completed by this lab (Hurley *et al.*, PNAS 111: 16995-17002, 2014) and others has identified 10-40% of the *Neurospora* genome that is
circadianly regulated under glucose metabolizing conditions. When cultured on pure cellulose, over 100 genes are upregulated, cellulases
are produced in large quantities, and additional biochemical pathways such as the unfolded protein response (UPR) are activated (Tian *et al.*, PNAS 106: 22157-22162, 2009). While amongst the upregulated genes are many that have been identified as circadianly regulated
under glucose metabolizing conditions, confirmation of their circadian behavior during cellulose metabolism remains incomplete.
The goal of this work is to transcriptionally profile *Neurospora* cultures grown on 2% microcrystalline cellulose (Avicel) over a 48 hour high
resolution time course to identify circadian sense and anti-sense transcripts. Samples collected at two hour intervals and assessed for
circadian behavior of core clock genes and *ccgs* by real-time PCR provide the RNA for subsequent high-throughput sequencing; analysis
of primary data with JTK cycle detects rhythmic changes in abundance of both sense and anti-sense transcripts.
These data will provide the first global assessment of circadian transcripts under cellulose metabolism conditions, and of circadian
regulation of anti-sense transcripts from *Neurospora*. Understanding the differential regulation of core clock components and downstream
*ccgs* under cellulose metabolism will be valuable for optimization of biofuel production.

**381F Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.** A. K. Dubey¹, N. Luria¹, S. Barad¹,², D. Kumar¹, E. A. Espeso³, *D. Prusky*¹ 1) Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Rishon LeZion, IL; 2) Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel; 3) Department of Molecular and Cellular Biology, Centro de Investigaciones Biológicas (C.I.B.), Madrid, Spain.

Growth of *Colletotrichum gloeosporioides* in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of *Aspergillus nidulans* to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of *A. nidulans* SltA and CrzA were identified in *C. gloeosporioides*. The *C. gloeosporioides* CrzA homolog is a 682-amino acid protein, which contains a C₂H₂ zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The *C. gloeosporioides* SltA homolog encodes a 775-amino acid protein with strong similarity to *A. nidulans* SltA and *Trichoderma reesei* ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of *C. gloeosporioides crzA* (?crzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calcium-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of *C. gloeosporioides sltA* (?sltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ?crzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca²⁺ supplementation. Inhibition of appressorium formation in ?sltA mutants was accompanied by downregulation of the MAP kinase *pmk1* and carnitine acetyl transferase (*cat1*), genes involved in appressorium formation and colonization, which was restored by Ca²⁺ supplementation. Furthermore, exposure of *C. gloeosporioides ?crzA* or ?sltA mutants to cations such as Na⁺, K⁺ and Li⁺ at concentrations that the wild type *C. gloeosporioides* is not affected, had further adverse morphogenetic effects on *C. gloeosporioides* which were partially or fully restored by Ca²⁺. Overall results suggest that both genes modulating alkali cation homeostasis, have significant morphogenetic effects that reduce *C. gloeosporioides* colonization.

**382W You don't know what you've got until it's gone: novel RNAi components in *Cryptococcus neoformans*.** *Shelby Priest*,
Blake Billmyre, Joseph Heitman Duke University Medical Center, Durham, NC.

Whole genome sequencing from *Cryptococcus deuterogattii*, a species responsible for an ongoing outbreak in immunocompetent
individuals, revealed loss or significant truncation of fourteen conserved genes relative to the *Cryptococcus* pathogenic species
complex. The fourteen genes include three canonical RNAi components and previous studies have shown that six more of these genes are
involved in mitotic-induced silencing (MIS), sex-induced silencing (SIS), or both. We investigated the role of two additional genes identified
from this comparative genomics approach, *MEH1*, a microsomal epoxide hydrolase, and *CDP1*, a chromodomain protein. We initially
hypothesized that *CDP1* would likely play a role in at least one of the silencing pathways, while *MEH1* would not. However, assaying for
MIS and SIS through fluctuation assays and genetic crosses, respectively, in genetic deletion mutants of *MEH1* and *CDP1* revealed roles
for both genes in MIS and SIS. These unexpected phenotypes drove us to investigate the genomic landscape of *MEH1*. Interestingly,
there is significant overlap between the 3' UTRs of *MEH1* and its neighboring upstream gene, *RNH70*, an RNA exonuclease. Unlike the
previously observed silencing phenotypes, independent *MEH1* deletion mutants produced a modest increase in MIS and a large increase in
SIS during bilateral crosses. Conversely, deletion of *RNH70* resulted in complete abolishment of MIS and decreased SIS during unilateral
crosses. Bilateral crosses between *rnh70Δ* mutants generated primarily bald basidia and sparse basidiospores, suggestive of a meiotic
defect. These new findings represent a case of a neighboring gene effect, where off-target effects of a gene deletion may be responsible for
the observed phenotypes, and provide an opportunity to investigate how 3' UTRs of convergently oriented genes contribute to genetic
regulation.

**383T Plant homeodomain (PHD) finger proteins regulate cryptococcal yeast-hypha transition.** *Yumeng Fan*, Yunfang Meng, Xiaorong Lin Biology, Texas A&M University, College Station, TX.

*Cryptococcus neoformans*, an opportunistic human fungal pathogen, is responsible for a million infections and half a million deaths each
year globally. This fungus can undergo morphological transition from the yeast to the hypha form. Like other dimorphic fungal pathogens,
this morphological transition of *Cryptococcus* is tightly linked to its virulence. Previously, our lab identified a transcription factor Znf2 as the
master regulator that bridges cryptococcal morphology and its virulence potential. However, the regulatory circuit controlling this potent anti-
virulence factor is still unclear.

Morphotype transition indicates remodeling of gene expression. Epigenetic factors as well as genetic factors are shown to be critical in
regulating morphogenesis in other fungal species. The post-translational histone modifications can impact gene expression by altering
chromatin structure or recruiting histone modifiers. However, nothing is known about epigenetic regulation in cryptococcal yeast-hypha
transition. The plant homeodomain (PHD) finger proteins are known as epigenetic effectors in plants and mammals by reading and effecting
histone modifications. We deleted and characterized 15 PHD finger genes and found that five PHD finger containing proteins affect switching to the filamentous form dramatically. The *rum1Δ* and the *znf1Δ* mutants both showed enhanced initiation of filamentation but failed to maintain filamentous growth. The *bye1Δ* and the *phd11Δ* mutants exhibited enhanced filamentation while the *set302Δ* mutant displayed reduced filamentation. Ectopic overexpression of these five PHD genes in the corresponding null mutants partially or completely restored the defect in filamentation. Thus, these PHD finger proteins are activators or repressors of filamentation. We further demonstrated that Phd11 and Set302 regulate yeast-hypha transition through the known master morphogenetic regulator Znf2. Collectively, these findings indicate the existence of novel pathways to control cryptococcal morphogenesis. Currently, I am focusing on dissecting the detailed mechanism of epigenetic regulation of filamentation. Exploring this layer regulation will not only help understand *Cryptococcus* development, but also identify targets for therapeutic interventions and against this fatal pathogen.

**384F** *Pas3 regulates cryptococcal morphogenesis through Bre1/Npl3-Mediated alternative splicing.* Y. Zhao, X. Lin Biology, Texas A&M University, College Station, TX.

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

**385W** ***Aspergillus flavus aswA*** regulates sclerotial development and biosynthesis of sclerotium-associated secondary metabolites. *P. Chang*¹, *L. Scharfenstein*¹, *R. Li*², *J. Diana Di Mavungu³* 1) Southern Regional Research Center, New Orleans, ARS, USDA, Louisiana, USA; 2) Animal Genomics and Improvement Laboratory, ARS, USDA, Maryland, USA; 3) Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.

Proteins with a Zn(II)₂Cys₆ DNA-binding domain are pathway-specific regulators. They bind to promoter regions of controlled genes and up-regulate gene expression via a transcriptional activation domain. *Aspergillus flavus aswA* (AFLA_085170) is a gene encoding such a protein. Deletion of *aswA* yielded strains that made a truncated gene transcript and produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white), different from mature *A. flavus* sclerotia, which are oval and pigmented (dark brown to black). Transcriptomic analysis of the *ΔaswA* strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analysis of non-pigmented sclerotia confirmed that they contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites plus three asparasone analogs and dihydroxyaflavinine. Impairment in *aswA* gene function can stall normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites occurring at the maturation stage.

**386T** **Interspersed homology patterns influence the detection of unpaired DNA during meiosis in *Neurospora crassa*.** N. Rhoades¹, P. Manitchotpisit¹, A. Boyd¹, T. Malone¹, T. Reed¹, E. Gladyshev², *T. Hammond*¹ 1) School of Biological Sciences, Illinois State University, Science Laboratory Building, Normal, Illinois, 61790, USA; 2) Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, USA.

*Neurospora crassa* possess a phenomenon known as meiotic silencing by unpaired DNA (MSUD). MSUD has the remarkable ability to identify gene-sized fragments of DNA that are not at the same location on each member of a pair of homologous chromosomes. It is as if MSUD is searching for genes that lack pairing partners. If a gene is found to lack a pairing partner, it is silenced for the duration of meiosis. Thus far, it has been shown that the identification of pairing partners depends on the level of sequence identity between the partners and on the proximity of the partners to each other on the homologous chromosomes. Here, we report that interspersed homology patterns are also critical for the pairing-partner detection process. Our results are similar to those reported recently for the homology search process of repeat induced point mutation (RIP). RIP scans the *N. crassa* genome for repeated gene-sized fragments of DNA. Our findings raise the intriguing possibility that the homology search processes of MSUD and RIP are mechanistically and evolutionarily related.

**387F** **Regulation of fungal effector gene expression through chromatin de-condensation.** *M. Kramer*¹, D.E. Cook¹,², M.F. Seidl¹, B.P.H.J. Thomma¹ 1) Laboratory of Phytopathology, Wageningen University & Research, Wageningen, NL; 2) Department of Plant Pathology, Kansas State University, Manhattan, KS.

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

**388W Validation and characterization of development-related microRNA-like RNAs in the mushroom-forming fungus *Coprinopsis cinerea***
Yuet Ting Lau¹, Xuanjin Cheng¹ & Hoi Shan Kwan¹*.
School of Life Sciences, The Chinese University of Hong Kong¹, Shatin, New Territories, Hong Kong. *Correspondence: hoishankwan@cuhk.edu.hk.
Y.T Lau, H.S Kwan School of Life science, The Chinese University of Hong Kong, Hong Kong, HK.

Introduction. *Coprinosis cinerea* is a model organism for the study of homobasiidiomycete fungi due to its short life cycle and easy cultivation in the laboratory. The function and regulation mechanism of microRNA (miRNA) of animals and plants have been extensively studied. Although emerging studies have suggested that microRNA-like RNAs (milRNAs) are present in fungi and their biogenesis pathway might not be identical as conventional miRNA in plants and animals, milRNAs are still elusive for. The lack of studies on the role of milRNAs in fungi hinders the improvements of mushroom strains.

Aim. We aim to understand the development of mushroom-forming fungi at the molecular level, including RNA-mediated and protein-mediated regulation.

Methods. The presence of milRNAs has been validated experimentally through Northern blotting and Stem-loop Reverse Transcription Real-time PCR. Computational methods have been applied subsequently to characterize the roles of milRNAs in the development of mushrooms, especially during the transition from mycelium to primordium stage.

Results. We have identified, from *Ccinerea* genome, 16 putative milRNAs candidates with sizes around 21nt and a group of Dicer homologs (CC1G_00230, CC1G_03181, CC1G_13988) that are specific to mushroom-forming fungi. The PZA domain is present in the Dicer-like (DCLs) proteins of mushroom, while it is absent in all other fungal DCLs. Besides, for these 16 putative milRNAs candidates, one of them showed higher expression in mycelium stage and 15 showed higher expression in primordium stage.

Discussion. The mushroom-specific PAZ domains were grouped closely with the animal PAZs and plant PAZs and the Dicer_dimer domain sequences alone could distinguish fungal DCLs from their homologs in animals and plants. The DCLs domains of mushroom suggest Dicer genes duplicated and diversified independently in early evolution of all three multicellular kingdoms-animals, plants and fungi. A better understanding of the biogenesis pathways and regulatory mechanisms milRNAs in *Ccinerea* could improve the development of homobasiidiomycete fungi, such as edible ad medicinal mushrooms, contributing to evolutionary studies of miRNA among kingdom in the near future.

**389T Transcriptomic analysis of the development of basidiocarp in *Ustilago maydis*.**
C. Leon-Ramirez, J. Cabrera-Ponce, D. Martínez-Soto, J. Sánchez-Arreguín, J. Ruiz-Herrera, J. Cabrera-Ponce Genetics engeniering, CINVESTAV - CIE6010281U2, Irapuato, Guanajuato, MX.

*Ustilago maydis* is a Basidiomycota pathogenic fungus of corn and teozintle. The fungus has a saprophytic life cycle, and other virulent infective stage which carries out within its host. This phase ends when teliospores germinate with formation of a phragmobasidium that gives rise to four meiotic products. Although *U. maydis* has been classified as a non-forming basidiocarp species, Cabrera-Ponce et al 2012, showed that when *U. maydis* is grown in a dual culture with an embryogenic maize callus under controlled growth conditions, it has the ability to form basidiocarps. The developed structures showed the presence of a hymenium composed of skeletal hyphae, generative hyphae, clamp connections, hyphal pore, and more impressive, holobasidia (not phragmobasidia), to give rise to basidiospores and thus complete this sexual cycle.

One of the tools mostly used to identify the genes involved in the regulation of development are microarray analyses. Using several online programs such as MIPs, FunCat, JGI, Smart; Pfam, NCBI, we used this technique to analyze the transcriptomic changes occurring when the fungus is transformed from the yeast stage to young basidiocarp, and its further establishment as a mature basidiocarp. 2002 genes were found to be regulated in the first step, and 1064 in the second step. Classification of the genes using FunCat software gave ten different categories. The most highly represented categories were Unclassified (31%), and Metabolism and energy with about 25% of the total regulated genes. Search of *U. maydis* homologous regulated genes to those previously described as important in the formation of fruit bodies in other fungal models gave positive results for a number of them. Some genes important for the process encoded a MAPkk, transcription factors Ace2, Foxo3a, Mig3, Pro1, and TEC1; and copper, ABC and MFS transporters, some cytochromes, and hydrophobins. These results indicate the complexity of the developmental process, that show important similarities to fruiting body formation by other fungi.

**390F The histone methyltransferases FfSet2 and FfAsh1 deposit H3K36 methylation at specific loci, having a major impact on vegetative growth, sporulation, secondary metabolism and virulence in *Fusarium fujikuroi*.**
S. Janevska¹, L. Baumann¹, J. Ulrich², J. Kaemper², B. Tudzynski¹ 1) Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, DE; 2) Institute for Applied Biosciences, Karlsruhe Institute of Technology, Karlsruhe, DE.

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

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

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

**391W New regulatory tricks for an old toxin cluster.** Daren W. Brown¹, Alessandra Villani², Antonella Susca², Antonio Moretti², Robert H. Proctor¹, Susan P. McCormick¹ (1) Mycotoxin Prevention and Applied Microbiology Research Unit, USDA/ARS, Peoria, IL; 2) Institute of Sciences of Food Production, CNR, via Amendola 122/0, 70126 Bari, Italy.

Trichothecenes are among the mycotoxins of greatest concern to food and feed safety and are produced by at least two lineages of *Fusarium*: the *F. sambucinum* (FSAMSC) and *F. incarnatum-equiseti* (FIESC) species complexes. Trichothecene biosynthesis begins with the formation of a cyclic sesquiterpene followed by up to eight oxygenation and four acylation reactions. Most trichothecene biosynthetic genes (*TRI*) are co-regulated and located in a cluster. All *Fusarium* *TRI* cluster homologs characterized to date include two regulatory genes that encode a $C_2H_2$ zinc finger (*TRI6*) and a fungal transcription factor domain (*TRI10*). Functional analysis of FSAMSC members indicate that *TRI6* regulates expression of structural genes in the *TRI* cluster, while *TRI10* regulates *TRI6* expression. Here, we characterized a novel gene, *TRI21*, located in the *TRI* cluster of FIESC but not FSAMSC, which encodes a $Zn_2Cys_6$ transcription factor. Gene deletion and precursor feeding analyses with FIESC isolates indicate that *TRI21* is required for expression of two *TRI* cluster genes (*TRI11* and *TRI13*) responsible for two later oxygenation reactions, but not for expression of other cluster genes. In contrast, *TRI6* is required for expression of genes required for earlier and some later steps in trichothecene biosynthesis but not for *TRI11* and *TRI13* expression. These results indicate fundamental differences in the regulation of the *TRI* cluster in FIESC and FSAMSC. Furthermore, the presence of a pseudogenized *TRI21* in some members of FSAMSC indicates that the evolution of *TRI* cluster regulation in FSAMSC has included loss of *TRI21* and expansion of the role of *TRI6* in trichothecene biosynthesis.

**392T Transcriptional profiling and phenotypic analysis to identify genes involved in stress response and conidiogenesis in *Fusarium graminearum*.** C. Blaschke, Z. Bilton, K. Lambert, J. Hamilton, R. Manspeaker, *J. Flaherty* Coker College, Hartsville, SC.

