstrate that nitric oxide affects germination, DNA replication and cell cycle.** Francisco Anta¹, Daniela Santander¹,⁴, Wilson Acosta¹, Rodrigo Santamaría², Pedro San Segundo³, José María Díaz-Mínguez¹, Ernesto P. Benito¹ 1) CIALE-UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN; 2) DPTO. DE INFORMÁTICA Y AUTOMÁTICA, FAC. DE CIENCIAS, UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN; 3) INSTITUTO DE BIOLOGÍA FUNCIONAL Y GENÓMICA, CSIC-UNIVERSIDAD DE SALAMANCA, SALAMANCA, SPAIN; 4) FAC. DE CIENCIAS AGROPECUARIAS Y AMBIENTALES, UNIVERSIDAD TÉCNICA DEL NORTE, IBARRA, ECUADOR.

Nitric oxide (NO) is a highly reactive molecule with fundamental roles in the biology of all living systems. The participation of NO in developmental processes has been described in several fungal species. However, the nature of the mechanisms and factors being affected by NO in fungi is poorly characterized. *Botrytis cinerea* is a plant pathogenic fungus which has attracted much attention given its wide host range and its necrotrophic life style. The production of NO by the fungus has been demonstrated as well as its detoxification by means of a flavohemoglobin enzyme encoded by gene *Bcfhg1*. It has been suggested that the physiological functions of the flavohemoglobin could be related to its involvement in the modulation of endogenous NO levels produced by the fungus during specific developmental stages. Pharmacological studies in which germinating spores and mature mycelium were exposed either to NO donors or to NO scavengers indicate that NO affects germination. From the data obtained, in combination with the results derived from the analysis of the response of Δ*Bcfhg1* germinating spores to NO, it can be concluded that NO exerts and immediate and transitory effect on germination efficiency, on germ tube elongation and on nuclear division rate. Global expression analysis of Δ*Bcfhg1* in these conditions detected major changes in the expression pattern with about one third of the genes predicted in the *B. cinerea* genome responding to exposition to NO. Functional enrichment analysis allowed to identify links between exposition to NO, growth arrest and down-regulation of "DNA replication", "nucleolus" and "cell cycle" genes.

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

**99F Regulation of hyphal guidance by Pxl1 and Cst20 in *C. albicans*.** M.C. Almeida¹, E.M. Morrison¹, J. Craven², N.A.R. Gow¹, A.C. Brand¹ 1) MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, Aberdeen, GB; 2) Department of Molecular Biology and Biotechnology, The Krebs Institute, University of Sheffield, Sheffield, UK.

*Candida albicans* lives as a commensal yeast in humans but can cause life-threatening bloodstream infections in susceptible patient groups. The formation of invasive hyphae is a characteristic virulence trait of *C. albicans* and we have shown that the penetration of host tissue depends on the ability of hyphae to steer as they grow. We are investigating the regulatory mechanisms of how hyphae sense guidance cues in the environment and translate these signals to re-orient the direction of growth. We have identified two types of 'steering-locked' mutants and the proteins associated with them. A wavy hyphal phenotype is generated by deletion of Pxl1, a Paxillin-like homologue, and its putative regulatory kinase, Ptk2 (homologue of Focal Adhesion Kinase). In contrast, truncation of Cst20 (Cst20¹⁻⁵⁴⁹) or hyper-activation of the small GTPase, Rho1, cause hyphae to grow uni-directionally. Both the wavy and unidirectional phenotypes correlate with an inability to respond normally to directional cues. Pxl1-GFP localises to bud tip and bud neck in yeast and to the growing tip in hyphae, but not to the septa, suggesting that Pxl1 is involved in the transport or localisation of a specific subset of cell-wall remodelling proteins. RFP-Ptk2 localises throughout the cytoplasm. Although the wavy and unidirectional phenotypes are strikingly different, the Pxl1 and Cst20¹⁻⁵⁴⁹ strains exhibit other phenotypes in common. In yeast, TEM analysis showed both mutants have cell separation defects. In hyphae, they exhibit aberrant intracellular structures. These shared phenotypes suggest that Pxl1 and Cst20 are involved in the same pathway that governs cell growth. We hypothesise that Cst20, a homolog of PAK that negatively regulates Paxillin in humans, may have a similar function in *C. albicans*, suggesting that constant turnover of the Pxl1 complex is required for hyphal steering responses.