*Fusarium graminearum* is an important plant pathogen that causes major yield-limiting diseases like head blight of wheat and ear rot of maize. *F. graminearum* persists in the soil in the absence of a host and reproduces asexually through the production of conidia (asexual spores) to initiate repeating cycles of infection. Our project involves undergraduate students on both gene discovery in *F. graminearum* using bioinformatics approaches and phenotyping analysis of mutant strains. Candidate genes putatively involved in stress responses/conidiogenesis have been identified through two gene expression projects. RNAseq data obtained from *F. graminearum* PH-1 and two mutant strains, all grown in conditions either conducive or non-conducive for conidiation, was processed using cluster analysis (based on unbiased measures of similarity rather than sorting objects into predefined categories). Attributes for clustering genes from each strain were locus and change in expression. Analysis of this data provides a robust way of examining gene expression patterns to identify candidate genes and gene clusters for future investigation. Towards characterizing specific candidate genes, a series of mutant strains were confirmed as targeted disruption mutants and four of these, found to be closely linked *F. graminearum* genes FGSG_12704, _12705, _12744, and _05476, were interrogated for phenotypes related to growth and development. The characterization of candidate genes identified in this study will help to fill gaps in our understanding of fungal development and facilitate building models depicting developmental processes required for the survival and dissemination of *F. graminearum*.

**393F *FgSRPK1*, a SR protein kinase is involved in vegetative growth, sexual reproduction and virulence in *Fusarium graminearum*.** G. Wang¹, Z. Gong¹, P. Sun¹, J. Xu², Z. Wang¹ (1) Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, China; 2) College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China.

The wheat scab fungus *Fusarium graminearum* has 116 protein kinases that are involved in various aspects of growth, development, stress responses, sexual reproduction and pathogenicity. *FgSRPK1* is homologous to mammalian SR protein kinase *SRPK1*, which is involved in mRNA alternative splicing. In a previous study, the *FgSRPK1* kinase gene was firstly identified as a fungal virulence factor. In this study, we further characterized the roles of *FgSRPK1* gene. Here we show that the deletion of *FgSRPK1* significantly reduced the growth and DON production, and completely blocked sexual reproduction and plant infection. In *FgSRPK1*-GFP transformants, GFP signals were mainly localized to the cytoplasm in conidia and hyphae, but we also observed that GFP signals were localized in nucleus in only a small proportion of hyphae. Interestingly, in both conidia and hyphae FgSrk1 were specifically localized to the center of septal pores, and the same result was observed in rice blast fungus, suggesting that the *FgSRPK1* may have specific functions in filamentous fungi. The *FgSRPK1* gene contains two conserved kinase domains (K1 and K2) that are separated by a spacer domain. We found that expressing of *FgSRPK1* lacking the spacer domain caused nuclear localization of FgSrk1 in conidia and hypha and significantly restored the defects of *FgSrk1* mutant in growth, sexual reproduction and plant infection indicating that the spacer domain may function as an anchor fastening FgSrk1 in cytoplasm, and FgSrk1 has constitutive kinase activity and performs its specific functions by changing subcellular localization. Furthermore, we found that the K2 domain contributes the septal pores localization, because exclusively expressing K2-GFP led to GFP localization in septal pores. Furthermore, we detected that the FgSrk1 lacking spacer domain can interact with SR proteins FgNpl3, FgHrb1 and FgSrp1 respectively by BiFC assays. Recently, we are trying to identify the *SRPK1*-interacting proteins in cytoplasm, nucleus and septal pore by protein affinity purification.
**394W Characterization of the ADAT genes in *Fusarium graminearum*.** Z. Bian¹, H. Liu², Y. Li¹, L. Chen², J. Xu¹,² 1) Botany and Plant Pathology, Purdue University, west Lafayette, IN; 2) College of Life Sciences, Northwest A&F University, yanglin, shanxi.

RNA editing is an event that recodes hereditary information by changing the nucleotide sequence of mRNA molecules. In animals, Adenosine Deaminase Acting on RNA (ADAR) enzymes catalyze the A to I mRNA editing. The wheat scab fungus *Fusarium graminearum* lacks ADAR orthologs but has genome-wide RNA editing specifically during sexual reproduction. The preference of edited As in the loop indicates that fungal A-to-I editing is similar to editing of tRNA in anticodon loop catalyzed by Adenosine Deaminase Acting on RNA (ADAT) enzymes. Three predicted ADAT genes in *F. graminearum*, *FGRRES_16992*, *FGRRES_11590*, and *FGRRES_01444* are orthologous to yeast *TAD1*, *TAD2*, and *TAD3*, respectively. Deletion of *TAD1* had no detected phenotypes but *TAD2* and *TAD3* appeared to be essential genes. Both *TAD2* and *TAD3* were increased during sexual reproduction in *F. graminearum*. Overexpression of *TAD3* had no effects on vegetative growth but resulted in defects in ascospore morphogenesis and releasing. A number of point mutations that affected ascosporogenesis and release but not vegetative growth or perithecium formation were identified in *TAD3*. Further characterization of these point mutations in *TAD3* on A-to-I RNA editing is under the way. Several proteins interacting with Tad3 specifically during sexual reproduction were identified by the affinity approach. Their functions in RNA editing and sexual reproduction also will be presented.

**395T Epigenetics of a cereal killer: fungal pathogen *Fusarium graminearum*.** Christopher T Bonner¹,², Owen Rowland¹, Rajagopal Subramaniam² 1) Biology, Carleton University, Ottawa, Ontario, CA; 2) Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Ca.

*Fusarium graminearum* is a fungal pathogen of cereal crops and causes fusarium head blight disease in wheat. Identifying how *F. graminearum* regulates gene expression is of the utmost importance in understanding pathogenicity. Recent literature has indicated that epigenetics, such as histone modification, plays a key role in regulating genes responsible for producing secondary metabolites. This study focuses on the role that DNA epigenetics, specifically DNA methylation, has on regulating gene expression under changing environmental conditions. Expression of key secondary metabolism genes, such as the deoxynivalenol toxin pathway regulator gene, TRI6, changes rapidly with changing environmental conditions. We hypothesize that changes in DNA cytosine methylation patterns may help regulate environmentally responsive genes, including many secondary metabolite related genes. DNA methylation was assessed through whole genome bisulfite sequencing (WGBS). Methylation sites on genes of interest were validated by methylation specific PCR. This study has identified DNA methylation in *F. graminearum*. Furthermore, methylation patterns change rapidly in response to environmental conditions such as under nutrient rich vegetative growth or nutrient poor, stress growth. Preliminary data indicates that targeted gene deletions of DNA methyltransferase enzymes result in altered morphology, growth, sporulation and pathogenicity on susceptible wheat. Future efforts involve characterizing DNA methyltransferase mutants, and their role in the infection process. The purpose of this study of DNA methylation is to help identify mechanisms of rapid gene regulation in response to changing environmental conditions in the fungal pathogen *F. graminearum*, as well as in other filamentous ascomycete fungi.

**396F Functional analyses of the RNAi pathway required for sexual development in the cereal pathogen *Fusarium graminearum*.** Da-Woon Kim, Hee-Kyoung Kim, Sung-Hwan Yun Dept Med Biotech, Soonchunhyang Univ, Asan, Chungnam, KR.

*Fusarium graminearum*, the causal agent of Fusarium head blight in cereal crops, produces sexual progeny (ascospore) as an important overwintering and dissemination strategy for completing the disease cycle. This homothallic ascomycetous species carries two opposite mating-type (MAT) loci in a single nucleus to control sexual development. Recently, we have identified putative target genes or regulatory pathways controlled by the MAT proteins, among which a RNA interference (RNAi) pathway has been suggested to control a late stage of sexual development. Using several molecular strategies, we have determined the functions of several genes involved in the putative RNAi pathway such as *FgSAD-1*, *FgSAD-3*, *FgDCL-1*, *FgDCL-2*, *FgRecQ-2*, *FgQIP*, *FgSMS-2*, *FgQDE-3*, *FgQDE-2* and *FgQDE-1*. In particular, we have focused on the role of *FgSMS-2* encoding an Argonaute-like protein, which is a part of the RNA-induced silencing complex for specific cleavage of target mRNAs, in *F. graminearum*. Both gene deletion- and gene overexpression-strains of *FgSMS-2* were defective in ascospore/asci maturation. A GFP-tagging analysis showed that FgSMS-2 was specifically localized on perinuclear regions inside the immature asci. A BiFC analysis revealed that FgSMS-2 was able to bind to a Dicer-like protein, FgDCL-1 in cytoplasmic region during the early stage of sexual development. However, three isoforms of the AGO2 protein from *Drosophila melanogaster* were not able to complement the defects caused by *FgSMS-2* in *F. graminearum*, indicating an evolutionary divergence of the AGO-2 like proteins between these two eukaryotes. In addition, we identified a putative target gene (*TAG1*) of FgSMS-2, which showed an unknown protein similarity. The overexpression of *TAG1* led at least 12 h-delay of ascospore formation in *F. graminearum*. Overall, it is likely that the a RNAi pathway plays an important role during the sexual development in *F. graminearum*, particularly the Argonaute-like protein, FgSMS-2 protein, controls a set of mRNAs that might be unnecessary during the meiotic event in *F. graminearum*.

**397W The cyclase-associated protein FgCap1 has both PKA-dependent and -independent functions during DON production and plant infection in *Fusarium graminearum*.** Cong Jiang¹, Tao Yin¹, Qiang Zhang¹, Jianhua Wang², Huiquan Liu¹, Chenfang Wang¹, Jin-rong Xu¹,² 1) Northwest A&F University, Yangling, CN; 2) Purdue University, West Lafayette, US.

*Fusarium graminearum* is a causal agent of wheat scab and a producer of trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (*TRI*) genes and DON production are regulated by the cAMP-PKA pathway and two pathway-specific transcription factors (*TRI6* and *TRI10*). Interestingly, mutants deleted of *TRI6* had reduced expression of several components of cAMP signaling, including the *FgCAP1* adenylate binding protein gene that has not been functionally characterized in *F. graminearum*. In this study, we showed that FgCap1 interacted with Fac1 adenylate cyclase and deletion of *FgCAP1* reduced the intracellular cAMP level and PKA activities. The *Fgcap1* deletion mutant was defective in vegetative growth, conidiogenesis, and plant infection. It also was significantly reduced in DON production and *TRI* gene expression, which could be suppressed by exogenous cAMP, indicating that a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild type but not *tri6* mutant had increased levels of intracellular cAMP and *FgCAP1* expression under DON producing conditions. Furthermore, the promoter of *FgCAP1* has one putative Tri6-binding site that was important for its function during DON biosynthesis but dispensable for hyphal growth, conidiogenesis, and pathogenesis. In addition, FgCap1 has an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was
identified by phosphoproteomics analysis. The S353A mutation in *FgCAP1* had no effects on its functions during vegetative growth, conidiation, and DON production. However, expression of the *FgCAP1*<sup>S353A</sup> allele failed to complement the defects of *Fgcap1* mutant in plant infection, indicating the importance of phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggested that *FgCAP1* is involved in the regulation of DON production via cAMP signaling and subjected to a feedback regulation by *TRI6*, but phosphorylation of FgCap1 at S353 is likely unrelated to the cAMP-PKA pathway because S353A mutation only affected plant infection.

**398T Nucleosome dynamics in the plant pathogen *Fusarium graminearum*.** E. ZEHRAOUI¹, M. MONTIBUS¹,², N. PONTS¹ 1) UR 1264 - MycSA, INRA, Villenave d'Ornon Cedex, FR; 2) Laboratoire de Biologie FCBA, Bordeaux, France.

Nucleosome dynamics are the first level of regulation of all eukaryotic molecular processes that use genomic DNA as a template, including gene expression. Abundant literature on various organisms, notably in yeasts, reports that the position of nucleosomes and their relative stabilities are major parameters influencing gene expression. Changes in nucleosome positioning during cell differentiation and growth are commonly observed in eukaryotes in general. In the yeast *Candida albicans*, such mechanisms were shown to be involved in morphological plasticity that plays a role in virulence.

Here, we report the investigation of nucleosome dynamics during the development *in vitro* of the mycotoxin-producing phytopathogen *Fusarium graminearum*. Nucleosome landscapes were investigated using MNase-Assisted Isolation of Nucleosomal Elements coupled to deep sequencing, or MAINE-seq. The general nucleosomal organisation extensively described in various organisms appears conserved in *F. graminearum*, with most nucleosomes arrayed and well-positioned relative to start and stop codons of genes. In the details, nucleosome dynamics were observed between three and four days of culture, i.e., when growth speeds up and toxin production becomes detectable in our conditions of culture. The observed events regard mostly differences in nucleosome stability, sometimes referred to as occupancy. Additional transcriptomics data provide leading information regarding the significance of these observations.

**399F Identification of novel genes involved in the photoregulation of carotenoid biosynthesis in *Fusarium oxysporum*.** O. Parra-Rivero, J. Avalos, M.C. Limon Department of Genetics, Faculty of Biology, University of Seville, Seville, ES.

The genus *Fusarium* comprises hundreds of saprophytic and pathogenic fungal species widely distributed in nature. In addition to their roles as pathogens, these fungi stand out for their complex secondary metabolism, which includes the production of carotenoids, terpenoid pigments providing the characteristic orange colour to their colonies when grown in the light. As a regulatory model of secondary metabolism in filamentous fungi, our group investigates the molecular mechanisms underlying the control of carotenoids production by *Fusarium*. The stimulation of carotenoid biosynthesis by light is mainly achieved through the induced transcription of three structural genes, *carRA*, *carB*, and *carT*, presumably mediated by the White collar protein WcoA/Wc1. Besides the up-regulation by light, the pathway is down regulated by the RING-finger protein CarS, as indicates the enhanced expression of the structural genes and the high carotenoid accumulation in the *carS* mutants. The *carS* gene is preceded by a 4-kb sequence with no predicted ORFs, where we have identified by RNAseq analysis a novel 1.2-kb transcript. Its sequence lacks significant ORFs and it was disregarded in the annotation of the *F. oxysporum* genome, suggesting that the transcript is a long non-coding RNA (lncRNA). Deletion of this gene, whose orientation was determined by northern and PCR assays, results in an albino phenotype and in a major loss of photoinduction of the structural *car* genes. Due to its presumed role in photoregulation, we named this putative lncRNA *carP*. Interestingly, the *carP* mutant exhibits higher levels of *carS* mRNA, suggesting a connection between *carP* and photoregulation through *carS* expression. At the 5' end of the 4-Kb *carS* upstream region, and divergently transcribed from *carP*, there is a gene coding for a protein of unpredicted function that we called *carF*. Deletion of *carF* causes a decrease in carotenoid production in the light. However, the photoinduction of the structural *car* genes in the *carF* mutant was at least as efficient as in the wild type, suggesting a post-transcriptional regulatory function. In conclusion, this communication suggests the occurrence in *Fusarium* of a *car* regulatory cluster, consisting of gene *carS* and at least two novel regulatory genes participating in the control by light of carotenoid biosynthesis in *Fusarium* through novel regulatory mechanisms, involving the mediation of at least a long non-coding RNA.

**400W A Mitogen-Activated Protein Kinase Gene (*VmPMK1*) is Involved in Virulence and Regulates Pectinase Activity in *Valsa mali*.** Y. Wu, L. Xu, L. Huang College Of Plant Protection, NORTHWEST A&F UNIVERSITY, Yangling, shaanxi, CN.

Mitogen-activated protein kinases (MAPKs) play a critical role in signaling pathways and are involved in the transduction of a variety of extracellular signals and regulation of different developmental processes such as appressorium-formation, conidiation, infectious growth, and regulation of hydrolytic enzymes in many fungal phytopathogens. In the present study, a FUS3/KSS1 MAPK superfamily gene, *VmPmk1*, has been identified and characterized in Valsa mali. *VmPmk1* null mutants show a reduced growth phenotype (-33.2 %) on PDA. When inoculated onto apple leaves and twigs, ΔVmPmk1 mutants showed a decrease in lesion size by about 80 %. Furthermore, transcript levels of pectinase genes were decreased in mutants during infection. VmPmk1 deletion mutants also showed a significant reduction of biomass and enzyme activity on pectin medium. Meanwhile, conidiation have not been found in PDA or 30 % ABA medium, and sensitivity to H₂O₂ were promoted significantly in mutants. In conclusion, VmPmk1 could affect virulence and regulated the pectinase and essential for conidiation and oxidative stresses in *V. mali*.

**401T Terbinafine affects transcription pattern of an ERAD encoding gene in *Trichophyton rubrum*.** T.A. Bitencourt, P.R. Sanches, V.M. Oliveira, A. Rossi, N. M. Martinez-Rossi University of São Paulo, Ribeirão Preto Medical School, Department of Genetics, Ribeirão Preto, SP, Brazil.

The genome of *Trichophyton rubrum*, the main causative agent of dermatophytosis worldwide, was recently sequenced, enlarging knowledge about its genetic structure. Pre-mRNA splicing events occur as an adaptive genetic response to a myriad of cellular stressors. After translation, the newly synthesized proteins are loaded into the endoplasmic reticulum (ER) lumen, where they undergo folding and posttranslational modifications. ER stress is the consequence of the imbalance between protein synthesis and folding, accumulating misfolded proteins, or even triggered by disturbs of the cell wall integrity. Under this stress, the cells switch on the unfolded protein response (UPR), which regulates the ER-folding and ER-associated degradation (ERAD) machinery. The homeostasis of these processes is required to cytoprotection and apoptosis prevention. In this context, we aimed to assess the transcription pattern of an ERAD encoding gene in *T. rubrum* (TERG_04442), and also to examine the retention of intron-3 during exposure of *T. rubrum* to the sub-lethal dose of
terbinafine. To assess gene modulation and intron retention we employed RT-PCR and qRT-PCR techniques by exposing *T. rubrum* mycelia to terbinafine for 12h (MIC70). Our results show that terbinafine exposure leads to a decrease in transcript levels of ERAD encoding gene, and an up-regulation of intron-3 retention. Due to intron-3 retention, the Der1-like domain of the protein is interrupted, probably leading to conformational changes, and possibly interfering with the affinity for the substrates. These data may suggest that an adaptive strategy assures cytoprotection toward an antifungal compound that acts on the ergosterol pathway, which in turn can cause cell wall damage, implying in a fine regulation of mRNA processing and mRNA decay.

Financial support: FAPESP (Process number: 2015/23435-8 and 2014/03847-7), CAPES, CNPq, FAEPA.

**402F Drug resistance via RNAi-dependent epimutations in *Mucor circinelloides*.** Z. Chang¹, B. Billmyre¹, S.C. Lee², J. Heitman¹
1) Duke University, Durham, NC; 2) University of Texas at San Antonio, San Antonio, TX.

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

**403W Comparative analysis of LysM genes in the dermatophyte *Trichophyton rubrum*.** L. Lopes¹, E. A. S. Lang¹, N. T. A. Peres², A. Rossi¹, N. M. Martinez-Rossi¹
1) Department of Genetics, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil; 2) Department of Morphology, University of Sergipe, Aracaju, Sergipe, Brazil.

*Trichophyton rubrum* is a dermatophyte that shows a high incidence of cutaneous infections in humans, are persistent and difficult to treat. Therefore, studies are crucial to elucidate the mechanisms involved in pathogenicity and the disease process. The genome of dermatophytes presents a high number of genes encoding proteins with the LysM domains that bind chitin and related carbohydrates. However, it is unclear whether these proteins are related to chitin ligation in the fungal cell wall, and if this interaction could be involved in the relationship between dermatophytes and the host. Our aim is to characterize these LysM genes in *T. rubrum* using gene expression studies and in silico analysis. Our analysis revealed that LysM proteins showed signal peptide and hydrophobic regions, suggesting a possible anchorage in the cytoplasmic membrane. Furthermore, there was no accumulation of LysM transcripts in the conidia and, during growth for 48, 72 and 96 hours, some genes were expressed only in later times of cultivation, in different keratinous substrates, suggesting that they have function preferentially at alkaline pH. These results provide new information and contribute to the better understanding of the functions of LysM genes in dermatophytes.

Financial Support: FAPESP, CNPq, CAPES, and FAEPA.

**404T New insights into the transcriptional regulation of carotenoid genes in *Fusarium fujikuroi*.** Steffen Nordzieke, Macarena Ruger-Herreros, M. Carmen Limón, Javier Avalos Department of Genetics, University of Seville, Sevilla, ES.

Carotenoids include over 600 different terpenoid pigments, produced by bacteria, plants, and fungi. Their diverse individual abilities include coloration, protection against light induced or oxidative damage, and their use as a source for other physiologically active compounds. All structural genes of the carotenoid pathway (*car* genes) in *Fusarium fujikuroi* have been identified, while their regulation remains to be elucidated. The *car* genes are organized both in the form of a cluster and autonomously, and their light induced expression leads, among others, to the production of neurosporaxanthin.

CarS, a major regulator of the cluster in this fungus, down-regulates transcription of all involved car genes despite its inability to bind directly to the corresponding promoters. Thus, we postulate the participation of other intermediating proteins as part of the regulation process. By analyzing mutant phenotypes in combination with promotor-binding studies, Y2H, and co- immunoprecipitation experiments, we aim to describe a network of car-interacting proteins and transcription factors: The analysis of the proteins binding the promoters of the car genes show that transcriptional regulation of the carotenogenic pathway in *F. fujikuroi* is likely to include transcription factors of the high-mobility group. Interestingly, the identification of different sets of putative binding proteins suggest that different mechanisms govern the parallel biosynthetic pathways leading to the production of Neurosporaxanthin and retinal. Other results suggest that the impact of CarS on the transcription of the *car* genes is highly regulated through alternative splicing, an infrequent event in filamentous fungi, known to influence only 1.3 to 4.3% of transcripts. Using a combined RNAseq / RT PCR approach, we were able to identify four different splicing variants of CarS in *F. fujikuroi*, leading to proteins with alternative C-terminal parts. However, the functional significance of this alternative splicing process remains to be investigated.

**405F Identifying carbohydrate sensing pathways in *Neurospora crassa*.** L.B. Huberman, S.T. Coradetti, N.L. Glass Plant and Microbial Biology, UC Berkeley, Berkeley, CA.

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

**406W Regulation of Sulfur Metabolism in Neurospora crassa.** J.V. Paietta Biochemistry and Molecular Biology, Wright State University, Dayton, OH.

The sulfur regulatory system of *Neurospora crassa* is composed of a set of structural genes involved in various aspects of sulfur metabolism that are controlled by the CYS3 transcriptional activator and SCON (sulfur controller) negative regulators. We are analyzing the entire set of structural genes involved in sulfur metabolism for control by the CYS3/SCON regulators. While previous work has focused on transsulfuration, we examine here in detail two steps connected to this important metabolic process. The *met-5*\+ and *met-8*\+ genes were cloned and characterized to this regard. *met-5*\+ encodes homoserine O-acetyltransferase which produces O-acetylhomoserine from homoscysteine and acetylCoA. O-acetylhomoserine can be subsequently converted into cystathionine (which is central to transsulfuration). *met-8*\+ encodes methionine synthase which generates methionine from 5-methyl tetrahydrofolate and homocysteine (a product of transsulfuration). Using Northern blot analysis, transcripts were detectable under high sulfur growth conditions for *met-5*\+ and for *met-8*\+; however, under low sulfur growth conditions the transcript levels were substantially up-regulated. Further experiments using a *cys-3* deletion mutant showed a loss of the up-regulation observed under the low sulfur (or derepressing) conditions for both *met-5*\+ and *met-8*\+; while maintaining basal transcript levels. Additional experiments detected the presence of CYS3 activator binding sites on the promoters of the *met-5*\+ and *met-8*\+ genes. The data further adds to our understanding of the regulatory dynamics involved in sulfur metabolism.

**407T Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in pre-mRNA splicing, alternative splice-site choice and stress-regulated splicing.** G. Melangath¹, T. Sen¹, P. Bawa², R. Kumar¹, S. Banerjee¹, N. Vijaykrishna¹, S. Srinivasan², U. Vijayraghavan¹ ¹) Microbiology/Cell Biology Labs, Indian Inst Sciences, Bangalore, Karnataka 560012, IN; ²) Institute of Bioinformatics and Applied Biotechnology, Bangalore Karnataka 560100, IN.

The fission yeast genome with numerous short introns, degenerate splice site elements and factors for alternative splicing is an apt model for studies on splicing by intron-definition as relevant to most fungi. Here, we report *Schizosaccharomyces pombe* SpSlu7 and SpPrp18 have essential, wide-spread but intron-specific splicing functions. Our analyses using strains with conditional expression of *SpSlu7* or *SpPrp18* and in mis-sense mutants reveal their genome-wide splicing functions. Statistical analysis of these global data ascribe multiple intronic features such as branch nucleotide-to-3' ss distance and A/U content at the 5' end co-relate with an intron's dependence on SpSlu7. However, features like polypyrimidine tracts between 5'ss and branch nucleotide and presence of non-consensus nucleotides in the 5'ss co-relate with a role for SpPrp18. Primer extension analyses, on *tflld+* intron 1 mini-transcript show the absence of either factor causes arrest with high levels of pre-mRNA and little or no lariat intron-exon 2 splicing intermediate. These data and the genetic interactions of *spprp18* and *spslu7* with mutants in other early-acting factors show novel early splicing functions for *SpSlu7* and *SpPrp18* that differ from the role of their budding yeast orthologs in the later step of second step 3'ss cleavage.
Use of alternative 5' or 3'ss in introns of a transcript can create spliced isoforms that vary in their relative abundance. Our studies on *ats1+* isoforms in wild type cells and in mutants show SpSlu7 can influence the exon 3 skipped mRNA isoform levels. Whereas SpPrp18 affects the abundance of the intron 2 retained isoform. Environmental stresses too alter the ratio of *ats1+* transcript isoforms where all but the fully spliced mRNA are RNA species destined for degradation. Overall, our data imply substrate-splicing factor relationships in the multi-intron transcripts of fission yeast differ from that in budding yeast and our data show alternative spliced isoforms allow for an additional level gene regulation in fission yeast.

**408F Bacteria induce pigment formation in the basidiomycete *Serpula lacrymans*.** J. Tauber¹, V. Schroeckh², D. Hoffmeister¹, E. Shelest², A. Brakhage² ¹) Friedrich-Schiller-University, Jena, Germany; ²) Leibniz Institute for Natural Product Research and Infection Biology– Hans Knöll Institute, Jena, Germany.

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

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

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

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

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

**409W Codon Usage is an Important Determinant of Gene Expression Levels Largely Through its Effects on Transcription.** Zhipeng Zhou¹, Yunkun Dang¹, Mian Zhou¹,³, Lin Liu², Chien-Hung Yu¹, Jingjing Fu¹, She Chen², Yi Liu¹ ¹) The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA; ²) National Institute of Biological Sciences, 7 Life
Science Park Rd., Changping District, Beijing 102206, China ; 3) State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237.

Codon usage biases are found in all eukaryotic and prokaryotic genomes and preferred codons are more frequently used in highly expressed genes. The effects of codon usage on gene expression were previously thought to be mainly mediated by its impacts on translation. Here we show that codon usage strongly correlates with both protein and mRNA levels genome-wide in the filamentous fungus Neurospora. Gene codon optimization also results in strong up-regulation of protein and RNA levels, suggesting that codon usage is an important determinant of gene expression. Surprisingly, we found that the impact of codon usage on gene expression results mainly from effects on transcription and is largely independent of mRNA translation and mRNA stability. Furthermore, we show that histone H3 lysine 9 trimethylation is one of the mechanisms responsible for the codon usage-mediated transcriptional silencing of some genes with non-optimal codons. Together, these results uncovered an unexpected important role of codon usage in open reading frame sequences in determining transcription levels and suggest that codon biases are adaptation of protein coding sequences to both transcription and translation machineries. Therefore, synonymous codons not only specify protein sequences and translation dynamics but also help determine gene expression levels.

**410T The transcriptional activators RhaR, AraR and GaaR co-regulate pectin degradation in Aspergillus niger.** J. E. Kowalczyk, R. J. M. Lubbers, E. Battaglia, R. P. de Vires Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, the Netherlands.

*Aspergillus niger* produces an arsenal of extracellular enzymes that allow step-by-step degradation of organic matter found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act sequentially. Production of pectinolytic enzymes in *A. niger* is highly regulated, which allow flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in *A. niger*. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I (RG-I) [1]. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of RG-I [2]. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin [3]. This project aims to understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of those regulators and analyzed their growth phenotype and pectinolytic gene expression in *A. iger* grown on different pectins.

1. Gruben, B.S., Zhou, M., Wiebenga, A., Ballering, J., Overkamp, K.M., Punt, P.J., and de Vries, R.P. (2014) *Aspergillus niger* RhaR, a regulator involved in L-rhamnose release and catabolism. *Applied Microbiol Biotechnol* 98(12): 5531-5540.

2. Battaglia, E., Visser, L., Nijssen, A., van Veluw, J., Wösten, H.A.B., and de Vries, R.P. (2011) Analysis of regulation of pentose utilisation in *Aspergillus niger* reveals evolutionary adaptations in the Eurotiales. *Stud Mycol* 69: 31-38.

3. Alazi, E., Niu, J., Kowalczyk, J.E., Peng, M., Aguilar Pontes, M.V., Van Kan, J.A.L. et al. (2016) The transcriptional activator GaaR of *Aspergillus niger* is required for release and utilization of D-galacturonic acid from pectin. *FEBS Letters* 590(12): 1804-1815.

**411F The effects of modification of the cellulase regulator ace3.** Mari Valkonen, Marika Vitikainen, Markku Saloheimo, Tiina Pakula Industrial Biotechnology, VTT Technical Research Centre of Finland Ltd., Espoo, Finland.

The *ace3* gene was originally identified from transcriptome data of *Trichoderma reesei* cultivated in the presence different carbon sources and it was found to be co-induced with cellulase/hemicellulase genes. Deletion of the gene was detrimental to the production of cellulase activity, reduced hemicellulase gene expression as well as expression of the activator *xyr1*. Overexpression of *ace3*, which is located next to a co-regulated β-glucosidase gene (*cel1b*) and a candidate sugar transporter gene in its original locus, resulted in a significantly increased production of cellulase and xylanase activity as compared to the parental strain. Therefore, *ace3* can be considered to code for a novel master regulator of cellulase expression and a modulator of xylan degrading enzyme expression. Increased cellulase production was obtained by overexpression of the *ace3* according to the predicted gene model in the QM6A.

However, the gene model predicted later for Rut-C30 is different. The Rut-C30 gene model contains an additional short exon separated by a very long intron (>400bases) in the 5'end of the gene, so that the DNA binding domain is split by the intron. In the QM6A model the DNA binding domain is partial. RNA seq data supports the idea that both short and long form may exist, the gene region for the N-terminal exon gives weaker signals.

We have tested the effects of the overexpression of the different predicted *ace3* forms in *T. reesei* and were able to conclude that the QM6A annotated gene model improves cellulase activity production the most. The gene model with longer 5' end of *ace3* gene has little effect to cellulase activity in the strains studied. Expression profiling experiments show that deletion of *ace3* causes large amount of differences in the data. In the data, both negative and positive effects of *ace3* on gene expression were seen.

**412W RNA-Editing sites controlling CAZyme production in fungus Coprinopsis cinerea.** J. Chang, H. S. Kwan School of life sciences, The Chinese University of Hong Kong, Sha Tin, NT, HK.

Our goal is to elucidate the regulatory process of the enzymatic degradation of different lignocellulosic substrates by the model mushroom Coprinopsis cinerea, and identify RNA editing sites involved in lignin degradation regulation on different substrates or inducers in Coprinopsis cinerea by in silico analyses.

C. cinerea is a model organism in studies of homobasidiomycetes biology for its short life cycle under laboratory conditions. This fungus has been a target to study carbohydrate active enzymes (CAZymes) that take part in lignocellulose degradation, an important step in biomass to biofuel process. We know little about the regulation of CAZymes production and signal transduction pathways in fungi growing on different lignocellulosic substrates. Supported by next generation sequencing, there are many studies on the transcriptome studies of mushroom forming fungi. Our laboratory has studied the biology of fungi for more than 15 years. We have recently obtained RNA-seq gene expression profiles of 46 single spore isolates of C. cinerea and conducted eQTL analysis. Unlike general belief that gene expression varies with different genotypes of the transcription factors, we could not observe SNPs in transcription factors that associate with CAZymes expression. On the other hand, we found that gene expression is associated with the RNA editing sites on several transcription factors. Based on this
observation, we developed an expression Quantitative Trait RNA editing sites (eQTR) strategy to identify regulators harboring RNA editing sites. eQTR represents a recoding base in the RNA different from its template DNA. Such variation between individuals could affect mRNA abundance, a quantitative gene expression trait. The basic model included the gene expression level as a response variable, with the RNA editing type as the predictor. We have used the *C. cinerea* genome sequences and RNA sequences to call RNA editing sites. Together with the gene expression profile from the same set of samples, some transcription factors were identified that link and may regulate CAZyme expression.

To our knowledge, this would be the first functional study on RNA editing sites for roles in CAZymes production in mushroom forming fungi. Identification and functional characterization of regulators in CAZymes production will contribute to our understanding of the fungal lignocellulose degradation with potentials to benefit enzyme production industry.

**413T** *LaeA* regulation of a MAK-1 sensor complex is required for cellular communication in *Aspergillus flavus*. *x. zhao*¹,², *J. Sparker*³, *J. Bok*¹, *Z. He*², *N. Keller*¹,³ ¹) medical microbiology and immunology, University of Wisconsin-Madison, Madison, WI; ²) MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; ³) Department of Plant Pathology, University of Wisconsin - Madison, Madison, WI 53706-1598, USA.

*Aspergillus flavus* is a saprophytic soil fungus that poses a serious threat worldwide as it contaminates many food and feed crops with the carcinogenic mycotoxin called aflatoxin. *Aspergillus flavus* survives as sclerotia in soil which enables survival in harsh environmental conditions. Sclerotial formation by *A. flavus* depends on successful cell communication and hyphal fusion events. Loss of *LaeA*, a member of the conserved developmental Velvet Complex, abolishes sclerotial formation in this species whereas overexpression (OE) of *laeA* results in enhanced sclerotia production. Here we demonstrate that sclerotial loss and inability to form heterokaryons in *A. flavus* ? *laeA* is mediated by a MAK-1 sensor complex encoded by homologs of the *Neurospora crassa* ham (hyphal anastomosis mutants) genes termed *hamF-I* in *A. flavus*. *LaeA* positively regulates *ham* gene expression with near loss in ?*laeA* and higher expression in an OE::*laeA* strain. Phenocopying ?*laeA*, deletion of *hamF*, *G*, *H*, or *I* results in both heterokaryon and sclerotial loss. This is accompanied by a significant decrease in aflatoxin synthesis in the *ham* mutants, presumably due to sclerotial loss and decreased heterokaryon formation.

**414F** Light controls dimorphic transitions in the human fungal pathogen *Histoplasma capsulatum*. *S. Beyhan*¹,², *M. Voorhies*², *A. Sil*² ¹) J. Craig Venter Institute, La Jolla, CA; ²) University of California, San Francisco, San Francisco, CA.

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

**415W** Light conservatively regulates the ergosterol biosynthetic pathway in the mold pathogens *Aspergillus* and *Fusarium*. *Kevin Fuller*¹, *Mike Zegans*², *Robert Cramer*³, *Jennifer Loros*⁴, *Jay Dunlap*¹ ¹) Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH; ²) Department of Ophthalmology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; ³) Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH; ⁴) Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH.

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

**416T** The expression of the rice blast effector *PWL2* is induced during appressorium-mediated and hyphal penetration into live plant cells. *J. Zhu*, D.W. Kim, C.H. Khang Department of Plant Biology, University of Georgia, Athens, GA.