**100W Rsr1 GTPase regulation is required for hyphal steering and differential effector binding in *Candida albicans* yeast and hyphae.** T. Bedekovic, A. Brand University of Aberdeen, Aberdeen, GB.

*Candida albicans* is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised patients, where the formation of invasive hyphal filaments contributes to fatal levels of sepsis and organ failure. Hypha-mediated tissue damage depends on directional growth responses that are regulated by the small N-Ras-like GTPase, Rsr1. In *S. cerevisiae*, Rsr1 is K-Ras-like and specifies the yeast bud site where it is activated by its guanine-nucleotide exchange factor (GEF), Bud5, and de-activated by its GTPase activating protein (GAP), Bud2. However, its role in *C. albicans* hyphae is less understood. To elucidate the role of Rsr1 in hyphae, we generated mutant strains expressing a single copy of Rsr1 in a GDP-locked (Rsr1K16N) or GTP-locked (Rsr1G12V) form and compared their phenotypes with those of the *rsr1Δ*, *bud5Δ* and *bud2Δ* null strains. Deletion or dysregulation of Rsr1 caused large, rounded yeast cells and shorter hyphae that were unable to steer round obstacles or penetrate agar. Deletion of Rsr1 and Bud2 caused yeast and hyphae to become multinucleate. Strikingly, these defects were seen in the strain expressing Rsr1K16N but not in the *bud5Δ* null, suggesting that Rsr1 may be regulated by a second GEF that functions in hyphal steering responses. The site of Rsr1 activity is thought to be regulated by the localization of the Bud5 and Bud2 regulators at the tip and subapical region of hyphae, respectively. Here we show that YFP-Rsr1K16N and YFP-Rsr1G12V were differentially localised. YFP-Rsr1K16N appeared as patches on the plasma membrane whereas YFP-Rsr1G12V was uniformly localised to the plasma membrane, similar to WT. Early proteomics results using GST-tagged versions of these proteins expressed in yeast and hyphae show that activity state and cell morphology differentially determines the binding partners of Rsr1. These findings suggest a more complex role for the N-Ras Rsr1 GTPase in *C. albicans* than has been described for the K-Ras Rsr1 GTPase in the yeast, *S. cerevisiae*.

**101T Two cell wall glycosyl hydrolases are required for the maturation of the N. crassa conidial cell wall.** Jie Ao, Stephen Free Dept Biological Sci, SUNY Univ, Buffalo, Buffalo, NY.

Using a proteomic analysis, we identified 35 cell wall proteins in mature conidia. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared. These shared proteins were cell wall biosynthetic proteins or cell wall structural proteins. We analyzed deletion mutants for 34 of the genes for phenotypes indicative of conidial cell wall defects. We found that mutants for two cell wall glycosyl hydrolases, the beta-1,3-glucanase CGL-1 (NCU07523) and the exochitinase NAG-1 (NCU10852), have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. We generated promoter::RFP or promoter::GFP constructs for the 19 genes encoding what we thought might be conidia-
specific proteins. We demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation.

**102F Very high expression and incorporation of specific cell wall proteins secures fast surface expansion during apical growth of Ashbya gossypii.** R. Rischatsch¹, S. Voegeli¹, M. Finlayson¹, A. Martinez³, Q. Yuan Yin³, C. de Koster³, F. M. Klis³, P. Philippsen¹, P. W. J. de Groot²,³ 1) Molecular Microbiology, Biozentrum, University of Basel, 4056 Basel, Switzerland; 2) Regional Center for Biomedical Research, University of Castilla-La Mancha, Spain; 3) Swammerdam Institute for Life Sciences, University of Amsterdam, 1018 WV Amsterdam, The Netherlands.