Rice blast, caused by the filamentous fungus *Magnaporthe oryzae*, is one of the world's most devastating agricultural diseases. During plant infection, the fungus produces various secreted proteins, called effectors, to modify plant cell structure and function to facilitate colonization. Given the significant roles played by effectors, there is surprisingly little information about how the expression of effector genes is regulated. To investigate effector gene expression at the cellular level, we generated transgenic *M. oryzae* strains expressing a fluorescent reporter gene under control of the endogenous promoter of the *M. oryzae* effector gene *PWL2*. Using this method, we revealed that the expression of *PWL2* was strongly induced in the appressorium upon plant penetration and subsequently decreased during the proliferation of invasive hyphae (IH) in the first invaded rice cell. Interestingly, *PWL2* expression was induced again when IH moved into adjacent rice cells. This up-down-up expression pattern was consistent with our qRT-PCR data. We further determined that strong induction of *PWL2* expression is not host specific, but requires hyphal penetration into live plant cells. A promoter deletion study determined that a 388-bp sequence upstream of the *PWL2* coding sequence was sufficient for high-level promoter activity in planta. Taken together, we propose that *M. oryzae* recognizes an unknown live plant-derived cue(s) during plant penetration to induce the effector gene expression.

**417F** Dissecting the molecular basis of temperature response in *H. capsulatum*. *D. Assa*, M. Voorhies, A. Sil Sil Lab, Microbiology and Immunology Dept., Univ. of California, San Francisco, San Francisco, CA.

*Histoplasma capsulatum* is the causative agent of histoplasmosis, the most common endemic mycosis in the United States. Thermally dimorphic fungi use temperature as a critical signal to sense whether they are growing in the soil or in a mammalian host: environmental temperatures trigger a growth form that is specialized for soil colonization whereas mammalian body temperature triggers a growth form that is specialized for growth in the host. These organisms utilize unknown signal transduction pathways to dramatically alter cell shape and gene expression programs in response to temperature. I hypothesize that temperature change triggers a conformational change in an unknown sensor molecule(s), resulting in activation of a thermosensory pathway that allows the fungus to adapt to growth in the environment or in the host. This conformational change could affect the activity or stability of the sensor molecule. I propose a multipronged approach to explore aspects of thermodetection in *H. capsulatum*, by (1) inspecting a potential thermosensory mechanism in the transcript of *RYP1*, a master regulator of morphology, (2) conducting a forward genetic screen to identify previously unknown regulators that participate in the immediate response to temperature change, and (3) taking an unbiased, whole genome approach to identify RNA molecules that alter their conformation in vivo in response to temperature and partake in temperature response.

**418W** Investigating how a thermally dimorphic fungal pathogen, *Histoplasma capsulatum*, senses and responds to its host environment. *Lauren Rodriguez*, Sarah Gilmore, Sinem Beyhan, Anthony Myint, Mark Voorhies, Anita Sil Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, Ca.

Thermally dimorphic fungal pathogens utilize temperature as a signal to distinguish between growth in the environment and growth within a mammalian host. Growth at 37°C triggers alterations in morphology and gene expression. We study *Histoplasma capsulatum*, which exists in a multicellular hyphal phase in the soil that transitions to a unicellular yeast phase after inhalation by a mammalian host. Here we describe the identification of insertion mutants that are locked in the yeast phase independent of temperature. These mutants are either defective in sensing temperature or transducing the temperature signal. One insertion mutation disrupts the regulation of the *MSB2* gene, whose ortholog encodes a putative osmosensor in *Saccharomyces cerevisiae*. Gene expression profiling of the *MSB2* mutant revealed that, unlike wild-type cells, the transcription factor Efg1 fails to be induced at room temperature in the yeast-locked mutant. Since Efg1 promotes filamentation in *C. albicans*, we explored the role of Efg1 in filamentation in *H. capsulatum*. We determined that overexpression of Efg1 is sufficient to promote filamentation even at 37°C. Taken together, these data suggest that Msb2 may be required for Efg1 induction to promote filamentation in response to temperature. We are currently characterizing the role of *MSB2* in temperature response pathways in *H. capsulatum*.

**419T** Evidence for post-transcriptional control of alternative oxidase expression in Neurospora crassa. *Natasa Bosnjak*, Iman Asaria, Frank Nargang Biological Sciences, University of Alberta, Edmonton, Alberta, CA.

In the event of a disruption of the standard mitochondrial electron transport chain, the nuclear-encoded *aod-1* gene is induced in *Neurospora crassa*. *Aod-1* encodes an alternative oxidase (AOX) that provides a branch point off the standard chain. When present, AOX transfers electrons from coenzyme Q directly to oxygen. In normal (non-inducing) conditions, AOX is transcribed at very low levels and no protein is detectable. We previously identified a strain of *N. crassa* that carries a mutation in the tyrosinase gene (T), that produces relatively high amounts of *aod-1* transcript in non-inducing conditions. However, we are unable to detect AOX protein in the strain. Growth of the strain in inducing conditions results in an increase in transcription and production of the protein. Two questions arise from these findings. First, why are there *aod-1* transcripts present in this strain non-inducing conditions and second, why is there no AOX protein present in these cells? Genetic analysis has shown that the tyrosinase mutation is not responsible for the increased levels of *aod-1* transcript. In addition, we have shown that there are no significant alterations in the *aod-1* promoter region of the strain nor in the transcription factors controlling *aod-1* expression. Thus, we suggest that an unknown factor(s) results in the transcription of the *aod-1* gene in this strain under non-inducing conditions. Similarly, an unknown factor(s) prevents the transcript from being translated under non-inducing conditions.

**420F** Circadian clock control of translation in Neurospora crassa. *K.D. Castillo*, S.Z. Caster, D Bell-Pedersen Biology, Texas A&M University, College Station, TX.

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

**421W** **Changes in the proteome of Neurospora crassa in Response to Quinic Acid.** Kayla Brown¹,², Katie Allen¹,², Dana Tirabassi¹,², Gary Walker¹,², David Asch¹,² 1) Biological Sciences, Youngstown State University, Youngstown ; 2) Center for Chemical Biology.

The filamentous fungus *Neurospora crassa*, serves as an ideal model for eukaryotic organisms. Like many fungi, *N. crassa* is able to utilize many different carbon sources for energy. This requires the presence genes that encode for metabolic pathways which are not always needed. One such pathway is the pathway to metabolize quinic acid. The genes coding for this pathway, the quinic acid (*qa*) gene cluster, have been studied for many years as a model of eukaryotic gene regulation. Recently it has been noted that the expression of many other genes outside the *qa* gene cluster is affected by the presence of quinic acid. In this study we have compared the proteomes of *N. crassa* grown on sucrose with that of *N. crassa* grown on quinic acid. We have identified several proteins that are present during growth on quinic acid but not present during growth on sucrose. We are presently confirming these observations using qRT-PCR.

**422T** **DNA sequence homology triggers cytosine-to-thymine mutation by a heterochromatin-related pathway.** Eugene Gladyshev, Nancy Kleckner Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA.

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

**423F** **Circadian clock regulation of translation initiation through eIF2α phosphorylation** Shanta Karki and Deborah Bell-Pedersen. Center for Biological Clocks Research, Department of Biology, Texas A&M University, College Station, TX

77840. Shanta Karki Dept of Biology, Texas A&M University, College Station, TX.

Roughly half of eukaryotic mRNAs accumulate with a circadian rhythm, demonstrating the profound impact of the clock on gene expression. In addition, mounting evidence supports a role for the circadian clock in controlling mRNA translation, extending the influence of the clock on gene expression beyond its role in rhythmic transcription control. However, the mechanisms and extent of translational regulation by the clock are largely unknown. Previous studies in our lab revealed that the clock, through rhythmic activation of the stress-associated p38 MAPK pathway, regulates the activity of translation elongation factor eEF-2 in Neurospora crassa. This discovery prompted us to also examine if the clock controls translation initiation, where we first focused on determining if the activity of eIF2α, a conserved component of the translation initiation machinery, is clock-controlled. In fungi, phosphorylation of eIF2α is accomplished by GCN2 kinase, which is activated in response to nutrient starvation. While phosphorylation of eIF2α blocks translation initiation of most mRNAs, some mRNAs are actively translated, including mRNAs encoding proteins involved in adaptation to stress. We discovered that phosphorylation of eIF2α is clock-controlled, peaking during the subjective day, and that the Neurospora eIF2α kinase CPC-3, a homolog of GCN2, is necessary for phosphorylation of eIF2α. In addition, we showed that the levels of *cpc-3* mRNA and CPC-3 protein accumulate rhythmically. Consistent with these data, we found using *in vitro* translation assays that mRNA translation is reduced using cell-free translation extracts made from WT cells harvested at the peak of phospho-eIF2α levels, as compared the trough. As predicted, this translation rhythm was abolished in clock mutant and Δ*cpc-3* cells. Experiments are in progress to determine the impact of rhythmic phospho-eIF2α levels on mRNA translation and rhythmic protein accumulation using ribosome profiling, coupled with RNA-seq, in wild type versus Δ*cpc-3* cells.

**424W** **Transgenic Editing of Neurospora Crassa as a Tool for Studying Oscillatory Reproductive Cycle Synchronization.** Brooke Hull¹, Michael Judge¹, Zhaojie Deng², Carmen Rodriguez¹, James Griffith¹, Jonathan Arnold¹, Leidong Mao² 1) Genetics, University of Georgia, Athens, GA; 2) Regenerative Bioscience Center, University of Georgia, Athens, GA.

Circadian rhythms are a well-characterized emergent property in Neurospora crassa with known responses to external factors such as temperature and light, but the effect of intercellular communication on this system is not understood. Our group has recently demonstrated synchronicity of cells over time in interactive cell populations, but not in those which are physically barred from cell-to-cell interaction. We therefore hypothesize cells can communicate circadian time to each other, resulting in synchronization of cell clock phase. To directly measure the synchronicity of cells over time we have engineered a second construct with yellow fluorescence. Venus, a yellow fluorescent protein was introduced into the genome in codon optimized form under the control of the promoter of the *clock* controlled gene 2 using CRISPR Cas9 guided homologous recombination. When used in tandem with an existing red fluorescence construct, this new strain allows visualization and direct study of circadian synchronicity in *N. crassa* on both a single cell and on a population scale.
**425T** The RUV-1/2 proteins have a functional role in heat shock response in *Neurospora crassa*. Pablo Acera Mateos, Jonatas Erick Campanella, Fernanda Freitas, *Maria Célia Bertolini* Departamento de Bioquímica e Tecnologia Química, Instituto de Química, UNESP, Araraquara, BR.

In a previous work, using a combination of pulldown assay and mass spectrometry, we identified the *Neurospora crassa* RUV-1 protein that binds to STRE (STress Response Element), a motif present in promoters of stress-responsive genes. This protein, together with its paralogue RUV-2, belongs to the AAA⁺ ATPase protein family, and is annotated in the *N. crassa* database as an ATP-dependent DNA helicase. The proteins have been identified as components of several macromolecular complexes being implicated in many cellular processes. Both paralogous proteins are described in different organisms (*Homo sapiens* and *Saccharomyces cerevisiae*) as partner proteins that interact with each other in different complexes, such as histone acetyltransferase Tip60 complex, the chromatin remodelling complexes Ino80 and SWR-C, and the telomerase complex, among others. In this work, we demonstrate by EMSA that recombinant RUV-1 binds specifically to the STRE motif, confirming our previous results. Gene expression assays showed that the *ruv-1* transcript and the RUV-1 protein are up-regulated in the fungus submitted to heat stress. The *ruv-2* transcript is not regulated under heat stress; however, *ruv-2* mRNA undergoes an alternative splicing (intron retention) under heat stress (45°C), in which a 75 bp intron is not removed leading to the synthesis of a truncated protein having the 97 N-terminal amino acid residues. Cellular localization analyses showed that both proteins translocate to nucleus under heat stress. Experiments to investigate if both proteins interact each other are currently underway. Financial Support: CNPq and FAPESP

**426F** Effects of *mus-52* gene disruption and extracellular phosphate availability in the regulation of an ATP Synthase in *Neurospora crassa*. Maira Martins, *Eriston Gomes*, Pablo Sanches, Wellington Pedersoli, Nilce Martinez-Rossi, Antonio Rossi Ribeirão Preto Medical School, Genetics Department, University of São Paulo, SP – Brazil.

The completion of *N. crassa* genome sequence and the availability of a set of mutant strains individually knocked-out in particular genes, such as the *mutagen sensitive-52* (*mus-52*), provide an efficient tool for gene targeting. *N. crassa mus-52* (NCU00077) is homologous to human *KU80*, which is involved in telomere maintenance; apoptosis process; DNA replication; gene transcription regulation; and especially in non-homologous end-joining of double-stranded DNA breaks. In the absence of *mus-52* the homologous recombination is considerably increased, a molecular event and its consequences that are yet not well understood. This work aimed to estimate the effects of inorganic phosphate (Pi) availability and the *mus-52* deletion in gene transcription regulation, using two *N. crassa* strains (74A [wild-type] - FGSC#2489 and *Δmus-52* - FGSC#9568). All strains were maintained on Vogel's minimal medium, pH 5.8, at 30°C. Conidia from each strain were germinated for 5 h at 30°C in an orbital shaker (200 rpm), in low-Pi (10µM) and high-Pi (10mM) media. *N. crassa* hypothetical protein (NCU06977), which showed over than 70% of identity with a *Colletotrichum graminicola* ATP synthase F0 subunit, was used as gene reference. Thus, when the wild-type strain was subjected from low-Pi to high-Pi, the relative expression was up-regulated; interestingly, when the mutant *Δmus-52* was submitted to the same condition, the expression was down-regulated. When compared the expression fold-change between the wild-type and *Δmus-52* in low-Pi, the mutant strain showed an up-regulation, but in high-Pi condition, the expression was down-regulated. These results indicate that the *N. crassa* wild-type strain modulates the ATP synthase transcription according to with Pi availability. But the absence of *mus-52* indeed affects the regulation of this gene, being oppositely modulated in both strains and all conditions tested probably by the directly binding of Mus-52 or the reducing access of the enhancers and transcription factors to the ATP synthase promoter region.

**Financial Support:** FAPESP, CNPq, CAPES, and FAEPA.

**427W** Epigenetic suppression of transposons co-opted as global regulator of virulence gene expression in a fungal phytopathogen. Adam P. Taranto, Megan C. McDonald, Peter S. Solomon Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, Australia.

Many fungal plant-pathogens display a rapid transcriptomic switch in gene expression upon infection of a susceptible host. We have explored the role of epigenetic suppression in regulation of this shift towards virulence in the wheat-pathogen *Parastagonospora nodorum*. We have characterised the methylome of *P. nodorum* via whole genome bisulfite sequencing and assessed the contribution of DNA-methylation to gene expression by RNA-seq. Genomic regions subject to epigenetic control were enriched in transposable elements, effector-like genes, and putatively horizontally acquired genes. Key necrotrophic effectors, *ToxA*, *Tox1* and *Tox3* occur in close proximity to transposons. Molecular disruption of epigenetic silencing mechanisms active in these regions (H3K9me3, H3K27me3, and 5mC DNA methylation) allowed for partial recapitulation of the early-infection transcriptomic profile of *P. nodorum*. Similar epigenetic reprogramming of pathogen gene expression was achieved through application of chemical inhibitors of epigenetic pathways.

**428T** The photoreceptor WcoB interacts with carotenogenic proteins in the cytoplasm of *Phycomyces blakesleeanus*. A. Miralles Durán, MA Sánchez-Romero, LM Corrochano Genetics, University of Seville, Seville, Sevilla, ES.

*Phycomyces blakesleeanus* is sensitive to environmental signals such as light, wind, gravity and pressure. Light modifies the direction of growth of the fruiting body, sporangiophore, (phototropism), stimulates the production of beta-carotene in the mycelium and regulates the development of the sporangiophores. Blue light is sensed through the Mad complex, a transcription factor complex composed of MadA and MadB. MadA and MadB are homologs of WC-1 and WC-2 from *Neurospora crassa*. The *Phycomyces* genome has three genes homologous to *wc-1: madA*, *wcoA* and *wcoB*; and four genes homologous to *wc-2: madB*, *wctB*, *wctC* and *wctD*. WcoB contains a LOV domain for chromophore binding, but lacks the Zn finger domain. We have characterized the localization of WcoB in the mycelium of Phycomyces using an antibody raised against a peptide of WcoB. The gene *wcoB* is induced by light in vegetative mycelia producing an increase in the amount of WcoB. In order to identify the cellular localization of WcoB we performed cellular fractionations using cultures grown in the dark or exposed to 30 min of light. We detected WcoB in the cytoplasmic fraction of cellular extracts, while the nuclear fraction was devoid of WcoB. Immunofluorescence assays with spores or germinating mycelia showed that WcoB was detected as localized patches in the cell membrane. Our results suggest that WcoB does not act as a transcription factor and is located in the cell membrane. In order to identify proteins that interact with WcoB we performed immunoprecipitation assays. Candidate proteins were excised from the electrophoresis gel and sequenced. Several proteins were immunoprecipitated with WcoB: HMG-CoA, CarRA, CarB and CarS. To
corroborate the location of WcoB and other WC proteins in vivo, we have used *Aspergillus* as a model due to the absence of an efficient transformation system for *Phycomyces*. In addition we have confirmed the interactions between WcoB and *Phycomyces* enzymes using the Bimolecular Fluorescence Complementation System (BiFC) in *Aspergillus*. Our results suggest that WcoB is a photoreceptor for the regulation of the enzymes that participate in the biosynthesis of beta-carotene and pheromones.

**429F Comparative proteomics and phosphoproteomics to decipher signal transduction in Botrytis cinerea.** J. Kilani<fnref n="1" />,³, M. Davanture², M. Zivy², S. Fillinger<fnref n="1" /> 1) UMR BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France; 2) PAPPSO, UMR GQE, INRA, CNRS, Univ Paris-Sud, AgroParisTech, Université Paris-Saclay, Gif-sur-Yvette, France; 3) Univ. Paris-Sud, Université Paris-Saclay, France.