The phytopathogen *Ashbya gossypii* and the yeast *Saccharomyces cerevisiae* are phylogenetically closely related. It is not known how *A. gossypii* has evolved an exclusively hyphal growth mode with very rapid apical extension requiring cell wall expansion rates that are up to 40-fold faster compared to *S. cerevisiae*.

The *A. gossypii* genome codes for 44 putative GPI proteins, 10 without a homolog in *S. cerevisiae*. It also reveals amplification of several cell wall protein-encoding genes, notably *CWP1*. Transcriptome studies show that one third of the CWP-encoding genes are expressed at higher levels than ribosomal protein genes. Mass spectrometric analysis of protein extracts from purified walls of rapidly growing hyphae resulted in the identification of 14 covalently bound cell wall proteins (CWPs). Some CWPs that are common in hemiascomycetes are missing in *A. gossypii*. On the other hand, the chitin deacetylase Cda1/Cda2 was identified in addition to three novel proteins (Agp1, Awp1, and Sod6), all without homologs in baker's yeast (NOHBYs). Phenotypic analysis confirmed the importance of these NOHBYs for cell wall integrity. Interestingly, hyphal walls of *A. gossypii* contain very little chitin, and orthologs of genes required for cell wall remodeling and degradation of septa during cell division in *S. cerevisiae* show low expression or are absent. We conclude that loss of distinct cell wall genes, acquisition of novel genes, and amplification as well as increased expression of evolutionary conserved fungal cell wall genes led to the evolution of fast polar surface expansion of *A. gossypii* hyphae.

**103W Some like it on the rocks: recurring stresses select for rock-inhabiting fungi with manifold protective pigments.** Nicole Knabe¹, Romy Breitenbach¹,², Polina Dementyeva¹, Felix Heeger³, Camila Mazzoni³, Anna A. Gorbushina¹,² 1) Federal Institute for Materials Research and Testing (BAM), Department Materials and the Environment, Berlin, Germany; 2) Freie Universität Berlin, Department of Biology, Chemistry and Pharmacy & Department of Earth Sciences, Berlin, Germany; 3) Berlin Center for Genomics in Biodiversity Research, Berlin, Germany.

Black ascomycetous microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in deserts as well as ubiquitous in other wide-spread terrestrial ecosystems including man-made materials such as solar panels. The ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance. Also, applied research is necessary to find ways of preventing MCF from colonising and degrading solar panels and historic monuments.

Carotenoids and melanin are amongst the protective pigments that contribute to the robustness of MCF. We studied their role in stress resistance of the non-pathogenic rock-inhabiting fungus *Knufia petricola* (Chaetothyriales) strain A95. Several knock-out mutants have been produced in melanin- (A95ΔSDH and A95ΔPKS) as well as carotenoid- (A95ΔPDG) synthesis and a double mutant (A95ΔPKS/ΔPDG) also exists. Disruption of melanin synthesis affects not only oxidative-stress resistance but also the extracellular polysaccharide (EPS) matrix and lipid composition of the cell membrane. Exposure to ≤ 30 mM H₂O₂ did not affect the growth rate of A95ΔPKS as compared to wild type cells. Almost twice as much water-soluble EPS was present in cultures of the A95ΔPKS mutant compared to the wild type and A95ΔPDG strains. Comparative gene expression analyses of the wild type and the A95ΔPKS mutant identified genes regulated under oxidative stress conditions. These data will help elucidate mechanisms of cell wall maturation and oxidative stress defence strategies.

**104T Spatiotemporal regulation of clock associated mRNA in Neurospora crassa.** Bradley M. Bartholomai¹, Samantha E.R. Dundon², Amy S. Gladfelter³, Jennifer J. Loros⁴, Jay C. Dunlap¹ 1) Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH; 2) Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT; 3) Department of Biology, University of North Carolina, Chapel Hill, NC; 4) Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH.