Adaptation to changing environmental conditions takes place rapidly in all living organisms: cells must respond to a wide variety of signals. Through successive protein phosphorylation and de-phosphorylation steps, signal transduction cascades mediate between the perception of the extracellular conditions and the adaptation of intracellular processes.

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

In *Botrytis cinerea*, the fungicide fludioxonil activates the Sak1 (Hog1-like) and Bmp3 (Slt2-like) MAPKs, which are respectively involved in osmoregulation, cell wall integrity, development and pathogenicity. In order to trace the transduction of fludioxonil to the MAPK pathways, we have undertaken a comparative proteomics and phosphoproteomics study of the *B. cinerea* osmosensing pathway challenged by the fungicide fludioxonil. We compared mutants of the sensor histidine-kinase Bos1 and of the MAPK Sak1 to the parental wild-type. Strains were exposed (or not) to fludioxonil for 15 min during exponential *in vitro* growth. Shotgun proteomics revealed considerable differences in protein content among the strains, but no treatment effect. These results indicate a strong transcriptional and/or translational regulation under Bos1 and Sak1 control, respectively, under standard conditions. One of the most relevant result is the abundance of 263 proteins controlled by Bos1 and Sak1 independently. Proteins of the oxidative stress response are under negative control of Bos1, while some proteases are positively regulated. In addition, proteins involved in translation are specifically regulated by Bos1. It appears that Bos1 is important in different biological processes. Sak1, on its turn, controls the abundance of proteins involved in oxidative stress response, early secretome, protein folding, primary metabolism and secondary metabolism.

Quantitative phosphoproteomics is ongoing and it will permit to verify a clear response to the fludioxonil treatment as well as (de)phosphorylation events controlled independently by Bos1 or Sak1, or by both kinases.

**430W Identifying targets of Autophagic mRNA decay in S. cerevisiae.** J.F. Garcia<fnref n="1" />, R Parker²,³ 1) Molecular Biology, Colorado College, Colorado Springs, CO; 2) Dept. of Biochemistry, CU Boulder, Boulder, CO; 3) HHMI.

Autophagy is conically thought of as mechanism by which to degrade proteins. However, the autophagy of cytoplasmic RNA granules and rRNAs, and the existence of a ribonuclease in the lysosome or yeast vacuole, suggests that autophagy may also act as a novel mRNA decay pathway. To test this, we performed RNA-seq on total RNA isolated from *S. cerevisiae* that harbor deletions of vacuolar ribonuclease, Rny1, or, a key autophagy factor, atg15, to identify potential mRNA targets of autophagic mRNA decay. Our analysis of mRNAs isolated from exponentially growing rny1? cells revealed that mRNA levels remained unchanged by the removal of Rny1. When mRNA was isolated from rny1? cells grown past the post-diauxic shift, a subset of mRNAs increased in abundance when Rny1 was not present. This suggested that Rny1 acts to modulate levels of mRNAs in a temporal manner. Approximately 189 mRNAs with increased levels in rny1? and atg15? post-diauxic cells were identified as potential targets for autophagic mRNA decay. Interestingly 40% of mRNAs within this subset are mRNAs that encode for proteins associated with mitochondria function. Further computational analysis showed that this subset of potential mRNA targets tended to be preferentially translated on the mitochondria surface, or localized to mitochondria. Lastly, rny1? displays slight respiration defects. Though further analysis into the mechanism by which Rny1 and autophagy selectively regulate mRNAs that encode for mitochondrial proteins needs to be done, our current findings suggests that mRNAs associated with the mitochondria are preferentially degraded by Rny1 during the post-diauxic phase through a novel autophagic mRNA decay mechanism.

**431T Evidence for a demethylase-independent role of KdmB, a chromatin regulator of secondary metabolism in Aspergillus nidulans.** S. Bachleitner<fnref n="1" />, L. Studt<fnref n="1" />, A. Gacek-Matthews<fnref n="1" />, M. Sulyok², J. Strauss<fnref n="1" /> 1) Division of Microbial Genetics and Pathogen Interactions, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Science Vienna, Campus Tulln Austria; 2) Center for Analytical Chemistry, Department of Agrobiotechnology, BOKU-University of Natural Resources and Life Science Vienna, Campus Tulln, Austria.

Fungi produce a plethora of secondary metabolites (SMs), low-molecular-weight compounds involved in cellular protection, defence and signalling. The genes involved in the biosynthesis of these substances are usually physically linked in gene clusters and tightly regulated to prevent an unnecessary use of resources. Efficient transcription of the clusters involves posttranslational modifications (PTMs) of histones which influence the status of chromatin in and around these gene clusters. The exact molecular mechanisms by which histone PTMs influence silencing and activation of SM genes, however, remains largely unknown. Previously, we showed that KdmB, a member of the KDM5-family of jumonji histone demethylases is a regulator of genome-wide H3 lysine 4 trimethylation (H3K4me3) and functions in both gene repression and activation. Specifically, normal induction of several SM-associated genes depend on KdmB. To determine whether the demethylase domain of KdmB is required for SM gene activation we expressed a KdmB mutant carrying point mutations in the highly conserved catalytic JmjC domain and subsequently analysed SM-gene expression and chromatin marks in selected gene clusters and their associated SM metabolite levels. H3K4me3 marks did not differ much between wild type and the JmjC mutant at the selected SM gene loci in agreement with the generally low levels of this PTM found previously by ChIP-seq even in active SM genes<fnref n="1" />. Interestingly, the integrity of the JmjC-demethylase domain is required for the activation of most studied SM gene clusters suggesting a structural role of this domain for the putative activation regions containing PHD-type and other Zn-fingers. However, KdmB-mediated SM gene repression was also found to be independent of the JmjC domain suggesting that KdmB also acts as a recruitment platform for other repressors.

<footnote n="1">¹Gacek-Matthews et al., 2016 PLoS Genet. 12:e1006222</footnote>
**432F** A CRE1-regulated cluster is responsible for light dependent production of Dihydrotrichotetronine in *Trichoderma reesei*. Alberto Alonso Monroy¹, Eva Stappler¹, Andre Schuster², Stefan Böhmdorfer³, Rainer Schuhmacher⁴, Michael Sulyok⁴, Monika Schmoll¹ 1) AIT - Austrian Institute of Technology GmbH, Health & Environment, Bioresources, Konrad-Lorenz-Straße 24, 3430 Tulln, AUSTRIA; 2) TU Wien, Institute of Chemical Engineering, Research Area Molecular Biotechnology, Gumpendorfer Straße 1a, 1060 Vienna; 3) University of Natural Resources and Life Sciences Vienna, Department of Chemistry, Division of Chemistry of Renewable Resources, Konrad-Lorenz-Straße 24, 3430 Tulln, AUSTRIA; 4) University of Natural Resources and Life Sciences Vienna, Department for Agrobiotechnology (IFA-Tulln), Center for Analytical Chemistry, Konrad-Lorenz-Straße 20, 3430 Tulln, AUSTRIA.

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse *Trichoderma reesei* (syn. *Hypocrea jecorina*), regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Investigation of genes regulated in response to light in *T. reesei* and their distribution within the genome revealed several genomic clusters. One of them comprises genes involved in secondary metabolism and is regulated by the carbon catabolite repressor CRE1 in a light dependent manner as well as by photoreceptors. High performance thin layer chromatography (HPTLC) revealed clear differences in secondary metabolite production in light and darkness as well as changing patterns in mutants of this cluster. Two polyketide syntheses as well as one monooxygenase were found to be involved in production of Dihydrotrichotetronine and Trichodimerol in a light dependent manner. Accordingly, these genes show a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing mechanism for the products of these enzymes. The transcription factor residing within the cluster regulates the cluster genes in a light dependent manner, but transcriptome analysis shows that also other secondary metabolism related clusters are targeted. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected that suggests sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

**433W** ChIP-seq analysis of the velvet protein PcVelA from *Penicillium chrysogenum*: Methyltransferase PcLlmA is a downstream regulator of fungal development. U. Kück¹, K. Becker¹, S. Ziemons¹, K. Lentz¹, M. Freitag² 1) Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Bochum, DE; 2) Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA.

A velvet multisubunit complex was previously described for *Penicillium chrysogenum*, the major industrial producer of the beta-lactam antibiotic penicillin. A core component of this complex is *velvet A* (PcVelA), a global regulator, which is highly conserved within filamentous euascomycetes. In *P. chrysogenum*, PcVelA regulates secondary metabolite production, hyphal morphology, conidiation, and pellet formation. Here we present a detailed chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of PcVelA. We provide experimental evidence that PcVelA acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in *P. chrysogenum*, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major applied relevance.

Becker K, Ziemons S, Lentz K, Freitag M, Kück U (2016) Genome-wide ChIP-seq analysis of the velvet protein PcVelA identifies methyltransferase PcLlmA as a novel downstream regulator of fungal development. mSphere 1(4):e00149-16

**434T** Genome-wide ADAR-independent A-to-I RNA editing is generally adaptive for sexual reproduction in *Neurospora crassa*. Huquan Liu¹, Yang Li², Daipeng Chen¹,², Qinhu Wang¹, Cong Jiang¹, Jin-Rong Xu² 1) Plant Pathology, Northwest A&F University, Yangling, Shaanxi, P. R. China; 2) Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America.

ADAR enzymes mediated A-to-I editing is the most prevalent RNA modification in animals. Recently, A-to-I editing was reported to occur specifically during sexual reproduction in *Fusarium graminearum*. Here, we identified over 41000 A-to-I sites that specifically occurred during sexual stages in the model filamentous fungus *Neurospora crassa* by strand-specific RNA-seq with hyphae, wild-type perithecia harvested 3, 4, 5, and 6 days-post-fertilization (dpf), and 5-dpf perithecia of the *stc1* and *sad-1* mutants that are defective in ascus or ascospore development. RNA editing events were found in premeiotic stage but increased significantly during ascus development and ascospore formation. Many phase-specific A-to-I sites and sites with different editing levels across sexual stages were also identified, suggesting that these may affect stage-specific functions. Over 81% of the A-to-I sites in CDS regions are nonsynonymous recoding events, which is significantly higher than synonymous one (18.7%). In addition, the editing level of recoding sites is also significantly higher than that of synonymous sites, with the tendency of the higher the editing level, the higher percentage of recoding events. These results indicate that recoding events are generally beneficial, and editing sites with high editing levels may be more beneficial than those with low editing levels. RNA-seq analysis also showed that A-to-I editing occurred in sexual stage of *N. tetrasperma*. Approximately 52% of the *N. tetrasperma* editing sites are conserved in *N. crassa*, of which 454 were also conserved in *F. graminearum*. The conserved editing sites tend to have higher editing levels and frequency of recoding events, suggesting that the conserved recoding events are likely functionally important and have adaptive advantages during evolution. Consistent with this observation, recoding editing was found to be enriched in genes important for sexual reproduction and meiotic cell cycle. Furthermore, most of the genes encoding enzymes or proteins important for RNA silencing, DNA methylation, and histone modifications are subjected to extensive recoding editing, suggesting that the RNA editing may have important regulations to other known epigenetic phenomena in *N. crassa*. Overall, *N. crassa* has great genetic resources to further characterize the molecular mechanism and function of RNA editing in filamentous fungi. Several genes with conserved editing sites were selected for functionally characterization.
**435F** ChIP-seq analyses of the α-domain transcription factor MAT1-1-1 in *Penicillium chrysogenum* point towards a dose-dependent regulation of target genes. *T.A. Dahlmann*, K. Becker, S. Krevet, U. Kück Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44801 Bochum, Germany.

The filamentous ascomycete *Penicillium chrysogenum* is the main industrial producer of the β-lactam antibiotic penicillin and is therefore of great economical and medical importance. Like many related fungi, it has been considered as asexual for more than a century. However, it was recently demonstrated that *P. chrysogenum* is able to complete a heterothallic sexual life cycle [1]. In *Penicilli*, sex is determined by the mating-type ideomorphs *MAT1-1* and *MAT1-2*, each characterized by presence of a single gene, called *MAT1-1-1* and *MAT1-2-1* [2]. Deletion and overexpression of these genes resulted in various phenotypic changes and functional characterization of both proteins revealed functions beyond sexual development. Most interestingly, deletion and overexpression of *MAT1-1-1* resulted in similar phenotypes. To identify target genes of transcription factor MAT1-1-1 (MATα1) and to enlighten the genetic reasons for the observed phenotypes, ChIP-seq experiments of a GFP-tagged MAT1-1-1 protein were performed. Up to now, these studies were only performed under overexpression conditions using a strong constitutive promoter [3]. We have now extended our analysis using the native MAT1-1-1 promoter. Under high overexpression conditions 238 MAT1-1-1 target sites were identified [3]. 139 of these were now validated and 98 additional targets were found exclusively under control of the native promoter. These results point towards a dose-dependent regulation of MAT1-1-1 target genes by this important regulator representing a master switch for development and metabolism.

[1] Böhm J, Hoff B, O'Gorman CM, Wolfers S, et al.: Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus *Penicillium chrysogenum*. PNAS 2013, 110:1476-81

[2] Hoff B, Pöggeler S, Kück U: Eighty years after its discovery, Fleming's *Penicillium* strain discloses the secret of its sex. *Eukaryot Cell* 2008, 7:465-70

[3] Becker K, Beer C, Freitag M, Kück U: Genome-wide identification of target genes of a mating-type alpha-domain transcription factor reveals functions beyond sexual development. *Mol Microbiol* 2015, 96:1002-22

**436W** The root endophyte *Piriformospora indica* modulates growth and stress response in *Glycine max*. *R. Bajaj*, K.E.

Bushley Department of Plant and Microbial Biology, University of Minnesota, Saint Paul, MN.

*Piriformospora indica* is an endophytic symbiotic fungus, which was isolated from rhizosphere of the xerophytic woody shrubs from the Thar deserts of Rajasthan, India. This fungus colonizes plant roots endophytically to promote plant growth while also providing additional benefits of biotic (e.g. pathogens and herbivores) and abiotic (e.g. drought, salinity, heat) stress tolerance to a wide range of host plants. This study investigated the effects of *P. indica* on soybean (*Glycine max*) in a biological growth chamber study that included a uninoculated Control (C), 2% (w/w) *P. indica* (Pi), and 2% (w/w) *P. indica* + Nutrient Hoaglands solution (Pi + N). Plants were harvested 60 days after germination and root colonization was determined using lactophenol cotton blue. Transcriptome analysis was performed on roots of two biological replicates for each treatment plus *P. indica* grown in liquid PDA. Differential gene expression analyses were performed using Tophat and Cuffdiff. GO ontology (GO) and metabolic pathway enrichment analyses were performed using Soybase tools and SoyCyc, respectively. Results confirmed 41.47% and 28.89%, colonization in Pi and Pi + N treatments, respectively. Colonization with *P. indica* also significantly impacted plant growth and nutrient status, increasing shoot dry weight while decreasing root dry weight and increasing macronutrients (P, N, K) in shoots. Differential expression analyses showed that biosynthetic pathways and GO categories pertaining to lignin biosynthesis and regulation of iron transport were highly enriched in the C vs. Pi comparison. The C vs. Pi + N comparison also yielded significant upregulation of genes and pathways pertaining to cell wall biogenesis/lignin biosynthesis, iron/transition metal homeostasis, as well as response to drought and salinity stress. Higher colonization rates in the C vs. Pi treatment were associated with downregulation and over-representation of various plant defense categories, namely response to chitin, respiratory burst, and salicylic acid and jasmonic acid signaling/biosynthesis as well as genes involved in response to heat and high-light intensity. Overall, these results suggest that, in the presence of the root endophyte *P. indica*, the soybean plant induces a positive growth and abiotic stress response while down-regulation major plant defense pathways, potentially indicative of a measured accommodation to the endophytic symbiont.

**437T** Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing. Yunkun Dang¹, Jiasen Cheng², Xianyun Sun³, Zhipeng Zhou¹, Yí Liu¹ 1) UT Southwestern Medical Center, Dallas, TX. USA; 2) Huazhong Agricultural University, Wuhan, Hubei, China; 3) Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We previously showed that antisense transcription triggers Dicer-independent small interfering RNA (disiRNA) production and disiRNA loci DNA methylation (DLDM) in *Neurospora crassa*. Here we show that the conserved exonuclease ERI-1 is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and to recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that in the cytoplasm ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long non-coding RNAs-mediated epigenetic modifications and identify ERI-1 as an important regulator of co-transcriptional gene silencing and post-transcriptional RNA metabolism.

**438F** The SAGA complex mediates the transcriptional up-regulation of antiviral RNA silencing in the chestnut blight fungus. Ida Bagus Andika, Atif Jamal, Hideki Kondo, *Nobuhiro Suzuki* Institute of Plant Science and Resources, Okayama University, Kurashiki, JP.

RNA silencing, which is conserved across eukaryotic organisms, functions as an innate antiviral defense with the key components including Dicer with RNase III-like dsRNA cleavage activities and Argonaute with RNase H-like activities. Although RNA silencing-related genes, if not all, are known to be transcriptionally up-regulated upon virus infection in some host organisms, little is known about its induction mechanism. In this regard, a phytopathogenic ascomycete, *Cryphonectria parasitica* (chestnut blight fungus) and its viruses provide a particularly advantageous system to study RNA silencing activation, because its infection by certain RNA viruses highly induces the transcription of dicer-like 2 (*dcl2*) and argonaute-like 2 (*agl2*), two major antiviral silencing players. In this study, we developed a screening protocol to identify host factors governing activation of antiviral RNA silencing in *C. parasitica*. The protocol entails multiple
sequential transformations of the host fungus with a cDNA copy of a mutant of the prototype hypovirus lacking the RNA silencing suppressor (CHV1-Dp69), a reporter construct consisting of an enhanced green fluorescent protein gene under the control of the *dcl2* promoter, and a random mutagenic construct with a drug resistance gene. Screening for GFP-negative colonies allowed for identification of *sgf73*, a component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, known to be a transcriptional co-activator of a subset of genes. Knockout of other known SAGA components showed that the histone acetyltransferase module regulates transcriptional induction of *dcl2* and *agl2*, while the histone deubiquitinase is involved in regulation of only *agl2*. Interestingly, full-scale induction of *agl2* and *dcl2* by CHV1-Dp69 required both DCL2 and AGL2, whereas that by another RNA virus, mycoreovirus 1 (MyRV1), required only DCL2, supporting the previously proposed positive feedback by DCL2 and AGL2 to this RNA silencing activation. Taken together, these results showed that SAGA mediates the regulation of virus-induced activation of RNA silencing in *C. parasitica*.

**439W Nuclear accumulation of the *Aspergillus nidulans* GATA transcription factor AreA is independent of DNA binding.** *C. Hunter*¹, M. Hynes², J. Fraser², D. Clarke², M. Davis², R. Todd¹ 1) Department of Plant Pathology, Kansas State University, Manhattan, KS; 2) Department of Genetics, The University of Melbourne, Melbourne, AU.

The primary regulator of nitrogen metabolic genes in fungi is the GATA transcription factor AreA. Subcellular localization of AreA in *A. nidulans* is dependent on nutrient availability. When glucose and nitrogen sources are available, AreA is found throughout the cytoplasm and the nucleus. When the cell becomes nitrogen starved, AreA accumulates in the nucleus due to blocked AreA nuclear export. We have recently found redundancy in the nuclear localization signals (NLSs) of AreA. Five conserved canonical NLSs and one conserved noncanonical arginine-based bipartite NLS within the zinc-finger DNA binding domain all function together to mediate nuclear import. Only mutation of the noncanonical bipartite NLS within the DNA binding domain had any significant effect on AreA function, completely abolishing transcriptional activity. In order to determine whether AreA DNA binding affects the intracellular localization of AreA we have HA-epitope-tagged and analyzed the two classical DNA binding mutants; an altered DNA binding specificity mutant, AreA102, and a non-binding mutant, AreA217. The AreA102HA mutant protein showed a similar pattern of subcellular localization to wildtype AreA except when transferred to uric acid (a nitrogen source the *areA102* mutant cannot utilize). On uric acid AreA102HA accumulated in the nucleus as observed during nitrogen starvation. The AreA217HA non-binding mutant protein accumulated in the nuclei of nitrogen-starved cells, demonstrating that DNA binding is not required for AreA nuclear accumulation. AreA217HA does not show nuclear accumulation when ammonium is present. In contrast to wildtype, AreA217HA accumulated in the nucleus when alternative nitrogen sources were available. These findings suggest that while nuclear accumulation of AreA is independent of DNA binding, nuclear export is dependent on AreA DNA binding function. We show using nitrogen metabolism mutants that this is likely due to the requirement of AreA for metabolic signaling of nitrogen availability.

**440T Elucidation of the role of the truncated version of Cre1 (Cre1-96) in cellulase production in *Trichoderma reesei*.** *Alice Rassinger*, Thiago Mello-de-Sousa, Robert Mach, Astrid Mach-Aigner Institute of Chemical Engineering, Vienna, AT.

Rut-C30 is a cellulase-hyperproducing *Trichoderma reesei* (*T. reesei*) strain and, consequently, became the ancestor of most industry strains used in the production of plant cell wall-degrading enzymes, in particular cellulases. Due to three rounds of undirected mutagenesis its genetic background differs from the wild-type QM6a in many ways, of which two are the lack of a 83 kb large sequence in scaffold 15 and the partial lack of the gene encoding the Carbon catabolite repressor 1 (Cre1). Nonetheless, it remained still unclear, whether the truncated protein Cre1-96 has remained with any functional feature of a transcription factor and if its role remained repressing or changed to an activator. The truncated protein Cre1-96 has been left with a zinc finger domain and a nuclear localization signal *in silico*. On the one hand, the DNA binding was investigated within the Cre1-target genes (*xyr1*, *cbh1* or *xyn1*). On the other hand, trans-regulatory effects were further elucidated such as differences in cellulase activity and chances in the DNA accessibility in cellulase encoding promoters (e.g. *pcbh1* and *pcbh2*). To this end, it should further the understanding of the function of a putative new transcription factor in *T. reesei*. The gained knowledge could be used for further strain improvement.

**441F RNA-editing on transcription factors in model mushroom *Coprinopsis cinerea*.** *Y. Xie*, J. Chang, H.-S. Kwan School of Life Sciences, the Chinese University of Hong Kong, Shatin, NT, HK.

Introduction. RNA-editing is an unusual form of post-transcriptional event which can recodes the hereditary information by changing the nucleotide sequence of RNA molecules. It diversifies the transcriptome and proteome of the organism by several means. To date, research on RNA-editing in fungi is rare. *Coprinopsis cinerea*, a typical mushroom, is one of the model organisms to study homobasidiomycetous fungi. It has a short life-cycle and is easy to be cultivated in laboratory. The complete genome sequence of *C. cinerea* has been released. Aims. To identify RNA-editing sites and editing pattern in *C. cinerea*. Also, to determine the influence of RNA-editing on transcription factors (TFs) and the expression of downstream genes.

Methods. We first sequenced and analyzed the total RNA collected from mycelium of medium cultured *C. cinerea* to predict RNA-editing candidates. The RNA was then reverse transcribed with specific primers which target on the predicted sites. cDNA prepared were sequenced separately using Sanger method. To confirm the RNA-editing event, we aligned the sequencing read to the reference genome. Expression level is measured by real-time PCR.

Results. *In silico* analysis of RNA-seq data revealed that several TFs, involving in *CAZymes* expression, kinases expression and other cell activities, are likely to have RNA-editing. We have identified one TF that one of the base of its mRNA is edited from A to I. This editing event changes the expression level of downstream genes. We also looked into the mechanism of the editing event. However, no ADAR gene was found. The genome of *C. cinerea* has 3 predicted ADAT genes but the function has not been confirmed.

Discussion. In this study, we uncovered one A-to-I RNA-editing site on the transcription factor of *C. cinerea*, which is the first report of RNA-editing in this model mushroom. Here, we have not analyzed the editing pattern of the whole genome or different developmental stages. RNA-editing can help to improve the diversity of gene expression products in an organism, which means that it may help the creature to survive under stress environments or provide extra diversities in protein coding genes. Stage-specific RNA-editing events and stress-stimulated RNA-editing events worth further studies.
**442W Crosstalk of LaeA and CreA in controlling virulence and secondary metabolism in *Penicillium expansum*.** *J. Tannous*¹, X. Luo², D. Kumar³, S. Barad³,⁴, Y. Chen⁵, A. Dubey³, N.G. Matana³,⁴, S. Tian⁵, B. Li⁵, D. Prusky³, N. Keller¹,²,⁶ 1) Department of Medical Microbiology and Immunology, University of Wisconsin - Madison, Madison, WI 53706-1598, USA; 2) Department of Plant Pathology, University of Wisconsin - Madison, Madison, WI 53706-1598, USA; 3) Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel; 4) Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel; 5) Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; 6) Department of Bacteriology, University of Wisconsin - Madison, Madison, WI 53706-1598, USA.

The plant pathogenic fungus *Penicillium expansum* is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Although the patulin gene cluster has been recently identified and characterized, little is known on how it is regulated. Here we demonstrate that LaeA, a global regulator of mycotoxins in other fungi, is also required for the production of not only patulin but also other putative toxic secondary metabolites (SMs) produced by *P. expansum* as well as virulence on apple. We find that an increase of the sucrose molarity in the culture medium from 15 to 175 mM positively impacts *creA* expression, but conversely, decreases the expression of *laeA* and the accumulation of patulin by 5 and 175 fold, respectively. The increase in sucrose also negatively influences the expression of most of the SM synthase genes in *P. expansum* Pe-D1 strain. Independently, LaeA was deemed as a positive regulator of at least 6 SM gene clusters. Here we address the hypothesis that CreA regulation of *laeA* mediates many of the ΔlaeA phenotypes including regulation of SM clusters and virulence on apple.

**443T AP-1-type transcription factor PcYap1 is involved in regulation of penicillin biosynthesis in *Penicillium chrysogenum*.** *W. Perez Perez*¹, U. Carrasco Navarro¹, J. Barrios Gozalez¹, M. Gutierrez Ruiz², F. Fierro Fierro¹ 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.

AP-1 transcription factors are found in eukaryotes from yeasts to mammals, they contain a bZIP domain, regulate transcription of genes in response to a variety of stimuli and their highest affinity binding sequence is TGACTCA. In yeasts, a family of eight transcription factors, named Yap, was described which showed structural similarities to AP-1 factors and whose preferred DNA binding sequence is TTACTAA. *Yap1* is involved in the oxidative stress response, and orthologs of this gene has been described in filamentous fungi participating in the same process. In *Penicillium chrysogenum*, a transcriptional activator of the penicillin biosynthetic gen *pcbAB* was detected by electrophoretic mobility shift assay (EMSA) which bound the sequence TTAGTAA in the promoter of the gene, but its identity remained unclear. In this work, we cloned the *Yap1* ortholog of *P. chrysogenum* (*PcYap1*), expressed and purified the His-tagged *PcYap1* protein from *Pichia pastoris* and tested its ability to bind the *pcbAB* promoter region containing the sequence TTAGTAA. We also attenuated the expression of *PcYap1* by RNAi and studied its effect on different processes.

**Results:** The PcYap1 transcription factor, purified by Nickel-affinity chromatography, bound and caused a mobility shift to a 28 bp DNA probe from the *pcbAB* gene promoter containing the TTAGTAA sequence. No binding was observed when a probe containing the mutated sequence CTAGTCA was used. Then we analyzed the role of PcYap1 in the cell by attenuating its expression through the RNAi pathway. For this purpose, a 458 bp DNA fragment from the exon 2 of *PcYap1* was introduced in the vector pGpdPki-RNAi to generate a dsRNA in *P. chrysogenum* transformants. The transformants showed reduced conidiation, lower tolerance to H₂O₂ and decrease in penicillin production. **Conclusion:** We have identified the transcription factor PcYap1 of *P. chrysogenum* as the transcriptional activator binding to the TTAGTAA sequence in the *pcbAB* promoter. Our results suggest that PcYap1 mediates regulation of *pcbAB* transcription by oxidative stress.

**444F Deciphering the regulatory network between SREBP pathway and secretory pathway in *Neurospora crassa*.** *L. Qin, M Reilley, V Wu, N.L. Glass* Energy Biosciences Institute, The University of California, Berkeley, California, USA 94720.

Sterol regulatory element binding proteins (SREBPs) are first found in mammalian cells and their major function is regulating cholesterol uptake and synthesis. Recent studies demonstrate the SREBP pathway is conserved in fungi. In addition to regulate the sterol homeostasis, fungal SREBPs have been reported to be involved in hypoxia adaptation and virulence. Furthermore, our previous study demonstrates that disruption of SREBP pathway in *Neurospora crassa* and *Trichoderma reesei* results in hyper production of cellulases. However, how fungal SREBPs mediate the protein secretion is unknown and has never been investigated so far. To address this question, in this study, we identified the homolog components of SREBP pathway in *N. crassa* and utilized the next generation RNA-Seq technology to compare the transcriptional profiling of WT, Δsre-1, Mclr-2 and MΔsre-1 strains under sucrose in which the expression of cellulases genes are repressed in WT and Δsre-1 but induced in Mclr-2 and MΔsre-1. Our results demonstrated that the function of SREBP in ergosterol biosynthesis and hypoxia response was conserved in *N. crassa*. In addition, our data revealed that SREBP pathway was highly activated in cellulase induction condition and the activation of SREBP limited the expression of genes encoding cellobiose dehydrogenase and most of the copper-dependent polysaccharide monooxygenase. Moreover, a set of genes which potentially involved in protein modification, sorting, and trafficking were repressed by sre-1. Furthermore, disruption of SREBP pathway can restored the growth defect of ?hac-1 mutant under cellulose condition. The analyses presented here demonstrated new insights into how SREBPs regulated protein secretion and provided new clue for genetic engineering of filamentous fungal organisms to improve their production of extracellular proteins.

**445W Introns regulate gene expression in *Cryptococcus neoformans*.** *G. Janbon* Institut Pasteur, Paris, FR.

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

**446T Analysis of Clp1-dependent UPR-modulation in *Ustilago maydis*. N. Pinter**, K. Heimel Department of Molecular Microbiology and Genetics, Georg-August-University, Göttingen, Lower Saxony, DE.

The unfolded protein response (UPR) is conserved eukaryotic signaling pathway to counteract endoplasmatic reticulum (ER) stress through extensive restructuring of the secretory pathway. UPR activation is mediated by unconventional splicing of *cib1* mRNA, encoding the central UPR regulator in the corn smut fungus *Ustilago maydis*. During the life cycle of *U. maydis* the UPR is inactive during saprophytic growth but specifically activated after successful penetration of the host. We identified Cib1 (Clp1 interacting bZIP1) to physically interact with Clp1, an important developmental regulator of *U. maydis* and the decisive factor for the induction of fungal proliferation after successful host penetration. Both genes are crucial for pathogenic development and deletion strains are blocked in development after penetration of the leaf surface. Cib1-Clp1 complex formation results in stabilization of both Clp1 and Cib1 and alters the transcriptional program of the UPR, rendering cells hyper-resistant towards ER-stress. Since a constitutive active UPR is deleterious for cell growth, we hypothesize that Clp1-mediated modulation of the UPR prevents deleterious UPR hyperactivation to facilitate long-term UPR activation during biotrophic growth in planta. Transcriptional profiling of Clp1-mediated UPR modulation and subsequent gene deletion analysis identified a previously uncharacterized UPR target gene, being specifically required for biotrophic growth of *U. maydis*. While growth under axenic conditions and ER-stress resistance is not affected, deletion strains are fully avirulent in infection assays. Our results suggest that the induction of proliferation in planta and modulation of the UPR pathway are connected processes to re-shape the secretory pathway and align fungal development with the cellular physiology.

**447F Transcriptional Regulation in haploid cells of *Ustilago maydis* may tie together the High Affinity Ammonium Transporter, Ump2, and the mating program. R. Margaret Wallen**, Kirsten Richardson, Madison Furnish, Allison Dentinger, Michael H. Perlin Biology, University of Louisville, Louisville, KY.

*Ustilago maydis* is a biotrophic pathogen of maize. In order to cause disease in the host, the fungus must undergo a dimorphic switch from a yeast-like, budding state to a filamentous state that allows for plant penetration and propagation of the infection. Different environmental cues can induce filamentous growth and some, specifically low nitrogen availability, are essential for mating to occur in nature. In haploid wild type strains of *U. maydis*, low nitrogen availability also results in a non-infectious filamentous growth response. Mating is governed by the coordinated expression of genes from the mating loci in *U. maydis*, the *a* locus and the *b* locus. The *b* locus in particular must play an additional role in haploid cells, as deletion of the locus prevents filamentation due to nitrogen limitation. Previous work found that the *U. maydis* genome encodes two ammonium transports (AMTs), Ump1 and Ump2. While deletion of *ump1* does not yield an observable phenotype, deletion of *ump2* in haploids results in the loss of filamentous growth as a response to low nitrogen, similar to what is observed with deletion of the *b* locus. Overexpression of the high affinity ammonium transporter, Ump2, results in filamentation under non-inducing conditions and can rescue the filamentation defect of the *b* locus deletion mutant. In light of this discovery, we hypothesized that Ump2, like its homolog in *Saccharomyces cerevisiae*, acts as a transceptor, both transporting ammonium across the membrane and signaling low ammonium availability; furthermore, we here present evidence that functional Ump2 is required for transcriptional regulation related to mating, and that there is interaction at the transcriptional level between *ump2* and the *b* locus.

**448W The master is switched off, long live the new master: phase specific hierarchical transcriptional control in *Ustilago maydis*. J. Ulrich¹, J. Fortenbacher¹, K. Heimel¹,², F. Finkernagel³, J. Kämper¹**
1) Karlsruhe Institut of Technology, Institute for Applied Biosciences, Karlsruhe, GER; 2) Georg-August-University-Göttingen, Institute for Microbiology and Genetics, Göttingen, GER; 3) Philipps-University Marburg, Institute for Molecular Biology and Tumor Research, Marburg, GER.

In *Ustilago maydis*, the change from yeast-like saprophytic growth to the formation of pathogenic filamentous hyphae is controlled by a closely interconnected and cross-controlled network of transcription factors. The network integrates pheromone signaling during the mating-associated cell fusion events as well as the subsequent onset of the biotrophic development by formation of the bE/bW heterodimeric transcription factor complex. bE/bW activation triggers the expression of Rbf1, the master regulator for most bE/bW-regulated genes. bE/bW expression alone is sufficient to trigger pathogenic development, ectopic Rbf1 expression at least initiates pathogenic development, stressing the central role of both factors. During plant penetration, function of both proteins is inhibited by the interaction with Clp1; at later stages, expression of the “master regulator” Rbf1 is not detectable.
Via a combination of microarray-data and ChipSeq analysis, we show the complex combinatorial control of bE/bW, Rbf1 and Clp1 of genes during the switch to pathogenic development on various promoters. Both promoter-binding as well as expression data show that at later stages the two transcription factors Biz1 and Hdp2, both essential for plant infection, overtake the central function of Rbf1. Apparently, *U. maydis* uses distinct sets of regulators to integrate and/or differentiate the different signals before and after infecting its plant host.

**449T Utilizing next generation sequencing to revitalize a forward genetic screen for mutants deficient in the production of stergigmatocystin in *Aspergillus nidulans*. B. T. Pfannenstiel¹, K. J. Affeldt², J. Bok², R. A. E. Butchko³, T. Choera², G. J. Fischer¹, B. P. Knox², F. Y. Lim², X. Luo⁴, A. A. Soukup¹, J. E. Spraker⁴, K. Throckmorton¹, P. Wiemann², N. P. Keller²,⁵, J. M. Palmer⁶**
1) Department of Genetics, University of Wisconsin-Madison, Madison, WI, 53706, USA; 2) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, 53706, USA; 3) Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA; 4) Department of Plant Pathology, University of Wisconsin-Madison, Wisconsin 53706; 5) Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA; 6) Center for Forest Mycology Research, Northern Research Station, U.S. Forest Service, Madison, WI 53706, USA.