*Neurospora crassa* has long been an important model for circadian biology. However, the spatiotemporal dynamics of the *N. crassa* clock components at the level of individual hyphae have remained elusive. The core oscillator in the *Neurospora* clock is a negative feedback loop in which WC-1 and WC-2 acting together as a transcription factor drive expression of *frequency* (*frq*) whose product, the Intrinsically Disordered Protein FRQ, dimerizes, complexes with the DEAD-Box Helicase FRH and with CK1, and depresses the activity of WC-1/WC-2. We set out to investigate whether *frq* mRNA is non-randomly positioned in the cytoplasm. We hypothesized that *frq* mRNA translation may be spatially controlled through the association with an RNA-binding protein. To examine if *frq* transcript is spatially patterned, we employed single molecule RNA FISH coupled with quantitative image processing. The image analysis suggested that there is non-random clustering of *frq*, indicating that there is spatial regulation of mRNA position. We are currently using bacteriophage stem-loop and coat protein based techniques to discover *frq* mRNA binding proteins that participate in regulating localization and to visualize clock related mRNA in living cells.

**105F A novel signaling network regulates circadian adaptation in Rice Blast.** N.I. NAQVI¹, Y.Z DENG², Z QU¹ 1) Temasek Life Sciences Laboratory, Singapore; 2) South China Agricultural University, China.

Phototropic response and circadian rhythms are critical for growth, differentiation and environmental adaptation in fungi. However, the precise regulators of the intracellular metabolic response during such dark-light rhythms have not been identified/characterized yet. The blast fungus, *Magnaporthe oryzae*, initiates conidiation in response to phototropic and nutritional cues. We found that autophagy is induced specifically upon exposure to light and is essential for asexual development in *M. oryzae*. Comparative RNAseq analyses provided insights into novel signal transduction cascades underlying such phototropic induction of conidiation and in adaptation to the host milieu. We identified a histone acetyltransferase that negatively regulates light- and nitrogen-starvation induced autophagy during conidiation *in planta*. Furthermore, we identified a novel circadian-regulated *Twilight* (*TWL*) function essential for phototropic response and pathogenesis in *M. oryzae*. Expression of *TWL* showed a distinct circadian rhythm, and its transcript level peaked at subjective twilight. Mutually exclusive
phosphorylation acetylation cycles governed the intracellular dynamics and regulation of the Twl protein. Acetylated GFP-Twl remained
cytosolic in the dark, whereas the light-induced phosphorylated (via Snf1 kinase) version translocated to the nucleus. Twilight function was
required for regulating the mRNA levels of several important transcription/repair factors, including *TFB5*. Overexpression of *Tfb5*
significantly suppressed the conidiation defects associated with the *twlΔ* mutant. Tfb5-GFP translocated to the nucleus during the
phototropic response and under redox stress, whereas it failed to do so in the *twlΔ* mutant. Lastly, we provide mechanistic insight into Twl-
based regulation of nutrient and redox homeostasis in the adaptation of the pathogen to the host milieu; and propose a regulatory
mechanism that underlies the induction of autophagy by important environmental clues such as light and nutrients in the rice blast
pathosystem.

**106W Analysis of predicted essential genes in the MAT type of *Cryptococcus neoformans* and insights into the mechanisms of mitochondrial uniparental inheritance.** Giuseppe laniri¹, Shen Sun¹, Alex Idnurm², Joseph Heitman¹ 1) Molecular Genetics and Microbiology, Duke University, Durham, NC; 2) School of BioSciences, University of Melbourne, VIC 3010, Australia.

The *MAT* locus of *Cryptococcus neoformans* has a bipolar system and it is characterized by an unusually large structure, spanning over 100 kb. The majority of the *MAT* genes have been characterized by functional genetics as being involved in sexual reproduction, virulence and mitochondrial uniparental inheritance (UPI). However, classical gene replacement failed to achieve mutants for five *MAT* genes (*RPL22*, *RPO41*, *MYO2*, *PRT1*, *RPL39*) of *C. neoformans*, indicating that they are likely playing an essential role. In order to infer their function, targeted gene replacement was performed in a diploid background for both the α and a alleles of these genes. Homologous recombination (HR) was achieved using a NAT-based split marker approach coupled with the use of W7 hydrochloride, a chemical that increases HR by inhibiting the non-homologous end joining pathway. While the study of *RPO41a/α* and *PRT1a/α* is in progress, Mendelian analysis of haploid progeny failed to recover the *NAT* marker from heterozygous mutants *RPL22a/rpl22aΔ*, *RPL22α/rpl22aΔ*, *RPL39a/rpl39aΔ*, and *RPL39α/rpl39aΔ*, consistent with an essential function. Strikingly, while *myo2aΔ* is inviable, *NAT* resistant progeny was obtained for *MYO2a/myo2aΔ*. Ongoing studies seek to test the hypotheses that *MYO2α* is either a non-essential gene or it is an evolvable essential gene, the last being a phenomenon recently described in *Saccharomyces cerevisiae* and that consists in mechanisms of adaptive evolution that overcome the loss of gene function by changes in the ploidy status of the cell. Moreover, in yeast *Myo2* is a myosin motor involved in actin-based transport of cargos, including mitochondria. To function, it is bound by Ypt11 and it requires components of the ERMES complex Mmm1, Mdm10, Mdm12, and Mdm34. Mutants for genes encoding these proteins are tested for mitochondrial UPI through genetic crosses, and mitochondrial distribution through mitotracker staining and Hem15-GFP localization. Our study will contribute to elucidate the functions of the *MAT* locus in *C. neoformans* and how it influences mitochondrial UPI.

**107T Role of microfilament motor, MYO5 in the D-amino acid utilization.** Ami Khanal Lamichhane, Yun Chang, Kwon-Chung June National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD.

The ability to utilize D-proline as sole Nitrogen source has been used as a diagnostic test to distinguish *Cryptococcus gattii* from its sister species, *C. neoformans*. *MYO5* gene was identified as one of the gene required for utilization of D-proline in *C. gattii* strain by screening insertional mutants library. Deletion of *MYO5* gene in *Cryptococcus gattii* compromised its growth in D-proline and complementation of gene was able to reconstitute the phenotype as wild type. Increasing concentrations of D-proline inhibited its growth suggesting the toxic effect of increasing amount of D-proline with deletion of *MYO5* gene. As previous studies suggested D-amino acid oxidase gene 2 (*RDAO2*) is a major gene involved in utilization of many D-amino acids, we checked if deletion of *MYO5* gene affects the expression of D-amino acid oxidase gene. Deletion of *MYO5* gene resulted in increased expression of *RDAO2* in D-proline and D-alanine. *MYO5* gene was tagged with mNG marker and compared its localization with actin using lifeact-RFP marker. *MYO5* was seen to be colocalized with most of actin structures such as cortical actin patches and ring structures. Our ongoing study suggest possible role of microfilament motor protein, *MYO5* in utilization of D-amino acids through vesicles trafficking of D-amino acid oxidase.

**108F Elucidating fluconazole based aneuploidy in *Cryptococcus neoformans*.** D.Sophie. Altamirano, D. Fang, C. Simmons, L. Kozubowski Genetics and Biochemistry, Clemson University, Clemson, SC.

*Cryptococcus neoformans* is a pathogenic yeast that causes lethal cryptococcal meningitis in immunocompromised patients. One of the challenges in treating cryptococcosis is the development of resistance to azole antifungals. Previous studies linked the azole resistance to the elevated copies of critical resistance genes in aneuploid cells. However, how aneuploidy is formed in the presence of azole drugs remains unclear. We found that treatment with inhibitory concentrations of an azole drug, fluconazole, led to a gradual and non-uniform depletion of ergosterol from the plasma membrane with concomitant progressive inhibition of budding and failure of cytokinesis. On the other hand, fluconazole had relatively less inhibitory effect on the progression through the cell cycle, as indicated by the premature formation of the mitotic spindle and continued nuclear division. To investigate which part of cytokinesis fails in the presence of fluconazole, we monitored the dynamics of two cytokinetic components: 1) non-muscle myosin II, *Myo1*, which is an essential constituent of the actomyosin ring (AMR) and 2) the septin Cdc10, which is one of the four septins that assemble into a higher order complex at the mother-bud neck. Our data show heterogeneous responses to fluconazole in terms of timing and rate of constriction of the AMR. Following the constriction of the AMR, septum was usually formed between the mother and daughter cells. Septin Cdc10 localization and dynamics were not significantly perturbed. Our findings suggest that in the presence of inhibitory concentrations of fluconazole, the AMR does constrict and septin assembly is normal. However, a final degradation of the septum between mother and daughter is affected, resulting in a lack of complete separation between the cells. Despite a defect in daughter cell separation, cell cycle continues leading to an increase in ploidy, potentially contributing to the formation of aneuploidy and fluconazole resistance.