The study of aflatoxin regulation in *Aspergillus* spp. has warranted the attention of many researchers due to its carcinogenic properties and frequency as a food and feed contaminant. Significant progress on the regulation of the AF cluster has been conducted in the model organism *Aspergillus nidulans* by assessing regulation of the stergigmatocystin (ST) gene cluster as ST is the penultimate precursor of AF. A screen using a chemical mutagen identified 23 loci involved in regulating ST production. Only six of these loci were characterized from this screen using classical mapping (*mcsA*) and complementation with a cosmid library (*laeA*). Recently the remaining mutants were
backcrossed and sequenced using an Ion Torrent PGM™. Each mutant contained one or more SNPs in predicted genes. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 out of the 17 remaining mutants. Three of the causative mutations were in uncharacterized genes unknown to be involved with ST production, of which two appear to regulate the ST cluster via transcriptional regulation of its cluster specific transcription factor, *aflR*. Based on protein domains and homologs, the remaining uncharacterized gene has a predicted role in the plasma membrane fusion. Nine mutations were in genes already known to affect ST synthesis (*laeA*, *mcsA*, *fluG*, *stcA*).

**450F Local rates of nucleosome turnover are influenced by transcription and chromatin modifications in Neurospora crassa.** M. Kamei, A. Courtney, Z. Lewis Department of Microbiology, University of Georgia, Athens, GA.

Nucleosome turnover occurs when old nucleosomes are removed from the chromatin fiber and replaced with new nucleosomes. Turnover happens regularly during nuclear processes such as transcription, DNA replication, and DNA repair. Several studies have shown that different genomic regions exhibit different rates of nucleosome turnover, yet the molecular basis of differential histone turnover is poorly understood. We developed a transgenic strain of *N. crassa* that enables us to measure rates of new histone incorporation across the genome. Specifically, we replaced the *N. crassa*-1 locus with a single, epitope tagged copy of histone H3 (H3-FLAG) under control of the *tcu-1* gene promoter. Expression of the H3-FLAG transgene is tightly regulated by the presence or absence of copper in the growth medium. We induced H3-FLAG for 0, 2, 4, or 8 hours and performed ChIP-seq (Chromatin-immunoprecipitation followed by Illumina sequencing) using anti-FLAG antibodies to enrich for sites that preferentially incorporate newly synthesized H3-FLAG. Induction of the transgene led to rapid incorporation of H3-FLAG in the promoters and +1 nucleosomes of highly expressed genes, indicating high rates of nucleosome turnover at these sites. As expected, the rate of nucleosome turnover in heterochromatin was low. Heterochromatin formation depends on DIM-5, an H3K9 methyltransferase. We found that dim-5 strains exhibit increased chromatin accessibility in silent heterochromatin domains and discrete heterochromatic loci displayed increased rates of nucleosome turnover relative to wildtype. Currently, we are analyzing nucleosome turnover rates in additional mutants that are defective for specific chromatin modifying activities. We will summarize our progress in identifying chromatin determinants of nucleosome turnover.

**451W Pervasive adenine N6-methylation of active genes in fungi.** S.J. Mondo¹, R.O. Dannebaum¹, R.C. Kuo¹, A. Gryganskyl², J. Magnuson³, T.Y. James⁴, M.A. O'Malley⁵, J.E. Stajich⁶, J.W. Spatafora⁷, A. Visel¹, I.V. Grigoriev¹ 1) Fungal Genomics, Joint Genome Institute, Walnut Creek, CA; 2) L. F. Lambert Spawn Co, Coatesville, PA; 3) Pacific Northwest National Laboratory, Richland, WA; 4) Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI; 5) Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA; 6) Department of Plant Pathology and Microbiology, University of California, Riverside, California; 7) Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.

Epigenetics plays a critical role in regulation and expression of genetic information. Many important epigenetic marks are encoded as DNA-based modifications, for example 5-methylcytosine (5mC) or N6-methyldeoxyadenine (6mA). 6mA is a non-canonical eukaryotic DNA base modification reported genome-wide in only four species: an alga, *Chlamydomonas reinhardtii* and three animals, *Drosophila melanogaster*, *Caenorhabditis elegans* and *Mus musculus*. However, the genome-wide abundance of 6mA in these organisms ranges from low (0.4% of all A's in *C. reinhardtii*) to extremely low (0.001%-0.07% in *D. melanogaster*). Despite low levels, 6mA is critical for proper development, as it plays an important role in regulation of gene expression. Regrettably, exploration of 6mA thus far has been limited to single model eukaryotes and therefore its relevance in many major branches of Eukaryota is still unknown. Here we conduct the first kingdom-wide exploration of both 6mA and 5mC, where we interrogate the Fungi, an ancient and extremely diverse eukaryotic lineage. We found heavy utilization of 6mA in early-diverging fungi where up to 2.8% of all adenines were methylated, vastly exceeding the levels observed in other eukaryotes and more derived fungi, whereas 5mC was primarily in the CpG context and restricted to repeats. Interesting, with one exception 6mA and 5mC were mutually exclusive (i.e. lineages with high 6mA had no 5mC and vice versa). 6mA occurred symmetrically at ApT dinucleotides, where it concentrated in dense 6mA 'islands' at gene promoters. Islands were positioned nearby promoter thymine-blocks and ~95% of methylated genes were expressed. Our results demonstrate the importance of 6mA as a broadly conserved epigenomic mark in eukaryotes as well as identify key components involved in positioning of genomic 6mA. Furthermore, our results implicate 6mA as an epigenomic mark transmissible across nuclear division and suggest its involvement in nucleosome organization. We anticipate that our study will provide a foundation for deeper exploration of 6mA not only in fungi, but across all Eukaryota.

**452T Genome-wide analysis of the GATA transcription factor AreA in Aspergillus nidulans.** A. Li¹, Z. Miao¹, D.J. Downes², G.Y. Busot², R.B. Todd², K. Wong¹ 1) Faculty of Health Sciences, University of Macau, Macau, CN; 2) Department of Plant Pathology, Kansas State University, KS.

In *Aspergillus nidulans*, nitrogen utilization is precisely regulated at the gene expression level. Expression of genes encoding enzymes to utilize alternative nitrogen sources or to scavenge nitrogen compounds during nitrogen starvation is dependent on the GATA transcription factor AreA. In general for typical nitrogen metabolic genes, AreA-dependent activation is minimal during nitrogen sufficiency, increased when nitrogen is limiting, and strongest during nitrogen starvation. Although many AreA target genes are known, the genome-wide targets of AreA and AreA DNA binding properties remain unknown. To address this, we profiled AreA DNA binding under nitrogen sufficiency, nitrogen limitation and nitrogen starvation via Chromatin Immuno-precipitation followed by Sequencing (ChIP-seq) and identified more than 1,600 promoters bound by AreA. Interestingly, AreA binds to more than 400 promoters under nitrogen sufficiency, when its protein level in the nucleus is low. Expression analysis shows that less than 5% of these targets are affected by *areA* deletion. In contrast, under nitrogen starvation expression of more than 40% of AreA bound genes is significantly altered by *areA* deletion. These results indicate that AreA binds to many target promoters poised to activate upon appropriate signaling. Comparisons of targets among the different nitrogen conditions revealed many (~200) common binding sites as well as nitrogen source-specific AreA binding events, indicating that AreA targets different promoters according to nitrogen sources and availability. De novo motif discovery analysis showed that besides "GATA", several other motifs are also enriched among AreA bound regions, consistent with cooperation of AreA with pathway-specific transcription factors. In addition to nitrogen metabolism, Gene Ontology analysis of AreA target genes suggests roles in development and stress response. Moreover, our data also provides compelling evidence for positive feedback control of AreA on the co-repressor NmrA that
negatively regulates its function. Therefore, this work not only reveals the genome-wide functions of AreA, but also uncovers a new
regulatory loop for nitrogen metabolism repression in *A. nidulans*.

**453F Evidence for inducer function of 2-keto-3-deoxy-L-galactonate in D-galacturonic acid induced gene expression in Aspergillus niger.** *E. Alazi*¹, *C. Khosravi*², *T.G. Homan*¹, *M. Arentshorst*¹, *M. Di Falco*³, *M. Peng*², *M.V. Aguilar Pontes*², *A. Tsang*³, *J. Visser*¹, *R.P. de Vries*², *A.F.J. Ram*¹ 1) Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, The Netherlands; 2) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, The Netherlands; 3) Centre for Structural and Functional Genomics, Concordia University, Canada.

Pectin is an important carbon source for the saprotrophic fungus *Aspergillus niger*, which is an efficient producer of pectin-degrading enzymes. D-galacturonic acid (GA) is the main product of pectin degradation. In *A. niger*, GA is transported into the cell by the sugar transporter GatA and subsequently catabolized into pyruvate and glycerol through a pathway consisting of four enzymes: GaaA, D-galacturonate reductase; GaaB, L-galactonate dehydratase; GaaC, 2-keto-3-deoxy-L-galactonate aldolase; and GaaD, L-glyceraldehyde reductase. It has been shown that GA or a derivative of GA is required for the induction of the genes needed for pectin degradation, GA transport, and GA catabolism. In order to identify the GA derivative that acts as an inducer, we constructed GA catabolic pathway deletion mutants (?gaaA, ?gaaB, ?gaaC and ?gaaD) anticipating that the mutants would accumulate the substrate of the deleted enzyme when grown on GA. The growth of both ?gaaB and ?gaaC was abolished on GA pointing out that there are no redundant enzymes replacing GaaB and GaaC. ?gaaB and ?gaaC, pregrown in fructose medium and then transferred to GA medium, accumulated pathway intermediates L-galactonate and 2-keto-3-deoxy-L-galactonate, respectively. Northern analysis showed that the expression of the GA induced genes is drastically reduced in ?gaaB and highly increased in ?gaaC compared to the wild type strain. Genome wide gene expression analysis via RNA-seq indicated that the accumulation of 2-keto-3-deoxy-L-galactonate in ?gaaC results in the induction of the GA induced genes. Identification of the inducer of the pectinase genes, together with the recent identification of the transcriptional activator-repressor module controlling pectinase expression would facilitate the industrial use of *A. niger* in pectinase production.

**454W Multidrug resistance caused by a gain-of-function mutation in a novel fungal specific transcription factor in Sclerotinia homoeocarpa.** *H. Sang*¹, *J. Hulvey*², *R. Green*¹, *T. Chang*³, *G. Jung*¹ 1) Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA; 2) Biology Department, University of Massachusetts, Amherst, MA; 3) School of Ecology and Environmental System, Kyungpook National University, South Korea.

The ongoing emergence of multidrug resistance (MDR) in human and plant pathogenic fungi threatens global human and plant health. To combat multidrug resistant fungal pathogens, understanding the MDR mechanisms is a crucial step, but is limited in the filamentous plant pathogenic fungi. In this study, we used genomics and molecular genetic tools to uncover the key factors for MDR mechanisms in the filamentous ascomycete fungus *Sclerotinia homoeocarpa* (the causal agent of dollar spot on turfgrass). We showed evidence that overexpression of cytochrome P450s (CYP450s) in drug metabolism and ATP-binding cassette (ABC) transporters in drug secretion are involved in MDR. Moreover, a gain-of-function mutation in a fungal specific transcription factor detected from the field MDR isolates is responsible for constitutive and induced overexpression of the CYP450s and ABC transporters, resulting in resistance to structurally different chemicals. Our establishment of a molecular mechanism for the MDR pathway in this fungal pathosystem has implications for controlling plant pathogenic fungi and possibly human pathogens.

Pathogenic and Mutalistic Interactions

**455T Hidden in Plain Sight: Heterogeneity in Asexual Spores of Aspergillus fumigatus.** *S.E. Kang*, *M. Momany* Department of Plant Biology, University of Georgia, Athens, GA.

*Aspergillus fumigatus* spores are found ubiquitously in the environment. The asexual clonal spores (conidia) produced in the environment are inhaled where they can germinate and cause high mortality rates in immunocompromised patients. Germination of conidia is the most crucial step in the development of aspergillosis. Given that conidia are produced in a variety of environments, we tested whether sporulation conditions impact the ability of conidia to break dormancy and grow. Spores from a single colony were bulked on complete media and cultured with environmentally- and medically-relevant stress conditions. Spores from each condition were collected and transferred to all other conditions. Germination and growth kinetics were analyzed for 25,000 conidia from each condition swap experiment using flow cytometry. We found that clonal populations of conidia are heterogeneous for germination and growth phenotypes and the stress increases this phenotypic heterogeneity.

**456F Aspergillus fumigatus carbon catabolite repression is essential for virulence in established infection microenvironments.** *S. Beattie*¹, *K. Mark*², *L. Reis*³, *S. Dhingra*¹, *C. Black*⁶, *C. Cheng*²,⁴,⁵, *G. Goldman*³, *R. Cramer*¹ 1) Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH USA; 2) Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH USA; 3) Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil; 4) Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH USA; 5) Institute for Quantitative Biomedical Sciences, Geisel School of Medicine at Dartmouth, Lebanon, NH USA; 6) Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH USA.

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

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

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

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

**457W The A. fumigatus Fumiquinazoline C is potentially cytotoxic to macrophages and soil amoeba Dictyostelium discoideum.** Marina Campos Rocha¹, Taicia Pacheco Fill², Juliana Issa Hori³, Lilian Pereira Silva⁴, João Henrique Tadini Marilhano Fabri ¹, Anderson Ferreira da Cunha¹, Gustavo Henrique Goldman⁴, *Iran Malavazi*¹ 1) Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, Brazil. ; 2) Instituto de Química, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. ; 3) Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil. ; 4) Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

Fungi remarkably produce a variety of secondary metabolites as a consequence of different environmental stimuli. These compounds can ultimately provide fitness attributes to the producing organism. Recently, we characterized two components of the *A. fumigatus* cell wall integrity pathway (CWI), *pkcA* and *rlmA* and observed that in addition to the cell wall related-phenotypes, the perturbation of the signaling circuit coordinated by the PkcA-MpkA-RlmA module impacts on the production of fumiquinazolines (Fq). FqC is the major Fq produced by *A. fumigatus* which accumulation was associated with conidia formation. Here we show that *pkcA*<sup>G579R</sup> and Δ*rlmA* mutant strains produce lower FqC (24.7% and 27.9%, respectively) and that FqC concentrations were 10.5- fold lower in the Δ*mpkA* strain. This decrease is accompanied by global down-regulation in mRNA expression of the Fq cluster genes during the asexual development. Aiming to understand if other cell stresses could influence the production of FqC, we performed a screening using different null mutants and found that the deletion of the transcription factor SebA, (primarily involved in heat shock and oxidative stress) overproduced FqC (about 4.5-fold increase) indicating that *sebA* is a negative regulator of FqC production. *A. fumigatus* is sensitive to FqC and this tolerance is decreased in the CWI pathway mutants and increased in the Δ*sebA* strain. In addition, FqC can induce pore formation on the membrane of macrophages and highly stimulates the secretion the pro-inflammatory cytokine TNF-α by this cell type. We also used the soil amoeba Dictyostelium discoideum to study the phagocytic interaction of this organism with conidia from the Δ*sebA* strain. Interestingly, conidia of the Δ*sebA* were significantly less phagocytized by *D. discoideum* and the opposite occurred when conidia from the CWI pathway mutants were tested. Our results suggest that Fq production is regulated at different levels in *A. fumigatus* and that FqC can serve as a defense compound against other microorganisms or soil predators. Although we could not detect FqC in the lung of infected mice, this molecule is potentially cytotoxic to fungi and mammalian cells.

**458T Culture independent detection of Aspergillus fumigatus Cyp51A in human lung.** Aiah Khateb¹,², Michael Bromley², Paul Bowyer² 1) Faculaty of Applied Sciences, Medical Laboratory Technology, University of Taibah, Medina, Kingdom of Saudi Arabia ; 2) Fungal Infection Group, Centre for Respiratory Medicine and Allergy, Faculty of Biology, Medicine and Health,Institute of Inflammation and Repair, Manchester Academic Health Science Centre, The University of Manchester and University Hospital of South Man.

**Background** *Aspergillus fumigatus* is the main causative agent of aspergillosis. Traditional culture methods recover a small fraction of all the lung resident species and therefore resistant isolates are usually missed. Long-term triazole oral therapy, particularly itraconazole and voriconazole has been used in treatment of chronic pulmonary Aspergillosis or allergic aspergillosis. Azole resistance is associated with poorer disease outcome in allergic and chronic disease and has increased in recent years. This study aims to detect resistant mutations in *cyp51A* in the context of uncultured *Aspergillus* strains present in clinical samples.

**Methods** DNA was extracted directly from 38 BAL samples (8 healthy controls, 9 ABPA, 8 SAFS, 7 SA and 3 MA). The *cyp51A* gene was directly amplified from extracted DNA and amplicons sequenced using illumina Miseq. Sequence analysis was done using the following software pipeline; trimmomatic 0.36>wbowtie2>samtools1.2>bcftools htslib 1.3.1.>VarScan.

**Results** 61 SNPS were identified, 29 resulted in amino acid change. Three SNPs matched cyp51A mutations that have previously been shown to confer azole resistance; M220I/M220T and F46L. 9 further mutations occurred only in azole treated patients; N218T, F219Y, N218Y, M220T, M220I, I217S, A62C, I135T, T112I and 17 were common to sequences obtained from patients with no azole treatment history.

**Conclusions** This study has identified new *cyp51A* mutations in the healthy, chronically colonized patients. It also broadened our understanding of *cyp51A* resistance mechanisms in the context of uncultured *Aspergillus* strains present in clinical samples.