**109W Elucidating septin-based mechanisms of cytokinesis in *Cryptococcus neoformans*.** P. Stempinski, E. Huey, E. McCormack, A. Rubin, J. Zielinski, L. Kozubowski Genetics and Biochemistry, Clemson University, Clemson, SC.

*Cryptococcus neoformans* is a basidiomycetous yeast, able to infect immunocompromised individuals due to its ability to grow at host temperature. Septins are conserved filament forming GTP-binding proteins that assemble as a complex at the site of cell division and contribute to cytokinesis. In *C. neoformans*, septins are not required for cytokinesis at 24°C but are essential at the host temperature of 37°C. Moreover, septins become essential at 24°C when the calcium-dependent protein phosphatase calcineurin is inhibited. While the role
of septins in cytokinesis is conserved in most eukaryotes, their exact contribution to this process remains elusive. Additionally, mechanisms of cytokinesis in *C. neoformans* are largely unknown. To define the roles of the septin complex and to identify other proteins involved in cytokinesis in *C. neoformans*, we performed a screen for genes necessary for growth at 37°C based on a collection of ~4,500 strains containing single deletions of non-essential genes. We searched for strains that at 37°C or in the presence of calcineurin inhibitor exhibited elongated bud morphology, a phenotype characteristic of septin mutants and indicative of cytokinesis failure. Our screen revealed 21 candidate genes, including 4 that were previously associated with cytokinesis and/or stress response (non-muscle myosin II, chitin synthase regulator, RAM pathway component, and the kinase PDK1), and 4 that encode hypothetical proteins. We are currently testing candidate genes identified in the screen for synthetic interaction with the septin deletion to probe for the potential involvement in septin pathway. In addition, we are testing if the candidate genes are involved in septin complex assembly and dynamics by introducing fluorescently tagged septin to the identified deletion strains. Our research will provide a better understanding of cytokinesis in *C. neoformans* and may lead to identification of novel antifungal drug targets.

**110T The role of mitochondrial fission in cyclophilin D-dependent mitophagy in the filamentous ascomycete *Podospora anserina*.** A. Hamann, P. Kramer, A. Jung, H.D. Osiewacz Molecular Biosciences, J.W. Goethe University, Frankfurt am Main, DE.

During aging of the filamentous ascomycete *Podospora anserina*, the mitochondrial peptidyl prolyl-cis, trans-isomerase cyclophilin D, PaCYPD, accumulates. This protein regulates the opening of the mitochondrial permeability transition pore (mPTP) which plays a key role in programmed cell death and induction of autophagy. Concordantly, we found that overexpression of *PaCypD* leads to increased general autophagy and mitophagy, while deletion prevents the previously demonstrated age-related induction of autophagy. Moreover, PaCYPD is required for a beneficial, so-called mitohormetic response to mild oxidative stress which results in lifespan extension. From these data we conclude that during aging, PaCYPD exerts a pro-survival function via induction of autophagy and mitophagy. However, excessive autophagy and mitophagy as it is observed upon constitutive strong *PaCypD* overexpression is detrimental and accelerates aging. To investigate the underlying mechanism, we analyzed whether mitochondrial fission, a prerequisite for the selective sequestration of damaged or dysfunctional mitochondria, is required for PaCYPD-dependent mitophagy. To this end, we used a *PaDnm1* deletion mutant which lacks the mitochondrial fission protein PaDNM1 resulting in impaired mitochondrial fission. Interestingly, although basal mitophagy does not depend on PaDNM1, preliminary data suggest that PaCYPD-induced mitophagy is PaDNM1-dependent. Thus, fission-dependent and - independent forms of mitophagy seem to exist.

**111F 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 ($H_2S$). 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 $H_2S$ addition and removal. Collectively, our study has implications in the areas of MT regulation, responses to hypoxia, gaseous signaling, and potentially fungal pathogenesis.

**112W 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.

**113T Adaptation of endocytosis to fast hyphal surface growth.** D. Nordmann¹, K. Masai³, H. P. Helfer³, R. Kurre², *P. Philippsen*³, H. P. Schmitz¹ 1) AG Genetik University of Osnabrück, 49090 Osnabrück, Germany; 2) Center for Advanced Light Microscopy University of Osnabrück, 49090 Osnabrück, Germany; 3) Molecular Microbiology, Biozentrum, University of Basel, 4056 Basel, Switzerland.

Intracellular traffic is influenced by the efficiency of endocytosis. This process is best characterized in budding yeast. Over 50 proteins cooperate in 1-2 minutes lasting multistep reactions from initiating membrane internalization to scission of the actin-coated vesicles. Endocytosis is enriched in growth zones e.g. yeast buds where polarity factors, exocyst components and excess of membranes have to be internalized to maintain polar surface expansion. We found a much accelerated endocytic process in growth zones of *Ashbya gossypii*, which is phylogenetically closely related to budding yeast but exerts up to 40x higher rates of polar surface expansion. Orthologs for all yeast endocytic proteins, except Sla2, are encoded in the *A. gossypii* genome, yet several of them fulfill their function in much less time than the budding yeast orthologs. On average, it takes less than 30 sec from the selection of an endocytic site to the abscission of an actin-coated vesicle. These vesicles have a fairly homogeneous size as shown by electron tomography (Gibeaux et al. Euk Cell 2013). Remarkably, Clathrin is not involved at any step of endocytosis. We also could demonstrate that the density of endocytic events at the growth zone increases approximately proportionally to the increase in polar surface expansion rate.

**114F Developmental genetics of a fungal monstrosity: comparative analyses of the secotiard and agaricoid forms of Lentinus triginus.** A. Knudson¹, Z. Xu², B. Wu¹, N. Chen², K. Krizsán³, R. Riley⁴, K. LaButti⁴, A. Lipzen⁴, W. Schakwitz⁴, C. Pennachio⁴, R. A. Ohm⁵, A. Carlson¹, I. V. Grigoriev⁴, L. G. Nagy³, D. Hibbett¹ 1) Dept of Biology, Clark University, Worcester, MA; 2) Institute of Applied Mycology, Huazhong Agricultural University Wuhan, 430070 China; 3) Synthetic and Systems Biology Unit, Institute of Biochemistry, BRC-HAS, Szeged 6726, Hungary; 4) US Department of Energy (DOE) Joint Genome Institute, Walnut Creek CA 94598, United States; 5) Microbiology, Department of Biology, Utrecht University, Utrecht 3584 CH, The Netherlands.

Lentinus triginus is a wood-decaying basidiomycete that occurs in two distinct forms: an "agaricoid" form, which has exposed gills that release spores into the air, and a "secotiard" form, which has its gills enclosed by a layer of tissue that traps the spores, much like a puffball. Prior genetic studies suggested that the secotiard form is conferred by a recessive allele at a single locus (sec). In an attempt to identify the genetic basis of the secotiard form, we produced draft genome sequences of two monokaryons, one sec+ and one sec-. We performed a cross between the monokaryons and generated progeny that we pooled by genotype (sec- vs. sec+), and subjected to Illumina sequencing to identify regions that segregate with the secotiard phenotype. We also produced six dikaryons, three homozygous sec- and three homozygous sec+, and obtained transcriptomes from developing fruiting bodies at four developmental stages (mycelium, primordium, and young and mature fruiting bodies). Preliminary analyses identify approximately 20 SNPs that co-segregate with the secotiard phenotype, and 27 genes that have differential expression in homozygous agaricoid vs. secotiard dikaryons.