**459F Experimental evolution of A. fumigatus in hypoxia results in increased virulence.** C.H. Kowalski¹, A.K. Caffrey², S.R. Beattie¹, J.J. Obar¹, J.E. Stajich³, R.A. Cramer¹ 1) Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; 2) Department of Immunology & Infectious Diseases, Montana State University, Bozeman, Montana, USA; 3) Department of Plant Pathology & Microbiology and Center for Plant Cell Biology, University of California-Riverside, Riverside, California, USA.

**Purpose:** The ability of microbes to thrive in hypoxic environments is important for virulence. Hypoxia fitness of *Aspergillus fumigatus* strains strongly correlates with virulence in murine models of invasive aspergillosis (IPA). Despite this observation, the molecular mechanisms of hypoxia adaption and its mechanistic link to virulence remain elusive. Here we aim to utilize an experimental evolution approach to elucidate underlying mechanisms of hypoxia and host fitness.

**Methods:** Conidial populations of a low virulent, hypoxia fitness deficient strain of *A. fumigatus* were passaged in hypoxia for 80 days on glucose minimal medium. Single spore clones from the population were isolated and phenotypic, virulence, genomic and transcriptomic sequencing, and metabolic analyses were carried out in comparison to ancestral strains.

**Results:** After 80 days in hypoxia, an experimentally evolved strain, EVOL20, was isolated that displays increased fitness in hypoxia highlighted by metabolic derepression, altered cell wall composition, and acidification of the culture media. Interestingly, EVOL20 is more
virulent in murine models of IPA and has altered immunopathogenesis in immune competent mice. Transcriptomic analysis reveals large scale expression changes in EVOL20 compared to the ancestor in hypoxia; with expression changes greater than 4-fold in 172 genes (55 increased, 112 decreased). Genome sequence analysis of EVOL20 identified 106 synonymous SNPs and only 3 nonsynonymous SNPs in 3 uncharacterized genes. Molecular genetic characterization of these genes in the context of hypoxia fitness and fungal virulence is underway.

**Conclusions:** Experimental evolution in hypoxia of a low virulent *A. fumigatus* strain with reduced hypoxia fitness dramatically improved *A. fumigatus* virulence compared to the ancestor strain. We conclude that hypoxia selection pressure may promote evolution of *A. fumigatus* virulence mechanisms.

## 460W Full length and five short segments of TRI6 RNA interference constructs that reduce deoxynivalenol production in Fusarium graminearum reveal consistent patterns of siRNA and different silencing efficiencies.

Thomas Baldwin, Phil Bregitzer National Small Grains Germplasm Research Facility, USDA-ARS, Aberdeen, Idaho, United States of America.

Deoxynivalenol (DON) contamination of small grains caused by Fusarium head blight is a problem of economic and health importance that may be addressed by RNAi interference (RNAi)-based host-induced gene silencing (HIGS). RNAi in higher eukaryotes, including fungi, involves processing double stranded RNA (dsRNA) into small interfering RNA (siRNA) that silence genes based on homology. Six transgenic *F. graminearum* (strain PH1) mutants containing the RNAi vector pTRM-TRI6 were studied, each containing a full length inverted repeat of *TRI6* inserted in a different genomic position. *TRI6* is a transcription factor that positively regulates DON production. The mutant lines showed drastic reductions of DON production in infected plant tissues and in TBI media, and reduced virulence on wheat. The sRNA populations of three mutant lines had abundant siRNA species, not present in PH1, that mapped to *TRI6*, and ~22 nt siRNA was the most abundant size class. There was a discontinuous and repeatable pattern among the mutants of *TRI6*-specific siRNA. The 5'-most base of 83% of siRNAs was uracil, indicative of dsRNA processing by a fungal dicer. Subsequent experiments, were conducted with additional mutants in which one of five shorter (200-250 nt) *TRI6*-specific inverted repeats (each targeting a different region of *TRI6*). The pattern of siRNA processing for each individual inverted repeat was similar to the pattern of the full-length construct for the corresponding section of *TRI6*. All mutants with short constructs showed reduced expression of a *TRI6*-regulated gene, *TRI5*, on TBI media, but not as much as observed for the full length construct. Dicer patterns for dsRNA processing have implications for design of efficient RNAi silencing vectors. Understanding the siRNA profiles that result from RNAi constructs is critical to optimizing RNAi applications that are designed to reduce pathogenicity and mycotoxin production in the field.

## 461T The fungal nitrooxidative stress response suppresses rice innate immunity during blast disease.

Margarita Marroquin-Guzman, *Richard Wilson* Plant Pathology, University of Nebraska at Lincoln, Lincoln, NE.

Understanding how microbes manipulate plant innate immunity and colonize host cells is a major goal of plant pathology and crop health improvement. Here, we report that the *Magnaporthe oryzae* nitrooxidative stress response quells host defenses and facilitates fungal growth and development in rice cells. Nitronate monooxygense (Nmo) catalyzes the oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrite. The *M. oryzae* genome carries five *NMO* genes. *NMO2* expression was subject to nitrogen metabolite repression by Nut1, and carbon catabolite repression by Tps1. Deleting *NMO2* revealed, counter intuitively, that the resulting Δ*nmo2* mutant strain was nitrate (NO₃⁻) and nitrite (NO₂⁻) non-utilizing. We determined this was because Δ*nmo2* strains, relative to wild type (WT), could not mitigate nitrooxidative cellular damage caused by reactive nitrogen species by-products of NO₃⁻ and NO₂⁻ metabolism. Δ*nmo2* mutant strains were also sensitive to the NO generator nitroprusside and reactive oxygen species (ROS). On plants, Δ*nmo2* mutant strains formed functional appressoria that penetrated rice cuticles, but growth of invasive hyphae (IH) in underlying epidermal cells was restricted and accompanied by granular depositions in rice cells, host ROS accumulation and the induced expression of rice pathogenesis-related (PR) genes *PBZ1* and *PR-1*. Δ*nmo2* strains expressing Bas4 fused to GFP and the cytoplasmic effector Pwl2 fused to mCherry:NLS showed that both effectors were expressed in rice cells. This signified that the elevated host defense responses elicited by Δ*nmo2* mutant strains did not kill the fungus. However, whereas Pwl2:mCherry:NLS expressed in WT IH localized to a single, punctate blast interfacial complex (BIC) (as reported previously), Pwl2:mCherry:NLS in Δ*nmo2* was localized to multiple foci throughout IH. Inhibiting the host ROS burst by treatment with diphenyleneiodonium (DPI), or quenching host ROS with sodium ascorbate, avoided triggering the rice immune response in cells infected with Δ*nmo2* mutant strains. This allowed Δ*nmo2* mutant strains to grow and develop normal IH with single BICs per host cell. Thus, *NMO2* is required for maintaining redox balance in host cells in order to avoid triggering plant defenses that restrict the growth and development of *M. oryzae* in rice. This work provides new knowledge on molecular plant-microbe interactions and, more broadly, identifies a new requisite for NO₃⁻ metabolism in fungi.

## 462F A novel Botrytis cinerea MFS transporter provides tolerance to glucosinolate breakdown products.

D. Vela-Coria, A. Dafa-Berger, O. Barda, M. Levy Hebrew Univ Jerusalem, Plant Pathology & Microbiology, Rehovot, Israel.

Glucosinolates are secondary metabolites that accumulate mainly in cruciferous plants. Glucosinolates and their hydrolytic products have antifungal properties and play a role in plants' resistance against pathogens. Botrytis cinerea, a necrotrophic pathogen, has variable sensitivity to glucosinolates. Here we studied the mechanisms of tolerance to glucosinolates in the fungus B. cinerea. Exposure of B. cinerea to glucosinolate breakdown products induced expression of Botrytis cinerea major facilitator superfamily transporter suggested to be involved in Isothiocyanates detoxification (Bcmfsl). B. cinerea inoculated on wild-type Arabidopsis thaliana or on IQD1<sup>OE</sup> plants, transgenic for high glucosinolate level, activated Bcmfsl expression to higher levels than B. cinerea on glucosinolate-deficient A. thaliana mutants cyp79B2/79B3. B. cinerea strains lacking functional MFSI (ΔBcmfsl) were more sensitive to glucosinolate breakdown products in vitro and in planta. ΔBcmfsl strains were less virulent on wild-type Arabidopsis plants, but not on glucosinolate-deficient mutants cyp79B2/B3. Furthermore, Saccharomyces cerevisiae expressing the Bcmfsl gene was more resistant to the fungitoxic glucosinolate breakdown products. We demonstrate here that the B. cinerea MFSI transporter is a virulence factor that increases tolerance of the pathogen to glucosinolates. We also demonstrate that Bcmfsl can confer tolerance to hydrolytic products of glucosinolate in the yeast S. cerevisiae.
**463W Antifungal activity of the human gut metabolome.** C.E. Garcia-Rangel¹, F Tebbji¹, MC Daigneault², NN Liu³, JR Ko'hler³, E Allen-Vercoe², A Sellam¹ 1) Infectious Diseases Research Centre-CRI, CHU de Québec Research Center, Quebec City, Quebec, Canada; 2) Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada; 3) Boston Children's Hospital/Harvard Medical School, Boston, MA, USA.

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

**464T Phagosomal neutralization by the fungal pathogen *Candida albicans* induces macrophage pyroptosis.** S. Vylkova¹,², Michael Lorenz³ 1) ZIK Septomics, Friedrich-Schiller University, Jena, Thuringia, DE; 2) Hans-Knöll Institute, Jena, Thuringia, DE; 3) University of Texas Health Science Center at Houston, Houston, TX, USA.

The interaction of *Candida albicans* with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that *C. albicans* co-opts amino acid catabolism to generate and excrete ammonia, which raises extracellular pH, both in *vitro* and *in vivo* and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking *STP2*, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungal-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking *AHR1* were defective in growth, alkalization and ammonia release on amino acid-rich media, similar to *stp2*? and *ahr1Δstp2Δ* cells, and occupied more acidic phagosomes. Notably, *ahr1*? and *stp2*? strains did not induce pyroptosis as measured by Caspase-1-dependent IL-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that *C. albicans*-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of Caspase-1 mediated macrophage lysis.

**465F Mapping host-pathogen transcriptional dynamics using sorted cell populations of *Candida albicans*-infected macrophages.** J.F. Munoz¹, T. Delorey², C.B. Ford¹, D.A. Thompson¹, R.P. Rao², C.A. Cuomo¹ 1) Broad Institute of MIT and Harvard, Cambridge, MA; 2) Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA.

Encounters between immune cells and *Candida albicans*, a member of the healthy human microbiome and a major pathogen in immunocompromised individuals, determine the course of infection. Phagocytes such as macrophages are key players during *C. albicans* infection, and their interactions have diverse outcomes based on microscopic observation; however, little is known about the molecular contribution of these heterogeneous subpopulations to the infection process. To characterize the variability that may be important for infection outcome, we developed a system to sort distinct subpopulations during *C. albicans*-macrophage interactions using fluorescent markers, and simultaneously profile the transcriptional responses of the host and pathogen using RNA-sequencing. Here we show we can successfully isolate macrophages and yeast cells interacting extracellularly and macrophages with phagocytized live or dead fungus. Using this system, we trace the phenotypic and expression variability for distinct infection fates. We have identified specific sets of *C. albicans* genes in sorted subpopulations that show a coordinated response needed for the commensal to pathogenic transition, and the adaptation and survival of the fungus in the nutrient-limited and stressful environment within the macrophages. Simultaneously, we examined the transcriptional profile of macrophage subpopulations, and characterize the coordinate host immune response, including several pathways and regulatory genes needed for the migration, pathogen recognition, activation of engulfment, cellular communication, and phagocytosis. This system offers finer scale analysis compared to bulk population studies and enables examination of different host-pathogen phenotypes and their distinct expression variability to better understand the evolution of drug resistance, host adaptation and immunomodulatory effects.

**466W Functional genomic analysis reveals fungal modulators of host immune responses.** Teresa O'Meara, Kwamaa Duah, Leah Cowen Molecular Genetics, University of Toronto, Toronto, Ontario, CA.

*Candida albicans* is a leading human fungal pathogen that causes life-threatening systemic infections. The first line of defense against this pathogen is the innate immune response. Macrophages readily engulf *C. albicans*, and the engulfed *C. albicans* cells subsequently undergo a morphogenetic switch from yeast to a filamentous growth. This change in morphology is temporally coupled to the induction of macrophage pyroptosis, an inflammatory host cell death program mediated via activation of NLRP3 and caspase-1. Recently, we discovered that it is the remodeling of the fungal cell wall in response to the macrophage environment that exposes the trigger of host cell
death, not the filament itself. Further, we found that heat-killed previously phagocytized *C. albicans* cells could drive macrophage lysis, unlike their counterparts that had not been phagocytized prior to killing, suggesting that this fungal cell wall remodeling is sufficient to drive macrophage lysis, and that the remodeling occurs specifically in response to the macrophage phagosome. However, the specific trigger and mechanisms involved remain enigmatic.

To address this question, we developed a high-content imaging platform to monitor interactions between *C. albicans* and macrophages, and have leveraged this platform to identify *C. albicans* mutants that are defective in pyroptosis. Using murine macrophages expressing an ASC-fluorescent protein (FP) reporter, we quantified ASC-FP paranuclear specks, which are indicative of NLRP3 inflammasome activation. Our primary screen allows us to simultaneously quantify not only pyroptosis, but also *C. albicans* filamentation within macrophages. Genes required for filamentation in the macrophage cannot be predicted *a priori* given that distinct genes regulate filamentation in response to distinct cues. Our initial analysis of 96 filamentation-competent conditional expression strains that enable transcriptional repression of cell wall genes identified 34 genes that are important for activation of pyroptosis. This included genes that encode glucan biogenesis enzymes, mannosyltransferases, and GPI-anchored proteins, and their potential regulators. Together, we have identified novel circuitry regulating fungal induction of host immune cell death.

**467T The active site of trehalose-6-phosphate synthase in *Candida albicans* impacts growth at elevated temperatures, hyphal growth in serum, and biofilm formation.** J.L. Tenor¹, Yi Maio²,³, D.L. Toffaletti¹, S.A. Maskarinec¹, R.G. Brennan², J.R. Perfect¹ 1) Division of Infectious Diseases, Department of Medicine, Duke University School of Medicine, Durham, NC; 2) Department of Biochemistry, Duke University School of Medicine, Durham, NC; 3) Present address: Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA.

The trehalose pathway is commonly found in fungi with putative roles in glycolytic regulation, signaling, protection against stressors, and energy by serving as a carbon source. The enzyme, trehalose-6-phosphate synthase (Tps1), is responsible for the first committed step in the trehalose biosynthetic pathway. In *C. albicans*, TPS1 affects hyphae formation in serum, growth at elevated temperatures, and survival within macrophages. We have recently solved the crystal structure of CaTps1. Structural analysis of CaTps1 has identified key amino acid residues predicted to impact its activity. Site-directed mutagenesis of 4 residues of the active site were created (Y89F, K285A, D379A, D387A) and resulted in loss or reduced ability to grow at elevated temperatures, defective hyphal growth in serum at 37°C, and abnormal biofilm formation. Mutagenesis of 2 residues predicted to effect dimerization, E341 (E341R) and E364 (E364R), exhibited wild-type phenotypes. The absence of the trehalose pathway in humans and the importance of this pathway for *C. albicans* and other fungal pathogens to cause disease provides us with an attractive target for antifungal drug development.

**468F *Debaryomyces hansenii* may antagonize *Candida* yeasts in the human gut.** H.E. Hallen-Adams, N. Banjara Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE.

Over a century of studies, more than 250 species of fungi have been reported from the human gut. Fungi detected in the healthy human gut vary widely between individuals, and within the same individuals at different time points. However, much of this variation is illusory, as many of the fungi detected in fecal samples are environmental or foodborne and incapable of growth under the conditions in the human GI tract. A much smaller pool of organisms is commonly detected in the human gut, and these are the taxa to consider as true members of the gut ecological community. In addition to colonizing fungi, some species may be unable to persist long term but nevertheless be commonly detected and play a role in gut ecology due to dietary or environmental exposure. *Saccharomyces cerevisiae*, reported in 20 of 37 studies published between 1917 and 2016, is the classic example. The foodborne yeast *Debaryomyces hansenii* was reported in 7 of 37 studies, and in 18 of 85 fecal samples profiled individually, where its presence was negatively correlated with that of *Candida albicans*, *C. tropicalis* and other *Candida* species. More than half of the *D. hansenii* strains we have isolated from cheese show killer activity against *C. albicans* and *C. tropicalis* in vitro. *D. hansenii* neither grows nor produces killer toxin at 37 C and crude extracts of the toxin lose their killer activity after a few hours at elevated temperature, presumably due to proteolysis. Killer toxin in cheese naturally colonized with *D. hansenii* retains its anti-*Candida* activity for several days, suggesting a mechanism by which *D. hansenii* could affect gut fungal dynamics.

**469W Purification and characterization of a symbiosis-induced endocellulase from the ectomycorrhizal symbiont *Laccaria bicolor*.** F Zhang¹, C Champion², M Haon², G Anasontzis², M Kemppainen³, A Pardo³, Y Daguerre¹, A Deveau¹, C Veneault-Fourrey¹, A Kohler¹, MN Rosso², B Henrissat⁴, JG Berrin², F Martin¹ 1) UMR 1136 INRA-Université de Lorraine 'Interactions Arbres/Microorganismes', Laboratoire d'Excellence ARBRE, Centre INRA-Lorraine, 54280, Champenoux, France; 2) UMR 1163 INRA-Biodiversité et Biotechnologie Fongiques, Polytech Marseille, Faculté des Sciences de Luminy, 13 009, Marseille, France; 3) Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Provincia de Buenos Aires, Argentina; 4) CNRS, UMR 7257 & Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Marseille, France.

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