**115W Functional analysis of class II hydrophobin HFB4 in *Trichoderma harzianum* and its sibling species *T. guizhouense*.** Feng Cai¹,², Mohammad Rahimi², Gunseli B. Ackapinar¹, Civan Yagtu², Agnieszka Przylucka², Guan Pang¹, Qirong Shen¹, Irina S. Druzhinina² 1) Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, China; 2) Institute of Chemical Engineering, TU Wien, Vienna, Austria.

Hydrophobins are the small secreted cysteine-rich proteins common in filamentous fungi. They have a distinct ability to self-assemble into stable amphiphilic layers at hydrophobic-hydrophilic interfaces and/or attach to various surfaces modulating their hydrophobicity. Thus, they can be involved in a diversity of functions related to the development of the fungus, its resistance to biotic and abiotic stress factors and in interactions with other organisms in the environment. The genome of the mycoparasitic filamentous fungus *Trichoderma* (Ascomycota, Dikarya) encodes a particularly large diversity of hydrophobins ranging from 6 up to 12 genes. In this work we have studied the function of hydrophobin 4 (HFB4) that is universally present in all *Trichoderma* groups. For this purpose, we have constructed libraries of HFB4 deletion and overexpressing mutants for *T. harzianum* CBS 226.95 and its sibling species *T. guizhouense* NJAU 4742 and also heterologously expressed the protein in *Pichia pastoris* cell factory. Results confirmed the involvement of HFB4 in hyphal growth in liquid medium. However, the impact was different in two *Trichoderma* species. *T. guizhouense* deletion mutants were not able to overcome the liquid surface tension and did not form aerial mycelia on liquid media, while *T. harzianum* deletion mutants did not express such deficiencies. More remarkably, HFB4 deletion mutants of *T. guizhouense* had essentially increased capacity to colonize tomato roots but their antagonistic potential in interactions with other fungi was reduced. Contrary, HFB4 deletion mutants of *T. harzianum* have shown reverse phenotypes. The results will be presented in a context of the analysis of the complete hydrophobome expression for both species.

**116T Knockdown of PaMus10 coding for a putative mitochondrial E3 ligase of *Podospora anserina*: impact on mitochondria and aging.** A. T. Jung, H.D. Osiewacz Institute for Mol. Biosciences, J. W. Goethe University, Frankfurt am Main, DE.

*Podospora anserina* is a filamentous fungus used as a model organism to unravel molecular mechanisms of organismal aging. In this system, aging has a strong mitochondrial etiology. Pathways involved in respiration, ROS generation and scavenging, and in particular in surveillance of a population of "healthy" mitochondria (e.g. mitochondrial biogenesis, dynamics and proteostasis) strongly affect aging and lifespan. In order to analyze the contribution of the ubiquitin proteasome system (UPS) to mitochondrial quality control, we examined the role of the putative mitochondrial ubiquitin E3 ligase PaMUS10, which is a possible functional homolog of the human E3 ligase PARKIN. We found that homokaryotic *?PaMus10* ascospores display mitochondrial impairments. The developing mycelia show a hyper-branching phenotype and stop growth soon after spore germination. Since these severe impairments do not allow to efficiently investigate the function of MUS10 in *P. anserina*, we developed an approach allowing to knockdown the expression of *PaMus10* in cultures originating from heterokaryotic *?PaMus10/PaMus10* spores. Cultivation on medium supplemented with hygromycin resulted in a gradual depletion of nuclei containing *PaMus10* and leads to reduced fertility and a strong reduction in lifespan. This phenotype correlates with a pronounced alteration of the mitochondrial morphology from long reticular networks to fragmented mitochondria. In addition, we found that the amount of the mitochondrial outer membrane protein PaPORIN strongly depends on the PaMUS10 level indicating that PaPORIN is a potential substrate of this E3 ligase. Since the human homolog of PaPORIN, VDAC, is a substrate of PARKIN this finding supports the assumption that